IIIHWIHIIHHHIHHHIlfilllilHWIWHHHHIMWI llllllllllllllMl lll ll// 3 1293 00688 LIBRARY Michigan State University This is to certify that the thesis entitled INVESTIGATIONS OF ISOZYMES IN SUGARBEET presented by Lauri Diane Aicher has been accepted towards fulfillment of the requirements for M.S. degree In Plant Breeding and Genetics/Crop and Soil Science MW Major professor Date i[0/21/39 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution MSU RETURNING MATERIALS: Place in book drop to LIBRARIES remove this checkout from -3_ your record. FINES will be charged if book is returned after the date , ,stamped below. 0.2535"- @553; l" 'L 2214;} NH"; 54:93"? but Y5 in: } r-éikfi HA) " i All»; 7' - *zsm 7 ‘M’ari gr? ' ~ , .- INVESTIGATIONS OF ISOZYMES IN SUGARBEET (BETA VULGARIS L.) By Lauri Diane Aicher A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Crap and Soil Science 1988 ABSTRACT INVESTIGATIONS 0F ISOZYHES IN SUGARBEET By Lauri Diane Aicher In this study, eight new isozyme loci were identified (Mdh-l, Hdh- 2, Mdh-B, Hdh-l}, Hdh-S, Pgn-l, Pgi-l and Skdh-l) and the inheritance and linkage relationships between four of these loci and two marker loci was determined. The Pgi-l locus had normal F1, but skewed F2 segregation ratios. It was hypothesized that linkage of the Pgi locus to a self- incompatibility locus caused these deviations from monogenic segregation. Two additional loci, HE-l and ME-Z, were proposed, however, more segregation data will be necessary to confirm their inheritance. Isozyme electrophoresis was also effective in almost completel distinguishing thirty-two plants using only seven marker enzymes. A new dwarf sugarbeet mutant was also observed in the progeny r f of the/frosses examined for isozyme analysis. This mutant, named spinach leaf appeared to be due to a single recessive gene which could not be overcome by application of gibberellic acid. ACKNOWLEDGEMENTS I would like to thank my major professor, Dr. Joseph Saunders, for supporting my work and for sharing with me his incredible energy and enthusiasm for science. I would also like to thank my commitee members, Dr. James Hancock and Dr. J. Clair Theurer, for their input, resources and editorial assistance. I am grateful to my parents for their support and for encouraging me to strive for whatever would make me happy. I would especially like to thank to my partner Sharon for her love and wisdom, and for putting up with me while I completed this project. 11 TABLE OF CONTENTS Page LI ST 0? TABLE 8 O O I O O 0 O O O O O O O O O O O O O O O O O O O O I v LIST OF FIGURES. O O O I O O O O O O O O O O O O O O O O O O O O O O v11 CHAPTER I INHERITANCE AND LINKAGE OF SOME ISOZYME LOCI IN SUGARBEET I mODUCTI ON C O O O O O O O O O O O O O O O I O O O O O O O O O O O 1 b MATERIALS AND “mops. O C O O O O O O O O O O O O O O O O O O O O O PRODUCTION OF A DOUBLED HAPLOID. . . . . . . . . . . . . . . . . . 4 CROSSES._,....1._....._T.‘.....................5 {4 7STARCH GEL ELECTROPEORESIS *.- . . . . . . . . . . . . . . . . . . 6 /l‘ j Sample preparation . . . .l. . . . . . . . . . . . . . . . . . . 6 Systems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 \\e Staining . . . . .—. . . . . . . . . . . . . . . . . . . . . . . 8 NOMENCLATURE 0F ENZYMES. . . . . . . . . . . . . . . . . . . . . . 10 STATISTICS . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 RESULTS AND DISCUSSION 0 O O O O O O O O O O O O O O O O O O O O O 0 11 CROSSES O O O O O O I O O O O O O O O O O O 1 1 PRELIMINARY ISOZYME STUDIES.. .. .. .. .. .. .. .. .. .. 14 ISOZYME INHERITANCE. . . . . . . . . . . . . . . . . . . . . . . . 14 Malate dehydrogenase . . . . . . . . . . . . . . . . . . . . . . 14 Phosphoglucomutase . . . . . . . . . . . . . . . . . . . . . . . 20 Phosphoglucose isomerase . . . . . . . . . . . . . . . . . . . . 21 Malic enzyme . .. . .. . .. .. .. .. .. .. .. .. .. . 28 Shikimate dehydrogenase. . . . . . . . . . . . . . . . . . . . . 31 6-Phosphogluconate dehydrogenase . . . . . . . . . . . . . . . . 31 Hypocotyl color and annualism. . . . . . . . . . . . . . . . . . 33 LINKAGE O O O O O O O O O O O O O O O O C O O O O O O O O O O O O O 33 SUMMARY. . . . . . . . . . . . . . . . . . . . . . . . . . . . ... 39 "Q CHAPTER II FINGERPRINTING OF GENOTYPES USING ISOZYMES INTRODUCTION 0 O O O O O O O O O O O O O O O O O O 0 C O O O O O I O 4 1 111 MATERIALS AND METHODS. . . . . . Gemplasm C O O O O O O O O O 0 Starch gel electrOphoresis . . RESULTS AND DISCUSSION . . . . . Preliminary experiments. Malate dehydrogenase . . Phosphoglucose isomerase Malic enzyme . . . . . . Shikimate dehydrogenase. Glutamate dehydrogenase. Phosphoglucomutase . . . Isocitrate dehydrogenase Fingerprinting of lines. Summary. . . . . . . . . CHAPTER III 0 O O O O O O O O 0 DESCRIPTION OF A SPINACH LEAF MUTANT INTRODUCTION . . . . . . . . . . MATERIALS AND METHODS. . . . . . Crosses. . . . . . . . . . . . Gibberellic acid experiments . RESULTS AND DISCUSSION . . . . . Description of a dwarf mutant. Gibberellic acid experiments . . . . Inheritance of the spinach leaf mutant sumry O C O O O O O O O O O C O I O O BIBLIOGRAPHY . . . . . . . . . . . . . . iv 43 43 43 46 46 49 51 52 52 54 S4 54 55 55 56 58 58 58 59 59 6O 63 7O 71 Table 1.1 1.2 1.3 1.6 1.7 1.8 1.10 1.11 1.12 1.13 1.14 1.15 LIST OF TABLES Page Desription of crosses used for isozyme segregation analysis. . 6 Summary of electrophoretic materials and methods . . . . . . . 9 Percent seed germination and segregation of the F1 progeny at the hypocotyl color marker locus . . . . . . . . . . . . . . . 12 The number of F1 individuals which set self seed over two consecutive seasons. .. . .. . .. . .. . .... . . . . . . 13 Chi-square analysis of allelic segregation of Mdh-l in F1 and F2 progenies of Beta vulgaris. .. . .. .... .. . .. .. . 17 Chi-square analysis of allelic segregation of Mdh-3 in F1 and F2 progenies of Beta vulgaris.. . .. . .. . .. . .. . .. 20 Chi-square analysis of allelic segregation of Pgm-l in F1 and P2 progenies of Beta vulgaris.. . .. . .. . .. . .. . .. 21 Chi-square analysis of allelic segregation of Pgi-1 in F1 and P2 progenies of Beta vulgaris. .. . .. . .. . .. . .. ... 22 Frequency of Pgi banding types in segregating F1 and F2 progeny O O O O O O O O O O O O O O I O O O O O O O O O O O O O 23 Possible F2 genotypes using 172 recombination between the Pgi-1 and self-incompatibility loci. . . . . . . . . . . . . . 24 Results of crosses for the tentative locus ME-Z. . . . . . . . 30 Results of crosses for Skdh-l. . . . . . . . . . . . . . . . . 32 Chi-square analysis of allelic segregation of R and B in F1 . and F2 progenies of Beta vulgaris. . . . . . . . . . . . . . . 34 F2 segregation ratios of Mdhl, Pgml, R and B, paired for linkage analysis . .. . .. . .. . .. .. . . . .l.. . . . 35 Analysis of linkage between Pgi-1, B and R using recombination frequencies and adjusted chi-square values . . . . . . . . . . 36 Table Page 1.16 Analysis of linkage between Pgi-l, Mdh-l and Pgmrl using recombination frequencies and adjusted chi-square values . .. 37 1.17 Analysis of linkage between Pgi and Pgm using F1 segregation data. C O O O O O O O O O O O O O O O O O O O O O O O O O O O O 39 1.18 Summary of the identified isozyme loci and their important Characteristics 0 O O O I O O O O O O O I O O O O O O O I O O O 40 2.1 Germplasm used in fingerprinting study . . . . . . . . . . . . 45 2.2 Summary of all the isozymes and systems examined . . . . . . . 47 2.3 Isozyme genotypes for clonal fingerprints. . . . . . . . . . . 48 . . . . . . 49 2.4 Frequencies of genotypes at each isozyme locus . 3&1 Emergence date difference between normal and spinach leaf progeny of the F1 line 4-7 .. .. .. .... .. .. .. .l.. 60 3.2 Comparison of the number of spinach leaf seedlings from untreated and GA treated seed balls. .. . .. . .. .. .. . 63 3.3 Summary of initial F1 crosses which gave spinach progeny . . . 66 3.4 Spinach leaf segregation in three separate sowings of F2 seed. 67 3.5 Summary of spinach ratios from the Fl's and Fz's . . . . . . . 68 3.6 Possible F2 phenotypes given 201 recombination between the spinach leaf and self-incompatibility loci . . . . . . . . . . 69 vi LIST OF FIGURES Figure Page 1.1 The three zones of activity (left) and five (right) loci of Mdh. This gel was resolved with system II and shows SS, SF, and FF types for both Mdh-1 and Mdh-3. Using this system the Mdh-2 band comigrated with the slow (SS) Mdh-1 band. When system I was used the single band of Mdh-2 comigrated with the fast (FF) Mdh-1 band . .. . .. . .. . .. . .. . . . . 15 1,2 Starch gel zymograms showing the banding genotypes at 4 isozyme loci: a) FF, 3-banded SF and SS banding genotypes at Mdh-1, b) Comparison of leaf (A) and pollen (B) samples from an Mdh-1 SF plant, c) FF, 3-banded SF and SS banding genotypes at Mdh93, d) SS, 2-banded SF and FF banding genotypes at Pgm-1, e) SS, 3-banded SF and FF banding genotypes at Pgi-1, f) Comparison of leaf (A) and pollen (B) samples from a Pgi-1 SF plant.. .... .. . .. . .. . .. . 16 1.3 Graph of lines describing the SS, SF and FF banding types from 02 to 502 recombination. Combined data and data from each of the F2 families in Table 139 are plotted on the SS and FF lines . .. . .. . .. . .. . . . .... . . .l.. . . 26 1.4 a) This Malic enzyme zymogram was resolved using system II and shows the parents (Types I + II) and segregating F2 progeny of a type B, F1 individual. b) diagram showing the structures of the tetramers composing the three banding patterns. a, b and b represent the polypeptides produced by the alleles from the proposed loci ME-l, and ME-2.. .. .... 29 2.1 a) Mdh banding patterns using system I: (L to R) FF; band from EW-53; SS; SF; FF and band from WB 222-1. b) Mdh banding patterns using system II: (L to R) Mdh-1 88; bands from.WB 222-1; and two Mdh-1 SS types. Note Mdh-5 band is missing in WB 222-1. c) Comparison of Pgi zymograms using systems I (bottom) and II (top). d) Pgi zymogram (system II) showing NB 222-1 in lane 2 .. .. .. .. .. .. .. .. .. .. .. 50 2.2 a) Malic enzyme banding patterns: I, II, III and IV. b) Shikimate dehydrogenase banding patterns: 8 and F. c) Glutamic dehydrogenase banding patterns: S and F. d) Idh banding patterns: I and II . .. . .. . .. . .. . .. . .. 53 Figure Page 3.1 3.2 a) Germinating normal and spinach leaf seedlings. b) Comparison of spinach 1eaf'(A) and normal (B) seedlings of the same age. c) Comparison of six week old spinach leaf (A) and normal (B) p1ants.. .. .. .. .. .. .. .. .. .. . 