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This is to certify that the

dissertation entitled

Genetic Inheritance and Molecular Cloning of

Anthracnose Resistance Genes in Common Bean

presented by

Maeli Melotto

has been accepted towards fulfillment
of the requirements for

Ph.D. Plant Breeding and Genetics

degree in

 

 

 

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GENETIC INHERITANCE AND MOLECULAR CLONING OF
ANTHRACNOSE RESISTANCE GENES IN COMMON BEAN

By

Maeli Melotto

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Plant Breeding and Genetics Program
Department of Crop and Soil Sciences

1999

ABSTRACT

GENETIC INHERITANCE AND MOLECULAR CLONING OF ANTHRACNOSE
RESISTANCE GENES IN COMMON BEAN

By

Maeli Melotto

Resistance to anthracnose caused by Colletotrichum lindemuthianum appears to
be controlled by independent complex loci. New sources of resistance have been
identified and genetic and molecular characterization indicated that there are fewer loci
with multiple allelic series than expected. Two diverse loci conditioning resistance to
anthracnose in common bean (Phaseolus vulgaris L.) were investigated. Genes
conditioning resistance in the Andean bean cultivars Kaboon and Perry Marrow were
shown to be allelic to the Co-I gene present in Michigan Dark Red Kidney (MDRK). The
gene symbols Co-IZ and Co-13 were assigned to these alleles. Other putative alleles were
recognized at this complex locus. The second locus, Co-42, present in the Middle
American differential cultivar G 2333 was characterized at the molecular level. A RAPD
marker, OASl3950, tightly linked to the C0-42 was identified. A SCAR marker (SASlB),

developed from the OASl3950 RAPD marker, was dominant and polymorphic similar to

the original RAPD, and supported the tight linkage between the marker and the Co-42
allele. The markers were present in two other differential cultivars, not previously
characterized, and in four navy bean cultivars suggesting the existence of a gene family
for anthracnose resistance at or near the C0-4 locus. Molecular markers, tightly linked to
resistance genes, were used to facilitate the identification of an uncharacterized resistance
gene for which no discriminating race of the pathogen is currently known. The SASI3
marker was used as a starting point for cloning gene sequences associated with the C0-4
locus. A contig developed from genomic clones flanking the marker region revealed an
1110 bp open reading frame, named COK-4. The predicted COK-4 protein contains a
serine-threonine kinase domain with a highly hydrophobic membrane-spanning region.
Specific primers designed to amplify the COX-4 region, were used to clone and sequence
COK-4 homologs from different bean cultivars. Single nucleotide polymorphisms (SNP)
found between the homologous sequences were fiirther confirmed with three restriction
enzymes. Restriction patterns were polymorphic among three bean cultivars known to
possess different alleles at the Co-4 locus confirming the presence of different alleles at
anthracnose resistance loci. These results strongly suggest that the COX-4 gene is a
member of the complex Co-4 locus conditioning resistance to anthracnose in bean. COK-
4, a gene ortholog of the Pto in tomato, is the first disease resistance gene successfully
cloned from legumes. Molecular cloning of resistance genes should facilitate studies on

plant-pathogen interaction and ultimately facilitate genetic improvement of crop species.

DEDICATION

T o my husband, Jorge, for his unconditional love, friendship, and encouragement.

iv

ACKNOWLEDGMENTS

I would like to express gratitude to all individuals that in some way helped me to
achieve my doctoral degree:

Dr. James D. Kelly, my academic advisor, for his guidance, encouragement and
openness to discuss my research project at any time during my Ph.D. study.

Drs. Amy Iezzoni, David Douches, Ray Hammerschmidt, and George Hosfield,
the members of my guidance committee, for their helpfill advice during my studies and
for the time and effort spent on reviewing this dissertation.

The Department of Crop and Soil Sciences for two years of financial support and
the Brazilian Ministry of Education and Culture, CAPES for one year of financial
support.

This research was supported in part by the grant DAN 1310-G-SS-6008-00 from
the USAID Bean/Cowpea Collaborative Research Support Program and the Michigan
Agricultural Experimentation Station. Partial support was also obtained from CIAT,

Centro Intemacional de Agricultura Tropical, Cali, Colombia.

PREFACE

Chapter 1 was formatted and submitted to the scientific journal Euphytica.

Chapter 2 was published in Theoretical and Applied Genetics (1998) 96:87-94.

Chapter 3 was formatted and submitted to the scientific journal Molecular Plant
Microbe Interaction.

Appendix Al was published in Genome (1996) 39: 1216-1219.

Appendix A2 is an updated version of an article published in the Annual Report of
the Bean Improvement Cooperative (1998) 41 19-10.

Appendix A3 was published in the Annual Report of the Bean Improvement

Cooperative (1998) 41 :64-65.

vi

TABLE OF CONTENTS

LIST OF TABLES ................................................................................. ix

LIST OF FIGURES ................................................................................ xi

GENERAL INTRODUCTION ................................................................... 1
GENERAL OBJECTIVES .................................................................. 16
REFERENCES ................................................................................ 18

CHAPTER ONE:

AN ALLELIC SERIES AT THE Co-I LOCUS CONDITIONING

RESISTANCE TO ANTHRACNOSE IN COMMON BEAN .............................. 25
ABSTRACT ................................................................................... 25
INTRODUCTION ............................................................................ 27
MATERIAL AND METHODS ............................................................. 29
RESULTS ...................................................................................... 32
DISCUSSION .................................................................................. 38
REFERENCES ................................................................................. 41

CHAPTER TWO:

MARKER-ASSISTED DISSECTION OF THE OLIGOGENIC
ANTHRACNOSE RESISTANCE IN THE COMMON BEAN

CULTIVAR, G2333 ............................................................................. 43
ABSTRACT .................................................................................. 43
INTRODUCTION ........................................................................... 45
MATERIAL AND METHODS ............................................................ 47
RESULTS ..................................................................................... 50
DISCUSSION ................................................................................ 54
REFERENCES ............................................................................... 6O

vii

CHAPTER THREE:

THE COX-4 GENE IS PART OF A COMPLEX LOCUS
CONDITIONING RESISTANCE TO ANTHRACNOSE

IN COMMON BEAN .............................................................................. 63
ABSTRACT ..................................................................................... 63
INTRODUCTION .............................................................................. 65
MATERIAL AND METHODS ............................................................... 67
RESULTS ....................................................................................... 70
DISCUSSION ................................................................................... 81
REFERENCES .................................................................................. 85

APPENDICES:

APPENDIX A1: DEVELOPMENT OF A SCAR MARKER LINKED
TO THE I GENE IN COMMON BEAN .................................................... 89

APPENDIX A2: GENETIC CHARACTERIZATION OF
THE Co-7 GENE PRESENT IN THE BEAN CULTIVAR G 2333 ................... 102

APPENDIX A3: SCAR MARKER LINKED TO THE RUST DISEASE
RESISTANCE GENE Ur-5 IN COMMON BEAN ...................................... 109

viii

LIST OF TABLES

Table l Anthracnose differential series, resistance genes, host gene pool, and the
binary number of each cultivar ............................................................ 5

Table 1.1 Gene pool origin, described anthracnose resistance genes, and disease
reaction of bean cultivars inoculated with different races
of C. lindemuthianum .................................................................. 33

Table 1.2 Crosses, races, disease reaction, and generations used for the
genetic characterization of anthracnose resistance in Kaboon .................. 34

Table 1.3 Observed and expected segregation ratios of progenies for their reaction
to C. lindemuthianum in SxR crosses ............................................... 37

Table 1.4 Allelism test for the genetic characterization of the resistance to
C. lindemuthianum in Kaboon and Perry Marrow cultivars ..................... 37

Table 2.1 Chi-square analysis for OAS13950 RAPD marker locus segregating in
two F2 populations, two-point chi-square analysis, and linkage
estimates for the marker locus and the Co-42 resistant allele .................... 51

Table 2.2 SCAR primer sequences derived from the RAPD marker OASI3950
linked to the Co-42 allele in common bean ......................................... 51

Table 3.1 Chi-square analysis of the SASl3 marker locus segregating
in Black Magic/SEL 1308 F2 population, two-point
chi-square and linkage estimates for the marker locus and the Co-42
resistance allele ........................................................................ 71

ix

Table 3.2 Sequence of the specific primer set designed to amplify the COK-4 gene
in the vicinity of the Co-42 locus .................................................... 76

Table A1.1 SCAR primer sequences derived from the RAPD marker OW13690
linked to the I gene in common bean ............................................. 94

Table A1.2 Linkage analyses of common bean F2 populations segregating
for the molecular markers and the dominant 1 allele.
Data for OW13690 from Haley et al. 1994a ........................................ 95

Table A1.3 Presence (+) or absence (-) of the I allele and the SW13
marker in bean genotypes from Andean and Middle
American gene pools (Singh et a1. 1991) .......................................... 99

Table A2.1 Nine genotypes segregating in the SEL 1360 x G 2333 F2
population. Frequency, phenotype to race 521, and
presence/absence of the SASl3 marker were determined ..................... 104

Table A2.2 Disease evaluation on genotypes carrying anthracnose
resistance genes derived from G 2333. Six plants of each

genotype were inoculated .......................................................... 105

Table A3.1 Molecular marker, PCR file, and linkage between the
Ur-5 gene and the $119 marker ................................................... 110

Table A3.2 SCAR primer sequences derived from the RAPD marker
0119460 linked to the Ur-5 gene in common bean .............................. 111

Figure 2.1

Figure 3.1

Figure 3.2

LIST OF FIGURES

DNA amplification of eight bean cultivars using A) the OASl3950

RAPD marker with Stoffel DNA polymerase, B) the OASl3950 RAPD

marker with Taq DNA polymerase, C) the SASl3 SCAR marker.

Lanes (1) 100 bp DNA ladder, (2) Resistant DNA bulk (Co-42/Co-42),

(3) Susceptible DNA bulk (co-42/co-42), (4) SEL 1308 (Co-42/Co-42),

(5) SEL 1360 (co-42/co-42,Co—5/Co-5), (6) G 2333 (Co-42/Co-42,

Co-5/Co-5), (7) Catrachita (Co-6/Co-6), (8) TO (Co-4/Co-4),

(9) Black Magic (co-42/co-42) ........................................................ 52

Diagram of the genomic region containing the Co-42 locus. A) Linkage
map showing position of molecular markers. B) Conti g developed
from overlapping genomic clones. Arrows indicate COK—4 specific
primers and restriction sites are letter coded, B=BamHI, E=EcoRI,
H=HindIII, K=KpnI, Me=MseI, Mp=MspI. C) Detail of the COK-4
amino acid sequence showing color coded putative domains: red =
N-myristoylation site, green = N-glycosylation site, pink = CAMP

and cGMP-dependent protein kinase phosphorylation site, blue =
protein kinase ATP-binding region signature, underlined amino acids =
primary transmembrane region, waved underlined amino acids =
secondary transmembrane region, 0 = stop codon.

The original SAS13 marker site included COK-4 amino

acids 1 through 173 .................................................................... 72

Alignment from regions of similarity between the COK-4 protein

sequence and reported protein sequences. Identical amino acids are in red

and similar amino acids are in blue. a) COK-4 protein (GenBank

accession no. AF 1 53441); b) disease resistance protein

kinase Pto gil430992; c) serine/threonine protein

kinase Pto (Lycopersicon esculentum) gil 1 80925 7; d) putative

serine/threonine protein kinase, Fen gene (L. esculentum) gil557882;

e) TMK (Oryza sativa) gnllPID|e267533; f) extracellular S domain

of Brassica oleracea gnllPIDlel 172841 ............................................ 74

xi

Figure 3.3 Restriction analysis of the COX-4 amplified in several genotypes.
A) Undigested; B) Kpn I; C) Mse I; D) Msp I. Lane (1) 100-bp
DNA ladder, (2) SEL 1308 (Co-42/Co-42), (3) Heterozygous
resistant F2 plant (Co-42/co-42), (4) Homozygous resistant
F2 plant (C o-42/Co-42), (5) Homozygous susceptible F2
plant (co-42/co-42), (6) TO (Co-4/Co-4), (7) Black Magic
(co-42/c0-42) ............................................................................ 77

Figure 3.4 DNA sequence showing single nucleotide polymorphisms (SNPs) in red
underlined letters among three cultivars possessing different
COK-4 homologs, A) SEL 1308, B) Black Magic, and C) TO ................. 79

Figure 3.5 Southern analysis of EcoR I digested DNA hybridized with the
SASl3 marker. Lane (1) SEL 1308, (2) SEL 1360, (3) G2333, (4)
Cardinal, (5) TO, (6) Seafarer. Molecular weight is
indicated in kb .......................................................................... 80

Figure A1.1 DNA amplification of ten bean cultivars using the
SW13 SCAR marker. The PCR product were detected
by direct ethidium bromide staining in microtiter plates
(top) and by gel electrophoresis (bottom). (1) N84004 (1/1),
(2) Michelite (i/i), (3) G91201 (1/1), (4) Alpine (i/i), (5)
Seafarer (1/1), (6) UI-114 (i/i), (7) Chinook (I/I), (8) Sierra (i/i),
(9) Montcalm (1/1), (10) negative control (no template
DNA was added to the PCR reaction), (11) molecular weight
marker (A Hind III/ EcoR 1; size of bands
indicated in base pairs) .............................................................. 96

xii

GENERAL INTRODUCTION

Anthracnose is a seed-bome disease of common bean (Phaseolus vulgaris L.)
caused by the hemibiotrophic fungus Colletotrichum lindemuthianum (Sacc. & Magnus)
Lams.-Scrib., that is found particularly in sub-tropical and temperate zones. Temperature
and humidity are the most important environmental factors for anthracnose infection and
expression of symptoms (Pastor-Corrales and Tu 1989). Spores, produced in acervuli, are
surrounded by a water-soluble gelatinous matrix. Temperature ranging from 18 to 22°C is
the optimum for spore formation and moderate rainfall and wind are essential for spore
dissemination. The rainfall dissolves the water-soluble matrix and the wind carries the
spores splashed by the rain. Relative humidity of at least 92% and free water on the
foliage for 12 hours are required for spore germination (Tu 1982). These environmental
conditions are related with the physiological needs of the pathogen.

Anthracnose symptoms initially appear on the lower surface of bean leaves and
petioles as angular, dark, brick red lesions, which become dark brown to black. On the
leaf veins, lesions appear as a slightly sunken canker. Lesions also appear on branches,
main stems, and pods. Infection of pods produces flesh to rust-colored circular spots and
the margin of the lesions is slightly raised. The center of the lesion contains a pink mass

of spores in acervuli, and as the pod matures, these spore masses dry to gray, brown, or

black granulations. Plants in all stages of growth are subject to anthracnose. Infected
seeds may display black cankers (Pastor-Corrales and Tu 1989).

Several control measures can be taken for control of anthracnose in the short-
terrn. High priorities are given to selection of resistant cultivar, crop rotation, seed
quality, and pesticide application. Crop rotation is important because spores can survive
on plant debris for 12-18 months (Tu 1992). After harvest infected bean debris has to be
incorporated into the soil, and beans should be in rotation with other crops for at least two
years. Seed is the major source of primary inoculum. Thus, production of clean seed of
cultivars resistant to the prevalent races of anthracnose in semi-arid regions and
distribution to growers through a certified seed program are essential. Colletotrichum
lindemuthianum can survive at least 5 years on pods and seeds that were air-dried and
stored at 4°C (Tu 1983). Infected seed can be treated with fungicides such as benomyl or
thiophanate methyl to effect partial control. Seed treatment can reduce disease incidence
down to 5%. Use of fungicides can prevent or reduce anthracnose if they are applied
early in the epidemic and coverage is thorough. Critical growth stages for fimgicide
application are 15 days after plant emergence and prior to flowering. Early infections
may result in complete yield loss because secondary cycles of the disease are the most
important for disease progress. Usually infection has to be monitored by the appearance
of symptoms prior to scheduling a fungicide spray. Continued use of fungicides may
favor the development of resistant biotypes of the pathogen (Pastor-Corrales and Tu
1989). To prevent disease spread, activity should be restricted in bean fields when plants
are wet. The movement of animals, humans, and equipment can disperse spores,

especially in fields planted under high-density (Pastor-Corrales and Tu 1989).

Complete yield loss can occur depending on weather conditions, susceptible
cultivars, agronomic poor management of the crop, and disease pressure (Singh et al.
1991b). In the 1950's and early 1960's, anthracnose was a serious problem in Michigan
causing severe yield loss. Efforts to control this disease were concentrated on the use of
disease-free certified seed produced in semi-arid regions and the development of resistant
cultivars through breeding programs. Although under control, anthracnose can always
become a problem because of the pathogen variability and the potential for new races to
appear. Thus, special attention should be paid at the appearance of symptoms in the field
and attempts to detect races occurring in the field prior to onset of epidemic potential. In
1976, an outbreak occurred in Ontario that affected yield, seed quality, and marketability
of beans (Tu et a1 1984). The origin and appearance of new races remains unknown.
Outbreaks of anthracnose have been reported in Michigan and Ontario in 1993 and new
races of the causal organism, C. lindemuthianum have been identified (Kelly et al.
1994a). In 1997, the same race reappeared in two Michigan locations, Bay County and
Montcalm County. Cyclic outbreaks are in part explained by the high variability of the
pathogen and the occurrence of newly evolved or introduced races, favorable weather

conditions, and grower laxity in crop rotation and seed selection.

Pathogen Variability

Isolates of C. lindemuthianum, known to exist in all continents, can be classified
into distinct physiological races (Pastor-Corrales and Tu 1989). Although races of C.
lindemuthianum were first described in the beginning of the 20th century (Barrus 1911,
1918), initially pathogenic variability was not thought to be great since sexual
recombination is rare in the fungus population and dispersal over great distance is limited
(Beebe and Pastor-Corrales 1991). However, in the last decade many new races have
been identified in bean-growing countries throughout the world (Menezes and Dianese
1988, Pastor-Corrales and Tu 1989, Tu 1994, Kelly et al. 1994a). The full extent of the
pathogen variability was difficult to assess because research groups in many countries
have used local codes rather than the original Greek letters to identify races. Despite the
tentative equivalency of described races in different countries, data collected using local
hosts as differentials has limited the knowledge of the variability present within C.
lindemuthianum, worldwide. A standard differential series of 12 bean cultivars of diverse
origin and a binary system based on the position of each cultivar within this series were
established (Table 1) to better understand and classify the variability and structure in C.
lindemuthianum populations (Drifhout and Davis 1989, Pastor-Corrales 1991). The
adoption of this standard procedure allows comparison and compilation of data from
different research groups and a fuller characterization of the wide variability present in C.
lindemuthianum. The re-characterization of races previously assigned Greek letters or

local codes suggests an overestimation of variability reported for this pathogen.

Table 1. Anthracnose differential series, resistance genes, host gene pool, and the binary

number of each cultivar.

 

 

Differential Cultivar Host GenesT Gene PoolII Binary Number"I
Michelite -- MA 1
MDRK Co-1 A 2
Perry Marrow C 0-13 A 4
Cornell 49242 C o-2 MA 8
Widusa -- A 16
Kaboon Co-12 A 32
Mexico 222 Co-3 MA 64
PI 207262 -- MA 128
TO Co-4 MA 256
TU Co-5 MA 512
AB 136 Co-6 MA 1024
G 2333 C0-42, Co-5, Co-7 MA 2048

 

I Host resistance genes. Not all resistance genes have been characterized.

" MA — Middle American gene pool, A — Andean gene pool (Singh et al 1991a).

