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This is to certify that the
dissertation entitled

INHERITANCE STUDIES AND DEVELOPMENT OF RAPD
MARKERS FOR MAJOR ANTHRACNOSE RESISTANCE
GENES IN COMMON BEAN

presented by

Roberto Antonio Young Bustillo

has been accepted towards fulﬁllment
of the requirements for

Doctoral Crop & Soil Sciences -

de eein
gr PIant Breeding 8: Genetics

 

 

Major professor

/

Date {’3 ‘95,

 

MS U is an Afﬁrmatiw Action/Equal Opportunity Institution 0-12771

 

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Michigan State
Unlverslﬁy

 

 

 

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DATE DUE DATE DUE DATE DUE

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INHERITAN CE STUDIES AND DEVELOPMENT OF RAPD MARKERS FOR
MAJOR ANTHRACNOSE RESISTANCE GENES IN COMMON BEAN

By

Roberto Antonio Young Bustillo

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

1995

ABSTRACT

INHERITAN CE STUDIES AND DEVELOPMENT OF RAPD MARKERS FOR
MAJOR ANTHRACNOSE RESISTANCE GENES IN COMMON BEAN

By

Roberto Antonio Young Bustillo

The inheritance of the genetic resistance to bean anthracnose in genotypes,
Catrachita and SEL 1360, derived from two differential cultivars, AB 136 and G 2333, is
described. Segregation data indicated that a single dominant gene is responsible for the
anthracnose resistance in Catrachita, and the gene was situated at a diﬂ‘erent locus from
previously characterized resistance genes: A, Are, Mexique 1, Mexique 2 and Mexique 3.
It is proposed that the single dominant resistance gene present in Catrachita (AB 136) be
assigned the gene symbol Are 1024 corresponding to its ranking in the bean anthracnose
diﬁ‘erential cultivar series. Segregation data suggested that a single dominant gene was
conditioning resistance to anthracnose in SEL 1360, and the gene is independent from the
A, Are, Mexique 1, and Mexique 2 genes. However, the dominant gene in SEL 1360 did
not segregate independently from the resistance gene Mexique 3 in the differential cultivar
TU, demonstrating that both dominant alleles are located at the same locus. Independent
segregation of the two dominant genes was also observed between Catrachita and SEL

1360, indicating that these genes are independently inherited.

Ah

Six random ampliﬁed polymorphic DNA (RAPD) markers linked to the Are, A,
Are 1024 and Mexique 3 loci were developed using near-isogenic lines, bulked segregant
analysis, and heterogeneous inbred populations either singly or in combination. A RAPD
marker designated OQ4mo, was found tightly linked in coupling-phase with the Are gene.
OQ41440, mapped at 2.0 :1: 1.4 centimorgans (cM) from the Are allele in the Andean
genetic background and at 5.5 i 2.3 cM in the Middle American background. A second
coupling-phase RAPD marker B355woo, mapped at 5.4 :t 2.3 cM fiom the Are allele in the
Andean genetic background and at 7.7 i: 2.7 cM in the Middle American background.
Based on a recombination distance of 7.0 d: 1.9 cM between the two RAPD markers,
OQ41440 and 33551000 appear to ﬂank the Are gene. The bracketing molecular markers
allowed tagging of the Are allele with a selection fidelity of 99%. The OF10530 RAPD was
tightly linked in repulsion-phase to the A allele (1.9 i 1.4 cM). A survey of bean
genotypes for the presence or absence of the marker locus, revealed that OFlOm RAPD
would facilitate the introgression of the A gene across Phaseolus gene pools. A coupling-
phase RAPD marker OAH 1m and a repulsion-phase RAPD marker OAK20390,
cosegregated with alleles at the Are 1024 locus in three different segregating populations.
The OAHlm and OAKZOsgo RAPD markers were found linked (12.3 :1: 1.9 and 7.3 i 1.5
cM, respectively) and ﬂanked the Are 1024 locus. Coupling and repulsion-phase RAPD
markers when used as a codominant pair showed higher selection eﬂiciency, for the
identiﬁcation of homozygous (Are 1024 Are 1024) F2 individuals, as opposed to individual
selection for either a coupling-phase or a repulsion-phase marker. The RAPD marker
OAB3450 cosegregated with the Mexique 3 allele in two different populations. The

OAB3450 RAPD was found linked at 5.9 :t 1.7 in coupling with the Mexique 3 allele.

Pyramiding four major anthracnose resistance genes using marker-assisted selection is
now feasible in common bean, and the effective durability of the resistance to C.
Iindemuthianum could be increased if Andean and Middle American genes were

combined.

DEDICATION

To my wife, Nane, with admiration and love.

To my children, Gabriel, Rebeca, Paulo Roberto and Luis Eduardo, I hope that this

achievement will serve as an inspiration to give your best in life.

01

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10

ACKNOWLEDGMENTS

I would like to express my sincere gratitude to all those individuals that in one way
or another helped me to complete my doctoral program:

Dr. James D. Kelly, my academic advisor, for his thoughtful guidance throughout
my research project, his willingness to share his wealth of experience as a scientist, his
openness to discuss (at any time) all the different aspects of my research work, and for the
friendship of his family.

Drs. Amy Iezzoni, Brian Diers and Patrick Hart, for serving on my guidance
committee, giving helpful advise during my research studies and for reviewing my
dissertation.

The bean group: Lucia Afanador, Jerry Taylor, Norman Blakely, Ricardo
Balardin, and Kristin Schneider, for the various help in the lab and greenhouse, for good
challenging discussions and for their friendship.

My family: Paco and Lety, Joel and Teresinha, for their unconditional support and
love.

Latin American Scholarship program of American Universities (LASPAU), for
ﬁnancial support.

To the Escuela Agricola Panemericana, El Zamorano, for supporting my candidacy
to come to MSU to pursue a PhD. degree.

Last but not least: To you Lord God, my strength and inspiration.

TABLE OF CONTENTS

LIST OF TABLES ........................................................................................................ ix
LIST OF FIGURES ....................................................................................................... xi
GENERAL INTRODUCTION ....................................................................................... 1

LIST OF REFERENCES .......................................................................................... 22
CHAPTER ONE:

CHARACTERIZATION OF THE GENETIC RESISTANCE TO
COLLETOYRICHWLINDEMUYHIANW IN COMMON BEAN

DIFFERENTIAL CULTIVARS ................................................................................ 3 1
ABSTRACT. ............................................................................................................ 3 1
INTRODUCTION .................................................................................................... 33
MATERIALS AND METHODS .............................................................................. 35
RESULTS ................................................................................................................ 41
DISCUSSION .......................................................................................................... 45
LIST OF REFERENCES .......................................................................................... 52
CHAPTER TWO:
RAPD MARKERS FLANKING THE ARE ANTHRACNOSE RESISTANCE
GENE IN COMMON BEAN .................................................................................... 55
ABSTRACT. ............................................................................................................ 55
INTRODUCTION...............................‘ ..................................................................... 56
MATERIALS AND METHODS .............................................................................. 58
RESULTS ................................................................................................................ 62
DISCUSSION .......................................................................................................... 68
LIST OF REFERENCES ................................................................................... . ....... 73
CHAPTER THREE:
RAPD MARKERS FOR MAJOR ANTHRACNOSE RESISTANCE GENES IN
COMMON BEAN .................................................................................................... 76

vii

ABSTRACT. ............................................................................................................ 76

INTRODUCTION .................................................................................................... 78
MATERIALS AND METHODS. ............................................................................. 81
RESULTS. ............................................................................................................... 83
DISCUSSION. ......................................................................................................... 96
LIST OF REFERENCES ........................................................................................ 105
APPENDIX ................................................................................................................ 109

viii

I:

T;

T;

 

 

 

LIST OF TABLES

Table 1.1 Phenotypic characterization of parental, original (AB 136, G 2333) and
derived (Catrachita, SEL 1360, SEL 1308) sources of resistance for
their reaction to different races of C. Iindemuthianum. ................................ 38

Table 1.2 Crosses, generation and race of C. Iindemurhianum used for the genetic
characterization of the resistance in Catrachita (AB 136) and SEL

1360 (G 2333) ............................................................................................ 39
Table 1.3. Phenotypic characterization and observed ratio of F2 progeny and F23
families for their reaction to Collelotrichum Iindemuthianum. ...................... 42

Table 1.4. Allelism test for the genetic characterization of the resistance to
Colletom'chum Iindemuthianum in Catrachita (AB 136) and SEL 1360
(G 2333). .................................................................................................... 44

Table 2.1. Two-point chi-square 0(2) analyses and linkage estimates for marker
loci (OQ4mo and 33552000) and the anthracnose resistant Are allele ............. 63

Table 2.2 Chi-square (X2) analysis for OQ4mo and B35510.» RAPD markers and
the Are allele segregating in Middle American and Andean F2 and F213

populations. ................................................................................................ 65
Table 2.3 Efﬁciency of RAPD marker(s) linked to the Are allele for marker-based

selection within and across common bean gene pools ................................... 67
Table 3.1 Chi-square (x2) analysis for 01:10,30 RAPD marker and the A allele

segregating in the N85006 S/N 85006 R F2 and F 2;; population. ................... 86

Table 3.2 Survey of common bean genotypes from different market classes and
gene pools for the presence of the A locus and the OFlOm RAPD
marker. ....................................................................................................... 87

Table 3.3 Chi-square (X2) analysis for OAHlm and OAKZOxgo RAPD markers
and the Are 1024 allele segregating in three different F2 and F23

populations. ................................................................................................ 91
Table 3.4 Two point Chi-square (X2) analysis and linkage estimates for marker

loci (OAHlm and OAK20m) and the Are 1024 locus ................................. 92
Table 3.5 Chi-square (X2) for the Mexique 3 allele and the OAB3450 RAPD

marker segregating in two different F2 and F223 populations .......................... 95

ix

Table 3

Table 3

 

 

 

 

 

 

 

 

Table 3.6 Two point Chi-square (X2) analysis and linkage estimates for OAB3450
RAPD marker and the Mexique 3 allele. ...................................................... 95

Table 3.7 Selection efﬁciency (%) observed based on selection for a coupling
RAPD OAHlm and against a repulsion RAPD OAK20290, and for
both RAPD markers scored as one codominant marker (OAHlm-
OAK20290) in MAS for homozygous resistant individuals for the Are
1024 resistance gene segregating in three diﬂ‘erent F2 populations ................ 99

 

 

 

 

 

Figure I

Figure I

 

Irigure 3

 

LIST OF FIGURES

Figure 2.1 Ampliﬁcation of genomic DNA using OQ4mo RAPD marker. Key to
individuals: (1) anthracnose-susceptible Andean parent, (2)
anthracnose-resistant Andean parent, (3) susceptible Andean F2
individual, (4) resistant Andean F2 individual, (5) anthracnose-
susceptible Middle American DNA bulk, (6) anthracnose-resistant
Middle American DNA bulk, (7) susceptible Andean cultivar
(Montcalm), (8) resistant Andean cultivar (Isles), (9) anthracnose-
susceptible Middle American parent (Ex Rico 23), (10) anthracnose-
resistant Middle-American parent (OAC Rico), (11) anthracnose-
susceptible Middle American cultivar (Mayﬂower), (12) anthracnose-
resistant Middle American cultivar (Harokent), (13) molecular weight
marker ( l Hind III / EcoRl; size of bands indicated in bp). .......................... 66

Figure 3.1 Ampliﬁcation of genomic DNA using OF 10530 RAPD marker. Key to
individuals: (1) anthracnose-susceptible NIL N85006-S, (2)
anthracnose-resistant NIL N85006-R (3) homozygous resistant F2
individual, (4) heterozygous resistant F2 individual, (5) homozygous
susceptible F2 individual, (6) Andean cultivar with A gene (Michigan
Dark Red Kidney), (7) Middle American cultivar with A gene
(Sanilac), (8) Andean cultivar with A gene (Montcalm), (9) Middle
American cultivar without A gene (Cornell 49242), (10) negative
control (no template DNA added to PCR ampliﬁcation), (11)
molecular weight marker (7L Hind III/EcoRI: size of bands indicated in
bp) .............................................................................................................. 85

Figure 3.2 Ampliﬁcation of genomic DNA using OAHlm RAPD marker. Key to
individuals: (1) anthracnose-susceptible Black Magic/Catrachita DNA
bulk, (2) anthracnose-resistant Black Magic/Catrachita DNA bulk, (3)
susceptible parent, Black Magic, (4) resistant parent, Catrachita, (5)
susceptible parent, Raven, (6) resistant cultivar, AB 136, (7)
susceptible parent, Blackhawk, (8) homozygous resistant F2 individual,
(9) heterozygous resistant F2 individual,(10) homozygous susceptible
F2 individual, (11) negative control (no template DNA added to PCR
ampliﬁcation), (12) molecular weight marker (1 Hind HI/EcoRI: size
of bands indicated in bp) .............................................................................. 89

r1,

Figurre 3.3 Ampliﬁcation of genomic DNA using OAKZOm RAPD marker.
Key to individuals: (1) anthracnose-susceptible Black
Magic/Catrachita DNA bulk, (2) anthracnose-resistant Black
Magic/Catrachita DNA bulk, (3) susceptible parent, Black Magic, (4)
resistant parent, Catrachita, (5) susceptible parent, Raven, (6)
resistant cultivar, AB 136, (7) susceptible parent, Blackhawk, (8)
homozygous resistant F2 individual, (9) heterozygous resistant F2
individual,(lO) homozygous susceptible F2 individual, (11) negative
control (no template DNA added to PCR ampliﬁcation), (12)
molecular weight marker (1 Hind III/EcoRI: size of bands indicated
in bp) ............................................................................................................. 90

Figure 3.4 Ampliﬁcation of genomic DNA using OAB3450 RAPD marker. Key
to Individuals: (1) anthracnose-susceptible Raven/SEL 1360 DNA
bulk, (2) anthracnose-resistant Raven/SEL 1360 DNA bulk, (3)
homozygous resistant F2 individual,(4) heterozygous resistant F2
individual, (5) homozygous susceptible F2 individual, (6) resistant
parent with Mexique 3 gene, SEL 1360, (7) resistant cultivar, with
Mexique 3 gene, TU, (8) resistant cultivar with Mexique 3 gene, G
2333, (9) resistant line without Mexique 3 gene, SEL 1308, (10)
resistant cultivar without mexique 3 gene, AB 136, (11) negative
control (no template DNA added to PCR ampliﬁcation), (12)
molecular weight marker (A Hind III/EcoRI: size of bands indicated
in bp) ............................................................................................................. 94

xii

 

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GENERAL INTRODUCTION

Biotic stresses are among the main biological constraints affecting common bean
(Phaseolus vulgaris L.), production worldwide. Bean anthracnose, caused by
Colletotrichum lindemuthianum (Sacc. & Magnus) Lambs.-Scrib., is recognized as one of
the most wide spread and economically important fungal diseases of common bean
(Hubbeling, 1957; Goth and Zaumeyer, 1965; Zaumeyer and Meiners, 1975; Chaves,
1980; Pastor-Corrales and Tu, 1989). Anthracnose is a seed-transmitted disease and when
conditions of high humidity (> 92%) and moderate temperatures (17-24 °C) are met,
complete yield losses can be induced on susceptible genotypes (Zaumeyer and Thomas,
1957; Tu and Aylesworth, 1980; Pastor-Corrales and Tu, 1989).

The pathogen is capable of infecting different parts of the bean plant during all
stages of growth. The initial symptom of anthracnose infection is a dark brown to black
lesion with purplish red margins along the veins of the lower surface of leaves. Similar
symptoms can be observed on leaf petioles and stems. On bean pods, symptoms appear as
circular, reddish-brown lesions, which develop into sunken cankers where pink to ﬂesh
colored fruiting structures (acervuli) containing spores (conidia) of the ﬁingus develop
(Saettler and Hart, 1983). After invading the pod, mycelia and conidia of the fungus infect
the seed coat and become established. Infected seeds are discolored and may develop

brown to black cankers which can extend into the cotyledons, resulting in losses in yield,

 

 

 

 

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and seed quality (Zaumeyer and Thomas, 1957; Saettler and Hart, 1983; Tu, 1992; Pastor-

Corrales and Tu, 1989).

Pathogenic variation. The host range of C. Iindemuthianum is largely restricted
to bean (P. vulgaris), cowpea (Vigna unguiculata) and tepary bean (P. acutifolius)
(Zaumeyer and Thomas, 1957). Mthin the P. vulgaris species, C. Iindemuthianum has
shown extensive physiological differentiation. The existence of pathogenic races was ﬁrst
observed by Barrus (1911), who distinguished two distinct isolates which differed in their
ability to infect a group of bean genotypes. The two characterized biological strains were
designated as alpha and beta (Barrus, 1918). Since then, the occurrence of other races has
continuously been reported in the literature: race gamma (Burkholder, 1923), delta
(Andrus and Wade, 1942), epsilon (Charrier and Bannerot, 1970), alpha-Brazil (F ouilloux,
1975), lambda (Hubbeling, 1976), kappa (Kruger et al., 1977), jota (Hubbeling, 1977). In
general, much broader pathogenic variation of C. lindemuthianum has been observed in
Mexico, Central America, to a lesser extent, the Andean region, than has been observed in
Europe and North America (Beebe and Pastor-Corrales, 1991). For instance, in Mexico
races are assigned to ﬁve distinct groups (Groups I, II, III, IV and alpha) based on their
reaction on a set of genotypes. Using this differential set, Yerkes and Ortiz (1956),
reported the existence of race alpha, beta and gamma and ten new races Aof the pathogen
designated MA-l through MA-lO. Recently, Garrido-Ramirez and Romero-Cova (1989),
reported the identiﬁcation of eight new races, designated MA-23 to MA-30; adding to the
collection of 22 previously described races of anthracnose in MexiCo. In Central America,
using the anthracnose differential cultivar set proposed by CIAT (Centro Internacional de

Agricultura Tropical, Cali, Colombia) (CIAT, 1988; Pastor-Corrales, 1991), Rava et al.,

 

 

 

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(1993) reported the identiﬁcation of seven different pathotypes, six of which were alpha-

Brazil type while the remaining one belonged to the Mexican Group I. Pathogenic
variation, in Brazil, was classiﬁed into groups of races: alpha, delta and Brazilian I and II;
and individual races are identiﬁed as ‘BA’ followed by a number (Oliari et al., 1973; Pio-
Ribeiro and Chaves, 1975). Seven physiologic races identiﬁed by Oliari et al., (1973)
were classiﬁed as belonging to the Brazilian groups I and H, alpha group and Mexican
group I. More recently, Menezes and Dianese (1988), identiﬁed races alpha, delta,
epsilon, kappa and lambda and the new races zeta, eta, teta and mu, ﬁom a comprehensive
collection of isolates ﬁ'om different parts of Brazil. Race characterization have also been
conducted in Colombia where race beta, delta, kappa, alpha-Brazil, Mexico group II and
new isolates not previously reported, were observed (Cobo-Soto, 1986; Pastor-Corrales
and Tu, 1989). In Africa, Leakey and Simbwa-Bunnya (1972), reported ﬁve new races
among 19 Uganda isolates. Races resembling alpha, beta, delta and gamma pathotypes
were also observed in this country. Apart ﬁ'om beta and delta, gamma was the most
abundant race observed in Malawi; where three new races of C Iindemuthianum were also
reported (Ayonoadu, 1974).