61 Comparison of the percent germination (averaged over 2 reps) of untreated and gibberelic acid treated seed of the F1 spinaCh carrier RCA-5. O O O O O O O O O O O O O I O O O O O 0 64 viii CHAPTERI mmmmorsmrsozmwcrnm CHAPTERI INHBITAICEANDLMOPSMISOZMIDCIINSUGARBEET INTRODUCTION In the past 200 years, the sugarbeet (Beta vulgaris In) has been developed from the mangel beet. Over this period great improvements have been made in many important characters such as yield, sugar percentage and disease resistance. As a result of these and other improvements, sugarbeet production in 1980 accounted for 422 of the world's sugar supply (Smith, 1987). Despite the enormous progress in breeding, study of the basic genetics of the sugarbeet has been limited in comparison with other crops. Smith (1980) described 44 loci in sugarbeet. .Although inheritance studies have been conducted on most of these loci, only four of them, hypocotyl color (R), yellow pigment (Y), annual growth habit (B) and monogerm (n), have been studied intensely. Of the loci which have been studied for linkage, ten are associated with the Y-R-B linkage group. Half of these ten loci encode betacyanin pigmentation patterns. Four other linkage groups have also been proposed, however; three of these have only one marker gene (Theurer, 1968). This accounts for possibly five of the nine possible linkage groups in the diploid.(n-18) sugarbeet. ‘None of these linkage groups have been mapped to specific chromosomes. This can be contrasted with other plant species such as maize or tomato, in which hundreds of loci have been identified . representing.all of their chromosomes (Goodman, Stuber and Newton, 1982; Bernatzky and Tanksley, 1986). There are many reasons why progress in increasing genetic information in sugarbeets has been slow. One reason is a long generation time, due to the biennial nature of most sugarbeet lines. Many loci also show variable penetrance or expressivity. The segregation ratios obtained for these loci can be inconsistent from one environment to another. In addition to this, most of the marker loci which have been studied represent pigmentation, chlorophyll deficient or foliar mutants. Due to lethality of some mutants and problems distinguishing phenotypes of some of the foliar mutants, it has been difficult to obtain new information on the linkage of multiple loci. An increase in genetic and linkage information could be of great value in sugarbeet research. Therefore, it would be highly desirable to identify new loci which are easy to distinguish and represent all of the linkage groups. These loci would also be helpful as markers in breeding and genetic studies. A method whereby this could be accomplished is isozyme electrophoresis. Isozymes, first defined in 1959, are recognized as multiple molecular forms of enzymes with the same catalytic function (Markert and Moller, 1959). Although isozymes can be distinguished by many techniques, starch gel electrophoresis (SGE) is by far the most common and practical method used to acquire genetic and linkage information in plant species. One of many advantages of SGE is that genotypes can be identified without progeny testing since isozymes are generally codominant. This is valuable in F2 linkage analysis since much smaller numbers of progeny are necessary when all genotypic classes are identified (Allard, 1956). - Another advantage of SGE is that horizontal slicing of the starch gel permits quantification of the same samples for several different enzymes. This allows the study of many different isozyme loci at one time. The status of isozyme research in over 24 plant species has been recently reviewed (Tanksley and Orton, 1983). Although a large number of plant species have been studied using isozymes, very little isozyme research has been conducted in sugarbeets. The research which has been done tends to fall into 4 categories: (1) attempts to correlate a tissue culture trait or an adult plant character, such as ploidy, 1 sucrose, or fresh weight, with an isozyme pattern (Spettoli, Cacco and Ferrari, 1976; Spettoli, Bottacin and Cacco, 1980; Lehnhardt, Wiedmann and Gunther, 1981; Revers, Coumans, Degreef, Hoffinger and Gaspar, 1981; Revers, Coumans, Degreef, Jacobs and Gaspar, 1981), (2)4A general description of variability and banding patterns for different enzymes (Van Geyt and Smed, 1984, Monastreva, Reimers and Levites, 1982), (3) Use of bending patterns to distinguish cultivars (Itenov and Kristensen, 1985), interspecific hybrids (Oleo, Van Geyt, Lange and DeBock, 1986), and monosomic addition lines (Jung, Wehling and Loptein, 1986), (4) Inheritance of isozyme loci (Maletskii and Ronalov, 1985). We initiated a research project to assess the potential of increasing the number of marker loci and linkage relationships in sugarbeet using isozymes, and to obtain a general idea of their usefulness in identifying individual clones and lines. Other objectives of this research included an assessment of the genetic diversity of the East Lansing breeding population and the creation of markers for tissue culture and somaclonal variation studies. The present study was conducted to elucidate the inheritance of isozyme variability in six different enzyme systems and to probe possible linkage relationships between the isozymes and with two other marker loci. MATERIALSANDHETHODS FIODDCTIOI OF A DWBLED BAPLOID A diploid plant, presumably completely homozygous, was produced from a haploid via an in vitro colchicine technique which was adapted from Bussey and Hepher, 1978. ‘The haploid plant, n50 X 34, was detected in F1 seedlings of a cross between a monogerm biennial plant and an extremely multigerm, annual, male sterile individual from the cytoplasmic male sterile Owens Annual (OA) line. The haploid plant resembled the female parent in every aspect except that it was smaller. Shoot cultures of n50 X 34 were established from axillary buds on the flower stalk and were multiplied on Murashige-Skoog (MS) medium with 1.1 uM 6-benzy1adenine (BA) (Saunders, 1982). Fifty small shoots, 4 mm in length, were soaked.in.a lzlautoclaved colchicine solution for 50 minutes and another 50 shoots were soaked in the same for 100 minutes. After soaking, the shoots were rinsed in sterile water and returned to the multiplication medium. When the shoots were approximately 3 cm tall they were placed on MS medium supplemented with 16.0 uM 1- naphthaleneacetic acid (NAA) to be rooted. Rooted shoots were transplanted into peat pots and grown in the greenhouse. All.of the plants which resulted from this technique were male- sterile annuals which bolted under incandescent lights. The bolting plants were exposed to pollen.shedding plants nearby to see if any plants or branches set plentiful seed. This tested for chromosome doubling due to the colchicine treatment, since only a doubled haploid, (functionally a diploid) would have been able to set plentiful seed. Two functional diploids were detected and then multiplied by in vitro shoot culture from axillary buds as described earlier. These two plants were given the clonal designations doubled haploid A and B (DH-A and DH-B). They were homozygous for the green hypocotyl color (rr), annualism (BB), male fertility maintainer (xx,zz) and self-fertility (stf) alleles and also had the sterile (S) cytoplasm. CROSSBS Six crosses were made in Fall, 1984, to produce F1 seed for an inheritance and linkage study of several isozyme loci (Table 1.1). DH-A and DH-B were the female parents of three of these crosses. The male parents of these crosses, 1380, 138D and 13811, were diploid, biennial plants which were at least partial male fertility restorers. These plants had their female parents in common and were also carriers of a dwarf crinkly leaf mutant character called spinach leaf. The male parents were crossed with the DH-A and DH-B females and were also pair crossed (Table 1.1). These crosses were labeled with the letters E.C. (enzyme cross) followed by a number. In Spring, 1985, seed was sown from each of the crosses and F1 families were obtained. In Fall, 1985, plants from three F1 families were grown in the greenhouse under artificial long days (incandescent lights on a 18:6 LD cycle) where they bolted, were scored for pollen fertility and were then bagged to produce F2 seed. F2 progeny from selected self-fertile F1 individuals and many of the plants from the F1 families were examined for isozyme segregation ratios via starch gel electrophoresis. Data on segregation at the annualism and hypocotyl color loci was also recorded for all of the F1 and F2 progenies. Table 1.1. Description of crosses used for isozyme segregation analysis EC # Cross Cenotypes" ECl DH-A X 138D rrBB X RRbb EC4 DH-A X 138C rrBB X Rrbb EC6 138C X 138D Rrbb X RRbb E08 138D X 138C RRbb X Rrbb EC1O 138H X 138C RRbb X Rrbb EC12 DH-B X 138D rrBB X RRbb * genotypes were hypocotyl color: RR or Rr-red and rr-green; annualism: BB or Bb-annual and bb-biennial. STARCH GEL ELECTBDPHORESIS Sample preparation Young healthy leaves, approximately 5 cm long, were collected from greenhouse plants immediately before they were used. Three 1 cm diameter leaf discs were excised from.each leaf blade, excluding the midvein, using a #5 cork borer. The discs were homogenized using a glass pestle in porcelain spot plates containing 4 drops of extraction buffer. The extraction buffer consisted of 0.1 M tris-HCl, pH 7.0 buffer, containing 182 glycerol, 52 soluble polyvinylpyrrolidone (PVP- 40T), 0.5! Triton X-100 and 1.02 2-mercaptoethanol which was added just before use. The spot plates were placed on blocks of ice in trays and kept covered to maintain temperatures near 0°C throughout sample preparation. Pollen.from individual plants was also used for samples. The pollen was collected when the plants were in flower and frozen in glass vials until needed. A mound of pollen, approximately 3 mm in diameter, was placed in the spot plate, and ground in three drops of extraction buffer in the same manner as the leaf discs. Systems The apparatus was a slight modification of that described by _n--—--vv~.._,-..r,u. O'Malley, Wheeler and Guries (1980). Where used to resolve all of the enzyme systems examined. System’I consisted of a 0.065 M L-histidinf, 0.02 M gitligflacidmmonoh‘yggte, pH 5.7 electrode buffer and a gel buffer that was a 1:6 dilution of the electrode buffer (Cardy, Stuber and Goodman, 1980). C..