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Flavonoids are secondary metabolites associated with plant seed coat and flower color. These compounds provide health benefits to humans as anti-inflammatory and antioxidant compounds. The expression of the late biosynthetic genes in the flavonoid pathway is controlled by a ternary MBW protein complex consisting of interfacing MYB, beta-helix-loop-helix (bHLH), and WD40 Repeat (WDR) proteins. P, the master regulator gene of the flavonoid expression in common bean (Phaseolus vulgaris L.), was recently determined to encode a bHLH protein. The T and Z genes control the distribution of color in bean seeds and flowers and have historically been considered regulators of the flavonoid gene expression. T and Z candidates were identified using reverse genetics based on genetic mapping, phylogenetic analysis, and mutant analysis. Domain and AlphaFold2 structure analyses determined that T encodes a seven-bladed β-propeller WDR protein, while Z encodes a R2R3 MYB protein. Deletions and SNPs in T and Z mutants, respectively, altered the 3D structure of these proteins. Modeling of the Z MYB/P bHLH/T WDR MBW complex identified interfacing sequence domains and motifs in all three genes that are conserved in dicots. One Z MYB motif is a possible beta-molecular recognition feature (β-MoRF) that only appears in a structured state when Z MYB is modeled as a component of a MBW complex. Complexes containing mutant T and Z proteins changed the interaction of members of the complex in ways that would alter their role in regulating the expression of genes in the flavonoid pathway., Competing Interests: Conflicts of interest The authors declare no conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

Bacterial brown spot (BBS) caused by Pseudomonas syringae pv. syringae ( Pss ), common bacterial blight (CBB) caused by Xanthomonas axonopodis pv. phaseoli ( Xap ) and Xanthomonas fuscans subsp. fuscans ( Xff ), and halo bacterial blight (HBB), caused by Pseudomonas syringae pv. phaseolicola ( Psph ), are major bacterial diseases that severely affect common bean yields and global food security. Andean-origin dry beans, representing large-seeded market classes, are particularly susceptible. Using 140,325 SNPs, a multi-locus GWAS was conducted on subsets of the Andean diversity panel (ADP) phenotyped for BBS in South Africa, CBB in Puerto Rico, South Africa, and Zambia, and HBB in South Africa, through natural infection, artificial inoculation, or both. Twenty-four QTL associated with resistance were identified: nine for BBS, eight for CBB, and seven for HBB. Four QTL intervals on Pv01, Pv03, Pv05, and Pv08 overlapped with BBS and HBB resistance. A genomic interval on Pv01, near the fin gene, which determines growth habit, was linked to resistance to all three pathogens. Different QTLs were detected for BBS and CBB resistance when phenotyped under natural infection versus artificial inoculation. These results underscore the importance of combining phenotyping methods in multi-GWAS to capture the full genetic spectrum. Previously recognized CBB resistance QTL SAP6 and SU91 and HBB resistance QTL HB4.2, and HB5.1, were observed. Other common (MAF >0.25) and rare (MAF <0.05) resistance QTL were also detected. Overall, these findings enhance the understanding and utilization of bacterial resistance present in ADP for the development of common beans with improved resistance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Soler-Garzón, Mulube, Kamfwa, Lungu, Hamabwe, Roy, Salegua, Fourie, Porch, McClean and Miklas.)

Bean common mosaic virus (BCMV) and bean common mosaic necrosis virus (BCMNV) have a damaging impact on global common bean (Phaseolus vulgaris L.) cultivation, causing potential yield losses of over 80%. The primary strategy for controlling these viruses is through host plant resistance. This research aimed to identify and validate structural variations for the bc-u d gene as revealed by long-read sequencing, develop an efficient DNA marker to assist selection of bc-u d in snap and dry beans, and examine the interactions between the bc-u d allele and other BCMV resistance genes. A gene (Phvul.005G125100) model on chromosome Pv05, encoding a vacuolar protein-sorting 4 (Vps4) AAA+ ATPase endosomal sorting complexes required for transport (ESCRT) protein, was identified as the best candidate gene for bc-u d . An 84-bp repetitive insertion variant within the gene, exhibited 100% co-segregation with the bc-u d resistance allele across 264 common bean accessions. The 84-bp repetitive insertion was labeled with an indel marker IND&#95;05&#95;36225873 which was useful for tracking the bc-u d allele across diverse germplasm. A different single nucleotide polymorphism variant within the same candidate gene was associated with the bc-4 gene. Segregation in F 2 populations confirmed bc-u d and bc-4 were alleles, so bc-4 was renamed bc-u r to fit gene nomenclature guidelines. The interactions of bc-u d and bc-u r with other resistance genes, such as bc-1 (receptor-like kinase on Pv03) and bc-2 (Vps4 AAA+ ATPase ESCRT protein on Pv11), validated gene combinations in the differential "host groups" effective against specific BCMV/BCMNV "pathogroups." These findings increase our understanding of the Bc-u locus, and enhance our ability to develop more resilient bean varieties through marker-assisted selection, reducing the impact of BCMV and BCMNV., (© 2023 The Authors. The Plant Genome published by Wiley Periodicals LLC on behalf of Crop Science Society of America. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Sclerotinia sclerotiorum is a necrotrophic fungal pathogen causing white mold on many important economic crops. Recently, some mycoviruses such as S. sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1) converted S. sclerotiorum into a beneficial symbiont that helps plants manage pathogens and other stresses. To explore the potential use of SsHADV-1 as a biocontrol agent in the United States and to test the efficacy of SsHADV-1-infected United States isolates in managing white mold and other crop diseases, SsHADV-1 was transferred from the Chinese strain DT-8 to United States isolates of S. sclerotiorum . SsHADV-1 is readily transmitted horizontally among United States isolates of S. sclerotiorum and consistently conferred hypovirulence to its host strains. Biopriming of dry bean seeds with hypovirulent S. sclerotiorum strains enhanced resistance to white mold, gray mold, and Rhizoctonia root rot. To investigate the underlying mechanisms, endophytic growth of hypovirulent S. sclerotiorum in dry beans was confirmed using PCR, and the expression of 12 plant defense-related genes were monitored before and after infection. The results indicated that the endophytic growth of SsHADV-1-infected strains in plants stimulated the expression of plant immunity pathway genes that assisted a rapid response from the plant to fungal infection. Finally, application of the seed biopriming technology with SsHADV-1-infected hypervirulent strain has promise for the biological control of several diseases of wheat, pea, and sunflower., Competing Interests: The author(s) declare no conflict of interest.

