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Bisognin has been accepted towards fulfillment of the requirements for Ph.D. degree inPlant Breeding & Genetics 3%. Major professor Date % é 0400/ v MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 6/01 c:/ClRCJDateDue.p65—p.15 INTROGRESSION OF LATE BLIGHT RESISTANCE FROM WILD SPECIES AND UNADAPTED GERMPLASM TO CULTIVATED POTATO by Dilson A. Bisognin A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Crop and Soil Sciences 2001 ABSTRACT Introgression of Late Blight Resistance from Wild Species and Unadapted Germplasm to Cultivated Potato by Dilson A. Bisognin Since the mid-1990’s, the United States and Canada have experienced late blight (Phytophthora infestans (Mont) de Bary) epidemics caused by new, more aggressive and metalaxyl resistant races that impose new disease management strategies in potato (Solanum tuberosum L.). Breeding offers the opportunity to identify and release advanced germplasm with late blight resistance. The general objective of this research was to introgress late blight resistance from unadapted germplasm and wild species to cultivated potato. The first effort was to combine late blight resistance from eight (BO718-3, Bertita, Bzura, Greta, Libertas, Stobrawa, Tollocan and Zarevo) unadapted cultivars with tuber quality and marketable maturity found in cultivars/advanced breeding clones adapted to North America. A total of 408 field selected clones from 95 crosses were evaluated in single- and eight-hill plots for tuber quality (tuber appearance, specific gravity and chip color). The same clones were assessed for foliar late blight reaction using a mixture of complex races of USS/A2 mating type of P. infestans isolates in greenhouse and field studies in 1998 and 1999. The late blight resistant parents differ in their ability to transmit late blight resistance and tuber quality to the offspring. In addition, late blight resistance can be combined with marketable maturity and tuber quality. Moderate selection intensity for tuber quality traits can be initiated at the single- hill generation before testing for late blight resistance. The second effort was to select, within plant introduction accessions, clones with high levels of late blight resistance in greenhouse. A total of 60 selected clones representing South American species, hybrids between wild and cultivated species and cultivars/advanced breeding clones were than evaluated for their genetic diversity based on isozymes and simple sequence repeats (SSR). There is a high level of genetic diversity within and between accessions, species and ploidy levels of the late blight resistant gerrnplasm from S. microdontum Bitter, S. berthaultii Hawkes and S. sucrense Hawkes that should be introgressed and combined in a breeding. The last effort was to map quantitative trait loci (QTL) conferring late blight resistance and other agronomic traits using isozymes and SSR markers in a S. microdontum derived population. Progeny of 110 clones and parents were field tested for foliar late blight reaction in 1999 and 2000, and for maturity, tuber number and size, yield and tuber quality in 2000. High phenotypic correlation (r = 0.89, P < 0.0001) was found for late blight reaction between years and no correlation was found between late blight with other evaluated trait. There was only one marker linked with late blight resistance and another trait (tuber size). Solanum microdontum has a QTL associated with foliar late blight resistance that is not associated with late maturity or any poor tuber quality traits that explains 70% of the phenotypic variance of two years of field testing. A SSR marker closely linked to the QTL that can be followed through polyploidization is suitable for using in a marker assisted selection strategy. iv Dedicated to my family ACKNOWLEDGEMENTS I would like to express a very special thanks to my major professor and mentor Dr. David S. Douches for believing in my potential and giving me the opportunity to pursue the Ph.D. degree in his breeding program. His incredible enthusiasm, creativity and friendship have been a great source for encouragement making the Ph.D. program much more enjoyable and productive. I am very thankful for the committee members Dr. James Kelly, Dr. Amy Iezzoni, and Dr. Raymond Harnmerschmidt for their input and contribution to my research and publication of results. Even not being part of my committee, Dr. William Kirk gave major contributions to all steps of late blight testing. A special thanks to the “potato family”, Lynn Buszka, Kim Walter-Felcher, Kaz Jastrzebski, Joe Coombs, Chris Long, Anne Lund, Beth Leary, Kelly Zarka and Jarred Driscoll. Lynn’s contribution was decisive for completing the mapping studies. Kim was critical in discussing and reviewing the manuscripts. Kaz helped me to fill up my gaps of gerrnplasm development and crossing block strategies. Joe’s expertise brought easy and efficient solutions to all computer issues. Joe’s and Chris’ friendship were very important since my first day at MSU. After all, every one contributed in doing some serious work or being funny during those many hard days of work. I will keep those memories. I am indebted forever with my family for being very supporting and understanding. My wife quit the job to follow my dream and now stop her career to take care of our adorable son. My parents gave their time to raise and teach me the values of love, friendship and perseverance to pursue my goals. My parents in law raised my lovely wife and wonderfirl mother of our son. Thanks for your support and encouragement. Thanks also to my good friends of both the Latin American and the Brazilian Communities for being part of these important moments and supporting us here. Besides fiiends, I also had the extraordinary experience to work with Luis Velasquez and Dr. Irvin Widders with cucurbits, my small but important dream. Afier all, the Ph.D. program is gone, but good friends and good memories last forever. I am very thankful to the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), Ministry for Science and Technology of Brazil for all the financial aid and to the Department of Horticulture, Federal University of Santa Maria for the opportunity of taking my Ph.D. program. Also, thanks to the MSU Plant Breeding and Genetics Program for being part of this highly prestigious group. vi TABLE OF CONTENTS LIST OF TABLES ........................................................................... x LIST OF FIGURES .......................................................................... xii CHAPTER I General introduction ............................................................................. 1 Origin and importance of potato .......................................................... 1 Use of potato gerrnplasm in breeding .................................................... 3 Late blight and its constraints to potato production and breeding .................... 5 Mapping potato for late blight resistance and other important traits .......... . 9 Objectives and dissertation contents ..................................................... 12 Literature cited .............................................................................. 13 CHAPTER I] Half-sib progeny evaluation and selection of potatoes resistant to the USS genotype of Phytophthora infestans from crosses between resistant and susceptible parents 21 Abstract ...................................................................................... 21 Introduction ................................................................................. 22 Material and methods ..................................................................... 24 Crosses, segregating populations and evaluated clones .......................... 24 Characterization of P. infestans isolates and inoculum preparation ............ 27 Late blight reaction in greenhouse tests ............................................ 28 Late blight reaction in field tests .................................................... 28 Maturity and tuber quality evaluations ............................................. 29 Statistical analysis ..................................................................... 30 Results ....................................................................................... 31 Identification of superior parents for late blight resistance breeding ........... 31 vii Grouping and selection of recombinant progeny ................................. 34 Discussion ................................................................................... 38 Literature cited .............................................................................. 42 CHAPTER 111 Early generation selection for potato tuber quality in progeny of late blight resistant parents ............................................................................................ 45 Abstract ...................................................................................... 45 Introduction ................................................................................. 46 Material and methods ..................................................................... 48 Results ....................................................................................... 50 Progeny performance for tuber quality ............................................. 50 Selection of clones with acceptable tuber quality in single-hill generation 55 Identification of clones with acceptable chip-processing or tablestock quality ................................................................................... 59 Discussion ................................................................................... 59 Literature cited .............................................................................. 65 CHAPTER IV Genetic diversity in diploid and tetraploid potato late blight resistant gerrnplasm ..... 68 Abstract ...................................................................................... 68 Introduction ................................................................................. 69 Material and methods ..................................................................... 71 Results and discussion ...................................................................... 75 General genetic diversity in the late blight resistant gerrnplasm ................ 75 Sources of unique allozyme alleles ................................................. 76 Sources of unique microsatellite fragments ........................................ 77 Genetic similarity among late blight resistant clones ............................. 81 Improvements of late blight resistant cultivars with broad genetic base ....... 83 Literature cited .............................................................................. 86 CHAPTER V Mapping late blight resistance and other agronomic traits in a {[(Solanum tuberosum x S. chacoense) x S. phureja] x S. microdontum} population 89 Abstract ...................................................................................... 89 viii Introduction ................................................................................. 90 Material and methods ..................................................................... 92 Selection of resistant parent and mapping population ............................ 92 Late blight reaction in field tests .................................................... 93 Vine maturity and tuber evaluations ................................................ 94 Marker technology ..................................................................... 94 Statistical analysis ..................................................................... 96 Results and discussion ...................................................................... 97 Phenotypic evaluations ............................................................... 97 Marker and linkage analyses ......................................................... 101 Quantitative trait loci analysis ......................................................... 106 Literature cited .............................................................................. 117 CHAPTER VI General discussion and conclusions ........................................................... 121 Literature cited .............................................................................. 127 APPENDICES .................................................................................. 130 LIST OF TABLES TABLE 2.1. Late blight resistant parents and susceptible adapted cultivars/advanced breeding clones were crossed to generate segregating populations ....................................................................................... TABLE 2.2. Number of evaluated clones and foliar late blight reaction for family mean based upon greenhouse and field testing in 1998 ................................... TABLE 2.3. Number of evaluated clones and foliar late blight reaction for family mean and parents based upon greenhouse and field testing in 1999 ..................... TABLE 2.4. Scott-Knott cluster groups of clones and resistant parents differing in late blight resistance in the 1999 field testing and the respective range of relative area under the disease progress curve ........................................................ TABLE 2.5. Foliar late blight reaction, maturity and tuber quality performance of advanced selected clones in the 2000 field evaluation .................................... TABLE 3.1. Progeny performance of eight late blight resistant parents for tuber quality in single-hill plot in 1997 ............................................................. TABLE 3.2. Progeny performance of eight late blight resistant parents for tuber quality in eight-hill plot in 1998 ............................................................... TABLE 3.3. Correlations at the clonal level between single- and eight-hill generations for individual late blight families and combined for all families .......... TABLE 3.4. Percentage comparison of clones with acceptable chip-color quality in single- and eight-hill clonal generations ..................................................... TABLE 3.5. Percentage comparison of clones with acceptable tuber appearance quality in single- and eight—hill clonal generations ........................................ TABLE 3.6. Percentage comparison of clones with acceptable specific gravity quality in single- and eight-hill clonal generations ........................................ TABLE 4.1. Pedigree of Solanum accessions or cultivars used in this study and their origin, ploidy level and clone identification .......................................... 26 32 33 36 37 52 53 54 56 57 58 72 TABLE 4.2. Number of evaluated clones per accession or group of genotypes, number of alleles/locus, proportion of polymorphic loci and expected heterozygosity in the potato late blight resistant gerrnplasm ............................. TABLE 4.3. Unique allozymes present in the unadapted late blight resistant germplasm and absent in cultivated potato ................................................. TABLE 4.4. Unique microsatellite fragments present in unadapted late blight resistant gerrnplasm and absent in cultivated potato ....................................... TABLE 5.1. Phenotypic values of the parents, progeny size and descriptive statistics for foliar late blight reaction and other agronomic traits of a S. microdontum population ........................................................................ TABLE 5.2. Coefficients of correlation among foliar late blight reaction in 1999 and 2000 and other agronomic traits in 2000 ............................................... TABLE 5.3. Simple sequence repeat markers used to map a S. microdontum derived population ............................................................................... TABLE 5.4. Markers associated with multiple evaluated traits in a S. microdontum derived population .............................................................................. TABLE A.1. Late blight families, number of crosses and evaluated clones per family, number and percentage of selected clones with late blight resistance ......... TABLE A.2. Foliar late blight reaction in the greenhouse for the percentage of infected area and in the field for the relative area under the disease progress curve and maturity in 1999 ........................................................................... TABLE A.3. F oliar late blight reaction in the field based on the relative area under the disease progress curve and Scott-Knott cluster groups differing in resistance level in 1999 and 2000 ......................................................................... xi 78 79 80 99 100 102 105 131 138 144 LIST OF FIGURES FIGURE 4.1. Genetic similarity among diploid and tetraploid potato gerrnplasm with reported late blight resistance based on 35 allozymes, encoding ll isozyme loci, and 42 polymorphic DNA fragments in nine pairs of microsatellite primers FIGURE 5.1. Quantitative trait loci associated with foliar late blight resistance in the field of a S. microdontum derived population on linkage group 21 in 1999, 2000 and combined data analysis ................................................................... FIGURE 5.2. Quantitative trait loci associated with vine maturity of a S. microdontum derived population on linkage group 3 in 2000 ............................. FIGURE 5.3. Quantitative trait loci associated With tuber appearance of a S. microdontum derived population on linkage group 1 in 2000 ............................ FIGURE 5.4. Quantitative trait loci associated with specific gravity of a S. microdontum derived population on chromosome III in 2000 ........................... FIGURE 5.5. Quantitative trait loci associated with chip color of a S. microdontum derived population on chromosomes VIII and X in 2000 ................................. FIGURE 5.6. Simple sequence repeat marker Stm0020 fragments separated in 3% metaphor agarose gel run at 100 V for 4.5 hours. A) Parents, bulks and ten resistant and susceptible progeny clones, respectively. Stm0020a linked with resistance and Stm0020b linked with susceptibility, both from resistant parent S. microdontum. B and C) SSR marker Stm0020a followed through polyploidization. Parents of the diploid population, diploid resistant clone of the progeny and tetraploid parent with tetraploid progeny clones ....................................................................... FIGURE AL A) 1998 greenhouse late blight (LB) reaction evaluated as the percentage of foliage infected area for 408 clones from crosses between late blight resistant parents and susceptible ones. B) Comparisons among LB resistant families and check cv. Atlantic. Means followed by the same letter do not differ significantly using Tukey’s multiple range test at P = 0.05 .............................. xii 82 108 109 111 112 113 116 134 FIGURE A2 A) 1998 field foliage late blight (LB) reaction expressed as the relative area under the disease progress curve for 408 clones from crosses between late blight resistant parents and susceptible ones. B) Comparisons among LB resistant families and check cv. Atlantic. Means followed by the same letter do not differ significantly using Tukey’s multiple range test at P = 0.05 ....................... FIGURE A3. A) 1999 greenhouse late blight reaction (LB) evaluated as the percentage of foliage infected area for 119 clones from crosses between late blight resistant parents and susceptible ones. B) Comparisons among LB resistant families and check cv. Atlantic. Means followed by the same letter do not differ significantly using Tukey’s multiple range test at P = 0.05 .............................. FIGURE A4. A) 1999 field foliage late blight (LB) reaction expressed as the relative area under the disease progress curve for 119 clones from crosses between late blight resistant parents and susceptible ones. B) Comparisons among LB resistant families, parents and check cv. Atlantic. Means followed by the same letter do not differ significantly using Tukey’s multiple range test at P = 0.05 FIGURE A.5. Intercrossing scheme to combine late blight resistance from different sources with other important characteristics ................................................ FIGURE A.6. Frequency distribution of a S. microdontum population for foliar late blight reaction in the field in 1999 and 2000 ................................................ FIGURE A.7. Correlation of S. microdontum mapping population for foliar late blight reaction in the filed between 1999 and 2000 (r = 0.82, P = 0.0001) ............. FIGURE A.8. Frequency distribution for vine maturity of a S. microdontum mapping population on a scale 1 to 5 of increasing lateness in 2000 .................... FIGURE A.9. Frequency distribution for yield . hill'1 (kg) of a S. microdontum mapping population in the field in 2000 ..................................................... FIGURE A.10. Frequency distribution for number of tubers . hill'1 of a S. microdontum mapping population in the field in 2000 .................................... FIGURE A.1l. Frequency distribution for tuber appearance of a S. microdontum mapping population on a scale 1 to 5 of increasing defects in 2000 ..................... FIGURE A.12. Frequency distribution for tuber size (g) of a S. microdontum mapping population in the field in 2000 ..................................................... FIGURE A.13. Frequency distribution for specific gravity of a S. microdontum mapping population in the field in 2000 ..................................................... FIGURE A.14. Frequency distribution for chip color of a S. microdontum mapping population on a scale 1 to 9 of increasing darkness in 2000 .............................. xiii 135 136 137 141 142 143 147 148 149 150 151 152 153 CHAPTER I General Introduction Origin and Importance of Potato Potato (Solanum tuberosum L.) belongs to the family Solanaceae section Petota Dumortier. The origin and domestication of the potato occurred in the highlands of Peru and Bolivia 7,000 to 10,000 year ago. Solanum stenotomum Juz. et Bukasov (2n = 24) was the first domesticated species (Hawkes, 1990). There were at least four wild species involved in the evolution of domesticated potato, with S. sparsipilum (Bitter) Juz. et Bukasov (2n = 24) being the most important. A hybridization between S. sparsipilum and S. stenotomum followed by chromosome doubling formed S. tuberosum subsp. andigena Hawkes (2n = 48) (Hawkes, 1990; Hawkes, 1994). Solanum tuberosum subsp. andigena was brought to Chile and to Europe, where it became adapted to long day length evolving into S. tuberosum subsp. tuberosum Hawkes (Hawkes, 1994). There are seven cultivated potato species: two diploid (2n = 24), two triploid (2n = 36), two tetraploid (2n = 48), and one pentaploid (2n = 60) (Hawkes, 1990). Solanum tuberosum is the most important cultivated species with two subspecies. Solanum tuberosum subsp. tuberosum has long-day adaptation with widespread cultivation in temperate and l subtropical climates. Solanum tuberosum subsp. andigena has short-day adaptation and its cultivation is restricted to the Andes of South America (Correll, 1962; Hawkes, 1990). Although potato originated in South America, its worldwide distribution was from Europe at the end of the 16th century (Hawkes, 1990; Hawkes, 1994). In this research, cultivated potato refers to S. tuberosum subsp. tuberosum. In a time frame of about 300 years, cultivated potato arose from a little known crop in the Andes area of South America to become one of the most important food crops (Hawkes, 1990; Hawkes, 1994). The importance of the potato as a food crop relies on its high yield and calorc production, excellent nutrient source, and high proportion of edible biomass (Niederhauser, 1993). The United Nation’s Food and Agriculture Organization (FAO) estimated during the 1996-1998 period that an average of more than 299 million metric tons of potatoes were harvested per year, compared with wheat (595 million metric tons), maize (592 million metric tons) and paddy rice (571 million metric tons). Europe accounted for 50% of the total potato production and Russia, Poland and Germany were responsible for 48% of the European production. Asia accounted for 32% of the total production and China and India were responsible for 75% of that production. South America accounted for 4% of the total world production and Brazil, Argentina, Colombia and Peru were responsible for 89% of that production. North and Central America