__System Allliizonsisted of a 0.125 M 91?..31521913919: pH 7.0 electrode buffer modified from Cheliak and Pitel (1984). The gel buffer was a 0.125 M DL-liigtldlneaflm, 1.4 mM ,NaQEDM'IfAnbuffer adjusted to pH 7.0 with tris. Gels were prepared with 500 ml gel buffer, 62.5 g potato starch (Sigma Chemical Company) and for system I gels only, 15 g sucrose. The heated and aspirated gel solution was poured into 9.1193;3]:§9§.§?§Pl¢ fl which had these dimensions: 21.7 cm long x 14.0 cm wide x 1.5 cm deep. The gels were allowed to cool for 2 hours, and were then covered with 2 layers of plastic wrap and refrigerated overnight. Wigkfiflmhatman Electrophoresis Paper No. 3MM), 4X15 mm in size, were soaked in leaf extract, blotted on we; tgwgling, and inserted into a slice in the starch gel either 10 or 6 cm from the cathodal side of the gel, for systems I and II, respectively. After loading the samples, the gel was placed in a firefrtlgfiratnr...§£wéqg. 11.69,,~ mgwere placed on top of the gels and the gels were run at 20 watts constant power. After 25 minutes the wicks were removed and electrOphoresis resumed for a total of 4 1/2 hours for system I and 6 hours for system II. The enzymes which were resolvable using system I were malate dehydrogenase (Mdh), phosphoglucomutase (Pgm), phosphoglucose isomerase (Pgi) and 6-phosphogluconic acid dehydrogenase (6-Pgdh). The enzymes which were resolvable using system II were Mdh, Malic enzyme (ME), Shikimate dehydrogenase (Skdh) and Pgm. A summary of these methods can be found in Table 1.2. Staining After electrophoresis, gels were sliced into 1/16" thick slices using W and a flingiateelsmmins drawn taut on a hacksaw. Although 9 or 10 thin slices were obtained from a single gel, the top and bottom slices were discardegfbecause they gave poor band resolution. The remaining 7 or 8 slices were immersed in stain solutions in the order given in Table 1.2, for 1 hour at 37°C. The Pgi, Pgm and ME enzyme stains were adapted from Cardy, Stuber and Goodman (1980). The Pgi assay solution contained 50 mg MgClz, 100 mg D-fructose-6-phosphate, 40 units G6PDH (glucose-6-phosphate dehydrogenase), 10 mg NADP (G-nicotinamide adenine dinucleotide phosphate), 10 mg NBT (nitro blue tetrazolium) and 1.5 mg PMS (phenazine methosulfate) in 50 ml 0.1 M tris-HCl pH 8.0 buffer. The Pgm stain was made with 50 ml 0.1 M tris-HCl pH 8.5 buffer, 100 mg MgClz, 50 mg NazEDTA ((ethylenedinitrilo)-tetraacetic acid disodium salt), 250 mg glucose-l-phosphate, 40 units G6PDE, 10 mg NADP, 10 mg NBT and 1 mg PMS. The ME stain contained 60 ml 0.1 M tris-BCl pH 8.5 buffer plus 40 ml 1.2 M DL-malate pH 8.0 solution, 200 mg MgClz, 52 mg NADP, 40 mg NBT and 4 mg PMS. The Mdh, Skdh and 6-Pgdh enzyme stains were adapted from Cheliak and Pitel (1984). Mdh was stained using 50 ml 0.5 M DL-malate pH 7.0 solution, 25 mg NAD (B-nicotinamide adenine dinucleotide), 20 mg NBT and 2 mg PMS in 50 ml 0.1 M tris-HCl pH 8.0 buffer. Skdh was stained with 40 mg shikimic acid, 10 mg NADP, 10 mg NET and 1 mg PMS in 50 ml 0.1 M tris-HCl pH 8.0 buffer. The 6-Pgdh stain was made with 50 mg MgClz, Table 1.2. Summary of electrophoretic materials and methods System I8 System IIb pH 5.7 700 Electrode 0.065 M L-histidine 0.125 M tris adjusted Buffer 0.020 M citric acid to pH 7.0 with citric (EB) adjust pH with_ci£rate. ”asidlanhxdrous ? 7 Gel Buffer 0.009 M L-histidine 0.05 M.DL-histidine-HC1 (GB) 0.903 M citric acid (1:6 dilution of EB) Gel 62.5 g potato starch and 15 g sucrose in 500 ml GB Origin 10 on Power 20 watts (50 mAmps, 400 V) Duration 4.5 hours Enzymesc 1) Mdh 2) Pgm 3) Pgi 4) 6Pgdh 0.0014 M EDTA to pH 7.0 with_tris2 . k sk>~~~zyr m. } 62.5 g potato starch in 500 ml GB :_ i, ”if". ,r if! 1‘? ,113'1, l l i. g "I ’ ( ‘\ " h (" I'll 1 ‘l ,‘l' 6 cm 20 watts ' ' ’ ' (75 mAmps, 270 V) ‘ 'vuw <.A~«;,* 6 hours 1) Mdh 2) ME 3) Skdh 4) Pgm a Cardy, Stuber and Goodman, 1980. b adapted from Cheliak and Pitel, 1984. c gel slices stained in this order, starting from bottom slice. 10 20 mg 6-phosphogluconic acid, 10 mg NADP, 10 mg NBT and 1 mg PMS in 50 ml 0.1 M tris-HCl pH 8.0 buffer. Gels were kept in the dark during staining to prevent excess background staining and to protect light sensitive reagents. Mdh, ME and 6-Pgdh stained gels were left overnight at room temperature to darken bands. Zymograms were scored and photographed for later reference. mamas 01' 1802138 Loci and alleles, determined by band segregation, were designated using the IUPAC-IUB guidelines (1978). Each locus was named using the enzyme name followed by a number. The loci were numbered consecutively with the lowest number given to the locus which encoded the band with the highest mobility toward the anode. The designations slow (SS) and fast (FF) represented the two major bands or alleles seen at a locus and their mobilities relative to one another. The most anodal band was considered the fast isozyme. Banding patterns which showed both SS and FF bands and sometimes an intermediate band, were designated SF. STATISTICS Chi-square values were calculated to determine the inheritance of individual loci and linkage between loci. Data from two or more crosses was only combined if it was determined to be homogeneous by the chi- square homogeneity test (Little and Hills, 1978). Independence of loci was also determined by calculating the recombination frequencies and standard errors of F2 progeny data using the maximum liklihood method (Allard, 1956). RESULTS AND DISCUSSION CROSSES Crosses were made in Fall, 1984, to produce F1 seed for genetic analysis of isozyme and marker loci. The seed from six F1 crosses was sown in Summer, 1985. Where possible, the hypocotyl color locus was used as a marker to determine the fidelity of the F1 crosses. At this locus, red hypocotyl color (RR, Rr) is dominant to green hypocotyl color (rr) (Kajanus, 1917; Keller, 1936). This marker was chosen because the phenotypes can be characterized a few days after germination. Since no green progeny were observed, it was determined that no selfing or contamination by (r) pollen had occurred in the crosses labeled ECl, EC6 or EC12 (Table 1.3). An 11:9 segregation ratio of green to red seedlings implied fidelity of the EC4 cross. Self- incompatibility was relied on to control pollination in EC6, EC8 and EC10; since, when 1380 and 138D, the respective female parents in the crosses, were individually bagged they did not set self seed. One could not detect contamination of the EC8 and EC10 crosses using the hypocotyl color marker because the female parents were homozygous dominant red. Further generations of EC6, E68 and EC10 were not produced because all of the progeny were biennial. Seed germination percentages of the lines ranged from 29-1002 (Table 1.3). There were two problems, however, with these calculated values. First, they represented the number of seedlings which arose from multigerm seed balls and were not accurate measurements of the viability 11 12 Table 1.3. Percent seed germination and segregation of the F1 progeny at the hypocotyl color marker locus EC # Parents 2 germ cross* observed expected pink green pink green E01 DBrA X 138D 581 rr X RR 29 0 29 0 E04 DB-A X 1380 402 rr X Rr 11 9 10 10 E06 1380 X 138D 942 Rr X RR 47 O 47 0 E08 138D X 1380 >100! RR X Rr 56 0 56 0 E010 138E X 1380 1002 RR X Rr 50 O 50 0 E012 DH-B X 138D >100! rr X RR 49 O 49 0 * hypocotyl color phenotypes are RR or Rr:red and rr:green. of all.individual embryos. Second, it was impossible to determine the extent to which they'were affected by unfavorable environmental conditions during seed set. These conditions included low light intensity levels and an infestation of spider mites and aphids on the plants in the crossing bags. In Fall, 1985, the F1 plants which bolted under artificial long days (annuals) were isolated in selfing bags for F2 seed production. All of the annual Fl's had inherited one Sf allele at the self- incompatibility (S) locus from DR-A or DR-B. Pollen which contains this allele is compatible on any stigma, thereby allowing plentiful self seed production (Owen, 1942). These F1 plants had also inherited the male sterile cytoplasm from DH-A.or DH-B. 'This cytoplasm causes plants to be male sterile unless they have a dominant allele at either the X or Z fertility restorer locus (Owen, 1945). The number of fertile Fifs produced and the degree of fertility restoration were dependent upon the genotype of the male parents at the X and z restorer loci. Therefore, the ratio of male 13 sterile to male fertile Fl's should have indicated the genotypes of the male parents. Approximately half of the Fl's put in selfing bags in Fall, 1985, appeared to be male sterile. The other half of the Fl's set varying amounts of self seed. When the Fl's were bagged again in Fall, 1986, some plants which had set self seed in 1985 appeared sterile, and some plants which had not set seed in 1985 set plentiful seed (Table 1.4). Table 1.4. The number of F1 individuals which set self seed over two consecutive seasons Total # which set self seed Total EC # # progeny Fall 85+86 Fall 85 Fall 86 Total 1 plants setting seed E01 20 6 . 1 2 9 451 E04 19 10 O 1 11 58% E012 21 7 2 4 13 621 During both growing seasons many plants suffered from a severe insect infestation which interfered with pollen production and seed develOpment. Many of the E01, E04 and E012 Fl's died because of the infestation. Due to conflicting results over seasons, severe aphid infestations and the death of many F1 plants, no conclusions were drawn about the genotypes of the male parents at the X and Z loci. Segregation data for several isozyme and marker loci were collected from the surviving F1 progeny and F2 progeny of E01, E04, and E012 individuals. Some F1 and F2 progenies were also analyzed for linkage between loci. It is important to note that the death of many Fl's during bagging as well as possible linkage of isozyme loci to recessive alleles at X and 2 might have skewed the F1 segregation ratios. 14 MIMINART ISOZTME STUDIES Before the isozyme banding patterns were defined for any plant, all of the isozymes were examined under a variety of conditions. Clones of the plants 1380, 138D, 13811 and DH-A were examined extensively to insure their banding patterns were the same when different extraction buffers, gel and electrode buffers and enzyme stains were used. Samples from plants grown in the greenhouse over different seasons were also compared for consistency. Leaves of different sizes and ages were sampled over time to insure that banding patterns were not affected by plant growth conditions, plant age or leaf age. Wmmgg‘mgj 5933 {greet bandM—aefifiwrsndersall.,ef..