It is clear that worldwide C. lindemuthianum has demonstrated considerable
variation in pathogenicity. However, comparisons of race characterization studies
between regions becomes very difficult due to different sets of differential cultivars
commonly used by researchers. Currently, bean researchers are using the established
universal set of differential cultivars and the numerical system for classifying races (CIAT,
1988; Pastor-Corrales, 1991), to facilitate data exchange and breeding work. The

proposed set of anthracnose differential cultivars is composed of 12 genotypes, each

 

 

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differential cultivar has a ﬁxed position in the general order of the set, and is identiﬁed

with a numeric value. The genotypes and their binary values are: Michelite (1), Michigan
Dark Red Kidney (2), Perry Marrow (4), Cornell 49242 (8), Widusa (16), Kaboon (32),
Mexico 222 (64), PI 207262 (128), TO (256), TU (512), AB136 (1024), and G 2333
(2048). The numerical system (binary system) of nomenclature is used for classiﬁcation of
races, based upon the spectrum of pathogenicity exhibited by an isolate on the 12
differential genotypes. Values are assigned as the sum of the binary numbers of the

differentials on which the isolate is pathogenic.

Host genetic resistance. Genetic resistance to C. Iindernuthianam was ﬁrst
observed in the cultivar, Wells Red Kidney, which was later released for commercial
production (Barrus, 1911, 1915). It was determined that the anthracnose resistance in
Wells Red Kidney was governed by a single dominant gene which conferred resistance to
races alpha and beta (Burkholder, 1918; McRostie, 1919). At present, ﬁve major
anthracnose resistance genes have been genetically characterized. They include: A
(McRostie, 1919), Are (Mastenbroek, 1960) and Mexique 1, 2 and 3 (F ouilloux, 1979).
However, more complex genetic systems responsible for the resistance to bean
anthracnose have been observed in several studies. To explain the resistance to races beta
and gamma, Andrus and Wade (1942) proposed a system of ten genes in three
allelomorphic series, and gene interaction at three loci. Both duplicate and complementary
genes and one dominant gene for susceptibility. Evidence suggesting duplicate factor loci
and complementary factors with multiple allelism was observed to condition resistance to

races alpha, beta and gamma (Cardenas et al., 1964). In addition, linkages between genes

 

 

 

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from distinct duplicate and complementary factor sets was also reported. Independent and

complementary gene action at one or two different loci conferred resistance to races beta
gamma and delta (Muhalet et al., 1981). An allelomorphic series of three alleles was also
observed controlling resistance to race beta. It is noteworthy to indicate that studies on
the genetics of the resistance may differ depending. on the genotypes involved and/or the
races of C. Iindemuthianum used.

The single dominant Are gene which confers resistance to alpha, beta, gamma,
delta, epsilon and lambda races (Pastor-Corrales and Tu, 1989) has been the source of
resistance most widely utilized by plant breeders throughout the world (North America:
Tu and Aylesworth, 1980; Muhalet et al., 1981; Tu, 1988, 1992. Europe: Mastenbroek,
1960; Goth and Zaumeyer, 1965; Fouilloux, 1979. Latin America: Menezes and Dianese,
1988. Aﬁica: Leakey and Simbwa-Bunnya, 1972; Ayonoadu, 1974). Despite the broad
resistance shown by the Are gene, numerous races have been observed to be pathogenic
on Are, thus limiting its useﬁrlness in Latin America (Menezes and Dianese, 1988; Pastor-
Corrales and Tu 1989; Beebe and Pastor Corrales, 1991; Rava et al., 1993). Break down
of Are resistance by the kappa race has also been reported in Europe (Kruger et al., 1977),
and more recently races 73 and alpha-Brazil overcome the resistance in North America
(Kelly et al., 1994a; Tu, 1994). In addition to race 73, Kelly et al. (1994a) also reported
the ﬁrst appearance of race 7 which was virulent on genotypes carrying the A resistance
gene. Considering that most of the commercial cultivars in Michigan carry either A or Are
genes, bean production in this state is under potential threat from these two new races.

However, the combination of A and Are genes in a single genotype would afford

 

 

 

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protection to all pathogenic variation of C. Iindemuthianum currently observed in North

America including races 7, 73 and alpha-Brazil.

Preventing an anthracnose epidemic in the case of a break down of Are resistance,
bean breeders in Europe identiﬁed new sources of resistance to this disease. Three
sources of resistance were found in a collection of Mexican bean gerrnplasm, and the
sources were characterized as monogenic, dominant genes independent from Are. These
genes were described as Mexique 1, 2 and 3 (Fouilloux, 1976; 1979). Among the
European races of the pathogen, Mexique 2 and Mexique 3 genes conferred resistance to
all known races, while the Mexique 1 resistance was overcome by the alpha-Brazil race.
In Latin America, Beebe and Pastor-Corrales (1991), reported that the Mexique genes
were susceptible to fewer isolates of anthracnose than was the Are gene. These three
major resistance genes have been included as members of CIAT’s differential cultivars;
Mexico 222 (Mexique 1), TO (Mexique 2) and TU (Mexique 3).

In recent years, a major effort to evaluate gerrnplasm for anthracnose resistance
has been undertaken by scientists at CIAT. Thousands of gennplasm accessions ﬁ'om
CIAT’s gene bank were screened for new sources of anthracnose resistance using a
diverse collection of isolates of the pathogen (Schwartz et al., 1982; Pastor-Corrales and
Tu, 1989; Pastor-Corrales et al., 1995). Among the accessions that showed the broadest
anthracnose resistance were AB 136 and G 2333 cultivars both members of CIAT’s set of
differential genotypes. The differential cultivar AB 136, is a small red seeded bean from
Central America, where it has been used as a source of anthracnose resistance for cultivar
development. Recently, a breeding line RAB 205 derived from AB 136, developed by

CIAT, was released as the cultivar Catrachita in Honduras (Beebe and Pastor-Corrales,

 

 

 

1991)
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7
1991). Studies on the inheritance of the resistance of AB 136 were conducted by

Goncalves-Vldigal (1994). A series of crosses involving AB 136 and a group of
differential cultivars (Michelite, Perry Marrow, MDRK, CN 49242 and PI 207262) were
performed and segregating populations were evaluated using races alpha, delta and kappa
in independent inoculations. A single factor was found responsible for the resistance to all
three races. However, different genetic symbols (B, A’, Q) were assigned to this factor to

distinguished the resistance reaction to the different races used.

The ﬁnal differential cultivar G 2333, originating as the Mexican cultivar Colorado
dc Teopisca, has shown the broadest race-speciﬁc resistance to C. Iindemuthianwn.
Resistance has been conﬁrmed in ﬁeld tests in several countries and in greenhouse seedling
tests in Colombia (Schwartz et al., 1982; Pastor-Corrales and Tu, 1989; Pastor-Corrales
et al., 1994; 1995). Inheritance of the resistance in G 2333 was studied in a F2 and
backcross generations derived from a cross ICA Pijao/G 2333 (Pastor-Corrales et al.,
1994). It was shown that duplicate and independent factors controlled resistance in G
2333 to race 521. Neither the study of Goncalves-Vidigal (1994; AB 136) nor the study
of Pastor-Corrales et al. (1994; G 2333), reported on the independency of the individual
genes or the relationship of these dominant genes to other previously characterized

resistance genes.

Disease control. Breeding for anthracnose resistance has played a major role in
different parts of the world where the disease is a limiting production factor. However,

genetic resistance should be incorporated as part of an integrated disease management

8
strategy, to increase the effectiveness of the control. For instance, anthracnose was once

considered the most important disease in the bean-producing areas of the eastern U. S.
(Zaumeyer and Thomas, 195 7). However, the development of resistant varieties,
improved seed production and certiﬁcation programs signiﬁcantly reduced the threat of
this disease in this country (Saettler and Hart, 1983). An outbreak of anthracnose caused
by the delta race in southern Ontario, demonstrated that sole dependence on genetic
resistance to control this disease is not advisable (Tu and Aylesworth, 1980; Tu, 1981;
1994). As a result, control measures such as quarantine, pedigree seed inspection, seed
treatment, cultural practices and breeding for disease resistance were initiated in Canada as
part of a combined control strategy (Tu, 1992). It is noteworthy that in many parts of the
world most of the disease management procedures already mentioned are difﬁcult to
implement. Breeding for resistance to anthracnose is the most practical and convenient
strategy to pursue, assuming that resistance genes are available (Chaves, 1980; Silbemagel
and Zaumeyer, 1973; Zaumeyer and Meiners, 1975; Schwartz ct al., 1982).

Plant breeders recognized that breeding for vertical resistance against a highly
variable pathogen such as C. Iindemuthianum, is not the most desirable control strategy.
This is because of the potential short term longevity of the reistance. Horizontal
resistance, if available, seems to be more suitable for increasing the longevity of the
resistance. However breeding for horizontal resistance as opposed to breeding for vertical
resistance, is more complex since many genes might be involved and although selection for
individuals with desirable agronomic and resistance traits might be possible, it would
require a higher investment in time and resources. The use of multilines or cultivar

mixtures to increase longevity will have limited acceptance in developed nations, since

 

 

 

 

 

 

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9
uniformity of the crop is usually required, by farmers, the processing industry and

consumers. In some countries in Latin America and Aﬁica, multilines or cultivar mixtures
could have some useﬁrlness, but if an organized seed industry is non-existent, multilines
cannot be recommended. An easily adopted breeding strategy in most countries that
might be suitable for developing cultivars with durable resistance against variable plant
pathogens is gene pyramiding (Nelson, 1978). The concept of gene pyramiding is based
upon the incorporation of two or more genes into a single cultivar. In common bean,
combination of two or more vertical resistance genes has been previously suggested as a
means to achieve stable resistance (Silbemagel and Zaumeyer, 1973). Currently, the
development of DNA marker technology, has opened up the possibility of implementing

gene pyramiding as a breeding strategy for the control of plant pathogens.

Molecular markers. The identiﬁcation of individuals, which have accumulated a
number of desirable agronomic/economic characteristics during the breeding process,
constitutes the most important activity of a plant breeder. Several factors may interfere
with the successful incorporation of an important trait(s): low heritability, genetic
epistasis, undesirable linkages, pleiotropic effects, inadequate screening procedures.
Molecular markers have gained the attention of breeders, since, they can overcome some
of the limitations encountered by conventional selection procedures. Selection for a trait
is performed indirectly based on the phenotype of a marker that cosegregates with the
gene of interest. A useful marker(s) meets the following properties: easy recognition of all
possible phenotypes, early expression in the development of the plant, neutral eﬁ‘ect, ﬁee

of epistatic effects (Stuber, 1991, Arfls and Moreno-Gonzales, 1994). In addition to being

10
a useful breeding tool for indirect selection of a trait, molecular markers have been utilized

in genetic ﬁngerprinting to identify individuals, evaluate genetic relationships among
individuals and study the genetic variation within and among populations (Caetano-
Anollés et al., 1992; Smith and Chin, 1992; Thonnann and Osborn, 1992; Nienhuis et al.,
1994; )

T mes of genetic markers. Markers can be classiﬁed into morphological,
biochemical and DNA-based markers. Morphological trait markers have had limited
application in plant breeding when used for indirect selection of a trait, because of
undesirable phenotypic effects, unfavorable pleiotrophic interactions, dominance and late
expression, rare polymorphisms and the potential effect on ﬁtness or desirability of an
individual (Tanksley 1983; Weeden, 1994a). The inherent properties that distinguish
biochemical (isozymes, Tanksley and Orton, 1983) and/or DNA-based markers (RFLPs,
Botstein et al., 1980; RAPDs, Welsh and McClelland, 1990; Williams et al., 1990) over
morphological markers allows them greater utility in MAS breeding. Isozymes are based
on genetic differences of speciﬁc enzymes coding for proteins that can be separated by
electrophoresis. Isozymes, as opposed to DNA markers, are restricted by a limited
number of markers available and by the number of biochemical assays needed to detect
enzyme activity, which limits the number of genes able to be assessed (Stuber, 1991). The
potential of isozymes as markers for indirect selection remains to be developed.
Restriction ﬁ'agment length polymorphisms (RFLPs) are based on genetic diﬁ‘erences
expressed as length polymorphisms in fragments resulting from the restriction of genomic
DNA with endonucleases. Polymorphisms are detected by hybridizing labeled DNA clones

to Southern blots containing DNAs digested with restriction endonucleases. RFLPs have

11
become widely used in the development of linkage maps of many crop species and for the

mapping of genetic traits (Ganal et al., 1990; Tanksley et al., 1989). The RFLP assay,
however, is expensive, laborious, requires the use of radioactivity, and thus is not readily
adapted as a selection tool by most breeders working with large populations. Random
ampliﬁed polymorphic DNA (RAPDs), a polymerase chain reaction (PCR)-based assay,
detects nucleotide sequence polymorphisms within genomic DNA using a single short
oligodeoxynucleotide of arbitrary sequence, resulting in the ampliﬁcation of several
discrete DNA products. The reaction products are separated on agarose gels and then
visualized by ethidiun bromide staining. The RAPD protocol produces a dominant
marker, because RAPD polymorphisms are the result of either a nucleotide base change
that alters the primer binding site, or an insertion or deletion within the ampliﬁed region.
Polymorphisms are usually scored as the presence or absence of an ampliﬁcation product
ﬁom a single locus (Williams et al., 1990). The RAPD assay possess several advantages
over RFLP technology for MAS application: no requirement of DNA sequence
information, quick and simple to perform, uses ﬂuorescence instead of radioactivity,
nanograms quantities of DNA are required, and the technique is amenable to automation
(Tingey et al., 1992).

RAPD markers for major disease resistcmce genes. The development of RAPD
markers linked to major disease resistance genes has been successﬁrl in diﬁ‘erent crop
species. For example RAPDs linked to major resistance genes have been identiﬁed in
barley (Hordeum vulgare L., Barua et al., 1993; Borovkova et al., 1995), common bean
(Adam-Blondon et al., 1994; Haley et al., 1993; 1994a; 1994b; 1994c; Johnson and Gepts,

1994; Johnson et al., 1994; 1995; Jung et al., 1994; Miklas et al., 1993; Young and Kelly,

12
1994), lettuce (Lactuca sativa L., Kesseli et al., 1992; Michelmore et al., 1991; Paran et

al., 1991), cat (Avena sativa L., Penner et al., 1993), pea (Pisum sativum L., Weeden et
al., 1994b), rice (Oryza sativa L., Mohan et al., 1994), tomato (Lycopersicon esculentum
L., Klein-Lankhorts et al., 1991; Martin et a1, 1991; Williamson et al., 1994), and wheat
(T riticum aestivum L., Schacherrnayr et al., 1994).

Gene raging strategies. The generation of near-isogenic lines (NILs) through
backcrossing, offers the opportunity to ﬁnd markers linked to the introgressed segment of
the genome from the donor parent. In theory NILs are genetically identical except for a
small region that contains the gene that differentiates the isogenic lines. Thus,
polymorphisms observed between NILs represent putative linked markers to the gene of
interest. Near-isogenic lines have been used in conjunction with RAPD markers to tag
resistance genes in barley (Barua et al., 1993), common bean (Haley et al., 1994a; 1994b;
1994c) tomato (Klein-Lankhorts et al., 1991; Martin et al., 1991; ), lettuce (Paran et al.,
1991). The use of NILs, however, has the limitation of requiring a lengthy process to
develop the contrasting isogenic lines. ' The generation of false polymorphisms, not
associated with the gene of interest from the donor parent DNA segments even after
several rounds of backcrossing to the recurrent parent is a problem (Young and Tanksley,
1989). Another strategy that overcomes some of the disadvantages of developing NILs
was proposed by Michelmore et al. (1991). The method, called bulked segregant analysis
(BSA), involves comparing two samples of DNA bulked from individuals in a segregating
population which carry the same monogenic trait. All loci in the genome are regarded as
being in linkage equilibrium except for the region of the genome linked to the selected

locus. Polymorphic markers between the DNA bulks will be genetically linked to the loci

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13
determining the trait used to construct the bulks. Using BSA strategy, three RAPD

markers were found linked to the Din/8 downy mildew resistance gene in lettuce
(Michelmore et al., 1991). The rpg4 gene in barley which confers resistance to pathotype
QCC of Puccinia graminis f,’sp. tritici, was tagged with three RAPD markers using BSA
approach (Borovkova et al., 1995). In common bean, BSA has been used to develop
markers linked to different rust (Uromyces appendiculatus) resistance genes (B-190,
Haley et al., 1993; PI 181996, Johnson et al., 1995; Up2, Miklas et al., 1993). The
combination of NILs and BSA approaches have been used effectively for tagging disease
resistance genes in barley (Barua et al., 1993), common bean (Haley et al., 1993; Johnson
ct al., 1995; Miklas et al., 1993), lettuce (Michelmore et al., 1991). A new method for
targeting RAPD markers linked to disease resistance genes was proposed by Haley et al.
(1994c). The method entails the development of a form of NIL through a process
characteristic of line and cultivar development in many autogamous crop species. The use
of such NILs served to identify RAPD markers linked to disease resistance genes in

common bean (Haley et al., 1994a; 1994b; 1994c).

Application of RAPD markers. Marker-assisted selection (MAS) proposed by
Melchinger (1990), could greatly facilitate the introgression of a gene(s) when direct
selection for a phenotype is not practical or feasible. The application of MAS requires
tight linkage (< 5 cM) between marker and gene of interest or the use of two loosely
linked markers ﬂanking the desirable locus (Tanksley, 1983). During the gene tagging
process, coupling-phase as opposed to repulsion-phase markers are usually sought.

However, Haley et al. (1994a) showed that selection against a repulsion-phase RAPD, as

 

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opposed to selection for a coupling-phase RAPD, provided a greater proportion of

homozygous resistance individuals, and a lower proportion of both segregating and
homozygous susceptible genotypes. Further more, it was indicated that even a loosely
linked (7.1 cM) repulsion-phase marker provided greater selection eﬁiciency than a tightly
linked (1.9 cM) coupling-phase marker. Selection efficiency was improved using a
coupling and a repulsion-phase RAPD markers together as a codominant pair when
compared to selection for either of the markers alone (Johnson et al., 1995). The
identiﬁcation of double recombinants (a homozygous individual with or without the
marker locus) in a segregating population increased the selection efficiency of a
codominant RAPD marker over a repulsion-phase RAPD marker. Johnson et al. (1995)
indicated that the nature of the population with which the breeder is working, will
determine the type of RAPD marker needed to implement efficient MAS. For instance, in
BC.F2 and F2 or later generation populations, selection against a RAPD marker linked in
repulsion-phase to a dominant resistance allele, will discriminate between heterozygous
and homozygous recessive genotypes from the homozygous dominant progeny. In
contrast, in a traditional back cross breeding (BC.F 1) population, coupling-phase RAPDs
are necessary to obtain the heterozygous resistant individual using MAS. In a
recombinant inbred line population, selection for a coupling-phase or against a repulsion-
phase RAPD marker would have the same level of efﬁciency, since the level of
heterozygosity would be minimal.