accounted for 9% of the total production and United States was responsible for 79% of that production (FAQ, 1998). During the 1993-1997 period, the United States annually potato production averaged 20.9 million metric tons and Idaho and Washington responsible for 48% of the national production. Per capita consumption was 63.8 kg, from which 35% went to fresh market and 65% for processing as frozen (63%), chips (18%) and dehydrated (16%). In the 1992-1996 period, potato was an average of 2.4 billion-dollar businesses in the United States (National Potato Council, 1998). Use of Potato Germplasm in Breeding Potato probably has the widest genetic diversity among related wild species than any other cultivated crop plant (Hawkes & Jackson, 1992). Among the 232 potato species recognized by Hawkes (1990), there were 183 with a known number of chromosomes. The vast majority of the species are diploid (136 or 74%) with the rest being triploid (7), tetraploid (27), pentaploid (3), and hexaploid (10). Despite the low level of genome differentiation in most Solanum species (Hawkes, 1994), the gerrnplasm can not all be used directly for breeding due to a combination of ploidy level and endosperm balance number (EBN) incompatibility (Hawkes & Jackson, 1992). Diploid wild species can be directly crossed with dihaploids (2n = 2x = 24) of cultivated potato (Hermsen, 1984; Hermsen, 1994). Dihaploids occur as a result of parthenogenesis (haploid pollinator technique) (Jansky et al., 1990; Hermsen, 1994; Alfano et al., 1999) or anther culture (Tai, 1994). Dihaploid x wild species hybrids can be crossed with cultivated potato via unilateral sexual polyploidization (4x - 2x crosses) using 2n gametes (Hermsen, 1994; Hutten et al., 1994). Two dihaploid x wild species hybrids that produce 2n pollen and Zn eggs can also be crossed leading to bilateral sexual polyploidization (2x - 2x crosses) (Ortiz, 1998; Alfano et al., 1999; Hanneman, 1999). The EBN is an arbitrary cross compatibility classification system that is based on a 2:1 maternal to parental ratio for the endosperrn to be functional in a cross (Ortiz & Ehlenfeldt, 1992; Hanneman, 1999). The diploid species with l EBN are distributed in North America, Central America, while only a few species are found in South America. The diploid species with 2 EBN have restricted distribution in South America and only one species in Mexico. The cultivated S. tuberosum subsp. tuberosum is an autotetraploid species with 4 EBN (Correll, 1962; Hawkes, 1990). Therefore, the knowledge and use of endospenn balance number, dihaploids and Zn gametes made available a wide range of the Solanum genetic diversity accessible to the potato breeders (Peloquin et al., 1989; Hanneman, 1999). Part of the potato gerrnplasm has already been characterized for a number of economically important characteristics. Sources for disease (viruses, fungi, bacteria, and nematodes) and insect resistance, temperature stress (frost and heat), glycoalkaloids and vigor for vine and flowering were reported for 111 tuber-bearing Solanum species (Bamberg et al., 1994). The invaluable potato germplasm also includes sources for chip- processing direct from storage, high solids content, tuber shape and color, production of unreduced gametes, endosperrn viability and improvement of yield potential (Hanneman, 1989). Moreover, fine-screening evaluation has been done for more specific traits such as Colorado potato beetle resistance (Bamberg et al., 1996), tuber calcium (Bamberg et al., 1998), and late blight resistance (Douches et al., 2001) to identify resistant clones within accessions. Germplasm characterization gave the opportunity to develop cultivated primitive diploid and tetraploid potato populations using wild and cultivated species. These populations are sources of genetic diversity for characteristics present in wild species, but with a significantly increased breeding value (Mendoza, 1989). Despite the high genetic diversity that exists in the genus Solanum, cultivated potato has a narrow genetic base. A few introductions of S. tuberosum subsp. andigena with short day adaptation made the initial genetic base for long day adaptation of S. tuberosum subsp. tuberosum in Europe (Hawkes, 1994). Intense breeding efforts further narrowed the cultivated potato gene pool. High genetic similarity characterizes more than 130 potato cultivars released in North America between 1930 and 1970 (Mendoza & Haynes, 1974). The cultivars Katahdin, Early Rose and Garnet Chili (Early Rose’s parent) had a very high genetic contribution to modern North American cultivars (Mendoza, 1989; Plaisted & Hoopes, 1989). Genetic diversity studies using molecular markers confirmed the relatedness among North American potato cultivars (Douches et al., 1991; Demeke et al., 1996; Provan et al., 1996) and also among European potato cultivars (Provan et al., 1999). Therefore, it is very important to increase the genetic diversity of the cultivated potato gene pool. Late Blight and its Constraints to Potato Production and Breeding Late blight is caused by the firngal-like oomycete Phytophthora infestans (Mont) de Bar-y (Kamoun et al., 1999). Phytophthora infestans populations have very high genetic diversity and one-to-one ratio of mating types (A1 and A2) in central Mexico (Toluca Valley), supporting that area as its center of origin (Fry & Spiehnan, 1991). New evidences showed that central Mexico was the center of diversity, but it was not the center of origin (Ristaino et al., 2001). Late blight has assumed importance as a potato disease since the middle of the 18405 (Wastie, 1991), and more recently late blight is present in almost all potato growing areas (Ross, 1986; Henfling, 1987; Kamoun et al., 1999). Late blight is notable because it is the disease that caused the Irish potato famine (Fry & Goodwin, 1997a) and because late blight devastating speed and destructive potential were a major stimulus for the development of the science of plant pathology (Fry & Goodwin, 1997b). Phytophthora infestans causes both foliar destruction and tuber decay (Ross, 1986) and ranks as the most devastating potato disease worldwide (Fry & Goodwin, 1997a; Kamoun et al., 1999). World yield losses in potato caused by P. infestans were estimated to exceed $2 billion annually (Kamoun et al., 1999). Major changes occurred in the P. infestans population composition in the United States from 1991 to 1992 (Goodwin et al., 1995a) and in Canada in the mid-19908 (Peters et al., 2001), “including the appearance of more aggressive and metalaxyl resistant genotypes. The new U88 genotype of P. infestans was shown to be more aggressive on potato foliage (Johnson et al., 1997) and on tuber tissue (Lambert & Currier, 1997). Metalaxyl resistant genotypes were first identified in western Washington in 1990 (Deahl et al., 1993). Since then, the most complex and virulent isolates, including either Al or A2 mating type, were isolated fi'om that location (Deahl et al., 1993; Goodwin et al., 1995b). The occurrence of both A1 and A2 mating types can result in sexual reproduction thus increasing the potential of pathogen evolution and is also necessary for the formation of oogonia that develops into oospores (Deahl et al., 1995; Goodwin et al., 1995a). Oospores can survive in adverse conditions for months or even years (Fry & Goodwin, 1997a) and can be source of primary inoculum in the soil. Oospore formation may increase the amount of fungicide applied to foliage and seed tubers (Dorrance et al., 1999) and may change the actual disease management strategies (Umaerus & Umaerus, 1994). The A2 mating type became more common in United States populations during 1992 and 1993 (Goodwin et al., 1995b) and Canada in 1994 and 1995 (Peters et al., 2001). In the United States, the most aggressive and metalaxyl resistant U88 genotype was found in 23 states in 1994 and 1995 (Fry & Goodwin, 1997a). The late blight epidemic of 1994 in the United States was caused by the U88 genotype (Inglis et al., 1996), the most complex genotype collected in western Washington during 1996 and 1997, which three out of six isolates had ten virulence factors (Dorrance et al., 1999). The high aggressiveness of the U88 genotype of P. infestans was attributed to metalaxyl resistance and/or increase in parasitic fitness (Inglis et al., 1996). Moreover, greater aggressiveness (faster lesion expansion rate and sporulation time) of the U88 genotype of P. infestans compared with U81 genotype explains why US8 genotype required more protectant fungicide for adequate epidemic suppression than U81 genotype (Kato et al., 1997). The average number of fungicide applications for late maturing potato cultivars increased from 2.5 in 1994 to 82-123 in the northern and southern Columbia Basin in 1995, respectively. The total estimated cost to control late blight in Columbia Basin in 1995 was estimated at 30 million dollars (Johnson etal., 1997). An important tool in managing late blight resistance is host plant resistance. Vertical resistance (R-gene or specific) is conferred by R-genes and is effective against a narrow range of pathogen races (Henfling, 1987). Attempts to introgress vertical resistance from S. demissum Lind]. to cultivated potato were initiated in the early 19005 and pursued until at least the 1960’s. These efforts resulted in the development of many late blight resistant potato cultivars (Umaerus et al., 1983). A total of 11 R-genes can be recognized that were introgressed from S. demissum to cultivated potato (W astie, 1991). Vertical resistance offered no solution for the late blight problem because P. infestans evolved and overcame the resistance (Ross, 1986). Horizontal resistance (field, partial or general) is effective against a broad range of pathogen races, may be expressed in different stages in the pathogen cycle from initial infection to production of sporangia (Henfling, 1987; Umaerus & Umaerus, 1994) and seems to be the only durable type of resistance to P. infestans (Colon et al., 1995b; Umaerus et al., 1983; Kamoun et al., 1999). Horizontal resistance was first employed in breeding and was the only type of host resistance available before the identification of R-genes from S. demissum (Umaerus et al., 1983). The level of horizontal resistance to late blight in potato has been increased through recurrent selection (Henfling, 1987), which recombines genes from different sources of resistance to build stronger and more durable resistance to late blight (Colon, 1999). Since 8. demissum exhibits both vertical and horizontal resistance (Wastie, 1991), the presence of horizontal resistance to late blight in cultivated potatoes could be expected. The development of genetic resistance to late blight in potatoes is one of the major objectives of many breeding programs (Colon et al., 1995a). Besides horizontal resistance, breeding for late blight resistance presents more challenges due to the relationships among foliar resistance, tuber resistance and late maturity. No correlation between foliar and tuber resistance was found among progeny of parents differing in both resistances (Stewart et al., 1992), but high correlation was found when using a small sample of parents (Stewart et al., 1994). A strong positive correlation was found between foliar resistance (horizontal resistance) and late maturity (Ross, 1986; Umaerus et al., 1983). Foliar resistance and late maturity were mapped to the same region of chromosome V (Collins et al., 1999; Oberhagemann et al., 1999) and QTLs for early maturity were associated with late blight susceptibility (Ewing et al., 2000). However, QTLs conferring lower levels of resistance in both foliage and tubers were mapped in other regions of the genome and they were not associated with late maturity (Collins et al., 1999). These findings suggest that increasing resistance in the foliage is possible by accumulating genes located in regions of the genome not linked with tuber susceptibility and late maturity. Mapping Potato for Late Blight Resistance and other Important Traits Cultivated potato is highly heterozygous, exhibits tetrasomic inheritance and has inbreeding depression (Bradshaw, 1994). Potato dihaploids have high levels of self- incompatibility and genetic load, which eliminates the possibility of obtaining inbred lines (Ritter et al., 1990; Leonards-Schippers et al., 1994). To overcome inbreeding depression, crosses between highly heterozygous parents are used to develop segregating F1 mapping populations (Leonards—Schippers et al., 1994). The first attempts to map potato were done at the diploid level to avoid problems of interpretation associated with tetrasomic inheritance (Meyer et al., 1998). The available diploid potato maps were based on the F1 generation (e.g. Barone et al., 1990; Collins et al., 1999; Freyre & Douches, 1994; Pineda et al., 1993; Sandbrink et al., 2000; Van Eck et al., 1994) and backcrosses to a different genotype of one or both parents (e.g. Ewing et al., 2000; Van den Berg et al., 1996a; Van den Berg et al., 1996b). More recently, tetraploid potato maps were also produced based on F1 generation of crosses with tetraploid cultivated potato (Meyer et al., 1998) and backcrosses to different potato cultivars (Naess et al., 2000). High informative potato maps were produced using restriction fragment length polymorphism (RFLP) (Gebhardt et al., 1991), amplified fragment length polymorphism (AFLP) (Van Eck et al., 1995), a combination of morphological markers, isozymes, RFLPs, and transposons (Jacobs et al., 1995), and simple sequence repeats (SSRs) or microsatellites (Milbourne et al., 1998). Since late blight is an important constraint for potato production, late blight resistance has been the most common characteristic mapped in potato including R-genes and QTLs associated with resistance. Of the 11 known R-genes, four were already mapped. The specific resistant gene R1 was mapped on chromosome V (El-Kharbotly et al., 1994; Leonards-Schippers etal., 1992). R2 was mapped on chromosome IV (Li et al., 1998). R3, R6 and R7 were mapped in a cluster on chromosome XI (El-Kharbotly et al., 1996; El-Kharbotly et al., 1994). One non-identified R-gene was mapped on chromosome X (Ewing et al., 2000). Some QTLs conferring resistance to late blight were mapped on the same regions of R-gene based resistance as on chromosomes V (Collins et al., 1999; Oberhagemann et al., 1999; Sandbrink et al., 2000) and on chromosome XI (Oberhagemann et al., 1999). These results support the idea that genes for specific and general resistance are not independent (Umaerus & Umaerus, 1994) and may be controlled by alleles at the same genetic locus or by related alleles of closely linked loci (Meksem et al., 1995). Quantitative trait loci for resistance to late blight have been mapped on all potato chromosomes (Leonards-Schippers et al., 1994; Meyer et al., 1998; Collins et al., 1999; Oberhagemann et al., 1999; Naess et al., 2000; Ewing et al., 2000; Kuhl et al., 2000; Sandbrink et al., 2000; Pande et al., 2001) based on diploid and tetraploid populations having a variety of wild species as sources of resistance. Other important characteristics received much less attention on mapping studies than late blight resistance. Specific gravity, an important tuber quality characteristic for potato processing industry, has been shown to have 10 QTLs distributed on 6 10 chromosomes of the potato genome (Freyre & Douches, 1994). Long dormancy in tubers is important to avoid sprouting in storage and short dormancy is important for seed tubers used in areas with two growing seasons. Six QTLs conferring long dormancy were located on six chromosomes, being the most important on chromosome VII (F reyre et al., 1994). Among nine chromosomes controlling dormancy, a major QTL conferring long dormancy was mapped on chromosome H (Van den Berg et al., 1996a). As many as 11 QTLs on 7 potato chromosomes were associated with tuberization and tuber fresh weight per plant, with a major QTL explaining 27% of the phenotypic variation on chromosome V (Van den Berg et al., 1996b). Potato mapping studies suggest that QTLs for late blight resistance were also associated with other important traits. A major QT L affecting foliar and tuber late blight resistance, vine maturity and vigor were all mapped in the region of the RFLP markers GP21 and GP179 on chromosome V (Oberhagemann et al., 1999), and foliar late blight resistance in another population (Collins et al., 1999). Other resistant genes were mapped in the same position of the potato genome as the R1 specific gene for late blight resistance (El-Kharbotly et al., 1994; Leonards-Schippers et al., 1992), the Rx2 gene for potato virus X resistance (Ritter et al., 1991), and Gpa5 gene to Globodera pallida (Stone) resistance (Rouppe van der Voort et al., 2000). Association between QTLs for foliar late blight resistance, tuberization and vine maturity was found in four out of five chromosomes (Ewing et al., 2000). In the potato chromosome IV, a QTL confening resistance to G. pallida from S. tuberosum subsp. andigena was mapped (Bradshaw et al., 1998) and a SSR marker (Strn3016) was linked to both late blight and nematode resistance conferred from different parents (Pande et al., 2001). The knowledge of QTLs conferring late blight resistance in different sources and their association with undesirable ll characteristics will increase the efficiency of breeding for late blight resistance. Improved levels of more durable resistance to late blight may be achieved by pyrarniding QTLs from different sources of resistance. Objectives and Dissertation Contents The objective of this research was to study the introgression of late blight resistance from wild species and unadapted gerrnplasm to cultivated potato. The specific objectives were: 1) to evaluate the use of late blight resistant parents in cultivar development; 2) to identify recombinant clones possessing late blight resistance, acceptable tuber quality and maturity in early stages of selection; 3) to screen wild Solanum species in greenhouse using the U88 genotype A2 mating type of P. infestans to identify resistant clones; 4) to assess the genetic diversity of the potato gerrnplasm with reported late blight resistance using a set of isozyme loci and SSR markers; and 5) to map QTL conferring late blight resistance and other agronomic traits using isozymes, AF LP and SSR markers in a diploid S. microdontum Bitter derived population. The first attempt to introgress late blight resistance was from unadapted gerrnplasm, since resistant sources were already identified and could be directly crossed with cultivated potato. A total of eight parents with reported late blight resistance was used to cross with a set of susceptible parents possessing acceptable maturity and tuber 12 quality. Progeny evaluations of these crosses are discussed in chapters two and three. Phenotypic selected clones based on overall appearance and tuber number, shape, and internal defects were evaluated for late blight resistance, vine maturity and tuber quality characteristics (tuber appearance, specific gravity and chip color). The progeny evaluation was a multi-trait evaluation of selected clones that have a common late blight resistant parent (half-sib progeny). These chapters were accepted for publication in Euphytica. The chapter four describes the evaluation of genetic diversity in the diploid and tetraploid potato late blight resistant gerrnplasm. The gerrnplasm used in this study included Solanum wild species and hybrids between wild species and cultivated potatoes identified in a previous two-year greenhouse screening. Ten other cultivars or advanced breeding clones were also included that have reported late blight resistance. This chapter was accepted for publication in HortScience. Based on the screening and genetic diversity studies, clones from S. .microdontum were selected to develop mapping populations. Further selection was based on the segregation of 40 seedlings for late blight resistance. The mapping study of late blight resistance and other agronomic traits in the diploid S. microdontum derived population is discussed in the chapter five. The chapter six is dedicated to a general discussion and conclusions. Literature Cited Alfano, F. M., M. Cammareri, A. Errico, L. Frusciante & C. Conicella, 1999. 2n gametes in Solanum tuberosum dihaploids. Amer. J. Potato Res., 76:281-285. 13 Anonymous, 1998. FAO Production Yearbook 1998. FAO Statistics Series, v. 52. Anonymous, 1998. National Potato Council — Potato Statistical Yearbook. 80 p. Bamberg, J.B., M.W. Martin & J.J. Shartner, 1994. 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Ninety-five crosses were made between eight unadapted parents with reported late blight resistance (B0718-3, Bertita, Bzura, Greta, Libertas, Stobrawa, Tollocan and Zarevo) and susceptible parents (cultivars or advanced breeding clones) adapted to North American growing conditions. A total of 408 field selected clones were assessed for late blight resistance in the greenhouse and in the field using a mixture of US8 P. infestans isolates (A2 mating type, metalaxyl resistant) that overcame all known R-genes except R8 and R9. Clones with S 10% infected foliar area in the greenhouse test or S 0.30 RAUDPC (relative area under 21 v.13 the disease progress curve) value in the field in 1998 were re-tested in 1999. A total of 118 (29% of 408) putative late blight resistant clones were selected. The eight late blight resistant parents differed in both the ability to transmit late blight resistance and in the level of resistance transmitted to the progeny. The Tollocan and 80718-3 families (half- sib progeny) had the greatest degree of resistance and frequency of resistant clones. Scott-Knott cluster analysis ranked 79 clones (67% of 118) in the high and moderate late blight resistant groups. Among these 79 clones, 19 clones had vine maturity equal to or earlier than mid-season combined with acceptable tuber quality. Further selection in 2000 resulted in eight advanced selected clones (six fi'om Tollocan and two from 30718- 3 families) with the same level of resistance as the parent combined with vine maturity and tuber quality equivalent to Atlantic, a standard cultivar for chip processing in North America. The results indicate that this breeding approach can be used to select parents for late blight resistance breeding and to identify superior clones with high levels of late blight resistance and marketable vine maturity and tuber quality. Introduction Late blight, caused by the fungal-like oomycete Phytophthora infestans (Mont) de Bary, is present in almost all potato (Solanum tuberosum L.) growing areas (Ross, 1986; Henfling, 1987; Kamoun et al., 1999). Late blight epidemics result from rapid asexual reproduction of the pathogen in potato tissue (I-Ienfling, 1987). Phytophthora infestans can complete an asexual cycle from initial infection to production of sporangia in less than five days and sporangia can be washed fi'om foliage into soil where the spores 22 can infect tubers (Fry & Goodwin, 1997). Infected tubers may rot in storage or become a primary source of inoculum for the following season if used as seed. Yield losses in potato caused by P. infestans were estimated to exceed $2 billion annually worldwide (Kamoun et al., 1999). Challenges to the development of late blight resistant cultivars include the association between resistance and late maturity, durability of resistance and poor tuber quality in the resistant parents. Late blight resistant cultivars are more likely to have late maturity and indeed the most significant quantitative trait locus for foliar resistance across three environments was mapped in the same position as late maturity (Collins et al., 1999). Horizontal resistance is a form of durable resistance to P. infestans (Colon et al., 1995; Umaerus et al., 1983), is effective against a broad range of pathogen races, and should be pursued in breeding for late blight resistance (Umaerus & Umaerus, 1994). Recent breeding efforts have resulted in the identification of potato late blight resistant sources (Douches et al., 1997), evaluation of the resistant phenotype stability (Haynes et al., 1998) and in the release of late blight resistant gerrnplasm (Goth and Haynes, 1997). However, the majority of the late blight resistance sources are not adapted to North American growing conditions, because of late maturity and marginal tuber characteristics (appearance, specific gravity, sugar level, defects, etc). Although efforts have been made to develop late blight resistant cultivars, two- thirds of 147 North American cultivars and breeding clones were classified as very susceptible (Douches et al., 1997) and no cultivar currently grown has an adequate level of resistance (Helgeson et al., 1998). Late blight susceptible cultivars have early maturity, good tuber appearance and specific gravity (Douches et al., 1996), and good chip processing quality (Love et al., 1998). As late blight resistance is not considered a 23 characteristic that confers enough advantage for a clone to become a successful cultivar (Umaerus et al., 1983), new cultivars must combine resistance with acceptable maturity and tuber quality characteristics for tablestock and processing markets. Progeny evaluation has been suggested as a means to study the inheritance of quantitative traits and to identify superior parents for breeding (Bradshaw & Mackay, 1994). Progeny evaluation can also reduce the time for each cycle of recurrent selection if parents with good general combining ability are identified shortly after hybridization (Bradshaw et al., 1995). An extension of progeny evaluation is a multi-trait evaluation of half-sib progeny. The research reported here is a half-sib progeny evaluation of late blight resistant parents crossed with a set of susceptible parents possessing acceptable maturity and tuber quality. The objectives of this research were to evaluate the use of late blight resistant parents in cultivar development and to identify superior clones possessing moderate to high resistance combined with acceptable maturity and tuber quality. Materials and Methods Crosses, segregating populations and evaluated clones In this study, eight late blight resistant parents were crossed with susceptible parents to develop 95 segregating populations (Table 2.1). The parents BO718-3, Bertita, Bzura, Greta, Libertas, Stobrawa, Tollocan, and Zarevo have reported resistance to late blight (Goth & Haynes, 1997; Douches et al. 1997; Haynes et al., 1998), but were not well adapted to North American growing conditions. The susceptible parents were 24 cultivars or advanced breeding clones evaluated in previous field trials (Douches et al., 1996) and greenhouse studies (Douches et al., 1997). All susceptible parents are adapted to North American growing conditions. For each cross, 50 seedlings (4,750 seedlings total) were transplanted at the Michigan State University Montcalm Experiment Station, Entrican, Michigan in 1997 with 75 cm within-row spacing between plants. At harvest, approximately 10% of the best clones from each cross were selected based on overall appearance and tuber number, shape, and internal defects. These selected clones were tested in greenhouse and field trials for late blight reaction in 1998 (Table 2.2). Clones with S 10% infected foliar area in the greenhouse test or S 0.30 relative area under the disease progress curve (RAUDPC) in the field in 1998 were re-tested in 1999 (Table 2.3). Advanced selected clones possessing late blight resistance combined with acceptable maturity and tuber quality were further evaluated in 2000. The value of each late blight resistant parent was determined by the performance of its half-sib progeny. For the remainder of this paper, family refers to half-sib progeny. 