**9. Applications of MAS**

The key success of integrating MAS into breeding programmes lies in identifying applica‐ tions in which markers offer real advantages over conventional/classical breeding methods or complement them in a novel way. MAS offer significant advantages in cases where phe‐ notypic screening is expensive, difficult or impossible or traits are of low heritability and/or the selected trait is expressed late in plant development. Also, for incorporating genes for resistance to diseases or pests that cannot be easily screened due to special requirements for the genes to be expressed; the expression of the target gene is recessive; there is a need to accumulate multiple genes for one or more traits within the same cultivar, or improving per‐ ennial/biennial crops with long life cycle using a process called gene pyramiding [13, 20, 32, 33]. The success of MAS depend upon the distance between the markers and the target gene, the number of target genes to be transferred, the genetic basis of the trait, the number of in‐ dividuals that can be analyzed and the genetic background in which the target gene has to be transferred, the type of molecular marker (s) used and the availability of specific technical facilities [20, 21].

Conventional breeding has been successfully applied in several crops' breeding pro‐ grammes and a large number of varieties or lines possessing multiple attributes have been produced. However, the difficulties associated with this method are due to the dominance and epistasis effects of genes governing the target disease resistances, for example, the CBB resistance in case common bean. Therefore, MAS has been especially suggested for increas‐ ing the selection efficiency and timely delivery of cultivars in the particular case of breeding for resistant cultivars. The benefits from the use of genomics tools include (i) more effective‐ ly identify, quantify and characterize genetic variation from all available germplasm resour‐ ces; (ii) tag, clone and introgress genes and/or QTLs that are useful for enhancing the target trait using either genetic transformation, facilitating pyramiding or recurrent selection, by differentiating and selecting particular genotypes in breeding populations [20, 32].

#### **10. Molecular markers assisted selection for bean diseases**

Breeders used to rely on visual screening of genotypes to select for traits of economic impor‐ tance. However, successful application of this method depends on its reproducibility and heritability of the trait. Therefore, the use of molecular markers in the bean breeding pro‐ grammes has improved the accuracy of crosses to carry out and allowed breeders to pro‐ duce germplasm with combined traits that were hazardous and difficult before the advent of DNA technology [13].

#### **10.1. Angular leaf spot**

Resistance genes against *Phaeoisariopsis griseola* the causal agent of ALS are controlled by ma‐ jor genes, that are either dominant or recessive, acting singly or duplicated and which may interact in an additive manner with or without epistasis [56]. Inheritance of resistance is con‐ trolled by a single recessive gene [57], but in an earlier study, resistance to ALS was reported to be controlled by a single dominant gene. This shows that inheritance of ALS resistance is complex, involving both dominant and recessive genes that may be or may not be independ‐ ent. Major and minor genes mediate angular leaf spot (ALS) resistance in beans (*P. vulgaris)* and a number of sources for these resistance genes have been identified [56]. Diverse sour‐ ces of resistance to angular leaf spot in bean genotypes have been reported [58]. Examples of resistant cultivars include A 75, A 140, A 152, A 175, A 229, BAT 76, BAT 431, BAT 1432, BAT 1458 and G5686, MAR 1, MAR 2 [59]. In reference [60] found the ALS resistance in AND 277 to race 63:23 to be conferred by a single dominant gene (*Pgh-1*). Cornell 49-242 has *Pgh-2* which confers resistance to *P. griseola* pathotype 31:17 [61] while [41] found that resist‐ ance to ALS in Mexico 54 is due to a single dominant gene that confers resistance to patho‐ type 63:63 and G06727 has resistance to *P. griseola* pathotype 63:59. In reference [120] reported that 'Ouro Negro' had resistance to 8 pathotypes, including *P. griseola* race 63:63 from Brazil. G5686 and Mexico 54 display fairly good levels of resistance to nearly all races [59]. These cultivars are not only good sources of resistance to *P. griseola* but could also serve as reliable indicators of new races of the pathogen in the future [62]. Mexico 54 has shown to be resistant to all *P. griseola* isolates characterized in Africa [63]. Resistance in G5686 is con‐ ditioned by two dominant epistatic genes and Amendoim by two recessive genes [64]. Re‐ sistance to specific isolates of *P. griseola* has been reported to be simply inherited and molecular markers have been identified for some of these resistance genes [14, 41, 65, 66]. Sources of resistance reported from Africa include GLP 24, GLP X-92, GLP - 806 and GLP 77 [59]. Resistance to various diseases is monogenically determined, but cases of duplicate, complementary and other interactions have been reported [67]. The breed for ALS resist‐ ance, molecular markers linked to angular leaf spot resistance genes have been identified in beans*.* SCAR markers for selecting for genes for resistance to ALS include SH13 for *phg-1* gene in linkage group *6 [68]* and SNO2 for phg-2 gene in linkage group 8 [69, 61]. Others include, SAA19 [68], SBA16 [68] and SMO2 [68] which is ouro negro dominant gene.