&.1;§§$SOD‘B‘yREW“ ,. .amnl," considered for inheritance studies. . _..,-,-,Wr:r~=pt:« « It 3:41“ *g-‘f r 'i‘wfla-I .. «'- "1" *' '.' .1;-.a-;mr.:tw;mmnwzuqu; 1. . Isozm IIHERITAICE Malate dehydrogenase Gels stained for Mdh showed 3 distinct regions of handing activity. (Figure 1.1). The region which contained the fastest migrating bands showed three isozyme banding patterns: SS, FF and a three banded, SF pattern (Figure 1.2a). It was hypothesized that these bands were the products of a single locus, Mdh-1. The inheritance of these bands was determined by examining the segregation ratios of F1 and F2 progeny. Crosses between two F]? individuals resulted in all FF progeny (Table 1.5). Three SS X FF crosses resulted in progeny which uniformly expressed the SF genotype. F2 progeny from five selfed SF individuals segregated in the 18$:ZSF:1FF expected ratio with a greater than .7 probability (Table 1.5). The segregation data of one F2 line fit this ratio with a probability of .2-.3. The segregation data confirmed that the bands were the result of two alleles from a single locus, Mdh-1. 15 .onon also: Ammv anew one saw: mouaumaaoo also: we mean oamnum emu mom: on: H auumhm can: .mmmn also: Ammv moan emu sue: muuuuwuaoo mean also: on» Seaman menu wean: .nlnmz one also: :uon new momhu am one .mm .mm ozone one an auumhm no“: mo>uomuu no: How 35. .5: no 33 9.38 2,3 2:. Good .3332. we 3:3 ooh: 2; .3 35E 16 Figure 1.2. Starch gel zymograms showing the banding genotypes at 4 isozyme loci: a) FF, 3-banded SF and SS banding genotypes at Mdh-1, b) Comparison of leaf (A) and pollen (B) samples from an Mdh-1 SF plant, c) FF, 3-banded SF and SS banding genotypes at Mdh-3, d) SS, 2-banded SF and FF banding genotypes at Pgm-1, e) SS, 3-banded SF and FF banding genotypes at Pgi-1, f) Comparison of leaf (A) and pollen (B) samples from a Pgi-1 SF plant. Table 1.5. Chi-square analysis of allelic segregation of Mdh-l in F1 and F2 progenies of Beta vulgaris ac # Cross Gen@ ss sr FF N x2 P E018 38 x FF r1 - 20 -- 20 -- -- E012 88 x FF F1 - 20 -- 20 -- -- EC4 ss x rr r1 -- 18 -- 18 --- -- E06 FF x FF r1 -- - 52 52 --- -- E010 FF x FF r1 -- -- 25 25 -—- -- EC4-9* sr x sr r2 2 13 4 19 3.000 .2-.3 E01-22 S? x sr F2 10 23 14 47 0.702 .7-.8 EC4-5 53 x 5? F2 23 53 25 101 0.326 .8-.9 EC4-18 39 x 33 F2 19 38 15 72 0.670 .7-.8 EC4-19 33 x 3? F2 11 23 14 48 0.458 .7-.8 EC4-20 sr x sr F2 10 17 7 34 0.529 .7-.8 @ Gen: Generation, N: Total progeny, P: Probability, SS: slow homozygote, SF: heterozygote, FF: fast homozygote. * only spinach leaf mutant segregates were examined & E01: DEA X 138D, EC4: DEA X 1380, E06: 138D X 1380, E010: 1388 X 1380, E012: DEB X 138D. The existence of a three banded SF type provided strong evidence that the locus encoded a dimeric enzyme. In addition, the pollen of these SF individuals showed activity for the slow and fast bands, but not for the intermediate band (Figure 1.2b). When gels which contained pollen samples from SF plants were stained for a variety of dimeric enzyme systems, they did not show activity for the intermediate band (Tanksley, Zamir and Rick, 1981). It was determined that Mdh-1 was a single locus with.2.alleles which encoded a dimeric enzyme. A band was observed which always comigrated with one of the Mdh-1 bands (Figure LJ). When Mdh was resolved using system I, this band migrated to the same position as the intermediate band of Mdh-1. When system II was used, this band migrated to the same position as the fast band of Mdh-1. This band appeared regardless of the Mdh-1 genotype of 18 the plant from which the sample was taken; however, it was obscured in SF and FF types when system 11 was used. .All parents and progeny of the crosses showed activity for this band, but pollen did not. It was determined that this band is produced by a locus distinct from Mdh-1. This locus was designated Mdh-2 and was represented by only one allele. The second region of banding activity was only resolvable with system II. This region contained 3 banding types, 88, FF and a three banded, SF type (Figure 1d). It was hypothesized that these bands were the products of a single locus, Mdh-3. Each of the banding types was often coordinately expressed with several faint, faster migrating bands (Figure 1J9. Before determining if the major bands represented a new locus, it was necessary to examine the nature of the faint bands. The faint bands showed variability in activity in different samplings of the same individual. In some instances the bands were inactive:in.individuals which had previously shown activity for these bands. In addition, the bands that were associated with the FF band, never appeared with the SS band, and vice versa. Since they did not associate independently it was unlikely that the faint bands and the major bands were the products of different loci. It was also unlikely that the faint bands were due to intra- or interlocus heterodimers, since they were not located in a position intermediate to any two bands or loci. The phenomenon of associated Mdh bands was also observed in maize and was assumed to be due to secondary modification (McMillin and Scandalios, 1980). Other researchers were able to eliminate faint, trailing, Mdh bands in Maize by purification of the soluble Mdhs (Goodman and Stuber, 1983). The faint bands were determined to be artifactual, therefore they were ignored in the later collection of data for this study. 19 A cross between two SS plants resulted in all 88 progeny (Table 1.6). No SS X FF crosses produced progeny which gave a three banded, SF phenotype. This three banded pattern was similar to that observed in the Mdh-1, SF types (Figure 1.2c). F2 progeny from two, selfed SF types segregated in the expected lsS:ZSF:1FF ratio with a greater than 0.7 probability (Table 1.6). Since pollen was not active at this locus, comparisons of pollen and leaf tissue could not be used to confirm the dimeric nature of this enzyme. Nonetheless, the existence of an intermediate band was adequate to conclude that the Mdh-3 locus encoded a dimeric enzyme for which two alleles existed. The third region of Mdh activity (observed only when system II was used) showed less activity than the other two regions. This region consisted of two, thin (less active) bands located close to the origin on the gel (Figure 1.1). These isozymes were always active except in pollen samples. When cross progeny were examined the bands never segregated or interacted. In many species, MDH is known to be compartmentalized in microbodies, mitochondria and the cytosol (Ting, Fuhr, Curry and Zschoche, 1975). The activity of microbody Mdh in maize was shown to be much lower then in the activity of the cytosolic and mitochondrial isozymes (Yang and Scandalios, 1975b). The similarities between the Mdh zymograms in maize and sugarbeet imply that the thin bands are the products of isozymes localized in microbodies. It was determined that these bands were the products of two loci, Mdh-4 and Mdh-5 each of. which had only one allele. 20 Table 1.6. Chi-square analysis of allelic segregation of Mdh-3 in F1 and F2 progenies of Beta vulgaris 110 # Cross Gen@ ss 31‘ FF N x2 P rc1‘ 36 x ss 31 16 - -- 16 -.. __ EC6 ss 2 rr r1 -- 17 -- 17 -- -- E04 ss x FF r1 -- 7 -- 7 --- _- EC4-22 sr x sr rz 10 15 9 34 0.529 .7-.8 5 20 0.300 .8-.9 EC4-9 SF X SF F2 6 9 9 Gen: Generation, N: Total progeny, P: Probability, SS: slow homozygote, SF: heterozygote, FF: fast homozygote. * only spinach leaf mutant segregates were examined 8 801: DEA X 138D, EC4: DEA X 1380, E06: 138D X 1380. Phosphoglucomutase Gels stained for Pgm showed two regions of activity; however, only the more anodal (faster) region was resolvable. In gels resolved using system I, the slower region virtually'disappeared. The bands in this region never varied and also appeared to comigrate with the bands of the faster region. Stuber and Goodman (1983) observed a similar phenomenon and concluded that each allele of the Pgm locus was associated with a pair of isozyme bands. Since system I was primarily used in this study, the slower region of activity was simply ignored. The faster region contained three isozyme banding patterns: SS, FF and a two handed SF pattern (Figure 1.2d). It was hypothesized that these bands were the products of a single locus, Pgm-1. Three FF X SF crosses segregated in the expected 18F:1FF segregation ratio (Table 1.7). The data from two crosses between SF types and five selfs of SF individuals fit the 188:28F:1FF expected ratio. The SF individuals showed activity for the S and F bands but did not 21 show activity for an intermediate band (Figure 1.2d). The banding patterns from pollen versus leaf extracts were identical for SS, SF and FF individuals. The lack of an intermediate band in the heterozygotes indicated that the locus encoded a monomeric enzyme. It was determined that the Pgm-1 locus encoded a monomeric enzyme for which twolalleles were observed. Table 1.7 Chi-square analysis of allelic segregation of Pgmrl in F1 and F2 progenies of Beta vulgaris E0 # Cross Gene 88 SF FF N XZ P 801‘ FF x SF F1 -- 6 13 19 2.579 .1-.2 E012 PP 8 SP P1 -- 9 12 21 0.429 .5-.6 EC4 PP x SP F1 - 11 4 15 3.267 .05-.1 E010 SP x SP Fl 9 12 4 25 2.040 .3-.4 806 * SP x SP Fl 15 27 10 52 1.039 .5-.6 EC4-9 SP x SP P2 3 11 4 18 1.000 .6-.7 801-22 SP 2 SP P2 11 31 5 47 6.319 .05-.1 EC4-5 SP x SP P2 28 49 24 101 0.406 .8-.9 EC4-18 SP 2 SF P2 13 35 24 72 3.420 .1-.2 EC4-20 SP x SP P2 10 14 10 34 1.059 .5-.6 @ Gen: Generation, N: Total progeny, P: Probability, SS: slow homozygote, SF: heterozygote, FF: fast homozygote. * only spinach leaf mutant segregates were examined 8 E01: DEA X 138D, EC4: DEA X 1380, E06: 138D X 1380, E010: 1388 X 1380, E012: DEB X 138D. Phosphoglucose isomerase After separation with system II, gels stained for Pgi showed two distinctly stained regions, neither of which could be clearly resolved into bands. Pollen was active only in the slower of the 2 regions. Gels separated with system I showed only one clearly resolvable region of activity. This region.showed pollen.sctivity and contained three isozyme banding patterns: SS, FF and a three banded SF pattern. It was 22 hypothesized that these bands were encoded by a single locus, Pgi-1 (Figure 1.2e). Three FF X SF crosses resulted in F1 progeny which segregated in the expected lSF:lFF ratio. F1 progeny, from a SS X.SF cross, segregated