Phillip N. Miklas

A reference genome for common bean and genome-wide analysis of dual domestications

Common bean (Phaseolus vulgaris L.) is the most important grain legume for human consumption and has a role in sustainable agriculture owing to its ability to fix atmospheric nitrogen. We assembled 473 Mb of the 587-Mb genome and genetically anchored 98% of this sequence in 11 chromosome-scale pseudomolecules. We compared the genome for the common bean against the soybean genome to find changes in soybean resulting from polyploidy. Using resequencing of 60 wild individuals and 100 landraces from the genetically differentiated Mesoamerican and Andean gene pools, we confirmed 2 independent domestications from genetic pools that diverged before human colonization. Less than 10% of the 74 Mb of sequence putatively involved in domestication was shared by the two domestication events. We identified a set of genes linked with increased leaf and seed size and combined these results with quantitative trait locus data from Mesoamerican cultivars. Genes affected by domestication may be useful for genomics-enabled crop improvement.

Common bean breeding for resistance against biotic and abiotic stresses: From classical to MAS breeding

SummaryBreeding for resistance to biotic and abiotic stresses of global importance in common bean is reviewed with emphasis on development and application of marker-assisted selection (MAS). The implementation and adoption of MAS in breeding for disease resistance is advanced compared to the implementation of MAS for insect and abiotic stress resistance. Highlighted examples of breeding in common bean using molecular markers reveal the role and success of MAS in gene pyramiding, rapidly deploying resistance genes via marker-assisted backcrossing, enabling simpler detection and selection of resistance genes in absence of the pathogen, and contributing to simplified breeding of complex traits by detection and indirect selection of quantitative trait loci (QTL) with major effects. The current status of MAS in breeding for resistance to angular leaf spot, anthracnose, Bean common mosaic and Bean common mosaic necrosis viruses, Beet curly top virus, Bean golden yellow mosaic virus, common bacterial blight, halo bacterial blight, rust, root rots, and white mold is reviewed in detail. Cumulative mapping of disease resistance traits has revealed new resistance gene clusters while adding to others, and reinforces the co-location of QTL conditioning resistance with specific resistance genes and defense-related genes. Breeding for resistance to insect pests is updated for bean pod weevil (Apion), bruchid seed weevils, leafhopper, thrips, bean fly, and whitefly, including the use of arcelin proteins as selectable markers for resistance to bruchid seed weevils. Breeding for resistance to abiotic stresses concentrates on drought, low soil phosphorus, and improved symbiotic nitrogen fixation. The combination of root growth and morphology traits, phosphorus uptake mechanisms, root acid exudation, and other traits in alleviating phosphorus deficiency, and identification of numerous QTL of relatively minor effect associated with each trait, reveals the complexity to be addressed in breeding for abiotic stress resistance in common bean.

Tagging and mapping of genes and QTL and molecular marker-assisted selection for traits of economic importance in bean and cowpea

Bean/Cowpea Collaborative Research Support Program (B/C CRSP) scientists have successfully developed integrated consensus maps of the 11 linkage groups (LGs) in both bean (Phaseolus vulgaris L.) and cowpea (Vigna unguiculata L. Walp). The bean map is approximately 1200 cM with some 500 markers and an additional 500 markers shared with other bean maps. The cowpea map spans 2670 cM with over 400 markers. In addition to molecular markers, both maps include map locations of defense genes and phenotypic traits for disease and insect resistance, seed size, color and storage proteins, pod color and those traits associated with the domestication syndrome in bean. Since the bean and cowpea maps were developed independently, LGs with the same number probably refer to non-syntenic groups. Map locations of major resistance genes in bean are revealing gene clusters on LGs B1, B4, B7, and B11 for resistance to bean rust, anthracnose, common bacterial blight and white mold. Gene tagging and marker-assisted selection for disease resistance has progressed to a point where the indirect selection for resistance to a number of major diseases is now routine in bean breeding programs both in the US and overseas. # 2003 Elsevier Science B.V. All rights reserved.

Identification of RAPD markers linked to a major rust resistance gene block in common bean.