25 .... .4............... 71.1.4... 1.4.7.1.... .4 ~22. 7...... .:£~ v:?..:.: :2..~.J,.\V.LL :TQQQ but. a c .M .333. 3-852 Boo $8 5552 ES, $88280 chm? 200 :83.» Bow 82> :25 Steam E?» ES, 885 2.22 am 32222.0. 253 353. 538m 250$ 852$ 22622 200 owed 8mm Seam Same? SE 8:2 «gum 53-9632 $ch 0225.2 Rm? $582 25 332 2282 2-252 22,85. 58% 22,85. 8:2 2.82%: 883 33%: seam 2-38m: .3380 2.2892 5552 38%: 288m 3832 3:2 3882 22,85. 28 new; 34.03%: 28 as; 8-8%: $288: 8:2 $832 89380 @832 25%: $882 «-882 $8822 2.223%: 3352 2.3%: Bongo 322m: 1282 @552 steam: 336m: 3282 323/82 2.89%: 5&2? 38mm: 3282 38mm: 5.2%: «.4882 3282 2-232 2-83m: 2.39%: 2282 2-89%: 320m: 320m: ream: 2.82%: 805 58:3 $885 asap: see Sam steam 228m .mcouflzaoq mesmwouwom 883% 8 @888 803 95208 8:20 @2685 wooggfimazuao Bang 03:33.3 can A38 no: 3:23 28368 szn 223 .mm 2an 26 Characterization of P. Infestans isolates and inoculum preparation All P. infestans isolates were obtained from late blight infected potato crops in Michigan and were characterized as U88/A2 mating type, the most common and aggressive genotype of P. infestans currently present in United States (Fry & Goodwin, 1997). Genotype of these isolates was determined by restriction fragment length polymorphism using RG57 as the probe (Goodwin et al., 1992) and by the two allozyme loci glucose-6-phosphate isomerase (Gpi) and peptidase (Pep) (Goodwin et al., 1995). Growing isolates of unknown mating type in the presence of known A1 and A2 mating types and monitoring oospore production determined the mating type (Galindo & Gallegly, 1960; Honour & Tsao, 1974). The presence of avirulence genes in each isolate was evaluated using detached-leaf assays on a series of R-gene potato differentials. The mixture of isolates (MS94-1, MS94-4, MS95-7 and MS97-2) overcame all known R- genes except R8 and R9 in detached-leaf assays. In the field, the isolates overcome all Black’s differentials except R8 and R9, which were weakly pathogenic. Cultures of P. infestans were grown on rye agar plates in the dark at 15 °C and started about 20 days prior to each inoculation. Sporangia were harvested from Petri dishes by rinsing the mycelia/sporangia mat in cold (4 °C) sterile, distilled water and scraping the mycelia/sporangia mat from the agar surface with a rubber policeman. The mycelia/sporangia suspension was strained through four layers of cheesecloth and the concentration of sporangia was adjusted to about 1 x 10‘5 sporangia ml'l using a hemacytometcr. The suspension was stored at 4 °C for four hours to stimulate zoospore release prior to inoculation. 27 \\ fit U31 [at Res. Were Late blight reaction in greenhouse tests Plants were grown fi'om sprouted tuber pieces for about 6 weeks in the greenhouse with natural light supplemented by high-pressure sodium lamps (16h day length). Prior to flowering, plants were transferred to a mist chamber of approximately 3 m3. The chamber was situated within a greenhouse and covered with 0.6 mm transparent polyethylethene plastic sheets. Relative humidity was maintained at 90% by misting the chamber atmosphere for 15 minutes every hour (6 liters of deionized water per 24-hour period) with gravity-fed humidifiers (Herrmidifier Series 500 - Trion, Stanford, NC). Plants were inoculated in the evening, by spraying the plants with 50 ml of inoculum per m2 using a hand-held bottle sprayer. Temperature within the chamber was maintained between 18 °C and 25 °C. Infected foliar area was estimated based on a visual observation of the diseased area of stems and leaves at seven days afier inoculation. The experimental unit was a single plant in one pot (16 cm diameter). In 1998, 408 clones were tested in 2 replications in a completely random design. In 1999, 118 clones were tested in 4 replications in a randomized complete block design. All tests were carried out from January to April of each year and the late blight susceptible cultivar Atlantic was used as standard. Late blight reaction in field tests The field tests were carried out at the Michigan State University Muck Soils Research Farm, Bath, Michigan in a randomized complete block design. No fungicides were applied on the plants. The 408 selected clones tested in the greenhouse in 1998 were planted in 2 replications as single-hill plots on June 15th and inoculated on July 22nd. The 118 advanced selected clones tested in the greenhouse in 1999 along with the eight 28 late blight resistant parents were planted in 3 replications of four-hill plots on May 27th and inoculated on July 22'”. In 2000 the evaluated clones were planted in 3 replications of four-hill plots on June 9th and inoculated on July 26th. Inoculation was done through a permanent sprinkle irrigation system in the early evening and high humidity was maintained in the canopy through periodic irrigations throughout the season. A visual estimation of the percentage of stem and leaf infected area was scored at three to five day intervals from inoculation until the most susceptible clones reached 100% infection. The area under the disease progress curve (AUDPC) was calculated as described by Shaner & Finney (1977) and divided by the maximum AUDPC (e.g. 3300 for 33 days after inoculation) converting the value to relative AUDPC (RAUDPC), with 1.0 being the maximum RAUDPC value. See more details in the appendix. Maturity and tuber quality evaluations The selected clones evaluated in 1999 were also planted in non-replicated 20-hill plots at the Michigan State University Lake City Experiment Station, Lake City, Michigan for vine maturity and tuber quality evaluations. Advanced selected clones were planted in non-replicated 40-hill plots at the Michigan State University Montcalm Experiment Station, Entrican, Michigan in 2000. From this point on, tuber quality refers to a combination of tuber appearance, specific gravity and chip color. Vine maturity was evaluated in the field when the standard commercial cultivar Atlantic had a rating of 3 on a 1 to 5 scale (1 = early, as cultivar Superior and 5 = late, as cultivar Ontario). Tuber appearance was evaluated on a l to 5 scale of increasing defects (1 = excellent, as cultivar Atlantic; 2 = very good; 3 = acceptable; 4 = poor; and 5 = very poor). Chip color was evaluated on a l to 9 scale of increasing color darkness (1 - 2 = excellent; 3 = very 29 good, as cultivars Atlantic and Snowden; 4 = acceptable; 5 = unacceptable; and 6 - 9 = poor). Specific gravity was measured on a minimum 2 kg sample using the formula [dry weight / (dry weight — wet weight)]. Statistical analysis The percentage of infected foliar area in the greenhouse and RAUDPC in the field was analyzed using analysis of variance. Family means were calculated as the average of the clone values for each replication. Family means were compared by Fisher’s least significance difference (LSD) at a = 0.05 for greenhouse and field tests in 1998 and 1999. Fisher’s LSD (at = 0.05) was also used to compare the standard susceptible cultivar Atlantic and the eight late blight resistant parents in the 1999 field testing. For greenhouse and field 1999 data, Dunnett’s T test (a = 0.05) was used to compare clones with Atlantic. Pearson correlation analysis was done to compare greenhouse and field testing for 1999 data. Scott-Knott cluster analysis was used to rank clones, resistant parents and Atlantic in discrete groups differing in late blight reaction based on field testing in 1999 (Scott & Knott, 1974). Fisher’s LSD (a = 0.05) was used to compare Atlantic, Tollocan, BO718-3 and the advanced selected clones in the 2000 field testing. All analyses were done following the procedures of SAS (SAS Institute, 1995). 30 [u “I 31‘ far gr 96:1? [651113; Results Identification of superior parents for late blight resistance breeding The greenhouse testing in 1998 showed a wide range of infection for all families and the susceptible cultivar Atlantic had a foliar infection of 42% at seven days after inoculation (Table 2.2). Clones with less infection than Atlantic were identified in all families. The Tollocan family had the lowest mean infection at 17%, which was significantly less than any other family mean. High mean infection levels (over 35%) were found in the B0718-3, Bertita, Libertas, and Bzura families. See Fig. A.1 in the appendix. Foliar infection in the field showed a large range of RAUDPC values for all families (Table 2.2). However, all evaluated clones had RAUDPC values lower than Atlantic. Again, the Tollocan family had the lowest RAUDPC mean infection. The BO718-3 family had the second lowest RAUDPC mean infection, but was not significantly different from the Bzura family. See Fig. A2 in the appendix. Of the 408 selected clones, those with S 10% infected foliar area in the greenhouse test or S 0.30 RAUDPC value in the field test in 1998 were re-tested in 1999, resulting in 118 advanced selected clones (29% of 408) with putative resistance to late blight. The Bertita family had the lowest (4) and the Tollocan family had the highest (45) number of evaluated clones in 1999 (Table 2.3). Tollocan (63%), Zarevo (36%) and B0718-3 (29%) families had the highest percentage of selected clones, based on greenhouse and field tests in 1998. The Bzura family was not represented in the 1999 testing, because there was only one selected clone. 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In general, clones with less infection than Atlantic were identified in all families, but only the Tollocan and B0718-3 families had clones with less than 10% infection. The 30718-3 family had the lowest family mean infection with 16%, and the Tollocan family had the second lowest family mean infection (33%), but the Tollocan family was not significantly different from the Stobrawa, Greta, and Libertas families. See F ig. A3 in the appendix. In the 1999 field testing, the B0718-3 and Tollocan families showed a wide range of RAUDPC values (Table 2.3). In a comparison among family means, Fisher’s LSD test differentiated the eight families into three groups. The Tollocan family had the lowest RAUDPC value, the 30718-3 family had intermediate and the Greta, Libertas, Stobrawa, Bertita and Zarevo families had the highest mean RAUDPC values. There was a positive association between family means and parents, since the best parents produced the best family means (lower RAUDPC). Tollocan was the best parent with the best family mean. Also, the most resistant clone in each family tended to have an RAUDPC similar to its resistant parent. There were significant differences among late blight resistant parents and all resistant parents had a significantly lower RAUDPC than Atlantic. Also, there was a positive significant correlation (r = 0.56, P < 0.001) between greenhouse and field tests in 1999. See Fig. AA in the appendix. Grouping and selection of recombinant progeny The Scott-Knott cluster analysis based on the field test in 1999 ranked the advanced selected clones, the resistant parents and Atlantic in three groups differing in late blight reaction (Table 2.4). A total of 24 clones were ranked in the resistant group 34 (RAUDPC from 0.020 to 0.183), 63 clones in the moderately resistant group (RAUDPC from 0.222 to 0.560), and 40 clones in the susceptible group (RAUDPC from 0.565 to i 0.777). The late blight resistant parents Tollocan and B0718-3 were ranked in the resistant group, while Bzura, Greta, Libertas, Stobrawa, Bertita, and Zarevo were ranked in the moderately resistant group. Atlantic was ranked in the susceptible group. Assuming the moderately resistant group as a threshold, 79 advanced selected clones (67% of 118) could be further advanced in a breeding program. These selections represent seven of the eight families. Maturity and tuber quality evaluations in 1999 showed that 19 of the 79 advanced selected clones possessing high or moderate late blight resistance had a maturity rating as early mid-season or as mid-season. Of these advanced selected clones with marketable maturity, 5 were from the resistant and 14 were from the moderately resistant group (Table 2.4). The resistance of these 19 advanced selected clones came from 5 parents (12 from Tollocan, 3 from B0718-3, 2 from Stobrawa, 1 from Libertas, and 1 from Zarevo). Moreover, these 19 advanced selected clones also had acceptable tuber quality [(5 had chip processing quality (chip color 5 4, tuber appearance 3 3 and specific gravity 2 1.080) and 14 had tablestock quality (tuber appearance 5 3) (data not shown)]. These advanced selections possessing late blight resistance combined with acceptable maturity and tuber quality were further evaluated in 2000. The 2000 field evaluation showed that there were advanced selected clones (eight) with the same level of late blight resistance as their resistant parent that also combined maturity and tuber quality equivalent to Atlantic, a standard cultivar for chip processing in North America (Table 2.5). Only two families were represented in these new advanced selected clones; Tollocan with six and 30718-3 with two clones. See also Table A2 in the appendix. 35 :1: Iq-.II¢..7II.<-I.- I tut-5‘. - 2:..7. 1:... 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Based on the percentage of selected clones in 1998, the parents Tollocan, Zarevo and B0718-3 transmitted resistance to a higher percentage of their offspring than Greta, Libertas and Stobrawa. Tollocan had the lowest family mean infection in both greenhouse and field testing. Of the 118 selected clones identified in 1998, Tollocan and BO718-3 also had the highest percentage of selected clones in 1999. In terms of family means, B0718-3 had the lowest and Tollocan the second lowest mean infection in greenhouse testing in 1999. In the field testing, Tollocan had the lowest and BO718-3 the second lowest RAUDPC values, for family mean and parent. The advanced clones selected in 2000 were exclusively progeny from Tollocan and B0718-3 families. Tollocan and BO718-3 not only had higher levels of late blight resistance, but also transmitted resistance to a higher percentage of their progeny compared with any other parent. This permitted the selection among their progeny for acceptable maturity and tuber quality. Some attempts have been made to separate qualitative treatments in distinct, non- overlapping groups to give a reasonable threshold for selection. The Scott-Knott cluster analysis method uses principal component and cluster analysis to group treatment means (Scott & Knott, 1974). Gates & Bilbro (1978) showed that Scott-Knott cluster analysis was more effective at ranking genotypes into distinct groups than the Duncan multiple range test and Fisher’s LSD, even in experiments with a small coefficient of variation. The no treatment overlap property of Scott-Knott cluster analysis increases the Type I 38 error, but detects considerably smaller differences than Fisher’s LSD test does (Willavize et al., 1980). The use of principal component and cluster analysis (Platt & Tai, 1984) and residual maximum likelihood (Platt & Tai, 1998) was reported as effective for ranking potato clones in groups differing in late blight resistance. In this study, we used Dunnet’s T-test and Scott-Knott cluster analysis to group clones. Dunnett's T-test showed a clear threshold for selection, but this test identified only clones with high levels of resistance (four clones in the greenhouse test and 46 clones in the field test in 1999). The strategy of selecting only clones possessing high levels of late blight resistance may severely reduce the genetic base and thereby restrict the possibility of further selection for other important traits. However, Scott-Knott cluster analysis was able to rank the clones into three discrete groups differing in level of resistance. The highly resistant clones were ranked with the resistant parents Tollocan and B0718-3, the moderately resistant clones were ranked with the resistant parents Bzura, Greta, Libertas, Stobrawa, Bertita and Zarevo, and the susceptible clones were ranked with the susceptible cultivar Atlantic. These levels of resistance in the resistant parents were also detected by Fisher’s LSD which also showed significant differences between late blight resistant parents and the susceptible cultivar Atlantic. Moreover, the field testing and Scott-Knott cluster analysis results were in concordance with a previous study based on a multi-state field trial. In that study, Haynes et a1. (1998) ranked BO718-3 (3“), Bzura (5m), Greta (6m), Libertas (7th), Bertita (8th), and Stobrawa (10“) among 16 cultivars previously identified as resistant. Therefore, Scott-Knott cluster analysis provided a means to determine a threshold for selecting clones possessing high and moderate levels of resistance to late blight that can be further selected for other important traits. 39 Combining field and greenhouse resistance testing with field determinations of vine maturity and tuber characteristics provided a good measurement of the breeding potential of the late blight resistant parents and permitted a multi-trait selection for the identification of recombinant clones. Over a three-year period, beginning with unselected crosses, clones were identified that possessed late blight resistance combined with acceptable maturity and tuber quality. These selected clones can be advanced for further evaluation or used as parents in the next cycle of recurrent selection. The advanced selected clones were from the Tollocan and BO718-3 families suggesting that the late blight resistance in these parents should be highly heritable. See Fig. A5 in the appendix. Despite using a mixture of P. infestans isolates in these tests, the type of resistance present in Tollocan and BO7l8-3 has not been resolved. There were a few clones that did not show infection in the greenhouse tests. There was one clone from each Tollocan and BO718-3 families in 1998 and two clones from Tollocan family in 1999 in the greenhouse tests, but these clones were infected in the field tests. The clones with no infection in greenhouse tests may have R8 or R9 genes, since the P. infestans isolates used were not pathogenic in detached-leaf assays and only weakly pathogenic on the R8 and R9 differentials in the field tests. All the six advanced selected clones having Tollocan and the two advanced selected clones having 80718-3 as source of resistance were infected in the greenhouse tests. Moreover, the resistance does not appear to be associated with late maturity, since selected clones demonstrated mid-season maturity in two years of evaluations. Since late blight resistance associated with late maturity was mapped on Chromosome V (Collins et al., 1999), the resistance present in the advanced selected clones should be located in other regions of the potato genome. The resistance 40 in Tollocan and B0718—3 may not be solely vertical. BO718-3 and the most promising Tollocan-derived selections have been included in greenhouse and field tests at Michigan State University since 1997 and have showed consistently high levels of resistance to late blight. High levels of horizontal resistance to late blight from different wild species associated with more simple inheritance has recently been mapped in the cultivated potato background. The cultivar Stirling has S. demissum Lindl. as source of resistance (Meyer et al., 1998), in which one major quantitative trait locus (QTL) explaining 30% of the phenotypic variance was mapped to the chromosome IV (Pande et al., 2001). A major QTL explaining 62% of the phenotypic variance for the resistance of S. bulbocastanum Dunal was mapped to the chromosome VIII (Naess et al., 2000). Therefore, the strong and highly heritable resistance conferred by Tollocan and B0718-3 should not be designated as R-gene based resistance at this time. The main objective of our breeding effort was to combine resistance genes from different sources to broaden the genetic base and thus increase the degree and durability of late blight resistance. Before intercrossing these resistance sources, it was necessary to combine late blight resistance with acceptable maturity. Selected clones possessing high levels of resistance to late blight from Tollocan and BO718-3 are being crossed with selected clones possessing moderate resistance from Libertas, Stobrawa and Zarevo. Intercrossing these late blight resistant clones should also increase the probability of recombinants carrying foliar and tuber resistance, since Libertas (Platt & Tai, 1998) and Zarevo (Douches et al., 2001) are reported to transmit tuber resistance. Also, pyramiding genes from different sources (and in this case different levels of resistance) may build more durable resistance to late blight (Colon, 1999). Combining genes from different 41 sources will be more effective when the QTLs for late blight resistance are mapped in the parents. In summary, progeny evaluation was valuable to identify parents to use in breeding for late blight resistance. Moreover, the combination of greenhouse and field testing for late blight with field evaluations for maturity and tuber quality gave the possibility for multi-trait selection that resulted in the identification of recombinant clones after three years of evaluation. These selected clones can be used as parents in recurrent selection for combining sources of resistance and can continue being evaluated for gerrnplasm release. The results indicate that this breeding approach can be used to select parents for late blight resistance breeding and to identify superior clones with high levels of late blight resistance and marketable vine maturity and tuber quality. Literature Cited Bradshaw, J.B. & G.R. Mackay, 1994. Breeding strategies for clonaly propagated potatoes. In: J.B. Bradshaw & G.R. Mackay (ed.) Potato Genetics. CAB International, Cambridge. p. 467-497. 