Key Message: R-BPMV is located within a recently expanded TNL cluster in the Phaseolus genus with suppressed recombination and known for resistance to multiple pathogens including potyviruses controlled by the I gene. Bean pod mottle virus (BPMV) is a comovirus that infects common bean and legumes in general. BPMV is distributed throughout the world and is a major threat on soybean, a closely related species of common bean. In common bean, BAT93 was reported to carry the R-BPMV resistance gene conferring resistance to BPMV and linked with the I resistance gene. To fine map R-BPMV, 182 recombinant inbred lines (RILs) derived from the cross BAT93 × JaloEEP558 were genotyped with polymerase chain reaction (PCR)-based markers developed using genome assemblies from G19833 and BAT93, as well as BAT93 BAC clone sequences. Analysis of RILs carrying key recombination events positioned R-BPMV to a target region containing at least 16 TIR-NB-LRR (TNL) sequences in BAT93. Because the I cluster presents a suppression of recombination and a large number of repeated sequences, none of the 16 TNLs could be excluded as R-BPMV candidate gene. The evolutionary history of the TNLs for the I cluster were reconstructed using microsynteny and phylogenetic analyses within the legume family. A single I TNL was present in Medicago truncatula and lost in soybean, mirroring the absence of complete BPMV resistance in soybean. Amplification of TNLs in the I cluster predates the divergence of the Phaseolus species, in agreement with the emergence of R-BPMV before the separation of the common bean wild centers of diversity. This analysis provides PCR-based markers useful in marker-assisted selection (MAS) and laid the foundation for cloning of R-BPMV resistance gene in order to transfer the resistance into soybean., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

White mold (WM), caused by the ubiquitous fungus Sclerotinia sclerotiorum, is a devastating disease that limits production and quality of dry bean globally. In the present study, classic linkage mapping combined with QTL-seq were employed in two recombinant inbred line (RIL) populations, "Montrose"/I9365-25 (M25) and "Raven"/I9365-31 (R31), with the initial goal of fine-mapping QTL WM5.4 and WM7.5 that condition WM resistance. The RILs were phenotyped for WM reactions under greenhouse (straw test) and field environments. The general region of WM5.4 and WM7.5 were reconfirmed with both mapping strategies within each population. Combining the results from both mapping strategies, WM5.4 was delimited to a 22.60-36.25 Mb interval in the heterochromatic regions on Pv05, while WM7.5 was narrowed to a 0.83 Mb (3.99-4.82 Mb) region on the Pv07 chromosome. Furthermore, additional QTL WM2.2a (3.81-7.24 Mb), WM2.2b (11.18-17.37 Mb, heterochromatic region), and WM2.2c (23.33-25.94 Mb) were mapped to a narrowed genomic interval on Pv02 and WM4.2 in a 0.89 Mb physical interval at the distal end of Pv04 chromosome. Gene models encoding gibberellin 2-oxidase proteins regulating plant architecture are likely candidate genes associated with WM2.2a resistance. Nine gene models encoding a disease resistance protein (quinone reductase family protein and ATWRKY69) found within the WM5.4 QTL interval are putative candidate genes. Clusters of 13 and 5 copies of gene models encoding cysteine-rich receptor-like kinase and receptor-like protein kinase-related family proteins, respectively, are potential candidate genes associated with WM7.5 resistance and most likely trigger physiological resistance to WM. Acquired knowledge of the narrowed major QTL intervals, flanking markers, and candidate genes provides promising opportunities to develop functional molecular markers to implement marker-assisted selection for WM resistant dry bean cultivars., (© 2023 The Authors. The Plant Genome published by Wiley Periodicals LLC on behalf of Crop Science Society of America.)

Key Message: Quantitative Trait Loci "hotspots" for drought tolerance were identified on chromosomes Pv06, Pv07 and Pv10 of common bean. Drought is a major production constraint of common bean (Phaseolus vulgaris L.) worldwide. The objective of this study was to identify the Quantitative Trait Loci (QTL) for drought tolerance in an Andean population of Recombinant Inbred Lines (RILs). A total of 155 F 5:7 RILs derived from a cross between Kijivu (drought tolerant) and Bukoba (drought susceptible) were evaluated for drought tolerance in field and pot experiments. Four field experiments were conducted at three locations in Zambia in 2020 and 2021. All field trials were conducted in the dry season under irrigation. The 155 RILs were genotyped with 11,292 SNPs, and composite interval mapping was conducted to identify QTL for drought tolerance. Seed yield for Kijivu under drought stress was consistently higher than for Bukoba across all four field trials. A total of 60 QTL were identified for morphological, agronomic, and physiological traits under drought stress and non-stress conditions. However, the majority of these QTL were specific to drought stress. QTL "hotspots" for drought tolerance were identified on chromosomes Pv06, Pv07, and Pv10. Extensive co-localizations for agronomic and morpho-physiological traits under drought stress were observed at the three drought-tolerance QTL hotspots. Additionally, these three QTL hotspots overlapped with previously identified QTL for drought tolerance, while several others identified QTL are novel. The three identified QTL hotspots could be used in future marker-assisted selection for drought tolerance in common bean., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Common beans (Phaseolus vulgaris L.) provide important protein and calories globally. Anthracnose (Colletotrichum lindemuthianum (Sacc. & Magnus) Briosi & Cavara, 1889) is a major disease in common bean and causes significant yield losses in bean production areas. Screening for markers linked to known disease resistance genes provides useful information for plant breeders to develop improved common bean varieties. The Kompetitive Allele Specific PCR (KASP) assay is an affordable genetic screening technique that can be used to accelerate breeding programs, but manual DNA extraction and KASP assay preparation are time-consuming. Several KASP markers have been developed for genes involved in resistance to bean anthracnose, which can reduce yield by up to 100%, but their usefulness is hindered by the labor required to screen a significant number of bean lines. Our research objective was to develop publicly available protocols for DNA extraction and KASP assaying using a liquid handling robot (LHR) which would facilitate high-throughput genetic screening with less active human time required. Anthracnose resistance markers were used to compare manual and automated results., Results: The 12 bean anthracnose differential cultivars were screened for four anthracnose KASP markers linked to the resistance genes Co-1, Co-3 and Co-4 2 both by hand and with the use of an LHR. A protocol was written for DNA extraction and KASP assay thermocycling to implement the LHR. The LHR protocol reduced the active human screening time of 24 samples from 3h44 to 1h23. KASP calls were consistent across replicates but not always accurate for their known linked resistance genes, suggesting more specific markers still need to be developed. Using an LHR, information from KASP assays can be accumulated with little active human time., Conclusion: Results suggest that LHRs can be used to expedite time-consuming and tedious lab work such as DNA extraction or PCR plate filling. Notably, LHRs can be used to prepare KASP assays for large sample sizes, facilitating higher throughput use of genetic marker screening tools., (© 2023. BioMed Central Ltd., part of Springer Nature.)