I" Binary number: 2", where n is equivalent to the place of the cultivar within the series.
The sum of cultivars with susceptible reaction will give the binary number of a specific

race (Pastor-Corrales 1991). E. g., race 17 = virulent on Michelite ( 1) + Widusa (16).

Combining virulence and molecular analyses has led to a better understanding of
the variability present in C. lindemuthianum. Molecular analysis of entire genomes can
reveal the extent of variability in one species. Random Amplified Polymorphic DNA
(RAPD) markers have been used to identify isolates of C. lindemuthianum (Balardin et
al. 1997). Sequence analysis of specific regions of the genome, such as ribosomal DNA,
is a powerful approach and complements studies on intraspecific diversity (Melotto et a1.
1999). A specific set of primers, PN3 and PN10, was developed to amplify the rDNA
region between the 188 and 28S genes in C. lindemuthianum, that includes Internal
Trancribed Spacer (ITS) 1, 5.88 rRNA gene, and ITS 2 (Fabre et a1. 1995). Polymerase
Chain Reaction (PCR) amplification of this region produced a single reproducible 580 bp
DNA fragment in all 57 isolates of C. lindemuthianum including C. orbiculare (Balardin
et al. 1999). Digestion with the endonucleases Hae III and Msp I generated two patterns
(F abre et al. 1995, Sicard et a1. 1997, Balardin et al. 1999). Polymorphism in rDNA does
not appear to be linked to a specific virulence phenotype or structured with the
geographic origin of races or host gene pool (Fabre et al. 1995). A variable level of
genetic similarity among races and the lack of association of geographic origin with
polymorphism in the rDNA region is clear evidence of the high level of molecular
variability within C. lindemuthianum. In addition, insufficient support for parallel
evolution of C. lindemuthianum and its host P. vulgaris is indicated by the high sequence
similarity of the ITS region among Andean and Middle American races (Balardin et al.
1999). For instance, genetic distance among races belonging to different gene pools did

not exceed the genetic distance among races of the same gene pool (Balardin et a1. 1999).

Intra-race polymorphism, determined using RAPD and AFLP (Amplified
Fragment Length Polymorphism) markers, was observed within several isolates of
different races of C. lindemuthianum collected in different countries (Balardin et al. 1997,
Sicard et al. 1997, Gonzales et al. 1998). For instance, isolates of race 65, characterized
in the United States, showed a different RAPD pattern, whereas isolates from Brazil were
monomorphic (Balardin et al. 1997). Isolates of race 0, collected in Mexico, showed
distinct AF LP patterns (Gonzales et al. 1998) and isolates of races 7, 17, 31, and 73 from
different countries showed polymorphism (Balardin et al. 1999). The intra-race
polymorphism observed using molecular markers suggested a high level of molecular
variability within C. lindemuthianum and emphasized the limitation of virulence analysis
for assessing variability. The significance of such intra-race variability on pathogen
population structure is poorly understood, but it suggests independent evolution of

specific virulence phenotypes, such as race 73, in different geographic regions.

Host Genetic Resistance

Although unproven, resistance to anthracnose is likely to follow the gene-for-gene
theory (Flor 1947). Resistance genes have been described in common bean (Melotto et al.
1999), but avirulence genes were never isolated or studied in C. lindemuthianum. Race-
cultivar specificity is well known in the P. vulgaris/C. lindemuthianum pathosystem (Tu
1992) and hypersensitive reaction (HR) is characteristic of the incompatible interaction.

Morphological, biochemical, and molecular markers confirm the existence of a
Middle American and an Andean gene pool within P. vulgaris (Gepts 1988, Singh et al.

1991, Haley et al. 1994b). These gene pools originated from divergence of the ancestral

population prior to domestication. Unique molecular and biochemical markers detected
within wild bean populations from northern Peru and Ecuador, suggest the presence of an
ancestral third gene pool within P. vulgaris (Kami et a1. 1995). Host resistance genes are
classified as either Middle American or Andean depending on the gene pool origin of the
host cultivar. Races of C. lindemuthianum are similarly classified as either Middle
American or Andean depending on the gene pool of the host cultivar for which each was
isolated. Races of C. lindemuthianum virulent to Middle American hosts show greater
diversity in pathogenicity by attacking gerrnplasm from both gene pools, whereas races
virulent to Andean hosts are mostly pathogenic on Andean gerrnplasm (Beebe and
Pastor-Corrales 1991, Balardin and Kelly 1998). Races alpha (race 17) and delta (race
23) were found to be more pathogenic on small-seeded, indeterminate (Middle American
gene pool) cultivars, whereas races beta (race 130) and gamma (race 102) were
pathogenic on large-seeded, determinate cultivars (Andean gene pool). Reciprocal
selection of resistance genes in P. vulgaris and virulence genes in C. lindemuthianum
might occur naturally.

To date, six independent dominant genes, which confer resistance to anthracnose
in common bean, have been characterized. The Andean A gene reported by Burkholder
(1918) and recently renamed Co-I (Kelly and Young 1996), and five Middle American
genes, Are (Co-2) (Mastenbroek 1960), Mexique 1 (Co-3), Mexique 2 (Co-4), Mexique 3
(Co-5) (Fouilloux 1979), and Co-6 (Young and Kelly 1996a).

The Andean Co-l gene was the first major gene utilized to develop anthracnose
resistant cultivars of common bean (Burkholder 1923). Prior to 1978, the Co-I gene was

used as the only source of resistance in navy beans grown in Michigan and Ontario. After

the appearance of race delta (race 23) in Ontario (Tu 1988) and the United States (Goth
and Zaumeyer 1965), the Middle American Co-2 gene, characterized in a black bean
from Venezuela (Mastenbroek 1960), became the main source of anthracnose resistance
in the North America. The identification of race 73 in Michigan and race alpha-Brazil
(race 89) in Ontario, however, limited the further utilization of the Co-2 gene (Kelly et al.
1994a, Tu 1994). Combining the Co-I and Co-2 genes in single cultivars was suggested
as the best short-terrn protection against all known races currently present in North
America (Kelly et al. 1994a, Young and Kelly 1996a).

The Co-2 gene was the first predominant source of resistance to be used
worldwide. The appearance of races 31, 63, and 89 virulent to the Co-2 gene, however,
emphasized the need for new sources of resistance (Kruger et al. 1977). In France, new
resistance genes Co-3 (Mexique I), Co-4 (Mexique II) and Co-5 (Mexique III) were
characterized in a collection of bean germplasm from Mexico (Fouilloux 1979). The C0-
4 and Co-5 genes conferred resistance to current races virulent to the Co-2 gene, whereas
the Co-3 gene was overcome by race 89. Virulence of C. lindemuthianum has been
monitored in different countries, and evolving races have continually overcome resistant
gerrnplasm. Durability of resistance depends on how efficiently resistance against the
newest race can be incorporated into commercial gerrnplasm as new predominant

virulence phenotypes evolve within the pathogen population.

Breeding for Anthracnose Resistance

For long-term control of bean anthracnose, development of resistant cultivars is
the only practical approach. Sources of anthracnose resistance in different bean genotypes
are well known. There is resistance in navy, black, and kidney bean gerrnplasm. Other
bean classes such as pinto, great northern, small reds, pinks, and cranberries have little or
no resistance to anthracnose. Resistance from Andean kidney beans was incorporated in
Middle American bush navy beans including Sanilac, Seafarer, Gratiot, Kentwood,
Fleetwood and black beans such as Raven and is still effective in North America afier 20
years. New races, however, have been isolated from Isabella (light red kidney),
Blackhawk (black) and Aztec (pinto) that overcome this resistance (Kelly et al. 1994a).
Cultivars, such as Isles and Newport (Kelly et al. 1994b, 1995) that are resistant to all
current races in Michigan, have been recently developed. These cultivars have combined
sources of resistance from kidney and black beans.

The information on variability in C. lindemuthianum and the specialization of
specific races on one host gene pool is invaluable in breeding for resistance. Numerous
studies have indicated that resistance to C. lindemuthianum in common bean is controlled
by major genes acting singly (Young and Kelly 1996a), as duplicate or complementary
factors (Cardenas et al. 1964, Muhalet et al. 1981) or as members of an allelic series
(Fouilloux 1979, Young et al. 1998, Melotto and Kelly 1999). The most resistant
differential cultivar, G 2333, first thought to possess two dominant genes (Pastor-
Corrales et a1. 1994), was shown to possess three independent genes Co-42, Co-5, and
Co-7 (Young and Kelly 1997, Young et al. 1998). Pyramiding major resistance genes

may be the most appropriate breeding strategy for long-term resistance in P. vulgaris

10

(Young and Kelly 1997). Knowledge of gene complementarity has been suggested as a
strategy to improve the efficiency of pyramiding genes for durable resistance (Duvick
1996). If pathogens were specialized on one of the host gene pools, pyramiding resistance
genes from different gene pools may provide a more durable anthracnose resistance
(Young and Kelly 1997). For instance, the incorporation of Andean resistance genes in
bean breeding populations in Honduras where Middle American races predominate; or
Middle American resistance genes in gerrnplasm in the Dominican Republic where
Andean races prevail could result in more durable resistance in each country (Balardin
and Kelly 1998). The reduced virulence of isolates from Andean regions compared to
isolates from Middle American regions suggests that deployment of resistance genes
between gene pools should extend host resistance (Gepts 1988, Balardin and Kelly 1998).

Despite the large number of studies indicating that resistance to anthracnose is
controlled by either major dominant (Kelly and Young 1996a) or recessive resistance
genes (Cardenas et al. 1964, Muhalet et al. 1981, Alzate-Marin et a1. 1997), other
resistance mechanisms have been reported (Pastor-Corrales et al. 1985). The cultivar,
ICA Llanogrande is susceptible to anthracnose in seedling assays, but is resistant to the
same isolates under field conditions in widely different geographic regions of South
America and Africa. Under controlled greenhouse testing, plants of ICA Llanogrande
exhibit progressively greater resistance with age, but the mechanism or mode of
inheritance is unknown (Beebe and Pastor-Corrales 1991). Under field conditions, the
Brazilian cultivar Rio Negro, which carries the defeated Co-2 gene, exhibits resistance

against races of C. lindemuthianum to which it is known to be susceptible in seedling

ll

assays. The possibility of combining nonspecific resistance with major gene resistance

for anthracnose may, therefore, exist (Beebe and Pastor-Corrales 1991).

Marker Assisted Selection

Selection for cultivars carrying pyrarnided genes is most effective when indirect
selection assisted by the use of molecular markers tightly linked to the gene of interest is
practiced. The efficient pyramiding of epistatic resistance genes with the aid of linked
molecular markers has been demonstrated in common bean (Kelly and Miklas 1998).
Marker-assisted selection (MAS) can only be practiced if robust and reliable markers are
available (Melotto et al. 1996). Choice of the marker for MAS mostly depends on the
ease of the analysis. PCR-based markers are the common choice because only a small
amount of DNA is needed in addition to be cost-effective. More elaborate markers such
as RFLP, AF LP, usually require well-equipped laboratories and are time-consuming.
However, these markers, once identified, can be partially sequenced to design specific
primers and the marker can be transformed into a sequence characterized amplified
region (SCAR) marker. SCAR markers are usually dominant in nature and a quick
minus/plus assay can be applied to detect the PCR product. As suggested by Gu et a1.
(1995), ethidium bromide is added to the amplification reaction and strong fluorescence
is observed in samples containing amplified DNA, whereas weak fluorescence is
observed in samples in which no fi'agment is present. Direct staining with ethidium
bromide can be used in breeding programs when genotyping a large number of samples is
required, to facilitate the development of more disease-resistant cultivars using marker

assisted breeding (Gu et al. 1995, Melotto et al. 1996).

12

Genetic maps are the source of markers used to locate single or quantitative trait
loci (QTL; Adam-Blondon et al. 1994). Morphological and agronomic traits present in
the bean linkage map include the I gene for BCMV resistance, flower and seed color
genes (V allejos et al. 1992, Nodari et al. 1993a), a seed size gene (Vallejos and Chase
1991), the Co-2 gene for anthracnose resistance, the M38 gene conferring male sterility,
the SGou gene for pod-shape character (Adam-Blondon et al. 1994), and the QTLs
related to common bacterial blight and nodule number (Nodari et al. 1993b). Three
linkage maps of common bean are available (Vallejos et al. 1992, Nodari et al. 1993a,
Adam-Blondon et al. 1994) based on RF LPs, RAPD, isozymes, proteins, and phenotypic
markers, but none of these maps is completely saturated and the number of linkage
groups does not correspond to the number of bean chromosomes. In a recent publication
(Freyre at al. 1998), the three bean linkage maps were integrated to form a complete,
more accurate map of common bean based on different types of markers. Now the
number of linkage groups corresponds to the number of chromosomes in common bean.
The current bean linkage map still has limited genetic information on agronomic traits
usefill for breeders. However, breeders who want to tag a specific locus may use the
markers available in the map to screen their populations.

Many loci important for breeding have been tagged using bulked segregant
analysis (Michelmore et al. 1991). This technique allows rapid identification of tightly
linked markers useful for MAS. For instance the I gene has been tagged with the RAPD
marker OWl3690 (Haley et al. 1994a), which was later transformed into the SCAR
marker SW13 (Melotto et a1. 1996). Markers of the bean genome must be tested in a

collection of common bean genotypes representing both Andean and Middle American

13

gene pools (Singh et al. 1991a) because markers may not be polymorphic across gene
pools or races within gene pools. The presence or absence of the SW13 marker
corresponded to the presence or absence of the I gene indicating broad utility of this
marker across bean gene pools. In previous gerrnplasm surveys of common bean, certain
RAPD markers have been shown to be very useful as tools for indirect selection in
populations derived from both gene pools (Haley et al. 1994c, Young and Kelly 1996b),
whereas the application of other markers was restricted to a specific gene pool (Miklas et

al. 1993) or to a certain race within a gene pool (Haley et al. 1993).

Map-Based Cloning of Resistance Genes

Knowledge of gene location in the plant genome allows the isolation of that
particular gene and in depth study of resistance gene function. Isolation of disease
resistance genes is not only important for direct transformation of susceptible cultivars,
but also to understand the mechanisms of resistance, which may lead to novel approaches
for disease control. Several approaches exist to clone disease resistance genes. In the
early 1990’s, the Cf-9 gene, which confers resistance to Cladosporium fulvum in tomato,
was isolated by transposon tagging (Jones et al. 1994). An alternative approach is to use a
tightly linked molecular marker for chromosome walking to the gene. Map-based cloning
has been used to isolate the Pto gene of tomato (Martin et al. 1993). Since then, many
other resistance genes have been cloned from different plant taxa. Some examples are
Xa21 of rice (Song et al. 1995), RPSZ and RPM] of Arabidopsis (Bent et al. 1994,
Mindrinos et al. 1994, Grant et al. 1995), N of tobacco (Whitham et al. 1994), and L6 of

flax (Lawrence et al. 1995).

14

The classic ‘gene-for-gene’ theory proposed by Flor (1947) explains that a
complementary pair of dominant genes, one in the host and another in the pathogen, is
necessary for resistance to occur. This model suggests a receptor-like function for the
resistance gene product, whereas the avirulence gene product would be the ligand. Afler
50 years, Flor’s theory has been shown to exist at the molecular level in the
tomato/Pseudomonas syringae interaction. The Pto protein directly binds to the avrPto
protein triggering the defense reaction (Scofield et al. 1996, Tang et al. 1996). Analysis
of proteins encoded by cloned resistance genes has revealed a striking similarity and
these proteins appear to have key role in plant/pathogen interaction (Bent 1996).
Independent of the plant taxa or the pathogen involved, resistance gene-encoded proteins
possess a few conserved functional domains, leucine rich repeats (LRR), nucleotide
binding sites (NBS), leucine zippers (L2), and protein kinases (Hammond-Kosack and
Jones 1997). Each resistance gene may encode. one or more domains. For instance, Pto
encodes a serine threonine kinase (STK) and RPM] encodes a three-domain protein with
LRR, NBS and L2 consensus sequences. Leucine-rich repeat proteins could be
responsible for recognition specificity as it can bind corresponding ligands from the
pathogen. Nucleotide binding sites, L2 and kinases may be involved in signal
transduction pathways to activate gene transcription. These domains are usually involved
in activating other proteins, which may trigger a chain of events leading to defense
response (Bent 1996, Hammond-Kosack and Jones 1997).

Based on the characteristics of disease resistance genes, several research groups
have been using the DNA sequence information of conserved regions to map and isolate

disease resistance related genes from other plant organisms. In common bean and

15

soybean, resistance genes analogs (RGAs) were identified using primers specific to
conserved regions of known resistance genes and mapped close to known disease
resistance locus (Kanazin et al. 1996, Yu et al. 1996, Geffroy et al. 1998, Rivkin et al.
1999). Mapping of RGAs has been the only approach used to isolate known resistance
gene sequences from common bean. RGAs, however, are not always closely associated
with a resistance phenotype and may be loosely linked to known resistance locus limiting
their value in chromosome walking to the gene. Additional approaches, such as map-

based cloning are still needed to isolate resistance gene candidates in crop species.

GENERAL OBJECTIVES

Genetic inheritance of anthracnose resistance is well studied. Major dominant
resistance genes are involved in controlling this devastating disease and have been
extensively used in breeding programs worldwide. However, the fimgus C.
lindemuthianum, the causal agent of anthracnose, is highly variable and new pathogenic
types that overcome cultivar resistance continue to evolve. Therefore, breeders always
need new sources of resistance as well as a fast and reliable approach to incorporate this
resistance into elite breeding lines. In this dissertation, the objectives were to characterize
new Andean sources of anthracnose resistance, to identify molecular markers (RAPD and
SCAR) tightly linked to the Co-4 locus, and use these markers as a starting point for

chromosome walking towards the gene.

16

In order to successfully achieve these objectives, the research project was divided
into three main sections, which are described in each chapter of this dissertation. The
objective in Chapter 1 was to study the genetic inheritance of the anthracnose resistance
present in the bean cultivars Kaboon and Perry Marrow and describe the relation of this
resistance to other independent loci conditioning resistance to anthracnose. The
objectives in Chapter 2 were to find a RAPD marker tightly linked to the Co-42 gene
present in the bean breeding line SEL 1308 and transform the marker into a SCAR. This
molecular marker would be used to dissect the genetic resistance of G 2333, which
carries three anthracnose resistance genes, Co-42, Co-5, and Co- 7. The first two genes
have been isolated in two different bean lines and the Co-7, however, could not be
isolated because it is hypostatic to the Co-42 gene. Tagging the Co-42 was the only
approach to further characterize the Co-7 gene. The objectives in Chapter 3 were to fine
map the Co-42 gene using a large F2 segregating population and clone DNA sequences
involved in disease resistance. The Co-42 gene is a valuable resistance gene as it confers
resistance to many highly virulent races of anthracnose. Once cloned, Co-42 could be
used to transform susceptible bean cultivars to rapidly improve genetic resistance to

anthracnose.

l7

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24

CHAPTER ONE

AN ALLELIC SERIES AT THE Co-I Locus CONDITIONING RESISTANCE To

ANTHRACNOSE IN COMMON BEAN

ABSTRACT

In this study, the genetic resistance of the Andean bean cultivar Kaboon and itS
relation to other sources of anthracnose resistance in common bean was characterized.
Based on the segregation ratio (3R: 1 S) observed in two F2 populations it is concluded that
Kaboon carries one dominant gene conferring resistance to races 7 and 73 of
Colletotrichum lindemuthianum. This gene in Kaboon is independent from the Co-Z gene
and is an allele of the C o-I gene present in Michigan Dark Red Kidney (MDRK). Support
for allelism is based on the differential reaction of these genotypes to a range of
anthracnose races. Therefore, the symbol C 0-1 2 for the major dominant gene in Kaboon is
proposed. Among the Co anthracnose resistance genes characterized in common bean, the
Co-I is the only gene of Andean origin. When inoculated with a less virulent race, the
Andean race 5, segregation ratio in the F 2 progeny of Cardinal and Kaboon was 57R:7S
(p=0.38) indicating that Kaboon may have other dominant resistance genes with
complementary mode of action, since Cardinal possesses no known genes for anthracnose
resistance. Therefore, at least two of the three dominant genes segregating in this cross

must come from Kaboon. Perry Marrow, a second Andean genotype which conditions

25

resistance to a different group of races, appears to have another resistance allele at the Co-
] locus and the gene symbol Co-13 was assigned. In R x R crosses between Perry Marrow
and MDRK or Kaboon, no susceptible F2 plants were found when inoculated with race
73. These findings have major implication in developing breeding strategies for durable
anthracnose resistance. If different alleles occur at the same locus, breeders may have
limited sources of Andean resistance genes available for gene pyramiding and many of

these genes may reside at the Co-I locus.