The large genetic variation observed in common bean is associated to two main
centers of domestication, known as Andean and Middle American gene pools (Singh et al.,

1991). Inter gene pool introgression of useful genes is commonly practiced among bean

breede
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15
breeders. The usefulness of RAPD marker for MAS application in common bean, is best

determined after selection for a marker linked to the gene of interest is practiced in
diﬂ‘erent genetic backgrounds. Gene pool speciﬁc RAPD markers linked to rust resistance
genes (PI 181996, Up2) have been described (Johnson et al., 1995; Miklas et al., 1993).
The most desirable marker is one which can be used for indirect selection of a resistance
gene regardless of genetic background. Gene pool non-speciﬁc RAPD markers were
found for the rust resistance B-190 gene (Haley et al., 1993) and the I gene for bean
common mosaic virus (BCMV) resistance (Haley et al., 1994b). To overcome the
limitation of gene pool speciﬁcity of a RAPD marker, Miklas et al. (1995), proposed the
use of recombinant individuals as parents in MAS for disease resistance. For instance, a
coupling-phase marker is selected against, when using a recombinant individual that is
resistant and without the coupling RAPD, to introgress the gene into susceptible lines
having the coupling marker. However, the implementation of this strategy can only be
justiﬁed in those cases where an important major resistance gene needs to be introgressed
into a particular genetic background which other wise would be impossible in its absence.
Some of the concerns about the applicability of RAPD technology in plant
breeding are related to reliability or reproducibility of the assay in different laboratories.
The reliability of RAPD markers was investigated by Weeden et al. (1992). Some aspects
of the RAPD assay to consider were related to the purity and concentration of the DNA
template, primer and Mg+2 concentration, and scoring errors due to contamination of
template DNA and /or faintness of an ampliﬁed band. However, they concluded that the
RAPD technique should be very useﬁrl for gene tagging. The problem of band reliability

could be Overcome with the use of sequence. characterized ampliﬁed regions (SCARs)

(Kesse
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16
(Kesseli et al., 1992; Paran and Michelmore, 1993) or allele-speciﬁc associated primers

(ASAPs) (Weeden et al., 1992). Both, SCAR and ASAP, are genomic DNA fragments at
a single genetically deﬁned locus that are identiﬁed by PCR ampliﬁcation using a pair of
single speciﬁc oligonucleotide primers (24-mers). These two molecular markers are
advantageous over RAPD markers because they detect only a single locus and their
ampliﬁcation is less sensitive to reaction conditions. Since only one PCR-product is
generated, post-ampliﬁcation electrophoresis could be eliminated because the ampliﬁed
DNA can be visualized by either staining with ethidium bromide (Gu et al., 1994), or by
measuring DNA. concentration in the reaction mixture using an ELISA reader or other

rapid scanning devises capable of measuring absorbance at 260 nm (Weeden et al., 1992).

MAS in breeding for disease resistance. Durability of the genetic resistance in a
released cultivar is a major concern of breeders working with variable plant pathogens.
Nelson (1978), suggested pyramiding resistance genes as a strategy to provide durable
resistance to plant pathogens. Gene pyramiding involves the accumulation in a single
genotype of two or more resistance genes which are effective against the same strain of
the pathogen. However, incorporating disease resistance genes using conventional
breeding procedures could be laborious, time-consuming and expensive (Melchinger,
1990; Pedersen and Leath, 1988). One of the constraints of pyramiding genes is related to
epistatic interactions between resistance genes. Since gene pyramiding requires that both
epistatic and hypostatic genes be combined into a single genotype, breeders have no
convenient way to select for the hypostatic gene without using multiple inoculations with

different races or test crosses with a susceptible genotype (Kelly et al., 1993). In some

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17
instances, quarantine regulations may restrict the introduction of a strain(s) of the

pathogen which is needed to select for an epistatic gene. Again, if a new strain(s) is
introduced, it may required special manipulation and green house space (usually limited)
to avoid escapes. The pyramiding of resistance genes can be greatly facilitated through
MAS, providing markers tightly linked to the gene(s) of interest are available (Kelly, 1995;
Melchinger, 1990; Stuber, 1991). In the absence of the pathogen, molecular markers
allow the selection of a resistance gene based on the marker phenotype. Since RAPD
markers are free of epistatic effects, the presence of a hypostatic gene can be identiﬁed
indirectly by selecting directly for the linked marker. Pyramiding major resistance genes
using MAS to increase the longevity of the resistance against variable plant pathogens has
been suggested in barley (Barua et al., 1993; Borovkova et al., 1995), common bean
(Kelly, 1993; 1995; Stavely et al., 1989), and wheat (Autrique et al., 1995). Currently, up
to four major rust resistance genes (Up2, Ur-3, 8-190 and PI 181996) could be
incorporated into a single common bean cultivar via MAS. Noteworthy is that the
combination of Up2, Ur-3 and B-190 genes affords resistance to 63 of the 65 bean rust
races characterized in the US. Dept. of agriculture collection (Kelly et al, 1994b).
Similarly, complete protection to all known strains of BCMV is realized by combining the
epistatic bc-3 resistance gene with the hypostatic I gene in common bean. This
combination is possible using MAS, since RAPD markers linked to both resistance genes

are available (Haley et al., 1994a; 1994b).

Gene pyramiding and durable resistance. Attaining disease resistance that is

both uniform against all current and future races of a pathogen is the primary objective of

 

efforts
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18
eﬁ‘orts to pyramid host genes. Several researchers have observed eﬁ‘ective and stable

resistance to plant pathogens and have attributed this lasting control to the combination of
major resistance genes present in contemporary cultivars. For example, resistance to
stripe rust (Puccinia striiformis West. var. striifonnis) observed in several winter wheat
cultivars grown in Western Europe during more than 15 years, suggested that the cause of
the lasting resistance was due to the presence of three fiilly effective race-speciﬁc genes
pyramided into single cultivars (Parlevliet, 1993). Similarly, Schafer and Roelfs (1985),
indicated that the increase in durability of resistance to wheat stem rust (Puccinia
graminis Pers. f. sp. tritici) in the northern plains was associated with multiple race-
speciﬁc genes (6) pyramided into a single genotype. The successﬁrl control of rust by
gene pyramiding also maybe related to eradication of the barberry (Berbres vulgaris L.)
(Roelfs, 1982). Durability of the resistance was related to the low probability of mutation
to virulence occurring at various loci in the pathogen population. The major assumption is
that the pest lacks sufﬁcient genetic variation to overcome multiple resistance. However,
the mutability of the required virulence genes is only one aspect of the adaptation process
of the pathogen p0pulation to an increased resistance level. Parlevliet and Zadocks
(1977), suggested that another aspect is the exploitation of the newly produced virulence
genes, since such genes can only spread through the pathogen when they increase the
ﬁtness of the population. During this exploitation phase an increase in gene ﬁ'equencies of
virulence genes from very low to high would be observed. Differences in longevity of
resistance can therefore be expected to result from the inability of the pathogen population

to produce the required virulence gene combinations, or from the inability to exploit the

19
gene or genes that are present in low frequencies, due to a reduction in pathogen ﬁtness

(Green and Campbell, 1979; Parlevliet and Zadocks, 1977; Van Der Plank, 1975).
According to Person et al. (1976), a population large enough to maintain single mutants
would be capable of producing recombinants by sexual or parasexual processes.
Recombination of the virulence genes with high ﬁtness is an essential part of the pathogen
population adaptation process. The higher the recombination frequency, the easier new
virulence genes can be incorporated in gene combination. Therefore, stability of the
resistance is assumed to be highest when many resistance genes and so pathogenicity
genes are involved, and when recombination in the pathogen is strongly restricted
(Parlevliet and Zadocks, 1977). Other potential mechanisms responsible for the durability
of resistance gene pyramids is the associated chance discovery of single resistance genes
or combinations of small numbers of resistance genes that are more durable than average
(Mundt, 1990, 1991). It would appear that breeding for gene pyramids will result in an
increase of durability of resistance, regardless of the mechanism involved. However, there
is no clear correlation between the number of resistance genes and the durability of the
resistance (Mundt, 1991) which suggests that some gene combinations might be more
eﬁ‘ective than others. Green and Campbell (1979), indicated that stable resistance to stem
rust in spring wheat was due to both the larger number and certain combinations of
resistance genes. Johnson (1981, 1984), suggested that a large number of resistance genes
deployed singly in a cultivar or genetic factors other than race-speciﬁc genes, might be

involved in increasing durable resistance.

20
General objectives. Pyramiding major resistance genes into a cultivar has been

proposed as an effective control strategy against variable plant pathogens. The
accumulation of major race-speciﬁc genes into a genotype may increase the longevity of
the genetic resistance. Marker-assisted selection offers the opportunity of gene
pyramiding, since molecular markers used as breeding tools are able to overcome
limitations of conventional breeding procedures. Colletotrichum Iindemuthianum is a
highly variable pathogen, and major anthracnose resistance genes incorporated singly into
bean cultivars have been defeated by virulent races in different parts of the world.
Traditionally pyramiding of major anthracnose resistance genes was not feasible, since
molecular markers linked to resistance genes were not available. The general objective of
this doctoral dissertation was to identify RAPD markers linked to major anthracnose
resistance genes, and to propose breeding strategies to increase the effectiveness of gene
pyramiding seeking durable anthracnose resistance in common bean. In order to achieve
these goals, the research project was divided in three major stages, each with a speciﬁc
objective. In Chapter one, the inheritance of the resistance derived from two differential
cultivars, AB 136 and G 2333 to different races of C. Iindemuthianum was the main
objective. The relationship of the resistance factors in both differential cultivars to other
previously characterized resistance genes was analyzed. Strategies for developing long-
term durability of genetic resistance were also discussed. RAPD markers linked to the Are
gene were sought in Chapter 2. Comparison of recombination distance betvveen RAPD
marker(s) and the Are allele cosegregating in two segregating populations (Andean and

Middle American) were analyzed. In Chapter 3, research to identify RAPD markers

21
linked to three major anthracnose resistance genes, A, Are 1024 and Mexique 3, is

described. The implementation of RAPD markers in MAS for gene pyramiding, seeking

durable resistance in common bean is discussed.

22
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and RFLP marker linked to a dominant gene (Are) conferring resistance to
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Andrus, C. F, and Wade, B. L. 1942. The factorial interpretation of anthracnose resistance in
beans. U.S. Dep. Aglic. Tech. Bull. No. 810.

Arts, P., and Moreno-Gonzélez, J. 1993. Marker-assisted selection. Pages 314-331 in: Plant
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Ayonoadu, U. W. U. 1974. Races of bean anthracnose in Malawi. Turrialba 24(3):311-314.

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Barrus, M. F. 1915. An anthracnose resistant red kidney bean. Phytopathology 5:303-311.

Barrus, M. F. 1918. Varietal susceptibility of beans to strains of Colletotrichwn
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Beebe, S. E. and Pastor-Corrales, M. A 1991. Breeding for disease resistance. Pages 561-617
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Botstein, D., White, R L. Skolnick, M., and Davis, R W. 1980. Construction of a linkage map
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23

Burkholder, W. H. 1923. The gamma strain of Colletotrichum Iindemuthiamon (Sacc. &
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Burkholder, W. H. 1918. The production of an anthracnose-resistant white marrow bean.
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Caetano-Anollés, G., Bassan, B. J., and Gresshoﬁ: P. M. 1992. DNA ampliﬁcation
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Cardenas, F., Adams, M W., and Anderson, A L. 1964. The genetic system for reaction of
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Colletom'chum lindemuthiamon. Euphytica 13: 178-186.

Chanier, A, and Bannerot, H. 1970. Contribution a l’e’tude des races phisiologiques de
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Chaves, G. 1980. Anthracnose. Pages 37-54 in: Bean production problems: disease, insect, soil
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Cobo-Soto, F. 1986. Variacion patoge'nica y ﬁrentes de resisténcia a Colletotrichwn
lindemuthianam (Sacc. et Magn.) Scrib., patogeno de la antracnosis del ﬁijol en
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Fouilloux, G. 1975. L’anthracnose du haricot: etude des relations entre les pathotypes anciens
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Fouillomg G. 1976. Bean anthracnose: new genes of resistance. Ann. Rep. Bean Irnprov.
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Fouilloux, G. 1979. New races of bean anthracnose and consequences on our breeding
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Meyer, eds.

Ganal, M W., Martin, G. B., Messeguer, R, and Tanksley, S. D. 1990. Application of RFLPs,
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24

Garrido-Ramirez, E. R., and Romeo-Cova S. 1989. Identiﬁcacion de razas ﬁsiologicas dc
Colletotrichwn lindemuthianum (Sacc. et Magn.) Scrib. en Mexico y busqueda dc
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Chapter One
CHARACTERIZATION OF THE GENETIC RESISTANCE TO

COLLET 0T RICH UM LINDEMU THIAN UM IN COMMON BEAN
DIFFERENTIAL CULTIVARS

ABSTRACT.

The inheritance of genetic resistance to bean anthracnose in genotypes, Catrachita
and SEL 1360, derived from two anthracnose differential cultivars, AB 136 and G 2333, is
described. Segregation data ﬁ'om three different F2 populations and their respective F23
families indicated that a single dominant gene is responsible for the anthracnose resistance
in Catrachita. In the test for allelism, Chi-square analysis conﬁrmed that the single
dominant resistance gene in Catrachita was situated at a different locus ﬁ'om previously
characterized resistance genes: A, Are, Mexique 1, Mexique 2 and Mexique 3. It is
proposed that the single dominant resistance gene present in Catrachita (AB 136) be
assigned the gene symbol Are 1024 corresponding to its ranking in the bean anthracnose
diﬁ‘erential cultivar series. Segregation in the three F2 populations where SEL 1360 was
used as the resistant parent, ﬁt a 3:1 (R:S) ratio and a 1:221 ratio in the F23 families.
Segregation data suggested that a single dominant gene was conditioning resistance to
anthracnose in SEL 1360. The test for allelism involving SEL 1360 indicated that the
single dominant gene in SEL 1360 is independent ﬁ'om A, Are, Mexique 1, and Mexique 2
genes. However, the dominant gene in SEL 1360 did not segregate independently ﬁ'om

the resistance gene Mexique 3 in the differential cultivar TU, demonstrating that both

31

32
dominant alleles are located at the same locus. Independent segregation of the two

dominant genes in Catrachita and SEL 1360, was also observed. Deployment of major
Middle American genes, such as Are 1024 and Mexique 3, in different combinations with
other characterized genes of Andean origin is possible and should contribute to more

durable anthracnose resistance in common bean.

3 3
INTRODUCTION

Anthracnose caused by Colletotrichum Iindemuthianam‘ (Sacc. & Magnus)
Lambs-Scrib., is one of the most wide spread and economically important fungal diseases
of common bean (Phaseolus vulgaris L.). Complete yield loss can occur with'susceptible
genotypes when conditions favorable for the pathogen are present during the growing
season (Zaumeyer and Thomas, 1957; Pastor-Corrales and Tu, 1989). Since C.
lindemuthianum is a seedbome pathogen which exhibits extensive physiological
variability, the use of genetic resistance has been the most effective control strategy
implemented in Europe and North America (F ouilloux, 1979; Tu, 1992; Kelly et al.,
1994a). In the US, the control of bean anthracnose by means of genetic resistance has
relied primarily on the deployment of two genes, A and Are (Kelly et al., 1994a). The
single dominant A gene was the ﬁrst genetically characterized gene reported to confer
resistance to race alpha (Burkholder, 1918; McRostie, 1919). The Are gene, which
conditions resistance to six distinct physiological races of C. lindemuthianum (Tu, 1992),
was described as a monogenic dominant factor by Mastenbroek (1960). In Europe,
Fouilloux (1979) identiﬁed three additional single dominant resistance genes in a
collection of Mexican gennplasm. Allelism analysis showed that these Middle American
genes segregated independently from each other and from the Are gene; and were
designated Mexique 1, Mexique 2, and Mexique 3. Inheritance studies using the most
common races (alpha, beta, delta, and gamma) and to a lesser extent, epsilon and kappa
races, have been conducted. In addition to independent Single dominant genes
conditioning resistance to alpha (McRostie, 1919; Burkholder, 1918; Mastenbroek, 1960;

Cardenas et al., 1964; Fouilloux, 1979; Del-Peloso et al., 1989; Goncalves-Vidigal, 1994),

. 34
two independent complementary dominant genes have also been reported to confer

resistance to the alpha race (Del-Peloso et al., 1989; Goncalves-Vidigal, 1994; Young and
Kelly see appendix). Resistance to races beta and gamma appears to be governed by the
same dominant genetic factors (Fouilloux, 1979; Mastenbroek, 1960) in addition to a
system of multiple alleles at two loci (Andrus and Wade, 1942; Cardenas et al., 1964;
Muhalet et al., 1981). Both, independent and complementary gene action condition
resistance to race delta (McRostie, 1919; Burkholder, 1918; Andrus and Wade, 1942;
Mastenbroek, 1960; Cardenas et al., 1964; Fouilloux, 1979; Goncalves-Vidigal, 1994),
while, independent dominant alleles conferred resistance to races epsilon (F ouilloux, 1979)

and kappa (Goncalves-Vidigal, 1994).

Breeding for anthracnose resistance in North America has relied on the
introgression of two major resistance genes, Are and A. Resistance break down, leading in
some cases to disease epidemics, caused by races of C. Iindemuthianum virulent on Are or
A resistance genes have been observed in different parts of the world, including North
America (Kruger et al., 1977; Tu,1981; Menezes and Dianese, 1988; Tu, 1994; Kelly et
al., 1994a). Due to the potential short-term protection confered by single genes, a need
for diverse sources of genetic resistance has been widely recognized among bean breeders.
Using a diverse collection of isolates of the pathogen, scientists at CIAT, Colombia,
screened several thousand gerrnplasm accessions and identiﬁed new sources of genetic
resistance (Schwartz et al., 1982; Pastor-Corrales and Tu, 1989; Pastor-Corrales et al.,
1995). . Two members of the differential set of cultivars, AB 136 and G 2333, used to

categorize isolates of C. Iindemuthianum, were consistently among the gerrnplasm

35
accessions that showed the broadest resistance (Pastor-Corrales, 1991). Independent

studies on the genetic characterization of these two differential cultivars have been
conducted. According to Goncalves-Vidigal (1994), a monogenic dominant factor present
in AB 136 was responsible for the resistance to races alpha, delta and kappa, while two
independent dominant genes conditioned resistance to race 521 in G 2333 (Pastor-
Corrales et al., 1994). Neither study reported on the independency of the individual genes
nor the relationship of these dominant factors to other previously characterized resistance

genes.

In the present study, the inheritance of the resistance to diﬁ‘erent races of bean
anthracnose in genotypes derived ﬁom two differential cultivars, AB 136 and G 2333, is
described. The relationship of the resistance factors in AB 136 and G 2333 with other
previously characterized resistance genes is analyzed. Finally, strategies for developing

long-term durability of genetic resistance against bean anthracnose are discussed.