White mold (WM) is a major disease in common bean ( Phaseolus vulgaris L.), and its complex quantitative genetic control limits the development of WM resistant cultivars. WM2.2, one of the nine meta-QTL with a major effect on WM tolerance, explains up to 35% of the phenotypic variation and was previously mapped to a large genomic interval on Pv02. Our objective was to narrow the interval of this QTL using combined approach of classic QTL mapping and QTL-based bulk segregant analysis (BSA), and confirming those results with Khufu de novo QTL-seq. The phenotypic and genotypic data from two RIL populations, 'Raven'/I9365-31 (R31) and 'AN-37'/PS02-029C-20 (Z0726-9), were used to select resistant and susceptible lines to generate subpopulations for bulk DNA sequencing. The QTL physical interval was determined by considering overlapping interval of the identified QTL or peak region in both populations by three independent QTL mapping analyses. Our findings revealed that meta-QTL WM2.2 consists of three regions, WM2.2a (4.27-5.76 Mb; euchromatic), WM 2.2b (12.19 to 17.61 Mb; heterochromatic), and WM2.2c (23.01-25.74 Mb; heterochromatic) found in both populations. Gene models encoding for gibberellin 2-oxidase 8, pentatricopeptide repeat, and heat-shock proteins are the likely candidate genes associated with WM2.2a resistance. A TIR-NBS-LRR class of disease resistance protein (Phvul.002G09200) and LRR domain containing family proteins are potential candidate genes associated with WM2.2b resistance. Nine gene models encoding disease resistance protein [pathogenesis-related thaumatin superfamily protein and disease resistance-responsive (dirigent-like protein) family protein etc] found within the WM2.2c QTL interval are putative candidate genes. WM2.2a region is most likely associated with avoidance mechanisms while WM2.2b and WM2.2c regions trigger physiological resistance based on putative candidate genes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Oladzad, Roy, Mamidi, Miklas, Lee, Clevenger, Myers, Korani and McClean.)

Beet curly top virus (BCTV), which is synonymous with curly top virus (CTV), causes significant yield loss in common bean (snap and dry beans) cultivars and several other important crops. Common bean cultivars have been found to be resistant to CTV, but screening for resistance is challenging due to the cyclical nature of epidemics and spotty feeding by the leafhopper that vectors the virus. We used an SNP dataset for the Snap Bean Association Panel (SnAP) agro-inoculated with CTV-Logan (CA/Logan) strain to locate the Bct gene region to a 1.7-Mb interval on chromosome Pv07 using genome-wide association study (GWAS) analysis. Recombinant lines from the SnAP were used to further narrow the Bct region to a 58.0-kb interval. A missense SNP (S07&#95;2970381) in candidate gene Phvul.007G036300 Exonuclease V (EXO5) was identified as the most likely causal mutation, and it was the most significant SNP detected by GWAS in a dry bean population (DBP) naturally infected by the CTV-Worland (Wor) strain. Tm-shift assay markers developed for SNP S07&#95;2970381 and two linked SNPs, S07&#95;2970276 and S07&#95;2966197, were useful for tracking different origins of the Bct EXO5 candidate gene resistance to CTV in common bean. The three SNPs identified four haplotypes, with haplotype 3-1 (Haplo3-1) of Middle American origin associated with the highest levels of CTV resistance. This SNP-haplotype assay will enable breeders to track resistance sources and to develop cultivars with better CTV resistance., Competing Interests: Authors DG and AT were employed by HM Clause Seed Company. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Soler-Garzón, Goldoff, Thornton, Swisher Grimm, Hart, Song, Strausbaugh and Miklas.)

The classic V (violet, purple) gene of common bean ( Phaseolus vulgaris ) functions in a complex genetic network that controls seed coat and flower color and flavonoid content. V was cloned to understand its role in the network and the evolution of its orthologs in the Viridiplantae. V mapped genetically to a narrow interval on chromosome Pv06. A candidate gene was selected based on flavonoid analysis and confirmed by recombinational mapping. Protein and domain modeling determined V encodes flavonoid 3'5' hydroxylase (F3'5'H), a P450 enzyme required for the expression of dihydromyricetin-derived flavonoids in the flavonoid pathway. Eight recessive haplotypes, defined by mutations of key functional domains required for P450 activities, evolved independently in the two bean gene pools from a common ancestral gene. V homologs were identified in Viridiplantae orders by functional domain searches. A phylogenetic analysis determined F3'5'H first appeared in the Streptophyta and is present in only 41% of Angiosperm reference genomes. The evolutionarily related flavonoid pathway gene flavonoid 3' hydroxylase (F3'H) is found nearly universally in all Angiosperms. F3'H may be conserved because of its role in abiotic stress, while F3'5'H evolved as a major target gene for the evolution of flower and seed coat color in plants., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 McClean, Lee, Howe, Osborne, Grimwood, Levy, Haugrud, Plott, Robinson, Skiba, Tanha, Zamani, Thannhauser, Glahn, Schmutz, Osorno and Miklas.)

Bean common mosaic virus (BCMV) is a major disease in common bean ( Phaseolus vulgaris L.). Host plant resistance is the most effective strategy to minimize crop damage against BCMV and the related Bean common mosaic necrosis virus (BCMNV). To facilitate breeding for resistance, we sought to identify candidate genes and develop markers for the bc-2 gene and the unknown gene with which it interacts. Genome-wide association study (GWAS) of the Durango Diversity Panel (DDP) identified a peak region for bc-2 on chromosome Pv11. Haplotype mapping narrowed the bc-2 genomic interval and identified Phvul.011G092700, a vacuolar protein-sorting 4 (Vps4) AAA+ ATPase endosomal sorting complexes required for transport (ESCRT) protein, as the bc-2 candidate gene. The race Durango Phvul.011G092700 gene model, bc-2 [UI 111] , contains a 10-kb deletion, while the race Mesoamerican bc-2 [Robust] consists of a single nucleotide polymorphism (SNP) deletion. Each mutation introduces a premature stop codon, and they exhibit the same interaction with the pathogroups (PGs) tested. Phvul.005G125100, another Vps4 AAA+ ATPase ESCRT protein, was identified as the candidate gene for the new recessive bc-4 gene, and the recessive allele is likely an amino acid substitution in the microtubule interacting and transport (MIT) domain. The two Vps4 AAA+ ATPase ESCRT proteins exhibit high similarity to the Zym Cucsa.385040 candidate gene associated with recessive resistance to Zucchini yellow mosaic virus in cucumber. bc-2 alone has no resistance effect but, when combined with bc-4 , provides resistance to BCMV (except PG-V) but not BCMNV, and, when combined with bc-u d , provides resistance to BCMV (except BCMV PG-VII) and BCMNV. So instead of different resistance alleles (i.e., bc-2 and bc-2 2 ), there is only bc-2 with a differential reaction based on whether it is combined with bc-4 or bc-u d , which are tightly linked in repulsion. The new tools and enhanced understanding of this host-virus pathogen interaction will facilitate breeding common beans for resistance to BCMV and BCMNV., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Soler-Garzón, McClean and Miklas.)

