Dermot P. Coyne

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.

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.

Contributions of the Bean/Cowpea CRSP to cultivar and germplasm development in common bean

Disease and abiotic stress are important factors limiting bean production wherever beans are grown. The development of bean cultivars having resistance to these stresses is a cost-effective and sustainable means to address these constraints. During the past 20 years, the Bean/Cowpea Collaborative Research Support Project (B/C CRSP) has supported common bean cultivar development and germplasm improvement programs in the USA and developing countries. Plant breeders have developed and released in Central America and the Caribbean bean cultivars and germplasm with one or more of the following traits; resistance to bean golden yellow mosaic virus (BGYMV), bean common mosaic necrotic virus (BCMNV), rust, web blight and common bacterial blight (CBB) and greater tolerance to high temperatures. In the highlands of Mexico and Ecuador bean cultivars with resistance to anthracnose, rust, root rots and bean common mosaic virus (BCMV), greater biological nitrogen fixation and improved adaptation to intermittent drought have been released. The bean breeding programs in East Africa have developed and released bean cultivars and germplasm with resistance to BCMNV, rust and bruchid seed weevils. Participation in the B/C CRSP has permitted USA bean breeding programs to develop and release bean cultivars and germplasm with resistance to BGYMV, BCMNV, anthracnose, rust, CBB, architectural avoidance to white mold and greater yield potential. Numerous plant breeders, plant pathologists and agronomists from developing countries have received advanced degree training in the USA, which has enhanced the capacity to develop improved bean cultivars for Latin America and Africa. The lack of sustainable seed production and delivery systems continues to limit the impact of the release of improved bean cultivars in many parts of Latin America and East Africa.

Expression of Human Lactoferrin cDNA Confers Resistance to Ralstonia solanacearum in Transgenic Tobacco Plants

ABSTRACT A construct containing a human lactoferrin cDNA was used to transform tobacco (Nicotiana tabacum) using an Agrobacterium-mediated DNA-transfer system to express this human protein in transgenic plants. Transformants were analyzed by Southern, Northern, and Western blots to determine integration of the cDNA into the plant genome and lactoferrin gene expression levels. Most transgenic plants demonstrated significant delays of bacterial wilt symptoms when inoculated with the bacterial pathogen Ralstonia solanacearum. Quantification of the expressed lactoferrin protein by enzyme-linked immunosorbent assay in transgenic plants indicated a significant positive relationship between lactoferrin gene expression levels and levels of disease resistance. Incorporation of the lactoferrin gene into crop plants may enhance resistance to other phytopathogenic bacteria as well.

Breeding and genetic studies of tolerance to several bean (Phaseolus vulgaris L.) bacterial pathogens

SummaryA breeding and genetic program to develop Phaseolus vulgaris bean varieties tolerant to the bacterial pathogens Pseudomonas phaseolicola, Xanthomonas phaseoli and Corynebacterium flaccumfaciens was conducted from 1962 to the present tme. The research progress is reported. Great Northern (GN) Nebraska #1, sel. 27 and PI 150414 dry bean lines were highly tolerant to races 1 and 2 of the halo blight bacterium (P. phaseolicola). The tolerant inoculated leaf, pod, and non-systemic chlorosis reactions to this bacterium were each controlled by different major genes. Coupling linkage was detected between genes controlling the leaf and systemic chlorosis reactions. GN Nebraska #1, sel. 27 and PI 207262 were tolerant to isolates (USA) of X. phaseoli, causal pathogen of common blight disease. Reaction to this bacterium was inherited quantitatively. Narrow sense heritability estimates of the disease reaction were low. Genes controlling late maturity and tolerant reaction were found to be linked in crosses with GN Nebraska #1, sel. 27 but linkage was not apparent in one cross with PI 207262. A different reaction of pod and foliage was observed in some bean lines. Susceptibility increased with the onset of plant maturity. PI 165078 was tolerant to C. flaccumfaciens and the disease reaction was simply inherited. The dry bean varieties ‘GN Tara’ and ‘GN Jules’, tolerant X. phaseoli, and ‘GN Emerson’ tolerant to C. flaccumfaciens and X. phaseoli were released in recent years. Breeding approaches to develop bean varieties tolerant to these bacterial pathogens is discussed.