552 pp. Bradshaw, J.B., H.E. Stewart, R.L. Wastie, M.F.B. Dale & M.S. Phillips, 1995. Use of seedling progeny tests for genetical studies as part of potato (Solanum tuberosum subsp. tuberosum) breeding programme. Theor. Appl. Genet. 90:899-905. Collins, 0., D. Milboume, L. Ramsay, R. Meyer, C. Chatot-Balandras, P. Oberhagemann, W. de Jong, C. Gebhardt, E. Bonnel & R. Waugh, 1999. QTL for field resistance to late blight in potato are strongly correlated with maturity and vigour. Mol. Breeding 5:387-398. Colon, L.T., L.J. Turkensteen, W. Prummel, D.J. Budding & J Hoogendoom, 1995. Durable resistance to late blight (Phytophthora infestans) in old potato cultivars. European J. Plant Pathology 101: 387-397. Colon, LT. 1999. Trends in late blight resistance breeding in Western Europe. In: Proceedings of the Global Initiative on Late Blight Conference. Quito, Ecuador, March 16—19. Vol. 1, p. 41-42. 42 Douches, D.S., D. Maas, K. Jastrzebski & R.W. Chase, 1996. Assessment of potato breeding progress in the USA over the last Century. Crop Science 36:1544-1552. Douches, D.S., W. W. Kirk, K. Jastrzebski, C. Long & R. Hammerschmidt, 1997. Susceptibility of potato varieties and advanced breeding lines (Solanum tuberosum L.) to Phytophthora infestans (Mont) de Bary in greenhouse screenings. Amer. Potato J. 74:75-86. Douches, D.S., W.W. Kirk, M.A. Bertram & B.A. Niemira, 2001. Foliar and tuber assessment of late blight (Phytophthora infestans (Mont) de Bary) reaction in cultivated potato (Solanum tuberosum L.). Amer. J. Potato Res. In Press. Fry, W. E. & S.B. Goodwin, 1997. Resurgence of the Irish Potato famine fungus. Bioscience, 47:363-371. Galindo, J .A. & M.E. Gallegly, 1960. The nature of sexuality in Phytophthora infestans. Phytopathology 50:123-129. Gates, C.E. & J.D. Bilbro, 1978. Illustration of a cluster analysis method for mean separation. Agronomy Journal 70:462-465. Goodwin, 8.8., A. Drenth & W.E. Fry, 1992. Cloning and genetic analysis of two highly polymorphic, moderately repetitive nuclear DNAs fiom Phytophthora infestans. Cur. Genet. 22(2): 107-1 15. Goodwin, S.B., R.E. Schneider & W.E. Fry, 1995. Use of cellulose-acetate electrophoresis for rapid identification of allozyme genotypes of Phytophthora infestans. Plant Disease 79:1 181-1 185. Goth, R. W. & K.G. Haynes, 1997. The gerrnplasm release of B0718-3 and B0767-2: two late blight resistant potato clones. Amer. Potato J. 74:337-345. Haynes, K.G., D.H. Lambert, B.J. Christ, D.P. Weingartner, D.S. Douches, J.B. Backlund, G. Secor, W. Fry & W. Stevenson, 1998. Phenotypic stability of resistance to late blight in potato clones evaluated at eight sites in United States. Amer. J. of Potato Res. 75:211-217. Helgeson, J.P., J.D. Pohlman, S. Austin, G.T. Haberlach, S.M. Wielgus, D. Ronis, L. Zambolin, P. Tooley, J.M. McGrath, R.V. James & W.R. Stevenson, 1998. Somatic hybrids between Solanum bulbocastanum and potato: a new source of resistance to late blight. Theor. Appl. Genet. 96:738-742. Henfling, J.W., 1987. El tizon tardio de la papa: Phytophthora infestans, 2 ed. Centro Intemacional de la Papa, Lima, Peru. 25 p. Honour, R.C. & P.H. Tsao, 1974. Production of oospores by Phytophthora parasitica in liquid medium. Mycology 66: 1030-1038. Kamoun, S., E. Huitema & V.G.A.A. Vleeshouwers, 1999. Resistance to oomycetes: a general role for the hypersensitive response. Trends Plant Science 4: 196-200. 43 Love, S.L., J. J. Pavek, A. Thompson-Johns & W. Bohl, 1998. Breeding progress for potato chip quality in North American cultivars. Amer. J. of Potato Res. 75:27-36. Meyer, R.C., D. Milboume, C.A. Hackett, J .E. Bradshaw, J.W. McNichol & R. Waugh, 1998. Linkage analysis in tetraploid potato and association of markers with quantitative resistance to late blight (Phytophthora infestans). Mol. Gen. Genet. 259:150—160. Naess, S.K., J.M. Bradeen, S.M. Wielgus, G.T. Haberlach, J.M. McGrath & J.P. Helgeson, 2000. Resistance to late blight in Solanum bulbocastanum is mapped to chromosome 8. Theor. Appl. Genet. 101:697-704. Pande, B., K. McLean, D. Milboume, C. Hackett, J. Brakshaw, H. Stewart, E. Isidore, G. Bryan, J. McNicol & R. Waugh, 2001. QTL analysis in tetraploid potato. Plant and Animal Genome IX Conference. San Diego, CA, January 17-19, 2001. p. 189. Platt, H.W. & G. C. C. Tai, 1984. Assessment and analyses for the interpretation of potato late blight response in field studies. Amer. Potato J. 61 :599-609. Platt, H.W. & G.C.C. Tai, 1998. Relationship between resistance to late blight in potato foliage and tubers of cultivars and breeding selections with different resistance levels. Amer. J. Potato Res. 75:173-178. Ross, H., 1986. Potato Breeding - Problems and Perspectives. Verlag Paul Parey, Berlin and Hamburg, 132 p. SAS Institute, 1995. The SAS system for windows. Release 6.12. SAS Institute, Cary, NC. Scott, A.J. & M. Knott, 1974. A cluster analysis method for grouping means in the analysis of variance. Biometrics 30:507-512. Shaner, G. & R.E. Finney, 1977. The effect of nitrogen fertilization on the expression of slow-mildewing resistance in Knox wheat. Phytopathology 67: 1051-1056. Umaerus, V., M. Umaerus, L. Erjefalt & B.A. Nilsson, 1983. Control of Phytophthora by host resistance: problems and progress. In: DC. Erwin, S. Bartnicki-garcia & P.H. Tsao (ed.). Phytophthora infestans: Its Biology, Taxonomy, Ecology, and Pathology. The American Phytopathological Society. St. Paul, Minnesota. p. 315- 326. Umaerus, V. & M. Umaerus, 1994. Inheritance of resistance to late blight. In: J. E. Bradshaw & G.R. Mackay (ed.) Potato Genetics. CAB International, Cambridge. p. 365-401. Willavize, S.A., S.G. Carmer & W.M. Walker, 1980. Evaluation of cluster analysis for comparing treatment means. Agronomy J. 72:317-320. CHAPTER III Early Generation Selection for Potato Tuber Quality in Progeny of Late Blight Resistant Parents Abstract Developing disease resistant cultivars is one of the major objectives for a potato (Solanum tuberosum 1L.) breeding program, but many resistant clones have not achieved commercial acceptance because of late maturity and non-marketable tuber characteristics. Selection for tuber quality should have greater emphasis in breeding disease resistant cultivars. The objectives of this study were to evaluate the ability of late blight (Phytophthora infestans (Mont) de Bary) resistant parents to transmit chip-processing (tuber appearance, specific gravity, and chip-color) or tablestock (tuber appearance) quality to the offspring and to compare selecting for tuber quality in single-hill versus eight-hill clonal generations. Crosses were made among eight unadapted potato cultivars (B071 8-3, Bertita, Bzura, Greta, Libertas, Stobrawa, Tollocan, and Zarevo) with reported late blight resistance with adapted susceptible cultivars/breeding clones to generate 95 populations (4,750 seedlings). Approximately 10% of the progeny from each cross were 45 selected from single-hill plots based on tuber appearance, number, shape, and internal defects. These selected clones (408) were evaluated for tuber appearance, specific gravity, and chip-color. The same evaluations in the following year were made on tuber samples from eight-hill plots. Libertas and Tollocan were the best parents for transmitting chip-color; B0718-3, Zarevo, and Tollocan for transmitting tuber appearance; and Bzura, Libertas, and Zarevo for transmitting high specific gravity to the highest percentage of the offspring. Overall, 50% and 56% of the clones based on single- and eight-hill clonal generations, respectively, were considered to possess chip- processing quality; over 90% of the clones had acceptable tablestock quality. A total of 71% of the clones possessing acceptable chip-processing and 95% of the clones possessing acceptable tablestock quality selected in both clonal generations were identified in single-hill plots. The evaluation of tuber quality characteristics in single-hill plot not only permitted the identification of clones with acceptable chip-processing and tablestock, but also increased the amount of clonal information for the following generation of selection. In crosses between late blight resistant and susceptible clones, selection for tuber quality traits can be initiated in single-hill clonal generation using a moderate selection intensity and precede late blight testing. Introduction Breeding for disease resistance is a common objective for many crop species. Overemphasis on improving disease resistance can limit yield and other important traits because of genetic bottlenecks (Kelly et al., 1998). In potato (Solanum tuberosum L.), 46 late blight (Phyt0phthora infestans (Mont) de Bary) is the most devastating disease worldwide (Fry & Goodwin, 1997; Kamoun et al., 1999) and genetic host plant resistance is one of the major objectives for breeding (Colon et al., 1995). Although breeding for late blight resistance has had greater priority in the last century than for any other pathogen, the potato market in many countries is dominated by late blight susceptible cultivars (Umaerus et al., 1983). As an example, among 147 cultivars and breeding lines evaluated against USS genotype of P. infestans in greenhouse experiments, two-thirds were classified as very susceptible (Douches et al., 1997). In fact, there is no North American cultivar currently in use that has an adequate level of late blight resistance (Helgeson et al., 1998). In contrast, potato breeders have made great progress over the last century for early maturity, chip-color, tuber appearance, and specific gravity (Douches et al., 1996) and cultivars with good chip-processing quality have been released (Love et al., 1998). Since late blight resistance is not the trait that confers enough advantage for a clone to become a successful cultivar (Umaerus et al., 1983), late blight resistance needs to be combined with tuber quality, acceptable maturity, and other agronomically important traits. In order to combine characteristics, a selection/evaluation procedure capable of identifying desirable clones at the early generation stage is required. Selection in single- hill plot reduces the cost for clonal maintenance and also permits the identification of superior parents for use in other cross combinations (Thill & Peloquin, 1995). In addition, an efficient selection strategy should be able to significantly reduce the number of selected clones and to keep superior ones for later generations of selection (Tai & Young, 1984). Since superior tuber quality has been the market-limiting trait that characterizes the cultivars released in the past century (Douches et al., 1996; Love et al., 47 1998), this trait should be evaluated and selected for as early as possible in a potato breeding program. The objectives of this study were to evaluate the ability of late blight resistant parents to transmit chip-processing (chip-color, tuber appearance, and specific gravity) or tablestock (tuber appearance) quality to the progeny and to compare selecting for tuber quality in single-hill versus eight-hill clonal generations. Material and Methods Eight unadapted cultivars (B07l8-3, Bertita, Bzura, Greta, Libertas, Stobrawa, Tollocan, and Zarevo) with reported late blight resistance were crossed with adapted susceptible cultivars/breeding clones to generate 95 populations segregating for late blight resistance, marketable tuber quality, and maturity as described in Chapter 2. For each cross, 50 seedlings (4,750 seedlings total) were transplanted at the Michigan State University Montcalm Experiment Station, MI in 1997 with 75 cm within-row spacing between plants. At harvest, approximately 10% of the best offspring from each cross were selected based on tuber appearance, number, shape, and internal defects, which resulted in 408 clones (8.9%). These clones were grouped according to their late blight resistant parent and were planted in 1998 in eight-hill plots with 30 cm within-row spacing. In this study, family refers to half-sib progeny of the respective late blight parent (i.e. B0718-3 family, Bertita family, etc). All tubers harvested from single-hill plots and a random sample of tubers (SJ-8.3 cm diameter) from eight-hill plots was used for tuber appearance and specific gravity 48 evaluations. Tuber appearance was evaluated on a scale 1 to 5 of increasing defects (1 = excellent, as cultivar Atlantic; 2 = very good; 3 = acceptable; 4 = poor; and 5 = very poor). Specific gravity was measured on a minimum 2 kg sample using the formula [weight in air / (weight in air - weight in water)] with 1.080 or greater considered acceptable for chip-processing. One tuber and five-tuber samples (two slices/tuber), respectively from single- and eight-hill plots, were used for chip-color evaluation. Chip- color was evaluated on a scale 1 to 9 of increasing color darkness (1 - 2 = excellent; 3 = very good, as cultivars Atlantic and Snowden; 4 = acceptable; 5 = unacceptable; and 6 - 9 = poor). From here after, tuber quality refers to the combination of tuber appearance, specific gravity, and chip-color. Tuber quality data were analyzed using a mixed model (SAS PROC MIXED) including families, years (single- and eight-hill clonal generations) and the interaction family x year as fixed effects. Variance components were estimated by restricted maximum likelihood. The same analysis was done considering only years as a fixed effect. The significance of fixed effects was tested by the F type III test. Family means were compared by Fisher’s least significance difference (LSD) at or = 0.05. Pearson correlation analysis was done to compare tuber quality data from single— and eight-hill clonal generations. Correlation analysis was done at the clonal level for individual late blight families and combined analysis of all families. Correlation analysis was also done at the family level (late blight family means). All these analysis were done following the procedures of SAS (SAS Institute, 1995). 49 Results Progeny performance for tuber quality Families, years (single- and eight-hill clonal generations) and the interaction family x year as fixed effects were significant (P < 0.01) for all tuber quality traits, except for specific gravity between years. When year was considered as a fixed effect, there was not a significant (P > 0.05) difference between years for chip-color and specific gravity. The performance of the late blight families for tuber quality in single-hill plot in 1997 showed that, on average, families had marginal chip-color (4.2), acceptable tuber appearance (2.6), and high specific gravity (1.087) (Table 3.1). The Libertas family had the best chip-color, but did not significantly differ fi'om Zarevo, Tollocan, and Stobrawa families. For tuber appearance, the BO718-3 family had the best ratings, but did not significantly differ from the Tollocan family. The Libertas family was superior for both specific gravity and chip-color and the Tollocan family for chip-color and tuber appearance. The average performance of the families was slightly better in eight-hill plots than in single-hill plot (Table 3.2). Greta and B0718-3 families had the best chip-color. The B0718-3 family had the best and the Zarevo had the second best average tuber appearance rating. For specific gravity, Libertas, Zarevo, and Bzura families had the highest values. In eight-hill plot, only the B0718-3 family performed the best for more than one trait (chip-color and tuber appearance). The correlation analysis at the clonal level showed significant coefficients between single- and eight-hill clonal generations for the combined analysis of all late blight families for all tuber quality traits (Table 3.3). Tuber appearance had the smallest 50 (0.27) and specific gravity had the highest correlation coefficient (0.67). Considering individual late blight families, significant correlations between single— and eight-hill clonal generations for all individual families were found only for chip-color. Libertas family for specific gravity and B0718-3, Greta, Libertas, Stobrawa, and Tollocan families for tuber appearance had no significant correlation between single- and eight-hill clonal generations. At the late blight family level, high correlation coefficients were found for tuber appearance (r = 0.78, P < 0.05) and for specific gravity (r = 0.92, P < 0.01), but no significant correlation was found for chip-color (data not shown). 51 Table 3.1. Progeny performance of eight late blight resistant parents for tuber quality in single-hill plot in 1997. Late Blight Evaluated Chip-Color2 Tuber Appearance3 Specific Gravity4 Familiesl Clones (ratings) (ratings) Libertas 52 3.7 a5 2.8 cdef 1.098 a Zarevo 92 4.0 ab 2.5 bc 1.094 b Tollocan 71 4.0 ab 2.4 ab 1.084 e Stobrawa 34 4.1 ab 2.6 bcd 1.089 cd Greta 28 4.3 be 3.0 def 1.088 cd 80718-3 59 4.3 be 2.2 a 1.077 f Bertita 40 4.3 be 3.1 f 1.080 f Bzura 32 4.7 c 2.6 bcd 1.091 bc Average 51 4.2 2.6 1.087 ' All evaluated clones are half-sibs in relation to the late blight resistant parent. 2 Evaluated as a scale 1 to 9 of increasing color darkness. 3 Evaluated as a scale 1 to 5 of increasing defects. 4 Formula [weight in air / (weight in air - weight in water)]. 5 Means in columns followed by the same letter are not significantly different using Fisher’s LSD at or = 0.05. 52 Table 3.2. Progeny performance of eight late blight resistant parents for tuber quality in eight-hill plot in 1998. Late Blight Evaluated Chip-Color2 Tuber Appearance3 Specific Gravity4 Families1 Clones (ratings) (ratings) Greta 28 3.2 a5 2.5 c 1.090 b B0718-3 59 3.7 ab 1.4 a 1.078 d Zarevo 92 3.8 bc 1.7 b 1.095 a Bertita 40 3.9 bcd 2.5 c 1.080 cd Libertas 52 4.0 bode 2.5 c 1.094 ab Bzura 32 4.2 cdef 2.5 c 1.097 a Tollocan 71 4.3 def 2.3 c 1.082 c Stobrawa 34 4.4 f 2.2 c 1.090 b Average 51 3 .9 2.2 1 .092 ‘ All evaluated clones are half-sibs in relation to the late blight resistant parent. 2 Evaluated as a scale 1 to 9 of increasing color darkness. 3 Evaluated as a scale 1 to 5 of increasing defects. 4 Formula [weight in air/ (weight in air - weight in water)]. 5 Means in columns followed by the same letter are not significantly different using Fisher’s LSD at or = 0.05. 53 Table 3.3. Correlations at the clonal level between single- and eight-hill clonal generations for individual late blight families and combined for all families. Late Blight Families Traits Specific Gravity (SG) Tuber Appearance (TA) Chip- Color (CC) B0718-3 Bertita Bzura Greta Libertas Stobrawa Tollocan Zarevo Combined SG TA CC SG TA CC SG TA CC SG TA CC SG TA CC SG TA CC SG TA CC SG TA CC SG TA CC 0.43" 0.56M 0.44* 0.59" 1'18 068*“ 0.30"“ 0.65*** 0.67" HS 0041* 0.48M IIS l'lS ns HS 025* 0.27" 0.28”“ 060*" 063*" 0.43* 0.38"I 0.72*** 0.36* 0.46*** 0.42“ ' ns = not significant, * P .<. 0.05, ** P s 0.01 and *** P 5 0.001. 54 Selection of clones with acceptable tuber quality in single-hill plot Libertas, Stobrawa, Zarevo, and Tollocan families had the highest percentage of clones with acceptable chip-color (Table 3.4). Greta and Bzura were the parents that transmitted chip-color to the smallest percentage of its offspring. A selection based on a light chip-color (ratings 5 3) in single-hill plot eliminated 18% of the clones that would be selected in eight-hill plot. Alternatively, using the chip-color rating of 4 (acceptable quality) as a threshold for selection in single-hill plot eliminated only 7% of the clones that had ratings 5 3 in eight-hill plot. The percentage of discarded clones with acceptable chip-color in eight-hill plots varied from 0% (Stobrawa family) to 25% (Greta family). Stobrawa, B0718-3, Zarevo, Tollocan, and Bzura transmitted acceptable tuber appearance (ratings .<. 3) to more than 90% of the progeny (Table 3.5). An average of 32% of the clones that would have been rejected from single-hill plot for tuber appearance had good tuber appearance (ratings 5 2) in eight-hill plot. However, a selection criterion of tuber appearance ratings 5 3 would have eliminated only 4% of the clones that would be selected in eight-hill plot. The percentage of discarded clones with acceptable tuber appearance in eight-hill plot varied from 0% (Bzura family) to 14% (Greta family). Bzura, Libertas, Zarevo, and Stobrawa transmitted acceptable specific gravity to more than 90% of the offspring (Table 3.6). The selection of clones with specific gravity 2 1.080 (acceptable range) in single-hill plot resulted in 82% selection and would have eliminated only 4% of the clones that would be selected in eight-hill plot. The percentage of discarded clones that had acceptable specific gravity in eight-hill plot varied from 0% (Bzura and Stobrawa families) to 11% (Greta family). 55 Table 3.4. Percentage comparison of clones with acceptable chip-color quality in single- and eight-hill clonal generations. Late Blight Chip-Color Ratings 5 32 Chip-Color Ratings 5 42 F arniliesl Selected Not Selected3 Selected Not Selected3 Libertas 44.2 9.6 80.8 1.9 Stobrawa 32.4 1 1.8 76.5 0.0 Zarevo 28.3 22.8 71.7 5.4 Tollocan 52.1 11.3 69.0 7.0 Bertita 32.5 17.5 60.0 7.5 80718-3 28.8 20.3 59.3 11.9 Greta 21.4 53.6 57.1 25.0 Bzura 21.9 9.4 56.3 3.1 Total 34.3 18.4 67.6 7.1 ‘ All evaluated clones are half-sibs in relation to the late blight resistant parent. 2 Evaluated on a scale 1 to 9 of increasing color darkness in single-hill plots. 3 Not selected with chip-color ratings 3 3 in eight-hill plot. 56 Table 3.5. Percentage comparison of clones with acceptable tuber appearance quality in single— and eight-hill clonal generations. Late Blight Tuber App. Ratings S 22 Tuber App. Ratings S 32 Families1 Selected Not Selected3 Selected Not Selected3 Stobrawa 35.3 50.0 100.0 0.0 B0718-3 67.8 28.8 94.9 3.4 Zarevo 48.9 40.2 93.5 3.3 Tollocan 49.3 22.5 91.5 1.4 Bzura 50.0 15.6 90.6 0.0 Libertas 34.6 34.6 86.5 9.6 Greta 21.4 35.7 82.1 14.3 Bertita 22.5 27.5 72.5 7.5 Total 44.4 32.1 90.0 4.4 ' All evaluated clones are half-sibs in relation to the late blight resistant parent. 2 Evaluated on a scale 1 to 5 of increasing defects in single-hill plots. 3 Not selected with tuber appearance ratings 5 2 in eight-hill plot. 57 Table 3.6. Percentage comparison of clones with acceptable specific gravity quality in single- and eight-hill clonal generations. Late Blight Specific Gravity 2 1.0802 Families1 Selected Not Selected3 Bzura 100.0 0.0 Libertas 98.1 1.9 Zarevo 96.7 1.1 Stobrawa 91 .2 0.0 Greta 89.3 10.7 Tollocan 76.1 8.5 Bertita 55.0 7.5 B0718-3 52.5 6.8 Total 82.1 4.4 1 All evaluated clones are half-sibs in relation to the late blight resistant parent. 2 Formula [weight in air / (weight in air - weight in water)]. 3 Not selected with specific gravity 2 1.080 in eight-hill plot. 58 Identification of clones with acceptable chip-processing or tablestock quality The identification of clones with acceptable chip-processing quality was based on the threshold for selection identified for each tuber quality characteristic (Tables 3.4, 3.5, and 3.6). The selection was first done for chip-color ratings .<. 