in the lSS:lSF expected ratio (Table 1.8). Pollen from SF individuals showed activity for the slow and fast bands but not the intermediate band. (Figure 1,2f). This is the kind of data that one would expect for a locus with.two.alleles, each encoding a subunit of a dimeric enzyme. Table 1.8. Chi-square analysis of allelic segregation of Pgi-1 in F1 and F2 progenies of Beta vulgaris 80 # Cross Gene ss SP PP N X2 P 801‘ PP 8 SP P1 - 9 10 19 0.052 .8-.9 8012 PP x SP P1 -- 13 8 21 1.190 .2-.3 804 PP 8 SP F1 -- 10 6 16 1.000 .3-.4 8010 SS x SP F1 11 14 -- 25 0.360 .5-.6 806 SP 8 SP F1 6 28 18 52 5.846 .05-.1 801-22 SP 8 SP F2 2 22 23 47 18.957 <.05 804-5 SP x SP F2 10 50 41 101 19.039 <.05 804-18 SP 8 SP F2 5 39 28 72 15.190 <.05 804-19 SP 8 SP F2 4 21 23 48 15.792 <.05 @ Gen: Generation, N: Total progeny, P: Probability, 58: slow homozygote, SF: heterozygote, FF: fast homozygote * only spinach leaf mutant segregates were examined & EC1: DEA X 138D, ECA: DEA X 1386, 306: 138D X C, EC10: 1388 X C, EC12: DEB X 138D. In contrast to this data, F1 progeny from a cross between two SF plants and F2 progeny from.selfs of four SF individuals did not segregate in the expected lSS:ZSF:1FF ratio (Tablealn8). The ratios were consistently skewed such that when the data was averaged, 8.42 of 23 the progeny were of the 88 type, and 41.61 of the progeny were of the FF type (Table 1.9). Only the SF type, found in a total of 50.02 of the progeny, was close to its expected value. Table 1.9. Frequency of Pgi banding types in segregating F1 and F2 progeny Family SS SF FF N EC4-5 .099 .495 .406 101 EC4-19 .083 .438 .479 48 EC1-22 .043 .468 .489 47 EC6 .115 .539 .346 52 -EC 4 F2 data only .086 .498 .416 221 All of the F1 plants from 361, EC4 and EC12 had received one normal self-incompatibilty allele from the original male parent and one Sf allele from the female parent. Pollen which contains the Sf allele is compatible on any stigma; however, pollen containing any other 8 allele may or my not be compatible. Overall the Beta vulgaris incompatibility system is extremely complex and contains either 2 or 4 loci with multiple alleles (Owen, 1942, Larsen, 1977). A simplified model was developed to explain the skewed Pgi ratio. This model was based on the premise that the self-compatability system involved was monogenically inherited. Assuming this premise to be true, the following was also true: (1) Pollen which contained the (8) allele contributed by the male parent was self-incompatible in the Fl's, and (2) The segregation ratios of any genes linked to the (S) locus were skewed in favor of the allele contributed by the female parent. 24 Expected Pgi segregation ratios were calculated for a range of recombination percentages. An example which was calculated following this model, using a value of 172 recombination.between the loci, can be found in Table 8. Table 1,10. Possible F2 genotypes using 172 recombination between the Pgiand self-incompatibility loci Hale Gametes Parental types Recombinant types F Sf S 81 F 81 S Sf Female Gametes .83 incompatible .17 .415 F Sf .3444 FF Sfo -- --- .0706 SF SfSl P .415 S 81 .3444 SF SfSl -- --- .0706 SS SfSl .085 F 81 .0706 FF SfSl --- --- .0144 SF SfSl R .085 S Sf .0706 SF Sfo -- --- .0144 SS Sfo Expected Pgi ratio .085 SS .500 SF .415 FF When the calculated values in Table 1.10 were compared to the combined values in Table 1.9, it was obvious that the frequencies of the banding types were very similar. Equations for lines describing the three banding types at percent recombinations (ZR) from O to 50 percent were calculated using the incompatibility/linkage model as a basis. These equations, with Y equal to the frequency of the banding types and X equal to the IR, are: 4 SS: Y - .OOSX SF: Y - .500 FF: Y - -.005X.+ .5 Figure 1.3 is a graph of these lines. If the ZR was 02, the ratio 25 Figure 1.3. Graph of lines describing the SS, SF and FF banding types from 02 to 502 recombination. Combined data and data from each of the F2 families in Table 1.9 are plotted on the SS and FF lines. 26 coonnEoooc N on o.- o.n om 9 H U H X “Elf mmlxl mmlml Kouenbeu; ogdfimusfi 27 of the banding frequencies would be .SSF:.5FF and there would be no SS types. If the IR was 502, the loci would be independent and the ratio of the banding frequencies would be .ZSSS:.SOSF:.25FF. When the SS and FF data from each of the crosses were plotted on these lines the data tended to cluster between 15 and 252 recombination. When the SS and FF equations were calculated using the banding frequency values from the combined data, the resulting ZR for both equations was 16.8%. It was also possible that the skewed Pgi-l ratio observed in this study'was entirely or partially due to linkage between Pgi-1 and an embryonic lethal locus. A similar situation was observed in Beta 'vulgaris in conjuction with the alcohol dehydrogenase (ADE) locus (Maletskii and Xonovalov, 1986). Segregating progeny from the self- pollinations of 13 sister lines were examined. Some lines segregated in the expected 1:2:1 ratio while others deviated from this ratio. They concluded that the ADE locus was linked to an incompatibility locus and a locus which behaved like an embryonic lethal. In our study, 138C and 138D were carriers of a dwarf crinkly leafed mutant phenotype named spinach leaf. Approximately 91 of the progeny from crosses between 1380 and 138D are of the spinach leaf phenotype. Spinach leaf seedlings have shortened hypocotyls, are slow to germinate and are often deformed and die (see Chapter 3). Assuming that this mutant is caused by a single gene, there may have been lethality of some of the embryos which would have been spinach leaf types. If this were the case, any locus linked to the spinach leaf allele would have a skewed segregation ratio. Partial lethality of pollen carrying the mutant allele would have had a similar effect, even in the F1 progeny. The evidence does not support this theory for the following 28 reasons: 1) F1 progeny from the original EC1, 4, 10 and 12 crosses did not segregate in the skewed ratios that they should have if there was gametophytic lethality involved (Table 1.8) and 2) Lines which did not produce F2 spinach segregates still gave skewed F2 Pgi ratios. Although the spinach mutant may have had an effect, the skewed Pgi ratio was probably due to linkage between the Pgi-1 and self-incompatibility loci. The map distance between the two loci was estimated to be 16.8 map units. It was determined that the Pgi-1 locus encoded a dimeric enzyme for which two alleles were observed. Nonetheless, some additional crosses should be analyzed to confirm these conclusions and to insure that any lethal affects of the mutant did not confound the segregation data. More information about the spinach leaf mutant can be found in Chapter 3. Malic enzyme Two distinct ME banding types were observed in the parental lines, each composed of 5 equally spaced bands. When the two types were compared, the most anodal bands of both were located at the same migration distance on the gel. The other four bands, however, were in different positions (Figure 1.4). These banding types were identified using the Roman numerals I and II. Pollen was active for only the most anodal band. A cross between between two type I individuals produced progeny which exhibited only banding type I (Table 1.11). All of the progeny from a type II X type I cross produced progeny which showed activity of both of the banding types plus some additional bands for a total of 15 bands. Individuals showing this pattern were labeled a hybrid (E) banding type (Figure 1.4a). A self of a hybrid type gave progeny which 29 .NIMS was .aluz HoOH vomoaoun unu Esau moHoHHm osu an couscous novuuaonhaon oz» uaomounousn was a .m .aauouusn mauvnsn omega osu mcuaomaoo muofisuuou oau mo nouauosuum oz» mausosu Esuwouv An .Hosuu>fivsfi an .m onmu a Mo hmowoun Nu wuauswoumon was AnH + H sommhv unsound onu macaw was HH saunas wows: vo>Honou as: Bounces» saunas swan: many As .e.H Penman annn AAAA \ 82 fin m N .n ‘ <2 6 ans» can. Emu” umdfl news can. $sss nous puss ”$0“ “6”” “66¢ 30 segregated in a 1:2:1 (type I : hybrid : type II) ratio with a greater than 0.8 probability (Table 1.11). Table 1.11. Results of crosses for the tentative locus ME-Z EC # Cross* Gen@ II E 1 N x? P 8018 II x I F1 - 16 -- 16 -- -- EC6 I x 1 F1 - -- 17 17 -.. -- 804-20 E x E F2 8 16 1o 34 0.353 .8-.9 * Type I and II represent banding patterns and the fast and slow alleles respectively of the hypothesized ME-Z locus. 8 Gen: Generation, N: Total progeny, P: Probability, SS: slow homozygote, SF: heterozygote, FF: fast homozygote. & EC1: DEA X 138D, EC4: DEA X 138C, EC6: 138D X 138C. From this data it was hypothesized that malic enzyme was controlled by two loci which interacted to form a five banded pattern, the result of a tetrameric enzyme. Based on this hypothesis the observed data could be explained by the following: 1) The fastest band of both type I and II patterns was the result of the same allele at the invariant ME-l locus, 2) The slowest band of both type I and II patterns represented two different alleles at the 1113-2 locus, slow (type II) and fast (type I), 3) The three intermediate bands of both patterns were a result of interlocus heterotetramers between ME—l and ME-2, 4) The hybrid banding pattern was caused by inter- and intralocus heterotetramers between the IKE-1 locus and both alleles of the ME-Z locus. The result of all of these bands was a 15 banded pattern, and 5) The 1:2:1 (type I : hybrid : type II) ratio was actually a lSS:ZSF:lFF segregation of the two alleles of the ME-Z locus. Figure 1.4b is a diagram of this hypothesis. Evidence in support of this hypothesis is provided by many other 31 species (maize for example) which have tetrameric malic enzyme isozymes (Goodman and Stuber, 1983). Researchers working with many other species have chosen not to study ME because of its complex banding patterns. In sugarbeets, Van Geyt and Smed (1984) described an article by Levites (1979) which proposed that the five HE isozymes observed in his study were the result of a tetrameric enzyme, controlled by one gene. Van Geyt and Smed felt that the starch gel electrophoresis method used by Levites did not adequately resolve the bands, therefore they disagreed with his proposal. Since neither author examined segregation data they could not establish the genetic basis of the observed zymograms. Although the segregation data (in our study) seemed to fit our hypothesis, more progeny should be analyzed before a definitive statement of ME inheritance is made. Shikimate dehydrogenase Only one Skdh band was observed in the parents of all the crosses. All of the progeny of four different F1 crosses had activity for this band (Table 1.12). Pollen was not active for the Skdh enzyme. The band was tentatively designated as a product of the Skdh-1 locus. A slightly faster band was discovered in a genotype which was not used in the crosses. This will be discussed in Chapter 2. 