26

INTRODUCTION

The existence of Middle American and Andean gene pools within common bean,
Phaseolus vulgaris (L.) has been demonstrated based on morphological traits, phaseolin
types, isozymes, molecular markers, and lethality of Middle American/Andean hybrids
(Gepts 1988, Singh et al. 1991, Haley et al. 1994). Unique molecular and biochemical
markers detected within wild bean populations from northern Peru and Ecuador, suggest
the presence of a third ancestral gene pool within P. vulgaris (Kami et al. 1995).
Likewise, host resistance genes and races of Colletotrichum lindemuthianum (Sacc. &
Magnus) Briosi & Cav., the causal agent of anthracnose in common bean, are classified
as either Middle American or Andean depending on the gene pool origin of the host
cultivar. Races of C. lindemuthianum virulent to Middle American hosts Show greater
diversity in pathogenicity by attacking germplasm from both gene pools, whereas races
virulent to Andean hosts are mostly pathogenic on Andean germplasm (Balardin and
Kelly 1998, Balardin et al. 1997). In general, Middle American hosts are more resistant to
Andean races and Andean hosts are resistant to Middle American races. For instance, the
Co-I gene (Andean) conditions resistance to race 73 (Middle American) and the Co-2
gene (Middle American) conditions resistance to race 7 (Andean). Combining Co-I and
Co-2 genes in the same cultivar, is the best strategy to control these two highly virulent
races currently present in North America. Broad-based resistance to bean anthracnose,
therefore, can be achieved if both Andean and Middle American genes are combined in
the same cultivar (Young and Kelly 1996ab). However, Andean sources of anthracnose

resistance have not been studied as extensively as Middle American sources. There is

27

limited information available on the independence of the individual genes present in
Andean cultivars or on the relationship of these genes to previously characterized
anthracnose resistance genes. To date, only one Andean anthracnose resistance gene has
been described, the Co-I gene (Burkholder 1918), which is found in the kidney bean
cultivars Michigan Dark Red Kidney and Montcalm (Young and Kelly 1997).

New sources of anthracnose resistance must always be sought because of the high
variability in the pathogen population and the occurrence of newly evolved virulent races.
In a recent study, Balardin and Kelly (1997) found that the Andean differential cultivar
Kaboon is resistant to all but two isolates of C. lindemuthianun collected from bean fields
throughout the Americas. Muhalet et al. (1981) suggested that Kaboon carries one
dominant and two complementary factors conferring resistance to race beta, one
dominant complementary factor conferring resistance to race gamma, and two
complementary factors conferring resistance to race delta. These authors also suggested
that Kaboon does not carry the dominant Co-2 gene. Kaboon is susceptible only to races
55 (lambda), 63 (iota), 99 (theta), 102 (gamma), and 357, from among races presently
characterized (Tu 1994, Balardin and Kelly 1997).

There is no information available on the independence of the individual genes
present in the Andean differential cultivar Kaboon and Perry Marrow or on the
relationship of these genes to other previously characterized resistance genes. In this
chapter, the genetic characterization of anthracnose resistance present in Kaboon and its
relation to other Andean cultivars such as Perry Marrow is discussed. Evidence for

existence of an allelic series at the Co-l locus in common bean is presented.

28

MATERIAL AND METHODS

Genetic material. Several segregating populations were developed to determine
the inheritance and independence of the anthracnose resistance genes present in Kaboon.
Kaboon is a large-seeded white cultivar of Andean origin and was used as the male parent
of all populations. The female parents were the Andean genotypes Cardinal, Perry
Marrow and Michigan Dark Red Kidney (MDRK) and the Middle American genotypes
Cornell 49-242 and Flor de Mayo M38 (Table 1). Cardinal possesses no known gene for
anthracnose resistance, whereas MDRK carries the Co-I gene and Cornell 49-242 carries
the Co-2 gene. Perry Marrow and Flor de Mayo M38 possess resistance to certain races
of C. lindemuthianum, however anthracnose resistance gene(s) have not been previously
described in these cultivars. Perry Marrow was crossed with MDRK to assess the genetic
independence of the resistance genes present in these two cultivars. F2 populations and
their respective F2.3 families were used to determine inheritance and independence of
Andean genes for anthracnose resistance. For the test of allelism, six F2 populations were
developed using cultivars carrying independently characterized genes MDRK (Co-1),
Cornell 49-242 (Co-2), Mexico 222 (Co-3), SEL 1308 (Co-43, SEL 1360 (Co-5), and

Catrachita (Co-6) (Young and Kelly 1996a).

Races of C. lindemuthianum and inoculum preparation. Three races of C.
lindemuthianun were chosen based on the differential phenotypic reaction observed in
parental genotypes (Table 2), the Andean race 5 (collected in Peru) and race 7 (ATCC

96390), and the Middle American race 73 (ATCC 96512). Race 7 is highly virulent on

29

Andean cultivars such as Perry Marrow and MDRK, whereas race 73 is highly virulent
on Middle American cultivars. Kaboon is resistant to all races used in this study.
Reaction of the parents to each race is described in Table 1. Identification of each race
was confirmed by the reaction observed on the cultivar differential series (Melotto et al.
1999). Each race was grown from monosporic cultures maintained in fungus-colonized
filter papers stored at —20° C. To prepare the inoculum, fungal culture was grown by
using infected tissue incubated under sterile conditions on modified Mathur’s culture
medium containing dextrose (8g/L), MgSO,. 7 H20 (2.5g/L), KH2P04 (2.7g/L),
neopeptone (2.4g/L), yeast extract (2g/L), and agar (16g/L). Spores were re-plated and
used as the source of inoculum. All cultures were incubated for 10 days in complete
darkness at room temperature. Spore suspensions were prepared by flooding culture
plates with 0.01% Tween 20 in sterile distilled water and scraping the culture surface.

Inoculum concentration was adjusted to 1.0 x 106 spores/ml using a hemacytometer.

Disease phenotypic evaluation. Spore suspension of the pathogen was evenly
sprayed on 10 day-old seedlings at the primary leaf stage. After inoculation, seedling
were maintained in a mist chamber with relative humidity higher than 95% for 48 hours
at 22-25° C. Symptoms were evaluated seven days afier inoculation. Plants were rated as
resistant when no lesions were observed and rated as susceptible when enlarged lesions
were observed on the lower leaf surface and stem. Resistant and susceptible cultivars

used in this study presented extreme disease reactions to the chosen races.

Segregation ratios and statistical analysis. Segregation ratios tested in the F2

populations were 3R:lS for a single dominant gene, 15R:1S for two independent

30

dominant genes, and 57R:7S for a single dominant gene plus two complementary
dominant genes. In the F2,3 families, the segregation ratio tested was 12221 to discriminate
between homozygous and heterozygous resistant F2 plants. All observed ratios were
compared to the expected ratios using the Chi-square test for goodness of fit.

Probabilities (P values) are shown in Tables 3 and 4.

31

RESULTS

Gene pool origin of bean cultivars and their disease reaction to the races used in
this study are presented in Table 1. Cornell 49-242 and Flor de Mayo M38 were used to
determine the independence of the Andean gene(s) present in Kaboon from Middle
American genes. The Andean bean cultivars Cardinal and MDRK were used to determine
the number of genes present in Kaboon, whereas the cultivars Perry Marrow and MDRK
were used to determine the resistance relationship of genes in Kaboon to other Andean
genes. All crosses, generation tested, and races used for the allelism test or genetic
inheritance of segregating genes, are presented in Table 2. In all inoculation experiments,
no intermediary reaction was observed. Susceptible plants died and resistant plants
Showed no symptoms after seven days of inoculation.

The segregation ratio 3R:lS (p=0.34) observed in the Cardinal x Kaboon F2
population (Table 3) indicates that Kaboon carries one dominant gene conferring
resistance to race 7 of C. lindemuthianum. In a mixed inoculation with races 7 and 73, the
same 3:1 ratio was supported, suggesting that the same single dominant gene conditions
resistance to these two widely dispersed Andean and Middle American races. To
determine whether Kaboon has other resistance genes, the F2 progeny of Cardinal and
Kaboon was inoculated with a less virulent race, the Andean race 5. The observed
segregation ratio 57R:7S (p=0.38) indicates that Kaboon may have other dominant
resistance genes with complementary mode of action, since Cardinal possesses no known
genes for anthracnose resistance. Therefore, at least two of the three dominant genes

segregating in this cross must come from Kaboon.

32

Table 1.] Gene pool origin, described anthracnose resistance genes, and disease reaction

of bean cultivars inoculated with different races of C. lindemuthianum.

 

Races of C. lindemuthianum

 

 

Bean Cultivar Gene Poola Known gene 5 7 73
Cornell 49-242 MA Co-2 Rb S R
FM 38 MA unknown R R R
Cardinal A none S S S
Perry Marrow A Co-13 S R S
MDRK A C 0-1 R R S
Kaboon A Co-l’ R R R

 

‘ A= Andean; MA= Middle American (Singh et al. 1991).

b R= resistant reaction, 8: susceptible reaction.

33

Table 1.2 Crosses, races, disease reaction, and generations used for the genetic

characterization of anthracnose resistance in Kaboon.

 

 

Cross Race Reactiona Generationb
MDRK x Kaboon 73 R x R P, F2
MDRK x Kaboon 7 S x R P, F2, F2.3
Cornell 49-242 x Kaboon 7 R x R P, F2
Cardinal x Kaboon 5 S x R P, F2
Cardinal x Kaboon 73 S x R P, F2
Cardinal x Kaboon 7 + 73 S x R P, F2
Flor de Mayo M38 x Kaboon 73 R x R P, F2
Perry Marrow x Kaboon 73 R x R P, F2
MDRK x Perry Marrow 73 R x R P, F2

 

‘R = resistant reaction and S = susceptible reaction.

bParental, F2 plants, F2,3 families, respectively.

34

Upon inoculating the MDRK x Kaboon F 2 population with race 7 (S x R cross),
segregation in the F 2 generation again suggested the presence of a single dominant gene
in Kaboon conferring resistance to race 7. These data were confirmed in progeny tests in
the F2,3 families, where progeny genotypes segregated 1:2:1 (p=0.57). Allelism test was
conducted in the same F2 population using race 73 (R x R cross). No segregation was
observed in 174 F2 individuals inoculated, suggesting that the gene in Kaboon, which
conferred resistance to races 7 and 73, was allelic to the Co-I gene in MDRK. Clearly the
genes have to be allelic since one (MDRK) conditioned resistance to only race 73 while
the other (Kaboon) conditioned resistance to both races 7 and 73. Support for allelism is
based on the differential reaction of these genotypes to a range of anthracnose races.

Independence of the gene present in Kaboon was tested in the F2 population
derived from a cross between Cornell 49-242 and Kaboon. Inoculation with race 7 (R x R
cross) resulted in a segregation of 15:1 Q3=0.51) suggesting the presence of two
independent dominant genes, one the Co-2 gene in Cornell 49-242 and the other an allele
of the Co-I gene (Table 4). With the confirmation that these loci were independent and
that the gene in Kaboon is allelic to the Co-I gene, no further allelic tests were conducted
with other independent loci (Co-3 to Co-6). It is proposed to name the gene which
conditions resistance to race 7 and 73 in Kaboon as the Co-I’ allele. The other
complementary dominant genes in Kaboon do not appear to offer a greater level of
resistance to more virulent races of anthracnose. There was no evidence for recessive
resistance in any of the crosses studied.

The anthracnose resistance gene(s) present in the bean cultivar Flor de Mayo M38

have not been characterized genetically. This cultivar is resistance to races 7 and 73. F2

35

populations derived from a cross with Kaboon (R x R) was inoculated with race 73. A
segregation ratio 15:1 (p=0.7) was observed, indicating that each of the parents carry a
dominant independent resistance gene. This result supports the independence of the Co-1 2
allele (Kaboon) from other Middle American resistance genes. The anthracnose resistance
present in Flor de Mayo 38 could have originated from its Middle American background
(Acosta-Gallegos et a1. 1995).

The Andean cultivar Perry Marrow was crossed with MDRK (Co-1) and Kaboon
(Co-1") for allelism test. No segregation was observed in 152 or 143 F2 individuals from
each population (Table 4). These three Andean cultivars have different levels of
resistance to anthracnose. For instance, at least seven races including races 5, 357, 1165
attacks Perry Marrow but not MDRK (Balardin et al. 1997). Therefore, the Co-I allele
present in Perry Marrow is different from the one in MDRK. In addition, Perry Marrow
does not possess the Co-lz allele because Kaboon is more resistant than Perry Marrow
(Table 1). These results suggest the presence of another allele at the Co-I locus. The gene
symbol Co-I3 was assigned to the major gene present in Perry Marrow that confers

resistance to race 73.

36

Table 1.3 Observed and expected segregation ratios of progenies for their reaction to C.

lindemuthianum in SxR crosses. Kaboon (K) is the male parent of all crosses.

 

 

 

Phenotypic Evaluation in the Phenotypic Evaluation in the
F 2 (no. plants) F 23 (no. plants)
Cross Race Exp ratio R- rr P value RR Rr rr P value
MDRK x K 7 3:1 80 22 0.42 21 55 25 0.57
Cardinal x K 5 57:7 60 10 0.38
Cardinal x K 73 3:1 125 49 0.34
Cardinal x K 7+73 3:1 70 28 0.41

 

Table 1.4 Allelism test for the genetic characterization of the resistance to C.

lindemuthianum in Kaboon and Perry Marrow cultivars.

 

Phenotypic Evaluation in the F2 (no. plants)

 

 

Cross Race R- rr Expected ratio P value
MDRK x Kaboon 73 174 0 all R 1.0
Cornell 49-242 x Kaboon 7 55 5 15:1 0.51
Flor de Mayo M38 x Kaboon 73 9O 5 15:1 0.70
Perry Marrow x Kaboon 73 152 0 all R 1.0
MDRK x Perry Marrow 73 143 0 all R 1.0

 

37

DISCUSSION

In this chapter, genetic evidence for the occurrence of different alleles at the Co-I
locus, which conditions resistance to anthracnose, is presented. Therefore, the symbol
Co-I" is proposed for the major dominant gene present in the Andean bean cultivar
Kaboon, which condition resistance to races 7 and 73. The symbol Co-13 is proposed for
the resistance gene present in Perry Marrow. Occurrence of more than one resistance
allele at C0 loci is not unique. Two different resistance alleles at the independent Co-3
locus, for instance, confer resistance to different races of the pathogen C. lindemuthianum
(Fouilloux 1979). In a more recent study (Young et al. 1998), a second allele in SEL 1308
at the Co-4 locus was identified and it is the broadest-based source of resistance among
all Co genes identified to date (Balardin and Kelly 1998). As new sources of resistance
are identified and characterized, there is increasing evidence for new alleles instead of
new resistance genes. These results alert us to the need for allelism tests every time a new
resistance source is recognized. All characterized anthracnose resistance genes have been
isolated in different bean lines, which facilitates allelism tests without the interference of
other epistatic genes.

In two published studies (Cardenas et al. 1964, Muhalet et a1. 1981) reverse of
dominance in populations segregating at the Co-I locus was observed. Change of
dominance was not adequately addressed in these papers. Depending on the genotypes
involved in those crosses the reverse of dominance observed could be explained by a
multiple allelic series at that Specific locus. For instance, if a race such as beta (race 130)

possesses multiple virulence genes such that it is virulent to some alleles of the resistance

38

genes but not all the alleles at the Co-I locus, either 3R:lS or 1R:3S ratios would be
observed depending on the specific combinations of alleles segregating in the different
crosses. In an R x R cross between the cultivar Tuscola and Kaboon, two Co-I alleles
segregated and no susceptible plants were found (Muhalet et al. 1981). When Tuscola
was crossed with Montcalm (Co-1 gene) and the F2 population was inoculated with the
same race 130, a 1R:3S ratio was observed. These results indicate that Montcalm carries a
Co-I allele dominant over the one in Tuscola. Race 130 defeats the Montcahn allele but
not the Tuscola allele. Clearly a complex multiple allelic series exists at the Co-I locus.
Breeding for durable anthracnose resistance has focused on gene pyramiding.
Since resistance is conferred mostly by major dominant genes and each gene controls
certain races of the pathogen, the logical choice for broad-based resistance is to combine
genes based on their complementary reaction to the pathogen. Gene complementarity for
durable resistance is achieved by combining Andean and Middle American genes in the
same cultivar. Andean genes are most useful to improving Middle American cultivars and
in some locations breeders may not have many choices of Andean sources to improve
local bean cultivars. Our findings indicate that many Andean sources are allelic and the
major locus for anthracnose resistance is Co-I. Other cultivars such as A-193 (Mendoza-
Herrera et al. 1999), Perry Marrow, MDRK, Tuscola, Seafarer, Montcalm appear to
possess alleles at C o-1 locus as indicated by allelism tests, range of resistance, and DNA
analysis (Young and Kelly 1996c). These cultivars do not possess the OF10530 RAPD
marker, which is linked in repulsion to the Co-l locus (Young and Kelly 1997). The
choice of Andean resistance is among alleles not genes as they confer resistance to

different races. Breeders will have to identify local races and deploy the appropriate

39

resistance allele to control them. For instance, the Co-I" gene present in Kaboon is the
choice for the bean breeding program in Michigan as it confers resistance to both Andean
and Middle American races of anthracnose recently identified at that location.

Increasing evidence suggests that anthracnose resistance genes, once characterized
as single independent dominant genes, are complex loci and occur as allelic series and
multigene families in the bean genome. These recent findings have major implications
not only in breeding programs as discussed above, but also in agricultural biotechnology.
Two independent anthracnose genes, Co-2 and Co-4 have been studied at the molecular
level and they appear to be part of complex loci (Geffroy et al. 1998, Young et al. 1998).
These loci most often harbor multiple open reading flames and molecular cloning may be
laborious and time-consuming. If geneticists choose to use cloned gene(s) for the
transformation of susceptible cultivars, multiple genes must be used to achieve the

desired level of resistance.

40

REFERENCES

Acosta-Gallegos, J.A., Castellanos, J.Z., Nunez-Gonzales, S., Ochoa-Marquez, R.,
Rosales-Sema, R., and Singh, SP. 1995. Registration of ‘Flor de Mayo M38’
common bean. Crop Sci. 35: 941-942.