Rust in bean (Phaseolus vulgaris L.), caused byUromyces appendiculatus (Pers.) Unger var.appendiculatus [ =U. phaseoli (Reben) Wint.], is a major disease problem and production constraint in many parts of the world. The predominant form of genetic control of the pathogen is a series of major genes which necessitate the development of efficient selection strategies. Our objective was focused on the identification of RAPD (random amplified polymorphic DNA) markers linked to a major bean rust resistance gene block enabling marker-based selection and facilitating resistance gene pyramiding into susceptible bean germplasm. Using pooled DNA samples of genotyped individuals from two segregating populations, we identified two RAPD markers linked to the gene block of interest. One such RAPD, OF10970 (generated by a 5′-GGAAGCTTGG-3′ decamer), was found to be closely linked (2.15±1.50 centi Morgans) in coupling with the resistance gene block. The other identified RAPD, OI19460 (generated by a 5′-AATGCGGGAG-3′ decamer), was shown to be more tightly linked (also in coupling) than OF10970 as no recombinants were detected among 97 BC6F2 segregating individuals in the mapping population. Analysis of a collection of resistant and susceptible cultivars and experimental lines, of both Mesoamerican and Andean origin, revealed that: (1) recombination between OF10970 and the gene block has occurred as evidenced by the presence of the DNA fragment in several susceptible genotypes, (2) recombination between OI19460 and the gene block has also occurred indicating that the marker is not located within the gene block itself, and (3) marker-facilitated selection using these RAPD markers, and another previously identified, will enable gene pyramiding in Andean germplasm and certain Mesoamerican bean races in which the resistance gene block does not traditionally exist. Observations of variable recombination among Mesoamerican bean races suggested suppression of recombination between introgressed segments and divergent recurrent backgrounds.

Identification and potential use of a molecular marker for rust resistance in common bean

SummaryThe Up2 gene of common bean (Phaseolus Vulgaris L.) is an important source of dominant genetic resistance to the bean rust pathogen [Uromyces appendiculatus (Pers. ex Pers.) Unger var ‘appendiculatus’ [syn U. Phaseoli (Reben) Wint.]. Up2 in combination with other rust resistance genes may be used to obtain potentially stable genetic resistance. It is difficult, however, to combine rust resistance genes effective against a single race due to epistatic interactions that frequently occur between them. A strategy that employed bulked DNA samples formed separately from the DNA of three BC6F2 individuals with Up2 and three without Up2 as contrasting near-isogenic lines (NILs) was used to identify random amplified polymorphic DNA fragments (RAPDs) tightly linked to the Up2 locus. Only 1 of 931 fragments amplified by 167 10-mer primers of arbitrary sequence in the polymerase chain reaction (PCR) was polymorphic. The RAPD marker (OA141100) amplified by the 5′-TCTGTGCTGG-3′ primer was repeatable and its presence and absence easy to score. No recombination was observed between OA141100 and the dominant Up2 allele within a segregating BC6F2 population of 84 individuals. This result suggests that OA141100 and Up2 are tightly linked. Andean and Mesoamerican bean germ plasm, with and without the Up2 allele, were assayed for the presence of OA141100. Apparently, the marker is of Andean origin because all Andean lines, with or without the Up2 allele, contained the marker, and the marker was absent in all Mesoamerican germ plasm except the lines to which Up-2 had been purposely transferred. These results suggest that OA141100 will be most useful for pyramiding Up2 with other rust resistance genes into germ plasm of Mesoamerican origin where the marker does not traditionally exist. The use of bulked DNA samples may have concentrated resources toward the identification of RAPDs that were tightly linked to the target locus. Marker-based selection may provide an alternative to the time-consuming testcrosses required to pyramid bean rust resistance genes that exhibit epistasis.

The role of RAPD markers in breeding for disease resistance in common bean

Diseases are regarded as the leading constraint to increased common bean (Phaseolus vulgaris L.) production worldwide. The range in variability and complexity among bean pathogens can be controlled with different single gene and quantitative resistance sources. Combining these resistance sources into commercial cultivars is a major challenge for bean breeders. To assist breeders, a major effort to identify RAPD markers tightly linked to different genes was undertaken. To date, 23 RAPD and five SCAR markers linked to 15 different resistance genes have been identified, in addition to QTL conditioning resistance to seven major pathogens of common bean. We review the feasibility of using marker-assisted selection (MAS) to incorporate disease resistance into common bean. Indirect selection of single resistance genes in the absence of the pathogen and the opportunity afforded breeders to pyramid these genes to improve their longevity and retain valuable hypostatic genes is discussed. The role of markers linked to the QTL controlling complex resistance and the potential to combine resistance sources using marker based selection is reviewed. Improving levels of selection efficiency using flanking markers, repulsion-phase linkages, co-dominant marker pairs, recombination-facilitated MAS and SCAR markers is demonstrated. Marker-assisted selection for disease resistance in common bean provides opportunities to breeders that were not feasible with traditional breeding methods.