#### **10.2. Common bacterial blight**

ing the selection efficiency and timely delivery of cultivars in the particular case of breeding for resistant cultivars. The benefits from the use of genomics tools include (i) more effective‐ ly identify, quantify and characterize genetic variation from all available germplasm resour‐ ces; (ii) tag, clone and introgress genes and/or QTLs that are useful for enhancing the target trait using either genetic transformation, facilitating pyramiding or recurrent selection, by

Breeders used to rely on visual screening of genotypes to select for traits of economic impor‐ tance. However, successful application of this method depends on its reproducibility and heritability of the trait. Therefore, the use of molecular markers in the bean breeding pro‐ grammes has improved the accuracy of crosses to carry out and allowed breeders to pro‐ duce germplasm with combined traits that were hazardous and difficult before the advent

Resistance genes against *Phaeoisariopsis griseola* the causal agent of ALS are controlled by ma‐ jor genes, that are either dominant or recessive, acting singly or duplicated and which may interact in an additive manner with or without epistasis [56]. Inheritance of resistance is con‐ trolled by a single recessive gene [57], but in an earlier study, resistance to ALS was reported to be controlled by a single dominant gene. This shows that inheritance of ALS resistance is complex, involving both dominant and recessive genes that may be or may not be independ‐ ent. Major and minor genes mediate angular leaf spot (ALS) resistance in beans (*P. vulgaris)* and a number of sources for these resistance genes have been identified [56]. Diverse sour‐ ces of resistance to angular leaf spot in bean genotypes have been reported [58]. Examples of resistant cultivars include A 75, A 140, A 152, A 175, A 229, BAT 76, BAT 431, BAT 1432, BAT 1458 and G5686, MAR 1, MAR 2 [59]. In reference [60] found the ALS resistance in AND 277 to race 63:23 to be conferred by a single dominant gene (*Pgh-1*). Cornell 49-242 has *Pgh-2* which confers resistance to *P. griseola* pathotype 31:17 [61] while [41] found that resist‐ ance to ALS in Mexico 54 is due to a single dominant gene that confers resistance to patho‐ type 63:63 and G06727 has resistance to *P. griseola* pathotype 63:59. In reference [120] reported that 'Ouro Negro' had resistance to 8 pathotypes, including *P. griseola* race 63:63 from Brazil. G5686 and Mexico 54 display fairly good levels of resistance to nearly all races [59]. These cultivars are not only good sources of resistance to *P. griseola* but could also serve as reliable indicators of new races of the pathogen in the future [62]. Mexico 54 has shown to be resistant to all *P. griseola* isolates characterized in Africa [63]. Resistance in G5686 is con‐ ditioned by two dominant epistatic genes and Amendoim by two recessive genes [64]. Re‐ sistance to specific isolates of *P. griseola* has been reported to be simply inherited and molecular markers have been identified for some of these resistance genes [14, 41, 65, 66]. Sources of resistance reported from Africa include GLP 24, GLP X-92, GLP - 806 and GLP 77 [59]. Resistance to various diseases is monogenically determined, but cases of duplicate,

differentiating and selecting particular genotypes in breeding populations [20, 32].

**10. Molecular markers assisted selection for bean diseases**

of DNA technology [13].