4 followed by tuber appearance ratings 5 3 and a specific gravity 2 1.080. Using this selection criterion, a total of 206 clones (50%) and 228 clones (56%) were identified in single— and eight-hill clonal generations, respectively, as possessing acceptable chip-processing quality. A total of 146 clones were selected in both clonal generations, which was 71% of the clones identified in single-hill plot. The identification of clones for tablestock was done based solely on tuber appearance ratings 5 3. A total of 367 clones (90%) and 387 clones (95%) were selected, respectively, in single- and eight-hill plot. Five parents (Zarevo, Stobrawa, B0718-3, Tollocan, and Bzura) transmitted acceptable tablestock quality to more than 90% of the progeny in both generations of selection. A total of 350 clones were selected in both clonal generations, which was 95% of the clones identified in single-hill plot. Discussion In this study, unadapted late blight resistant parents were crossed with adapted susceptible clones to select for tuber quality traits. Selection within these crosses was also done for foliar late blight resistance based upon greenhouse and field tests, in which 80 clones were identified as possessing moderate to strong late blight resistance to the USS genotype of P. infestans (Bisognin et al., 2001). Cultivar releases over the past 59 century suggest that tuber quality should be considered a market—limiting trait (Douches et al., 1996). Therefore, even in breeding disease resistant cultivars, tuber quality needs to be a high priority for selection and these results showed that selection for tuber quality could be initiated at the single-hill plot. Since Tollocan and B0718-3 transmit a higher level of late blight resistance to the highest percentage of the offspring (Bisognin et al., 2001), these two parents offer the best chance for combining resistance with tuber quality traits. Tollocan and B0718-3 are also the best candidates to apply the strategy proposed here, in which selection for tuber quality precedes selection for late blight resistance. The selection of parents for their potential to transmit important traits to the offspring is an important step in a potato breeding program (Tai & Young, 1984; Thill & Peloquin, 1995). In this study we identified late blight resistant parents that also transmit tuber quality traits to the offspring. B0718-3 and Tollocan families had the highest tuber appearance ratings in single-hill plot. The B0718-3 family also had the highest tuber appearance rating average and the highest percentage of selected clones with a tuber appearance rating S 2. Bzura, Zarevo, and Libertas families had the highest specific gravity in both generations of selection and the highest percentage of selected clones. The fact that late blight resistant parents were crossed with a different number and, in most cases, to different susceptible parents might have influenced family performance. Therefore, all family differences found in this study should not be attributed solely to the late blight resistant parent, but it was clear that those parents do differ in tuber quality traits transmitted to the offspring. Bzura and Stobrawa were crossed to the same susceptible parents and Greta was crossed to four out of five susceptible parents. Greta family had the highest chip-color rating and Bzura family had the highest specific gravity in eight-hill plot. Moreover, late blight family differences can not be attributed to the 60 phenotypic selection done at harvest time, since the same selection intensity was applied to all crosses. Considering only the percentage of selected clones, Libertas, Stobrawa, Zarevo, and Tollocan were the best parents for transmitting chip-color; B0718-3, Stobrawa, Zarevo, and Tollocan for transmitting tuber appearance; and Bzura, Libertas, Zarevo, and Stobrawa for transmitting specific gravity to the highest percentage of the offspring. The Stobrawa family, for chip-color, and the Stobrawa and Bzura families, for tuber appearance and specific gravity, had all selected clones identified in single-hill plot, but the Greta family had the highest percentage of non-selected clones for all traits. Stobrawa, Bzura and Greta have similar contribution of susceptible parents. Therefore, the percentage of selected clones was effective in showing differences among late blight families when selection for tuber quality was applied at the single— or eight-hill clonal generations. Interaction between parents with years would increase the percentage of discarded clones as in the case of Greta family. Attempting to breed for tuber quality traits in potato, a phenotypic selection based on tuber appearance, number, shape, and internal defects at harvest time in single-hill plot was able to reduce the number of evaluated clones from 4,750 to 408. Comparing with other traits considered for phenotypic selection, Tai (1975) determined that tuber appearance was the only trait to directly affect selection and Neele et a1. (1991) found tuber yield to be the decisive component for selection. From the 408 clones, 68% possessed acceptable chip-color, 90% possessed acceptable tuber appearance, and 82% possessed desirable specific gravity in single-hill plot. If a moderate selection, based upon tuber appearance ratings 5 3 for tablestock, were employed no more than 4% of the 61 clones discarded in eight-hill plot would have been selected for each trait (chip-color, tuber appearance, and specific gravity) in eight-hill plot. The high percentage of clones selected’in both clonal generations is supported by the significant coefficients of correlation obtained between single- and eight-hill clonal generations. Correlation was considered the best estimate to determine relationship between early generations of selection in potato breeding (Maris, 1988). Chip-color and tuber appearance had smaller correlation coefficients than specific gravity at the clonal level, while at the family level, there was no correlation between generations for chip- color. As a consequence, with higher correlation coefficients, 96% of clones with desirable specific gravity (2 1.080) in eight-hill could be identified in single-hill plot using the same selection criteria. High correlation was expected for specific gravity because this trait has been previously reported to have a small genotype x environment interaction (Killick & Simmonds, 1974). Haynes & Wilson (1992) also found high positive correlation for specific gravity between the two first generations in the field. The correlation between single- and eight-hill clonal generations suggests that moderate selection intensity should be applied in single-hill plot for tuber appearance and chip-color. Tuber appearance had lower correlation coefficients at the clonal level than chip-color and had no significant correlations between single- and eight-hill clonal generations for five individual families. Tuber appearance was also the only trait significantly affected by year (single- and eight-hill clonal generations). Chip-color had significant correlations for all individual late blight families, but small correlation (0.42) for all combined families. As opposed to specific gravity, selecting clones with desirable tuber appearance (ratings 5 2) and chip-color (ratings .<. 3) would eliminate a significant percentage of clones that would have desirable quality based on eight—hill plot. However, 62 selecting clones with acceptable tuber appearance (ratings 5 3) and chip-color (ratings 5 4) in single-hill plot would discarded a very small percentage of clones that would be selected in eight-hill plot with desirable quality. Tai (1975) also found low correlation for tuber appearance at the clonal level, but medium to high correlations at the family level. Neele et a1. (1991) found high heritability estimates for tuber appearance components such as tuber shape (0.61), regularity of tuber shape (0.60), skin color (0.86), eye depth (0.69), number of tubers (0.54), and average tuber weight (0.64). The fact that clones with desirable quality, for specific gravity, and clones with acceptable quality (moderate selection intensity), for chip-color and tuber appearance, can be applied at single-hill plot is very important, since there is a gain of one year in the selection process for tuber quality. Thill & Peloquin (1995) reported that selection decisions for cold chip-processing at the single-hill plot did not differ from those in late generations and could potentially save four years in the breeding cycle. A low to moderate selection pressure in early generations was found in other studies as the best choice to reach a balance between gain from selection and elimination of valuable clones (Tai & Young, 1984; Maris, 1988). Neele et al. (1989) determined that phenotypic selection in early generations was optimized when about 32% of the clones were selected in the first clonal generation. Anderson & Howard (1981) found a higher number of discarded than selected clones comparing the first two generations of selection. In comparison, the post-harvest selection used here reduced the initial number of clones from 4,750 to 206, through selection for chip-processing quality (4.3% of selected clones), and to 367, through selection for tablestock quality (7.7% of selected clones). However, the evaluation in single- and eight-hill clonal generations has some key differences that should be considered in the selection process. The performance of plants 63 grown either from small greenhouse tubers or from transplants is often very distinct from that of the same plants grown from regular sized seed tubers (Davies & Johnston, 1974). In addition, difference in the in-row spacing (75 cm vs. 30 cm) may influence plant competition. These factors can reduce the heritability in single-hill plot for many traits resulting in poor selection efficiency (Tai & Young, 1984). Sample size is another concern for evaluations in single-hill plot. The accuracy of the specific gravity estimation decreases rapidly for samples smaller than 10 tubers (Lulai & Orr, 1979). Different traits are of primary importance when developing cultivars with chip- processing or tablestock quality. Chip-color is the most important trait for the chip- processing industry (Thill & Peloquin, 1995) followed by tuber appearance (freedom from internal and external defects) and high dry matter. For tablestock cultivars, tuber appearance is the most important trait (Dale & Mackay, 1994). If the objective is to develop cultivars for chip-processing industry and tablestock, the tuber quality information from single-hill plot could be used to assist in making better decisions in later generations of selection for other traits including disease resistance. Also, multitrait selections based on data from different environmental conditions might increase the probability of identifying clones possessing an acceptable balance of key agronomic traits. Haynes & Wilson (1992) found that the probability of selecting the same clone in the later generation was 1.7 and 1.9 times higher for clones selected based on horticultural characteristics than on specific gravity. In summary, a moderate selection intensity for tuber quality traits (chip-color ratings .<. 4, tuber appearance ratings S 3 and a specific gravity 2 1.080) can be initiated at the single-hill plot in crosses to select for late blight resistance. The identification of superior clones for tuber quality in single-hill plot reduces each selection cycle in one 64 year and reduces the number of clones for late blight testing. Intermating selected clones, a higher percentage of clones possessing acceptable chip-processing or tablestock quality is expected in following cycles of genotypic recurrent selection. Moreover, Tollocan and B0718-3 are the best parents for improving late blight resistance and offer the best opportunity for the application of tuber quality selection in advance of disease resistance selection for combining desirable traits. The progenies of Tollocan and B0718-3 could also be combined with the offspring of other high valuable sources of late blight resistance for the development of cultivars with durable resistance. Literature Cited Anderson, J .A.D. & H.W. Howard, 1981. Effectiveness of selection in the early stages of potato breeding programmes. Potato Res. 24:289-299. Bisognin, D.A., D.S. Douches, K. Jastrzebski & W.W. Kirk, 2001. Half-sib progeny evaluation and selection of potatoes resistant to the U88 genotype of Phytophthora infestans fi'om crosses between resistant and susceptible clones. Euphytica (Accepted for Publication). Colon, L.T., D.J. Budding, L.C.P. Keizer & M.M.J. Pieters, 1995. Components of resistance to late blight (Phytophthora infestans) in eight South American Solanum species. European J. Plant Pathol. 101:441-456. Dale, M.F.B. & G.R. Mackay, 1994. Inheritance of table and processing quality. In: J. E. Bradshaw & G.R. Mackay (ed.) Potato Genetics. CAB International, Cambridge. pp. 285-315. 552 p. Davies, H.T. & G.R. Johnston, 1974. Reliability of potato selection in the first clonal generation. Amer. Potato J. 51 :8-1 1. Douches, D.S., D. Maas, K. Jastrzebski & R.W Chase, 1996. Assessment of potato breeding progress in the USA over the last Century. Crop Sci. 36:1544-1552. Douches, D.S., W.W.Kirk, K. Jastrzebski, C. Long & R. Hammerschmidt, 1997. Susceptibility of potato varieties and advanced breeding lines (Solanum tuberosum 65 L.) to Phytophthora infestans (Mont) de Bary in greenhouse screenings. Amer. Potato J. 74:75-86. Fry, W.E. & S.B. Goodwin, 1997. Resurgence of the Irish potato famine fungus. Bioscience 47:363-371. Haynes, K.G.& D.R. Wilson, 1992. Correlations for yield and specific gravity between potato tuberling and second year field generations. Amer. Potato J. 69:817-826. Helgeson, J.P., J.D. Pohlman, S. Austin, G.T. Haberlach, S.M. Wielgus, D. Ronis, L. Zambolin, P. Tooley, J.M. McGrath, R.V. James & W.R. Stevenson, 1998. Somatic hybrids between Solanum bulbocastanum and potato: a new source of resistance to late blight. Theor. Appl. Genet. 96:738-742. Kamoun, S., E. Huitema & V.G.A.A. Vleeshouwers, 1999. Resistance to oomycetes: a general role for the hypersensitive response? Trends Plant Sci. 4:196-200. Kelly, J.D., J.M. Kolkrnan & K. Schneider, 1998. Breeding for yield in dry beans (Phaseolus vulgaris L.). Euphytica 102:342-356. Killick, R.J. & N.W. Simmonds, 1974. Specific gravity of potato tubers as a character showing small genotype-environment interactions. Heredity 32:109-112. Love, S.L., J.J. Pavek, A. Thompson-Johns & W. Bohl, 1998. Breeding progress for potato chip quality in North American cultivars. Amer. J. Potato Res. 75:27-36. Lulai, E.C. & PH. Orr, 1979. Influence of potato specific gravity on yield and oil content of chips. Amer. Potato J. 56:379-390. Maris, B., 1988. Correlations within and between characters between and within generations as a measure for the early generation selection in potato breeding. Euphytica 37:205-224. Neele, A.E.F., H.J. Nab, M.J. de Jongh de Leeuw, A.P. Vroegop & K.M. Louwes, 1989. Optimizing visual selection in early clonal generations of potato based on genetic and economic considerations. Theor. Appl. Genet. 78:665-671. Neele, A.E.F., H.J. Nab & K.M. Louwes, 1991. Components of visual selection in early clonal generations of a potato breeding programme. Plant Breed. 106:89-98. SAS Institute, 1995. The SAS system for windows. Release 6.12. SAS Institute, Cary, NC. Tai, G.C.C., 1975. Effectiveness of visual selection of early clonal generation seedlings ofpotato. Crop Sci. 15:15-18. Tai, G.C.C. & D.A. Young, 1984. Early generation selection for important agronomic characteristics in a potato breeding program. Amer. Potato J. 61 :419-434. 66 Thill, C.A. & S.J. Peloquin, 1995. A breeding method for accelerated development of cold chipping clones in potato. Euphytica 84:73-80. Umaerus, V., M. Umaerus, L. Erjefalt & B.A. Nilsson, 1983. Control of Phytophthora by host resistance: problems and progress. In: Erwin, D.C., S. Bartnicki-garcia, P.H. Tsao (ed.). Phytophthora infestans: Its Biology, Taxonomy, Ecology, and Pathology. The American Phytopathological Society. St. Paul, Minnesota. p. 315-326. 67 CHAPTER IV Genetic Diversity in Diploid and Tetraploid Late Blight Resistant Potato Germplasm Abstract An understanding of the genetic relationship within potato gerrnplasm is important to establish a broad genetic base for breeding purposes. The objective of this study was to assess the genetic diversity of potato (Solanum tuberosum subsp. tuberosum Hawkes) gerrnplasm that can be used in the development of cultivars with resistance to late blight caused by Phytophthora infestans (Mont) de Bary. Thirty-three diploid and 27 tetraploid late blight resistant potato clones were evaluated for their genetic diversity based on 11 isozyme loci and nine microsatellites. A total of 35 allozymes and 42 polymorphic microsatellite fragments was scored for presence or absence. The gerrnplasm was clustered based on the matrix of genetic similarities and the unweighted pair group means analysis of the isozyme and microsatellite data, which were used to construct a dendrogram using NTSYS-pc version 1.7. Twenty-three allozymes and DNA fragments were unique to the wild species. The diploid Solanum species S. berthaultii Hawkes and S. microdontum Bitter formed two distinct phenetic groups. Within S. 68 microdontum, three sub-groups were observed. The tetraploid gerrnplasm formed another group, with S. sucrense Hawkes in one sub-group and the cultivated potato and Russian hybrids in another sub-group. Based upon the genetic diversity and the level of late blight resistance, S. microdontum and S. sucrense offer the best choice for improved late blight resistance from genetically diverse sources. This potato gerrnplasm with reported late blight resistance should be introgressed into the potato gene pool to broaden the genetic base to achieve stronger and more durable resistance. Introduction The cultivated potato and its wild relatives belong to the genus Solanum L. sect. Petota Dumort. There are seven cultivated and 225 wild potato species, according to the most recent taxonomic treatment of Hawkes (1990), which include diploid (2n = 24), tetraploid (2n = 48), hexaploid (2n = 72) and a few triploid (2n = 36) and pentaploid (2n = 60) cytotypes (Spooner & van den Berg, 1992). The cultivated potato S. tuberosum subsp. tuberosum Hawkes is an autotetraploid (2n = 4x = 48) that originated in South America. - Despite the wide genetic diversity that exists in the genus Solanum, the use of closely related gerrnplasm in breeding programs has resulted in high genetic similarity among more than 130 potato cultivars released in North America between 1930 and 1970 (Mendoza & Haynes, 1974). The pedigrees of most cultivars can be traced back to the cultivars Early Rose and one of its parents, Garnet Chili (Plaisted & Hoopes, 1989). 69 Moreover, cultivars released between 1950 and 1970 have a high genetic similarity and may have reached a yield plateau (Mendoza & Haynes, 1974). Molecular markers have been used to confirm the relatedness among North American potato cultivars. Coefficients of similarity ranged from 0.51 to 0.89 among 28 potato cultivars based on random amplified polymorphic DNA (RAPD) (Demeke et al., 1996) and from 0.44 to 0.81 among 18 potato cultivars from different origins based on simple sequence repeats (SSR) or microsatellites (Provan et al., 1996). An identical chloroplast DNA (T-type) restriction pattern was found among 10 historically important potato cultivars that traced back through Garnet Chili to Rough Purple Chili, indicating that there is only one maternal lineage (Douches et al., 1991). The predominance of the T-type cytoplasm derived from Rough Purple Chili was found in the modern European cultivated gene pool. Rough Purple Chili was introduced in Europe after the 1840’s late blight epidemic and was extensively used as female parent (Provan et al., 1999). Late blight, caused by the fungal-like oomycete Phytophthora infestans (Mont) de Bary, is the most devastating potato disease worldwide (Fry & Goodwin, 1997; Kamoun et al., 1999) and causes both foliar destruction and tuber decay (Ross, 1986). The development of genetic resistance to late blight in potatoes is one of the major objectives in many breeding programs (Colon et al., 1995a) and has resulted in the release of late blight resistant gerrnplasm (Goth & Haynes, 1997; Corsini et al., 1999). Essential studies on breeding potato for late blight resistance have been done, such as identification of resistance sources (Colon & Budding, 1988; Colon et al., 1995c; Douches et al., 2001a), components of resistance (Colon et al., 1995a,b,c), and phenotypic stability of resistance (Haynes et al., 1998). These reported late blight resistance sources are of different origin and ploidy levels and have variable levels of 70 resistance. Combining these sources in a breeding program will establish a broad genetic base in the cultivated potato from which the probability of selecting superior offspring is increased. Moreover, improvements in yield, adaptation, tuber quality and disease resistance can be achieved by broadening the genetic base of potato breeding populations (Mendoza & Haynes, 1974). The objective of this study was to assess the genetic diversity of this potato gerrnplasm with reported late blight resistance using a set of isozyme loci and microsatellite markers. These data can be used to characterize this gerrnplasm that can be introgressed into cultivated gene pools to enhance late blight breeding efforts and concurrently broaden the genetic base of cultivated potatoes. Material and Methods The potato late blight resistant gerrnplasm used in this study was identified using a greenhouse fine-screening technique (Douches et al., 2001a) and represents different origins and ploidy levels (Table 4.1). Of the total of 60 evaluated clones, 36 were from South America (2 species), 14 were tetraploid hybrids (wild x cultivated potato), and 10 were tetraploid advanced breeding clones or cultivars from North America (5), Poland (3), Sweden (1), and Russia (1). For simplification, all accessions or cultivars will only be referred to by their respective code identification (Table 4.1). 71 A3 .oEmMZ £5850 980E m3 05 5 8 2:8 05 a 898:: connocunofl one—o 2: :5 Stem £5 E nozaocceovm 83035.55 EaE .03N 9’0th 223 :38: a $383 a 8.58 x 553V: 5. 8:82 a 8529: a £30: a Seed a 78-qu 58:: e823 .8 e53.— §:e§.§a2 x 9832...): x «$3.352: x .85 seesaw «3885 .3 £8on 882m 32 5220 x 58:8. $4582 .3882 28.8.— x 8m 085 av mean—.8502 x Aofitocm x 055: x noficocm x «50:2: x 283—: 8:35 8:33 xv 533m Rates; MEBE x 83. item: x Enema—2: x 853 820 880 .3 228 5 an a. 338 x 852a: x g 72:2 8. 8-0.: x «2.8: 83m 83m 2.4 38.933 w. x .3654 53.33% .5 av 8:82 x genie: .833 88.985 .8 u o< @333. x $933. 835m 8.8m 5. <2 65325 . m xv 233. 8836: 38.88 E33 5:883 w. x 338:8 .m 8.2 Emma E> a xv 383. 8:89 x Aocoom x $52 .3 E2 x 558.883 M8 55. ommman M; e.~ n. 283. 25... 1322 a seam 85:38 .8 8E 22% ES _ 3 £83. 2:25 13854.. x 58888.8 .8 23. m _ man M5 2.. av «68.: 392.3 E: x $53.3 E2 x ._B_._om £883.63 .3 max Emma ~=> w 2: .3 23.3. 