MATERIALS AND METHODS

Plant material. For the genetic characterization of the resistance in AB 136 and G
2333, three breeding lines derived ﬁom crosses involving these two genotypes were used
in this study. A CIAT breeding line RAB 205 (BAT 1225/AB 136) released in Honduras
as the cultivar Catrachita, ‘was the source of the resistance from AB 136 (Beebe and
Pastor-Corrales, 1991). CIAT breeding lines SEL 1360 and SEL 1308 (Talamanca " 2/G
2333), were utilized as the source of the resistance genes from the differential cultivar G

2333 (O. Voysest, personal communication). The derived genetic materials were used

36
because of their adaptation to growing conditions present at East Lansing.

Synchronization of ﬂowering for crossing purposes was facilitated using Catrachita and
the SEL lines instead of the original parental genotypes which are photoperiod sensitive.
Catrachita, SEL 1360 and SEL 1308 each were crossed to three Michigan cultivars; Black
Magic (Kelly et al., 1987), Raven (Kelly et al., 1994b) and Blackhawk (Ghaderi et al.,
1990). Black Magic (NEP-Z/BTS) is susceptible to race 64 (alpha) and race 73; Raven
(NS4004/BSSOO9) carries the A gene for resistance to races 64 and 73 but is susceptible to
race 7; and Blackhawk (Tuscola/CN49-242/lBlack Magic/3/Midnight) carries the Are
gene for resistance to races 7, 23 (delta) and 64 but is susceptible to race 73. During the
present study, it was observed that SEL 1308 unlike SEL 1360 displayed a diﬁ‘erent
resistance reaction when challenged with a wide group of races of the pathogen,
suggesting that SEL 1308 carried a resistance factor(s) different fi'om SEL 1360. The
development of segregating populations for the characterization of the resistance in SEL
1308 is still under way and those ﬁndings will be reported elsewhere. A total of 17
segregating populations were developed for the genetic characterization of the resistance
in Catrachita and SEL 1360. Six F2 populations, three involving Catrachita (AB 136
resistance) and three involving SEL 1360 (G 2333 resistance) and their respective F23
families, were used to determine the inheritance of the genetic resistance in both derived
genotypes, while, ten F2 populations were developed for the allelism test, using Catrachita
and SEL 1360 in testcrosses with a series of genotypes carrying independently
characterized genes previously described in the literature (Raven, Blackhawk, Mex 222,

TO, and TU; which carry A, Are, Mexique 1, Mexique 2 and Mexique 3, respectively;

37
Kelly et al., 1994b; Ghaderi et al., 1990; Fouilloux, 1979). The genetic independence of

the new resistance sources was also evaluated in the cross of Catrachita/SEL 1360.
Preparation of races of C. Iindemuthianum. Inoculum of each race used in the
present study were obtained from monosporic cultures which had been grown and
maintained in stocks of ﬁrngus-colonized ﬁlter paper for long-term storage. The identity
of each race was ﬁrst conﬁrmed by the phenotypic reaction displayed with the anthracnose
differential set (Pastor-Corrales, 1991). The numerical system used to identify diﬁ‘erent
races is based on the sum of the binary values assigned to each of the 12 differential
cultivars on which the speciﬁc race is pathogenic (CIAT, 1988). Parental genotypes,
original (AB 136, G 2333) and derived (Catrachita, SEL 1360, SEL 1308) sources of
resistance where screened with a collection of races of the pathogen (Table 1.1). Based
on the different phenotypic reactions observed on these genotypes, speciﬁc races of C.
Iindemuthianum were chosen for the study of the genetic characterization of the resistance
and different races were used for the allelism test (Table 1.2). Race 64 (ATCC 18987),
race 23 (ATCC 18989) and race 73 (ATCC 96512) were selected to determine the
inheritance of the resistance in Catrachita and SEL 1360 when crossed to the cultivars
Black Magic, Raven and Blackhawk, respectively. Race 64 was selected to test for
allelism in all segregating populations, except in Mex 222/Catrachita and Mex 222/SEL
1360 populations where race 7 (ATCC 96390) was used. A second independent
inoculation, for the allelism test, was performed on TU/SEL 1360 F2 population using race
7. Inocula of all races, except race 73 which was grown in Mathur’s agar (Mathur et al.,
1950), were prepared by placing ﬁingus-colonized ﬁlter paper into Petri dishes containing

potato-dextrose-agar. All cultures were incubated for 10 days under complete darkness at

38

Table 1.1 Phenotypic characterization of parental, original (AB 136, G 2333) and derived
(Catrachita, SEL 1360, SEL 1308) sources of resistance for their reaction to different
races of C. Iindemuthianunr.

 

Racgrhenotypic reaction‘

 

Genotype 7 23 64 73 1545 2047
Black Magic S R S S S S
Raven S S R R R S
Blackhawk R R R S S S
Mex 222 R R S S R S
TO R R R R R S
TU R R R R S S
AB 136 R R R R S S
Catrachita R R R R S S
G 2333 R R R R R R
SEL 1308 R R R R R R

SEL 1360 R R R R S S

 

’ R = resistant; S = susceptible

Table 1.2 Crosses, generation and race of C. Iindemuthianum used for the genetic
characterization of the resistance in Catrachita (AB 136) and SEL 1360 (G 2333)

 

 

Cross Generation‘ Racey

Black Magic/Catrachita (S/R)x P, F1, F2, F2,; 64
Raven/Catrachita (S/R), (R/R) P, F2, F2, F2; 23, 64
Blackhawk/Catrachita (SIR), (R/R) P, F 2, F2, F2,; 73, 64
Mex 222/Catrachita M) P, F1. F2 7
I'D/Catrachita (R/R) P. Fl. F2 64
TU/C atrachita (R/R) P, Fl, F2 64
Black Magic/SEL 1360 (S/R) P, F1, F2, F23 64
Raven/SEL 1360 (S/R), (R/R) P, F 1, F2, F23 23, 64
Blackhawk/SEL 1360 (SIR), (R/R) P, F2, F2, F23 73, 64
Mex 222/SEL 1360 (R/R) P, F1, F2 7
TOISEL 1360 (R/R) P, F1, F2 64
TU/SEL 1360 (R/R), (R/R) P, F1, F2 64, 7
generate/SEL 1360 (NR) P, F1, F2 64

 

2 Parental, F1, F2 and F23 families respectively. ’ Race of C. Itndemuthianum used for
the genetic characterization and the allelism test respectively. " Parental resistant
(R) and/or susceptible (S) reaction to the races shown in column four.

40

24°C. Spore suspensions were prepared by ﬂooding the plates with 5 ml of 0.01% Tween
20 in distilled water and scraping the surface of the culture with a spatula.

Disease phenotjpic characterization. Parental material, F 2, F2 and F 2;; families
were sprayed inoculated with a spore suspension (1.2 X 106 spores ml") of the pathogen.
The race of C. lindemuthianum used to characterize the resistance factors segregating in
each cross and the respective generations tested is shown in Table 1.2. The protocol for
spore inoculation was as follows: the ﬁrst incompletely expanded trifoliate leaf of 15 to 18
day-old parental F2 and/or F2 plants was inoculated on the lower and upper leaf surfaces.
Spore suspension was applied with a camel-hair paint brush. Conversely F2 populations
for the allelism test and F23 families were spray inoculated with the spore suspension on
the abaxial and stem surface of unifoliate leaves (10 to 12 day-old seedlings). Different
inoculation methods were used because F2 individual plants (particularly susceptible
individuals), unlike F2z3 Plants and F2 populations used for the allelism test, were saved for
F3 seed production . Inoculated plants were placed in a mist chamber at 100% RH, 22-25
°C for 48 hrs, then transferred to greenhouse conditions. Seven days post-inoculation
disease phenotypic characterization was carried out. Disease reaction was rated on a 1-9
scale in which 1 = no visible symptoms and 9 = severely diseased or dead ( Van
Schoonhoven and Pastor-Corrales, 1987). Resistant (R) phenotype was assigned to plants

with scores 1-3. Individual plants with scores 4-9 were considered susceptible (S).

41

RESULTS

Original and derived sources of anthracnose resistance were evaluated for their
phenotypic reaction to a collection of races of the pathogen (Table 1.1). Diﬁ‘erent races
(7, 23, 64, 73, 1545 and 2047) representing a wide range of virulence, were used to
conﬁrm if Catrachita and SEL lines were comparable to the anthracnose resistance present
in the AB 136 and G 2333 differential cultivars. The cultivars Catrachita and AB 136
exhibited similar phenotypic patterns, both showed resistance to all races except races
1545 and 2047. None of the races tested were pathogenic on G 2333 and its derived line
SEL 1308. However, SEL 1360 exhibited a different phenotypic pattern to the G 2333
parent. Two races, 1545 and 2047, overcame the resistance factor in this derived line.
Since SEL 1308 and SEL 1360 were derived from G 2333 through backcrossing to the
susceptible variety Talamanca, it is assumed that the resistance factor(s) observed in both
lines came ﬁ'om the G 2333 parent. Since G 2333 carries two dominant resistance genes
(Pastor-Corrales et al., 1994), it is possible that only one of the major genes was inherited
in SEL 1360, thus a different phenotypic pattern was observed. Although similar disease
reactions were observed between SEL 1308 and G 2333, it is not certain, in the absence of
testcrosses, if SEL 1308 inherited both genes responsible for anthracnose resistance of G
2333.

Six F2 populations, three involving Catrachita and three involving SEL 1360, and
their respective F23 families were used to determine the inheritance of the anthracnose

resistance in different genetic backgrounds (Table 1.3). Eleven F2 populations were

42

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43
utilized to test for allelism between the derived sources of resistance with other

characterized genes (T able 1.4). The nature of the resistance in Catrachita (AB 136) and
SEL 1360 (G 2333) was determined after analyzing the observed segregating ratios
obtained from the disease phenotypic characterization of the three different F2 populations
and their respective F23 families (Table 1.3). In the three crosses involving Catrachita as a
parent (crossed with Black Magic, Raven and Blackhawk), the Chi-square test performed
on data collected from the F2 populations supported a ﬁt to the 3:1 expected ratio of
resistant to susceptible plants (R2S), respectively (Table 1.3). The Chi-square analysis
performed on the F2,; families conﬁrmed a 1:221 ratio in each F2 population indicating that
a single dominant gene is responsible for the anthracnose resistance in Catrachita.

The allelism study performed to test the independence of the single dominant
resistance gene in Catrachita with the other ﬁve reported genes (A, Are, Mexique 1,
Mexique 2 and Mexique 3) is shown in Table 1.4. In all ﬁve F2 populations, the observed
segregation ﬁt a 15:1 ratio (R:S), indicating that the single dominant gene conditioning
resistance to anthracnose in Catrachita segregated independently from the A, Are,
Mexique 1, Mexique 2 and Mexique 3 dominant genes.

Segregation in the three F2 populations where SEL 1360 was the resistant parent,
ﬁt a 3:1 ratio of R to S respectively. A 122:] ratio was conﬁrmed in F2; families from
each F2 population evaluated, suggesting that a single dominant gene was conditioning
resistance to anthracnose in SEL 1360 (Table 1.3). Within four F2 populations,-
segregation ﬁt a 15:1 (R: S) ratio for the crosses of SEL 1360 with Raven, Blackhawk,
Mex 222, and TO. This allelism test indicated that the single dominant resistance gene in

SEL 1360 is independent and located at a different locus when compared to A, Are,

44

Table 1.4. Allelism test for the genetic characterization of the resistance to
Colletotrichum Iindemuthianum in Catrachita (AB 136) and SEL 1360 (G 2333).

 

Anthracnose reaction

 

 

No. F2 plants
Cross' R- rr X3 y P
Raven/Catrachita" 1 14 3 2. 12 0. 15
Blackhawk/Catrachita" 1 19 14 3 .45 0.06
Mex 222/Catrachitaw 104 5 0.27 0.60
TO/Catrachita" 93 9 0.76 0.38
TU/Catrachita" 96 6 0.00 1.00
Raven/SEL 1360x 95 9 0.66 0.42
Blackhawk/SEL 13601‘ 69 4 0.00 1.00
Mex 222/SEL 1360‘"' 96 I 6 0.00 1.00
TO/SEL 1360" 80 4 0.11 0.74
TU/SEL 1360" 102 0 5.78 0.02
Catrachita/SEL 13601‘ 91 l l 2.85 0.09

 

’ Resistant/Resistant crosses in all cases. " 15:1 (resistant, R- : susceptible, rr) expected
ratio for two independently assorting genes; "" phenotypic reaction to races 64 and 7 of
CoIIetotrichum Iindemuthianum respectively; " phenotypic reaction to two independent
inoculations with races 64 and 7 of Colletotrichum lindemuthianum respectively.

45
Mexique 1 and Mexique 2 genes (Table 1.4). However, no segregation was observed in
the F2 population derived from the cross between TU/SEL 1360 after two independent
inoculations with races 64 and 7. This result indicated that the dominant gene in SEL
1360 is located at the same locus as the Mexique 3 gene in TU. In addition, similar
phenotypic reactions were also observed in the differential cultivar TU and SEL 1360
when evaluated with a group of anthracnose races (Table 1.1), supporting the ﬁnding of

the allelism test that SEL 1360 and TU both carry the Mexique 3 dominant allele.

The test of allelism performed on the F2 population of the cross Catrachita/SEL
1360 segregated in a ratio of 15:1 (P = 0.09), indicating that the dominant resistance gene

in Catrachita is independent from the resistance gene in SEL 1360 (Table 1.4).

DISCUSSION

Stable resistance to plant pathogens, such as C. Iindemuthianum with extensive
physiological variability, requires continual evaluation of gerrnplasm and eventual
introgression of diverse genetic resistance into commercial cultivars. The appearance of
new races of anthracnose in Michigan (race 7 and 73; Kelly et al., 1994a) and Canada
(alpha Brazil; Tu, 1994), which overcome Are (race 73 and Alpha Brazil) and A (race 7)
genes, has forced breeders to seek alternative sources of resistance. Studies on the
inheritance of anthracnose resistance in genetic material (Catrachita and SEL 1360)
derived from AB 136 and G 2333 were conducted, because the broad base resistance

observed in these differential cultivars could be of value in breeding for long-term

46
anthracnose resistance in beans (Schwartz et al., 1982; Menezes and Dianese, 1988;

Pastor-Corrales and Tu, 1989; Pastor-Corrales et al., 1995). A unique monogenic
dominant gene segregated in three different F2 populations derived from crosses between
resistant (Catrachita) and susceptible (Black Magic, Raven and Blackhawk) parents. It
was concluded that this single dominant gene in Catrachita was responsible for the
resistance to races 23 (delta), 64 (alpha) and 73. A coupling-phase RAPD marker tightly
linked to the resistant allele in Catrachita was also present in AB 136 and absent in all
susceptible parents (Black Magic, Raven and Blackhawk) (see Chapter 3), conﬁmiing that
the single dominant resistance gene in Catrachita is the same resistance gene present in AB
136. A monogenic factor controlling anthracnose resistance in AB 136 was also described
by Goncalves-Vidigal (1994). In that work, segregating populations derived ﬁ'om crosses
involving AB 136 as the resistant parent, were challenged with three different races of the
pathogen. The symbols B, A’ and Q were assigned to described a dominant gene
governing the resistance to alpha, delta and kappa respectively. It is assumed that B, A’
and Q symbols refer the same gene, since no experimental evidence suggests the contrary.
Duplicate independent and dominant genes were observed to segregate in all populations
‘ derived ﬁ'om crosses involving Catrachita and each of the genotypes (Raven, A;
Blackhawk, Are; Mex 222, Mexique 1; TO, Mexique 2 and TU, Mexique 3) which carry a
previously characterized resistance gene. In all instances, Chi-square tests conﬁrmed a
15:1 ratio, suggesting that the single dominant gene in Catrachita was situated at a
diﬁ‘erent locus ﬁ'om A, Are, Mexique 1, Mexique 2 and Mexique ‘3 resistance genes. In

addition, independent segregation of two dominant genes was also observed in the cross

47
Catrachita/SEL 1360, indicating that these genes are located at different loci. However,

Goncalves-Vidigal, (1994) showed no segregation for susceptibility in progeny (F2)
derived from a cross between the differential cultivars PI 207262 and AB 136 when
inoculated with races alpha, delta or kappa. She concluded that both cultivars cany a
similar gene(s) each conditioning resistance to a different anthracnose race. Support for
different alleles is based on the number of races which are virulent on P1 207262 and fail
to overcome AB 136 resistance (Pastor-Corrales et al., 1994), or conversely, race 1545
was pathogenic on AB 136 but avirulent on P1 207262 (Table 1.1). In addition a RAPD
marker linked to the AB 136 resistance allele was absent in P1 207262 (Chapter 3). Based

on this evidence, AB 136 and PI 207262 would carry different alleles at the same locus.

The characterization of the genetic resistance in Catrachita (AB 136) determined
that one dominant gene conditions resistance to bean anthracnose in this cultivar. The
major gene in AB 136 is independent ﬁ'om the characterized genes: A, Are, Mexique 1,
Mexique 2 and Mexique 3. In recognition of the fact that the Are gene, since it discovery
and publication by Mastenbroek (1960), has been widely utilized by breeders as a source
of resistance and extensively reported in the literature (gene tagging: Adarn-Blondon et
al., 1994; Young and Kelly, 1994; genetics: Fouilloux, 1979; Muhalet et al., 1981;
breeding: Bannerot, 1971; Menezes and Dianese, 1988; Tu, 1992); the gene symbol Are
has become synonymous with anthracnose resistance and is a descriptive symbol of that
function. Therefore, it is proposed that the single dominant resistance gene present in AB
136 be assigned the genetic symbol Are 1024 corresponding to its ranking in the

differential series.

48
The resistance factor present in SEL 1360 was shown to be a monogenic dominant

gene based on segregation ratios in three different F2 populations. Support for a
difference in inheritance between parent (G 2333) and offspring (SEL 1360) was expected
since G 2333 and SEL 1360 displayed different phenotypic patterns after inoculation with
anthracnose races 1545 and 2047 (T able 1.1). Considering that SEL 1360 is a selection
derived ﬁ'om two backcrosses with the anthracnose resistant parent G 2333, it can be
assumed that only one of the two dominant genes was transmitted to SEL 1360 offspring.
The test for allelism involving SEL 1360 demonstrated that the segregation ratio in four
populations (Raven, Blackhawk, Mex 222 and TO) followed a 15:1 (R: S) ratio indicating
that the single dominant gene in SEL 1360 is independent ﬁ'om A, Are, Mexique 1, and
Mexique 2 genes. However, the dominant gene in SEL 1360 did not segregate
independently ﬁ'om the resistance gene (Mexique 3) in the differential cultivar TU
indicating that both dominant alleles are located at the same locus. Since none of the
anthracnose races tested (Table 1.1) could effectively differentiate between the two alleles,
the data suggest that the resistance gene in SEL 1360 is the same dominant Mexique 3
gene present in TU. Additional evidence supporting this hypothesis comes from a
coupling phase RAPD marker linked to the SEL 1360 allele which was also present in TU
and the G 2333 parent (see chapter 3). Since G 2333 and TU belong to the same
Phaseolus gene pool (Middle American, Singh et al, 1991), it is possible that these
cultivars share common resistance genes. Based on the work of Pastor-Corrales et al.
(1994), race 521 discriminates between the resistance gene in TU and the two genes

present in G 2333. Results of the present study when compared with the results obtained

49
by Pastor-Corrales et al. (1994) suggest two possible genetic models. In the ﬁrst model

the dominant gene in SEL 1360 is the Mexique 3 allele and is a third independent allele
present in G 2333. This third allele was not detected by Pastor-Corrales et al. (1994)
since it did not condition resistance to race 521 used in that study. The second model
suggests that the dominant gene present in SEL 1360 is allelic to the Mexique 3 gene and
is one of the two dominant genes in G 2333 which condition resistance to race 521.
Support for this model is lacking based on the RAPD marker evidence and in the absence

of testing with race 521.