Coupling- and repulsion-phase RAPDs for marker-assisted selection of PI 181996 rust resistance in common bean

The Guatemalan black bean (Phaseolus vulgaris L.) plant introduction (PI) 181996 is resistant to all known US races of the bean rust fungus Uromyces appendiculatus (Pers. ex Pers.) Unger var. appendiculatus [syn. U. phaseoli (Reben) Wint.]. We report on two random amplified polymorphic DNA (RAPD) markers OAC20490 tightly linked (no recombinants) in coupling phase and OAE19890 linked in repulsion phase (at 6.2±2.8 cM) to PI 181996 rust resistance. These RAPDs, generated by single decamer primers in the polymerase chain reaction, were identified in near-isogenic bulks of non-segregating resistant and susceptible BC4F2 (NX-040*4/PI 181996) lines. Linkage of the RAPD markers was confirmed by screening 19 BC4F2 and 57 BC4F3 individuals segregating for PI 181996 resistance. Utility of the RAPDs OAC20490 and OAE19890 was investigated in a diverse group of common bean cultivars and lines. All cultivars into which the PI 181996 resistance was introgressed had the RAPD OAC20490. A RAPD similar in size to OAC20490, observed in some susceptible common bean lines, was confirmed by Southern blotting to be homologous to the RAPD OAC20490. Use of the RAPDs OAC20490 and OAE19890 in marker-assisted selection (MAS) is proposed. The coupling-phase RAPD is most useful for MAS of resistant BCnF1individuals during traditional backcross breeding. The repulsion-phase RAPD has greatest utility in MAS of homozygous-resistant individuals in F2 or later-segregating generations.

Genomics of Phaseolus Beans, a Major Source of Dietary Protein and Micronutrients in the Tropics

Common bean is grown and consumed principally in developing countries in Latin America, Africa, and Asia. It is largely a subsistence crop eaten by its producers and, hence, is underestimated in production and commerce statistics. Common bean is a major source of dietary protein, which complements carbohydrate-rich sources such as rice, maize, and cassava. It is also a rich source of minerals, such as iron and zinc, and certain vitamins. Several large germplasm collections have been established, which contain large amounts of genetic diversity, including the five domesticated Phaseolus species and wild species, as well as an incipient stock collection. The genealogy and genetic diversity of P. vulgaris are among the best known in crop species through the systematic use of molecular markers, from seed proteins and isozymes to simple sequence repeats, and DNA sequences. Common bean exhibits a high level of genetic diversity, compared with other selfing species. A hierarchical organization into gene pools and ecogeographic races has been established. There are over 15 mapping populations that have been established to study the inheritance of agronomic traits in different locations. Most linkage maps have been correlated with the core map established in the BAT93 x Jalo EEP558 cross, which includes several hundreds of markers, including Restriction Fragment Length Polymorphisms, Random Amplified Polymorphic DNA, Amplified Fragment Length Polymorphisms, Short Sequence Repeats, Sequence Tagged Sites, and Target Region Amplification Polymorphisms. Over 30 individual genes for disease resistance and some 30 Quantitative Trait Loci for a broad range of agronomic traits have been tagged. Eleven BAC libraries have been developed in genotypes that represent key steps in the evolution before and after domestication of common bean, a unique resource among crops. Fluorescence in situ hybridization provides the first links between chromosomal and genetic maps. A gene index based on some P. vulgaris 21,000 expressed sequence tags (ESTs) has been developed. ESTs were developed from different genotypes, organs, and physiological conditions. They resolve currently in some 6,500–6,800 singletons and 2,900 contigs. An additional 20,000 embryonic P. coccineus ESTs provides an additional resource. Some 1,500 M2 Targeting Local Lesions In Genomes populations exist currently. Finally, transformation methods by biolistics and Agrobacterium have been developed, which can be applied for genetic engineering. Root transformation via A. rhizogenes is also possible. Thus, the Phaseomics community has laid a solid foundation towards its ultimate goal, namely the sequencing of the Phaseolus genome. These genomic resources are a much-needed source of additional markers of known map location for marker-assisted selection and the accelerated improvement of common bean cultivars.