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Each accession or cultivar was sampled and run twice. Tissue processing, electrophoresis, staining, and nomenclature were done as described in Douches & Quiros (1988). Eleven isozyme loci of seven enzyme systems were scored according to Douches & Quiros (1988) and Douches & Ludlam (1991). Malate dihydrogenase (Mdh-l and Mdh-Z), 6- phosphogluconic dehydrogenase (6-Pgdh-3), phosphoglucose isomerase (Pgi-I), and isocitric acid dehydrogenase (Idh-I) were resolved with a histidine-citrate pH 5.7 buffer system (Stuber et al., 1988). Diaphorase (Dia-I and Did-2), glutamate oxaloacetate transaminase (Got-I and Got-2) and phosphoglucomutase (Pgm-I and Pgm-Z) were resolved with a lithium-borate pH 8.3 buffer system (Stuber et al., 1988). Nine of these isozyme loci have been previously mapped to six distinct potato linkage groups. Mdh-2 and Idh-l were mapped to linkage group I, Pgm-I to III, Pgm-Z to IV, Mdh-I, 6-Pgdh—3 and Dia-I to V, Got-2 to VII, and Got-1 to VIII (Bonierbale et al., 1988; Freyre & Douches, 1994). For statistical analysis, each allele was recorded as 1 for presence or O for absence. DNA amplification, using nine pairs of microsatellite primers, was carried out in a total volume of 20 ul containing 1X REDTaqTM PCR reaction buffer, 1 unit of REDTaqTM DNA polymerase (Sigma—Aldrich Co., St. Louis, MO.), 20 ng of each dNTP, 25 ng of each microsatellite primer, and 50 ng of template DNA. The sequence of eight pairs of primers (GZ8WXST, STPROINI, ST STP, STACCAS3, STWINIZG, POTM 1-2, ST13ST, and ST LS] ) are published (Ashkenazi et al., 2001). The other primer combination, potato 73 inhibitor IIK locus, was also used by Provan et a1. (1996) and Milboume et al. (1997), and the sequences are identified as STIIKA. Four microsatellites have a known position on the potato linkage map. The loci G28WXST, STACCAS3 and POTM 1-2 map to linkage groups VIII, VII and VI, respectively (Veilleux, personal communication). The potato inhibitor IIK locus maps to the linkage group III (Meyer et al., 1998). All amplifications were carried out on a Thermolyne Amplitron® (BamsteadTM Therrnoline Corporation, Dubuque, IA.) thermal cycler. The protocol was as follows: 1) initial denaturation at 94 °C for 4 min; 2) 40 cycles of denaturation at 94 °C for l min, annealing at 55 °C for 2 min, and extension at 72 °C for 1.5 min; and 3) final extension at 72 °C for 5 min. The completed reaction products were held at 4 °C until electrophoretic separation using a 3% MetaphorTM Agarose (FMC Bioproducts, Rockland, ME.) gel with TBE (90 mM tris-borate, 90 mM boric acid and 2 mM EDTA) buffer. The gels were run at 100V for 4 h at 10 °C, stained with ethidium bromide (1 ug . ml") for 45 min, visualized under UV light, and photographed. Each microsatellite fragment was scored as l for presence and O for absence. Fragment sizes were estimated using a 50 bp DNA ladder (Gibco BRL, Grand Island, NY.) in each gel. For statistical analysis, data were scored as the presence or absence of alleles (isozymes) or fragments (microsatellites). The mean number of alleles per locus, the proportion of polymorphic loci and the mean expected heterozygosity (Nei, 1972) were estimated per accession or group of clones based on allelic frequency data. For these parameters, we did not consider allele dosage for isozymes and we evaluated DNA fragments per pair of microsatellite primers. Genetic similarity was calculated using Nei and Li's (1979) computation: GSxy = 2ny / (Nx - Ny) 74 where Nx and Ny are the number of bands for each genotype, and ny is the number of bands in common between the two genotypes. The unweighted pair group means analysis (UPGMA) results were used to draw the dendrogram. The distance matrix and the dendrogram were constructed with NTSYS—pc version 1.7 (Rohlf, 1992). Cophenetic correlation coefficients were used to measure the distortion between the similarity matrix and the resultant dendrogram (Rohlf & Sokal, 1981). Results And Discussion General genetic diversity in the late blight resistant gerrnplasm The total mean number of alleles per locus for isozymes was 3.18 and for microsatellites was 4.67 (Table 4.2). To have a better understanding of the genetic diversity, we divided the germplasm by species or group of clones. For example, there were 27 S. microdontum Bitter clones that we sub-divided by P1 numbers. We also separated cultivated PI (Russian hybrids) from cultivated potato. Based upon both marker systems, S. sucrense Hawkes (wild tetraploid) had one of the lowest mean numbers of alleles per locus. On the other hand, Russian hybrids and cultivated potatoes showed the highest number of alleles per locus. Solanum sucrense also had the smallest proportion of polymorphic loci, indicating low genetic diversity among evaluated clones. In other marker analyses, S. sucrense was previously assessed to have higher levels of genetic diversity (Hosaka & Hanneman, 1991; Barnberg et al., 2000). An underestimation of S. sucrense diversity was expected, since only three clones of one accession were evaluated. The highest proportion of polymorphic loci was observed in S. 75 berthaultii Hawkes and Russian hybrids for isozymes and Russian hybrids and cultivated potato for microsatellites. No trend was observed to distinguish species or group of clones using mean of expected heterozygosity (Table 4.2). In general, there were accessions or wild species with a similar level of genetic diversity compared to cultivated potato. However, direct comparisons between diploid and tetraploid species should not be made, since tetraploids have the potential to have greater heterozygosity than diploids. In summary, genetic markers showed that a high level of genetic diversity is distributed among wild diploid, and wild tetraploid and cultivated Solanum species with reported late blight resistance. Sources of unique allOzyme alleles A total of 35 allozymes was detected in the late blight resistant gennplasm. The presence of allozymes that were observed in the cultivated group was similar to the Douches et al. (1991) study of 112 North American cultivars and advanced breeding clones. Evaluating the genetic diversity in 2379 accessions of S. tuberosum subsp. andigena Hawkes, Huaman et a1. (2000) identified 38 allozymes, however 2 allozymes had a frequency of only 0.02%. Isozyme analysis revealed numerous allozymes in the wild gerrnplasm that were not found in the cultivated potato group. Nine allozymes, (26%), absent from the cultivated potato group, were present in other tetraploid clones and diploid Solanum species (Table 4.3). Berl and ber2 had six alleles not present in cultivated potatoes and Pgi-15 was unique to S. berthaultii. Solanum microdontum had seven allozymes that were absent in cultivated potatoes. Allozymes unique to S. microdontum were present in mcd6 (Malia-16) and mcd5 (Pgm-Z'). Pgi-13 was found only in one clone (mcd3-7). 76 Solanum sucrense had only one allozyme (6-Pgdh-33) not found in the cultivated potato group. The Russian hybrids had three alleles not found in cultivated potatoes, but these alleles were found in only one clone and were also present in wild species. Sources of unique microsatellite fragments Forty-two of the 43 DNA fragments from nine pairs of microsatellite primers were consistently amplified and polymorphic. High levels of polymorphism were also found in other genetic studies using microsatellites in cultivated potatoes (Provan et al. 1996; Milboume et al., 1997; Meyer et al., 1998). Six fragments were absent in the cultivated potato group (Table 4.4). The Russian hybrids did not possess any unique fragments, but did have two fi'agments that were not found in the cultivated potato group. Solanum microdontum possessed five fragments absent from the cultivated potatoes and three fragments associated with three microsatellite loci (GZSWXST, POTMl-Z, and STLSI) were present in all evaluated clones. Of the 23 alleles and microsatellite fiagments absent in cultivated potatoes, three alleles and two DNA fragments were present in Russian hybrids. These results were, in part, expected since Russian hybrids are hybrids of wild and cultivated potato. However, only four hybrids evaluated here (K99, K101, K102, and K105) have common wild species in their pedigrees. Therefore, the isozyme and microsatellite analyses showed that Russian hybrids carry some genetic diversity from wild species and this diversity is much more accessible in the genetic background to combine with cultivated potatoes. 77 .33_ in 6 538% «aw - ~ n a: .masew 22953 no 203% £53, a: mo owfioiw 05 mm bfiowfieouon Buooaxm N .mmfiow no.“ 3. 033. com .3068on Became 8 page £0585: was 3:38;. .m. 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N 9533 252 383220 563% 18m 32 82 BE .32 mom 033:3 8583822 Can u :Nv Ema—mgow Eoiabah ANN H :8 36on 83:30” 2035 SEEwNE 2583828 2.3:: 05 go 35 £36365 Mo 2883 .Bfioa 333:3 E 283m 98 Emma—Eow :83me E33 23 voamwmcs E Havana and 35893 8523828 oscED 41V 2an Genetic similarity among late blight resistant clones The assessment of either 35 allozymes or 42 polymorphic microsatellites alleles was insufficient to completely discriminate diploid from tetraploid species. The cophenetic correlation coefficient between the similarity matrix and the dendrogram was 0.79 and 0.78, respectively for isozymes and microsatellites. Solanum microdontum, S. berthaultii and S. sucrense belong to the series T uberosa (wild) and S. tuberosum subsp. tuberosum belong to the series T uberosa (cultivated) in the subsection Petotae (Hawkes, 1994). In both dendrograms, clones of S. microdontum were clustered in different groups with S. berthaultii, S. sucrense or cultivated potatoes (data not shown). A combined data analysis from isozyme and microsatellite markers was able to distinguish diploid fiom tetraploid species and also separate S. microdontum from S. berthaultii. The cophenetic correlation coefficient between the similarity matrix and the dendrogram data was 0.82. The late blight resistant gerrnplasm formed three groups, two with each diploid wild species (S. berthaultii and S. microdontum) and one with tetraploid gerrnplasm (S. sucrense, Russian hybrids, and cultivated potatoes) (Fig. 4.1). The genetic similarity between S. berthaultii and S. microdontum groups, excluding mcd4, was 0.58. Within S. microdontum, four distinct sub-groups were formed according to accessions (mcdl, mcd2, and mcd3; mcd4; mods; and mcd6). Overall, the maximum genetic similarity (0.93) was between Mcd3—l and Mcd3-5. The tetraploid gerrnplasm group (group 2) formed two sub-groups: one with the wild tetraploid species (S. sucrense) and another with Russian hybrids and cultivated potatoes, with a genetic similarity of about 0.65. All cultivars were grouped with Russian hybrids with a genetic similarity of about 0.77. The cultivars B0718-3, Tollocan and AWN86514-2, reported as highly resistant to late blight, separated into three sub-groups among cultivated potatoes. 81 ber1 -3 _____{ ber1-8 bed-12 ban-16 hen-20 Group 1 l 2 = 2x beKZ-19 " son-16 sen-17 l rat-:3 80718-3 '—1 L Bzura .s K104~21 ‘L——+:: ”$627“ Tollocan Libggas K1 .5 r-l Bertita Group 3 Greta 2n = 4x ,—-———— K10349 _..| g—— Zarevo AWN86514-2 l Stobrawa - -L. ll X e Group 2 meme 2n=2x 11 $33; 0.58 0.67 0.75 0.84 ' 0.93 Genetic Similarity Figure 4.1. Genetic similarity among diploid and tetraploid potato germplasm with reported late blight resistance based on 35 allozymes, encoding 11 isozyme loci, and 42 polymorphic DNA fragments in nine pairs of microsatellite primers. Code for clones and pedigrees follow Table 4.1. 82 Besides the genetic diversity quantified using isozyme and microsatellite markers, different types of late blight resistance also could be present within wild and cultivated gerrnplasm. Several minor genes with additive effects are involved in the late blight resistance of S. tuberosum subsp. andigena (part of the pedigree of K102, Bertita, and Zarevo), whereas major resistant genes are involved in the resistance of S. sucrense (Colon & Budding, 1988). If the genetic diversity present in the tetraploid gerrnplasm might also include different genes for late blight resistance, clones fi'om different sub- groups should be hybridized as a strategy to combine potential resistance sources. Improvements of late blight resistant cultivars with broad genetic base Pedigree (Mendoza & Haynes, 1974; Plaisted & Hoopes, 1989) and chloroplast diversity analysis (Douches et al., 1991; Provan et al., 1999) showed that a high genetic similarity characterizes many potato cultivars released in the last century. High genetic uniformity can result in vulnerability to diseases, pests and abiotic factors, and reduces gain from selection (Mendoza, 1989). Consequently, increasing genetic diversity in the cultivated potato gene pool is a goal in many breeding programs. Moreover, improving genetic diversity for non-T-type cytoplasm is important to reduce breeding problems associated with male sterility (Provan et al., 1999). The species studied here can be used to enhance efforts to breed late blight resistant tetraploid gerrnplasm and to broaden the genetic base of the cultivated potato using simple crossing schemes (4x - 4x and 4x - 2x). The late blight resistant gerrnplasm differs in the level and source of resistance (Haynes et al., 1998; Douches et al., 2001a). Solanum microdontum (mcd3, mcd5, and mcd6) and S. sucrense had the highest level of resistance to the US8 genotype of P. infestans. Also, S. berthaultii and other S. microdontum accessions and the Russian 83 hybrids had moderate to high resistance (Douches et al., 2001a). The source of resistance for the hybrids K98 and K100 is Mexican Solanum species, whereas all other hybrids (K97, K99 and K101 - K105) have South American wild species in their pedigrees. The hybrid K102 is a cross between two South American Solanum species (S. berthaultii and S. tuberosum subsp. andigena) in which both parents could be contributing to the resistance. Among the cultivated gerrnplasm, AWN86514-2 has high foliar and partial tuber resistance and has S. acaule Bitter, S. demissum Lindley, S. phureja Juz. & Bukasov, S. microdontum, S. stoloniferum Scheldl. & Bouche, and S. tuberosum subsp. andigena in its pedigree (Corsini et al., 1999) which can be contributing to its resistance. B0718-3 has foliar resistance to late blight derived from an Indian S. tuberosum introduction (PI383470B) selected in Mexico (Goth & Haynes, 1997). Bertita and Zarevo have S. demissum and S. tuberosum subsp. andigena in their pedigrees, both well- known sources of late blight resistance and these cultivars along with Bzura, Bertita, Greta, Libertas, and Stobrawa exhibit foliar late blight resistance in field evaluations (Haynes et al., 1998). Libertas is considered to have no R-gene and have both foliar (Colon et al., 1995b) and tuber resistance (Platt and Tai, 1998), probably sharing the same resistant genes with Pimpemel, Robijn, Populair and Surprise (Colon et al., 1995b). The advanced clone MSG274-3 is directly descended from the Mexican cultivar Tollocan. Tollocan and MSG274-3 have high foliar resistance to late blight in greenhouse and in field evaluations (Douches et al., 2001b). The genetic diversity analysis showed that this gerrnplasm could offer unique opportunities for late blight resistance breeding and that conventional breeding strategies may be useful to introgress and combine these different resistance sources. For short term strategy, combining sources of high levels of late blight resistance from different 84 sub-groups such as MSG274-3 and Tollocan with AWN86514-2 or with Russian hybrids possessing moderate resistance to late blight would be more productive than combining with S. sucrense, a wild 4x species. A long term breeding strategy would be to introgress the resistance from S. microdontum and S. berthaultii. The fact that two clones from the same accession of S. microdontum had different quantitative trait loci (QTL) conferring late blight resistance (Sandbrink et a1. 2000) and that there are separate clusters in the dendrogram for the different S. microdontum accessions, suggest that multiple selections of S. microdontum should be used according to the clustering. This strategy should maximize the diversity of the late blight resistant sources. Within gerrnplasm having moderate to high resistance to late blight it is difficult to differentiate resistant individuals and almost impossible to select recombinant offspring based upon phenotypic tests. The association between markers and QTL permits the selection of individuals with desirable QTL from different parents (Meyer et al., 1998). Therefore, mapping QTL conferring late blight resistance is required to analytically pyramid genes from diverse genetic background. In summary, there was high genetic diversity within and between accessions, species, and ploidy levels of the late blight resistant gerrnplasm. Both wild diploid species S. microdontum and S. berthaultii had more genetic diversity between and, in some cases, within accessions than cultivated potatoes. This genetic diversity should be exploited using both short and long-term strategies to broaden the genetic base of the potato gene pool and to combine different sources of resistance in a breeding program to achieve stronger and more durable resistance in the offspring. 85 Literature Cited Ashkenazi, V., E. Chani, U. Lavi, D. Levy, J. Hillel & R.E. Veilleux. 2001. Development of microsatellite markers in potato and their use in phylogenetic and fingerprinting analysis. Genome 44:50-62. Bamberg, J. B., C. Singsit, A.H. del Rio &E.B. Radcliffe. 2000. RAPD analysis of genetic diversity in Solanum populations to predict need for fine screening. Amer. J. Potato Res. 77:275-278. Bonierbale, M.W., R.L. Plaisted, & S.D. Tanksley. 1988. RFLP maps based on common set of clones reveal modes of chromosomal evolution in potato and tomato. Genetics 120: 1095-1 103. Colon, L.T. & D.J. Budding. 1988. Resistance to late blight (Phytophthora infestans) in ten wild Solanum species. 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Genetic diversity of potato determined by random amplified polymorphic DNA analysis. Plant Cell Rpt. 15:662-667. Douches, D.S. & C.F. Quiros. 1988. Additional isozyme loci in tuber-bearing solanums: inheritance and linkage relationship. J. Hered. 79:377-3 84. Douches, D.S. & K. Ludlam. 1991. Electrophoretic characterization of North American potato cultivars. Amer. Potato J. 68:767-780. Douches, D.S., K. Ludlam, & R. Freyre. 1991. Isozyme and plastid DNA assessment of pedigrees of nineteenth century potato cultivars. Theor. Appl. Genet. 82:195-200. 86 Douches, D.S., J.B. Bamberg, W. Kirk, K. Jastrzebski, B.A. Niemira, J. Coombs, D.A. Bisognin, & K. Walters-Felcher. 2001a. Evaluation of wild Solanum species for resistance to the USS genotype of Phytophthora infestans utilizing a fine-screening technique. Amer. J. Potato Res. 78:159-165. Douches, D.S., W.W. Kirk, M.A. Bertram, & B.A. Niemira. 2001b. Foliar and tuber assessment of late blight (Phytophthora infestans (Mont.) de Bary) reaction in cultivated potato (Solanum tuberosum L.). Potato Res. (Submitted). Freyre, R. & D.S. Douches. 1994. Development of a model for marker-assisted selection of specific gravity in diploid potato across environments. Crop Sci. 34:1361-1368. Fry, WE. & S.B. Goodwin. 1997. Resurgence of the Irish Potato famine fungus. BioScience 47 2363-371. Goth, R.W. & K.G. Haynes. 1997. The gerrnplasm release of B0718-3 and BO767-2: two late blight resistant potato clones. Amer. Potato J. 74:337-345. Hawkes, JG. 1990. The potato: evolution, biodiversity and genetic resources. Smithsonian Institution Press. Washington DC. 259 p. Hawkes, J .G. 1994. Origins of cultivated potatoes and species relationships. In: J.E. Bradshaw and GR. Mackay (ed.). Potato Genetics. CAB International. pp. 3-42. Haynes, K.G., D.H. Lambert, R]. Christ, D.P. Weingartner, D.S. Douches, J.E. Backlund, G. Secor, W. Fry, & W. Stevenson. 1998. Phenotypic stability of resistance to late blight in potato clones evaluated at eight sites in United States. Amer. J. Potato Res. 75:211-217. Hosaka, K. & R.E. Hanneman Jr. 1991. Seed protein variation within accessions of wild and cultivated potato species and inbred Solanum chacoense. Potato Res. 34:419- 428. Huaman, Z. R. Ortiz, S. Zhang, & F. Rodriguez 2000. Isozyme analysis of entire and core collections of Solanum tuberosum subsp. Andigena potato cultivars. Crop Sci. 40:273-276. Kamoun, S. E. Huitema, & V.G.A.A. Vleeshouwers. 1999. Resistance to oomycetes: a general role for the hypersensitive response? Trends Plant Sci. 4:196-200. Mendoza, H.A. 1989. Population breeding as a tool for gerrnplasm enhancement. Amer. Potato J. 66:639-653. Mendoza, H.A. & F.L. Haynes. 1974. Genetic relationship among potato cultivars grown in the United States. HortScience 9:328-330. Meyer, R.C., D. Milboume, C.A. Hackett, J .E. Bradshaw, J .W. McNichol, & R. Waugh. 1998. Linkage analysis in tetraploid potato and association of markers with 87 quantitative resistance to late blight (Phytophthora infestans). Mol. Gen. Genet. 259:150-160. Milboume, D., R. Meyer, J.E. Bradshaw, E. Baird, N. Bonar, J. Provan, W. Powell, & R. Waugh. 1997. Comparison of PCR-based marker systems for the analysis of genetic relationships in cultivated potato. Mol. Breed. 3: 127-136. Nei, M. 1972. Genetic distance between populations. Amer. Natur. 1062283-292. Nei, M. & W-H Li. 1979. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc. Natl. Acad. Sci. 76:5269-5273. Plaisted, R.L. & R.W. Hoopes. 1989. The past record and future prospects for the use of exotic potato germplasm. Amer. Potato J. 66:603-627. Platt, H.W. & G.C.C. Tai. 1998. Relationship between resistance to late blight in potato foliage and tubers of cultivars and breeding selections with different resistance levels. Amer. J. Potato Res. 75:173-178. Provan, J ., W. Powell, & R. Waugh. 1996. Microsatellite analysis of relationships within cultivated potato (Solanum tuberosum). Theor. Appl. Genet. 92: 1078-1084. Provan, J., W. Powell, H. Dewar, G. Bryan, G.C. Machray, & R. Waugh. 1999. An extreme cytoplasmic bottleneck in the modern European cultivated potato (Solanum tuberosum) is not reflected in decreased levels of nuclear diversity. Proc. Royal Soc. London B. 266:633-639. Rohlf, E.J. & R.R. Sokal. 1981. Comparing numerical taxonomy studies. Systematic Zoology 30:459-490. Rohlf, J .F. 1992. NTSYS-pc: Numerical taxonomy and multivariate analysis system. Version 1.7 Exeter Software, Setauket, NY. Ross, H. 1986. Potato Breeding - Problems and Perspectives, Verlag Paul Parey, Berlin and Hamburg. 132 p. Spooner, D.M. & R.G. van den Berg. 1992. An analysis of recent taxonomic concepts in wild potatoes (Solanum sect. Petota). Genet. Res. Crop Evol., 39:23-37. Sandbrink, J.M., L.T. Colon, P.J.C.C. Wolters & W.J. Stiekema. 2000. Two related genotypes of Solanum microdontum carry different segregating alleles for field resistance to Phytophthora infestans. Mol. Breed. 6:215-225. Stuber, C.W., J.F. Wendel, M.M. Goodman, & J.S.C. Smith. 1988. Techniques and scoring procedures for starch gel electrophoresis from maize (Zea mays L.). Technical Bull. 286, North Carolina Agricultural Research Service, NCSU, Raleigh, North Carolina. 88 CHAPTER V Mapping Late Blight Resistance and other Agronomic Traits in a {[(Solanum tuberosum x S. chacoense) x S. phureja] x S. microdontum} Population Abstract A diploid potato population was developed with the objectives to map quantitative trait loci (QTL) conferring resistance to Phytophthora infestans (Mont.) de Bary and other agronomic traits using simple sequence repeats (SSR) and isozymes and to examine associations between late blight resistance and other agronomic traits. The mapping population was a cross between a late blight resistant selection of Solanum microdontum Bitter and a susceptible diploid advanced breeding clone. The progeny of 110 clones and the parents were tested at the Muck Soils Research Farm, Bath, MI in 1999 and 2000 for foliar late blight reaction using a mixture of complex races of USS/A2 mating type of P. infestans. Disease severity was quantified as the relative area under the disease progress curve based upon the percentage of foliar infection over time. The same population was also evaluated at Montcalm Research Farm, Entrican, M1 for maturity, tuber number and size, yield, tuber appearance, specific gravity, and chip color. This 89 clone of S. microdontum transmitted high levels of resistance to late blight to a high percentage of the offspring. High phenotypic correlation (r = 0.89, P < 0.0001) was found for late blight reaction between years and no correlation was found between late blight with any other evaluated trait. A major QTL associated with foliar late blight resistance was located at the same position in linkage group 21 in both years of field testing. A QTL associated with vine maturity was mapped to linkage group 3. A QTL associated with tuber appearance was mapped to linkage group 1, one QTL associated with specific gravity was mapped to chromosome III and two QTLs associated with chip color were mapped to chromosomes VII and X. The major QTL associated with late blight resistance is suitable for marker assisted selection to introgress a new source of resistance to P. infestans to the cultivated tetraploid gerrnplasm of potato. Introduction Potato (Solanum tuberosum L.) is the fourth most important food crop with an annual world production of about 300 million metric tons (FAO, 1998). Among seven species of cultivated potato that originated in South America, the tetraploid (2n = 48) S. tuberosum is the most important species (Hawkes, 1990). Potato probably has the widest genetic diversity among related wild species than any other cultivated plant (Hawkes & Jackson, 1992) with more than 70% of the wild species being diploid (2n = 48) (Hawkes, 1994) In the mid—1990’s, the United States and Canada experienced a late blight (Phytophthora infestans (Mont.) de Bary) epidemic caused by new, more aggressive and metalaxyl resistant races (Goodwin et al., 1995; Peters et al., 2001) that imposed new 90 disease management strategies. The development of genetic resistance is a major strategy in managing late blight in potatoes and has become a major priority in many breeding programs (Colon et al., 1995a). Horizontal (field, partial or general) resistance seems to be the only durable type of resistance to late blight (Colon et al., 1995b; Umaerus et al., 1983; Kamoun et al., 1999). The level of horizontal resistance in potato is increased through recurrent selection (Henfling, 1987), which enables genes to be recombined from different resistant sources to build stronger and more durable resistance to late blight (Colon, 1999). However, the effectiveness of selecting recombinant individuals depends on mapping the quantitative trait loci (QTL) associated with resistance in different genetic sources (Meyer et al., 1998). Diploid and tetraploid populations have been used for mapping QTLs associated with late blight resistance introgressed from a variety of wild species. Mapping potato at the diploid level avoids interpretation problems associated with tetrasomic inheritance (Meyer et al., 1998). A QTL associated with late blight resistance can also be associated with other traits. This is the case of chromosome V on which QTLs were mapped for foliar and tuber late blight resistance, vine maturity and vigor (Oberhagemann et al., 1999) and foliar late blight resistance in other populations (Collins et al., 1999; Sandbrink et al., 2000). Association between QTLs conferring foliar late blight resistance, tuberization and vine maturity was found in four out of five chromosomes (Ewing et al., 2000). Nematode and late blight resistances conferred by different parents were linked to the same microsatellite marker (Pande et al. 2001). Moreover, major and minor QTLs can be associated with late blight resistance. A major QTL from S. bulbocastanum Dunal explaining 62% of the phenotypic variance was mapped to chromosome VIII (Naess et al., 2000). A QTL from S. demissum Lindley explaining about 30% of the phenotypic variance was mapped to chromosome IV (Meyer et al., 1998; Pande et al., 2001). 