130 Plant Breeding from Laboratories to Fields

**10.1. Angular leaf spot**

The control of common bacterial blight (CBB) disease caused by *Xanthomonas axonopodis pv phaseoli (Xap)* is challenging due to its complexity and seed borne nature [67]. The number of genes involved in resistance to *Xap* range from one to several genes with varying degrees of action and interactions [70, 71]. Breeding for CBB resistance is complicated pathogen genetic diversity and coevolution [72, 73] different genes conditioning resistance in leaves, pods and seeds [16, 73, 74, 76] and linkage of resistance with undesirable traits [16, 76]. Resistance of CBB is quantitatively and qualitatively controlled depending on the source of germplasm with pod and leaf resistance being controlled by different genes [9, 67, 77]. Quantitative in‐ heritance was observed after making original interspecific crosses between resistant *P. acuti‐ folius* 'tepary 4' and susceptible *P. Vulgaris* [67]. Sources of resistance to *Xap* in common bean have been reported [66, 78]. Other sources of resistance have been identified in tepary bean (*P. acutifolius*) [79, 80], and runner bean, (*P. coccineus*) [81]. Resistance to common bacterial blight has been reported in *Phaseolus acutifolius* [77], *P. coccineus* and lines of *P. vulgaris* [82]. CIAT lines VAX 3, VAX 4, VAX 6, and XAN 159 have also been reported to have good level of resistance to common bacterial blight [67]. Increased resistance can be developed by se‐ lecting for horizontal resistance [83].

Albeit, genetic studies have shown that resistance to CBB is quantitatively inherited, it in‐ volves a few major genes [13]. The identification of QTL influencing resistance to CBB com‐ bined with phenotypic data implying the involvement of few genes, suggests that MAS may be useful in combining resistance sources to CBB in common bean. To date, SCAR markers used in selecting resistance to CBB are dominant and are scored as presence or absence of a single band on an agarose gel. SCAR markers available in screening are SU91, BC420, SAP 6, BAC 6, R7313 and R4865. SU91 is linked to a QTL for CBB resistance in bean in the linkage group B8 [16, 84]. BC420 is linked to a QTL for CBB resistance on bean linkage group B6. SAP 6 is for a major QTL in the linkage group B10 [84], BAC 6 for a major QTL in linkage group B10 [85] R7313 for a major QTL in linkage group B8 [86] and R4865 for another major QTL [86]. Thus, molecular markers allow distinct QTLs to be screened and consequently provide an opportunity to pyramid multiple QTL for CBB resistance into a single genotype.

#### **10.3. Bean common mosaic virus and bean common mosaic necrosis virus**

Genetic resistance to both potyviruses is conditioned by a series of independent multi-allelic loci in common bean is affected by four different loci: bc-1, bc-2, bc-3 and bc-u [87]. Resist‐ ance controlled by alleles at these loci is inherited as recessive characters [88]. In addition to the recessive bc genes, the dominant I gene in *P. vulgaris* confers resistance to BCMV and other potyviruses through a hypersensitive response [88, 89] and has also been the focus of positional gene cloning activities [90]. The *I* gene located on B2 [91], is independent of reces‐ sive resistance conditioned by three different *bc* genes. The *bc-3* gene is located on B6 [84, 92, 93], whereas the *bc-1*2 allele was mapped to B3 [84]. The non-specific *bc-u* allele, needed for expression of *bc-2*2 resistance, also resides on B3 based on the loose linkage with the *bc-1* locus [94].

The independence of the BCMV resistance genes provides opportunities to use gene pyra‐ miding as a strategy in breeding for durable resistance. Bean breeders recognize that the combination of the dominant *I* gene with recessive *bc* resistance genes offers durability over single gene resistance to BCMV and BCMNV, since the two types of genes have distinctly different mechanisms of resistance [95]. The dominant *I* gene is defeated by all necrotic strains, whereas the three most effective recessive genes (*bc-1, bc-2 and bc-3*) act constitutive‐ ly by restricting virus movement within the plant, probably through the virus movement proteins. The action of the dominant *I* gene is masked by the recessive *bc-3* gene, so as efforts to incorporate the *bc-3* gene into new germplasm proceed, the risk of losing the *I* gene in im‐ proved germplasm increases, since direct selection for the *I* gene is not possible. Linked markers offer the only realistic opportunity to maintain and continue to utilize the *I* gene as a pyramided resistance gene in future bean cultivars.