Bean common mosaic necrosis virus (BCMNV) is a major disease in common bean ( Phaseolus vulgaris L.). Host plant resistance is the primary disease control. We sought to identify candidate genes to better understand the host-pathogen interaction and develop tools for marker-assisted selection (MAS). A genome-wide association study (GWAS) approach using 182 lines from a race Durango Diversity Panel (DDP) challenged by BCMNV isolates NL-8 [Pathogroup (PG)-III] and NL-3 (PG-VI), and genotyped with 1.26 million single-nucleotide polymorphisms (SNPs), revealed significant peak regions on chromosomes Pv03 and Pv05, which correspond to bc-1 and bc-u resistance gene loci, respectively. Three candidate genes were identified for NL-3 and NL-8 resistance. Side-by-side receptor-like protein kinases (RLKs), Phvul.003G038700 and Phvul.003G038800 were candidate genes for bc-1 . These RLKs were orthologous to linked RLKs associated with virus resistance in soybean ( Glycine max ). A basic Leucine Zipper (bZIP) transcription factor protein is the candidate gene for bc-u . bZIP protein gene Phvul.005G124100 carries a unique non-synonymous mutation at codon 14 in the first exon (Pv05: 36,114,516 bases), resulting in a premature termination codon that causes a nonfunctional protein. SNP markers for bc-1 and bc-u and new markers for I and bc-3 genes were used to genotype the resistance genes underpinning BCMNV phenotypes in the DDP, host group (HG) differentials, and segregating F 3 families. Results revealed major adjustments to the current host-pathogen interaction model: (i) there is only one resistance allele bc-1 for the Bc-1 locus, and differential expression of the allele is based on presence vs. absence of bc-u ; (ii) bc-1 exhibits dominance and incomplete dominance; (iii) bc-1 alone confers resistance to NL-8; (iv) bc-u was absent from HGs 2, 4, 5, and 7 necessitating a new gene symbol bc-u d to reflect this change; (v) bc-u d alone delays susceptible symptoms, and when combined with bc-1 enhanced resistance to NL-3; and (vi) bc-u d is on Pv05, not Pv03 as previously thought. These candidate genes, markers, and adjustments to the host-pathogen interaction will facilitate breeding for resistance to BCMNV and related Bean common mosaic virus (BCMV) in common bean., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Soler-Garzón, McClean and Miklas.)

Identifying the molecular basis of resistance to pathogens is critical to promote a chemical-free cropping system. In plants, nucleotide-binding leucine-rich repeat constitute the largest family of disease resistance (R) genes, but this resistance can be rapidly overcome by the pathogen, prompting research into alternative sources of resistance. Anthracnose, caused by the fungus Colletotrichum lindemuthianum, is one of the most important diseases of common bean. This study aimed to identify the molecular basis of Co-x, an anthracnose R gene conferring total resistance to the extremely virulent C. lindemuthianum strain 100. To that end, we sequenced the Co-x 58 kb target region in the resistant JaloEEP558 (Co-x) common bean and identified KTR2/3, an additional gene encoding a truncated and chimeric CRINKLY4 kinase, located within a CRINKLY4 kinase cluster. The presence of KTR2/3 is strictly correlated with resistance to strain 100 in a diversity panel of common beans. Furthermore, KTR2/3 expression is up-regulated 24 hours post-inoculation and its transient expression in a susceptible genotype increases resistance to strain 100. Our results provide evidence that Co-x encodes a truncated and chimeric CRINKLY4 kinase probably resulting from an unequal recombination event that occurred recently in the Andean domesticated gene pool. This atypical R gene may act as a decoy involved in indirect recognition of a fungal effector., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Genetic resistance is the primary means for control of Bean golden yellow mosaic virus (BGYMV) in common bean ( Phaseolus vulgaris L. ). Breeding for resistance is difficult because of sporadic and uneven infection across field nurseries. We sought to facilitate breeding for BGYMV resistance by improving marker-assisted selection (MAS) for the recessive bgm-1 gene and identifying and developing MAS for quantitative trait loci (QTL) conditioning resistance. Genetic linkage mapping in two recombinant inbred line populations and genome-wide association study (GWAS) in a large breeding population and two diversity panels revealed a candidate gene for bgm-1 and three QTL BGY4.1, BGY7.1, and BGY8.1 on independent chromosomes. A mutation (5 bp deletion) in a NAC (No Apical Meristem) domain transcriptional regulator superfamily protein gene Phvul.003G027100 on chromosome Pv03 corresponded with the recessive bgm-1 resistance allele. The five bp deletion in exon 2 starting at 20 bp (Pv03: 2,601,582) is expected to cause a stop codon at codon 23 (Pv03: 2,601,625), disrupting further translation of the gene. A T m -shift assay marker named PvNAC1 was developed to track bgm-1 . PvNAC1 corresponded with bgm-1 across ∼1,000 lines which trace bgm-1 back to a single landrace "Garrapato" from Mexico. BGY8.1 has no effect on its own but exhibited a major effect when combined with bgm-1 . BGY4.1 and BGY7.1 acted additively, and they enhanced the level of resistance when combined with bgm-1 . T m -shift assay markers were generated for MAS of the QTL, but their effectiveness requires further validation., Competing Interests: The handling editor RP and the author PNM declare that they are affiliated with the INCREASE consortium on genetic resources in legumes. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Soler-Garzón, Oladzad, Beaver, Beebe, Lee, Lobaton, Macea, McClean, Raatz, Rosas, Song and Miklas.)

Multienvironment trials (METs) are widely used to assess the performance of promising crop germplasm. Though seldom designed to elucidate genetic mechanisms, MET data sets are often much larger than could be duplicated for genetic research and, given proper interpretation, may offer valuable insights into the genetics of adaptation across time and space. The Cooperative Dry Bean Nursery (CDBN) is a MET for common bean ( Phaseolus vulgaris ) grown for > 70 years in the United States and Canada, consisting of 20-50 entries each year at 10-20 locations. The CDBN provides a rich source of phenotypic data across entries, years, and locations that is amenable to genetic analysis. To study stable genetic effects segregating in this MET, we conducted genome-wide association studies (GWAS) using best linear unbiased predictions derived across years and locations for 21 CDBN phenotypes and genotypic data (1.2 million SNPs) for 327 CDBN genotypes. The value of this approach was confirmed by the discovery of three candidate genes and genomic regions previously identified in balanced GWAS. Multivariate adaptive shrinkage (mash) analysis, which increased our power to detect significant correlated effects, found significant effects for all phenotypes. Mash found two large genomic regions with effects on multiple phenotypes, supporting a hypothesis of pleiotropic or linked effects that were likely selected on in pursuit of a crop ideotype. Overall, our results demonstrate that statistical genomics approaches can be used on MET phenotypic data to discover significant genetic effects and to define genomic regions associated with crop improvement., (Copyright © 2020 MacQueen et al.)

The genetic improvement of economically important production traits of dry bean ( Phaseolus vulgaris L.), for geographic regions where production is threatened by drought and high temperature stress, is challenging because of the complex genetic nature of these traits. Large scale SNP data sets for the two major gene pools of bean, Andean and Middle American, were developed by mapping multiple pools of genotype-by-sequencing reads and identifying over 200k SNPs for each gene pool against the most recent assembly of the P. vulgaris genome sequence. Moderately sized B ean A biotic S tress E valuation (BASE) panels, consisting of genotypes appropriate for production in Central America and Africa, were assembled. Phylogenetic analyses demonstrated the BASE populations represented broad genetic diversity for the appropriate races within the two gene pools. Joint mixed linear model genome-wide association studies with data from multiple locations discovered genetic factors associated with four production traits in both heat and drought stress environments using the BASE panels. Pleiotropic genetic factors were discovered using a multi-trait mixed model analysis. SNPs within or near candidate genes associated with hormone signaling, epigenetic regulation, and ROS detoxification under stress conditions were identified and can be used as genetic markers in dry bean breeding programs., (Copyright © 2019 Oladzad et al.)