91 Different QTLs associated with late blight resistance were identified in two clones of S. microdontum Bitter from the same accession. From one clone a QTL was mapped to chromosome IV and the other clone a QTL was mapped to chromosome X (Sandbrink et al,2000) The South American diploid species S. microdontum has shown high levels of resistance to late blight (Colon & Budding, 1988; Colon et al., 1995 a,c; Douches et al., 2001). Dominant gene action was identified in some crosses with susceptible clones (Colon et al., 1995c). Strong hypersensitive reaction or infection efficiency, lesion growth rate and sporulation time were associated with high levels of resistance in S. microdontum (Colon et al., 1995a). Among 20 accessions, including Russian hybrids of Solanum species with S. tuberosum and four South American species, Douches et al. (2001) selected 56 clones that were resistant to the US8 genotype of P. infestans, from which 27 clones represented three accessions of S. microdontum. One of these clones was used as a parent to develop mapping populations. The objectives of this study were to map QTLs conferring late blight resistance and other agronomic traits using simple sequence repeats (SSR) and isozymes in a diploid S. microdontum derived population and to examine associations between late blight resistance and other agronomic traits. Material and Methods Selection of resistant parent and mapping population A total of 175 clones representing six accessions of S. microdontum were tested in greenhouse using US8/A2 genotype of P. infestans in 1997. The most resistant clones 92 were re-tested in 1998 and 27 highly resistant clones were identified (Douches et al., 2001). The selected late blight resistant parent S. microdontum was identified as P1595511-5 by Douches et al. (2001). The accession was very distinct (genetic similarity of 0.58) from cultivated potato and other S. microdontum accessions with reported late blight resistance (Bisognin & Douches, 2001). P1595511-5 was crossed with susceptible parents and segregation for late blight was tested in a progeny of 40 individuals. The mapping population chosen was a cross between Michigan State University diploid breeding clone MSAl33-57 [(S. tuberosum x S. chacoense) x S. phureja] with S. microdontum P1595511-5. A progeny of 110 clones that set tubers under greenhouse conditions (about 50% of total seedlings) was used for phenotypic evaluations and molecular analysis. Late blight reaction in field tests The P. infestans isolates (MS94-1, MS94-4, MS95-7 and MS97-2) were characterized as US8/A2 mating type as described in Bisognin et al. (2001). Those isolates overcame all known R-genes except R8 and R9 in detached-leaf assays. In the field, the isolates were weakly pathogenic only on the R8 and R9 of Black’s differential clones. The tests were carried out at the Michigan State University Muck Soils Research Farm, Bath, Michigan in a randomized complete block design. No fungicides were applied to the plants. Parents and progeny were planted in three replications of three-hill plots on May 27th and on June 9th and inoculated on July 22nd and July 26‘“, respectively in 1999 and 2000. Inoculation was done through a permanent sprinkle irrigation system in the early evening and high humidity was maintained in the canopy through periodic irrigations throughout the season. A visual estimation of the percentage of stem and leaf 93 infected area was scored at three to five day intervals from inoculation until the most susceptible clones reached 100% infection. The area under the disease progress curve (AUDPC) was calculated as described by Shaner & F inney (1977) and divided by the maximum AUDPC (e.g. 3300 for 33 days after inoculation) converting the value to relative AUDPC (RAUDPC), with 1.0 being the maximum RAUDPC value (Kirk et al., 2001). See appendix for more details. Vine maturity and tuber evaluations Parents, progeny and check varieties were planted in non-replicated three-hill plots at the Michigan State University Montcalm Research Farm, Entrican, Michigan on May 22'”, 2000. Vine maturity was evaluated on September 24th when the cultivar Atlantic had a vine maturity rating of 1 on a l to 5 scale of increasing lateness (l = early and 5 = late). All tubers used for evaluations were about 25 days afier harvesting. The number and size of tubers and yield - hill" was recorded. Tuber appearance was evaluated on a l to 5 scale of increasing defects (1 = excellent, as cultivar Atlantic; 2 = very good; 3 = acceptable; 4 = poor; and 5 = very poor). Chip color was evaluated on a l to 9 scale of increasing color darkness (1 - 2 = excellent; 3 = very good, as cultivars Atlantic and Snowden; 4 = acceptable; 5 = unacceptable; and 6 - 9 = poor). Specific gravity was measured using the formula [dry weight / (dry weight — wet weight)]. Marker technology Parents and progeny were genotyped using isozymes and SSR markers. The isozyme analysis was carried out using crude protein extraction from a newly expanded leaflet (approximately 120 mg), resolved in a horizontal 10% starch gel by electrophoresis with two buffer systems. Tissue processing, electrophoresis, staining, and 94 nomenclature were conducted as described in Douches & Quiros (1988). Nine isozyme loci of four enzyme systems were scored according to Douches & Quiros (1988) and Douches & Ludlam (1991). Malate dihydrogenase (Mdh-I), and phosphoglucose isomerase (Pgi-I) were resolved with a histidine-citrate pH 5.7, and glutamate oxaloacetate transaminase (Got-1 and Got-2) and phosphoglucomutase (Pgm-I and Pgm- 2) were resolved with a lithium-borate pH 8.3 buffer systems (Stuber et al., 1988). Total genomic DNA used as a template for SSR analysis was isolated from young leaves of greenhouse plants. Tissue was harvested, freeze dried and then ground with glass beads. DNA was isolated from 20 mg of tissue using the DNeasyTM Plant Mini Kit (Qiagen Inc., Germany) following manufacturer’s protocol. A total of 161 pairs of SSR primers were used. All primer pairs have been published in Provan et al. (1996), Milboume et al. (1998), and Sandbrink et al. (2000) as well as primers from 1 to 14 of Table 1A and all primers of Table 1D in Ashkenazi et al. (2001). All primer pairs were synthesized at Michigan State University and screened for polymorphism between the parents. DNA amplifications were carried out in a total volume of 20 pl containing 1X REDTaqTM PCR reaction buffer, 1 unit of REDTaqTM DNA polymerase (Sigma-Aldrich Co., St. Louis, MO.), 20 ng of each dNTP, 25 ng of each microsatellite primer, and 50 ng of template DNA. All amplifications were carried out on a Thermolyne Amplitron® (BarnsteadTM Thermoline Corporation, Dubuque, IA.) thermal cycler. The protocol was as follows: 1) initial denaturation at 94 °C for 4 min; 2) 40 cycles of denaturation at 94 °C for 1 min, annealing at 50, 55 or 60 °C for 2 min depending on each primer, and extension at 72 °C for 1.5 min; and 3) final extension at 72 °C for 5 min. The completed reaction products were held at 4 °C until electrophoretic separation. 95 Electrophoretic separation for SSRs was done in a 3% MetaphorTM Agarose (FMC Bioproducts, Rockland, ME) or a 5% Polyacrylamide gels (Sigma-Aldrich Co., St. Louis, MO) depending on the size of amplified fragment. Metaphor agarose gels were run at 100V from 3.5 to 4.5 h at 10 °C, stained with ethidium bromide (1 pg . ml") for 45 min, visualized under UV light, and photographed. Polyacrylamide gels (34.5 x 50 cm) were run at 90 W for 2 h and 30 min in a Sequi-Gen® GT Sequencing Cell (Bio-Rad, Richmond, VA) and stained with Silver Sequence"M DNA (Promega, Madison, WI) following respective manufacturers’ protocols. Fragment sizes were estimated using a 10 or 25 bp DNA ladder (Gibco BRL, Grand Island, NY) in each gel. Multiple loci of SSR markers were labeled by a letter after the marker designation. Statistical analysis For phenotypic data of late blight reaction, analysis of variance was done for the 1999 and 2000 data sets. Pearson correlation analysis was done to compare late blight data between years and late blight with other agronomic traits. Descriptive statistics was used to characterize population distribution for all evaluated traits. All those analysis were done following the procedures of SAS (SAS institute, 1995). Data from the presence or absence of alleles present (heterozygous) in one and absent in the other parent for SSR and isozymes were used for linkage analysis and QTL mapping. The x2 test for goodness-of-fit was used to test for deviations of the expected Mendelian segregation ratio of 1:1 (presence versus absence). Linkage analysis was done with JoinMap V2.0 (Stam, 1993) using a minimal LOD score of 3.0 and maximum recombination fraction of 0.49. Map distances are presented in centi Morgans (cM) calculated by the Kosambi fimction (Kosambi, 1944). The QTL mapping was done with QTL Cartographer V1.13 (Basten et al., 1999), including analysis of genotype x 96 Sq environmental interaction for late blight reaction. A QTL was declared significant based on threshold calculations of 1000 permutations (Churchill & Doerge, 1994). Results and Discussion Phenotypic evaluations There were significant differences (P S 0.0001) among progeny clones for foliar late blight reaction in the field tests in 1999 and in 2000, between years and also clones x years interaction. The 1999 test had a higher mean and median RAUDPC, for parents and progeny, and progeny RAUDPC range than the 2000 test (Table 5.1). The RAUDPC of S. microdontum was 0.021 and 0.019 compared with 0.529 and 0.175 of MSAl33-57, respectively for 1999 and 2000. There were clones in the progeny with 1.5-fold RAUDPC higher than MASl33-57 in both years of testing. Even with a higher artificial epidemic of P. infestans in the field in 1999 than in 2000, S. microdontum had almost the same RAUDPC values in both years, confirming its high resistance found in greenhouse tests (Douches et al., 2001). This S. microdontum clone had only a total of 10% infection in a late blight nursery in Toluca valley, Mexico, during the 2000 season (Lozoya- Saldafia, personal communication). See Fig. A6 and A7 and Table A3 in the appendix. There was a transgressive segregation for early vine maturity in the progeny (Table 5.1). For tuber quality evaluations, S. microdontum did not tuberize and seven progeny clones produced only very small tubers that were not enough for evaluation. The breeding clone MSAl33-57 did not have a high yield . hill", but set a small number of larger tubers with acceptable appearance and chip color and high specific gravity. Some progeny clones set a high number of tubers and, on average, the whole progeny had 97 smaller tuber size than MSAl33-57. However, there were progeny clones with over 5- fold higher yield and better tuber appearance, specific gravity and chip color than MSAl33-57, showing that wild species can be valuable sources even for high quality tuber traits. All traits had skewed distribution and only yield a hill" and tuber size had kurtosis similar to zero. A set of data with this level of skewness and kurtosis would usually be transformed to normality, but as high contrasting parents were used to develop the population, a mixture distribution was expected in the progeny (Doerge et al., 1997). See Fig. fi'om A8 to A.14 in the appendix. There was a high correlation (r = 0.82, P 5 0.0001) for late blight reaction between the two years of testing and there was no correlation between late blight reaction and any other evaluated trait (Table 5.2). Among the evaluated traits, significant correlations between late blight resistance and late maturity is probably the most undesirable correlation (Ross, 1986; Umaerus et al., 1983). Since the high level of resistance to late blight in S. microdontum was not correlated with late maturity, the late blight resistance genes may be located in other chromosomal regions of the genome. A highly significant correlation (r = 0.84, P 5 0.0001) was found between yield and tubers . hill". Negative correlations (P S 0.05) were found among maturity with yield and tubers . hill" and tubers . hill" with tuber size. Significant positive correlations were found for tuber appearance with tuber size and yield 0 hill". Yield and tuber size are components of tuber appearance. 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Li . ease LE . 22> been: 8-0332 as: Baesm doom a gas 288%“ see 2a 6335: 88 2a 82 a 8088 2me as 33 mega 8388 00 986580 .2 038. 100 Marker and linkage analyses From four isozyme systems, nine isozyme loci were polymorphic in the population. A total of 161 pairs of SSR primers was screened with DNA template from the parents and 125 were successful amplified producing readable bands. A total of 45 pairs of primers was polymorphic in metaphor agarose gels and resulted in 63 marker loci. An additional 25 pairs of primers were polymorphic in polyacrylamide gels and resulted in 46 marker loci, which totals 109 SSR marker loci (Table 5.3). A general trend could be observed between metaphor and polyacrylamide gels. Pairs of primers that amplified fragments with about 250 bp or smaller with a polymorphism between parents of about 15 bp or more could efficiently be separated in metaphor agarose gels. Pairs of primers that amplify either bigger fragments or with smaller difference between parents required separation in polyacrylamide gels. The isozyme and SSR analyses resulted in a tOtal of 118 marker loci in the population, of which 54 were heterozygous (band present) in S. microdontum and 64 in MSA133-57. In addition to polymorphic primers, eight pairs of SSR primers were also homozygous in both parents and these SSRs were used to confirm the F l status of the mapping populations. These eight SSR markers that were fixed in the parents were examined in the progeny for the presence of co-dominant bands. A total of 93 markers were grouped in 27 linkage groups, leaving 25 markers unlinked. The map covered 844.4 cM of the potato genome, with an average distance of 9.1 cM between markers and an average of 3.4 markers per linkage group. The length of the potato linkage maps varies from 690 cM (Gebhardt et al., 1989) to 1120 cM (Jacobs et al., 1995). The only S. microdontum map available was constructed using six sub- populations with total genome coverage length varying from 341 cM to 683 cM (Sandbrink et al., 2000). 101 Table 5.3. Simple sequence repeat markers used to map a S. microdontum derived population. Original Genome Fragment Separation Original Genome Fragment Separation Name1 Position2 Size (bp) System3 NameI Position2 Size (bp) System3 ST34 2 15 Metaphor Stm1021a IX 220 PAGE STS 6a 225 Metaphor Stm1021b IX 215 STS 6b 175 Strn 1024 VIII 172 PAGE ST56c 150 Stm1025a III 310 PAGE ST78 258 PAGE Stm1025b III 293 ST910a 230 PAGE Stm1025c III 283 ST91 Ob 180 Stm 1029 I 150 Metaphor ST910c 135 Stm1030 II 190 Metaphor SN 1 12a 306 PAGE Stm 1040 208 PAGE ST] 1 12b 290 Stm1041 V 95 Metaphor STl l 120 257 Stm1051 IX 225 Metaphor STl 1 12d 245 Stm 1052 250 Metaphor ST15 16a 450 Metaphor Stm1053 III 239 PAGE ST15 16b 400 Stm 1056a VIII 275 PAGE ST] 5 16c 240 Stm 1056b VIII 265 ST1920a 225 Metaphor Stm105 6c VIII 260 ST1920b 170 Stm 1064 II 224 PAGE ST2122a 242 PAGE Stm 1072a 300 PAGE ST2122b 235 Stm1072b 198 ST3 3 34 214 Metaphor Stm10720 190 ST3334b 207 Strn1097 95 Metaphor ST3 940 148 PAGE Stm 1 102a LIX 1 8O Metaphor ST4142 190 Metaphor Stm1102b I,IX 145 ST61 62a 500 Metaphor Stml 104 VIII 1 93 PAGE ST6162b 145 Stm1105 VIII 100 Metaphor STIIKA 250 PAGE Stm1106 X 140 Metaphor Stm0004a VII 1 90 PAGE Stm2003 1 83 PAGE Stm0004b VII 188 SthOOSa X1 245 PAGE Stm0004c VII 162 Stm2005b X1 200 Stm0007 XII 280 PAGE Stm2013 160 Metaphor Stm001 3a V 1 73 PAGE Stm2020 I 145 Metaphor Stm0013b V 155 Stm2022a II 210 PAGE Stm0015 l 80 Metaphor Stm2022b II 1 72 StmOO 1 9a VI 200 Metaphor Stm202 8a XII 450 Metaphor Stm0019b VI 175 Stm2028b XII 290 Stm0019c VI 145 Stm2028c XII 230 StmOOZOa 125 Metaphor Strn3 000 l l 5 Metaphor Stm0020b 100 Stm3003 350 PAGE Stm0024 VIII 1 3 5 Metaphor Stm3009 VII 1 25 Metaphor Stm0028 VII 1 50 Metaphor Stm301 1 II 140 Metaphor Stm0030a X11 1 50 Metaphor Stm301 2a IX 300 PAGE Stm0030b X11 1 10 Stm3012b IX 293 Stm0032 XII 140 Metaphor Stm3012c IX 270 102 Snn0038 Snn0046 Snn0051 Snn0052 Snn1002 San1003 Snn1004 Snn1009a Snn1009b Snn1009c Snn1009d Sun1016 11 VII VII VII VII,XI VII,XI VII,XI VII,XI 95 100 125 105 200 230 150 290 160 135 105 250 Metaphor Metaphor Metaphor Metaphor Metaphor PAGE Metaphor Metaphor Metaphor Stm3015 Stm3016a Stm3016b Stm3023 STPOAC58 LECAB9 LEGASTI STl 3STa ST] 3 S'I'b STRBC S l b STACCAS3 VIII IV Continued 105 130 l 15 190 95 80 120 90 80 250 145 Metaphor PAGE Metaphor Metaphor Metaphor Metaphor Metaphor Metaphor Metaphor ‘ Primer sequences of ST markers were published by Ashkenazi et al. (2001) , STIIKA by Provan et al. (1996), Stm by Milboume et al. (1997) and other primer sequences by Sandbrink et al. (2000). 2 Position in the potato genome as published in Milboume et a1. (1997). 3 More details about DNA separation (polyacrylamide and metaphor agarose gels) in the material and methods. 103 Simple linear regression showed that there .were four SSR marker loci on the linkage group 21 linked with late blight resistance in both years of field testing and combined analysis. There were two other markers linked with late blight resistance in 1999, one on linkage group 6 and another one on linkage group 16 and one locus linked with late blight resistance on linkage group 4 in 2000. Three loci linked with vine maturity mapped on each of these linkage groups 3, 8 and 25. Among tuber traits, five loci were linked with specific gravity on chromosome III. Three loci were associated with tuber size on each of linkage groups 6 and 17 and two loci on each of linkage groups 9 and 18. There were two loci linked with chip color that mapped on chromosomes III and X. All other loci linked with traits were single loci that mapped to different linkage groups (data not shown). Simple linear regression also showed that there were markers linked with multiple traits (Table 5.4). This analysis showed that there was no association between late blight resistance and late maturity in this S. microdontum population. One SSR marker (ST6162b) was linked with late blight resistance in 1999 and tuber size, in which an association in repulsion increases susceptibility to late blight and reduces tuber size. The SSR marker ST56c was also linked in repulsion and was linked with earlier maturity and lower specific gravity. For tuber traits, there were three markers (Strn0032, Strn1025c, and STRBCSlb) linked with three traits and six markers (STIH(Aa, Stm0030b, Stm0046, Stm2003, Stm2028c, and Stm2028d) linked with two traits (Table 5.4). 104 . . . . .. L c :.. 3.3.: .::.:t:: 5.3» fi.:......oc.¢.v.r. E9013: 61% Eat: .583 w a ti. E. .53 w a ... ._3 w a .. .83 w a . N .888 .3 a. .2333 3 £893 2. 33: .3 a 258:2 .3 cam .68: .a a 5.65 3 5:3 .38 a a $5.22 .3 8333 2.3 Hm 23350 .8558. SEE _ £083 .83 .33 £893 .83 .23 3835 :83 .23 owmomém £383 .33 88:3 .83 ....ooo.o .23 88:5 :83 .33 ogcfim 2.833 .33 .33 388% .23 .83 882.5 .33 .33 .533 .33 .53 .85 :33 N.33 38$ 830 ago 53.5 .m cam .23 85.0.8? 3. 7:2 . be; 1% . 22> 3.5.2 20:0 83 @832 dose—smog 003.80 53293335 .m m E 0:05 29:58 53» 083083 80x32 dam 030,—. 105 0 cu me chi Chr Chn Chrt Pop mic, link. Quantitative trait locus analysis A QTL in S. microdontum associated with foliar late blight resistance on the linkage group 21 was detected at the same position in both years of field evaluations (Fig. 5.1). This QTL explained 42% of the phenotypic variance in 1999 within the interval between 41 and 47 cM and explained 70% of the phenotypic variance in 2000 and combined analysis within the interval between 36 and 50 cM. The SSR marker Stm0020 was linked to late blight resistance in both years located within the region of highest LOD score of the QTL. This marker has two bands in S. microdontum, one segregating in the progeny with the resistant individuals and the other with the susceptible individuals. The average RAUDPC of resistant clones having the band linked with resistance was 0.156 and 0.070 and with susceptibility was 0.407 and 0.160, respectively for 1999 and 2000. Quantitative trait loci associated with late blight resistance have been mapped in wild Solanum species and in hybrids among wild species resistant to late blight. In the cultivar Stirling that has S. demissum as source of late blight resistance, a QTL was mapped on chromosome IV (Meyer et al., 1998; Pande et al., 2001), in S. berthaultii on chromosomes 1, III, VII, VHI and XI (Ewing et al., 2000), in S. bulbocastanum on chromosome VIII (Naess et al., 2000), and in S. microdontum on chromosomes IV, V and X (Sandbrink et al., 2000). In a wild species hybrid, QTLs were mapped on chromosomes III, V, VI and IX (Collins et al., 1999) and in another hybrid on chromosomes III, IV, V, VI, IX and XI (Oberhagemann et al., 1999). The mapping population used in this study was also a hybrid involving three wild species, but only S. microdontum contributed with the resistant alleles. One major QTL was found on linkage group 21 that had four SSR loci. Since none of these markers have been mapped 106 in potato, we were unable to assign this linkage group to a specific chromosome. However, this QTL should be different from the QTL mapped in the cultivar Stirling (Meyer et al., 1998) and the previous QTLs mapped in S. microdontum (Sandbrink et al., 2000). The SSR markers linked with late blight resistance in these previous studies (Strn3016 in the cultivar Stirling on chromosome IV and STPOAC58 in S. microdontum on chromosome V) were polymorphic in the S. microdontum derived population, but were not linked to late blight resistance. Moreover, the SSR marker ST13ST on the top of the linkage group 21 was not linked with late blight resistance in the S. microdontum population studied by Sandbrink et al. (2000). One QTL was associated with vine maturity on linkage group 3 and had three SSR markers that were not mapped on potato (Fig. 5.2). Quantitative trait loci associated with vine maturity have been mapped to different chromosomes and regions of the potato genome. In this S. microdontum population, late blight resistance and vine maturity were mapped on different linkage groups. The fact that late blight resistance is not associated with late maturity suggests that the introgression of resistance can be achieved without incorporating late maturity. Previous molecular analysis shows that there is a major region conferring late blight resistance and late maturity located on chromosome V (Collins et al., 1999; Oberhagemann et al., 1999) and there are QTLs associated with late blight susceptibility and early maturity that map to other regions of the genome (Ewing et al., 2000). 107 '4‘“ 333~ .3w82 300. n 20 .o.m 0800 Q04 Co 20:00:: 06 $500500 00: 00:03 Eomt0> .888 .3 “0 gravy—mm .3 Emmam can Ago: .3 00 038:2 3 gm .283 .3 H0 flan—0029a .3 00.333 0003 Hm 0000—38 he 0030300 008th .3303 83 3:588 03 ooom .002 E K 9.on 03%: co sown—anon 002000 55298038 .m. a mo 20m 05 E 0036600 Emzn 03— 8:8 53, 003683 ‘02 tab 0335330 ._.m 053.» 20 msm nomoocsm 20 0.3 womoogm 20 0.3 9.22% 20 o thmgm 6053500 25m .33 108 San1021a OcM ST56b 2.7 cM ST56c 22.8 cM Figure 5.2. Quantitative trait loci associated with vine maturity of a S. microdontum derived population on linkage group 3 in 2000. Primer sequences of markers ST were published by Ashkenazi et al. (2001) and Stm by Milboume et al. (1997). Vertical dashed line represents the threshold of LOD score 3.0. cM = centi Morgan. 