A marker tightly linked to the *I* gene [96] has been demonstrated in many laboratories to be effective across a wide range of germplasm from both gene pools. Breeders have used mark‐ ers linked to the *I* gene to develop enhanced germplasm with the *I* +*bc-3* gene combination. In addition, [92] developed SCAR markers from the OC11350*/*420 (ROC11) and OC20460 RAPD markers linked to the *bc-3* gene to improve their utilization. The use of these markers in MAS, however, has been limited due to a lack of polymorphism and reproducibility across diverse genetic backgrounds and gene pools of common bean [91].

Direct screening with strains of BCMV and BCMNV is still required to confirm the presence of the *bc-3* gene. To efficiently introgression the *bc-3* gene for resistance to BCMV and BCMNV into susceptible bean cultivars, there is a need to identify more robust DNA mark‐ ers tightly linked to the *bc-3* gene that will demonstrate reproducibility across laboratories and be functional in different genetic backgrounds. Similarly, the hypostatic *I* gene is re‐ tained in the presence of the *bc-3* gene by MAS for the SW13 SCAR [69, 96]. This combina‐ tion of a dominant and a recessive gene, likely possessing different resistance mechanisms, should provide more durable resistance to bean common mosaic virus.

At CIAT, bean cultivars have been bred which combine *I* gene and recessive resistance genes. These have been evaluated in areas of East Africa where BCMNV is known to occur [96]. Several commercial varieties combining the *I* gene and recessive resistance genes are now available [97, 99].

#### **10.4. Anthracnose**

Two new sources of anthracnose resistance within the Andean gene pool were identified in germplasm from Brazil [10, 100; 101]. The two independent genes were identified as Co-12 in Jalo Vermelho and Co-13 in Jalo Listras Pretas and represent unique resistance patterns. These are significant findings as the multiallelic Co-1 locus with five alleles was the only re‐ sistance sources previously known in Andean germplasm. This is particularly important given the recent breakdown of the Co-12 gene by race 105 in Manitoba. The rapid evolution of this new race underscores the need to monitor the pathogenic variability in different pro‐ duction areas. The availability of new resistance genes of Andean origin offers breeders more choices for pyramiding genes with the more common Middle American resistance sources.

#### **10.5. Root rots**

positional gene cloning activities [90]. The *I* gene located on B2 [91], is independent of reces‐ sive resistance conditioned by three different *bc* genes. The *bc-3* gene is located on B6 [84, 92, 93], whereas the *bc-1*2 allele was mapped to B3 [84]. The non-specific *bc-u* allele, needed for expression of *bc-2*2 resistance, also resides on B3 based on the loose linkage with the *bc-1*

The independence of the BCMV resistance genes provides opportunities to use gene pyra‐ miding as a strategy in breeding for durable resistance. Bean breeders recognize that the combination of the dominant *I* gene with recessive *bc* resistance genes offers durability over single gene resistance to BCMV and BCMNV, since the two types of genes have distinctly different mechanisms of resistance [95]. The dominant *I* gene is defeated by all necrotic strains, whereas the three most effective recessive genes (*bc-1, bc-2 and bc-3*) act constitutive‐ ly by restricting virus movement within the plant, probably through the virus movement proteins. The action of the dominant *I* gene is masked by the recessive *bc-3* gene, so as efforts to incorporate the *bc-3* gene into new germplasm proceed, the risk of losing the *I* gene in im‐ proved germplasm increases, since direct selection for the *I* gene is not possible. Linked markers offer the only realistic opportunity to maintain and continue to utilize the *I* gene as

A marker tightly linked to the *I* gene [96] has been demonstrated in many laboratories to be effective across a wide range of germplasm from both gene pools. Breeders have used mark‐ ers linked to the *I* gene to develop enhanced germplasm with the *I* +*bc-3* gene combination. In addition, [92] developed SCAR markers from the OC11350*/*420 (ROC11) and OC20460 RAPD markers linked to the *bc-3* gene to improve their utilization. The use of these markers in MAS, however, has been limited due to a lack of polymorphism and reproducibility

Direct screening with strains of BCMV and BCMNV is still required to confirm the presence of the *bc-3* gene. To efficiently introgression the *bc-3* gene for resistance to BCMV and BCMNV into susceptible bean cultivars, there is a need to identify more robust DNA mark‐ ers tightly linked to the *bc-3* gene that will demonstrate reproducibility across laboratories and be functional in different genetic backgrounds. Similarly, the hypostatic *I* gene is re‐ tained in the presence of the *bc-3* gene by MAS for the SW13 SCAR [69, 96]. This combina‐ tion of a dominant and a recessive gene, likely possessing different resistance mechanisms,