6-Phoaphogluconate dehydrogenase When parents of the crosses were stained for 6-Pgdh, they all exhibited the same four banded pattern. This pattern was composed of a single fast band followed by three evenly spaced, slower bands. All cross progeny showed the same four banded pattern. 32 Table 1.12. Results of crosses for Skdh-1 80 # Cross* Gene I N x2 P 8065 1 x I F1 17 17 -- - 808 I x I P1 2 2 -- -- 8010 I x I 81 1 1 --- -- 801 1 x I P1 2 2 -- -- * Type I represents the single band of the hypothesized Skdh-1 locus. 8 Gen: Generation, N: Total progeny, P: Probability. & EC1: DEA X 138D, EC6: 138D X 138C, EC 8: 138C X 138D, EC10: 138E X 1380. There are several possible explanations for this banding pattern. 1) Each band was caused by a different locus. 2) Some of the bands were caused by different loci and some were artifactual. 3) The pattern was caused by the three invariant loci, two of which interacted to form an interlocus heterodimer. In many plant species 6-Pgdh isozymes have been shown to be dimers encoded by two loci, one cytOplasmic and one associated with plastids (Gottlieb, 1981). Maize 6-Pgdh isozymes are encoded by two cytosolic loci which interact to form heterodimers (Goodman, Stuber, Newton and Weissinger, 1980b). ‘Eeterodimers are not formed between enzymes located in different cellular compartments. Since most species have been shown to have only two 6-Pgdh loci it is unlikely, but not impossible, that there are four different loci in sugarbeet. To confirm any of these explanations, however, it will be necessary to find an individual which has a different 6-Pgdh banding pattern and use it as a parent in crosses. Analysis of the ensuing segregating progeny can then be used to determine the correct explanation. 33 Eypocotyl color and annualism The hypocotyl color and annualism loci were used as marker loci in crosses and were tested with the isozyme loci for linkage. These loci encode monogenical 1y inherited traits and are known to be found on the same linkage group (Rajanus, 1917; Keller, 1936; Hunerati, 1931; Abegg, 1936). Red hypocotyl color is dominant to green, and annual flowering habit is dominant to biennial. The expected segregation of the alleles of one or both of these loci was observed in most of the lines which were also examined for isozyme segregation (Table 1.13). The data did not fit segregation ratios for one line (Table 1.13). This was probably due to poor penetrance of the (B) allele under adverse environmental conditions. LINKAGE Mdh-l was found to be unlinked to the Pgm-1, B and R loci when chi-square goodness of fit and recombination values were calculated (Table 1.14). Pgm—1 was also found to be independent of the R and B 1001. Mdh-3, the proposed loci and all of the non-segregating loci were not included in the linkage tests. The data from the EC 6 and F2 progeny (see Table 1.8) which segregated for Pgi-1 gave highly significant chi-square values when linkage of Pgi-1 to the the Mdh-1, Pgm-1, hypocotyl color and annualism loci was tested (Tables 1.15 and 1.16). When the recombination percentages were calculated using Al lard's maximum likelihood formulas, however, they were all very close to 502, which indicated independence. These conflicting results were most likely a result of the skewed segregation of the alleles at the Pgi-1 locus. The calculations done using Allard's formulas were not affected by the skewed Pgi-1 ratios, whereas the chi- 34 Table 1.13. Chi-square analysis of allelic segregation of R and B in F1 and F2 progenies of Beta vulgaris EC # Cross Gen@ A aa N X2 P R: Eypocotyl color EC1 rr X RR F1 29 0 29 -- -- EC12 ' F1 49 0 49 -- -- EC4 rr X Rr F1 11 9 20 0.200 .6-.7 EC6 Rr X RR F1 52 0 52 -- - EC8 RR X Rr Fl 56 0 56 -- -- EC10 ” F1 50 0 50 -- -- EC4-5 rr X rr F2 0 101 101 - -- EC4-18 Rr X Rr F2 56 16 72 0.296 .5-.6 EC4-19 ' F2 36 12 48 0.000 1.00 COMBINE F2's 4-18,4-19 92 28 120 0.178 .6-.7 EC4—9* rr X rr F2 0 32 32 -- -- EC4-20 ' F2 0 34 34 -- -- EC1-22 Rr X Rr F2 31 16 47 2.050 .1-.2 B: Annualism E01 BB X bb F1 29 0 29 -- -- EC4 " F1 20 0 20 -- -- E012 ' F1 49 0 49 -- -- EC6 bb X bb F1 0 52 52 -- -- EC8 ” F1 0 56 56 -- -- EC10 ' F1 0 50 50 - -- EC4-5 Bb X Bb F2 72 29 101 0.740 .3-.4 EC4-9* ” F2 22 10 32 0.667 .4-.5 ECl-22 ' F2 26 21 47 9.709 (.05 @ Gen: Generation, N: Total progeny, P: Probability, A;; dominant phenotype, as: recessive phenotype. * Only spinach leaf mutant progeny were examined. 35 .uuou oposumlano huuosomoao: mean: ssoosswoaos on on ozone an: aunt voHooa « douse—Now cog—30x: ass—«nos Found H< wean: venue—house nouns cum—Esau I\+ unmouoa soaussunaooou “a .oosovsomov:N mo huNHNAsnoum um .moamuosozn msooa o>asuooou "a .oahuooonn usuoa usssuaov u< .ouowanoao: anon ”m .ouomhsououos A: .ouowhsoaos Boas "m @ NN.N IN+NN N.nc. NN.N NH NN «N NN 86 NN NH oN oN NoN .8 on NN.-N. 6N.N N e N N N N N e N «N oNuN om N.ue. NN.N N N e «N NH N N Na N NN NNIN on NN.-N. NN.N N NH N Na «N «N N «N N Nos N14 om N.-N. NN.oN N NH N 6 Na N o N N N4 NNIN on Naumnsncz NN.N -\+N6 N.-N. oN.N N NN N ON N N NN Nan: om No.v NN.NN N N Na Na N oz N6 NN-N om enemwm NN.N IN+NN N.-N. NN.N N ON as an N NN oNN .4 on N.-N. «N.o N as N oN N N N: mane um N.ue. NN.N N N N oN N NN NN Nsue on N.-N. NN.N N N N Na N N Ne NNuN on muses: NN.N uN+NN N.-N. NN.N q oN NH NN N NN Nos Nne on nuNmuN NN.N -\+oN NN.-N. NN.o N Na Na NN .N .NN NON Nue on nusnuz N.N m.m N.N s.m m.m N.m N.N =.N N.N a N Nx 6.» <.e 0.: <.m 6.N w<.N z Noon auohasss owsxsua you oceans .m was a .Hawm .Hsvz mo mouuou souusmoumoo Nu .e~.~ canoe 36 .ooauou soausmouwou Hlawm assess you moussfiuu mo=Hs> ousswulanu wouuoaxo ¢ .uoou ouosvmlano muuosomoaon wows: osoosowoao: on so cacao as: some voHooe N .Noasauom voozNHoxNH Hosanna o.vusHH< News: vocwauouov nouns venomous I\+ soauoswnaouou "a .oosovsoaosz mo unquananoum um .aoahuocona usooa o>Nsnooou as .omhuososn osooH unusuaoc N< .ouowhnoao: unom um .ouommsououon A: .ouomhuoaon scan um @ N.-N. NN.N «N.NN NN.NN NN.NN NN.NN NN.N NN.N Nxo nasNNN NN.N IN+NN NN.v NN.NN «NH NN NN NN N N NNN «N NN No.v NN.NN N Na N NN N N Ne NNuN 0N No.v NN.NN N NN N NN N N NN NN-N UN No.v NN.NN NH NN N Na N N NN NNnN NN NueNNN N.-N. NN.N N.NN N.NN N.NN N.NN N.N N.N .Nxo Nuanva NN.N -\+NN No.v oN.NN N NN NN NN N N Hos nus NN NuNNNN a a Na N.N <.N a.: <.N N.N N<.N 2 Neon us:as>.oussvonuao voumafivs was nowososvouu souussuasooon wows: m can a .Haumm cassava omsxsua mo uaahass< .n~.H sunny 37 .moNuou scuummouwom Hawmm voaoxm you vouosfivo oosHo> onusrnluno wouooaxo ‘ .umou unwavNIuao muuosowoao: «can: nsoosowoaos as ou cacao no: «use voaooq « .umasauow voonqaoxwa asaaxua o.uuoad< wean: vosaauouov nouns unaccoua I\+ usoouom :ONuoanaooou "a .oosovsoqovsw no huuaunsaoun “m .ouomhuoao: unsm um .oquNNOHouo: "a .ouomhuoao: soda Nm 9 m.lm. n~.e o.w~ o.on o.wH o.~N N.nq c.HN o.n m.h o.m ans Hum um.m I\+Nm mo.v No.5m NN Nm ma oN ow MN 0 o n NNH he on mo.v ¢0.0N OH NH 0 Ha HN N m N o NN NNIN UM mo.v mm.HN NH cN a a nN ea m N m NON mic om no.v mm.NN n ma N N 0H c o N o he NNIH om N.IH. MN.HH m NH m n «a a N H m Nn e um Amumlauwm «.1». mm.m o.mN o.oc o.NN o.NN N.nn o.NN o.e m.a c.c «Ana nun N<.m I\+Nn no.v Nm.on eN we oN nN mm on m MN m HNN he on mo.v MN.eN m ON a a m m o e c we aalc om no.v aw.¢~ a fig c n NN Na N m A NN male on mo.v on.mN Na NN o Nd «N ma N e N Hod mle um mo.v NQJN .N ma .N m a o N o o TN NNIH UN anvzlawwm a m NM m.m =.m m.m m.= =.m m.m m.m m.m wm.m z Noon mosao>,oussvulano wouoshvo can nouusosvoum sowusswnaooou mafia: Huawm was also: .Nlumm cassava owsxsun «o mammass< .oN.H manna 38 square calculations were. To confirm if skewed Pgi-1 ratio was the cause of the high chi- square values in all of the pairs of loci which were tested, expected values were calculated which accounted for the skewed Pgi ratio. These adjusted expected values were calculated by multiplying the observed Pgi class frequency (0) by the expected class frequency of the non-skewed locus (E), times the number of progeny (n). Formulas used in these calculations, using the combined data values from Table 1.9 (as an example) are as follows: expected 2,? I O ( E ) X n expected S,S - .084 (.25) X n expected E,S - .500 (.25) X n expected F,E - .416 (.50) X n When the adjusted chi-square values were used to calculate the independence between the Pgi-1 and Mdh-1, Pgm-1, R and B loci, the probability of independence was above 0.60 in every case (Tables 1.15 and 1.16). These values were in agreement with the recombination percentages determined using Allard's equations on unadjusted data. Chi-square values for independence between the Pgi-1 and Pgm-1 loci were also calculated for three F1 lines (Table 1.17). These lines were significant since they did not have a skewed Pgi-1 ratio. The chi- square values for all three lines confirmed the independence of the Pgi- 1 and Pgm-1 loci. It can be implied that the high chi-square values which were observed in the EC6 and F2 data in Tables 1.15 and 1.16 were a result of the skewed Pgi ratio. It was determined that there was no linkage between Pgi-1 and any of the 1001 examined. As discussed earlier these crosses should be reproduced using parents which do not carry the spinach leaf mutant. 39 Table 1.17. Analysis of linkage between Pgi and Pgm using F1 segregation data Line N E;E@ E;F F;E F;F X2 P S,S E,S S,E E,E S,F E,F 801‘ 19 7 6 3 3 2.68 .4-.5 EC12 * 21 4 8 4 5 2.05 .5-.6 EC1+12 40 11 14 7 8 3.00 .3-.4 8 8: slow homozygote, E: heterozygote, F: fast homozygote, P: probability of independence. * pooled data was shown to be homogeneous. & ECI: DEA X 138D, EC10: 138E X 138C, EC12: DEB X 138D. SUIIAI! In summary, eight new loci were identified, four of which had more than one allele and segregated as expected (Table 1.18). None of these loci were found to be linked to one another or to the annualism or hypocotyl color loci. Two additional loci were proposed, however, more progeny must be analyzed to determine their inheritance. Pollen samples showed activity at only three of the eight loci. Of the segregating loci, three encoded dimeric enzymes and one encoded a monomeric enzyme. 