Two hypothesis can be formulated to explain the broad base resistance observed in
SEL 1308 (Table 1.1). First, SEL 1308 carries both resistance genes from G 2333; and
second, SEL 1308 inherited one of the major genes from G 2333 but is different from the
gene in SEL 1360. This second gene alone is able to confer resistance to races 1545 and
2047. Since SEL 1308 was derived from G 2333 (two backcrosses) it is more likely that
only one resistance gene was introgressed into this line as was the case with SEL 1360.
The absence of the RAPD marker, linked to the SEL 1360 allele (Mexique 3), in SEL
1308 indicates that the resistance to bean anthracnose in this line is conferred by a
different factor independent of the Mexique 3 locus. Therefore, the resistance factor(s) in
SEL 1308 becomes of major importance to breeders since they afford protection to the
highly virulent races of the pathogen. The ﬁnal characterization of the genetic resistance

in SEL 1308 would distinguish between the proposed hypothesis.

Gene pyramiding has been suggested as a potential strategy for maintaining long-

term durable disease resistance (Nelson, 1978). The accumulation of major resistance

50
genes into a cultivar would delay the appearance of new races of the pathogen. The basis

for the stabilization of the resistance resides in the reduction in ﬁtness of a pathogen when
the number of virulence genes necessary to overcome host resistance increases (Van der
Plank, 1968). The differential cultivar G 2333 constitutes an example of the effectiveness
of gene pyramiding in controlling bean anthracnose when more than one major resistance
gene is present in the host. A collection of 380 isolates of C. Iindemuthianum from
different parts of the world were avirulent on this genotype (Pastor-Corrales et al., 1994).
To date, there has not been any isolate reported with the ability to overcome G 2333
resistance. Diversity in the sources of resistance used would also contribute to the
stability of anthracnose resistance. For example, Kelly et al. (.1994a) suggested that
pyramiding A and Are genes would afford protection against all known anthracnose races
in the US. It is noteworthy that the A gene is of Andean origin, in contrast to the Are
gene of Middle American origin. The introgression of both Andean and Middle American
genes seems to be an effective genetic combination for broad base resistance. For
instance, Pastor-Corrales et al. (1994), reported that the A gene present in the differential
cultivar Michigan Dark Red Kidney conferred resistance to a group of highly virulent
races (73, 129, 133, 136, 385, 448, 521, 901, 905, 1409, 1473) of the pathogen. These
races and race 1545 (Table 1.1) are pathogenic on most of the resistant sources of Middle
American origin. It has been observed that some of the differential cultivars of Andean
origin showed susceptibility to several isolates fiom South America whereas resistance to
Middle American isolates was observed. In contrast, Middle American differential

cultivars were usually immune to isolates of Andean origin (Pastor-Corrales et al., 1995).

51
Therefore, deployment of major genes, such as Are 1024 and Mexique 3, in different

combinations with other characterized genes such as the A gene should contribute to more

durable anthracnose resistance in common bean.

52
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Wallingford, UK.

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

Van der Plank, J. E. 1968. Disease resistance in plants. Academic press, New York and
London. 206p.

Van Schoonhoven, A, and Pastor-Corrales, M. A. 1987. Standard system for the
evaluation of bean gerrnplasm. CIAT, Cali, Colombia.

Young, R. A, and Kelly, J. D. 1994. A RAPD marker for the Are anthracnose resistance
gene in beans. Annu. Rep. Bean Improv. Coop. 37177-78.

Zaumeyer, W. J, and Thomas, H. R. 1957. A monographic study of bean diseases and
methods for their control. U.S. Dep. Agric. Tech. Bull. No. 868.

Chapter Two

RAPD MARKERS FLANKING THE ARE ANTHRACNOSE RESISTANCE
GENE IN COMMON BEAN

ABSTRACT.

Incorporation of the Middle American and dominant Are gene, for bean
anthracnose resistance, into commercial cultivars, has been the main disease control
strategy adopted by breeders to effectively limit the potential damage of the pathogen. A
random ampliﬁed polymorphic DNA (RAPD) marker designated OQ4mo, generated by a
5'-AGTGCGCTGA-3' decamer primer, was found to be tightly linked in coupling with the
Are gene. OQ4mo mapped at 2.0 i 1.4 centiMorgans (cM) from the Arc allele in the
Andean genetic background and at 5.5 i 2.3 cM in the Middle American background. A
second coupling phase RAPD marker B35520“) generated by a 5'-GTATGGGGCT-3'
primer mapped at 5.4 i 2.3 cM from the Are allele in the Andean genetic background and
at 7.7 i 2.7 cM in the Middle American background. Based on recombination distance of
7.0 :t 1.9 cM between the two markers, OQ4mo and B3551000 RAPDs appear to ﬂank the
Are gene. Bracketing molecular markers allowed tagging of the Arc allele with a selection
ﬁdelity of 99%. Use of the OQ4mo and B3551000 RAPD markers for marker-based
selection will afford the opportunity to retain the Are anthracnose resistance gene in bean
germplasm, as other epistatic resistance genes are characterized, and incorporated into

contemporary bean cultivars.

55

56

INTRODUCTION

Anthracnose, caused by Colletotrichum Iindemuthianum (Sacc. & Magnus.)
Lama-Scrib., is a serious seedbome fungus capable of inducing complete yield loss in
susceptible bean (Phaseolus vulgaris L.) genotypes. Surveys in a wide range of
production areas have identiﬁed several distinct races, suggesting extensive pathogenic
variation on all continents (Pastor-Corrales and Tu, 1989). In North America, reported
races include alpha, alpha Brazil, beta, gamma, delta, epsilon, and lambda in Canada (Tu,
1992, 1994). Recently, Kelly et al. (1994) identiﬁed two new races of C. lindemuthianum
in the US. which are pathogenic on resistance genes currently deployed. The dominant
allele at the Are locus, which confers resistance to alpha, beta, gamma, delta, epsilon, and
lambda races of the fungus, was ﬁrst found in a black bean from Venezuela, Cornell 49-
242 (Mastenbroek, 1960) and has been used widely in North America and Europe as the
main source of resistance against anthracnose (Fouilloux, 1978; Tu, 1992).

Marker-assisted selection (MAS), using molecular markers such as random
ampliﬁed polymorphic DNA (RAPDs) as the selectable marker, has the potential to be a
useful breeding tool in common bean, where monogenic disease resistance genes have
been tagged (Adam-Blondon et al., 1994; Haley et al., 1993; Haley et al., 1994a, b, c;
Johnson and Gepts, 1994; Johnson et al., 1995; Jung et al., 1994; Miklas et al., 1993).
Conventional procedures for gene pyramiding are not practical for plant breeders due to
epistatic interactions between resistance genes. Because pyramiding requires that epistatic
and hypostatic genes be combined into a single genotype, breeders have no convenient

way to select genotypes with a hypostatic gene without inoculating with different isolates

57
of a pathogen or test crossing back to a susceptible genotype. However the identiﬁcation

of molecular markers tightly linked to resistance genes allows indirect selection because
the expression of the marker is not masked by epistatic interactions. The identiﬁcation of
a number of RAPD markers in common bean now permits gene pyramiding for disease
resistance For example, RAPD markers can be used to pyramid three rust (Uromyces
appendiculatus) resistance. alleles (Up2, Ur-3, B-190; Kelly, 1995) with other epistatic
resistance alleles ﬁ'om the plant introduction collection (Johnson et al., 1995). Similar
pyramiding opportunities for MAS have been suggested in breeding for resistance to bean
common mosaic virus (BCMV), since the I, bc-3 gene combination affords resistance to
all known strains of BCMV (Haley et al., 1994b). Efficiency in selection can be improved
by using a second marker either linked in repulsion with the resistance gene (Haley et al.,
1994a) or linked in coupling but ﬂanking the allele of interest (Tanksley, 1983).

Two gene pools exist in P. vulgaris, the Middle American and the Andean (Gepts
and Bliss, 1985; Gepts et al., 1986; Singh et al., 1991) and crosses between these have
been utilized to exploit breeding for anthracnose resistance in different bean seed and pod
types. RAPD markers linked to genes introgressed from gerrnplasm derived ﬁ'om a
diﬂ‘erent gene pool appear to have lower recombinational ﬁ'equencies and apparent tighter
linkages than recombinational distances measured between the same RAPD linked to the
resistance gene in the same gene pool (Haley et al., 1994b). Brown et al., (1989)
demonstrated that linkage between traits can. vary depending on the populations studied-
and the genetic similarity between parents. Because recombinational frequencies may be

underestimated in wide crosses (Paterson et al., 1990), map distances based on ﬁgures

58
from such crosses may not represent the actual distance between markers and genes in

more related genetic backgrounds with which breeders most ﬁequently work.

The objectives of the present research were to ﬁnd a RAPD marker linked to the
Middle American Are allele, and compare its utility with another previously characterized
marker (B3551000) in Andean and Middle American bean populations. Comparison of
recombinational distance between RAPD marker(s) and the Are allele in common bean

gene pools were analyzed.

MATERIALS AND METHODS

Genetic material. Two groups of nearly-isogenic lines (NILs) were used to screen
for RAPD markers linked to the Middle American Are gene in diﬁ‘erent genetic
backgrounds. A pedigree generated (F n) inbred line (designated K86002) heterogeneous
for the Are locus was the source of NILs in the Andean genetic background. The kidney
bean line K86002 was originally derived ﬁ'om introgression of the Arc gene through
backcrossing (CN49242/4*Montcaan/Charlevoix). The development of NILs from
advanced heterogeneous lines has been described previously (Haley et al., 1994c). The
DNA ﬁ'om K86002 lines, homozygous dominant and homozygous recessive for the Arc
locus respectively, constituted the contrasting NIL pair in the Andean genetic background.
The other NIL pair was obtained ﬁ'om a F2 population (A4512) derived by crossing two
nearly-isogenic Middle American cultivars (Ex Rico 23 / OAC Rico; Beversdorf, 1983),
which differed for the presence (OAC Rico) and absence (Ex Rico 23) of the Are gene.
Bulked segregant analysis (Michelmore et al., 1991) was used to prepare the DNA bulk-

NILs ﬁ'om the A4512 population in the Middle American genetic background. The two

59
contrasting DNA pairs (one pair Andean and one pair of Middle American origin) were

screened with random decamer primers to identify RAPD markers linked to the Are allele.
Progeny of 108 F2 (103 F23) individuals from the Andean (K86002) and 109 F2 (104 F23)
individuals ﬁ'om the Middle American (A4512) crosses were used as mapping populations.

Disease evaluations. Parental material used as sources of DNA for primer
screening were ﬁrst genotyped for the Are locus by inoculating with the appropriate race
of C. lindemuthianum. The choice of different races of C. lindemuthianum to screen for
the Are resistance gene was based on differential disease reaction of gennplasm from each
gene pool. To discriminate effectively between resistant and susceptible genotypes in the
segregating F2 and F23 populations, the beta race was used to screen the K86002
population and delta race was used to screen the A4512 population. Progeny tests of at
least 11 to a maximum of 18 plants from each F23 family from both mapping populations
were carried out to conﬁrm the disease expression of F2 individuals and separate
homozygous AreAre from heterozygous Areare genotypes. All pathogen inoculations
were conducted using a suspension of 1.2 x 10" spores ml". The ﬁrst incompletely
expanded trifoliate leaf of 15 to 18 day old F2 plants was inoculated on the lower and
upper leaf surfaces. Spore suspension was applied with an artist's camel-hair paint brush.
Conversely, F23 families were spray inoculated with the spore suspension on the abaxial
and stem surface of unifoliate leaves (10 to 12 day old seedlings). Diﬁ‘erent inoculation
methods were used because F2 (particularly susceptible individuals), unlike F23 Plants,
were saved for F3 seed production and leaf tissue sampling for DNA extraction.
Inoculated plants were placed in a mist chamber (100% RH) at 22-25 °C for 48 hr, then

transferred to the greenhouse. Disease ratings were made 7 days post-inoculation.

60
Disease reactions were recorded as resistant (R) for those plants with no visible disease

symptoms of anthracnose, and susceptible (S) for those plants with presence of enlarged
lesions or sunken cankers on the lower side of the leaf or hypocotyl.

RAPD analysis. Prior to inoculation with C. Iindemuthianum, tissue was collected
for DNA extraction from young primary leaves (4 to 7 days post-emergence) from
greenhouse grown plants. Two discs of leaf tissue were cut by clipping the leaf with a 1.5
ml microcentrifuge tube. A simple and quick extraction method developed by Afanador et
al., (1993) was used for DNA extraction. The extracted DNA was standarized to a
uniform concentration (10ng ul") using DNA ﬂuorometry (Hoefer TKO 100, Hoefer
Scientiﬁc, San Francisco, Calif).

The Polymerase Chain Reaction (PCR) cycling proﬁle consisted of 3 cycles of 1
min/94°C , 1 min/35°C, 2 min/72°C; 34 cycles of 10 s/94°C, 20 s/40°C, 2 min/72°C; 1 cycle
of 5 min/72°C; 1 s "Auto-segment Extension” (for extension phase of 34-cycle portion of
PCR). Ampliﬁcation was carried out in a Perkin Elmer Cetus DNA Thermal Cycler 480.
Description of the PCR reactions have been reported previously (Haley et al., 1993;
Miklas et al., 1993). Approximately 10 ng of genomic DNA template and 10 ng of a
decamer primer (Operon Technologies, Alameda, Calif.) were combined in an 18.8 111
reaction, containing 2 units of Stoffel Fragment Polymerase (Perkin Elmer CETUS,
Norwalk, Conn). Ampliﬁed DNA fragments were resolved by electrophoresis in 1.4%
agarose gel containing 0.5 ug ml'l ethidium bromide, 40mM Tris-acetate, and 1 mM
EDTA. Polymorphisms were scored as either presence or absenCe of a band (visualized

using UV light) between bulked DNA pairs with or without the Are allele, respectively.

61
Prior to screening the mapping populations, DNA from other known resistant and

susceptible genotypes were used to conﬁrm potential RAPD-Are locus linkages.

Linkage with a second RAPD marker, identiﬁed by Weeden (personal
communication) in recombinant inbred populations of Andean snap bean gennplasm
segregrating for the Are gene, was studied in the same two F2 populations K86002 and
A4512. The second RAPD marker was obtained using the B355 primer 5'-
GTATGGGGCT-3' which ampliﬁed a 1000 bp fragment linked in coupling with the Are
allele. The marker symbol B355 is the identiﬁcation used by the University of British
Columbia to describe the 10-mer primer. The RAPD marker B35520“, was identiﬁed
using the PCR protocol for DNA ampliﬁcation recommended by GibcoBRL. The cycling
proﬁle consisted of 35 cycles of 1 minute each / at 92°, 35°, and 72°C using 1.0 unit of
Taq DNA polymerase (GibcoBRL) as the thermal stable enzyme.

A simple dominant transmission of the Arc gene and the putative linked RAPD
markers were analyzed by Chi-square. The recombination ﬁ'equency between a RAPD
marker and the Are allele and between OQ4mo and B3 552000 RAPDs was calculated using

Linkage-l (Suiter et al., 1983) and Mapmaker (Lander et al., 1987).

62

RESULTS

A total of 346 random decamer primers were screened against the contrasting
DNA combinations of resistant and susceptible genotypes from Middle American and
Andean origin. Polymorphisms within at least one of the DNA pairs, were generated by
approximately 32% of the primers, while only 8% of the primers tested produced
polymorphic loci in both DNA pairs. The number of false positives generated between
DNA pairs alone was relatively high, but once they were compared to DNA controls, the
majority of them were eliminated. RAPD variability generated within the Middle
American (A4512) bulk-NILs was less frequent (13%) than that found within the Andean
(K86002) NILs (19%). Different levels of polymorphisms within Middle American versus

within Andean gene pools have been reported by Haley et al., (l994d).

Identiﬁcation of RAPD markers. Five putatively linked RAPD markers, two
apparently associated in coupling and three in repulsion phase with the dominant Are
allele, were identiﬁed across both populations. These RAPDs were assayed for co-
segregation with F2 genotypes characterized as resistant and susceptible in the two
segregating populations Only one RAPD marker, generated by a 5'-AGTGCGCTGA-3'
decamer primer and designated OQ4mo,- ‘O’ for the source of the primer (Operon
Technologies), Q4 for the speciﬁc kit (Q) and primer number (4), and 2440 for the size (bp)
of the polymorphic fragment; was found to be tightly linked with the Are allele. The
OQ4mo mapped at 2.0 :t 1.4 centiMorgans (cM) in the Andean genetic background and at
5.5 :1: 2.3 cM in Middle American background from the Are allele (Table 2.1). A second
coupling phase RAPD marker B35520“) mapped at 5.4 i 2.3 cM in the Andean genetic
background and at 7.7 i 2.7 cM in the Middle American background ﬁ'om the Are allele.

63

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64
Based on recombination distances of 7.0 3; 1.9 cM between the two markers across both

gene pools (5.4 i 2.3 cM in the Andean population, and 8.3 :t 2.9 cM in the Middle
American population), OQ4mo and 133551000 RAPDs appear to ﬂank the Arc allele (Table
2.1). Differences in recombination values were observed between populations, but these
were not statistically signiﬁcant (P s 0.05) for either marker. In each population,
segregation ratios consistent with dominant monogenic inheritance were observed for the
0Q4mo and 83551000 RAPD markers and the dominant Are allele (Table 2.2). The
ampliﬁcation of the 1440 bp DNA fragment by the OQ4mo RAPD marker, was scored
across a number of individuals with known genotype at the Are locus (Fig. 2.1). The
presence and absence of the RAPD marker was associated with resistant and susceptible
phenotypes of F2 individuals and commercial cultivars possessing or lacking the Are gene
respectively.