Pseudomonas syringae pv. phaseolicola ( Psph ) Race 6 is a globally prevalent and broadly virulent bacterial pathogen with devastating impact causing halo blight of common bean ( Phaseolus vulgaris L.). Common bean lines PI 150414 and CAL 143 are known sources of resistance against this pathogen. We constructed high-resolution linkage maps for three recombinant inbred populations to map resistance to Psph Race 6 derived from the two common bean lines. This was complemented with a genome-wide association study (GWAS) of Race 6 resistance in an Andean Diversity Panel of common bean. Race 6 resistance from PI 150414 maps to a single major-effect quantitative trait locus (QTL; HB4.2) on chromosome Pv04 and confers broad-spectrum resistance to eight other races of the pathogen. Resistance segregating in a Rojo × CAL 143 population maps to five chromosome arms and includes HB4.2. GWAS detected one QTL (HB5.1) on chromosome Pv05 for resistance to Race 6 with significant influence on seed yield. The same HB5.1 QTL, found in both Canadian Wonder × PI 150414 and Rojo × CAL 143 populations, was effective against Race 6 but lacks broad resistance. This study provides evidence for marker-assisted breeding for more durable halo blight control in common bean by combining alleles of race-nonspecific resistance (HB4.2 from PI 150414) and race-specific resistance (HB5.1 from cv. Rojo).

White mold, caused by the fungus Sclerotinia sclerotiorum (Lib.) de Bary, is a major disease that limits common bean production and quality worldwide. The host-pathogen interaction is complex, with partial resistance in the host inherited as a quantitative trait with low to moderate heritability. Our objective was to identify meta-QTL conditioning partial resistance to white mold from individual QTL identified across multiple populations and environments. The physical positions for 37 individual QTL were identified across 14 recombinant inbred bi-parental populations (six new, three re-genotyped, and five from the literature). A meta-QTL analysis of the 37 QTL was conducted using the genetic linkage map of Stampede x Red Hawk population as the reference. The 37 QTL condensed into 17 named loci (12 previously named and five new) of which nine were defined as meta-QTL WM1.1, WM2.2, WM3.1, WM5.4, WM6.2, WM7.1, WM7.4, WM7.5, and WM8.3. The nine meta-QTL had confidence intervals ranging from 0.65 to 9.41 Mb. Candidate genes shown to express under S. sclerotiorum infection in other studies, including cell wall receptor kinase, COI1, ethylene responsive transcription factor, peroxidase, and MYB transcription factor, were found within the confidence interval for five of the meta-QTL. The nine meta-QTL are recommended as potential targets for MAS for partial resistance to white mold in common bean.

White mold, caused by the necrotrophic fungus (Lib.) de Bary, is a major disease of common bean ( L.). WM7.1 and WM8.3 are two quantitative trait loci (QTL) with major effects on tolerance to the pathogen. Advanced backcross populations segregating individually for either of the two QTL, and a recombinant inbred (RI) population segregating for both QTL were used to fine map and confirm the genetic location of the QTL. The QTL intervals were physically mapped using the reference common bean genome sequence, and the physical intervals for each QTL were further confirmed by sequence-based introgression mapping. Using whole-genome sequence data from susceptible and tolerant DNA pools, introgressed regions were identified as those with significantly higher numbers of single-nucleotide polymorphisms (SNPs) relative to the whole genome. By combining the QTL and SNP data, WM7.1 was located to a 660-kb region that contained 41 gene models on the proximal end of chromosome Pv07, while the WM8.3 introgression was narrowed to a 1.36-Mb region containing 70 gene models. The most polymorphic candidate gene in the WM7.1 region encodes a BEACH-domain protein associated with apoptosis. Within the WM8.3 interval, a receptor-like protein with the potential to recognize pathogen effectors was the most polymorphic gene. The use of gene and sequence-based mapping identified two candidate genes whose putative functions are consistent with the current model of pathogenicity., (Copyright © 2016 Crop Science Society of America.)

Background: Common bean was one of the first crops that benefited from the development and utilization of molecular marker-assisted selection (MAS) for major disease resistance genes. Efficiency of MAS for breeding common bean is still hampered, however, due to the dominance, linkage phase, and loose linkage of previously developed markers. Here we applied in silico bulked segregant analysis (BSA) to the BeanCAP diversity panel, composed of over 500 lines and genotyped with the BARCBEAN&#95;3 6K SNP BeadChip, to develop codominant and tightly linked markers to the I gene controlling resistance to Bean common mosaic virus (BCMV)., Results: We physically mapped the genomic region underlying the I gene. This locus, in the distal arm of chromosome Pv02, contains seven putative NBS-LRR-type disease resistance genes. Two contrasting bulks, containing BCMV host differentials and ten BeanCAP lines with known disease reaction to BCMV, were subjected to in silico BSA for targeting the I gene and flanking sequences. Two distinct haplotypes, containing a cluster of six single nucleotide polymorphisms (SNP), were associated with resistance or susceptibility to BCMV. One-hundred and twenty-two lines, including 115 of the BeanCAP panel, were screened for BCMV resistance in the greenhouse, and all of the resistant or susceptible plants displayed distinct SNP haplotypes as those found in the two bulks. The resistant/susceptible haplotypes were validated in 98 recombinant inbred lines segregating for BCMV resistance. The closest SNP (~25-32 kb) to the distal NBS-LRR gene model for the I gene locus was targeted for conversion to codominant KASP (Kompetitive Allele Specific PCR) and CAPS (Cleaved Amplified Polymorphic Sequence) markers. Both marker systems accurately predicted the disease reaction to BCMV conferred by the I gene in all screened lines of this study., Conclusions: We demonstrated the utility of the in silico BSA approach using genetically diverse germplasm, genotyped with a high-density SNP chip array, to discover SNP variation at a specific targeted genomic region. In common bean, many disease resistance genes are mapped and their physical genomic position can now be determined, thus the application of this approach will facilitate further development of codominant and tightly linked markers for use in MAS.

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 bacterial blight (CBB), caused by Xanthomonas axonopodis pv. phaseoli (Xap), is a major yield-limiting factor of common bean (Phaseolus vulgaris L.) production around the world. Two major CBB-resistant quantitative trait loci (QTL), linked to the sequence characterized amplified region markers BC420 and SU91, are located at chromosomes 6 and 8, respectively. Using map-based cloning approach, four bacterial artificial chromosome (BAC) clones from the BC420-QTL locus and one BAC clone containing SU91 were sequenced by Roche 454 technique and subsequently assembled using merged assemblies from three different programs. Based on the quality of the assembly, only the sequences of BAC 32H6 and 4K7 were used for candidate gene marker (CGM) development and candidate gene (CG) selection. For the BC420-QTL locus, 21 novel genes were predicted in silico by FGENESH using Medicago gene model, whereas 16 genes were identified in the SU91-QTL locus. For each putative gene, one or more primer pairs were designed and tested in the contrasting near isogenic lines. Overall, six and nine polymorphic markers were found in the SU91- and BC420-QTL loci, respectively. Afterwards, association mapping was conducted in a breeding population of 395 dry bean lines to discover marker-trait associations. Two CGMs per each locus showed better association with CBB resistance than the BC420 and SU91 markers, which include BC420-CG10B and BC420-CG14 for BC420&#95;QTL locus, and SU91-CG10 and SU91-CG11 for SU91&#95;QTL locus. The strong associations between CBB resistance and the CGs 10 and 14 from BC420&#95;QTL locus and the CGs 10 and 11 from SU91&#95;QTL locus indicate that the genes 10 and 14 from the BC420 locus are potential CGs underlying the BC420&#95;QTL locus, whereas the genes 10 and 11 from the SU91 locus are potential CGs underlying the SU91&#95;QTL locus. The superiority of SU91-CG11 was further validated in a recombinant inbred line population Sanilac × OAC 09-3. Thus, co-dominant CGMs, BC420-CG14 and SU91-CG11, are recommended to replace BC420 and SU91 for marker-assisted selection of common bean with resistance to CBB.