A major QTL for common bacterial blight resistance derives from the common bean great northern landrace cultivar Montana No. 5

Knowledge of the evolutionary origin and sources of pest resistance genes will facilitate gene deployment and development of crop cultivars with durable resistance. Our objective was to determine the source of common bacterial blight (CBB) resistance in the common bean Great Northern Nebraska #1 (GN#1) and GN#1 Selection 27 (GN#1 Sel 27). Several great northern cultivars including GN#1, GN#1 Sel 27, and Montana No.5 (the female parent of the common x tepary bean interspecific population from which GN #1 and GN # 1 Sel 27 were derived) and known susceptible checks were evaluated for CBB reaction in field and greenhouse environments. These genotypes and CBB resistant and susceptible tepary bean including Tepary #4, the male parent and presumed contributor of CBB resistance toGN#1 and GN#1 Sel 27, were assayed for presence or absence of three SCAR markers tightly linked with independent QTLs conditioning CBB resistance. The parents and F2 of Montana No. 5/GN #1 Sel 27 and Montana No.5/Othello(CBB susceptible) were screened for CBB reaction and SCAR markers. CBB resistance in Montana No.5 was comparable to that of GN#1 and GN#1 Sel27. The SAP6 SCAR marker present in GN#1 and GN#1 Sel 27 was also present in Montana No.5, and it co-segregated (R2 =35%) with the CBB resistance in the Montana No.5/Othello F2 population. Although a few CBB resistant and susceptible transgressive segregants were found in the F2 of MontanaNo.5/GN #1 Sel 27 and later confirmed by F3 progeny tests, SAP6 SCAR marker was present in all progenies. None of the tepary bean specific CBB resistance-linked SCAR markers were present in GN#1, GN#1 Sel 27, or Montana No.5. A cluster analysis of 169 polymorphic PCR-based markers across three common bean and Tepary #4 indicated that GN#1, GN#1 Sel 27, and Montana No.5 were closely related, and not related at all with Tepary #4.Thus, these results clearly indicate Montana No.5, not Tepary #4, as the source of CBB resistance in GN#1 and GN#1 Sel 27.

Potential marker-assisted selection for bc-12 resistance to bean common mosaic potyvirus in common bean

Pyramiding epistatic resistance genes to improve long term disease resistance has challenged plant breeders. Indirect selection using tightly linked markers will often facilitate the breeding of desired epistatic resistance gene combinations. In common bean, the most effective strategy for broad spectrum control of the bean common mosaic virus disease is to combine I, bc-u, bc-12, bc-22, and bc-3 genes. We describe the use of near-isogenic lines and bulked segregant analysis to identify a marker tightly linked with the bc-12 gene. The recessive bc-12 gene conditions resistance to specific strains of bean common mosaic virus and bean common mosaic necrosis virus and is masked by the bc-22 and bc-3 genes. We identified a RAPD marker completely linked (0 recombinants) with bc-12, based on 72 F3 progeny generated from a cross between the contrasting near isogenic lines (I + bc-1/I + bc-12). Segregation in this I gene background revealed that bc-12 was dominant to bc-1 in conferring resistance to top necrosis in the allelic series Bc-1 > bc-12 > bc-1. To facilitate marker-assisted selection of bc-12 across breeding programs, the RAPD was converted to a SCAR marker, designated SBD51300. Tight linkage (0 recombinants) was confirmed in a second population of 58 F2 progeny co-segregating for SBD51300 and bc-12 gene from a different source. Based on a survey of 130 genotypes, the SCAR will be useful for MAS of bc-12 in most beans of Middle American origin and snap beans, but will have very limited utility in the case of kidney and cranberry beans. The SBD51300 marker mapped on linkage group B3, revealing independence of bc-12 from the I gene on B2 and bc-3 gene on B6, which supports the opportunity to readily combine genes for broad spectrum and pyramided resistance to bean common mosaic potyviruses in a single bean cultivar.