40 Table 1.18. Summary of the identified isozyme loci and their important characteristics Locus # subunits5 # alleles pollen system Mdh-1 2 2 active I or II Mdh-2 - 1 inactive I or II Mdh-3 2 2 inactive II Mdh-4 - 1 inactive II Mdh-5 - 1 inactive II Pgm-1 l 2 active I or II Pgi-1 2 2 active I Skdh-1 - 1 inactive II ME-1* 5 1 active II ME-2* 5 2 inactive II & - means # of subunits not known * these loci are only proposed Mill WOIWSUSINGW MII W 01' W8 USING 150m INTRODUCTION Isozyme electrophoresis has been used to distinguish cultivars and assess the genetic variation in a number of craps (Ignart and Weeden, 1984; Wu, Earivandi, Harding and Davis, 1984). Eorizontal starch gel electrophoresis is the most common method used since a large number of samples and enzymes can be assayed quickly and inexpensively. Even heterozygous lines can be distinguished using allelic frequencies of isozyme loci. Breeding lines and hybrids of many crops (maize for example) are highly inbred and homogeneous and have been readily distinguished using genotypic differences at isozyme loci (Cardy and Kannenberg, 1982). The relative ease by which cultivars of any species can be identified depends upon the isozyme uniformity within each line. Cultivars of species which have variable alleles at isozyme loci have been distinguished by comparing the allozyme frequencies of the cultivars (Nielsen, Ostergaard and Johansen, 1983). Of the few papers written about sugarbeet isozymes, only one dealt specifically with the use of isozymes for distinguishing cultivars (Itenov and Kristensen, 1985). There have also been papers about using isozymes to distinguish interspecific hybrids and monosomic addition lines (Oleo, Van Geyt, Lange and DeBock, 1986; Jung, Wehling and - Loptein, 1986). Most sugarbeet breeding lines and cultivars are heterogeneous due to their modes of pollination and hybrid seed production. This 41 42 heterogeneity should permit a group of unrelated plants to be easily distinguished if enough marker loci are examined. The ability to distinguish individuals could have a variety of uses in a breeding program. Some examples of these uses are: detecting pollen contamination in crosses, checking seed lot integrity, detecting mislabeling of seed, plants or tissue culture plates, and assessing the genetic diversity of a breeding population. The objective of this research was to determine if individual plants from seed or tissue culture could be distinguished by isozyme phenotypes or ”fingerprinting". WNW Germplasm A germplasm collection of individual plants was chosen which represented a diverse range of Beta vulgaris germplasm. This collection included plants sampled from sugarbeet breeding lines, mutant stocks, hybrid parents and a variety of Beta cultivars (Table 2.1). Plants were obtained from tissue culture stocks, or from random selections of single seedlings from a line. These plants were grown and maintained in the greenhouse where leaves were always available for sampling. A total of 32 different lines and 39 individuals were examined by starch gel electrophoresis. Only 1 individual was sampled for 30 of the lines, whereas, several individuals were sampled from the remaining 2 lines (EL 44 and FC 701/5). Isozyme phenotypes, for 5 loci and 2 enzyme systems, were recorded for almost every individual. Starch gel electrophoresis The banding patterns of all of the individuals were determined by gel electrophoresis using the procedures and stains described in Chapter 1. In addition to the enzyme stains which were previously described, two enzyme systems, isocitrate dehydrogenase (Idh) and glutamic dehydrogenase (Gdh), were also used. The stain for isocitrate dehydrogenase contained 100 ml 0.1 M tris-ECl pH 8.0 buffer, 100 mg MgClz, 600 mg DL-isocitrate acid, 40 mg NADP, 40 mg NBT and 2 mg PMS. Glutamic dehydrogenase was stained with 2 g L-glutamic acid, 20 mg NAD, 43 44 20 mg NBT and 1 mg PMS in 50 ml 0.1 M tris-ECl pH 8.0. 45 Table 2.1. Germplasm used in fingerprinting study Clone or Population Characteristics Source T4E57-4 trout leaf mutant J .C. Theurer, E. Lansing, MI 84MS-20 trout leaf mutant J .C. Theurer, E. Lansing, MI 4022-3 chlorina mutant J.C. Theurer, E. Lansing, MI M816-1 yellow root mutant J .C. Theurer, E. Lansing, MI 4E25-3 semi-dwarf mutant J .C. Theurer, E. Lansing, MI MO78-48 plaintain leaf mutant J.C. Theurer, E. Lansing, MI J-3 stigmoid mutant T. Xinoshita, Japan EL 36 Type O, monogerm USDA stock, E. Lansing, MI EL 40 multigerm USDA stock, E. Lansing, MI EL 44 Type O, monogerm USDA stock, E. Lansing, MI EL 45 Type O, monogerm USDA stock, E. Lansing, MI EL 48 monogerm USDA stock, E. Lansing, MI PC 506 Type 0, monogerm R. Zielke, Carrollton, MI PC 607 Type O, monogerm G.A. Smith, Ft. Collins, CO PC 708 Type O, monogerm R. Eecker, Ft. Collins, CO L53 some mangel background J .C. Theurer, E. Lansing, MI SP6822 multigerm USDA stock, E. Lansing, MI SP6926 Type 0 G. Coe, Beltsville, MD CMS B male sterile J .W. Saunders, E. Lansing, MI 80-111 Type O J .W. Saunders, E. Lansing, MI 80-60 Type 0 J .W. Saunders, E. Lansing, MI 138 spinach leaf source J .W. Saunders, E. Lansing, MI I436-3 Type O J .W. Saunders, E. Lansing, MI OA-l Owens Annual Tester J .W. Saunders, E. Lansing, MI FC 701/5 rhizoctonia resistance R. Eecker, Ft. Collins, CO F1003 low respiration D. Cole, Fargo, ND GWK mangel Cartons White Knight ‘ CEARD chard Fordhook Giant (Northrup King Seeds) EW table beet Early Wonder (Northrup King Seeds) WB 222 B. lomatogona M.E.Yu, Salinas, CA RESULTS AND DISCUSSION Preliminary experiments A variety of isozyme stains and electrophoresis buffer systems were examined to determine which ones fulfilled the criteria necessary for fingerprinting of individual plants. The criteria were: (1) the system and stain provided adequate resolution of the observed banding patterns, (2) the patterns contained enough variability to distinguish at least 2 individuals, and (3) the banding pattern of an individual was the same over a range of environmental conditions, leaf sizes and plant ages. The systems and enzyme stains which were examined are summarized in Table 2.2. Of all the systems and enzymes examined, only seven enzymes and.two buffer systems fulfilled the outlined criteria. A summary of the results of an isozyme survey of 32 diverse Beta genotypes is found in Table 2.3. As discussed in Chapter 1, crosses between a few of these genotypes were analyzed and loci were identified for four of the seven enzyme systems. These loci were Mdh-1, Mdh-3, Pgm-1, Pgi-l and Skdh-1. In addition to these loci, the enzymes ME, Idh and Gdh gave consistent, repeatable patterns which were used to distinguish several individuals. The frequencies of the banding genotypes among the 32 individuals were calculated for the four loci with more than one allele (Table 2.4). When these frequencies were calculated only one individual was randomly chosen from EL 44 and FC 701/5 to avoid possible skewing of the data. Since the 32 plants were not randomly selected, the frequencies in Table 46 47 omosswouoNnov ousuuuoouw :vH oooswaomsouu ouoamxo ousaousam uou oshnco swans o: unsounnmonm cuss no< oosuoaomu onoosamosamonn Nam onssomouuNnov ouoaousaw :vu unsusaoosawonmoozm awn omououmo uom omssowouvhnov «amass av: ooovuxouoa .uom ouosomouvhaov uuosoosawo:nmonnlo mac onssomouvanov Houuouoo :vm oossowouvhsov ousNoUNNm vNu ouncowouwhsov Honooum sv< omsaowouvhnov ouasamonmlelonoosaw moo oussomoqunov ouoauanm :vxm omovuuaon osaau osuusoa as; ououonnaosm osqasxao mx< snouosvou odousususum uHu soovuuoosam soon saunm snags—anon a: oossowouvmnov ouanaaonnlmlaouoomam vac noshuso no usowusu>ounn< w .NNNN .NNNNNN New :60NNN uen< .NNNN.N6NNN Nan sNNNuso ammo .omaa .soavoou was possum .Nvuoo “an use ol.ml.1002 5 10:1 1306* 1380 X 138D 49 47 962 7 6:1 8 Only 56 were transplanted. of the last 5 E08 seedlings transplanted, 4 were spinach. * second sowing F2 Progeny from 27 F1 individuals were also examined for spinach leaf segregation (Table 3.4). Only 7 out of 27 lines produced spinach leaf progeny. If the spinach leaf mutant is controlled by a single recessive gene, half of the F1 lines should have been carriers of the spinach leaf allele. For five of the 27 lines, however, less than 80 67 Table 3N4. Spinach leaf segregation in three separate sowings of F2 seed sown on 4.29.86 sown on 6.29.86 total # of EC # # sown # germ # sp # sown # germ # sp progeny 1-1 50 43 0 70 * 0 43* 1-6 50 43 0 0 - - 43 1-7 50 60 0 160 * 0 60* 1-20 50 51 0 0 - - 65 1-20@ 54 14 0 - - - - 1-22 50 51 0 155 116 0 252 1-22@ 105 85 0 - - - - 1-24 50 71 l 244 266 1 337 1-28 50 58 0 124 120 0 178 4-5 50 52 0 54 * 1 52* 4-6 50 63 0 87 126 0 189 4-7 50 79 0 136 184 4 486 4-7@ 104 223 10 0 - - - 4-7@ 50 96 2 0 - - - 4-8 50 62 0 79 108 1 170 4-9 50 59 0 220 326 32 385 4-10 50 57 0 230 - 519 14 576 4-14 50 74 0 97 * 0 74* 4-16 50 47 0 168 * 0 47* 4-18@ 110 255 0 0 - - 255 4-19 50 33 0 144 191 O 382 4-19@ 103 158 0 - - - - 4-20 50 24 0 0 - - 192 4-20@ 108 168 0 - - - - 12-9 50 55 0 86 131 0 317 12-9@ 109 131 0 - - - - 12-15 50 60 0 133 * 0 60* 12-16@ 100 128 0 0 - - 128 12-17 50 50 0 259 * 0 50* 12-21 50 51 0 96 * 0 51* 12-22 50 40 0 0 - - 40 12-25 50 75 0 0 - - 75 12-36 50 53 0 0 - - 53 12-37 50 46 2 53 184 6 230 9 seed from these lines were treated with 0A3 before they were sown on 12.26.86 * complete data not available 68 seedlings were available for spinach leaf segregation counts. This was not an adequate number to determine if the parent F1 was a mutant carrier. In addition, many of the F2 seedlings were deformed, died and could not be classified. This evidence indicated that although half of the lines may have been spinach carriers, underrepresentation of the spinach leaf seedlings caused misclassification of some lines. For all of the F1 and F2 progeny which segregated for spinach leaf, the normal to spinach ratio ranged from 6:1 to 264:1 (Table 3.5). It is difficult to explain why there was such a wide range of segregation ratios. Although the spinach leaf seedlings were definately weak and often inviable, this does not explain how'one line could give an 8:1 ratio and another give a 264:1 ratio. Table 3.5. Summary of spinach ratios from the Fl's and Fz's Line spinach normal germ 2 ratio fraction EC6* 3 44 942 15:1 .0638 E06 7 41 962 6:1 .1429 E08 5 51 1182 10:1 .0893 ECl-24 1 70 1422 69:1 .0143 ECl-24 1 265 1092 264:1 .0038 EC4-5 1 53 -- 52:1 .0189 