Common bean genotypes were grown in three different growing sites and analyzed for 17 mineral compositions. The influence of growing sites was observed on all seed mineral contents however, ratio of genotypic variance to genotype x environment variance indicated greater influence and stability of genetic factor on Ca and Sr. It was observed that the Zn concentration is highly correlated with S and Fe and Ca with Sr in common bean seed.

Beet curly top virus, often referred to as Curly top virus (CTV), is an important virus disease of common bean in the semiarid regions of the United States, Canada, and Mexico and the only effective control is genetic resistance. Our objective was to determine if dry bean landrace G122, which lacks the Bct gene for resistance to CTV, contains novel resistance to the virus. Two populations, GT-A and GT-B, consisting of 98 F5:7 recombinant inbred lines (RILs) in total were derived from a cross between G122 and the susceptible variety Taylor Horticultural and evaluated for phenotypic response to natural CTV field infection. Genetic analyses revealed random amplified polymorphism DNA (RAPD) markers associated with a major-effect quantitative trait loci (QTL) from G122 which exhibited stable expression across 3 years in both populations. Phenotypic variation explained by the QTL in GT-A (37.6%) was greater than in GT-B (20.4%). RAPD marker Q14.973 was converted to a sequence-characterized amplified region (SCAR) and designated SQ14.973. The SCAR was used to locate the QTL on linkage group 6 of the Phaseolus core map. A survey of 74 common bean cultivars and breeding lines revealed SQ14.973 would be widely useful for marker-assisted selection of the QTL. An additional minor-effect QTL from G122 was detected on linkage group 7. G122 was determined to possess novel resistance to CTV conditioned by at least two genes, one with major the other minor effect.

White mold, caused by the fungus Sclerotinia sclerotiorum, is a serious disease in common bean (Phaseolus vulgaris) causing significant yield loss. Few cultivars with high levels of physiological resistance to white mold have been described in common bean. The objectives of this study were to (i) determine variation in aggressiveness for the local S. sclerotiorum isolates and (ii) identify sources of resistance against local isolates using the greenhouse straw test. The evaluated materials included 199 accessions of a core collection established from the main bean gene bank in Spain and 29 known cultivars or lines, 5 of them described as resistant sources to white mold: G122, PC50, A195, Cornell 606, and MO162. Significant differences for aggressiveness among the four S. sclerotiorum isolates were detected. Generally, isolates 1 and 3 were more aggressive than isolates 2 and 4. In all, 19 genotypes exhibited a level of resistance equal to or significantly better than G122: 11 accessions from the core collection and 8 cultivars or lines from known materials, including the lines A195 and Cornell 606. To confirm resistance, 19 selected genotypes were tested using a more severe straw test with reactions evaluated 21 days after inoculation. Fifteen genotypes exhibited significantly less susceptibility than G122: eight accessions from the core collection and the known cultivars or lines AB136, Kaboon, BRB57, BRB130, Don Timoteo, and A195. The logical next step will be to evaluate the best genotypes for field reaction to white mold and conduct inheritance studies.

Common bean ( Phaseolus vulgaris L.) is an important source of dietary protein and minerals worldwide. Genes conditioning variability for mineral contents are not clearly understood. Our ultimate goal is to identify genes conditioning genetic variation for Zn and Fe content. To establish mapping populations for this objective, we tested mineral content of 29 common bean genotypes. Chemical analyses revealed significant genetic variability for seed Zn and Fe contents among the genotypes. Genetic diversity was evaluated with 49 primer pairs, of which 23 were simple sequence repeats (SSR), 16 were developed from tentative consensus (TC) sequences, and 10 were generated from common bean NBS-LRR gene sequences. The discriminatory ability of molecular markers for identifying allelic variation among genotypes was estimated by polymorphism information content (PIC) and the genetic diversity was measured from genetic similarities between genotypes. Primers developed from NBS-LRR gene sequences were highly polymorphic in both PIC values and number of alleles (0.82 and 5.3), followed by SSRs (0.56 and 3.0), and markers developed from TC (0.39 and 2.0). genetic similarity values between genotypes ranged from 14.0 (JaloEEP558 and DOR364) to 91.4 (MIB152 and MIB465). Cluster analysis clearly discriminated the genotypes into Mesoamerican and Andean gene pools. Common bean genotypes were selected to include in crossing to enhance seed Zn and Fe content based on genetic diversity and seed mineral contents of the genotypes.

The dominant resistance gene, Bct, in common bean (Phaseolus vulgaris) confers qualitative resistance to Beet curly top virus, a leafhopper-transmitted geminivirus in the genus Curtovirus. To determine whether this gene confers resistance to other geminiviruses, bean plants of a recombinant inbred population were sap-inoculated with Bean dwarf mosaic virus (BDMV), a whitefly-transmitted bipartite begomovirus in the genus Begomovirus. Results indicated that Bct (or tightly linked gene) is associated with quantitative resistance to BDMV; thus, the Bct locus is associated with resistance to a bean-infecting begomovirus and curtovirus. The difference in the nature of the resistance to these geminiviruses may indicate a role for minor genes in begomovirus resistance or differences in the virus-host interaction. The Bct locus, whether it acts alone or represents a cluster of tightly linked genes, will be useful in breeding for broad-spectrum begomovirus resistance in common bean.

Resistance to common bacterial blight in common bean is a complex trait that is quantitatively inherited. Combining QTL is the current strategy for improving resistance, but interactions among different QTL are unknown. We examined the interaction between two independent QTL present in dry bean breeding line XAN 159. The QTL were studied in a near isogenic population consisting of 120 BC6:F2 plants. Each BC6:F2 plant was evaluated for disease reaction at several time points after pathogen inoculation and the dominant SCAR markers linked with QTL on linkage groups B6 (BC420 approximately QTL) and B8 (SU91 approximately QTL) were interpreted as codominant markers using real time PCR assays. This enabled assignment of BC6:F2 plants to all nine possible genotypes. Reaction to CBB in BC6:F2 plants was characterized by an epistatic interaction between BC420 and SU91 such that: 1) the expression of BC420 was epistatically suppressed by a homozygous recessive su91//su91 genotype; 2) SU91//SU91 and SU91//su91 genotypes conditioned an intermediate disease reaction when homozygous recessive for bc420//bc420; and 3) the highest level of disease resistance was conferred by genotypes with at least a single resistance allele at both QTL (BC420//-; SU91//-). Segregation for resistance among BC6:F3 plants derived from BC6:F2 plants that were heterozygous for both QTL did not deviate significantly from expected ratios of 9 resistant: 3 moderately resistant: 4 susceptible. This is consistent with a recessive epistatic model of inheritance between two loci. These results indicate breeders will realize greatest gains in resistance to CBB by selecting breeding materials that are fixed for both QTL. This is a first report of a qualitative digenic model of inheritance discerning an interaction between two QTL conditioning disease resistance in plants.