109 u'i P31 Among tuber characteristics, all identified QTLs were associated with tuber quality traits (tuber appearance, specific gravity and chip color). A QTL for tuber appearance was mapped on linkage group 1 (Fig. 5.3), a QTL associated with specific gravity on chromosome 111 (Fig. 5.4) and two QTLs associated with chip color on chromosomes VII and X (Fig. 5.5). For tuber appearance, the three SSR loci of the linkage group have not been mapped yet. The QTLs associated with tuber quality traits were located on different chromosomes or linkage groups, indicating that each QTL can be independently selected in a marker assisted selection (MAS) program. Although there were marker loci linked to tuber quality traits in different linkage groups, only one QTL was found for tuber appearance and specific gravity and two QTLs for chip color. For specific gravity, Freyre & Douches (1994) found 10 QTLs distributed on six chromosomes from which one QTL was located within the region of the Pgm-l allozyme on chromosome III, the same region in which a QTL was mapped in this S. microdontum population. Douches & Freyre (1994) found six QTLs associated with chip color, but none were located on chromosome VII in the region of the allozyme Got-2. A QTL was found on chromosome X, but no connections can be done with the QTL mapped in S. microdontum, since chip color was linked to different marker loci. The identification and selection of QTLs associated with tuber quality is important because all cultivars released over the past century have superior tuber quality (Douches et al., 1996; Love et al., 1998). Therefore, it is not only important to combine different sources of late blight resistance, but also to combine resistance with early maturity and tuber quality traits. The identification of QTLs associated with late blight resistance in S. microdontum together with QTLs associated with vine maturity and tuber quality traits gives the possibility of pyramiding desirable QTLs in the progeny through MAS strategy. 110 StmlO72c 0 cM StmOOl9b 15.3 CM StmOOO4c 33.7 cM Figure 5.3. Quantitative trait loci associated with tuber appearance of a S. microdontum derived population on linkage group 1 in 2000. Primer sequences of markers Stm were published by Milboume et a1. (1997). Vertical dashed line represents the threshold of LOD score 3.0. cM = centi Morgan. 111 Pgml 0 cM STIIKAb 16.6 cM Strn10250 21.9 cM > STRBCSlb 38.9 cM Sun1053 I 51.0 cM Figure 5.4. Quantitative trait loci associated with specific gravity of a S. microdontum derived population on chromosome III in 2000. Primer sequences of markers Stm were published by Milboume et al. (1997), STIIKA by Provan et al. (1996) and STRBCSlb by Sandbrink et al. (2000). Vertical dashed line represents the threshold of LOD score 3.0. cM = centi Morgan. 112 Chromosome VII Chromosome X Got2 0 cM StmlO97 18.2 cM ST1516c 22.2 cM ST910c 24.8 cM STACCAS StmlOO4 26.2 cM Stm0052 30.6 cM Strn1003 37.2 cM Stm2003 0 cM . ST1516d 26.7 cM Stml 106 5.7 cM Figure 5.5. Quantitative trait loci associated with chip color of a S. microdontum derived population on chromosomes VIII and X in 2000. Primer sequences of ST markers were published by Ashkenazi et al. (2001), Stm by Milboume et al. (1997) and STACCAS3 by Sandbrink et al. (2000). Vertical dashed line represents the threshold of LOD score 3.0. cM = centi Morgan. 113 The mapping population used in this study combined late blight resistance in S. microdontum with yield and tuber quality traits in the diploid breeding clone MSA133- 57. All marker locus associated with late blight resistance were linked in coupling with S. microdontum, but the ST3334b allele from MSA133-57 contributed to a reduction in the average RAUDPC value of the combined years from 0.246 to 0.145. With the exception of chip color on chromosome X, all other marker loci linked with tuber quality traits were linked in coupling with MSA133-57, but, even on chromosome X, MSA133- 57 contributed alleles that improved chip color from an average rating of 5.9 to 4.9. The clone P1595511-5 of S. microdontum has a QTL associated with foliar late blight resistance located at the same position on linkage group 21 in both years of field testing. This QTL explained 42% of the phenotypic variance in 1999 within the interval between 41 and 47 cM and explained 70% of the phenotypic variance in 2000 and combined analysis within the interval between 36 and 50 cM of the linkage group. This IQTL associated with foliar late blight resistance is not associated with any other undesirable trait such as late maturity or poor tuber quality. There is a SSR marker (Strn0020) that maps at 57.5 cM from the top of linkage group 21 and can be used in a MAS program. In S. microdontum, Stm0020 showed two bands, one linked with resistance and one with susceptibility in the progeny (Fig. 5.6A). The presence of bands in both resistant and susceptible clones of the progeny makes selection easier based on the band fragment itself instead of band presence or absence. The band linked with resistance could also be followed through polyploidization using DLBl-150 as diploid parent (Fig. 5.6 B and C). From eight progeny individuals of the cross with MSAF313-3, two had the resistant band and the only individual of the progeny of the cross with Norvalley also had the resistant band. This is a very important finding as being the first 114 report that a genetic marker from a diploid clone can be followed through polyploidization and used in a marker assisted selection program. The addition of more linkage data is necessary to locate the QTL associated with foliar late blight resistance in S. microdontum on the potato genome to a specific chromosome and determine the relationship with QTLs mapped in S. bulbocastanum (Naess et al., 2000) and S. berthaultii (Ewing et al., 2000). The next step should be to pyramid these QTLs into specific genotypes to develop potato cultivars to improve levels and durability of resistance to late blight. 115 E 5. §'-B 3355 M 5 . .g ‘2 Q. .2 Ten RCSIStaflt Clones Ten Susceptible Clones m 8 g 052mm A 125bp 5 § ‘25 s s. sass s ..-. E 5’ >. gnU-ln L n | — a v— F! a t—v-IIC') Progeny .295% within the plant canopy. Plots were irrigated as necessary to maintain canopy and soil moisture conditions conducive for development of foliar late blight with turbine rotary garden sprinklers (Gilmour Group, Somerset, PA, USA.) at 1055 1 H20 ha/hr and managed under standard potato agronomic practices. Weeds were controlled by billing and with metolachlor at 2.3 l/ha 10 days after planting (DAP), bentazon salt at 2.3 l/ha, 20 and 40 DAP and sethoxydim at 1.8 l/ha, 58 - 60 DAP. Insects were controlled with imidacloprid at 1.4 kg/ha at planting, carbaryl at 1.4 kg/ha, 31 and 55 DAP, endosulfan at 2.7 1/ha, 65 and 87 DAP and permethrin at 0.56 kg/ha, 48 DAP. The dates of application were similar for all years. 132 Disease Evaluation and Data Analysis As soon as late blight symptoms were detected (about 7 days after inoculation, DAI), each plant within each plot was visually rated at 3 to 5 day intervals for percent leaf and stem (foliar) area with late blight lesions. The mean percent blighted foliar area per treatment was calculated. Evaluations continued until untreated plots of susceptible cultivars reached 100% foliar area diseased. Days after inoculation were used as key reference points for calculation of Relative Area Under the Disease Progress Curve [RAUDPC (1)]. For each plot and assessment date, the area under the disease progress curve (AUDPC) was estimated using the formula: AUDPC = (TM ‘71)*(—'—”—- where T was the time in days since inoculation and D was the estimated percentage of area with blighted foliage. As foliar late blight was assessed at various time intervals, the AUDPC was estimated with the area of a right triangle whose side lengths were based on the time interval and amount of late blight in the canopy. To accumulate AUDPC for the entire season and convert it to a rate over time, the formula was: 2 T Total *100 20‘... 4343:1121.) RA UDPC = Estimated AUDPC for each interval were summed, divided by the total number of days to the 100% diseased foliar area reference point in the non-treated susceptible controls, and multiplied by 100, resulting in an accumulated assessment of seasonal disease estimated as a fraction of one (RAUDPC). 133 50 A ‘35: 40 g 5‘ : a a § 30 : ff, :7 g . 8 (L; x .5 Lu 20 _ I. : ., '2‘ . I ” [3. g I ,;: L 7;. , I ‘ i ,3 § “I: 0 1 .. ,, - . 0-10 11- 20 21- 30 31- 50 51- 100 Foliage Infected Area (%) 50 B :1 80718-3 § 40 b b h |:] Bertita m 4 E Bzura E 30 _F— {as}. Greta O ’o'ii‘g‘ _ “E 20 -_ * leertas a) $3? \\\V Stobrawa Lg 10 ‘T t. 333% m Tollocan n. ,0 601 LL mix. 18888 Zarevo o m LB Resistant Families Atl. Figure A]. A) 1998 greenhouse late blight (LB) reaction evaluated as the percentage of foliage infected area for 408 clones from crosses between late blight resistant parents (shown) and susceptible ones (top). B) Comparisons among LB resistant families and the check cv. Atlantic (bottom). Means (bars) followed by the same letter do not differ significantly using Tukey's multiple range test at P = 0.05. 134 .1 Hum... 1'wuououm . . .. NNN\\\\\\\\\\N\. .444‘2141. <<<<<<<< . . . ,. a. ,r y 3bfifi¢0054bdbijr¢9fflv v 5 Karate. HIKIIIX IXXKIK .KKKKIK IIIKKKK 80 H 0 0 0 0 6 4 2 g9 @55on Egan 61-100 -60 46 45 31 30 RAUDPC x 100 16- 0-15 m Tollocan m Zarevo [:| B0718-3 Stobrawa [:] Bertita Bzura Greta Libertas e e 9 04940 949. ‘ o o o o o o o o 9. 09990.... . . 009.99.909.09.” 0%.? x.” .t .17 .x. Kuhn.» .3“. u .. L. ..\.w a“ 2: x 0mg; Mean Atl. LB Resistant Families Figure A2. A) 1998 field foliage late blight (LB) reaction expressed as the relative area under the disease progress curve (RAUDPC) for 408 clones from crosses between late blight resistant parents (shown) and susceptible ones (top). B) Comparisons among LB resistant families and the check cv. Atlantic (bottom). Means (bars) followed by the same letter do not differ 0.05. significantly using Tukey's multiple range test at P 135 N O A A i' 2% l6 :: D 12 I -'-1 i 8‘ I; I E 8 ': u é‘ _ i: 5 , II vfi ', g 4 _ i" it E2; 5:5 E “‘ :5 := :33: H *3 ‘ $533113: {Ara , 0 1 5" . 5 H §rii$1rzt IIZiE§3 “ 1"? 0-10 11-20 21-30 31-50 51-100 Foliage Infected Area (%) [:1 80718-3 [:1 Bertita Foliage Infected Area (%) LB Resistant Families Atl. Bzura Greta Libertas Stobrawa gm Tollocan m Zarevo Mean Figure A3. A) 1999 greenhouse late blight (LB) reaction evaluated as the percentage of foliage infected area for 119 clones from crosses between late blight resistant parents (shown) and susceptible ones (top). B) Comparisons among LB resistant families and the check cv. Atlantic (bottom). Means (bars) followed by the same letter do not differ significantly using Tukey's multiple range test at P = 0.05. 136 20 A A x 16 .~ v x" 9.1 >3 ": ea 0 xx “x :9: 5 12 2 z :3 :x :x it: 8 x: 4 ;.; . L-n xx “x O . L“ 8 * ‘ to: >\ x" x" .3 L '1 x: — ‘: :01 f, E :K :X 0.4 iv. «s 4 *" ~* 851 31:31 31%;? LL x x _ Io”... fig; "x “x :9; ,9, o e" ‘ "x "x :3" QM’I . .‘(Lv-i :x :x £1 go: ,0, e4 ,1: 0 ’3‘ “x ._ B 23‘. ,‘ 2'" 1101:“11; I I 0-15 16-30 31-45 46-60 6 -100 RAUDPC X 100 O O F—‘ X U 94 Q E LB Resistant Families LB Resistant Parents Atl. Mean E: B0718-3 [:| Bertita Bzura Greta Libertas Stobrawa m Tollocan m Zarevo Figure AA. A) 1999 field foliage late blight (LB) reaction expressed as the relative area under the disease progress curve (RAUDPC) for 119 clones from crosses between late blight resistant parents (shown) and susceptible ones (top). B) Comparisons among LB resistant families and the check cv. Atlantic (bottom). Means (bars) followed by the same letter do not differ significantly using Tukey's multiple range test at P = 0.05. 137 Table A2. Foliar late blight (LB) reaction in the greenhouse (GH) for the percentage of infected area and in the field for the relative area under the disease progress curve (RAUDPC) and maturity in 1999. Clone or LB Source LB Evaluation in the GH LB Evaluation in the Field Maturity C ultivar Foliage Infection (%) Cluster' RAUDPC Cluster“ Ratings’ Tollocan3 0.020 a5 1 1468-24 Tollocan 2.2 abcs 1 0.048 ab 1 4 1460-33 Tollocan 14.6 abcdefghij 1 0.051 ab 1 5 1461-23 Tollocan 0.1 a 1 0.053 ab 1 4 .1464-5‘ Tollocan 0.4 ab 1 0.064 ab 1 4 1461-1‘ Tollocan 16.4 abcdefghijk 2 0.071 abc l 3 1458-13 Tollocan 14.2 abcdefghij 1 0.074 abc 1 5 1457-43 Tollocan 44.4 bcdefghijklm 2 0.081 abc 1 4 1458-23 Tollocan 56.7 defghijklm 1 0.095 abc 1 3 1462-23 Tollocan 13.1 abcdefghi 1 0.106 abcd l 3 1468-53 Tollocan 24.4 abcdefghijkl 1 0.119 abode 1 5 1456-43 Tollocan 9.0 abcdefgh 1 0.132 abcdef 1 4 J466-43 Tollocan 16.5 abcdefghijkl 3 0.133 abcdcf l 5 1459-23 Tollocan 20.2 abcdefghijkl 1 0.140 abcdef 1 4 1457-23 Tollocan 9.1 abcdcfgh 1 0.144 abcdcf l 4 .1459-5’ Tollocan 23.1 abcdefghijkl 2 0.149 abcdcf 1 4 1453-43 Tollocan 9.9 abcdefgh 1 0.154 abcdefg 1 4 1459-33 Tollocan 14.7 abcdcfghij 1 0.157 abcdefg 1 3 1453-2‘ Tollocan 3.0 abcd 1 0.157 abcdefg 1 5 1459-43 Tollocan 46.1 bcdefghijklm 1 0.160 abcdcfg l 3 1306-5’ 80718-3 9.7 abcdefgh 1 0.160 abcdefg 1 5 1459-13 Tollocan 19.9 abcdefghijkl 1 0.172 abcdefgh l 4 13071833 0.175 abcdefghi 1 . 1307-13 BO7l8-3 7.2 abcdef 1 0.183 abcdefghij l 5 1464-43 Tollocan 15.5 abcdefghij 3 0.222 abcdefghijk 2 3 1306-33 BO718-3 35.4 abcdefghijklm 3 0.236 abcdefghijkl 2 5 1464-13 Tollocan 38.6 abcdefghijklm 3 0.237 abcdefghijkl 2 3 1467-21 Tollocan 46.9 cdefghijklm 1 0.238 abcdcfghijklm 2 4 Bzura’ 0.252 abcdcfghijklmn 2 .. 1466-33 Tollocan 37.8 abcdefghijklm 1 0.256 abcdcfghijklmno 2 3 1317-53 80718-3 7.9 abcdcfg 1 0.263 abcdefghijklmnop 2 5 1458-33 Tollocan 18.0 abcdefghijkl 1 0.265 abcdcfghijklmnopq 2 4 1455-43 Tollocan 43.7 abcdefghijklm 3 0.268 abcdefghijklmnopqr 2 3 1307-23 80718-3 9.4 abcdefgh 2 0.272 abcdefghijklmnopqr 2 4 J320-2‘ 80718-3 4.6 abcde 1 0.275 abcdefghijklmnopqrs 2 5 1465-13 Tollocan 35.4 abcdefghijklm 1 0.284 abcdefghijklrrmopqrst 2 4 J467-33 Tollocan 27.2 abcdefghijklm 2 0.294 bcdcfghijklrmopqrstu 2 4 1462-33 Tollocan 16.9 abcdefghijkl 1 0.303 bcdcfghijklnmopqrstuv 2 4 1462-13 Tollocan 43.7 abcdefghijklm 1 0.312 bcdefghijklmnopqrstuvw 2 5 1319-93 80718-3 22.6 abcdefghijkl 1 0.317 bcdefghijklmnopqrstuvw 2 4 1456-23 Tollocan 17.9 abcdefghijkl 3 0.342 cdefghijklmnopqrstuvwx 2 3 1348-73 Bzura 46.5 bcdefghijklm 1 0.371 defghijklmnopqrstuvwxy 2 5 1453-33 Tollocan 43.2 abcdefghijklm 2 0.373 defghijklrrmopqrstuvwxy 2 4 Greta3 0.377 defghijklrmopqrstuvwxyz 2 138 1319-13 1317-13 1448-13 1309-63 1468-13 1319-7: 1469-2’ 1332-1 Libertas 1314-3 1395-10 1471-5 1310-3 Stobrawa 1464-6 1476-1 Bertita 1404-5 Zarevo 1503-1 1488-2 1449-5 1365-2 1481 -1 1452-3 1497-1 1496-2 .1488-4 1501 -6 1320-1 1499-2 1365-8 1502-1 1492-2 1366-4 1491-3 1399-1 1324-2 1466-2 14524 1332-6 1467-6 1395-1 1364-1 1496-1 1462-5 1494-1 1482-1 1365-10 1400-3 8071 8-3 8071 8-3 Stobrawa 8071 8-3 Tollocan 8071 8-3 Tollocan Bertita 8071 8-3 Libertas Zarevo 8071 8-3 Tollocan Zarevo Libertas Zarevo Zarevo Stobrawa Greta Zarevo Stobrawa Zarevo Zarevo Zarevo Zarevo 8071 8-3 Zarevo Greta Zarevo Zarevo Greta Zarevo Libertas Bertita Tollocan Stobrawa Bertita Tollocan Libertas Greta Zarevo Tollocan Zarevo Zarevo Greta Libertas 16.0 10.7 20.8 10.1 39.6 6.2 50.2 42.4 7.5 27.7 14.0 7.7 39.5 41.7 45.8 38.1 12.9 22.8 29.7 67.6 35.0 68.8 42.4 23.5 57.7 11.4 32.2 31.3 59.6 58.9 30.9 56.4 33.2 62.7 88.1 51.9 79.7 80.2 19.0 40.5 29.9 28.3 20.6 64.5 34.5 63.9 abcdefghij abcdefgh abcdefghijkl abcdefgh abcdefghijklm abcdef cdefghijklm abcdefghijklm abcdefg abcdefghijklm abcdefghij abcdefg abcdefghijklm abcdefghijklm bcdefghijklm abcdefghijklm abcdefghi abcdefghijkl abcdefghijklm ghijklm abcdefghijklm hijklm abcdefghijklm abcdefghijkl defghijklm abcdefgh abcdefghijklm abcdefghijklm efghijklm efghijklm abcdefghijklm defghijklm abcdefghijklm fghijklm m cdefghijklm klm 1m abcdefghijkl abcdefghijklm abcdefghijklm abcdefghijklm abcdefghijkl fghijklm abcdefghijklm fghijklm u—ewN—nu—o—eu—n —N~fl —N—— 0378 0.382 0.388 0.391 0.395 0.423 0.439 0.444 0.446 0.449 0.450 0.452 0.455 0.466 0.468 0.472 0.473 0.484 0.485 0.486 0.488 0.498 0.500 0.501 0.502 0.504 0.505 0.509 0.516 0.519 0.524 0.526 0.528 0.531 0.536 0.539 0.539 0.545 0.547 0.549 0.551 0.553 0.553 0.560 0.565 0.568 0.574 0.576 0.579 0.581 139 Continued efghijklmnopqrstuvwxyz efghijklmnopqrstuvwxyz efghijklmnopqrstuvwxyz efghijklmnopqrstuvwxyz fghijklmnopqrstuvwxyz ghijklmnopqrstuvwxyza hijklnmopqrstuvwxyzab ijklmnopqrstuvwxyzab ijkln‘mopqrstuvwxyzab jklmnopqrstuvwxyzab jklmnopqrsmvwxyzab jklmnopqrstuvwxyzab klmnopqrstuvwxyzab klmnopqrsmvwxyzabc klmnopqrstuvwxyzabcd klnmopqrstuvwxyzabcd klmnopqrstuvwxyzabcd klmnopqrstuvwxyzabcd klmnopqrstuvwxyzabcd klrmopqrstuvwxyzabcd klmnopqrstuvwxyzabcd lmnopqrstuvwxyzabcd lmnopqrstuvwxyzabcd lmnopqrstuvwxyzabcd lmnopqrstuvwxyzabcd lmopqrstuvwxyzabcd lnmopqrsmvwxyzabcd mnopqrstuvwxyzabcde nopqrstuvwxyzabcde nopqrstuvwxyzabcde nopqrstuvwxyzabcde opqrstuvwxyzabcde pqrstuvwxyzabcde pqrstuvwxyzabcde qrstuvwxyzabcde rstuvwxyzabcde rstuvwxyzabcde stuvwxyzabcde tuvwxyzabcde tuvwxyzabcde tuvwxyzabcde tuvwxyzabcde tuvwxyzabcde uvwxyzabcde uvwxyzabcde vwxyzabcde vwxyzabcde wxyzabcde wxyzabcde wxyzabcde wwwwb)b.)NNNNNNNMNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN MMNNMMbU wumaf whawuaaai but M NM§W§§AUNMW§§W5M¥ Continued 1405-1 Libertas 46. l bcdefghijklm 3 0.581 wxyzabcde 3 3 1484-2 Zarevo 37.1 abcdefghijklm 0.591 xyzabcde 3 .. 1465-3 Tollocan 77.0 jklm 1 0.598 xyzabcde 3 4 1364-5 Greta 56.4 defghijklm 1 0.610 xyzabcde 3 3 1463-1 Tollocan 37.9 abcde fghijklm 1 0.610 xyzabcde 3 3 1365-6 Greta 34.2 abcdefghijklm 3 0.615 yzabcde 3 3 1501-1 Zarevo 34.8 abcdefghijklm 1 0.618 yzabcde 3 4 1476-5 Zarevo 62.5 fghijklm 1 0.619 yzabcde 3 4 1468-4 Tollocan 65.0 fghijklm 1 0.622 yzabcde 3 3 1326-5 Bertita 51.5 cdefghijklm 3 0.629 yzabcde 3 3 1497-4 Zarevo 23.7 abcdefghijkl 1 0.636 yzabcde 3 4 1492-4 Zarevo 42.2 abcdefghijklm 2 0.640 yzabcde 3 3 1493-2 Zarevo 36.6 abcdefghijklm 1 0.641 yzabcde 3 4 1315-1 80718-3 23.2 abcdefghijkl 1 0.642 yzabcde 3 4 Atlantic 49.3 cdefghijklm 1 0.648 zabcde 3 ... 1492-1 Zarevo 33.1 abcdefghijklm 2 0.668 abcde 3 3 1314-1 80718-3 54.6 cdefghijklm 1 0.670 abcde 3 4 1464-3 Tollocan 60.5 efghijklm 1 0.677 abcde 3 5 1487-1 Zarevo 26.4 abcdefghijklm 2 0.681 abcde 3 4 1482-2 Zarevo 56.2 defghijklm 1 0.690 abcde 3 4 1451 -3 Stobrawa 56.9 defghijklm 1 0.694 abcde 3 3 1456-3 Tollocan 53.7 cdefghijklm 1 0.694 abcde 3 3 1450-5 Stobrawa 37.5 abcdefghijklm 1 0.695 bcde 3 3 1494-4 Zarevo 51 .3 cdefghijklm 3 0.696 bcde 3 3 1501 -5 Zarevo 63.9 fghijklm 1 0.697 bcde 3 3 1456-1 Tollocan 33.8 abcdefghijklm 2 0.698 bcde 3 3 1495-2 Zarevo 29.4 abcdefghijklm 2 0.701 bcde 3 4 1492-6 Zarevo 54.2 cdefghijklm 3 0.734 cde 3 3 1489-1 Zarevo 75.2 ijklm 1 0.738 de 3 2 1483-1 Zarevo 27.1 abcdefghijklm 1 0.738 de 3 3 1487-5 Zarevo 65.0 fghijklm 1 0.777 e 3 3 1315-5 80718-3 7.2 abcdef 2 1455-1 Tollocan 41.2 abcdefghijklm 2 5 1487-3 Zarevo 61.6 efghijklm 2 3 Average 35.1 0.432 3.8 C .V.% 35.7 17.2 1 Scott-Knott cluster groups differing in late blight resistance: cluster group 1 = resistant, 2 = intermediate, and 3 = susceptible. 2 Evaluated in 20-hill plots at MSU - Lake City Research Farm, in a scale 1-5 of increasing lateness. 3' 4 Dunnett's T tests for alpha = 0.05 against Atlantic control in the field3 and in the greenhouse‘. 5 Numbers in columns followed by the same letter do not differ significantly using Tukey's multiple range test at P = 0.05. ... Not evaluated. 140 Strong L8 Intermediate LB Resistance Resistance Greta Chip-Quality 80718-3 Tuber Appearance Libertas A Specific Gravity Tuber LB Resist. 1 Stobrawa Tollocan Zarevo Specific Gravity T uber LB Resist. \ Figure A.5. Intercrossing scheme to combine late blight (LB) resistance from different sources with other important characteristics. 141 50 40- Frequency 10 3O 1- 20 '” 1999‘ 52000 , 00- 06- 11- 16- 21- 26- 31- 36- 41- 46- 51- 61- 71- 81- 05 10 15 20 25 30 35 4O 45 50 60 70 80 90 RAUDPCXIOO Figure A.6. Frequency distribution of a S. microdontum population for foliar late blight reaction in the field in 1999 and 2000. RAUDPC = relative area under the disease progress curve. Note changes in interval frequency. (RAUDPC for P1595511-5 = 0.021 and 0.019, MSA133-57 = 0.529 and 0.175, respectively for 1999 and 2000 field tests). 142 2000 Testing 0.30 0.25 . " o ' o ' 0 . o e 0.20 - 9 o 9 ' . C e . e ! 9": e 0 ' O O 0.15 . . . . o . . ' o o . 0.10 - ° ' ' ° C C . O 0.05 fig... 3. . . O 0.00 , . . . . 0.00 0.20 0.40 0.60 0.80 1999Testing 1.00 Figure A.7. Correlation of S. microdontum mapping population for foliar late blight reaction in the field between 1999 and 2000 (r = 0.82, P = 0.0001). 143 Table A3. Foliar late blight reaction in the field based on the relative area under the disease progress curve and Scott-Knott cluster groups differing in resistance level in 1999 and 2000. Parents and RAUDPC' Scott-Knott RAUDPC' Scott-Knott Progeny 1999 Groups2 2000 Groups2 176 0.025 1 0.010 1 002 0.024 1 0.016 1 P159551 1-5 0.021 1 0.019 1 004 0.018 1 0.019 1 185 0.038 1 0.021 1 195 0.033 1 0.021 1 225 0.071 1 0.025 1 095 0.034 1 0.026 1 068 0.038 1 0.026 1 209 0.238 5 0.026 1 1 17 0.048 1 0.026 1 122 0.066 1 0.027 1 073 0.072 1 0.027 1 169 0.097 1 0.030 1 124 0.014 1 0.030 1 005 0.101 1 0.030 1 213 0.019 1 0.031 2 153 0.050 1 0.031 2 125 0.074 1 0.031 2 123 0.092 1 0.032 2 134 0.503 5 0.034 2 220 0.125 2 0.034 2 170 0.033 1 0.035 3 065 0.030 1 0.035 3 097 0.024 1 0.036 3 217 0.031 1 0.037 4 182 0.075 1 0.037 4 206 0.096 1 0.038 5 152 0.125 2 0.039 6 173 0.064 1 0.039 6 140 0.007 1 0.040 6 179 0.171 2 0.040 6 086 0.093 1 0.041 6 109 0.040 1 0.041 6 216 0.041 1 0.041 6 040 0.044 1 0.041 6 053 0.21 1 5 0.042 6 062 0.065 1 0.042 6 084 0.185 4 0.042 6 104 0.365 5 0.043 6 144 103 193 177 156 155 205 099 157 096 121 214 145 199 092 050 150 003 166 044 223 019 218 108 224 020 057 048 188 107 131 120 192 154 159 210 128 222 172 167 091 203 MSA133-57 161 076 101 129 070 0.091 0.184 0.088 0.071 0.058 0.030 0.066 0.082 0.055 0.170 0.045 0.183 0.082 0.176 0.071 0.050 0.051 0.535 0.112 0.196 0.387 0.189 0.110 0.054 0.422 0.341 0.228 0.456 0.074 0.176 0.045 0.342 0.184 0.186 0.409 0.436 0.287 0.315 0.380 0.438 0.207 0.529 0.741 0.582 0.537 0.438 0.775 \lMU’IO‘t\JU!MMMMMMMMWMHNHMWMMHHMMMHMU—‘U—‘F-‘Nv-‘wt-‘N—‘F-‘V-‘I-‘v-‘t-‘V-‘u—t 145 Continued 0.043 0.043 0.044 0.047 0.047 0.047 0.048 0.052 0.053 0.053 0.055 0.057 0.058 0.060 0.061 0.066 0.066 0.092 0.094 0.094 0.1 1 1 0.1 16 0.117 0.1 19 0.120 0.122 0.127 0.128 0.130 0.130 0.144 - 0.145 0.146 0.150 0.150 0.164 0.165 0.165 0.170 0.172 0.173 0.175 0.177 0.179 0.179 0.179 0.180 \O\O\O\O\O\O\O\O\O\O\OOOOO\J\J\J\I\I\I\I\J\JO\O\ 10 Continued 111 0.833 10 0.182 22 158 0.519 5 0.184 22 219 0.309 5 0.186 22 080 0.582 6 0.189 23 051 0.565 6 0.190 23 113 0.547 5 0.192 23 102 0.463 5 0.195 24 207 0.640 6 0.198 25 012 0.451 5 0.206 26 198 0.417 5 0.208 26 164 0.514 5 0.210 26 094 0.571 6 0.211 26 115 0.807 9 0.223 27 200 0.747 7 0.225 28 078 0.615 6 0.228 29 178 0.680 7 0.231 30 221 0.462 5 0.232 30 171 0.647 6 0.241 31 058 0.789 8 0.254 32 061 0.675 6 0.261 33 008 0.606 6 0.262 33 105 0.593 6 0.263 34 183 0.563 5 0.268 35 045 0.393 5 0.271 35 100 0.102 1 0.273 36 Mean 0.260 0.106 L.S.D. 0.433 0.067 C.V.% 42.6 33.2 ' Relative area under the disease progress curve (maximum RAUDPC = l). 2 Scott-Knott cluster groups differing in late blight resistance. 146 70 60, 50- Frequency 20. 10- Figure A.8. Frequency distribution for vine maturity of a S. microdontum mapping 40- 30- population on a scale 1 to 5 of increasing lateness in 2000. (MSA133-57 = 2 and P1595511-5 = 5). M aturity 147 Frequency 003 0.3-0.6 0.6-0.9 0.9-1.2 1.2-1.5 1.5-1.8 Y1eld/hill(kg) Figure A.9. Frequency distribution for yield 0 hill'l (kg) of a S. microdontum mapping population in the field in 2000. (MSA133-57 = 0.242 kg). 148 Ut O 40 - 5‘ 1:. 30 - “:3 g 20 - DA 10 . 0 - . 0-8 9-16 17-24 25-32 33-40 Nunberof Tubes/hill Figure A.10. Frequency distribution for number of tubers . 11111'1 of a s. microdontum mapping population in the field in 2000. (MSA133-57 = 2). 149 50 40 2.22., ---- - 5‘ a 30 ,- - -, - <1) 5- 8 20 , ---. -- - LL. 10 -8- .2 2. - 0 J 1 2 3 4 5 TuberAppwranoe Figure A.11. Frequency distribution for tuber appearance of a S. microdontum mapping population on a scale 1 to 5 of increasing defects in 2000. (MSA133-57=3). 150 U.) Kl} Frequency A T HHNNU) OUIOUIOUIO 10-20 21-30 31-40 41-50 51-60 61-70 71-120 TuberSize® Figure A.12. Frequency distribution for tuber size (g) of a S. microdontum mapping population in the field in 2000. (MSA133-57 = 120.8 g). 151 211 O 40- 5‘ ,3 30 .- O) 5* 20 2 .. ILL 10- O < 1.07 1.07-1.08 1.08-1.09 1.09-1.10 > 1.10 Specific Gravity Figure A.13. Frequency distribution for specific gravity of a S. microdontum mapping population in the field in 2000. (MSA133-57 = 1.107). 152 35« 30 201 Frequency <30 4 5 6 >7 Grip Color Figure A.14. Frequency distribution for chip color of a S. microdontum mapping population on a scale 1 to 9 of increasing darkness in the field in 2000. (MSA133-57 = 2). 153