EC4-58 3 243 2462 81:1 .0123 EC4-7 4 180 1352 44:1 .0222 EC4-7 2 94 982 47:1 .0208 EC4-70 10 213 962 21:1 .0448 EC4-8 1 107 1372 106:1 .0093 EC4-9 32 294 1482 8:1 .1111 EC4-10 15 504 2262 33:1 .0294 E012-37 2 44 922 21:1 .0455 E012-37 8 176 1202 21:1 .0455 Total 92 2136 -- 23:1 .0413 * lines listed more than once indicate different sowings @ treated with GA 69 The percent germination of all the lines was greater than 902, however, this was not a reliable indicator since all of the seed was multigerm. The health of the plant may have contributed to the variability of the segregation ratios. Some plants were infested with aphids and spider mites during seed set, therefore weak seeds may have been aborted in an effort to conserve energy for the healthier seed. If this were the case, the spinach leaf embryos may have been preferentially aborted causing skewed progeny ratios. The skewed ratio might also be a result of linkage of the spinach leaf locus to the self-incompatibility locus. A 202 recombination frequency between the loci would result in a 9:1, normal to spinach leaf ratio (Table 3.6). This coupled with poor germination and variable health.of the Fads during seed set might explain the observed range of skewed ratios. Table 3.6. Possible F2 phenotypes given 202 recombination between the spinach leaf and self-incompatibility loci Male gametes Parental types Recombinant types 81 Sf s1 SI 81 SI 81 Sf Female Gametes .8 incompatible .2 .4 51 Sf* .32 8181 Sfo -- --- .08 Slsl Sfo P .4 s1 81 .32 Slsl SfSl --- --- .08 slsl SfSl .1 $1 $1 .08 8181 SfSl ---- --- .02 $181 SfSl R .1 s1 Sf .08 Slsl Sfo --- --- .02 slsl Sfo * Sl,sl: spinach leaf types; Sf,Sl: self-incompatibility types Expected spinach ratio: .9 normal leaf : .1 spinach leaf 70 Summary Although the results were inconclusive, the spinach leaf dwarf mutant was probably caused by a recessive allele at one locus. This is the mode of inheritance of the five known sugarbeet dwarf mutants as well as most of the dwarf mutants in other species (Abegg, 1940; Owen and Ryser, 1942; Theurer, 1983; Pelton, 1964). None of the evidence argues against this possibility and much of it supports a recessive single locus mode of inheritance. ‘The spinach leaf mutant also appears to be GA insensitive although GA.treatment may slightly improve seed germination. BIBLIOGRAPHY BIBLIOGRAPHY Abegg, F.A. 1936. A genetic factor for the annual habit in beets and linkage relationship. J. Agric. Res. 53:493-511. Abegg, F.A. 1940. List of characters and gene symbols reported for the species Beta vulgaris L. Proc. Am. Soc. Sugar Beet Technol. 2:109-113. Allard, R.W. 1956. Formulas and tables to facilitate the calculaton of recombination values in heredity. Eilgardia 24:235-279. Ashton, 0.0. and A.W.E. Braden. 1961. Serum beta-blobulin polymorphism in mice. Aust. J. Bio. Sci. 14:248-253. Bernatzky, R., and S.D. Tanksley. 1986. Toward a saturated linkage map in tomato based on isozymes and random cDNA sequences. Genetics 112:887- 898. Cardy, B.J., 0.W. Stuber, and M.M. Goodman. 1980. Techniques for starch gel electrophoresis of enzymes from maize (Zea mazs L.). Institute of Statistics Mimeo Series No. 1317, North Carolina State University, Raleigh. Cardy, BJ. and L.W. Kannenberg. 1982. Allozymic variability among maize inbred lines and hybrids: Applications for cultivar identification. Crop Sci. 22:1016-1020. Cheliak, W.M., and LA. Pitel. 1984. Genetic control of allozyme variants in mature tissues of white spruce trees. J. Eered. 75:34-40. Goodman, M.M., Stuber, 0.W., Newton K. and E.E. Weissinger. 1980b. Linkage relationships of 19 enzyme loci in maize. Genetics 96:697-710. Goodman, M.M., Stuber, 0.W. and R.J. Newton. 1982. p. 53-60. Isozyme loci in Maize. In, W.F. Sheridan (ed.) Maize for Biological Research. Plant Molecular Biology Association, Charlottesvil 1e, Virginia. Goodman, M.M. and 0.W. Stuber. 1983. Maize p. 1-33. In, S.D. Tanksley and TJ. Orton (eds.), Isozymes in plant genetics and breeding, part B. Elsevier Science Publishers, Amsterdam. Gottlieb, L.D. 1981. ElectrOphoretic evidence and plant populations. Progress in Phytochemistry. 7:1-46. Eussey, 0., and A. Eepher. 1978. Clonal prOpagation of sugar beet plants and the formation of polyploids by tissue culture. Ann. Bot. 42:477-479. 71 72 Ignart, F. and N. F. Weeden. 1984. Allozyme variation in cultivars of Cucurbita pepo L. Euphytica 33:779-785. Itenov, K., and K. Kristensen. 1985. Cultivar distinction in sugar beet (Beta vuliaris L.) by electrophorectic genotype determination. Tidsskr. Planteavl 89:1-10. IUPAC-IUB Commission on Biochemical Nomenclature (CBN) Recommendations. 1976. Nomenclature of multiple forms of enzymes. Eur. J. Biochem. Jung, 0., P. Wehling, and E. Loptien. 1986. Electrophoretic investigation on nematode resistant sugar beets. Plant Breeding 97:39- 45. Kajanus, B. 1917. On the color variation of Beta. [In German]. 2. Pflanzenzucht. 5:357-372. Keller, W. 1936. Inheritance of some major color types in beets. J. Agric. Res. 52:27-38. Kevers, 0., M. Coumans. W. De Greef, M. Eoffinger and Th. Gaspar. 1981. Eabituation in sugarbeet callus: auxin content, auxin protectors, peroxidase pattern and inhibitors. Physiol. Plant. 51:281-286. Kevers, 0., M. Coumans. W. De Greef, M. Jacobs and Th. Gaspar. 1981. Organogenesis in habituated sugarbeet callus: auxin content and protectors, peroxidase pattern and inhibitors. 2. Pflanzenphysiol. Bd. Larsen, K. 1977. Self-incompatibility in Beta vulgaris L. 1. Four gametophytic complementary S-loci in sugar beet. Eereditas 85:227-248. Lehnhardt, L., M. Wiedmann, and G.Gunther. 1981. Activity and multiple forms of hudrolytic enzymes in germinating sugar beets (Beta vulgaris L.). Biochem. Physiol. Pflanzen 176:638—658. Levites, E.V. 1979. Genetic control of NADP dependant malic enzyme in the sugarbeet (Beta vulgaris L.). Dokl. Acad. Nauk. SSr. 249:215-217. Little, T.M., and FJ. Eills. 1978. Agricultural experimentation. John Wiley & Sons, Inc., New York. Liu, P.B.W. and J.B. Loy. 1972. Inheritance and morphology of two dwarf mutants in watermelon. Amer. Soc. Hort. Sci. 97:745-749. Markert, C.L. and F. Moller. 1959. Multiple forms of enzymes: tissue, ontogenetic and species specific patterns. Proc. Natl. Acad. Sci. .USA. 45:753-763. Maletskii, 8.1., and A.A. Konovalov. 1986. Inheritance of alcohol dehydrogenase in sugar beet. 1. Analysis of deviations from monogenic segregation. Genetics 1205-1211. translated from Genetika 21:1527-1534, 1985. 73 McMillin, D.E., and J.C. Scandalios. 1980. Duplicated cytosolic malate dehydrogenase genes in Zea mays. Proc. Natl. Acad. Sci. USA 77:4866- 4870. Monastreva, L.E., Reimers, F.E., and E.V. Levites. 1982. Enzyme variability in the inbred lines of sugar beet. translated from Doklady Akademic Nauk SSR., 1982. 264:722-724. Munerati, O. 1931. Inheritance of the annual tendency in the common cultivated beet. [In Italian]. Z.Zuchtung, Reike, A., Pflanzenzuchtung 17:84-89. Nielsen, 0., Ostergaard, E. and E. Johansen. 1983. Cultivar identification by means of isoenzymes. z. Pflanzenzuchtng. 94:74-86. Oleo, M., J.P.C. Van Geyt, W. Lange and Th.S.M. DeBock. 1986. Investigations on an interspecific hybrid involving three species of the genus Beta, with special reference to isozyme polymorphisms. Theor. Appl. Genet. 73:261-266. Owen, F.V. 1942. Inheritence of cross and self-sterility and self- fertility in Beta vulgaris. J. of Agric. Res. 64(12):679-698. Owen, F.V. 1945. Cytoplasmically inherited male-sterility in sugar beets. Jo Agric. Res. 71:423-440. Owen, F.V. and G.K. Ryser. 1942. Some Mendelian characters in Beta vulgaris and linkages observed in the Y-R-B group. J. Agr. Res. 65:155- 171. O'Malley, D.M., N.C. Wheeler, and R.P. Guries. 1980. A manual for starch gel electrophoresis. Univ. of Wiscosin Madison staff paper no. 11. Dept. of Natural Resources. Pelton, J.S. 1964. Genetic and morphogenetic studies of angiosperm single-gene dwarfs. Bot. Rev. 30:479-512. Phinney, B.O. 1956a. Growth response of single-gene dwarf mutants in maize to gibberellic acid. Proc. Nat. Acad. Sci. 42:185-189. Saunders, J.W. 1982. A flexible in vitro shoot culture prOpagation system for sugarbeet that includes rapid floral induction of ramets. 0r0p Science 22:1102-1105. Smith, G. 1980. Sugarbeet. p.601-616. In W.R. Fehr and E.E Eadley (eds.) Hybridization of crop plants. Am. Soc. of Agron. Inc. Madison, Wis. Smith, G. A. 1987. Sugarbeet. p.577-625. In W.R Fehr, (ed.) Principles of cultivar develOpment, Vol. 2, Crop species. Macmillan Publishing 001, NOYO Soost, R.K. 1959. Effects of gibberellic acid on genetic characters in two tomato lines. Bot. Gaz. 121: 114-118. 74 Spettoli, P., Cacco, 0., and 0. Ferrari. 1976. Comparitive evaluation of the enzyme multiplicity in a diploid, a triploid and a tetraploid suger beet variety. J. Sci. Fd Agric. 27:341-344. Spettoli, P., Bottacin, A. and G. Cacco. 1980. Correlazione tra polimorfismo isoenzimatico e parametri techologici in barbabietole da zucchero a diverso stato di plodial. a L'Industria Saccarifera Italians. Stuber, 0.W., and M.M. Goodman. 1983. Inheritance, intracellular localization, and genetic variation of phosphoglucomutase isozymes in maize (Zea mazs L.). Bioch. Genet. 21:667-689. Tanksley, S.D., D. Zamir, and C.M. Rick. 1981. Evidence for extensive overlap of sporophytic and gametophytic gene expression in Lycopersicon esculentum. Science. 213:453-455. Tanksley, S.D. and T.J. Orton. (eds.) 1983. Isozymes in Plant Genetics and Breeding, part B. Elsevier Science Publishers, Amsterdam. Theurer, J.C. 1968. Linkage tests for mendelian male sterility and other genetic characters in sugarbeets, Beta vulgaris L. Crop Sci. 8:698-701. Ting, I.P., Fuhr, I., Curry, R. and W.C. Zschoche. 1975. Malate dehydrogenase isozymes in plants: preparation, properties and biological significance. p. 369-384. In C.L. Markert (ed.) Isozymes, Vol. 2. Van Geyt, J.P.C.F., and E. Smed. 1984. Polymorphism of some marker enzymes of the sugarbeet (Beta vulgaris L.) investigated by polyacrylamide gel electr0phoresis and starch gel electrophoresis. z. Pflanzenzuchtg. 92:295-308. Wu, L., Earivandi, A.E., Earding, J.A. and W.B. Davis. 1984. Identification of Kentucky bluegrass cultivars with esterase and phosphoglucomutase isoenzyme markers. 0r0p Sci. 24:763-768. Yang, N.S. and J.G. Scandalios. 1975b. 95 novo, synthesis and developmental control of the multiple gene-control led malate dehydrogenase isozymes in maize scuttella. Biochem. Biophys. Acta. 384: "4411441114144