Isolates of Uromyces appendiculatus with Specific Virulence to Landraces of Phaseolus vulgaris of Andean Origin

Five isolates of the bean rust fungus Uromyces appendiculatus were shown to be specifically virulent on bean genotypes of Andean origin. This specificity was demonstrated by the virulence of five pairs of isolates on a differential set of 30 Phaseolus vulgaris landraces. Each isolate pair was from a different country in the Americas and consisted of one Andean-specific isolate and one nonspecific isolate. Of the differential P. vulgaris landraces, 15 were of Middle American origin and 15 were of Andean origin. The Andean-specific rust isolates were highly virulent on Andean landraces but not on landraces of Middle American origin. Rust isolates with virulence to Middle American landraces were also generally virulent on Andean material; no truly Middle American-specific isolates were found. Random amplified polymorphic DNA (RAPD) analysis of the rust isolates also distinguished the two groups. Four of the Andean-specific rust isolates formed a distinct group compared to four of the nonspecific isolates. Two of the isolates, one from each of the two virulence groups, had intermediate RAPD banding patterns, suggesting that plasmagomy but not karyogamy occurred between isolates of the two groups.

Phenotypic and Genotypic Characterization of Uromyces appendiculatus from Phaseolus vulgaris in the Americas

Populations of 90 Uromyces appendiculatus isolates were collected from throughout the Americas and evaluated for virulence on 19 standard bean rust differentials, and also on 12 landraces of Phaseolus vulgaris from South and Central America. The landrace differentials represented geographical centers of bean domestication. Three groups were observed. Two groups were isolates from centers of bean domestication and a third heterogeneous group comprised isolates from countries in South and Central America. Molecular analysis using random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) was also conducted on these isolates. Cluster analysis of the molecular profiles showed three groups that corresponded to those obtained by virulence tests. These results show a clear differentiation of the pathogen population along similar lines as its host and suggest parallel evolution in the bean rust pathosystem.

Contributions of the Bean/Cowpea CRSP to management of bean diseases

Abstract Diseases are a major constraint to production of beans in developing countries, reducing yields and seed quality. Contributing factors include poor disease management, lack of resistant cultivars, and the limited availability of certified disease-free seed. From the outset the Bean/Cowpea Collaborative Research Support Program (CRSP) has emphasized integrated disease management, and breeding for resistance to bean rust ( Uromyces appendiculatus ), common bacterial blight ( Xanthomonas campestris (= X. axonopodis ) pv. phaseoli ) (CBB) and web blight ( Thanatephorus cucumeris (anamorph Rhizoctonia solani )) (WB). Later in the 1990s, angular leaf spot ( Phaeoisariopsis griseola ) (ALS), and virus induced bean common mosaic (BCM) and bean common mosaic necrosis (BCMN) became more widespread and epidemic. The research included development of disease-resistant germplasm and studies of pathogenic variation and epidemiology. During the early 1990s bean golden yellow mosaic (BGYM), became a major disease of common bean in the Caribbean and Central America. No cultivar resistant to the gemini virus causing BGYM was available in the Dominican Republic (DR) and pesticides were used to reduce transmission of the virus by white flies ( Bremisia tabaci ) biotype A. A change in the bean-production system to limit reproduction of the vector through a host-free period and concentrate the bean growing season to a four month period reduced BGYM significantly. Improved resistant cultivars and disease management resulted in high yields and self-sufficiency of beans in the DR. Evidence for co-evolution of the pathogens causing ALS, CBB, and rust in the two major bean gene pools (Andean and Middle American) was found. Variation in the WB pathogen on bean indicated independent genetic populations and the presence of different anastosmosis subgroups. WB disease management practices should be designed for the sub-group of the pathogen. Seed transmission was found to be a significant source of R. solani inoculum. Specific ( Ur-9 gene) and adult-plant resistance ( Ur-12 gene) to rust was identified for PC-50 (Andean origin) in the DR, and the genes were mapped. A mobile rust nursery was developed to monitor races of rust in a region and assist in resistance-gene deployment in bean germplasm and varieties. DNA based diagnostic methods were developed to differentiate X. campestris pv. phaseoli from X. campestris pv. phaseoli var fuscans (prevalent in East Africa) and P. griseola isolates. DNA hybridization methods also were developed to identify BGYM and bean golden mosaic viruses. Research in USA and East Africa has helped define bean common mosaic virus (BCMV) and bean common mosaic necrotic virus (BCMNV) as separate viruses and has demonstrated that BCMNV appears to be indigenous to Africa. Serological tools were developed that assist in the detection and identification of potyviruses, BCMV and BCMNV. These tools are now used worldwide.