Balardin, RS and Kelly, J.D.1998. Interaction among races of Colletotrichum
lindemuthianum and diversity in Phaseolus vulgaris. J. Amer. Soc. Hort. Sci. 123:
1038-1047.

Balardin, R.S., Jarosz A., and Kelly, JD. 1997. Virulence and molecular diversity in
Colletotrichum lindemuthianum from South, Central and North America.
Phytopathology 87: 1184-1 191.

Burkholder, W.H., 1918. The production of an anthracnose-resistant white marrow bean.
Phytopathology 8: 353-359.

Cardenas, F ., Adams, M.W., and Andersen, A. 1964. The genetic system for reaction of
field beans (Phaseolus vulgaris L.) to infection by three physiologic races of
Colletotrichum lindemuthianum. Euphytica 13: 178-186.

Geffroy, V., Creusot, F., Falquet, J., Sevignac, M., Adam-Blondon, A-F., Bannerot, H.,
Gepts, P., and Dron, M. 1998. A family of LRR sequences in the vicinity of the Co-2
locus for anthracnose resistance in Phaseolus vulgaris and its potential use in marker-
assisted selection. Theor. Appl. Genet. 96: 494-502.

Gepts, P., 1988. A Middle American and an Andean common bean gene pool. Pages 375-
390 in: Genetic resources of Phaseolus beans; their maintenance, domestication, and
utilization. P. Gepts, ed. Kluwer, London.

Haley, S.D., Miklas, P.N., Afanador, L., and Kelly, JD. 1994. Random amplified
polymorphic DNA (RAPD) marker variability between and within gene pools of
common bean. J. Amer. Soc. Hort. Sci. 119(1):122-125.

Kami, J., Velasquez, V.B., Debouck, D.G., and Gepts, P. 1995. Identification of
presumed ancestral DNA sequences of phaseolin in Phaseolus vulgaris. Proc. Natl.
Acad. Sci. USA 92: 1101-1104.

Kelly, J .D., Afanador, L., and Cameron, LS. 1994. New races of Colletotrichum
lindemuthianum in Michigan and implications in dry bean resistance breeding. Plant
Dis. 78: 892-894.

Melotto, M., Balardin, RS, and Kelly, JD. 1999. Host-pathogen interaction and

variability of Colletotrichum lindemuthianum. APS Press, St Paul, MN, USA. (in
press).

41

Mendoza-Herrera, A., Hernandez, F., Martinez, 0., Hernandez, S., Acosta, J., and
Simpson, J. 1999. Molecular mapping of one anthracnose resistance gene in the
common bean. Plant & Aninal Genome VII Conference, San Diego, CA. (Abstracts).
P240.

Muhalet, C.S., Adams, M.W., Saettler, A.W., and Ghaderi, A. 1981. Genetic system for
the reaction of field beans to beta, gamma, and delta races of Colletotrichum
lindemuthianum. J. Amer. Soc. Hort. Sci. 106: 601-604.

Singh, S.P., Gepts, P., and Debouck, D.G. 1991. Races of common bean (Phaseolus
vulgaris, Fabaceae). Econ. Bot. 45: 379-396.

Tu, J.C., 1994. Occurrence and characterization of the alpha-Brazil race of bean
anthracnose (Colletotrichum lindemuthianum) in Ontario. Can. J. Plant. Pathol. 16:
129-131.

Young, RA. and Kelly, J.D. 1996a. Characterization of the genetic resistance to
Colletotrichum lindemuthianum in common bean differential cultivars. Plant Dis. 80:
650-654.

Young, RA. and Kelly, J.D. 1996b. RAPD markers flanking the Are gene for
anthracnose resistance in common bean. J. Amer. Soc. Hort. Sci. 121: 37-41.

Young, RA. and Kelly, J.D. 1996c. Is the anthracnose resistance A gene the same in
cultivars belonging to both bean gene pools? Ann. Rep. Bean Irnprov. Coop. 39: 296-
297.

Young, R.A. and Kelly, JD. 1997. RAPD markers linked to three major anthracnose
resistance genes in common bean. Crop Sci. 37:940-946.

Young, R.A., Melotto, M., Nodari, R.O., and Kelly, J .D. 1998. Marker-assisted dissection
of the oligogenic resistance in the differential cultivar, G2333. Theor. Appl. Genet.
96: 87-94.

42

CHAPTER TWO

MARKER-ASSISTED DISSECTION OF THE OLIGOGENIC ANTHRACNOSE RESISTANCE IN

THE COMMON BEAN CULTIVAR, G 2333

ABSTRACT

Two independently assorting dominant genes conditioning resistance to bean
anthracnose were previously identified in an F2 population derived from the highly resistant
bean differential cultivar, G 2333. One gene was allelic to the Co-4 gene in the differential
cultivar TO and was named Co-4", whereas the second gene was assigned the temporary
symbol Co-7 until a complete characterization with other known resistance genes can be
conducted. A RAPD marker linked to the Co-42 allele was identified. OASl3950 and Co-4"
allele co-segregated with no recombinants in two segregating populations of 143 F2
individuals. A SCAR marker (SAS13), developed from the OASl3950 RAPD marker, was
dominant and polymorphic similar to the original RAPD, and supported the tight linkage
between the marker and the Co-4" allele. The markers were present in germplasm with
known resistance alleles at the Co-4 locus. The presence of the markers in two other
differential cultivars, not previously characterized, and in four navy bean cultivars suggests
the existence of a gene family for anthracnose resistance at or near the Co-4 locus. Since the
Co-7 gene was present only in germplasm which also possessed the Co-42 and Co-5 genes,

the SAS13 marker was used in combination with standard inoculation techniques to identify

43

F 3 lines in which the Co-7 gene was homozygous and the Co-42 allele was absent. A Similar
strategy of marker assisted dissection is proposed to identify resistant lines in which the Co-5
gene is absent and the C o-7 gene is present by selecting against the OAB3450 marker, shown
previously to be linked to the Co-5 gene. These genes cannot be distinguished using
traditional screening methods since all current races of the pathogen virulent to the Co-5
gene are avirulent to the Co-4" and C o-7 genes. The use of molecular markers, tightly linked
to resistance genes, to facilitate the identification of an uncharacterized resistance gene for

which no discriminating race of the pathogen is known, is described.

INTRODUCTION

One of the most severe, widespread diseases of common bean (Phaseolus vulgaris
L.) is anthracnose, caused by Colletotrichum lindemuthianum (Sacc. & Magnus) Lambs.-
Scrib. (Pastor-Corrales et al. 1995, Schwartz 1991, Tu 1992). Genetic variability of C.
lindemuthianum has been observed in different parts of the world where beans are grown
(Beebe and Pastor-Corrales 1991, Pastor-Corrales and Tu 1989), and many races of the
pathogen have been characterized (Balardin et al. 1997, Rava et al. 1993, Garrido-Ramirez
and Romero-Cova 1989, Pastor-Corrales and Tu 1989, Menezes and Dianese 1988, Yerkes
and Teliz-Ortiz 1956). Due to the highly variable nature of the pathogen and the continual
emergence of new races, genetic resistance in the host is not durable. Currently, races of C.
lindemuthianum are classified by inoculating isolates onto a universal set of 12 differential
cultivars. Races are assigned a cumulative numerical value for each susceptible differential
cultivar based on an established binary system (CIAT 1988, Pastor-Corrales 1991). Only six
of the 12 differential cultivars have been genetically characterized for anthracnose resistance:
Michigan Dark Red Kidney, Co-I gene (Burkholder 1918, Young and Kelly 1997); Cornell
49242, Co-2 gene (Mastenbroek 1960); Mexico 222, Co-3 gene; TO, Co-4 gene; TU, Co-5
gene (Fouilloux 1979); and AB 136, Co-6 gene (Young and Kelly 1996a). There is limited
evidence that some of the remaining six differentials, such as Perry Marrow and P1207262,
may carry more than one resistance factor (Cardenas et al. 1964, Muhalet et al. 1981,
Goncalves-Vidigal et al. 1997). For instance, using race 521, Pastor-Corrales et al. (1994)
demonstrated that anthracnose resistance in differential cultivar G 2333 was controlled by

two unknown resistance genes. In addition to these two unknown genes, Young and Kelly

45

(1996a) showed that G 2333 carried also the Co-5 gene, which was not detected by Pastor-
Corrales et al. (1994) since race 521 is virulent to the Co-5 gene. The presence of more than
one gene in G 2333 may account for its broad resistance to all known races of C.
lindemuthianum (Balardin et al. 1997, Pastor-Corrales et al. 1994, Schwartz et al. 1982). If
resistance in G 2333 is conditioned by three resistance genes (Young and Kelly 1996a), one
of which has been previously described (Co-5), the two other genes need to be characterized.
Since no discriminating race(s) of the pathogen is available to distinguish effectively
between Co-4" and Co-7 resistance genes, it is impossible to know which gene or gene
combination is being transmitted to the progeny. When combined in a single genotype, those
unknown resistance genes, that cannot be identified by traditional pathological techniques,
can be distinguished from each other using a marker tightly linked to one of the resistance
genes. The use of markers to dissect the genetic basis of complex traits in other crops has
been described (marker assisted dissection, Allard 1996).

In this study the use of tightly linked RAPD and SCAR markers to assist in the
identification of independently inherited resistance genes for which no discriminating races
of the pathogen are available is discussed. To demonstrate the application of marker assisted
dissection, the anthracnose resistance genes present in the bean cultivar G 2333 were studied.
RAPD and SCAR markers linked to the Co-4 gene were identified and used to isolate the

third resistance gene present in G 2333.

46

MATERIALS AND METHODS

Genetic material. The study of resistance to anthracnose in G 2333 was previously
investigated using two breeding lines, SEL 1360 and SEL 1308, derived through
backcrossing with G 2333. The cultivar G 2333 is highly photoperiod sensitive, which makes
it difficult to work with in temperate regions. Both lines were resistant to a number of races
of C. lindemuthianum, and each line was known to carry different resistance gene(s) since
they displayed different phenotypic reactions after inoculation with a wide array of races of
the pathogen (Young and Kelly 1996a). SEL 1360 possesses a single dominant resistance
gene characterized as Co-5. Resistance in SEL 1308, however, was concluded to be similar
to G 2333 parent, since no race was found to discriminate between the resistance genes
present (Young and Kelly 1996a). Crosses between SEL 1308 and the cultivars Catrachita
(Co-6 gene; Beebe and Pastor-Corrales 1991) and Black Magic (susceptible; Kelly et al.
1987) were made and these two segregating populations were used to characterize the genetic
resistance in SEL 1308 (Young et al. 1998). The Catrachita/SEL 1308 and Black Magic/SEL
1308 populations were composed of 72 and 99 F2 individual plants, respectively, and the F23
progeny of each individual F 2 plant were tested to discriminate homozygous from
heterozygous resistant genotypes. Inheritance and allelism tests have shown that SEL 1308
possesses a single dominant gene conditioning resistance to anthracnose, the Co-42 gene,
which is allelic to the Co-4 gene present in the differential cultivar TO (Young et al. 1998).
SEL 1360 (Co-5 gene) was crossed with the original parent G 2333 to generate a population

where the unknown genes would be segregating and the Co-5 gene would be fixed.

47

RAPD and SCAR analyses. Individual F2 plants from Catrachita/SEL 1308 and
Black Magic/SEL 1308 populations were used as two independent mapping populations to
test for putative linkages between RAPD markers and the resistance gene in SEL 1308.
Bulked segregant analysis (Michelmore et al. 1991) was used to form two independent and
contrasting DNA bulks composed of equal volumes of fluorometrically standardized DNA
from six F2 homozygous resistant and six F2 homozygous susceptible plants derived from
both mapping populations. The PCR procedure was similar to that described by Young and
Kelly (1996b) using random primers (Operon Technologies, Inc., Alameda, CA) and Stoffel
DNA polymerase (Perkin Elmer). The thermal profile used consisted of 3 cycles of 1min at
94° C, 1min at 35° C, 2min at 72° C; 34 cycles of 105 at 94° C, 203 at 40° C, 2min at 72° C,
followed by 1 cycle of 5min at 72° C. The RAPD amplification products linked to the Co-4"
gene were purified using the QIAquick gel extraction kit (Qiagen Inc., Chatsworth CA) and
cloned by means of the TA cloning system (Invitrogen Corporation, San Diego, CA). Cloned
amplification products were sequenced following the procedure described by Melotto et al.
(1996, Appendix A1). SCAR markers were generated from the RAPD markers and tested
in both mapping populations. Amplification of the DNA fragment by the SCAR primers was
carried out as described by Melotto et a1. (1996). The PCR procedure consisted of 34 cycles
of 105 at 94° C, 2 min 40 sec at 72° C, followed by 1 cycle of 5min at 72° C. The presence
or absence of DNA amplification was confirmed in the parental genotypes and in selected
samples from the two populations using Taq DNA polymerase (Gibco, BRL). In addition,
the markers were used for purposes of marker assisted selection to identify F2 individuals,

which did not possess the marker and to screen other differential and commercial cultivars.

48

Linkage analysis. Simple inheritance of the disease phenotype and the putative
linked RAPD and SCAR markers was confirmed using Chi-square tests. Linkage analysis
was performed using the program Linkage-1 (Suiter et al. 1983). Linkage estimates between
loci are expressed in centimorgans (cM), as calculated using Kosambi’s function by the

Linkage-1 computer program.

49

RESULTS

A total of 260 decarner primers were used to screen for RAPD markers linked to the
Co-42 gene and two linked RAPD markers were identified. One marker designated OAS13950
(generated by a 5'-CACGGACCGA-3' decamer; Figure 2.1A) co-segregated with the
resistance gene in 143 individuals in both populations and no recombinants were observed
(Table 2.1). This marker was present in SEL 1308, G 2333 and in the differential cultivars,
Widusa, PI 207262 and TO which carries the Co-4 gene. The marker was absent from all
other differential cultivars including SEL 1360 and those with characterized resistance genes.
In a survey of 24 commercial bean cultivars representing the two gene pools of P. vulgaris,
the OASl3950 RAPD marker was present in four related navy bean cultivars, Monroe,
Sanilac, Seafarer and Seaforth. Similar results were observed using Taq DNA polymerase

(Figure 2.1B).
The two specific 24-mer SCAR primers, synthesized based on the DNA fragment

sequence amplified by the OAS13_.,,50 RAPD marker, are shown in Table 2.2. The SCAR
marker SASl3 amplified a single DNA fragment of the expected size (950 bp) and appeared
as a single polymorphic band in agarose gels (Figure 2.1C). In addition, SASl3 followed the
same co-segregation with the Co-4" allele as the OAS13950 RAPD marker in both F2

populations, supporting the tight linkage between the marker and the resistance gene.

50

Table 2.1 Chi-square analysis for the OASl3950 RAPD marker locus segregating in two F 2
populations, two-point chi-square analysis, and linkage estimates for the marker locus and

the Co-4" resistant allele.

 

 

Population Locus tested Expected Observed X2 P3 cMb
ratio frequency (r :1: SE)

Catrachita/SEL 1308 OASl3950 3:1 47:19 0.32 0.57

Black Magic/SEL 1308 OAS l 3950 3: l 70:25 0.03 0.86

Catrachita/SEL1308 OASl3950/Co—42 3:6:3:l:2:1 16:29:02020zl7 62.0 0.0 0.0:tn.d.
BlackMagic/SEL1308 OASl3950/Co-4" 3:6:3:l:2:1 12:45:0:0:0:24 81.0 0.0 0.0:l:n.d.

Across populations OASl3950/Co-42 3:6:3:l:2:1 28:74:0z020z41 143.0 0.0 0.0in.d.

 

' P = probability estimated value.
b Linkage analysis based on 1:2:1 genotypic segregation ratio for the Co-42 locus and 3:1
ratio for the OASl3950 RAPD marker.

Table 2.2 SCAR primer sequences derived from the RAPD marker OAS 1 3950 linked to the

Co-4" allele in common bean.

 

SCAR marker Primer Sequenceal

 

SAS 1 3 SAS 1 3 .24XP 5 ’-CACGGACCGAATAAGCCACCAACA-3 ’

SAS 1 3 .24RP 5 ’-CACGGAC§GAGGATACAGTGAAAG-3 ’

 

alUnderlined sequences are the original OASl3,-,50 RAPD marker.

51

123456789

 

Figure 2.1 DNA amplification of eight bean cultivars using A) the OASl3950 RAPD marker
with Stoffel DNA polymerase, B) the OAS13950 RAPD marker with Taq DNA polymerase,
C) the SASl3 SCAR marker. Lanes (1) 100 bp DNA ladder, (2) Resistant DNA bulk (Co-
4‘7/Co-42), (3) Susceptible DNA bulk (co-42/co-4Z), (4) SEL 1308 (Co-42/Co-42), (5) SEL
1360 (co-42/co-42,C0-5/Co-5), (6) G 2333 (Co—42/C0-42,C0-5/C0-5), (7) Catrachita (Co-6/Co-
6), (8) TO (Co-4/Co-4), (9) Black Magic (co-4Z/co-42).

52

The OAS13950 and SASl3 markers were used to identify F 2 individuals, which did
not possess the Co-4" allele in the SEL 1360/G 2333 population. A progeny test of F,
families (derived from F2 individuals without the OASl3,,0 marker) was performed using
race 521 to discriminate homozygous resistant co-42co-42/Co- 7Co-7 genotypes from the
heterozygous co-42co-42/Co- 7co-7 individuals. Six F 2 individuals, in the SEL 1360/G 2333
population, were first identified as susceptible to race 521, thus are recessive for both genes
(Table 2.1). Only 55 individuals from the remaining 86 F 2 individuals were harvested due
to problems with photoperiod sensitivity in the cross. Among these, 14 individuals lacking
the OAS 13950 marker were identified. The F, progeny (consisting mainly of 15 individuals)
of these 14 F2 individuals were screened with race 521 and 3 homozygous resistant F,
families (co-42co-42/Co- 7Co-7) consisting of 15 individuals were identified (Table A2.l).
One of these F, families, named SEL 111 was chosen for crossing with the susceptible
cultivar Black Magic and members of the anthracnose differential series. The derived
populations will be used in allelism tests with other known resistance genes to fully

characterize the Co-7 gene (Appendix A2).

53

DISCUSSION

The characterization of the genetic resistance of common bean to C. lindemuthianum
in G 2333 is of major importance to breeders because of the broad based resistance exhibited
by this cultivar (Pastor-Corrales et al. 1994). The combination of resistance genes present
in G 2333 would be of great utility for long term resistance if bred into commercial bean
cultivars. In this study, we confirm that G 2333 carries three independently inherited
resistance genes, one of which is a second resistance allele, Co-42, at the Co-4 locus. The
finding of different alleles at the same locus conferring differential resistance to races of C.
lindemuthianum is not unique (F ouilloux 1979). The second allele at the Co-4 locus offers
improved potential in breeding for anthracnose resistance since it conditions resistance to a
broader array of pathogenic races, similar to the second allele at the bc—Z locus which
conditions resistance to bean common mosaic virus (Drij fhout 1991).