Selection efficiency. Based on actual observations, the efﬁciency of indirect
selection for the Are locus using OQ4mo and B3551000 RAPD markers was analyzed
within and across gene pools (Table 2.3). Selection efﬁciency is deﬁned here as the ability
of a RAPD marker, used alone or a combination of both markers, to effectively distinguish
between resistant (AreAre and Areare) and susceptible (areare) F2 individuals based on
the marker phenotype as selection criteria. Since OQ4mo RAPD mapped tighter to the
Are allele than the B355mo RAPD, efﬁciency of indirect selection for resistant and
susceptible F2 individuals was higher when the OQ4mo marker was used alone regardless
of genetic background (95.2 vs. 93.3% in Middle American and 98.1 vs. 95.1% in Andean
genetic background). However when both RAPD markers were utilized together,

selection efﬁciency within and across gene pools increased to 98.6% in the combined

 

65
Middle American and Andean populations. The largest improvement in selection

eﬁciency was observed using B3551000 alone (93.3%) when compared to the combination

of OQ4mo + B3551000 RAPDs (98.1%) in the Middle American genetic background.

Table 2.2 Chi-square (X2) analysis for 0Q41440 and B35510“) RAPD markers and the Are
allele segregating in Middle American and Andean F2 and F2; 3 populations.

 

 

napalm”? 13:; Generation 11mm 3:33; x1 Probability

Middle Amerlean Are l=2 3:1 78:31 0.52 0.47
Middle American Are l=23 1:2:1 30:43:31 3.14 0.21
Andean Are 1"2 3:1 86:22 1.00 0.32

Andean Are l"‘2:3 1:2:1 26:55:22 0.79 0.67

Middle American 0Q4m0 F2 3:1 73:36 3.33 0.07
Middle American 13355.000 F2 3:1 79:30 0.25 0.62
Andean 0Q4mo I"‘2 3:1 86:22 1.00 0.32

_ Andean 13355looo F2 3:1 89:19 2.78 0.10

 

* Middle American = Ex Rico 23/0AC Rico (A4512); Andean = K86002 S/K86002 R

66

12345678910111213

' a _. d t -

EEEazraaﬁamr

'“OO‘

0.... —- .-..

-——-----~_-—--o—

 

Figure 2.1 Ampliﬁcation of genomic DNA using 0Q4mo RAPD marker. Key to
individuals: (1) anthracnose-susceptible Andean parent, (2) anthracnose-resistant Andean
parent, (3) susceptible Andean F2 individual, (4) resistant Andean F2 individual, (5)
anthracnose-susceptible Middle American DNA bulk, (6) anthracnose-resistant Middle
American DNA bulk, (7) susceptible Andean cultivar (Montcalm), (8) resistant Andean
cultivar (Isles), (9) anthracnose-susceptible Middle American parent (Ex Rico 23), (10)
anthracnose-resistant Middle-American parent (OAC Rico), (1 l) anthracnose-susceptible
Mddle American cultivar (Mayﬂower), (12) anthracnose-resistant Middle American
Cultivar (Harokent), (l3) molecular weight marker ( l Hind III / EcoRl; size of bands

indicated in bp).

67

Table 2.3 Eﬂiciency of RAPD marker(s) linked to the Arc allele for marker-based
selection within and across common bean gene pools.

 

Gene Pools'

RAPD Marker(s) MA A MA + A
No. of Selection N o. of Selection No. of Selection
recomb. efﬁciency recomb efﬁciency recomb efficiency

 

(%) - (%) . (%)

0Q4mo 5 95.2 2 98.1 7 96.6
133551000 7 93.3 5 95.1 12 94.2
004,440+ 133551000 2 98.1 1 99.0 3 98.6

 

‘ MA= Middle American (Ex Rico 23/OAC Rico, A4512); A= Andean
(K86002 S/K86002 R).

68

DISCUSSION
Identiﬁcation and useﬁtlness of RAPD markers. In plant breeding, marker loci

tightly linked to genes of interest are needed for implementation of MAS strategies for
gene introgression. The most desirable markers are those that exhibit tight linkage (s 5
0M; Tanksley, 1983) with the resistance genes. However, many studies restrict the
application of markers to speciﬁc genetic population, so the usefulness of a linked marker
is not always demonstrated across a range of genetic backgrounds. In common bean,
RAPD markers may be gene pool speciﬁc and of limited value in selecting for resistance
genes in the same genetic background from which the genes are indigenous. This
limitation was demonstrated with the RAPD marker linked to the Up2 gene which could
not be used for selection for rust resistance in the Andean gene pool from which the Up;
gene originated (Miklas et al., 1993). Therefore the value of that marker was restricted to
the Middle American gene pool. This result is in contrast to that for the RAPD marker
0119450 linked to the Middle American B-190 gene which can be used in all gene pools
regardless of origin of the resistance gene (Haley et al., 1993). Similarly RAPD markers
associated with the rust resistance gene Ur—3 (Haley et al., 1994c) and the I gene for
BCMV resistance (Haley et al., 1994b) have broad application across gene pools and
genotypes. A RAPD marker found linked to the Arc allele in an Andean snap bean
POpulation was reported by Adam-Blondon et al. (1994). Since this molecular marker

Was not tested across different genetic backgrounds its usefulness for introgressing the Arc

gene into Middle American gerrnplasm using MAS remains to be conﬁrmed.

69
The use of DNA ﬁom the two distinct genetic populations (Andean and Middle

American) in the initial screening of primers, enabled us to identify a gene pool non-
speciﬁc RAPD marker (OQ4mo) for the Are gene. The second RAPD marker (B355mo)
linked in coupling with Are allele, exhibited similar differential recombination between the
two populations. The linkage distances were greater than those found for the OQ41440
marker but since the differences were non signiﬁcant, the B35520“, marker is equally
robust as a tool for indirect selection in populations of bean derived from the two gene
pools. The linkage distance between the two markers is greater than the distance of either
individual marker to the Are gene in both populations. Although the actual linkage
distance is less than the sum of the individual distances between individual markers and the
gene, the data clearly suggest that the markers ﬂank the Are locus.

Selection efﬁciency and recombination frequency. Flanking markers have been
shown to be more efﬁcient in selection for a trait of interest (Tanksley, 1983). Combining
marker data ﬁom 207 F2 individuals belonging to both segregating populations, the
bracketing RAPDs were able to select for the Are allele with 98.6% efﬁciency. Based on
recombination frequency of the these two RAPD markers the theoretical selection ﬁdelity
reaches 99.7%. The beneﬁt of bracketing markers for marker-assisted selection is not
fully appreciated when individual markers are tightly linked to the gene of interest, which
is the case of the present study. However with looser linkages, scoring for ﬂanking

tnal‘kers would showed greater selection efﬁciency over single (closely linked) marker
selection.
The introgression of the Are gene into the Andean gene pool from Middle

American gerrnplasm represents a wide cross. Haley et al., (1993), showed that genes

70
from the Middle American gene pool appear to exhibit tighter linkages with markers when

measured in the Andean genetic background. A similar trend has been observed with
Andean resistance genes when studied in Middle American segregating populations
(Miklas et al., 1993). Lower recombination values between the Are allele and both RAPD
markers were both expected and observed in the Andean mapping population. Although
differences in recombination frequencies were not statistically supported, it is noteworthy
that the two described RAPD markers were developed independently and both showed
trends similar to previously reported observations. Recombination has speciﬁc
mechanisms that are not clearly understood. Environmental eﬁ‘ects may play a role in
diﬁ‘erent recombination ﬁ'equencies observed between populations. The present study was
not designed to distinguish genotypic from environmental inﬂuences on estimates of
recombination values. However the apparent difference in linkage between populations
derived from the two gene pools might suggest that some form of recombinational
suppression is occurring in wide crosses of common bean. A larger population size and/or
a larger sample of crosses representing both gene pools might be needed to determine
diﬂ‘erences in recombination distance between gerrnplasm pools. One property of wide
Crosses is the incorporation of genetic material that may not be sumciently similar to
Permit the same degree of recombination as seen with crosses of more similar genetic
material. Paterson et al., (1990) observed a diminished reciprocal exchange, between
heterozygous chromosomal segments ﬂanked by homozygous regions, in wide crosses
I3etvveerl Lycopersicon species. They pointed out, that the lower likelihood of reciprocal

e><<3hange in a heterozygous chromosomal segment may be accentuated by positive

71
interference, ﬁom more frequent reciprocal exchanges in ﬂanking homozygous regions.

Any difference in the recombination value between gene pools of P. vulgaris might
suggest the need to conduct mapping studies in populations in which breeders are actively
working. Developing genetic maps using wide crosses may allow the mapping of a
number of traits but may be of limited value to applied plant breeders interested in using
markers to introgress an agronomically useful trait. Until linkages are generated and
tested among related materials, map-based breeding will probably be restricted to the
introgression of foreign genes.

Application of RAPD markers. The occurrence of two new races of C.
Iindemuthianum in North America has put in jeopardy the long-term strategy of relying on
single gene resistance for controlling bean anthracnose (Kelly et al., 1994). Race 73 and
alpha-Brazil overcome the resistance conferred by the Are gene, and race 7 is virulent on
genotypes carrying the A gene. Since the A gene conditions resistance to races 73 and
alpha-Brazil, Phaseolus breeders now need to pyramid the Are and A genes into new
cultivars as the only effective means of conferring resistance to all current North American
races of C. Iindemuthianum. With the characterization of additional anthracnose
resistance genes (Pastor-Corrales et al., 1994), MAS offers bean breeders a viable
alternative for developing cultivars with pyramided resistance genes as a means of
increasing the longevity of anthracnose resistance in common bean. As new broadly-based
epistatic anthracnose resistance genes are being introgressed into commercial bean-
cultivars, using more virulent races of the pathogen, the OQ41440 and B3 551000 RAPD
markers will be used to ensure the retention of the hypostatic Are gene in these materials.

Since selection for the hypostatic Are gene will be dependent on linked markers in the

72
absence of test crosses and multiple anthracnose inoculations, ﬂanking markers oﬂ‘er ‘ a

signiﬁcantly increased level of efﬁciency over single closely linked markers for marker
based selection. Thus, long terrn_durable anthracnose resistance in common bean could be

provided through the pyramiding of resistance genes using these RAPD markers.

73

LIST OF REFERENCES

Adam-Blondon, A F., Sevignac, M., Bannerot, H., and Dron, M. 1994. SCAR RAPD
and RFLP marker linked to a dominant gene (Are) conferring resistance to
anthracnose in common bean. Theor. Appl. Genet. 88:865-870.

Afanador, L. K., Haley, S. D., and Kelly, J. D. 1993. Adoption of a 'mini-prep' DNA
extraction method for RAPD marker analysis in common bean (Phaseolus
vulgaris). Ann. Rep. Bean Improv. Coop. 36:10-11.

Beaversdorf, W. D. 1983. OAC Rico ﬁeld bean. Can. J. Plant Sci. 64:753-755.

Brown, A. H. D., Lawrence, J., Jenkin, M, Douglass, J., and Gregory, E. 1989. Linkage
drag in backcross breeding in barley. J. Hered. 80(3):234-23 9.

Fouilloux, G. 1979. New races of bean anthracnose and consequences on our breeding
programs. Pages 221-235 in: Int. Symp. Dis. Trop. Food Crops. H. Maraite and J.A
Meyer, eds.

Gepts, P,. and Bliss, F. A. 1985. F 2 hybrid weakness in the common bean. J. Hered.
76:447-450.

Gepts, P., Osborn, T. C., Rashka, K., and Bliss, F. A. 1986. Phaseolin protein variability
in wild forms and landraces of the common bean (Phaseolus vulgaris): Evidence
for multiple centers of domestication. Econ. Bot. 40:451-468.

Haley, S. D., Miklas, P. N., Stavely, J. R, Byrum, J., and Kelly, J. D. 1993. Identiﬁcation of
RAPD markers linked to a major rust resistance gene block in common bean. Theor.
Appl. Genet. 86:505-512.

Haley, S. D., Afanador, L. K., and Kelly, J. D. 1994a. Selection for monogenic resistance
traits with coupling and repulsion-phase RAPD markers. Crop Sci. 34:1061-1066.

Haley, S. D., Afanador, L. K., and Kelly, J. D. 1994b. Identiﬁcation and application of a
random ampliﬁed polymorphic DNA marker for the I gene (Potyvirus resistance)
in common bean. Phytopathology 84:157-160.

Haley, S. D., Afanador, L. K., Miklas, P. N., Stavely, J. R., and Kelly, J. D. 1994c.
Heterogeneous inbred populations are useﬁll as sources of near-isogenic lines from
RAPD marker localization. Theor. Appl. Genet. 88:337-342.

74

Haley, S.D., P. N. Miklas, L. K. Afanador, and J. D. Kelly. 1994d. Random Ampliﬁed
Polymorphic DNA (RAPD) marker variability between and within gene pools of
common bean. J. Amer. Soc. Hort. Sci. 119:122—125.

Johnson, E., Miklas, P. N., Stavely, J. R, and Martinez-szado, J. C. 1995. Coupling and
repulsion phase RAPDs for marker-assisted selection of the PI 181996 rust resistance
in common bean. Theor. Appl. Genet. accepted.

Johnson, W. C., and Gepts, P. 1994. Two new molecular markers linked to bc-3. Ann.
Rep. Bean Impov. Coop. 37:206-207.

Jung, 6., Coyne, D. P., Skroch, P. W., Nienhuis, J., Arnaud-Santana, E., Bokosi, J.,
Kaeppler, S. M., and Steadman, J. R. 1994. Construction of a genetic linkage map
and locations of common blight, rust resistance and pubescence loci in Phaseolns
vulgaris L. using RAPD markers. Ann. Rep. Bean Improv. Coop. 37237-38.

Kelly, J. D., Afanador, L. K., and Cameron, L. S. 1994. New races of Colletotrichum
Iindemuthianum in Michigan and implications in dry bean resistance breeding.
Plant Disease 78:892-894.

Kelly, J. D. 1995. Use of random ampliﬁed polymorphic DNA markers in breeding for major
resistance to plant pathogens. HortScience 30(3):461-465.

Mastenbroek, C. 1960. A breeding programme for resistance to anthracnose in dry shell
haricot beans, based on a new gene. Euphytica 9(2): 177-184.

Michelmore, R. W., Paran, I., 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 88:9828-9832.

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

Pastor-Corrales, M. A, and Tu, J. C. 1989. Anthracnose. Pages 77-104 in: Bean production
problems in the tropics. H. F Schwartz, and M. A Pastor-Corrales, eds. Centro
Internacional de Agricultura Tropical (CIAT), Cali, Colombia.

Pastor-Corrales, M. A., Erazo, O. A., Estrada, E. I., and Singh, S. P. 1994. Inheritance of
anthracnose resistance in common bean accession G 2333. Plant Disease 78:959-
962.

 

75

Paterson, A. H., DeVerna, J. W., Lanini, B., and Tanksley, S. D. 1990. Fine mapping of
quantitative trait loci, using selected overlapping recombinant chromosomes, in an
interspeciﬁc cross of tomato. Genetics 1242735-742.

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

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

Tanksley, S. D. 1983. Molecular markers in plant breeding. Plant. Mol. Biol. Rep. 1(1):3-
8.

Tu, J. C. 1992. Colletotrichum Iindemuthiamnl on bean. Population dynamics of the pathogen
and brwding for resistance. Pages 203 -224 in: Colletotrichlan-Biology, Pathology and
Control. J. A Bailey and M. J. Jeger, eds. CAB. International, Wallingford, UK.

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

Chapter Three
RAPD MARKERS FOR MAJOR ANTHRACNOSE RESISTANCE GENES IN
COMMON BEAN
ABSTRACT.

Pyramiding three major anthracnose resistance genes using marker-assisted
selection (MAS) is now feasible in common bean. Four RAPD markers linked to the A,
Are 1024 and Mexique 3 loci were developed using bulk segregant analysis and
heterogeneous inbred population approaches. RAPD marker, OFlOm was tightly linked
in repulsion-phase to the A allele (1.9 i 1.4 cM). A survey of bean genotypes for the
presence or absence of the marker locus, revealed that OF 10530 RAPD would facilitate the
introgression of the A gene across Phaseolus gene pools. A coupling-phase RAPD
marker OAHl-no and a repulsion-phase OAKZOggo RAPD marker, cosegregated with
alleles at the Are 1024 locus in three different segregating populations. The OAHlm and
OAKZOm RAPD markers were found linked (12.3 i 1.9 and 7.3 :t 1.5 cM, respectively)
and ﬂanked the Are 1024 locus. Coupling and repulsion-phase RAPD markers when used
as a codominant pair showed higher selection efﬁciency, for the identiﬁcation of
homozygous (Are 1024 Are 1024) F2 individuals, as opposed to individual selection for a
coupling-phase or against a repulsion-phase RAPD phenotype. The RAPD marker
OAB3450 cosegregated with the Mexique 3 allele in two different populations. The
0AB3450 RAPD was found linked at 5.9 3: 1.7 in coupling-phase with the Mexique 3

allele. Pyramiding major anthracnose resistance genes using MAS is proposed, and the

76

77
eﬁ‘ective durability of the resistance to C. Iindemuthianunl could be increased if Andean

and Middle American genes were combined.

78

INTRODUCTION.

Colletotrichum Iindemuthianum (Sacc. & Magnus) Lambs-Scrib., the causal
agent of anthracnose in common bean (Phaseolus vulgaris L.), is considered one of the
most economically important fungal pathogens of the crop. The presence of the disease
has been reported in most parts of the world where beans are produced and when
favorable conditions for the establishment and grth of the pathogen exist (Pastor-
Corrales and Tu, 1989). The main control measure against bean anthracnose has been
genetic resistance. However, the extensive physiological variability exhibited by C.
lindemuthianum has continuously lead bean breeders and pathologists to seek alternative
genetic sources for long-term durability of the resistance (Beebe and Pastor-Corrales,
1991)

Gene pyramiding has been suggested as a strategy for stable resistance against
variable plant pathogens (Nelson, 1978). Incorporating more than one gene into a single
genotype is time-consuming and a difﬁcult task due to epistatic interactions between
resistance genes requiring extensive test crossing with diﬂ‘erent races of the pathogen,
while, ensuring that the genotype possesses several other agronomic traits. Often,
progeny testing might require screening With a new race of the pathogen to ensure the
presence of a gene during the breeding process. Working with introduced isolates
demands careful manipulation of the pathogen to avoid escapes and usually testing of
genotypes is carried out under greenhouse conditions where space is limited.

Molecular markers offer the opportunity to pyramid major genes into a cultivar,

since they are able to overcome the constraints usually encountered by conventional

79
breeding procedures (T anksley, 1983; Melchinger, 1990; Stuber, 1991). The use of

random ampliﬁed polymorphic DNA (RAPD), a polymerase chain reaction (PCR) based
marker (Welsh and McClelland, 1990; Williams et al., 1990), can facilitate the transfer of a
gene(s) when direct selection of a RAPD, closely linked to the gene of interest, is carried
out. Marker assisted selection (MAS) using RAPD markers has been suggested as a
potential useﬁll breeding tool for gene introgression in common bean. At present, MAS is
being implemented to pyramid major resistance genes affording complete protection to
bean common mosaic virus (BCMV) and bean rust [Uromyces appendiculatus (Pers. ex
Pers.) Unger var appendiculatus] (Kelly, 19958).