Soybean aphid (Aphis glycines) outbreaks occurring since 2000 have been associated with severe virus epidemics in snap bean (Phaseolus vulgaris) production in the Great Lakes region. Our objective was to identify specific viruses associated with the disease complex observed in the region and to survey bean germplasm for sources of resistance to the causal agents. The principle causal agent of the disease complex associated with extensive pod necrosis was identified as Clover yellow vein virus (ClYVV), designated ClYVV-WI. The virus alone caused severe mosaic, apical necrosis, and stunting. Putative coat protein amino acid sequence from clones of amplicons generated by reverse-transcription polymerase chain reaction was 98% identical to ClYVV strain no. 30 identified in Japan that has not been reported to cause pod necrosis. ClYVV-WI amplicons were 96% identical to a mild strain of ClYVV from Oregon. A distinguishing feature of this new strain is that it does not react with Potyvirus broad-spectrum monoclonal antibody PTY 1. A survey of common bean lines and cultivars revealed that, in addition to UI-31 and US1140 with known resistance to ClYVV, lines with the bc-3 gene for resistance to Bean common mosaic necrosis virus also were resistant to ClYVV-WI. An evaluation of 63 snap bean cultivars and breeding lines revealed just one, Roma 442, with a moderate level of tolerance to ClYVV-WI. Introgression of the bc-3 gene and resistances from UI-31 and US1140 into snap bean may offer a high level of resistance to extensive pod necrosis disease caused by ClYVV in the Great Lakes region.

Rust in common bean (Phaseolus vulgaris L.) is caused by Uromyces appendiculatus Pers.:Pers. (Unger) which exhibits a high level of pathogenic diversity. Resistance to this disease is conditioned by a considerable number of genes. Pyramiding resistance genes is desirable and could be simplified by the use of molecular markers closely linked to the genes. The resistance gene Ur-13, present in the South African large seeded cultivar Kranskop, has been used extensively in the local breeding program. The purpose of this study was the development of a molecular marker linked to Ur-13. An F(2) population derived from a cross between Kranskop and a susceptible (South African) cultivar Bonus was used in combination with bulked segregant analysis utilizing the amplified fragment length polymorphism (AFLP) technique. Seven AFLP fragments linked significantly to the rust resistance and five were successfully converted to sequence characterized amplified region (SCAR) markers. The co-dominant SCAR markers derived from a 405 bp EAACMACC fragment, KB 126, was located 1.6 cM from the gene. Two additional SCAR markers and one cleaved amplified polymorphic sequence marker were located further from the gene. The gene was mapped to linkage group B8 on the BAT 93/Jalo EEP 558 core map (chromosome 3).

ABSTRACT A strain of Bean common mosaic necrosis virus (BCMNV) from Idaho was identified by enzyme-linked immunosorbent assay using monoclonal antibodies and determined to be similar to the NL-3 D strain (of Drifjhout) by reaction of differential bean cultivars. However, this BCMNV strain (designated NL-3 K) caused earlier and more severe symptoms on bean plants representing host groups 0, 4, and 5. The nucleotide sequence encoding the predicted polyprotein of NL-3 K was 9,893 nucleotides (nt) in length, yielding a peptide with a molecular size of 362.1 kDa compared with a 9,626-nt, 350.9-kDa polyprotein for NL-3 D. Sequence analysis of the putative P1 protein suggests that the NL-3 K strain is a recombinant between NL-3 D and the Russian strain (RU1) of Bean common mosaic virus. The P1 protein of NL-3 K consisted of 415 amino acids compared with 317 for NL-3 D. The first 114 predicted amino acids of the NL-3 K P1 region were 98% identical with RU1. The remaining 301 amino acids of the protein shared only 34% identity with RU1 but were 98% identical with NL-3 D. Primers were designed that flanked the recombination point in the P1 coding sequence of NL-3 K. An amplicon of the expected size was produced by reverse-transcriptase polymerase chain reaction of total nucleic acid extracts of bean plants inoculated with NL-3 K, but not from those with NL-3 D or RU1. The increased symptom severity on selected common bean lines induced by NL-3 K suggests that the P1 gene may play a significant role in pathogenicity and virulence.

ABSTRACT A multiplex real-time polymerase chain reaction (PCR) assay was developed to simultaneously genotype plants for the I and bc-1(2) alleles, which condition resistance in beans to Bean common mosaic virus and Bean common mosaic necrosis virus. A segregating F(2) population was derived from the cross between pinto bean breeding line P94207-189A (bc-1 bc-1 I I) and Olathe (bc-12 bc-1(2) i i). Real-time PCR assays were developed that were specific for each allele, and a multiplex PCR reaction could unambiguously assign F(2) plants to one of nine genotypes. Remnant F(1) plants were used as a comparative reference sample. PCR results among this sample fit a normal distribution for both real-time PCR assays, and 99% probability distributions were determined for heterozygotes. F(2) plants were genotyped based on results relative to the probability distributions for heterozygotes. F(2) plants also were genotyped for the I and bc-1(2) alleles by performing F(3) family progeny tests for virus resistance. Agreement between the two methods was 100% (198/198) for the bc-1(2) allele, and 92.4% (183/198) for the I allele. Erroneous genotyping was due to recombination between the amplicon and the I allele. Realtime PCR assays provide a robust method for genotyping seedlings and, in some cases, may eliminate the need for progeny testing.

ABSTRACT A random amplified polymorphic DNA (RAPD) marker directly linked (0.0 cM) with a resistance gene was identified in a snap bean recombinant inbred population (Moncayo x Primo) consisting of 94 F(5:7) recombinant inbred lines that had uniform segregation for disease reaction to Beet curly top virus (BCTV) across three field locations. Resistance was conditioned by a single dominant allele tentatively designated Bct. Seven hundred and fifty decamer primers were screened to obtain the linked RAPD marker that was then converted to a sequence characterized amplified region (SCAR) marker SAS8.1550. The SCAR mapped within a cluster of resistance genes on linkage group B7 of the core map. A survey of 103 BCTV-resistant and -susceptible snap and dry bean genotypes was conducted using SAS8.1550. Results showed that the SCAR would be highly useful for marker-assisted selection of Bct in snap and dry bean originating from the Andean gene pool. Marker-assisted selection for Bct will expedite the development of BCTV-resistant cultivars and minimize the need for cumbersome pathogen tests.