The current study supports the importance of Mexican bean germplasm as a valuable
source of resistance to anthracnose. Five of the 12 differential cultivars either come directly
from Mexico or were derived through crossing with Mexican germplasm. Two Mexican
landrace cultivars, PI 207262 named Tlalnepantla 64 and G 2333 named Colorado de
Teopisca (Pastor-Corrales et al. 1994), are used directly as differentials. Fouilloux (1979)
identified the three Mexique genes (Co-3, Co-4, Co-5) in Mexican germplasm and transferred
them into the differential cultivars, Mexico 222, TO, and TU, respectively. G 2333 carries
additional resistance genes (Young and Kelly 1996a), whereas the resistance gene(s) in P1
207262 has not been characterized fully (Goncalves-Vidi gal et al. 1997). Although the Co-5

gene in G 2333 is the same Mexique [II gene in TU, the second gene in G 2333 is unique as

54

it is an allele of the Co-4 (Mexique II) gene with a broader resistance pattern. The third gene,
Co- 7, appears to offer equal potential in breeding for resistance since, in combination with
the Co-42 allele, it has not been overcome by any existing race of the pathogen (Balardin et
al. 1997, Pastor-Corrales et al. 1994). Bean breeders interested in controlling anthracnose
may wish to utilize Mexican germplasm as a source of new and useful Middle American
genes for resistance to anthracnose. This is analogous to the research on breeding for rust
resistance in common bean where germplasm from Guatemala appears to offer the best
potential as a source of resistance to that variable pathogen (Stavely 1990).

RAPD and SCAR markers linked to anthracnose resistance genes in common bean
have been reported previously (Adam-Blondon et al. 1994, Young and Kelly 1996b, 1997).
However, the finding Of the OASl3950 marker in the three differential cultivars, Widusa, PI
207262 and T0 was unexpected. Finding the markers only in G 2333 was anticipated so their
presence in the differential cultivar TO (Co-4 gene) raises some question about the linkage
with the Co-42 allele. Since no recombinants were observed between the OAS13950 marker
and the Co-42 allele in two populations, a tight linkage is assumed. Similar tight linkages
have been observed in common bean for the rust resistance Ur-4 gene and the OA14HO0
marker (Miklas et al. 1993). One major difference between these studies is that the OAS 13950
marker was detected in two populations (143 individuals) with similar genetic backgrounds,
i.e., Middle American gene in a Middle American background, whereas Miklas et al. (1993)
found no recombinants (70 individuals) between the OA14l 100 marker and the Andean Ur-4
gene. Recombination was restricted in the latter population because the Andean Ur-4 gene
had been backcrossed into genetically distant Middle American germplasm. Young and

Kelly ( 1996b) observed lower recombination values between RAPD markers and the Middle

55

American Co-2 gene expressed in an Andean background than in a Middle American
background. The presence of the OASl3950 marker in two cultivars, TO and SEL 1308,
which carry alleles at the Co-4 locus, is not a major limitation in breeding for resistance. The
markers can be used to indirectly select either allele at the Co-4 locus depending on the
germplasm available to the breeder. The presence of the markers in Widusa and P1207262
suggests that they possess a different allele at the Co-4 locus in addition to other resistance
gene(s). Since neither Widusa nor P1 207262 possess greater levels of resistance than TO,
they cannot carry either characterized Co-4 allele. Allelism tests among Widusa, PI 207262,
TO, and SEL 1308 are planned since the resistance gene(s) in Widusa and P1207262 has not
been characterized fully.

The presence of the markers in the navy bean cultivars, Monroe, Sanilac, Seafarer
and Seaforth has no obvious explanation. Other related navy bean cultivars such as
Michelite, Mayflower, C-20, Bunsi, Kentwood, Fleetwood, Harofleet, Harokent, OAC-Rico
were screened, but the marker was absent. The marker was absent from all other market
classes of dry bean and snap bean surveyed. Since all bean genotypes with the OASl3950
RAPD and SCAR markers possess different genes for resistance to anthracnose, possibly a
cluster of anthracnose resistance genes exists at or near the Co-4 locus. The existence of
clusters of resistance genes has been demonstrated in Lactuca and Lycopersicon spp
(Maisonneuve et al. 1994, Salmeron et al. 1996). Although the Co-l gene in Sanilac and
Seafarer, and the Co-2 gene in Seaforth are independent of the Co-4 locus, all these cultivars
carry additional resistance to anthracnose races beta and gamma (Cardenas et al. 1964). The
resistance in Monroe is unknown, but it may carry the same resistance to races beta and

gamma as its parent, Michelite. In common bean, Adarn-Blondon et al. (1994) mapped nine

56

genes of known function, the majority of which represent gene families such as the 3-10
genes for phaseolin storage protein, six genes for chalcone synthase, and four for
phenylalanine ammonia lyase. In all cases only one member of the gene family was mapped
(Adam-Blondon et a1. 1994). The presence of clustered genes for phaseolin supports the
theory that specific genes for anthracnose resistance may exhibit a similar clustering pattern
based on the presence of the marker in genotypes with different resistance genes. Additional
evidence for the presence of complex loci of linked resistance genes in common bean was
demonstrated for genes controlling resistance to bean rust at the Ur-5 locus (Stavely 1984,
Appendix A3).

Although the broad-based anthracnose resistance of G 2333 has been widely tested
and confirmed by different research groups (Balardin et al. 1997, Pastor-Corrales et al. 1994,
Schwartz et al. 1982), the identification of individual genes is needed to evaluate their
potential in breeding for resistance. In the absence of a race of the pathogen virulent on G
2333, the use of tightly linked markers, as suggested by Maisonneuve et al. (1994), to
identify and combine different genes was evaluated. In the F 2 population (SEL 1360/G 2333)
where the Co-5 gene is fixed and the Co-42 and Co-7 genes are segregating independently,
six of the nine possible genotypic combinations will carry at least one dominant Co-42 allele.
The other three genotypes would be homozygous for the recessive co-4‘? allele, one of which
is the double recessive co-42co-4‘7/co-7co- 7, identified by its susceptible reaction to race 521
which is also virulent to the Co-5 gene. All three genotypes were identified by selecting F2
individuals which do not possess the OASl3950 RAPD and SASlB SCAR markers linked in
coupling with the Co-42 allele. By eliminating the double recessive individuals based on

inoculation, the other two genotypes were identified. These genotypes possess the recessive

57

co-4" allele and differ at the Co-7 locus for homo- or heterozygosity. The homozygous
individuals were identified as pure breeding resistant F, families after inoculation with race
521. In addition, the OAS 1 3950 RAPD and SCAR markers were used to construct the resistant
and susceptible DNA bulks for screening to detect markers linked to the Co-7 gene.
Individuals included in the susceptible bulk were detected in the SEL 1360/G 2333
population after inoculation with race 521 of the pathogen. We confirmed that all members
of the susceptible bulk did not have the OAS13950 marker as expected. Since no race is
available to distinguish the C 0-4" and C o-7 genes, only those resistant individuals which do
not possess the OAS 1 3,50 markers linked to the Co-4" gene will be included as members of
the resistant bulk. This ensures that the resistance to race 521 can only be derived from the
C o-7 gene. Screening of the bulks with random decamer primers will be conducted to find
a coupling RAPD marker linked to the new resistance gene, since the resistant F,
heterozygotes were not distinguished from the F, homozygotes when the resistant bulk was
formed. Once detected, the marker can be used to ensure that the Co-7 resistance gene is
incorporated into different genetic backgrounds, independent of other anthracnose resistance
genes.

Finally, the use of this information in developing a better differential series for the
classification of new races of anthracnose is proposed. Despite its broad resistance, G 2333
is not the ideal differential cultivar since it carries three different resistance genes. One
suggestion would be to replace G 2333 with SEL 1308 shown to carry only the single Co-4"
allele and consider including upon identification, a genotype known to carry only the Co-7
gene. The development of lines in which the C o-7 gene is present alone becomes a priority.

The Co-7 gene represents a unique and important source of resistance to anthracnose and

58

should be invaluable, when fixed alone in a hOSt genotype, to better classify potential new
races of the anthracnose pathogen and be used in breeding durable anthracnose resistant

germplasm.

59

REFERENCES

Adam-Blondon, A-F., Sevignac, M., Dron, M., and Bannerot, H. 1994. A genetic map of
common bean to localize specific resistance genes against anthracnose. Genome 37:915-
924.

Allard, R.W. 1996. Genetic basis of the evolution of adaptedness in plants. Euphytica 92:1-
11.

Balardin, R.S., Jarosz, A.M., and Kelly, ID. 1997. Virulence and molecular diversity in
Colletotrichum lindemuthianum from South Central and North America. Phytopathology
87:1184-1191.

Bassett, M.J. 1996. List of genes. Ann. Rep. Bean Improv. Coop. 3921-19.

Beebe, SE, and Pastor-Corrales, M.A. 1991. Breeding for disease resistance. In: Van
Schoonhoven A, Voysest O (eds) Common Beans: Research for Crop Improvement.
Centro Intemacional de Agricultura Tropical, CIAT, Cali, Colombia, pp 562-617.

Burkholder, W.H. 1918. The production of an anthracnose-resistant white marrow bean.
Phytopathology 8:353-359.

Cardenas, F ., Adams, M.W., and Andersen, A. 1964. The genetic system for reaction of field
beans (Phaseolus vulgaris L) to infection by three physiologic races of Colletotrichum
lindemuthianum. Euphytica 13: 178-186.

CIAT Centro Intemacional de Agricultura Tropical. 1988. Annual Report of Bean Program,
Cali, Colombia, pp 173-175.

Drij flrout, E. 1991. Bean common mosaic In: Hall, R. (ed) Compendium of bean diseases.
APS Press, St Paul, Minnesota, pp 37-39.

Fouilloux, G. 1979. New races of bean anthracnose and consequences on our breeding
programs. In: Maraite H, Meyer JA (eds) Diseases of Tropical Food Crops. Universite
Catholique de Louvain-la-Neuve, Belgium, pp 221-235.

Garrido-Ramirez, ER, and Romero-Cova, S. 1989. Identificacion de razas fisiologicas de
Colletotrichum lindemuthianum en Mexico y busqueda de resistencia genetica a este
hongo. Agrociencia 77: 139-1 56.

Kelly, J.D., Adams, M.W., Saettler, A.W., Hosfield, G.L., Uebersax, M., and Ghaderi, A.
1987. Registration of 'Domino' , 'Black Magic' tropical black beans. Crop. Sci. 27:363.

60

Maisonneuve, B., Bellec, Y., Anderson, R, and Michelmore, R.W. 1994. Rapid mapping of
two genes for resistance to downy mildew from Lactuca serriola to existing clusters of
resistance genes. Theor. Appl. Genet. 89296-104.

Mastenbroek, C. 1960. A breeding programme for resistance to anthracnose in dry shell
haricot beans based on a new gene. Euphytica 9: 177-184.

Melotto, M., Afanador, L., and Kelly, J .D. 1996. Development of a SCAR marker linked to
the I gene in common bean. Genome 39:1216-1219.

Menezes, J.R., and Dianese, J.C. 1988. Race characterization of Brazilian isolates of
C olletotrichum lindemuthianum and detection of resistance to anthracnose in Phaseolus
vulgaris. Phytopathology 782650-655.

Michelmore, R.W., Paran, 1., and Kesseli, R.V. 1991. Identification of markers linked to
disease resistance genes by bulked segregant analysis: a rapid method to detect markers
in specific genomic regions using segregating populations. Proc. Natl. Acad. Sci. USA
8829828-9832.

Miklas, P.N., Stavely, J.R., and Kelly, JD. 1993. Identification and potential use of a
molecular marker for rust resistance in common bean. Theor. Appl. Genet. 85:745-749.

Muhalet, C.S., Adams, M.W., Saettler, A.W., and Ghaderi, A. 1981. Genetic system for the
reaction of field beans to beta gamma and delta races of Colletotrichum lindemuthianum.
J. Amer. Soc. Hort. Sci. 106:601-604.

Pastor-Corrales, M.A. 1991. Estandarizacién de variedades diferenciales y de designacién
de razas de Colletotrichum lindemuthianum. Phytopathology 81:694.

Pastor-Corrales, M.A., and Tu, TC. 1989. Anthracnose In: Schwartz I-IF, Pastor-Corrales
MA (eds) Bean production problems in the tropics. CIAT, Colombia, pp 77-104.

Pastor-Corrales, M.A., Erazo, O.A., Estrada, BI, and Singh, SP. 1994. Inheritance of
anthracnose resistance in common bean accession G 2333. Plant Dis. 78:959-962.

Pastor-Corrales, M.A., Otoya, M.M., Molina, A., and Singh, SP. 1995. Resistance to
Colletotrichum lindemuthianum isolates from Middle America and Andean South
America in different common bean races. Plant Dis. 79:63-67.

Rava, C.A., Molina, J., Kauffrnann, M., and Briones, I. 1993. Determinacién de razas

fisiolégicas de Colletotrichum lindemuthianum en Nicaragua. Fitopatol. Bras. 18:388-
391.

61

Salmeron, J.M., Oldroyd, G.E.D., Rommens, C.M.T., Scofield, S.R., Kim, H.S., Lavelle,
D.T., Dahlbeck, D., and Staskawicz, B.J. 1996. Tomato Prf is a member of the leucine-
rich repeat class of plant disease resistance genes and lies embedded within the Pto
kinase gene cluster. Cell 86:123-133.

Schwartz, HF. 1991. Anthracnose In: Hall, R. (ed) Compendium of Bean Diseases. APS
Press, St Paul, Minnesota, pp 16-17.

Schwartz, H.F., Pastor-Corrales, M.A., and Singh, SP. 1982. New sources of resistance to
anthracnose and angular leaf spot of beans (Phaseolus vulgaris L). Euphytica 31 :741-
754.

Stavely, JR. 1984. Genetics of resistance to Uromyces phaseoli in a Phaseolus vulgaris line
resistant to most races of the pathogen. Phytopathology 74:339-344.

Stavely, JR. 1990. Rust resistance of additional plant introductions. Ann. Rep. Bean Improv.
Coop. 33:185-186.

Suiter, K.A., Wendel, IF, and Case, IS 1983. Linkage-1: a PASCAL computer program
for the detection and analysis of genetic linkage. J. Hered. 74:203-204.

Tu, J.C. 1992. Colletotrichum lindemuthianum on bean. Population dynamics of the
pathogen and breeding for resistance In: Bailey JA, Jeger MJ (eds). Colletotrichum —
Biology, Pathology and Control. CAB International, pp 203-224.

Yerkes, W.D., and Teliz-Ortiz, M. 1956. New races of Colletotrichum lindemuthianum in
Mexico. Phytopathology 46:564-567.

Young, R.A., and Kelly, J.D. 1996a. Characterization of the genetic resistance to
Colletotrichum lindemuthianum in common bean differential cultivars. Plant Dis.
80:650-654.

Young, R.A., and Kelly, J.D. 1996b. RAPD markers flanking the Are gene for anthracnose
resistance in common bean. J. Amer. Soc. Hort. Sci. 121237-41.

Young, R.A., and Kelly, JD. 1997. RAPD markers linked to three major anthracnose
resistance genes in common bean. Crop Sci. 37:940-946.

Young, R.A., Melotto, M., Nodari, R.O., and Kelly, J .D. 1998. Marker-assisted dissection
of the oligogenic anthracnose resistance in the common bean cultivar, G 2333. Theor.
Appl. Genet. 96:87-94.

62

CHAPTER THREE

THE COX-4 GENE IS PART OF A COMPLEX LOCUS CONDITIONING RESISTANCE TO

ANTHRACNOSE IN COMMON BEAN, PHASEOLUS VULGARIS

ABSTRACT

Genetic mapping is a powerful approach to clone disease resistance genes. In this
study, a tightly linked SCAR marker, SAS13 tested in 1018 F, individual plants, was
used as a starting point for cloning gene sequences associated with the Co-4 locus, which
conditions resistance to the fungal pathogen Colletotrichum lindemuthianum in common
bean. A contig developed from genomic clones flanking the marker region revealed an
1110 bp open reading frame, named C 0K-4. Two essential eukaryote promoter elements,
TATA and CAAT boxes, and putative promoter sequences were found upstream of the
COK-4 gene. The predicted COK—4 protein contains a serine-threonine kinase domain
with a highly hydrophobic membrane-Spanning region. Specific primers designed to
amplify the COX-4 region, were used to clone and sequence COX-4 homologs from
different bean cultivars. Single nucleotide polymorphisms were found between the
homologous sequences and were further confirmed with three restriction enzymes.
Restriction patterns were polymorphic among three bean cultivars known to possess
different alleles at the Co-4 locus, Black Magic (co-4), TO (Co-4), and SEL 1308 (Co-4")

as indicated by genetic analysis. Perfect co-segregation between restriction patterns and

63

disease phenotype was observed in 1350 F, individuals. More than one copy of the COK-
4 gene analog exists in the bean genome as demonstrated by Southern analysis. These
results strongly suggest that the COK-4 gene is a member of the complex Co-4 locus
conditioning resistance to anthracnose in bean. COX-4, a gene ortholog of the Pto in
tomato, is the first disease resistance gene successfully cloned from legumes. Molecular
cloning of resistance genes should facilitate studies on plant-pathogen interaction and

ultimately facilitate genetic improvement of crop species.

 

 

 

 

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A199

INTRODUCTION

Genetic resistance is the most efficient way to control anthracnose, the disease
caused by the fungus Colletotrichum lindemuthianum, in common bean (Phaseolus
vulgaris L.). The high genetic variability observed in the pathogen population (Balardin
et al. 1997) is associated with different resistance genes present in the host (Balardin and
Kelly 1998). Seven independent dominant disease resistance genes (Co-I to Co-7)
controlling anthracnose in bean have been described (Balardin et al. 1997). Each of these
genes confers resistance to certain races of the pathogen strongly suggesting that
resistance to anthracnose in common bean follows the gene-for—gene theory (Flor 1947).
Certain resistance genes are more effective than others in controlling multiple races of the
pathogen (Balardin and Kelly 1998).

The bean breeding line SEL 1308 derived from the highly resistant differential
cultivar G2333, is known to possess the single dominant Co-4’ gene for anthracnose
resistance (Young et al.. 1998). When inoculated with 34 selected races of C.
lindemuthianum chosen to represent a diverse sample of the pathogen population, SEL
1308 demonstrated a resistance index (R1) of 97% (Balardin and Kelly 1998). The only
cultivar with a higher RI (100%) was G2333 known to possess the combination of three
independent resistance genes, Co-4", Co-5, and Co-7 (Young et al. 1998). This three-gene
combination confers resistance to all described races of the pathogen (Pastor-Corrales et
al. 1994). Among the reported resistance genes, the Co-42 gene in SEL 1308 exhibits the

broadest-based resistance in common bean (Balardin and Kelly 1998).

65

The C 0-4" gene is a valuable candidate gene for molecular cloning due to its broad
resistance and availability of a tightly linked marker (Young et al. 1998). To better
understand the mechanisms of disease resistance several disease-resistance genes have
been cloned from different plant species. Sequence analysis indicated that these genes
encode structurally similar proteins with conserved function across plant species (Bent
1996). The opportunity exists, therefore, to identify resistance gene candidates from
diverse plant taxa. In common bean and soybean, resistance genes analogs (RGAS) were
identified using primers specific to conserved regions of known resistance genes and
mapped close to known disease resistance locus (Kanazin et al. 1996, Yu et al. 1996,
Geffroy et al. 1998, Rivkin et al. 1999). Mapping of RGAS has been the only approach
used to isolate known resistance gene sequences from common bean. RGAS, however, are
not always closely associated with a resistance phenotype and may be loosely linked to
known resistance locus limiting their value in chromosome walking to the gene.
Additional approaches, such as map-based cloning are still needed to isolate resistance
gene candidates in crop species.

In the present study the fine mapping of the Co-42 locus using a tightly linked
molecular marker is described. A previously described SCAR marker (Young et al. 1998)
was used as the starting point for the cloning of gene sequences associated with the Co-42
anthracnose resistance gene. Cloned sequences were compared to known resistance gene

sequences and the function of candidate resistance genes in common bean is discussed.