The Are gene (Mastenbroek, 1960) is the ﬁrst major anthracnose resistance gene
for which molecular markers have been developed in common bean (Adam-Blondon et al.,
1994; Young and Kelly, 1994). The RAPD marker, described by Young and Kelly (1994),
linked in coupling to Are allele, could be used in MAS breeding to facilitate introgression
of the Are gene across Phaseolus gene pools. In contrast, the utility in diﬂ‘erent genetic
backgrounds, of the RAPD marker developed by Adam-Blondon et al. (1994) remains
unknown, since, this RAPD was mapped in a population of Andean origin and no other
gennplasm screening was reported. Limited usefulness for MAS have been observed in
other studies in common bean (Miklas et al., 1993; Haley et al., 1993; Johnson et al.,
1995), where RAPD markers linked to disease resistance genes were found to be gene
pool speciﬁc.

The Arc gene has provided long-term effective protection against bean anthracnose
in North America (Tu, 1992; Kelly et al., 1994a) and Europe (Fouilloux, 1979). However,

the greater pathogenic variability of the fungus observed in Latin America has limited the

80
useﬁllness of this gene for anthracnose resistance (Menezes and Dianese, 1988; Rava et

al., 1993). In the US, the introgression of the A gene, of Andean origin (McRostie,
1919) and the Are gene, of Middle American origin, into commercial cultivars, has now
been overcome in North America. Recently, Kelly et al. (1994a) reported the presence in
Michigan of two new races of C. lindemuthianum, races 7 and 73. Race 7 was virulent on
cultivars carrying the A gene but was non-pathogenic on genotypes carrying the Are gene.
Conversely, race 73 was vimlent on cultivars carrying the Are gene and non-pathogenic on
genotypes carrying the A gene. Concurrently, Tu (1994) reported the appearance of race
alpha-Brazil which was also capable of defeating the genetic resistance conferred by the
Are gene in Canada.

Two genotypes, AB 136 and G 2333, members of the anthracnose differential
series, have shown broad resistance to a large collection of isolates from Latin America
(Schwartz et al., 1982; Pastor-Corrales and Tu, 1989; Pastor-Corrales et al., 1995),
including races 7, 73 and alpha-Brazil. It was observed that a single dominant gene was
responsible for the resistance in AB 136 (Chapter 1), while, independent duplicate factors
accounted for the resistance in G 2333 (Pastor-Corrales et al., 1994). A breeding line,
SEL 1360, derived ﬁom G 2333, was found to carry one of two dominant genes present
in the resistant parent (chapter 1). The two characterized dominant genes designated Are
1024 (AB 136) and Mexique 3 (SEL 1360), have been incorporated into the bean
breeding lines at Michigan State University (MSU) as the new sources of resistance to
anthracnose.

In the present study, research to ﬁnd RAPD markers linked to three major

anthracnose resistance genes, A, Are 1024 and Mexique 3, was conducted. The

81
implementation of molecular markers in MAS for gene pyramiding, seeking durable

resistance to anthracnose in common bean, is discussed.

MATERIALS AND METHODS.

Genetic material. The MSU breeding line N8 5006 heterogeneous for the A locus,
was the source of two near-isogenic lines (NILs), N85006 S (homozygous recessive for
the A locus) and N85006 R (homozygous dominant for the A locus), which were assayed
to identify linked RAPD markers. The N85006 is an FM line derived from the cross of
MSU breeding lines N81058/N80038. The original source of the dominant A resistance
gene in N85006 traces back to the anthracnose resistance in the cultivar Sanilac (Andersen
et al., 1960). The development of NILs from advanced heterogeneous lines was described
by Haley et al., (1994b); and the effectiveness of this strategy in molecular tagging of the
Are resistance gene is discussed in Chapter 2. Progeny of 104 F2 (104 F23) individual
plants from the N85006 S/N 85006 R cross were used as the mapping population for the A

locus.

The cultivar Catrachita, an small red bean type ﬁ'om Honduras, was used as the
source of the Are 1024 resistance gene (Beebe and Pastor-Corrales, 1991). Three
different mapping populations (Black Magic/Catrachita, Raven/Catrachita and
Blackhawk/Catrachita) with an average of 107 F2 individual plants and their respective F22
families, each, were used to conﬁrm putative linkages between a RAPD marker and alleles

at the Are 1024 locus.

82
The source of the Mexiqtte 3 resistance gene was a CIAT (Centro Internacional de

Agricultura Tropical, Cali, Colombia) breeding line (SEL 1360) derived ﬁom the
differential cultivar G 2333 (Voysest et al., 1995). Testing of putative genetic linkages
between RAPD markers and alleles at the Mexique 3 locus was performed in two diﬁ‘erent
mapping populations (Black Magic/SEL 1360 and Blackhawk/SEL 1360) averaging 103
F2 individual plants with their respective F23 families. The bulked segregant analysis
approach (Michelmore et al., 1991) was used to form two contrasting DNA pairs
(resistant and susceptible bulks) when screening for markers linked to the Are 1024 and
Mexique 3 genes. The contrasting DNA bulks were composed of equal volumes of
standarized DNA from six F2 homozygous resistant and six F2 homozygous susceptible
individual plants. The F2 individual plants, used to form the DNA bulks were derived ﬁ'om
crosses between Black Magic/Catrachita and Raven/Catrachita segregating for the Are
1024 gene and Black Magic/SEL 1360 and Blackhawk/SEL 1360 segregating for the
Mexique 3 gene. The parental material used in the formation of mapping populations and
DNA bulks for Are 1024 and Mexique 3 genes was described in Chapter 1.

Disease evaluation. The genotype of the parental material, used as a source of
DNA for RAPD screening, for each locus under study was ﬁrst conﬁrmed by inoculation
with the appropriate race of the fungus. Alpha served as the discriminating race between
N85006 S and N85006 R parental lines, in the segregating F2 population and for progeny
testing the F23 families. Three different races of C. lindemuthianum were used for-
genotyping parental, F2 individual plants and F23 families, from crosses involving the Arc

1024 and Mexique 3 genes. Race alpha was used for Black Magic/Catrachita and Black

83
Magic/SEL 1360 populations; race delta for Raven/Catrachita population; and race 73 for

Blackhawk/Catrachita and Blackhawk/SEL 1360 populations. All progeny testing was
conducted by spray inoculating 12 to 18 (10 to 12 day-old seedlings) F223 individuals ﬁ'om
each F2 plant. The inoculation procedure and disease rating followed the description
provided in materials and methods in Chapter 1.

RAPD analysis. Similar protocols for DNA extraction, PCR reactions for DNA
ampliﬁcation and cycling proﬁle and visualization of PCR ampliﬁed products, used to tag
the Are gene were used in the current study (see chapter 2). For resolution enhancement
of a small polymorphic band generated by the primer OAB3, electrophoresis was
performed in 2.7 % agarose gel, containing 0.5 ug n'll'l ethidium bromide, in 0.5X TBE
buffer (0.045M Tris-borate and 0.001M EDTA). Primer amplifying products that
exhibited polymorphisms between contrasting NILs and/or DNA bulks were subsequently
evaluated in their respective segregating population. Simple inheritance of the disease
phenotype and the putative linked RAPD markers were conﬁrmed using Chi-square tests.
Linkage analysis were performed using the program Linkage-l (Suiter et al., 1983) and
MAPMAKER (Lander et al., 1987), version 3.0. A logarithm of the odds ratio (LOD)
score of 3.0 was established for linkage, and the Kosambi mapping function was used to

calculate map distance in centimorgans (cM.).

RESULTS.
A total of 572 random decamer primers were used to screen the NILs
heterogeneous for the A locus (N 85006 S and N85006 R). Three putative RAPDs, two in

repulsion (OF10530 and OAM4670) and one in coupling-phase (OAL7 530) with the A allele,

84
were polymorphic between the NIL pair. However, only one of these markers, OF10530

(generated by a 5'-GGAAGCTTGG-3' decamer; Fig. 3.1), was found tightly linked with
the A locus, at a distance of 1.9 :t 1.4 cM (Table 3.1). Dominant and monogenic
inheritance of the anthracnose resistance phenotype and the OF10530 RAPD is shown in
Table 3.1. The presence of the A locus and the OF10530 RAPD marker was surveyed in a
series of common bean genotypes belonging to different market classes from different
gene pools (Table 3.2). The OF10530 RAPD appeared to be a gene pool non-speciﬁc
marker, since, it was able to determine the genotype of the A locus in individuals
regardless of genetic origin. In Sanilac, the source of the A gene in the NILs used for
primer screening, the OF10530 marker locus was absent; indirectly conﬁrming the presence
of the A gene in this cultivar. In a few individuals, however, the OF10530 marker locus
was either present (Seafarer) or absent (Isabella, G 2333, Perry Marrow, Kaboon),
contrary to the expected genotype of the A locus.

A total of 216 decamer primers were used to screen the four contrasting DNA
bulks for the Are 1024 and the Mexique 3 genes. Three putative RAPD markers were
found to be linked to the alleles of interest. Polymorphisms between either contrasting
DNA bulk was ﬁrst conﬁrmed using a second DNA bulk, prepared with individuals from a
different F2 population (segregating for the same gene of interest). All polymorphic
band(s) which were consistent in both DNA bulks were also found cosegregating in the F2
population used to construct both bulks. Conﬁrming a putative linkage, by using a second
contrasting DNA bulk, before the actual mapping of a RAPD in a segregating population,
helped accelerate the process of primer screening, since false positives were readiliy

discarded saving time and resources.

85

 

Figure 3.1 Ampliﬁcation of genomic DNA using OF10530 RAPD marker. Key to
individuals: (1) anthracnose-susceptible NIL N85006-S, (2) anthracnose-resistant NIL
N85006-R (3) homozygous resistant F2 individual, (4) heterozygous resistant F2
individual, (5) homozygous susceptible F2 individual, (6) Andean cultivar with A gene
(Michigan Dark Red Kidney), (7) Middle American cultivar with A gene (Sanilac), (8)
Andean cultivar with A gene (Montcalm), (9) Middle American cultivar without A gene
(Cornell 49242), (10) negative control (no template DNA added to PCR ampliﬁcation),
(1 1) molecular weight marker (1 Hind III/EcoRI: size of bands indicated in bp)

86

Table 3.1 Chi-square (X2) analysis for OF10530 RAPD marker and the A allele segregating
in the N85006 S/N 85006 R F2 and F23 population.

 

 

 

Locus Generation Expected Observed X2 Probability
tested ratio frequenq
A F2 3:1 75:29 0.32 0.57
A F23 1:2: 1 30:45:29 1.90 0.39
OF10330 F2 3: 1 76:28 0.12 0.73
Linkage estimate2

a/ OF10330 x2 = 94.5 P = 0.00 1.9 i1.4 cM

' Two point Chi-square (X2) analysis and linkage estimate (3:613:12221) based on 122:1
genotypic segregation ratio for the A locus and 3:1 ratio for the OF10530 repulsion-phase
RAPD marker.

87

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88
population used to construct both bulks. Conﬁrming a putative linkage, by using a second

contrasting DNA bulk, before the actual mapping of a RAPD in a segregating population,
helped accelerate the process of primer screening, since false positives were readiliy
discarded saving time and resources

Two RAPD markers were found linked with alleles at the Are 1024 locus. The
RAPD marker OAHl-m (generated by a 5'-TCCGCAACCA-3' decamer primer) was
linked in coupling, whereas the RAPD marker Oak20890 (ampliﬁed by decamer 5’-
TGATGGCGTC-3’), was linked in repulsion with the Are 1024 resistance allele (Fig. 3.2
and Fig. 3.3). Segregation ratios, in three different populations, consistent with simple
monogenic inheritance were observed for the marker loci and the Are 1024 gene (Table
3.3). Linkage analysis showed a consistent linkage between the Arc 1024 gene and
OAHlm in three different populations (Table 3.4). The OAHlm RAPD mapped at 14.0
i 3.6, 10.7 i 3.1 and 12.2 i 3.3 cM from the Are 1024 resistance allele in the Black
Magic/Catrachita, Raven/Catrachita and Blackhawk/Catrachita populations, respectively.
No signiﬁcant differences in recombination values, calculated using the standard errors of
the estimates, were observed among the three F2 mapping populations. The repulsion-
phase marker OAK20290 was found linked to the are 1024 allele, at a distance between
loci of 6.5 :t 2.5 cM in the Black Magic/Catrachita, 12.5 :t 3.3 cM in the Raven/Catrachita
and 3.0 i 1.7 cM in the Blackhawk/Catrachita F2 populations (Table 3.4). Difference in
recombination ﬁequency (P = 0.01), however, was observed between the
Raven/Catrachita and the Blackhawk/Catrachita mapping populations. Recombination
values between the two marker loci, observed among the three mapping populations were

statistically not signiﬁcant. The map distance between RAPD markers ranged ﬁom 21.6 :t

89

1234 56789101112

 

Figure 3.2 Ampliﬁcation of genomic DNA using OAHlno RAPD marker. Key to
individuals: (1) anthracnose-susceptible Black Magic/Catrachita DNA bulk, (2)
anthracnose-resistant Black Magic/Catrachita DNA bulk, (3) susceptible parent, Black
Magic, (4) resistant parent, Catrachita, (5) susceptible parent, Raven, (6) resistant cultivar,
AB 136, (7) susceptible parent, Blackhawk, (8) homozygous resistant F2 individual, (9)
heterozygous resistant F2 individual,(lO) homozygous susceptible F2 individual, (11)
negative control (no template DNA added to PCR ampliﬁcation), (12) molecular weight
marker (3. Hind III/EcoRI: size of bands indicated in bp).

I (I! //ll

//./rr

/.

90

123456 789101112

OA K2000»

e947
<—830

 

Figure 3.3 Ampliﬁcation of genomic DNA using OAKZOago RAPD marker. Key to
individuals: (1) anthracnose-susceptible Black Magic/Catrachita DNA bulk, (2)
anthracnose-resistant Black Magic/Catrachita DNA bulk, (3) susceptible parent, Black
Magic, (4) resistant parent, Catrachita, (5) susceptible parent, Raven, (6) resistant cultivar,
AB 136, (7) susceptible parent, Blackhawk, (8) homozygous resistant F2 individual, (9)
heterozygous resistant F2 individual,(lO) homozygous susceptible F2 individual, (1 1)
negative control (no template DNA added to PCR ampliﬁcation), (12) molecular weight
marker (7. Hind III/EcoRI: size of bands indicated in bp).

91

Table 3.3 Chi-square (X2) analysis for OAHlm and OAK20290 RAPD markers and the
Are [024 allele segregating in three different F2 and F23 populations.

 

 

Population Locus Generation Expected Observed X2 Probability
tested ratio frequency

Black Magic/Catrachita Are 1024 F2 3 :1 74:32 1.26 0.26
Black Magic/Catrachita Are 1024 F23 1:2:1 23:50:32 1.78 0.41
Raven/Catrachita Are 1024 F2 3:1 76:34 1.75 0.19
Raven/Catrachita Are 1024 F23 1:2:1 28:47:34 2.73 0.26
Blackhawk/Catrachita Are 1024 F2 3:1 79:32 0.68 0.41
Blackhawk/Catrachita Are 1024 F22 1:2:1 23:55:31 1.18 0.55
Black Magic/Catrachita OAHlm F2 3 :1 74:31 0.92 0.34
Raven/Catrachita OAHlm F2 3 :1 75:34 1.91 0.17
Blackhawk/Catrachita OAHlm F2 3 :1 74:35 2.57 0.11
Black Magic/Catrachita OAK20290 F2 3 :1 85:20 1.68 0.20
Raven/Catrachita OAK20290 F2 3 :1 86:23 0.69 0.41

Blackhawk/Catrachita OAK20390 F2 3: l 87:22 I. 10 0.29

 

 

92

 

 

 

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93
4.6 cM in the Black Magic/Catrachita, 18.9 :t 4.3 cM in the Raven/Catrachita and 15.2 i

3.8 cM in the Blackhawk/Catrachita F2 population (Table 3.4). Linkage estimates
calculated across mapping populations (323 F2 individuals) showed that the OAHlm
RAPD was located at 12.3 :1: 1.9 cM from the Are 1024 allele, OAKZOm RAPD mapped
at 7.3 i 1.5 cM ﬁom the are 1024 allele and 15.8 :I: 2.3 cM separated both RAPD marker
loci (Table 3.4). Using the data across populations MAPMAKER analysis showed that
the RAPD markers were ﬂanking the Are 1024 locus.

The third RAPD marker, designated OAB3450 (generated by a 5'-TGGCGCACAC-
3' decamer), identiﬁed using bulked segregant analysis, was found linked in coupling with
the Mexique 3 allele (Fig. 3.4). Monogenic inheritance of the Mexique 3 gene was
conﬁrmed in two segregating populations (Black Magic/SEL 1360 and Blackhawk/SEL
1360), where the OAB3430 marker segregated as a single factor (Table 3.5).
Cosegregation analysis revealed that OAB3450 RAPD was linked to the Mexique 3 allele,
at a distance of 7.2 i 2.6 cM in the Black Magic/SEL 1360 population; and at 4.4 :t 2.1
cM in the Blackhawk/SEL 1360 population (Table 3.6). Combining data from all P;
individuals (206) in both populations, the linkage estimate for OAB3450 RAPD marker and
the Mexique 3 allele was 5.9 i 1.7 cM (Table 3.6). No difference in recombination value
among mapping populations was observed between the marker locus and the resistance

allele.

94

OAB3450»

 

Figure 3.4 Ampliﬁcation of genomic DNA using OAB3450 RAPD marker. Key to
Individuals: (1) anthracnose-susceptible Raven/SEL 1360 DNA bulk, (2) anthracnose-
resistant Raven/SEL 1360 DNA bulk, (3) homozygous resistant F2 individual,(4)
heterozygous resistant F2 individual, (5) homozygous susceptible F2 individual, (6)
resistant parent with Mexique 3 gene, SEL 1360, (7) resistant cultivar, with Mexique 3
gene, TU, (8) resistant cultivar with Mexique 3 gene, G 2333, (9) resistant line without
Mexique 3 gene, SEL 1308, (10) resistant cultivar withoutM exique 3 gene, AB 136,
(l 1) negative control (no template DNA added to PCR ampliﬁcation), (12) molecular
weight marker (1 Hind III/EcoRI: size of bands indicated in bp).

 

95

Table 3.5 Chi-square (X2) for the Mexique 3 allele and the OAB3450 RAPD marker segregating
in two diﬂ‘erent F2 and F23 populations.

 

 

Population Locus Generation Expected Observed X1 Probability
tested ratio frequency
Black Magic/SEL 1360 Mexique 3 F2 3:1 83:27 0.00 1.00
Black Magic/ SEL 1360 Mexique 3 F23 1:2:1 29:54:27 0.11 0.95
Blackhawk/SEL 1360 Mexique 3 F2 3:1 83:28 0.00 1.00
Blackhawk/SEL 1360 Mexique 3 F23 1:2:1 33:48:28 2.01 0.41
Black Magic/SEL 1360 OAB3450 F2 3:1 76:33 1.35 0.25
Blackhawk/SEL 1360 OAB3450 F2 3:1 74:23 0.03 0.86

 

Table 3.6 Two point Chi-square (X2) analysis and linkage estimates for OAB3450 RAPD
marker and the Mexique 3 allele.