Variation in common bean (Phaseolus vulgaris L.) was investigated by sequencing intron 1 of the dihydroflavonol 4-reductase (DFR) gene for 92 genotypes that represent both landraces and cultivars. We were also interested in determining if introns provide sufficient variation for genetic diversity studies and if the sequence data could be used to develop allele-specific primers that could differentiate genotypes using a standard PCR assay. Sixty-nine polymorphic sites were observed. Nucleotide variation (pi/bp) was 0.0481, a value higher than that reported for introns from other plant species. Tests for significant deviation from the mutation drift model were positive for the population as a whole, the cultivar and landrace subsets, and the Middle American landrace set. Significant linkage disequilibrium extended about 300 nucleotides. Twenty haplotypes were detected among the cultivated genotypes. Seven recombination events were detected for the whole population, and six events for the landraces. Recombination was not observed among the landraces within either the Middle American or Andean gene pools. Evidence for hybridization between the two gene pools was discovered. Five allele-specific primers were developed that could distinguish 56 additional genotypes. The allele-specific primers were used to map duplicate DFR genes on linkage group B8.

During 2003, a new disease, dry bean phyllody (DBPh), was observed in the Columbia Basin of Washington in dry bean (Phaseolus vulgaris L.) cultivars of Andean origin grown in Mattawa and Paterson, WA that caused great reduction in dry bean production. Symptoms of DBPh became apparent during mid-to-late pod development and were characterized by leafy petals (phyllody) and aborted seed pods resembling thin, twisted, and corrugated leaf-like structures. Deformed sterile pods that were small, sickle-shaped, upright, and leathery were also observed. The infected plants generally exhibited chlorosis, stunting, or bud proliferation from leaf axils. Symptoms of DBPh were indicative of possible infection by phytoplasmas. Restriction fragment length polymorphism (RFLP) and phylogenetic analyses of amplified 16S rDNA sequences were used for phytoplasma identification. Four symptomatic bean plants were analyzed and tested positive for phytoplasma infection on the basis of results of initial polymerase chain reaction (PCR) and subsequent nested-PCR amplifications (2). RFLP analyses of 16S rDNA sequences with restriction enzymes, MseI, AluI, HhaI, RsaI, and HpaII indicated that the phytoplasma strains associated with DBPh belonged to the clover proliferation group (16SrVI) subgroup A (16SrVI-A) (2). This subgroup currently consists of three members, clover proliferation (CP; GenBank Accession No. AY500130), potato witches'-broom (PWB; GenBank Accession No. AY500818), and vinca virescence (VR; GenBank Accession No. AY500817), a strain of beet leafhopper-transmitted virescence agent (BLTVA) phytoplasmas (1,2). The taxonomic affiliations of the DBPh phytoplasma strains were confirmed by phylogenetic analysis of cloned 16S rRNA gene sequences (GenBank Accession Nos. DBPh2, AY496002; DBPh3, AY496003). Among the existing members of subgroup 16SrVI-A, the four DBPh strains were closely related to the VR strain with 99.7% 16S rDNA sequence homology and to the CP strain with 99.2% sequence homology. To gain further evidence on the role of 16SrVI-A phytoplasma strains in DBPh disease, a modified test of Koch's postulates was conducted. Infected tissue from one phytoplasma-positive dry bean sample was grafted onto three Pinto UI-114 bean seedlings in the greenhouse. Within 60 days, the bean seedlings exhibited corrugated leaf-like structures from aborted seedpods, a lack of flower formation, general chlorosis, and stunting similar to the original diseased plants. The lower leaves of the inoculated bean plants became epinastic and leathery. The transmitted phytoplasma was detected in each of the grafted symptomatic seedlings, and the RFLP patterns of its 16S rRNA gene sequences were identical to those of the phytoplasmas in the scions. A high correlation between the presence of disease symptoms and the presence of subgroup 16SrVI-A phytoplasmas in the bean plants suggests that these phytoplasmas play an etiological role in DBPh disease. To our knowledge, these findings provide the first confirmed case of phytoplasma-associated DBPh in the United States. References: (1) D. A. Golino et al. Plant Dis. 73:850, 1989. (2) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998.

ABSTRACT A previously unrecognized recessive resistance gene (or allele) was identified in three host group (HG) 3 common bean (Phaseolus vulgaris) cvs. Olathe, Victor, and UI 37, based on genetic analysis of plants from five populations screened with the NL-3 K strain of Bean common mosaic necrosis virus (BCMNV). The gene (or allele) was associated with resistance to leaf stunting and deformity and reduction in plant height. The gene (or allele) provides similar, but slightly better resistance than the bc-1(2) gene that is characteristic of HG 3 cultivars. Traditional HG 3 cultivars like Redlands Greenleaf B with bc-1(2) are susceptible to NL-3 K, whereas this newly identified gene (or allele) conditions resistance to NL-3 K. Other slight variations in disease reaction pattern to a wide array of bean common mosaic (BCM)-inducing strains were noted among HG 3 differentials, indicating that additional resistance to BCM exists in common bean that remains to be exploited. To gauge the full breeding value of this newly identified gene (or allele), allelism tests with existing genes, namely bc-1(2), and further characterization of responses to all Bean common mosaic virus (BCMV) and BCMNV strains need to be conducted. Meanwhile, breeders should consider introgressing this more effective gene (or allele) into susceptible cultivars while plant pathologists continue to decipher the genetic variability present among HG 3 differential cultivars.

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.

The development and use of RAPD markers for applications in crop improvement has recently generated considerable interest within the plant breeding community. One potential application of RAPDs is their use for "tagging" simply-inherited (monogenic) pest-resistance genes and enabling more efficient identification and selection of genotypes carrying specific combinations of resistance genes. In this report, we propose and describe the use of heterogeneous inbred populations as sources of near-isogenic lines (NILs) for targeting RAPD markers linked to major pest resistance genes. The development of these NILs for RAPD marker analyses involved a sequence of line and mass selection during successive generations of inbreeding. DNA bulks derived from the NILs were used to identify a RAPD marker (designated OK14620, generated by 5'-CCCGCTACAC-3' decamer) that was tightly linked (2.23±1.33 centiMorgans) to an important rust [Uromyces appendiculatus (Pers.) Unger var. appendiculatus] resistance gene (Ur-3) in common bean (Phaseolus vulgaris L.). The efficiency of this approach was demonstrated by a low rate of false-positives identified, the tightness of the linkage identified, and the ability to detect polymorphism between genomic regions that are representative of the same gene pool of common bean. This method of deriving NILs should find application by researchers interested in utilizing marker-assisted selection for one or more major pest resistance genes. The identification of OK14620 should help to facilitate continued use of the Ur-3 resistance source and will now enable marker-assisted pyramiding of three different bean rust resistance sources (two previously tagged) to provide effective and stable resistance to this important pathogen.

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.

The Up 2 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.]. Up 2 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 Up 2 and three without Up 2 as contrasting near-isogenic lines (NILs) was used to identify random amplified polymorphic DNA fragments (RAPDs) tightly linked to the Up 2 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 Up 2 allele within a segregating BC6F2 population of 84 individuals. This result suggests that OA141100 and Up 2 are tightly linked. Andean and Mesoamerican bean germ plasm, with and without the Up 2 allele, were assayed for the presence of OA141100. Apparently, the marker is of Andean origin because all Andean lines, with or without the Up 2 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 Up 2 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.