66

MATERIALS AND METHODS

Genetic analysis of the segregating population. The bean breeding line SEL
1308 obtained from CIAT was used as the source of the Co-42 gene. This line was
derived from a backcross between the anthracnose susceptible cultivar Talamanca and the
recurrent parent Colorado de Teopisca (accession number G2333). SEL 1308 was crossed
to Black Magic, a susceptible black bean cultivar. Hybrid seeds were advanced to the F,
generation and a population of 1018 F, individuals was developed. Progeny tests were
performed in 96 F, derived F, families to discriminate homozygous from heterozygous
resistant genotypes. Race 73 (ATCC 96512) of C. lindemuthianum was chosen to confirm
the dominant inheritance of the Co-4" gene in SEL 1308. Black Magic, the susceptible
parent of the mapping population, dies in five days after inoculation. Inoculum
preparation, inoculation methods, and disease characterization of the segregating
population were conducted as described by Young and Kelly (1996). Individual F, plants
from Black Magic/SEL 1308 population were screened with the SCAR marker SAS13
previously found to be linked to the Co-42 gene. Procedures for SCAR analysis are
described elsewhere (Melotto et a1. 1996). Additional RAPD markers flanking the Co-42
gene were sought by using bulked segregant analysis (Michelmore et al. 1991).
Inheritance of the disease phenotype and molecular markers was confirmed in 1018 F,
plants using the Chi-square test. Linkage analysis was performed using the Linkage-1
software (Suiter et a1. 1983) and distance between marker and resistance gene expressed

in centiMorgan (cM) was calculated using Kosambi’s function in the Linkage-1 program.

67

Southern analysis. Genomic DNA from bean cultivars was digested with EcoR 1
according to the manufacturer (Boehringer Mannheim, Indianapolis, IN). Electrophoresis
and blotting were conducted using standard techniques (Sarnbrook et a1. 1989). The
SASl3 marker was labeled using the Gibco RadPrime kit (Life Technologies, Inc.,
Rockville, MD) and used as a probe for hybridization. Stringency washes were performed
in 2x SSC, 0.1% SDS and 0.1x SSC, 0.1% SDS solutions. Both washes were conducted

twice for 30 minutes at 60°C.

Long distance PCR and primer walking. DNA clones flanking the SAS13
marker were generated using the Universal GenomeWalkerTM kit (Clontech Laboratories,
Inc., Palo Alto, CA). DNA from SEL 1308 was purified using phenol and chloroform,
and digested with five restriction enzyme, Dra I, EcoR V, Pvu 11, Sea 1, and Stu I.
Adaptors were ligated to restricted DNA samples for PCR amplification with adaptor-
Specific primers. PCR reactions were carried out in 50ul solution containing 1x
Advantage Genomic Polymerase Mix (Clontech Laboratories, Inc., Palo Alto, CA), 1.1
mM Mg(OAc),, 10 mM of each dNTP, 10 pM of each adaptor-specific and SAS13
primers, and 50 ng of DNA template. PCR reactions were placed in a 9600 Thermocycler
(Perkin Ehner Applied Biosystems) and the PCR file consisted of 7 cycles of 2 seconds at
94°C, 4 minutes at 70°C, followed by 32 cycles of 2 seconds at 94°C, 4 minutes at 65°C
and an extension cycle of 7 minutes at 65°C. Long distance PCR (LD-PCR) amplification
products were cloned using the TOPOTM TA Cloning kit (Invitrogen Corp., San Diego,
CA). Both strands of cloned DNA fragments were sequenced using an Applied

Biosystems 377 DNA Sequencer (Perkin Elmer Applied Biosystems) as previously

68

described by Melotto et al. (1996). New primers were designed based on those sequences

to walk in uncloned genomic DNA as proposed by Siebert et al. (1995).

Sequence analysis. The sequences obtained by primer walking were aligned and
a conti g was generated using the Sequencher 3.0 software (Gene Codes Corporation, Ann
Arbor, MI). The consensus sequence was compared to other sequences available in the
computer database using BLAST search programs (Altschul et al. 1997). The amino acid
sequence was deduced from the consensus DNA sequence and was analyzed for putative
firnction and domains using the computer search programs PROSITE (Hofinann et al.

1999), SOSUI (Hirokawa et al. 1998), and BLASTP (Altschul et al. 1990).

Restriction analysis of specific PCR products. PCR primers were designed to
amplify specific DNA fragments near the SASl3 marker region. The PCR amplification
reaction contained 50 ng of genomic DNA, 10 mM of each dNTP, 10 pmol of each
forward and reverse primers, 1x enzyme buffer containing MgSO4 and IU of Pfu DNA
polymerase (Promega, Madison, WI). PCR reactions were placed in a 9600 Therrnocycler
(Perkin Elmer Applied Biosystems) and the PCR file consisted of 34 cycles of 20 seconds
at 95°C, 30 seconds at 55°C, and 4 minutes at 72°C, followed by an extension cycle of 7
minutes at 72°C. The amplification product was used in a digestion reaction containing 1x
enzyme buffer, 10U of restriction enzyme and 10p] of PCR reaction. Digestion of DNA
fragment was carried out for four hours at 37°C. Restriction patterns were observed on

0.8% ethidium bromide-stained agarose gel.

69

RESULTS

The original SAS13 SCAR marker, which amplified a single 950-bp fi'agment in
the resistant parent, co-segregated with the Co-42 resistance gene in the segregating
population of 1018 F, individuals (Figure 3.1A). Four recombinant individuals were
detected and the distance between the marker and the Co-42 locus was estimated at 0.39
cM (Table 3.1). Three susceptible individuals possessing the SAS13 fragment and one
resistant line lacking the fragment were observed. The 950-bp DNA fragment generated
by the SAS13 SCAR marker (Young et al. 1998) was sequenced and analyzed for
similarities to sequences of known disease resistance genes. The alignment obtained by
using BLAST search software (Altschul et al. 1997) revealed a high similarity to serine-
threonine kinase (STK) domains such as the ones encoded by the disease resistance gene
Pto (gi|430992; gill809257; Martin et al. 1993) and Fen gene (gi|1098334; Martin et al.
1994) in tomato. Other proteins Similar to the SAS13 DNA fragment included receptor-
like kinases (RLK) from other organisms including Arabidopsis thaliana, Brassica Sp,
0022a sativa, and Zea mays. Based on these results, the SAS13 marker was used as a
starting point for primer walking in genomic DNA. Four overlapping clones extending
the original SAS13 950-bp fragment were obtained and the full length of the contig
included 3,371 bp (Figure 3.13). Primer pairs were designed to test whether the generated
clones were contiguous in the plant genome. A11 primer sets amplified a single band of

the predicted size (data not shown).

70

Table 3.1 Chi-square analysis of the SAS13 marker locus segregating in Black
Magic/SEL 1308 F, population, two-point chi-square and linkage estimates for the

marker locus and the C o-4" resistance allele.

 

 

Locus Expected Ratio Observed Frequency P* cM (r t SE)1
SASl3 321 7532265 0.45

Co-4" co-4” 321 7512267 0.37

SAS13 / Co-4° 9232321 75021232264 0.0 0.39 i 3.14

 

*P = probability estimated value
I Linkage analysis based on 12221 genotypic segregation ratio of the Co-42 and OBB14

marker loci and 3:1 ratio for OAL9, SAS13, and OH18 markers.

71

A)

 

 

 

 

2.24 CM 1.11 cM 5.8 an
1 L I I
I A ' I
031314 SASI3 C042 OH18 OAL9
3 c 8
B) 9 E if 8 § E
h, I 0 u— N ('1
2 M 2 a: an m‘
—> 1 I 1 g l I -
| I I I T I I l
“’P 3,371 bp
SASBnnma
ORF
C)
MFLNCVGMCCSKPTTNTTSSQRQFPTLIEELCHQFSLTDLE‘I-TJ-C‘NNFDQKRV

IGSGLFSEVYKGCLQHDGASDYTVAIK

RFDYQGWAAFNKEIELLCQLRHPRCVSLIGFCNHE
NEKILVYEYMSNGSLDKHLQEGQLSWKKRLEICIGVARGLHFLHTGAKRSI
FHCILGPGTVLLDDQMEPKLAGFDASEQGSRFMSKQ

KQINVIVFWVIFVLLYELTHCHDF

LWIKLSLLFVIGCRGYTATDYLMDGIITAK

WDVFSFGFLLLEVVCRRMFYLIT
LTKKECLENPVEERIDPIIKGKIAPDCWQVFVDMMVSCLKYEPDERPTIGEVEVQLEHALSMQE

 

 

 

QSDITNSNSEYTLLSKTIISLGVKKCKO

Figure 3.1 Diagram of the genomic region containing the Co-42 locus. (A) Linkage map
showing position of molecular markers. (B) Contig developed from overlapping genomic
clones. Arrows indicate C 0K-4 specific primers and restriction sites are letter coded,
B=BamHI, E=EcoRI, H=HindIII, K=Kpnl, Me=MseI, Mp=MspI. (C) Detail of the COK-
4 amino acid sequence showing color coded putative domains: red = N-myristoylation
sites, green = N-glycosylation sites, pink = CAMP and cGMP-dependent protein kinase
phosphorylation site, blue = protein kinase ATP-binding region signature, underlined
anrino acids = primary transmembrane region, waved underlined amino acids = secondary
transmembrane region, . = stop codon. The original SAS13 marker site included COK-4
amino acids 1 through 173.

72

Sequence analysis of the contig revealed an open reading frame (ORF) of 1110
bp, which was named COK-4. Two essential eukaryote promoter elements, TATA and
CAAT boxes, and putative promoter sequences were found upstream Of the COK-4 gene.
The predicted amino acid sequence of COX-4 has a high degree of similarity with
expressed sequences generated by the Pto gene from Lycopersicon pimpinellifolium and
L. esculentum (38% identity, 53% similarity and 15% gap; Martin et al. 1993), TMK
protein from rice (29%, 45%, and 16%; van der Knaap et al. 1996), extracellular S-
domain from Brassica oleracea (30%, 45%, 15%; PID2g2598271) (Figure 3.2), S-domain
receptor-like protein kinase from Z. mays (33%, 49%, 14%; PID:g3445397), and leucine-
lich repeat (LRR) transmembrane protein kinase 2 from Z. mays (28%, 45%, 19%; Li and
Wurtzel 1998). The protein encoded by the COK-4 was analyzed for possible functional
domains. The COK-4 protein has a STK domain, which includes a protein kinase ATP-
binding region signature (amino acids 53 to 79), a primary transmembrane domain
(amino acids 202 to 224), putative sites for N-myristoylation and N-glycosylation, and a

CAMP and cGMP-dependent protein kinase site (amino acids 41 to 44) (Figure 3.1C).

73

a)
b)
C)
d)
e)
f)

a)
b)
C)
d)
e)
f)

1 MFLNCVGMCCSKPTTNTTSSQRQFPTLIEELCHQFSLTDLRKATNNFDQKRVIGSGLFSEVYKGCLQHDG
9 TNSINDALSSSYLVPFESYRVPLVDLEEATNNFDHKFLIGHGVFGKVYKGVLR-DG
1 MGSKYSKATNSISDASNSFES —————— YRFPLEDLEEATNNFDDKFFIGEGAFGKVYKGVLR—DG
27 YRVPFVDLEEATNNFDDKFFIGEGGFGKVYRGVLR-DG
564 NVNGGAAASETYSQASSGPRDIHVVETGNMVISIQVLRNVTNNFSDENVLGRGGFGTVYKGEL-HDG
513 ATNNFSSANKLGRGGFGTVYKGRLL-DG
ASDYTVAIKRFDY ----- QGWAAFNKEIELLCQLRHPRCVSLIGFCNHENEKILVYEYMSNGSLDKHL-———QEG
AKVALKRRTPESS ----- QGIEEFETEIETLSFCRHPHLVSLIGFCDERNEMILIYKYMENGNLKRHL----YGS
TKVALKRQNRDSR ----- QGIEEFGTEIGILSRRSHPHLVSLIGYCDERNEMVLIYDYMENGNLKSHL----TGS
TKVALKKHKRESS ----- QGIEEFETEIEILSFCSHPHLVSLIGFCDERNEMILIYDYMENGNLKSHL----YGS
TK---IAVKRMEAGVMGNKGLNEFKSEIAVLTKVRHRNLVSLLGYCLDGNERILVYEYMPQGTLSQHL---—FEW
KEIAVKRLSKMSL ----- QGTDEFKNEVKLIARLQHINLVRLIGCCIDKGEKMLIYEYLENLSLDSHIFDITRRS
Q —————— L ----- SWKKRLEICIGVARGLHFLHTGAKRSIFHCILGPGTVLLDDQMEPKLAGFD---ASEQGSRF
D ------ LPTMSMSWEQRLEICIGAARGLHYLHT——-RAIIHRDVKSINILLDENFVPKITDFG---ISKKGT-—
D ------ LPSM—-SWEQRLEICIGAARGLHYLHT---NGVMHRDVKSSNILLDENFVPKITDFG---LSKTRPQ-
D ------ LPTMSMSWEQPLEICIGAAPGLHYLHT---NGVIHRDVKCTNILLDENFVPKITDFG---ISKTMPBL
KEHNLRPL ----- EWKKRLSIALDVARGVEYLHSLAQQTFIHRDLKPSNILLGDDMKAKVADFGLVRLAPADGKC
N ------ L ----- NWQMRFDITNGIARGLVYLHRDSRFMIIHRDLKASNVLLDKNMTPKISDFG---MARIFGRD
MSKQKQINVIVFWVIFVLLYELTHCHDFLWIKLSLLFVIGCRGYTATDYLMDGIITAKWDVFSFGFLLLEVVCR

-------------------- ELDQTH------LSTV-VKGTLGYIDPEYFIKGRLTEKSDVYSFGVVLFEVLCA
------------------ LYQTTD-------------VKGTFGYIDPEYFIKGRLTEKSDVYSFGVVLFEVLCA
-------------------- DLTH--------LSTV~VRGNIGYIAPEYALWGQLTEKSDVYSFGVVLFEVLCA
VSVETRL ------------------------------- AGTFGYLAPEYAVTGRVTTKADVFSFGVILMELITG
DAEANTRK ----------------------------- VVGTYGYMSPEYAMDGIFSMKSDVFSFGVLLLEIISG
R—M ---------- FY ------- LIT-LTKK --------- E---CLEN—PVEERIDPII-—K-GKIAPDCWQVF-—
R-S ---------- AI ------- VQS-LPREMVNLAEWAVE---SHNNGQLEQIVDPNL-—A-DKIRPESLRKF—-
R-S ---------- AM ------- VQS-LPREMVNLAEWAVE---SHNNGQLEQIVDPNL--A-DKIRPESLRKF—-
RPA ---------- LY ------- LSE-MMSS --------- DDETQKMG-QLEQIVDPAI--A:AKIRPESLRMF-—
R-KALDETQPEDSMH ------- LVTWFRRM --------- Q---LSKD-TFQKAIDPTI--DLTEETLASVSTV--
K-KNNG ------- FYNSNQDLNLLA-LVWR --------- K---WKEG-KWLEILDPIIIDS-SSSTGQAHEILRC

VDMMVSCLKYEPDERPTIGEVEVQLEHALSMQEQSDITNSNSEYTLLSKTIISLGVKKCK 369

GDTAVKCLALSSEDRPSMGDVLWKLEYALRLQE 318
GETAVKCLALSSEDRPSMGDVLWKLEYALRLQE 308
GETAMKCLAPSSKNRPSMGDVLWKLEYALCLQE 312
AELAGHCCAREPHQRPDMGHAVNVLSTLSDVWKPSDPDSDDS 902
IQIGLLCVQERAEDRPVMASVMVMI 792

Figure 3.2 Alignment from regions of similarity between the COK—4 protein sequence and
reported protein sequences. Anrino acid identity is indicated in red and anrino acid
similarity is indicated in blue. Numbers in the sequence indicate the first and last amino
acids aligned. a) COK—4 protein (GenBank data base accession no. AF 153441); b) disease
resistance protein kinase Pto gi|430992; c) serine/threonine protein kinase Pto
(Lycopersicon esculentum) gi| 1809257; (1) putative serine/threonine protein kinase, Fen
gene (L. esculentum) gil557882; e) TMK (Oryza sativa) gnllPID|e267533; f) extracellular
S domain ofBrassica oleracea gnllPIDlel 172841.

74

Two specific primers were designed to amplify the COK-4 gene (Table 3.2). PCR
analysis using those primers confirmed the presence of a single 1150 bp DNA fragment
in the bean genome, which contains the COX-4 gene (Figure 3.3A). Both parents of the
mapping population and all individual bean cultivars tested possessed the COK-4 gene.
To search for nucleotide polymorphisms between the resistant and susceptible parents,
the 1150 bp DNA fragments were cloned and sequenced. Alignment of these two
sequences revealed a nucleotide identity of 98% between resistant and susceptible parents
(Figure 3.4). The predicted amino acid sequence from the susceptible parent was
interrupted by several stop codons. Based on the sequence data a restriction map at that
region was predicted (Figure 3.1B) and confirmed with 15 different restriction enzymes
in the resistant parent SEL 1308 and susceptible parent Black Magic (data not shown). To
further demonstrate the nucleotide polymorphism between the parental genotypes and
confirm the DNA sequence, COK-4 was digested with Specific restriction enzymes. Three
restriction enzymes, Kpn I, Mse I, and Msp I were polymorphic between the resistant and
susceptible parents of the mapping population (Figure 3.3B-D). All three enzymes
restricted the COK-4 of SEL 1308 at one site and the COK-4 of Black Magic at two or
more sites. Co-segregation of COK-4 restriction patterns with disease phenotype was
confirmed in 75 F, individual plants. All susceptible and heterozygous plants (genotyped
as F, families) had more than one restriction site Similar to Black Magic, whereas all
homozygous dominant individuals had only one restriction site Similar to SEL 1308.
Restriction analysis of the bean cultivar TO, known to possess a different resistance allele
at the Co-4 locus revealed a third restriction pattern (Figure 3.3B-D). The COK-4

homolog found in T0 was sequenced and aligned with SEL 1308 and Black Magic

75

homologs (Figure 3.4). T0 and SEL 1308 nucleotide sequences were 95% identical
whereas the alignment of the amino acid sequences revealed 86% identity, 94%
similarity, and 1% gap. TO and Black Magic nucleotide sequences were 94% identical.
To determine the copy number of the COK-4 gene in different bean cultivars,
EcoR I-restricted DNA was probed with the SAS13 marker (Figure 3.5). The resistant
cultivars SEL 1308, 62333, and Seafarer possessed two major homologous DNA
sequences of 1.5 and 9 kb in Size, whereas the susceptible cultivars SEL 1360 and
Cardinal possessed multiple homologous sequences of various sizes. Again, TO

possessed a unique RFLP pattern with only one 9-kb DNA fragment.

Table 3.2 Sequence of the specific primer set designed to amplify the COK-4 gene in the

vicinity of the Co-42 locus.

 

 

Primer Nucleotide position Sequence (5’-3’)
I 405-426 GTA TGG TAA GTG ACA AGT GAG A
2 1578-1556 ACC TGG TCA CTT ACA TTT CTT CA

 

76

 

Figure 3.3 Restriction analysis of the COX-4 amplified in several genotypes. (A)
Undigested; (B) Kpn I; (C) Mse I; (D) Msp 1. Lane (1) lOO-bp DNA ladder, (2) SEL 1308
(Co-42/Co-4’), (3) Heterozygous resistant F, plant (Co-42/c0—4’), (4) Homozygous
resistant F, plant (Co-42/Co-4’), (5) Homozygous susceptible F, plant (co-42/co-4’), (6)
TO (Co-4/Co-4), (7) Black Magic (co-42/co-4’).