 

 

Population' Observed x2 Probability cM
frequency’ 0‘ i SE)
Black Magic/SEL 1360 28:47: 1 : l 15:27 78.5 0.00 7.2 i 2.6
Blackhawk/SEL 1360 282432301222 77.0 0.00 4.4 i: 2.1
Across populations" 56:90:4z1:6:49 153.9 0.00 5.9 i 1.7

 

’ Anthracnose evaluation conducted on F23 progenies. ’ Linkage analysis, expected
ratio = 3:613:12211, based on 122:] genotypic segregation ratio for the Mexique 3
allele and 3:1 ratio for the 0.483450 RAPD marker. " Includes data ﬁ'om Black
Magic/Se] 1360 and Blackhawk/SEL 1360 populations.

96

DISCUSSION.

Based on our data, bean breeders have the opportunity to pyramid up to four
major anthracnose resistance genes (A, Are 1024 and Mexique 3, this Chapter and; Are,
Chapter 2) using RAPD markers for MAS. The combination of speciﬁc resistance genes
would aﬂ‘ord complete protection against all existing races of C. Iindemuthianum in North
America, and in regions of Latin America where higher pathogenic variability has been
observed. The value of the A, Are 1024 and Mexique 3 major genes and the application
of the linked RAPD markers in breeding for anthracnose resistance using MAS is now
possible.

A gene. The Andean A gene was the ﬁrst major gene utilized to develop
anthracnose resistant cultivars in common bean (Burkholder, 1918; McRostie, 1919).
With the discovery of the Middle American Are gene by Mastenbroek (1960), which
confers resistance to six races of C. lindemuthianum (Tu, 1992), the utilization of the A
gene as the main genetic source for anthracnose resistance began to decline. However,
the A gene regained importance after Kelly et al. (1994a), reported the appearance of two
new races of anthracnose in Michigan, and proposed pyramiding A and Are genes as a
suitable breeding strategy for controlling the emerging pathogenic variability. The
combination of Andean and Middle American resistance genes seems to be a sound
strategy for the control of bean anthracnose. For instance, it was observed, that several
highly virulent isolates of C. Iindemuthianum pathogenic on resistance genes of Middle
American origin, were avirulent on the Andean A gene (Pastor-Corrales et al., 1994);

conversely, isolates of Andean origin have been observed avirulent on Middle American

 

 

97
resistance genes (Pastor-Corrales et. al., 1995). Hence, the A gene could be a valuable

source for anthracnose resistance in regions where most of the contemporary cultivars in
use are derived from Middle American gerrnplasm, e. g. Mexico, Central America and
Brazil (Rava et al., 1993; Voysest et al., 1994). The tightly linked (1.9 :t 1.4 cM)
repulsion-phase OFlOm RAPD marker can be used in MAS, selecting against the
presence of the RAPD, to identiﬁy homozygous dominant individuals for the A locus. It is
also noteworthy that the OFlO primer was found polymorphic and linked in coupling with
a major rust resistance (B-l90) gene block in common bean (Haley et. al., 1993).
Although the OFIO RAPD marker had limited application within the Middle American
gene pool for selection of the B-19O gene, it offers the unique opportunity for
simultaneously introgressing two major resistance genes (A and B-190) into the great
northern and pinto bean market classes. There seemed to be no restriction for the
implementation of OF10530 RAPD for introgressing the A gene across gene pools.
However, in the cultivar Seafarer, which is known to carry the A gene (see appendix), the
marker locus was present. The OFIOm RAPD marker was absent in Isabella, G 2333,
Perry Marrow and Kaboon cultivars, but there is insufﬁcient genetic information regarding
the presence or absence of the A gene in these genotypes. However, more than one
genetic factor conditions resistance to the alpha race in Isabella (see appendix), G 2333
(Pastor-Corrales et al., 1994), Perry Marrow (Del-Peloso et al., 1989; Goncalves-Vidigal,
1994) and Kaboon (Muhalet et al., 1981) and the marker data suggests that the A gene
could be one of them.

Are 1024 gene. The anthracnose differential cultivar AB 136, a small red bean

type of Middle American origin, was the original source of the Are 1024 gene. The

98
genetic resistance conferred by Are 1024 has proven to be effective to a large number of

Latin American isolates of C. lindemuthianum, and is an important resistance source used
by breeding programs in Central America (Schwartz et al., 1982; Pastor-Corrales and Tu,
1989). Finding two RAPD markers in opposite orientation and ﬂanking the Are 1024
allele provides the opportunity to compare different selection strategies based on the
phenotype of individual markers or the combination of both as a codominant pair. A
comparison of selection efﬁciency for homozygous resistant individuals, using actual
observations, by means of MAS using a coupling-phase, repulsion-phase and/or a
codominant marker is shown in Table 3.7. Using a codominant marker, scoring
individuals simultaneously with both coupling (OAHl-m) and repulsion (OAKZOsgo)
RAPD markers, a level of 95% selection efﬁciency was observed, while 92% eﬂiciency
was obtained by selecting against the OAKZOm repulsion-phase marker alone. The
increased effectiveness of genotype selection observed using RAPDs as codominant
markers versus repulsion marker-based selection, was due to the ability of the codominant
marker as opposed to the repulsion-phase marker to distinguish, thus discard, double
recombinant individuals. Recognition of double recombinants by RAPD markers used as a
codominant pair has been cited as the main reason for the increased level of selection
efﬁciency over a repulsion-phase marker in MAS (Johnson et al., 1995). Tight linkages (s
5 cM) between markers and genes of interest is required for efﬁcient marker-based
selection (Tanksley, 1983). Linkages greater than ﬁve centimorgans, however, can

efﬁciently be used in MAS providing markers ﬂanking the locus of interest are available.

99

Table 3.7 Selection efﬁciency (%) observed based on selection for a coupling RAPD
OAHl-no and against a repulsion RAPD OAKZOsgo, and for both RAPD markers scored
as one codominant marker (OAHl-m- OAKZOsgo) in MAS for homozygous resistant
individuals for the Are 1024 resistance gene segregating in three different F2 populations.

 

 

 

Genotypic No. of Coupling Repulsion Codominant
frequency recombinants OAHl-m OAK2099. OAHlm-OAKZO”.
(C:R)'
RR-74 2:4 32 92 95
Rr-152 19:3 59 5 5
rr-97 18:2 8 3 0
No of selected F2 individuals 226 65 63

 

‘ Number of recombinant individuals observed using C = coupling-phase OAHl-m RAPD
marker and R = repulsion-phase OAKZOsgo RAPD marker.

This fact was conﬁrmed with the OAH 1m and OAKZOsgo RAPDs which were considered
loosely linked markers (12.3 :t 1.9 and 7.3 .+. 1.5 cM, respectively). Since OAHl-m and
OAK20390 RAPDs ﬂanked the Are 1024 locus and could be combined as a codominant
marker, higher levels of selection eﬁiciency over selection based on single coupling or
single repulsion-phase RAPD phenotype were possible (Table 3.7). Coupling-phase
RAPD linkages are usually sought during primer screening for molecular tagging.
Selection against a repulsion-phase RAPD marker, however, resulted in an increased-
selection efﬁciency of homozygous resistant individuals (92%) as opposed to 33%
selection efﬁciency for the coupling-phase RAPD in a F2 population segregating for the

Are 1024 resistance gene (Table 3.7). Greater selection efﬁciency of repulsion-phase over

100
coupling-phase RAPD markers for MAS has been demonstrated in common bean for

BCMV resistance (Haley et al., 1994b) and bean rust resistance (Johnson et al., 1995).
Estimates of recombination ﬁ'equency for the OAK20290/Are 1024 loci between the
Raven/Catrachita and Blackhawk/Catrachita mapping populations, were statistically
diﬁ‘erent. Factors affecting the rate of recombinational events among plants of a particular
population, may be genetically or environmentally inﬂuenced (Brown et al., 1989; Beavis
and Grant, 1991). Although the segregating populations are genetically related and were
developed during the same season and under the same environmental conditions, the
present research was not designed to discriminate between such factors which alter
recombination. The development of three different segregating populations for estimating
genetic linkages, helped to increase the level of accuracy when determining map distance
between loci of interest. Concurrently, testing cosegregation of RAPD markers and
resistance loci over different genetic backgrounds served to test the potential useﬁilness of
the marker, for ﬁxture MAS application, in genetically diverse breeding populations.
Mexique 3 gene. The genetic characterization of the Middle American Mexique 3
gene, present in the differential cultivar TU, was reported by Fouilloux (1979) in Europe
where it has been used extensively as a source of anthracnose resistance. It was shown, ,
that the Mexique 3 gene was present in SEL 1360, a black bean, derived ﬁ'om the
differential cultivar G 2333 (Chapter 1). The OAB3450 RAPD marker found linked in
coupling-phase to the Mexique 3 allele (5.9 i 1.7 cM) served to conﬁrm the presence of
the gene in TU, SEL 1360 and G 2333 and its absence in the other anthracnose differential
genotypes (data not shown). In Chapter 1 we proposed that G 2333 may in fact carry

three factors conditioning resistance to bean anthracnose, rather than the two proposed by

101
Pastor-Corrales et al., (1994). Since the previous authors used race 521 to screen their

segregating population, the Mexique 3 gene would have not been detected because it is
overcome by race 521. We have clearly demonstrated in genetic (Chapter 1) and DNA
analysis (marker) that G 2333 carries the Mexique 3 gene. Since G 2333 possesses the
most eﬂ‘ective race-speciﬁc resistance to C. Iindemuthianum currently observed, ﬁnding
markers linked to the other resistance genes will be complicated by the presence of more
than one segregating factor. Research to separate and tag these resistance factors in G
2333 is underway, while the OAB3450 RAPD marker will be used to assist in the
introgression and pyramiding of the Mexique 3 gene into commercial bean cultivars.

Gene pyramiding. The importance of incorporating a diverse group of resistance
sources and/or combining Andean and Middle American genes for stable resistance to
bean anthracnose has been suggested (Pastor-Corrales and Tu, 1989; Beebe and Pastor-
Corrales, 1991; Kelly et al., 1994a; Pastor-Corrales et al., 1995). Major resistance genes
such as A, Are, Are 1024 and Mexique 3, which have shown broad-based resistance, could
be pyramided into a single genotype, since, RAPD markers linked to alleles at these loci
can now be used in MAS breeding. If gene pyramiding is aiming to increase the longevity
of anthracnose resistance in common bean, then the number of genes really needed to
attained durable resistance becomes important. Based on the resistance to stripe mst
(Puccinia strirformis West. var. strirformis) observed in several winter wheat cultivars
grown in Western Europe during more than 15 years, Parlevliet (1993), suggested that the
cause of the lasting resistance was due to the presence of three fully eﬁ‘ective race-speciﬁc
genes pyramided into single cultivars. Similarly, Schafer and Roelfs (1985), indicated that

the increase in durability of resistance to wheat stem rust (Puccinia graminis Pers. f. sp.

 

102
tritici) in the northern plains was associated with multiple race-speciﬁc genes (6)

pyramided into a single genotype. Durability of the resistance was related to the low
probability of mutation to virulence occurring at various loci in the pathogen population.
Other potential mechanisms suggested responsible for the durability of resistance gene
pyramids are associated with: the reduced ﬁtness of a pathogen race which has
accumulated a large number of virulence genes (Van Der Plank, 1975; Green and
Campbell, 1979); the diﬂiculty of combining multiple virulence mutations with high
pathogen ﬁtness; and the chance discovery of single resistance genes or combinations of
small numbers of resistance genes that are more durable than average (Mundt, 1990,
1991). It would appear that breeding for gene pyramids will result in the increase of
durability of resistance, regardless of the mechanism involved. However, there is no clear
correlation between the number of resistance genes and the durability of the resistance
(Mundt, 1991) which suggests that some gene combinations might be more effective than
others. A large number of resistance genes deployed singly in a cultivar or genetic factors
other than race-speciﬁc genes, might be involved in increasing durable resistance
(Johnson, 1981).

We propose combining two genes into different genotypes and cultivar deployment
of such genetic combinations, would be more practical and realistic to eﬂiciently provide
long-term control of bean anthracnose at a reasonable cost and in a relatively short period
of time. Genetic diversity of resistance sources, suggested as a means of expanding host
resistance and increased durability, would be attained if the Andean A gene is combined
with any of the other three Middle American genes (Are, Are 1024 and Mexique 3) in

diﬁ‘erent combinations. Ideally, when breeding for a particular region, the breeder should

 

 

103
carefully select a gene pair that if deployed singly, would confer resistance to all known

races in that region. For example, in the US. and Canada the latter can be accomplished if
Are 1024 and Mexique 3 genes are selected for gene pyramiding. However for other areas
a suitable genetic pair and correspondence molecular markers might not always be
available. For instance, Are, Are 1024 and Mexique 3 genes as opposed to the A gene,
may have limited use in some areas of Central America due to presence of virulent races of
the pathogen that overcome the resistance conferred by these three Middle American
genes individually (Rava et al., 1993). For those situations, it would be advisable to
choose genes with a comprehensive race coverage and complementary to each other.
Thus, pyramiding A with any of the Middle American genes as a complementary gene pair
would be the best strategy to follow for complete protection to bean anthracnose in that
region. A similar complementary genetic pair could also be generated for the control of
the disease in North America. Two commercial bean cultivars (Isles and Newport) with
the gene combination (A and Are ) have already been released in Michigan (Kelly et
al.,1994b, 1995b); so the durability of the resistance in these cultivars remains to be
determined. The useﬁrlness and application of MAS as a breeding tool for gene
pyramiding directed towards durable disease resistance will differ between breeding
programs. In some instances, the presence of a gene combination could be detected using
discriminating races of the pathogen alone, or require the use of a linked molecular marker
to select for a hypostatic gene combined with race inoculation to ensure the selection of

the epistatic gene in segregating populations. Plant breeders will need to assess when to

104
use MAS as a strategy based on cost, time, space, and the availability of races to

effectively discriminate between resistance genes masked by epistasis.

 

 

105

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r
A...

 

APPENDIX

109

APPENDIX

Is the Anthracnose Resistance ‘A ’ Gene the Same in Cultivars Belonging to Both
Bean Gene Pools?

The A gene was one of the ﬁrst anthracnose resistance genes reported in the
literature (McRostie, 1919). Since its discovery, this single dominant gene, which
conditions resistance to the alpha race of Colletotrr'chum Iindemuthianum (Sacc. &
Magnus) Lams.- Scrib, has been incorporated into cultivars ﬁ'om both gene pools.
Currently, it is not clear if resistance to the alpha race observed in contemporary
Mesoamerican and Andean cultivars, traces back to a single origin.

One of the ﬁrst bean cultivars known to carry a resistance gene effective against
the alpha race was Wells’ Red Kidney (Barrus, 1915). Crosses between this kidney bean
and selections of Michigan Robust (a white navy bean), were carried out by McRostie to
introduce anthracnose resistance into the navy bean. Based on this evidence, one might
speculate that the alpha resistance gene in Andean and Mesoamerican cultivars has a single
origin in Wells’ Red Kidney. Genetic resistance to the alpha race in Emerson 847, a navy
bean breeding line, and Dark Red Kidney was shown to be governed by a dominant gene
at the A locus (Cardenas et al., 1964). This raises two important questions: is the source
of the A gene the same for cultivars ﬁom both gene pools? and is there only a single
dominant gene that conditions resistance to the alpha race? Certainly, many of the current

commercial cultivars have either Emerson 847 or Dark Red Kidney in their pedigree. For

l 10
instance, Sanilac and Seafarer (navy bean cultivars), derives their resistance to alpha from

Emerson 847 (Andersen et al., 1960); whereas Montcalm (a kidney bean cultivar), derives
its resistance to alpha from Michigan Dark Red Kidney (Copeland and Erdmann, 1977).

In order to determine if the same alpha resistance gene is present in navy and
kidney bean cultivars, the following cross was made: Montcalm x Seafarer. Parental
material, a susceptible check and 103 F2 individual plants derived ﬁ'om this cross were
inoculated with a spore suspension of the alpha race. Apart from symptoms on the
susceptible check, none of the segregating F2 plants and neither parental genotype showed
any anthracnose symptoms. These results suggest that both Seafarer and Montcalm carry
identical genes corresponding to the A locus.

In order to verify if other kidney bean cultivars carried the same A gene, a second
cross between Isabella x Seafarer was made. Isabella, a light red kidney bean, was
released by MSU as an alpha resistance cultivar (Kelly et al., 1987), but no known
anthracnose resistance parent is recognized in its pedigree (‘Redkloud/Mecosta’). The
disease evaluation of the F2 population of the Isabella x Seafarer cross generated a
diﬁ‘erent outcome. Among the 177 F2 individual plants tested, 21 plants were rated as
susceptible (disease symptoms present), whereas 156 plants were rated as resistant
(disease symptoms absent) to the alpha race. Chi-square tests performed on this data
supported a 57:7 ratio (P = 0.78), suggesting the segregation of three factors (3: 1, A gene
in Seafarer; 9:7, duplicate complementary factors in Isabella). The data clearly showsthat
a different genetic system conditions resistance to the alpha race in Isabella. Progeny tests

performed in the F3 generation conﬁrmed the F2 observations. The source of the

 

1 1 1
resistance to alpha in Isabella is unknown, and, as demonstrated, is not controlled by the A

gene.

The ﬁnding that Isabella carries different alpha resistance factors, was recently
conﬁrmed by Kelly et al. (1994), using two new anthracnose races reported in Michigan.
They found that one of these races (Race 73), in addition to defeating the Are resistance
gene, was also pathogenic on Isabella. However, Race 73 was non pathogenic on
Seafarer, Montcalm, Sanilac and Michigan Dark Red Kidney cultivars, which all carry the
A gene.

For years, anthracnose resistance in North America has relied on the protection
conferred by the Are gene. The appearance of race 73 has put the useﬁrlness of the Are
gene in jeopardy, whereas the A gene has regained importance. Hence, pyramiding Are
and A genes is a sound breeding strategy for developing anthracnose resistant bean

cultivars where variability in C. Iindemuthianum is a potential threat.

Literature cited.

Andersen, A.L., Down, EB. and Whitford, 6.1960. The Sanilac pea bean. Michigan
Quarterly Bulletin 43(2):2l4-236

Barrus, M.F. 1915. An anthracnose-resistant red kidney bean. Phytopathology 52303-311

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

Copeland, L0. and Erdmann, M.H. 1977. Montcalm and Mecosta (Halo blight tolerant
kidney bean varieties for Michigan). Cooperative Extension Service. Michigan
State University. No 957

 

112

McRostie, GP. 1919. Inheritance of anthracnose resistance as indicated by a cross
between a resistant and a susceptible bean. Phytopathology 9(3): 141-148

Kelly, J.D., Adams, M.W., Saetler, A.W., Hosﬁeld, G.L., Uebersax, MA. and Ghaderi, A.
1987 . Registration of ‘Isabella’Light Red Kidney Bean. Crop Sci. 27:363-364

Kelly, J.D., Afanador, L. and Cameron, LS. 1994. New races of Colletotrichum
Iindemuthianum in Michigan and implications in dry bean resistance breeding.
Plant disease 78(9):892-894

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