Molecular markers linked to genes for specific rust resistance and indeterminate growth habit in common bean

Bulked segregant analysis was utilized to identify random amplified polymorphic DNA (RAPD) markers linked to genes for specific resistance to a rust pathotype and indeterminate growth habit in an F2 population from the common bean cross PC-50 (resistant to rust and determinate growth habit) × Chichara 83-109 (susceptible to rust and indeterminate growth habit). Six RAPD markers were mapped in a coupling phase linkage with the gene ( Ur-9) for specific rust resistance. The linkage group spanned a distance of 41 cM. A RAPD marker OA4.1050 was the most closely linked to the Ur-9 gene at a distance of 8.6 cM. Twenty-eight RAPD markers were mapped in a coupling phase linkage with the gene ( Fin) for indeterminate growth habit. The linkage group spanned a distance of 77 cM. RAPD markers OQ3.450 and OA17.600 were linked to the Fin allele as flanking markers at a distance of 1.2 cM and 3.8 cM, respectively. The RAPD markers linked to the gene for specific rust resistance of Andean origin detected here, along with other independent rust resistance genes from other germplasm, could be utilized to pyramid the different genes into a bean cultivar for durable rust resistance.

Inheritance and linkage relations of reaction to Xanthomonas phaseoli (E. F. Smith) Dowson (common blight), stage of plant development and plant habit in Phaseolus vulgaris L.

SummaryThe inheritance of the reaction to Xanthomonas phaseoli (E. F. Smith) Dowson Nebraska isolate Xp-816, cause of common blight disease of beans Phaseolus vulgaris L. was studied in crosses between the late flowering, indeterminate, blight tolerant dry bean PI 207262 (Colombia) and susceptible cvs. GN 1140, an early flowering and indeterminate dry bean; Dark Red Kidney, a late flowering and determinate dry bean; and Gallatin 50, an early and determinate green bean. The tolerant disease reaction was dominant in the F1. A continuous distribution of disease reaction ratings, skewed in the direction of dominance, occurred in the F2 derived from the first 2 crosses while a slight bimodal distribution was observed in the F2 of the last cross. A low narrow sense heritability estimate of 14% was calculated by the regression of F3 progeny means on individual F2 plants, in the cross GN 1140×PI 207262. The occurrence of a small number of nonsegregating families in a low number of F3 families indicates that a small number of major genes were involved in controlling the disease reaction. Linkage did not appear to be involved between genes controlling early flowering (early maturity) and common blight tolerance. Coupling linkage occurred between genes controlling determinate plant habit and early flowering. A crossover value of 8.4% was estimated. Recombinants for early maturity, determinate habit, and blight tolerance were obtained. Transgressive segregation for early flowering and common blight susceptibility occurred in progeny derived from the cross of the two late-flowering blight tolerant lines, PI 207262 and GN Nebraska 1, sel. 27, indicating that the parents possessed different genes controlling these traits.