77

A)
B)
C)

A)
B)
C)

A)
B)
C)

A)
B)
C)

A)
B)
C)

A)
B)
C)

A)
B)
C)

A)
B)
C)

A)
B)
C)

A)
B)
C)

A)
B)

l

121

181

241

301

361

421

481

541

601

atgtttctgaattgtgtgggcatgtgttgttcgaagcccacaacaaatacaacttcatct
atgtttctgaattgtgtgggcatgtgttgttcgaagcccacaacaaatacaacttcatct
atgtttctgaattgtgtgggcatgtgttgtacgaagcccacaacaaatacaacttcatct

cagagacagtttccaacgttgatagaagagctgtgccatcaattttctctcaccgatctt
cagagacagtttccaacgttgatagaagagctgtgccatcaattttctctcaccgatctt
cagagacagtttccaacgttgatagaagagctgtgccatcaattttctctcEccgatctt

aggaaagccaccaataactttgatcagaagagagtaataggaagtggattatttagtgaa
aggaaagccaccaataactttgatcagaagagagtaataggaagtggattatttagtgaa
aggaaagccagcaataactttgatcagaagagagtaataggaagtggattgtttagggaa

gtatacaaagggtgtctgcagcacgatggtgcttctgattacacggtcgcaataaagcga
gtatacaaagggtgtctgcagcaEgatggtgcttctgattacacggtcgcaataaagcga
gtataEEaagggtgtctgcagcaggatggtgcttctgattacacggtcgcaataaagcga

tttgattatcaaggatgggcagcgttcaacaaggaaatcgaattgctatgccagcttcgt
tttgattatcaaggatgggcagcgttcaacaaggaaatcgaattgctatgccagcttcgt
tttgattatcaaggatggggagcgttcaacaaggaaatcgaattgctatgccagcttcgt

caccctagatgtgtttctcttataggattctgcaaccacgaaaatgagaagattcttgta
caccctagatgtgtttctcttataggattcggcaaccacgaaaatgagaagattcttgta
caccctagatgtgtttctcttataggattctgcaaccacgaaaatgagaagattcttgta

tacgagtacatgtccaatggatctctagataaacacctacaagaaggtcaactatcatgg
tacgagtacatgtccaatggatctctagataaacacctacaagaaggtcaactatcatgg
tacgagtacatgtccaatggatctctagataaacacctacaagagggtgaactatcatgg

aagaagaggctggagatatgcataggagtagcacgtggactacacttccttcacaccgga
aagaagaggctagagatatgcataggagtagcacgtggactacactgccttcacaccgga
aagaagaggctagagatgtgcataggagtagcacgtggactacactgccttcacacgggt

gccaagcgttccatctttcactgtatcctcggtcctggtaccgtccttttggatgaccag
gccaagcgttccatctttcactgtatcctcggtcctggtaccgtccttttggatgaccag
gccaagcgttccatctttcactgtatcctcggtcctagtaccgtccttttggatgaccaa

atggagccaaaactcgctggtttcgatgctagcgagcagggatcacgttttatgtcaaag
atggagccaaaactcgctggtttcggtgctagcgagcagggatcacgttttatgtcaaag
atggagccaaaactcgctggtttcggtgttagcgggcagggatcacgttttatgtcaaag

cagaagcaaatcaatgt-gatcgtgttttgggtaatttttgttttgttgtatgagctcac
cagaagcaaatcaatgtggatcgtgttttgggtaatttttgttttgttgtatgagctcac
cagaagcaaatcaatgtggatcgtgttttgggtaatttttgttttgttgtatgagctcac

78

A)
B)
C)

A)
B)
C)

A)
B)
C)

A)
B)
C)

A)
B)
C)

A)
B)
C)

A)
B)
C)

A)
B)
C)

Figure 3.4 DNA sequence showing single nucleotide polymorphisms (SNPS) in underlined

red letters among three cultivars possessing different C 0K-4 homologs, A) SEL 1308, B)

660

720

780

840

900

960

1020

1080

tcactgccatgattttttgtggatcaaactaagct--tactctttgttataggttgtagggg
tcactgccatgattttttgtggatcaaactaagct--tactctttgttataggttgtggggg
tcactgcgatgagtttttgtggatcaaactaagctggtactctttgttataggtggttttgg

ctacacggctacggactatctcatggatggtatcatcacagctaaatgggatgttttctc
ctacacggctacggactatctcatggatggtatcatcacagctaaatgggatgttttctc
ctacgcggctacggactatgtcatggatggtagcatcacagctaaatgggatgttttctc

atttggtttccttctactagaagttgtgtgcaggaggatgttttatttaataactctgac
atttggtttccttctactagaagttgtgtgcaggaggatgttttatttgataactctgac
atttggtttccttctactagaagttgtgtgcaggaggatgttttatttgataactctgac

taaaaaagaatgtctggagaatcctgttgaggagagaattgatccgattatcaaaggaaa
taaaaaagaatgtctggagaatcctgttgaggagagaattgatccgattatcaaagggaa
taaaaaagaatgtctggagaatcctgttgaggagagaattgatccgattatcaaagggaa

gattgcaccagattgttggcaagtgtttgtagatatgatggtaagttgcttgaagtatga
gattgcaccagattgttggcaagtgtttgtagatatgatggtaagttgcttgaagtataa
gattgcaccagattgttggcaagtgtttgtagatatgatggtaagttgcttgaagtatga

accagatgagagaccaacaattggtgaagtggaggtgcaacttgagcatgctctatccat
accagatgagagaccaacaattggtgaagtggaggtgcaacttgagcatgctctatccat
accagatgagggaccaacaattggtgaagtggaggtgcaacttgagcatgctctatccat

gcaggaacaatctgatatcacaaactccaactctgagtataccttactctccaaaaccat
gcaggaacaagctgatatcacaaactccaactctgagtatacgttactgtccaaaaccat
gcaggaacaagctgatatcacaaactccaactctgagtataccttactgtccaaaaccat

tatttcccttggagtgaagaaatgtaagtga 1110
tatttccctgggagtgaagaaatgtaagtga
tatttccc

Black Magic, and C) TO.

79

—2

—'l5

 

Figure 3.5 Southern analysis of EcoR I digested DNA of bean cultivars hybridized with
the SASl3 marker. Lane (1) SEL 1308, (2) SEL 1360, (3) G2333, (4) Cardinal, (5) TO,

(6) Seafarer. Molecular weight is indicated in kb.

80

DISCUSSION

Two lines of evidence strongly suggest that the COK-4 gene, herein described, is
a member of the complex Co-4" locus conditioning resistance to anthracnose in common
bean. First, genetic analysis indicated co-segregation of the SASl3 SCAR marker with
the resistant phenotype in a population of 1018 F, individuals. Secondly, amino acid
sequence analysis of the COX-4 gene, which is located 462 bp downstream of the marker
(Figure 3.2B), revealed high similarity with previously cloned resistance genes and
protein domains known to play an important role in disease resistance. The putative
protein encoded by the COK-4 gene has the structure of STKs and RLKs. Receptor-like
kinases contain an extracellular domain possibly functioning in ligand binding and a
cytoplasmic domain responsible for signal transduction (Walker 1994). Unlike the Pto
protein, the COK-4 protein most likely is localized at the membrane because it contains
three highly hydrophobic regions characteristic of a transmembrane domain and has an
average hydrophobicity of —0.036 (calculated by the SOSUI software; Hirokawa et al.
1998). Alignment of the COX-4 amino acid sequence with the extracellular S-domain of
Brassica oleracea and LR transmembrane and RLK domain of Z. mays also supports
localization of the COK-4 protein in the cellular membrane. If the resistance gene product
is the receptor for the pathogen Avr gene product, it is expected that recognition occur at
the membrane level. Colletotrichum lindemuthianum is a hemibiotrophic fungus that
penetrates the bean cell wall (Bailey et a1. 1992). In addition, race specificity in C.
lindemuthianum is expressed after fungal penetration through the epidermal cell wall and

the primary hyphae of C. lindemuthianum remain external to the host plasma membrane,

81

which becomes invaginated around the fungus (Bailey et al. 1992). These Observations
suggest that the avirulence gene product maybe a host-specific elicitor located in the
membrane and pathogen recognition may occur at the surfaces of the infection hyphae
and host cell membrane.

Most of the disease resistance genes previously cloned confer resistance to
bacterial diseases and are localized in the cytoplasm. Bacterial Avr gene products are
known to be secreted in to the host cytoplasm through the type III secretory system (Bent
1996). However, little is known about the function of Avr proteins from fungal pathogens
and only a few fimgal Avr-generated Signals have been described. Race-specific elicitors
have been partially purified from two hemibiotrophic plant pathogens, C. fulvum and C.
lindemuthianum (Lamb et al. 1989). One well-studied example of race-cultivar specificity
is the Cladosporium fulvum / Lycopersicon pathosystem. Cf proteins possess extracellular
domains, which supposedly recognize the corresponding Avr proteins (Jones et al. 1994,
Dixon et al. 1996). Although Avr proteins of C. lindemuthianum have not been isolated,
occurrence of race-cultivar specificity suggests the presence of Avr-generated signal
triggering plant defense response. Based on the similarities between the C. fulvum/tomato
and C. lindemuthianum/bean pathosystems, one would expect that host-specific elicitors
and anthracnose resistance gene products are located at the membrane where the pathogen
is recognized.

Although the COK-4 region was amplified in both resistant and susceptible
parents of the mapping population, internal differences in nucleotide sequences exist as
indicated by restriction (Figure 3.4) and sequence analyses. Single nucleotide

polymorphisms (SNPS) identified in the COK-4 sequences of resistant and susceptible

82

bean lines and co-segregation of restriction patterns with disease phenotype, indicate that
the COK-4 gene is involved in anthracnose resistance. The four F, individuals found to be
recombinants between the SASl3 marker and the Co-4" gene, however, possessed the
COK-4 allele corresponding to the phenotype of the plant (data not shown). In tomato,
the Pto and Fen genes are present in bacterial Speck-susceptible and fenthion-sensitive
genotypes and encode a protein kinase 87 and 98% identical to the resistance alleles,
respectively (Jia et al. 1997). A SNP found in rice accounted for 80% of the variation in
amylose content (Ayres et al. 1997). Small variation in gene sequences, therefore, can
result in contrasting phenotypes.

Previous genetic studies indicated that the Co-4 locus is a complex gene family
(Young et al. 1998). Two resistance alleles have been described which mapped at the Co-
4 locus, one present in the bean cultivar TO and the other present in SEL 1308 as
supported by allelism test and DNA sequence analysis. TO showed a unique restriction
pattern and 44 nucleotide substitutions at the COX-4 region compared to SEL 1308.
Genetic analysis indicates a single gene segregating in the Black Magic/SEL 1308 F,
mapping population, however other genes may be tightly clustered at the Co-42 locus.
Bean cultivars appear to possess multiple copies of the COK-4 gene based on Southern
analysis. If the COK-4 homolog in T0 is non-functional and different from that in SEL
1308, clearly the filnctional Co-4 gene in TO must be linked to COX-4 and may be a gene
duplication based on the RFLP patterns. Another anthracnose resistance gene, Co-Z has
also been shown to be a complex multigene family (Geffroy et al. 1998). Sequence
analysis of a linked marker revealed multiple copies of LRR sequences clustered near the

Co-Z gene. Resistance genes appears to be clustered in the plant genome and may occur

83

 

in multiple copies spanning large regions of the plant genome (Kesseli et al. 1993,
Maisonneuve et al. 1994, Meyers et al. 1998).

These findings indicate that tightly linked molecular markers may be used to
identify disease resistance candidate genes. The SAS13 marker tagged the Co-4 locus as
it allowed the identification of different resistance alleles present in diverse bean
cultivars. The marker was used to clone the COK-4 gene from resistant cultivars as well
as homologs present in the susceptible cultivars. By comparing these homologs, SNPS
were identified and therefore, be more accurate than the SCAR marker in discriminating
the plant phenotype. The four recombinant F, individuals from the mapping population
between the marker and resistance locus, in fact, possessed the COK-4 allele
corresponding to the expected disease reaction confirmed by the restriction analysis. Most
important, SNPS could be used to identify three different alleles at the Co-4 locus. This
work represents the first report of the successful cloning of a disease resistance gene in
the plant family Leguminosae. The COX-4 gene that conditions resistance to a fiingal

pathogen is a gene ortholog of the Pto resistance gene present in tomato.

84

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Ayres, N.M., McClung, A.M., Larkin, P.D., Bligh, H.F.J., Jones, C.A., and Park, W.D.
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Balardin, R.S., Jarosz, A.M., and Kelly, JD. 1997. Virulence and molecular diversity in
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Balardin, RS. and Kelly, J .D. 1998. Interaction between Colletotrichum lindemuthianum
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Bent, AF. 1996. Plant disease resistance genes: function meets structure. The Plant Cell
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Dixon, M.S., Jones, D.A., Keddle, J.S., Thomas, C. M., Harrison, K., and Jones, J.D.G.
1996. The tomato Cf-2 disease resistance locus comprises two functional gene
encoding leucine-rich repeat proteins. Cell 84:451-459.

Flor, H.H. 1947. Host parasite interaction in flax rust — its genetics and other
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Geffroy, V., Creusot, F., Falquet, J., Sevignac, M., Adam-Blondon, A-F., Bannerot, H.,
Gepts, P., and Dron, M. 1998. A family of LRR sequences in the vicinity of the Co-2
locus for anthracnose resistance in Phaseolus vulgaris and its potential use in marker-
assisted selection. Theor. Appl. Genet. 962494-502.

Jia, Y., Loh, Y.T., Zhou, J., and Martin, GB. 1997. Alleles of Pto and Fen occur in

bacterial speck-susceptible and fenthion-sensitive tomato cultivars and encode active
protein kinases. Plant Cell 9261-73.

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Jones, D.A., Thomas, C.M., Hammond-Kosack, K.E., Balint-Kurti, P.J., and Jones,
J.D.G. 1994. Isolation of the tomato Cf-9 gene for resistance to Cladosporium fulvum
by transposon tagging. Science 266:789-793.

Hirokawa, T., Boon-Chieng, S., and Mitaku, S. 1998. SOSUI: Classification and
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Hofrnann K., Bucher P., Falquet L., and Bairoch A. 1999. The PROSITE database, its
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Kanazin, V., Marek, LE, and Shoemaker, RC. 1996. Resistance gene analogs are
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Kesseli, R.V., Witsenboer, H., Vandemark, G.J., Stangellini, ME, and Michehnore,
R.W. 1993. Recessive resistance to Plasmopara lactucae-radicis maps by bulked
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Lamb, C.J., Lawton, M.A., Dron, M., and Dixon, RA. 1989. Signals and transduction
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87

 

APPENDICES

88

APPENDIX A1

DEVELOPMENT OF A SCAR MARKER LINKED TO THE I GENE IN COMMON BEAN

ABSTRACT

Two 24-mer SCAR primers (SW13) were developed fi‘om a previously identified
10-mer RAPD primer (OW13690) linked to the I gene, which conditions resistance to bean
common mosaic virus (BCMV) in common bean. Linkage between SW13 and the I gene
was tested in three F, populations segregating for both SW13 and the I gene:
N84004/Michelite (1.0 i 0.7 cM), Seafarer/UI-114 (1.3 :1: 0.8 cM), and G91201/Alpine
(5.0 d: 2.2 cM). SW13 proved to be more specific and reproducible than the OW13690
RAPD marker. Using different heat stable DNA polymerases, SW13 amplified a single
690 bp fragment linked to the I gene which more consistently permitted the identification
of resistant plants. In addition, the presence of the I gene was detected using SW13 in
genotypes originating from different gene pools of Phaseolus vulgaris (L.), indicating a

broad utility of this marker for bean breeding programs.

89

INTRODUCTION

Bean common mosaic virus (BCMV) is a widespread and economically important
pathogen of common bean (Phaseolus vulgaris L.). The only effective way to prevent the
occurrence of the disease caused by this pathogen is to develop genetically resistant
cultivars (Kelly et al. 1995). Bean cultivars which carry the dominant I gene are resistant
to all known races of BCMV. However, when bean plants are infected with strains of
bean common mosaic necrosis virus (BCMNV), the presence of the I gene causes a lethal
hypersensitive reaction in the plant (Haley et al. 1994a). Combining the I gene with other
strain-specific recessive resistance genes (be-1, bc-2", and bc-3) will protect the plant
against BCMNV. However, using disease screening, it is not possible to detect the
presence of the I gene in the I/bc-3 gene combination (Morales and Komegay 1996),
because the recessive bc-3 gene epistatically masks the action of the I gene (Kelly 1995).
Hence, a reliable and specific molecular marker that can be used to indirectly select for
the hypostatic resistant I gene is needed to breed for BCMNV resistance.

A single random amplified polymorphic DNA (RAPD) marker (OW13690) has
been found to be tightly linked in coupling with the I gene (ranging from 1.3 i 0.8 to 5.0
i 2.2 cM) in five segregating populations (Haley et al. 1994a). However, use of RAPD
markers is restricted because they are very sensitive to variations in reaction conditions.
A precise match between the RAPD primer and the template DNA depends on the choice
of the heat-stable DNA polymerase and thermal cycler, the concentration of Mg”, and the

annealing temperature (Gu et al. 1995). Development of a specific repeatable protocol is

90

crucial for identifying robust polymorphisms and for obtaining consistent reproducibility
of RAPD analysis (Kelly 1995).

Recently, a more reliable and specific PCR-based marker known as sequence
characterized amplified region (SCAR) was developed. SCAR primers are longer than
RAPD primers and a highly stringent annealing temperature can be employed which
prevents mismatching in the priming Site during DNA amplification (Paran and
Michelmore 1993). Unlike RAPD, SCAR primers can amplify a single locus which
appears as a Single, easily-scored band in agarose gels.

In this research, the objectives were to (1) clone and sequence the DNA fragment
amplified by RAPD marker OW13690, (2) develop 24-mer SCAR primers with more
specificity than OW13690, (3) score the segregating bean populations developed by Haley
et al. (1994a) for presence or absence of both I gene and marker, (4) identify the
usefulness of these SCAR primers in amplifying the I locus across gene pools (Singh et
al. 1991), and (5) test the robustness of the SCAR primers with different heat stable DNA

polymerases.

91

MATERIALS AND METHODS

RAPD analysis. A RAPD marker linked to the I gene was identified using bulk
segregant analysis (BSA, Michelmore et a1. 1991) in near isogenic lines and linkage
between this RAPD marker and the I locus was determined in five F, segregating bean
populations. Development of these populations and their evaluation for resistance to

BCMV have been previously described (Haley et al. 1994a).

Cloning and sequencing of the RAPD fragment. The RAPD amplification
product linked to the I gene was purified and cloned using the PCR select spin column
following procedures of the Primer PCR Cloner system (5 prime-3 prime, Inc.). Cloned
amplification products were sequenced following the Sanger dideoxy-mediated chain
terminator method (Sanger et al. 1977). This process involved two steps: a) preparation of
DNA extension which consisted of 15 cycles of 20s at 94°C, 40s at 50°C, and 60s at 68°C
followed by 15 cycles of 205 at 94°C, and 60s at 68°C, and b) separation of fragments on
a polyacrylarnide gel using the ABI 373A DNA sequencer. Double-stranded DNA
sequencing was carried out using the -21M13 and M13 reverse fluorescently labeled

primers.