At CIAT, bean cultivars have been bred which combine *I* gene and recessive resistance genes. These have been evaluated in areas of East Africa where BCMNV is known to occur [96]. Several commercial varieties combining the *I* gene and recessive resistance genes are

Two new sources of anthracnose resistance within the Andean gene pool were identified in germplasm from Brazil [10, 100; 101]. The two independent genes were identified as Co-12 in Jalo Vermelho and Co-13 in Jalo Listras Pretas and represent unique resistance patterns.

across diverse genetic backgrounds and gene pools of common bean [91].

should provide more durable resistance to bean common mosaic virus.

a pyramided resistance gene in future bean cultivars.

locus [94].

132 Plant Breeding from Laboratories to Fields

now available [97, 99].

**10.4. Anthracnose**

Root rot of dry bean is a yield-limiting disease problem for growers in the North-Central re‐ gion of the U.S. [102]. In North Dakota and Minnesota, *Fusarium solani* was considered to be the most common causal agent of root rot followed by *Rhizoctonia solani* [103]. However, re‐ cent findings have highlighted the ability of other Fusarium species to cause root rot in dry beans [104, 105]. Little is known about the prevalence and virulence of the four subspecies of *Rhizoctonia solani* that are found on common bean. Crops grown in rotation with beans, such as sugar beets, are also hosts for *R. solani*. [106]) found low genetic diversity among 166 iso‐ lates of the Fusarium wilt pathogen from the U.S. Central High Plains using RAPD markers. Resistance to Fusarium wilt in race Durango dry beans CO 33142 and Fisher were controlled by a single dominant gene, whereas polygenic control (h2 ranged from 0.25 to 0.60) was found for resistance in race Mesoamerica cultivars Rio Tibagi and Jamapa [107, 108]. In ad‐ dition, limited research has been conducted on *Aphanomyces euteiches f.sp. phaseoli*, but this fungus occurs frequently in the sandy soils in the Upper Midwest.

#### **10.6. Rust**

Two new races of rust have been recently reported in Michigan and North Dakota. The new races have reoccurred in Michigan since 2007 and in North Dakota since 2008. Preliminary results are showing that both races are similar, but not identical [109]. Resistance to both races is conditioned by the Ur-5, Ur-11, and CNC genes. A new source of resistance was mapped to LG 4 near the Ur-5 and Ur-Dorado108 loci in black bean populations derived from Tacana [110]. Several new cultivars with different combinations of rust resistance genes have been released [111]. Salient among these are six unique great northern bean germplasm lines named BelDakMi-RMR-8, to -13. These are the first great northern beans that combine four genes for rust resistance and two genes for resistance to the two bean common mosaic potyviruses. These beans combine two Andean (Ur-4 and Ur-6) and two Middle American (Ur-3 and Ur-11) rust resistance genes [111]. Other rust resistant cultivars include great northern bean cultivars ABC-Weihing (Ur-3 and Ur-6) [112], and Coyne (Ur-3 and Ur-6) [113], and Pinto CO46348 (Ur-4 and Ur-11) [114]. In the case of soybean rust, the common bean lines Compuesto Negro Chimaltenengo (CNC) and PI 181996 were among the most resistant to all six isolates. Inheritance of SBR resistance in CNC was studied by crossing Mx309/CNC. Based on severity, the segregation for SBR resistance in the F2 popula‐ tion fit a 9 resistant to 7 susceptible ratio.

#### **11. Other case studies of MAS**

MAS has been proposed as the most practical and realistic approach to provide efficient long term control of bean anthracnose, ashly stem, bean common mosaic virus, common mosaic necrosis virus, bean golden mosaic virus [69], bean rust [115] and common bacterial blight [16, 64]. It has been or is being used to assist the simultaneous transfer of resistance genes for rust, anthracnose and angular leaf spot into Brazilian commercial cultivars [29]. Several lines resistant to rust [115, 116]; bean golden mosaic virus [69] and anthracnose [117] are being obtained using MAS.