SCAR analysis. Amplification reactions were carried out in 25111 solution
containing 2 units of Stoffel DNA polymerase, 1X buffer, SmM MgCl, , 200p.M of each

dNTP, 25ng of DNA template and 25ng of SCAR primer mixture. The PCR procedure

consisted of 34 cycles of 108 at 94°C, 40s at 67°C, and 2min at 72°C, followed by one

92

cycle of 5min at 72°C. Amplification products were detected by direct staining with 0.05
pg of ethidium bromide in microtiter plates and then observed in a 1.4% agarose gel.
Three out of five segregating pOpulations developed by Haley et al. (1994a)
(N84004/Michelite, Seafarer/UI-114, and G91201/Alpine) and several bean genotypes
were screened using the SCAR primers. The presence or absence of the DNA
amplification in selected samples of the three segregating populations was confirmed
either with Amplitaq DNA polymerase (Perkin Elmer) or T aq DNA polymerase (BRL

Gibco) using the same amplification protocol.

Linkage analysis. The recombination frequency between the SCAR marker and

the I allele was calculated using the Linkage-1 software (Suiter et al. 1983).

93

RESULTS AND DISCUSSION

Two specific 24-mer SCAR primers were synthesized based on the DNA
fragment sequence amplified by the RAPD marker. The 24—mer oligonucleotides
contained the ten original base pairs of the RAPD marker plus the internal 14 base pairs

(Table A1.l).

Table A1.1. SCAR primer sequences derived from the RAPD marker OW13690 linked to

the I gene in common bean.

 

 

SCAR marker Primer Sequence“
SW13 W13.7XP 5’-CACAGQGACATTAATTTTCCTTTC-3’

W1 3 .4RP 5 ’-CACAGCGACAGGAGGAGCTTATTA-3 ’

 

*underlined sequences are the original RAPD marker

The SCAR marker SW13 amplified DNA fragments of the expected size (690 bp)
and appeared as a single polymorphic band in agarose gel (Figure A1.1). The
polymorphic SCAR primers were then used to amplify DNA from the mapping
populations. The same polymorphism was observed in both RAPD and SCAR analysis in
two populations, Seafarer/UI-114 and G91201/Alpine. Nevertheless, SW13 amplification
product differed from that of OW13690 in the N84004/Michelite population. In this case,
the I gene in three resistant plants was amplified by the SCAR marker but not by the
RAPD marker (Table A1.2). These longer SCAR primers showed increased specificity

and sensitivity when compared to the RAPD primers. Other reports also demonstrated

94

that longer primers were more specific and sensitive than MPD, although in some cases
they amplified an alternate allele from both parents and the original RAPD polymorphism
was lost (Paran and Michelmore 1993), while in other reports RAPDS were converted
into a codominant marker (Adam-Blondon et al. 1994, Timmerrnan et al. 1994, Horvath

et al. 1995, Ohmori et al. 1996).

Table A1.2. Linkage analyses of common bean F, populations segregating for the

molecular markers and the dominant 1 allele. Data for OW13690 from Haley et al. 1994a.

 

 

 

Population Expected ratio“ I/OW13690 I/SW13
cM1SE observed cM¢SE observed
fieqLency frequency
Seafarer/UI-114 9232321 1,3 1- 0,8 1632221257 13 1- 0,8 1542221255
N84004/Michelite 9232321 2.5 i 1.1 1402520251 1.0 t 0.7 1331220251
G91201/Alpine 3:6:3:l:2:1 5,0 t 2,2 32243232022225 5.0 i 2.2 31243232022225

 

*Expected ratios: 9232321, 9 I/- + marker: 3 I - - marker: 3 i/i + marker: 1 i/i - marker;
32623212221, 3 [/1 + marker: 6 I/i + marker: 3 i/i + marker: 1 1/1 - marker: 2 I/i - marker: 1

i/i - marker.

95

12345678910

s. 2’98' :r . t.. _-
- r; _ »
. " J ‘ ‘,
~ ‘ .I .
.- .-.‘ v _ _~_ . ,
.
a

    

->

 

Figure A1.1. DNA amplification of ten bean cultivars using the SW13 SCAR marker.
The PCR product were detected by direct ethidium bromide staining in microtiter plates
(top) and by gel electrophoresis (bottOm). (1) N84004 (M), (2) Michelite (i/i), (3) G91201
(1/1), (4) Alpine (i/i), (5) Seafarer (I/I), (6) UI-114 (i/i), (7) Chinook (1/1), (8) Sierra (i/i),
(9) Montcalm (1/1), (10) negative control (no template DNA was added to the PCR
reaction), (11) molecular weight marker (A Hind III/ EcoR 1; size of bands indicated in

base pairs).

96

Due to the dominant nature of the SW13 marker we were able to apply a quick
minus/plus assay to detect the PCR product. AS suggested by Gu et al. (1995), ethidium
bromide was added to the amplification reaction. Strong fluorescence was Observed in
samples containing amplified DNA whereas weak fluorescence was observed in samples
in which no fragment was expected. Results of all ethidium bromide assays were
validated and confirmed by gel electrophoresis. Figure Al.l shows an example of
detection of SW13 product either in agarose gels or through directly staining in microtiter
plates. Direct staining with ethidium bromide can be used in breeding programs when
genotyping a large number of samples is required, to facilitate the development of more
disease-resistant cultivars using marker-assisted breeding.

Use of different DNA polymerases in RAPD analysis has contributed to a lack of
reproducibility across laboratories because each enzyme generates a specific banding
pattern (Kelly 1995). Our results indicated that three different DNA polymerases can be
used to demonstrate the expected polymorphism without any interference in the
amplification of the I gene when SW13 primers were used (data not shown).

SW13 was tested in a collection of common bean genotypes representing both
Andean and Middle American gene pools (Singh et al. 1991). In all cases, the presence or
absence of the SCAR marker corresponded to the presence or absence of the I gene
(Table A1.3), indicating broad utility of this marker across bean gene pools. In previous
germplasm surveys of common bean, certain RAPD markers have been shown to be very
useful as tools for indirect selection in populations derived from both gene pools (Haley
et al. 1994b, Young and Kelly 1996), whereas the application of other markers was

restricted to a Specific gene pool (Miklas et al. 1993) or to a certain race within a gene

97

pool (Haley et al. 1993). In the present work, no gene pool or race specificity was
observed. The SCAR marker SW13 is currently being used to pyramid the I gene with

other epistatic recessive genes in the Michigan State University Bean Breeding Program

for bean common mosaic virus resistance.

98

Table A1.3. Presence (+) or absence (-) of the I allele and the SW13 marker in bean

genotypes from Andean and Middle American gene pools (Singh et al. 1991).

 

 

 

 

Gene Pools
Middle American Andean

Genotype I allele* SW13 Genotype I allele* SW13
Alpine - - A1 93 ND -
Blackhawk + + Chinook + +
Black Magic + + Isabella - -
Bunsi + + Kaboon - -
C-20 + + MDRK - -
Fiesta - - Montcahn + +
Fleetwood + + Perry Marrow - -
G91201 + + Ruddy ND +
Harofleet + + Widusa + +
Harokent + +

Mayflower + +

Michelite - -

N84004 + +

OAC Rico + +

Raven + +

Redkloud + +

Seafarer + +

Seaforth + +

Sierra - -

UI-114 - -

 

*Presence or absence of the 1 allele determined through inoculation with NL 3 strain of

BCMV; ND=not determined.

99

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mildew fungus (Elysiphe pisi D.C.). Theor. Appl. Genet. 88: 1050-1055.

Young, R.A., and Kelly, J.D. 1996. RAPD markers flanking the Are gene for anthracnose
resistance in common bean. J. Amer. Soc. Hort. Sci. 121(1): 37-41.

101

APPENDIX A2

GENETIC CHARACTERIZATION or THE Co-7 GENE PRESENT

IN THE BEAN CULTIVAR G 2333

INTRODUCTION

The differential bean cultivar G 2333 possesses three independent dominant genes
conditioning resistance to anthracnose (Chapter 2). Two of these genes were isolated in
two different bean breeding lines derived from G 2333, SEL 1308 possesses the Co-4"
gene and SEL 1360 possesses the Co-5 gene. The third resistance gene, Co- 7, could not
be isolated in a bean line because the Co-42 gene is epistatic to the Co-7 and no race of
the pathogen that distinguishes these two genes has been identified. The Objective of this
study was to develop a bean line that carries only the Co-7 gene in order to determine its

usefulness in controlling anthracnose.

102

MATERIAL AND METHODS

The F, population SEL 1360 x G2333 was used to identify resistant genotypes
carrying Co-7 but not Co-42. The segregating population was inoculated with race 521 to
overcome the Co-5 gene that is fixed in this population. In addition, the SAS13 marker
(Young et al. 1998) was used to detect the Co-4" gene and select genotypes without the
marker (Table A2.1).

Genotypes 1 through 8 are resistant and genotype 9 is susceptible when inoculated
with race 521 (15R:1S segregation ratio). Genotypes 1 though 6 have the SAS13 marker
linked to Co-4" (3:1 ratio for the presence and absence of the marker). We are interested
on genotypes 7 and 8, where Co-7 is present but Co-42 is absent and these genotypes were
identified by selecting resistant plants lacking the SAS13 marker (Chapter 2). Those
plants were selfed to distinguish between the homozygous and heterozygous for the Co-7

gene.

103

Table A2.1 Nine genotypes segregating in the SEL 1360 x G 2333 F, population.

Frequency, phenotype to race 521, and presence/absence of the SASl3 marker were

 

 

determined.
Genotypes Segregating Genes“ Frequency Phenotype SAS13 marker
1 C0-4: Co-43/Co-7 Co-7 l R +
2 Co-42 Co-43/ Co-7 co-7 2 R +
3 Co-4" Co-4" / co-7 co-7 1 R +
4 Co-4: co-43 /Co-7 Co-7 2 R +
5 C0-4: co-4: /Co-7 co-7 4 R +
6 C04" co-42 /co-7 co-7 2 R +
7 co-42 co-43 /Co-7 Co-7 1 R -
8 co-42 co-42 / Co-7 co-7 2 R -
9 co-4: co-43 / co- 7 co-7 1 S -

 

*The Co-5 gene is fixed in this population.

RESULTS

Using standard inoculation techniques and a linked molecular marker, a F,.,
family homozygous for the Co-7 (genotype 7) was identified and named SEL 111. Each
F, seed was selfed and maintained separate. The F,4 families were inoculated with several
anthracnose races (Table A22) and crossed to the susceptible cultivar Black Magic (BM).
The derived F, population was screened with race 73 to confirm the presence of the two
genes (Co-5 and Co-7). The Black Magic/SEL 111 population unexpectedly segregated
3:1 (78R229S) suggesting the presence of one gene. The data suggest that the Co-7 gene

must have been overcome by race 73 since Co-5 is resistant to race 73. Support for this

104

hypothesis came indirectly from work conducted by Alzate-Marin et al. (1998) in Brazil.
In an F, population derived from the cross Rudy (susceptible)/G 2333 inoculated with
race 73, Alzate-Marin et al. (1998) observed a 1521 ratio suggesting that G 2333
possessed only two independent dominant genes. The third gene in the population tested
by Alzate-Marin et al. (1998), must have been defeated by race 73 and lends support to

the observation herein reported.

Table A2.2 Disease evaluation on genotypes carrying anthracnose resistance genes

derived from G 2333. Six plants of each genotype were inoculated.

 

 

 

Race
Genotype 7 73 449 521 1545 1929 2029 2047
G 2333 R* R R R R R R R
SEL 1308 R R R R R R R R
SEL 111 R R R R S S S S
Black Magic R S R S S S S S

 

*R=resistant reaction, S= susceptible reaction.

105

DISCUSSION

Based on recent findings suggesting that more alleles exist at previously described
locus, we did not discard the hypothesis that the Co-7 gene (the third resistance gene of G
2333) could be an allele instead of a new locus. The two major resistance genes defeated
by race 73 are the Co-2 and Co-3 genes. G 2333 does not possess the Co-2 gene since
markers linked to the C o-2 are absent in G 2333 and the original F, population inoculated
with race 521 at CIAT indicated the presence of genes other than Co-2, which is
overcome by race 521. Since the third gene is resistant to race 521 and susceptible to race
73, the most likely candidate would be the Co-3 gene or an allele at that loci. Current
plans are underway to inoculate the same F, population with race 521 which overcome
the Co-5 and should permit the identification of the third gene, since neither are
pathogenic on Co-3. In addition, to confirming the presence of two independent genes in
SEL 111, the same population will be inoculated with a race which is ineffective against
either the Co-3 or Co-5 genes.

In order to confirm the presence of the Co-3 locus in SEL 111, the line was
crossed to Mexico 222 (Co-3) and the F, population will be inoculated with a race that
confers R/R reaction to both parents. Race 521 will be selected since it overcomes the
Co-5 gene, but not the putative Co-3 gene in SEL 111. If SEL 111 and Mexico 222 prove
to be allelic, then there should be no segregation regardless of whether the Co-5 gene is
present or absent. This would give us insights into the identity of the third gene in G

2333. Markers for the Co-3 gene will need to be developed to confirm these results.

106

Markers for the Co-6 gene need to be screened in G 2333 and SEL 111 to eliminate this
resistance locus as a candidate for the third gene.

Preliminary results indicated that SEL 111 is susceptible to race 1929. If this is
confirmed then the theory that the third gene in G 2333 is Co-3 is unlikely unless SEL
111 carries a weaker allele at this locus. A weaker allele of the Co-3 gene was reported
(Fouilloux, 1979) in Mexico 227 (now extant). This allele can be discriminated fiom the
original Co-3 gene by race 31 (kappa). Co-3 is resistant to race 31 whereas, the second
allele is susceptible. Both alleles are susceptible to races 73 and 89, however, SEL 111
cannot be inoculated with race 31 to determine if it carries the Co-3 resistant gene or the
susceptible allele at the same locus, since SEL 111 also carries the Co-5 gene which
confers resistance to race 31.

In order to test this possibility, resistant F, progeny of the BM/SEL 111
pOpulation identified after inoculation with race 521 will be selected and screened against
the marker linked to the Co-5 gene to identify homozygous resistant individuals absent
for the Co-5 gene but carrying the third gene. Those individuals could be inoculated with
race 31 and crossed with Mexico 222, known to carry the Co-3 gene. Allelism tests will

be conducted to confirm if the third gene in G 2333 is in fact the Co-3 gene.

107

REFERENCE

Alzate-Marin, A.L., Baia, G.S., de Paula Junior, T.J., de Carvalho, G.A., de Barros, E.G.,
and Moreira, M.A. 1997. Inheritance of anthracnose resistance in common bean
differential cultivar AB 136. Plant Dis. 81:996-998.

Fouilloux, G. 1979. New races of bean anthracnose and consequences on our breeding
programs. Pages 221-235 in: Disease of tropical food crops. H. Maraite and J.A.
Meyer, eds. Louvain-la-Neuve, Belgium.

Young, R.A., Melotto, M., Nodari, R.O., and Kelly, J.D. 1998. Marker assisted dissection
of oligogenic anthracnose resistance in the common bean cultivar, G2333. Theor.
Appl. Genet. 96287-94.

108

APPENDIX A3

SCAR MARKER LINKED TO THE RUST DISEASE RESISTANCE GENE UR-5

IN COMMON BEAN

INTRODUCTION

In common bean (Phaseolus vulgaris L.), resistance to several pathogens is
controlled by major genes. Indirect selection for those genes would be greatly facilitated
if tightly linked molecular markers were available. SCAR (sequence characterized
amplified region) markers were chosen for selection purposes because they have proven
to be reliable, reproducible, robust, and easily scored when a single polymorphic band is
generated (Gu et al. 1995, Melotto et a1. 1996). Here, the development of a SCAR
markers linked to the major disease resistance gene Ur-5 is reported. This gene conditions
resistance to multiple races of bean rust caused by Uromyces appendiculatus (Pers.)

Unger var. appendiculatus (Stavely 1984).

109

 

MATERIALS AND METHODS

The source for the gene Ur-5 was the genotype B-190. Random amplified
polymorphic DNA (RAPD) marker linked to this gene was previously identified OIl9460
(Haley et al. 1993). The amplification products were cloned and sequenced following
procedures described by Melotto et al. (1996). SCAR primers were designed based on
those sequences and specific PCR (polymerase chain reaction) files were created to
amplify a single polymorphic band linked to the resistance gene of interest. Linkage
between the SCAR marker and the resistance gene was calculated based on the

recombination ratio observed in segregating populations.

RESULTS AND DISCUSSION

The SCAR marker developed, amplified a single polymorphic band of the same
size as the correspondent RAPD marker. In Table A3.1, the molecular marker, the PCR
file, and the linkage between the gene and the marker are specified. The SCAR primer

sequences are shown in Table A3.2.

Table A3.1 Molecular marker, PCR file, and linkage between the Ur-5 gene and the 8119

 

 

marker.
Gene RAPD marker SCAR marker PCR file Linkage
Ur-5 OIl9460 S119 94°/IOSCC; 67°/405ec; 72°/2min. 0 cM*

34 cycles

 

*No recombinants were found.

110

Identification of linked SCAR markers is particularly useful for marker-assisted
selection and can be readily used for locating resistance genes on linkage maps. The Ur-5
gene has been mapped to linkage group B4 of integrated bean linkage map (Freyre et al.
1998). Resistance genes often present epistatic interactions and are difficult to combine
with test crossing the progeny. The marker S119 has been used to facilitate the
pyramiding of Ur-5 resistance gene into previously resistant bean germplasm (Stavely
1999) in order to achieve broad long-term resistance to the variable pathogens U.

appendiculatus.

Table A3.2 SCAR primer sequences derived from the RAPD marker OIl9460 linked to

the Ur-5 gene in common bean.

 

 

Gene SCAR marker Primer sequence (5’-3’)* Primer length
Ur-5 SIl9 AATGCGGGAGATATTAAAAGGAAAG 25-mer
AATQCGGGAGTTCAATAGAAAAACC 25-mer

 

*Underlined sequence corresponds to the original 10-mer RAPD primer.

lll

REFERENCES

Freyre, R., Skroch, P.W., Geffroy, V., Adam-Blondon, A.-F., Shirmohamadali, A.,
Johnson, W.C., Llaca, V., Nodari, R.O., Pereira, P.A., Tsai, S.M., Tohme, J., Dron,
M., Nienhuis, J., Vallejos, and CE, Gepts, P. 1998. Towards an integrated linkage
map of common bean. 4. Development of a core linkage map and alignment of RF LP
maps. Theor. Appl. Genet. 97:847-856.

Gu, K.W., Weeden, N.F., Yu, J., and Wallace, DH. 1995. Large-scale, cost-effective
screening of PCR products in marker-assisted selection applications. Theor. Appl.
Genet. 91:465-470.

Haley, S.D., Miklas, P.N., Stavely, J .R., Byrum, J ., and Kelly, J .D. 1993. Identification of
RAPD markers linked to a rust resistance gene block in common bean. Theor. Appl.

Genet. 86:505-512.

Melotto, M., Afanador, L., and Kelly, J.D. 1996. Development of a SCAR marker linked
to the I gene in common bean. Genome 39:1216-1219.

Stavely, JR. 1984. Genetics of resistance to Uromyces phaseoli in a Phaseolus vulgaris
line resistant to most races of the pathogen. Phytopathology 74:339-344.

Stavely, JR. 1999. Bean rust in the United States in 1998. Ann. Rep. Bean Improv. Coop.
422 35-36.

112