Exploring the Mycovirus Sclerotinia sclerotiorum Hypovirulence-Associated DNA Virus 1 as a Biocontrol Agent of White Mold Caused by Sclerotinia sclerotiorum.

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.

Fine-mapping and evolutionary history of R-BPMV, a dominant resistance gene to Bean pod mottle virus in Phaseolus vulgaris L.

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.

Identification of quantitative trait loci for drought tolerance in Bukoba/Kijivu Andean mapping population of common bean.

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 F5:7 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.

The genetics and physiology of seed dormancy, a crucial trait in common bean domestication.

BACKGROUND: Physical seed dormancy is an important trait in legume domestication. Although seed dormancy is beneficial in wild ecosystems, it is generally considered to be an undesirable trait in crops due to reduction in yield and / or quality. The physiological mechanism and underlying genetic factor(s) of seed dormancy is largely unknown in several legume species. Here we employed an integrative approach to understand the mechanisms controlling physical seed dormancy in common bean (Phaseolus vulgaris L.). RESULTS: Using an innovative CT scan imaging system, we were able to track water movements inside the seed coat. We found that water uptake initiates from the bean seed lens. Using a scanning electron microscopy (SEM) we further identified several micro-cracks on the lens surface of non-dormant bean genotypes. Bulked segregant analysis (BSA) was conducted on a bi-parental RIL (recombinant inbred line) population, segregating for seed dormancy. This analysis revealed that the seed water uptake is associated with a single major QTL on Pv03. The QTL region was fine-mapped to a 118 Kb interval possessing 11 genes. Coding sequence analysis of candidate genes revealed a 5-bp insertion in an ortholog of pectin acetylesterase 8 that causes a frame shift, loss-of-function mutation in non-dormant genotype. Gene expression analysis of the candidate genes in the seed coat of contrasting genotypes indicated 21-fold lower expression of pectin acetylesterase 8 in non-dormant genotype. An analysis of mutational polymorphism was conducted among wild and domesticated beans. Although all the wild beans possessed the functional allele of pectin acetylesterase 8, the majority (77%) of domesticated beans had the non-functional allele suggesting that this variant was under strong selection pressure through domestication. CONCLUSIONS: In this study, we identified the physiological mechanism of physical seed dormancy and have identified a candidate allele causing variation in this trait. Our findings suggest that a 5-bp insertion in an ortholog of pectin acetylesterase 8 is likely a major causative mutation underlying the loss of seed dormancy during domestication. Although the results of current study provide strong evidences for the role of pectin acetylesterase 8 in seed dormancy, further confirmations seem necessary by employing transgenic approaches.

A common bean truncated CRINKLY4 kinase controls gene-for-gene resistance to the fungus Colletotrichum lindemuthianum.

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.

Application of in silico bulked segregant analysis for rapid development of markers linked to Bean common mosaic virus resistance in common bean.

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_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.

The alleles bc-ud and bc-ur (previously bc-4 gene), representing coding mutations within Vps4 AAA+ ATPase ESCRT protein, interact with other genes to condition resistance to BCMV and BCMNV in common bean.

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-ud gene as revealed by long-read sequencing, develop an efficient DNA marker to assist selection of bc-ud in snap and dry beans, and examine the interactions between the bc-ud 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-ud. An 84-bp repetitive insertion variant within the gene, exhibited 100% co-segregation with the bc-ud resistance allele across 264 common bean accessions. The 84-bp repetitive insertion was labeled with an indel marker IND_05_36225873 which was useful for tracking the bc-ud allele across diverse germplasm. A different single nucleotide polymorphism variant within the same candidate gene was associated with the bc-4 gene. Segregation in F2 populations confirmed bc-ud and bc-4 were alleles, so bc-4 was renamed bc-ur to fit gene nomenclature guidelines. The interactions of bc-ud and bc-ur 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.

T and Z, partial seed coat patterning genes in common bean, provide insight into the structure and protein interactions of a plant MBW complex.

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 ß-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 (ß-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.

GWAS of resistance to three bacterial diseases in the Andean common bean diversity panel.

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.

Automating high-throughput screening for anthracnose resistance in common bean using allele specific PCR.

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-42 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.

Linked candidate genes of different functions for white mold resistance in common bean (Phaseolus vulgaris L) are identified by multiple QTL mapping approaches.

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.

Integrating de novo QTL-seq and linkage mapping to identify quantitative trait loci conditioning physiological resistance and avoidance to white mold disease in dry bean.

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.

A robust SNP-haplotype assay for Bct gene region conferring resistance to beet curly top virus in common bean (Phaseolus vulgaris L.).

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_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_2970381 and two linked SNPs, S07_2970276 and S07_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.

Development of candidate gene markers associated to common bacterial blight resistance in common bean.

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_QTL locus, and SU91-CG10 and SU91-CG11 for SU91_QTL locus. The strong associations between CBB resistance and the CGs 10 and 14 from BC420_QTL locus and the CGs 10 and 11 from SU91_QTL locus indicate that the genes 10 and 14 from the BC420 locus are potential CGs underlying the BC420_QTL locus, whereas the genes 10 and 11 from the SU91 locus are potential CGs underlying the SU91_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.

New genomic regions associated with white mold resistance in dry bean using a MAGIC population.

Dry bean (Phaseolus vulgaris L.) production in many regions is threatened by white mold (WM) [Sclerotinia sclerotiorum (Lib.) de Bary]. Seed yield losses can be up to 100% under conditions favorable for the pathogen. The low heritability, polygenic inheritance, and cumbersome screening protocols make it difficult to breed for improved genetic resistance. Some progress in understanding genetic resistance and germplasm improvement has been accomplished, but cultivars with high levels of resistance are yet to be released. A WM multiparent advanced generation inter-cross (MAGIC) population (n = 1060) was developed to facilitate mapping and breeding efforts. A seedling straw test screening method provided a quick assay to phenotype the population for response to WM isolate 1980. Nineteen MAGIC lines were identified with improved resistance. For genome-wide association studies (GWAS), the data was transformed into three phenotypic distributions-quantitative, polynomial, and binomial-and coupled with ∼52,000 single-nucleotide polymorphisms (SNPs). The three phenotypic distributions identified 30 significant genomic intervals [-log10 (P value) ≥ 3.0]. However, across distributions, four new genomic regions as well as two regions previously reported were found to be associated with resistance. Cumulative R2 values were 57% for binomial distribution using 13 genomic intervals, 41% for polynomial using eight intervals, and 40% for quantitative using 11 intervals. New resistant germplasm as well as new genomic regions associated with resistance are now available for further investigation.

Improving Association Studies and Genomic Predictions for Climbing Beans With Data From Bush Bean Populations.

Common bean (Phaseolus vulgaris L.) has two major origins of domestication, Andean and Mesoamerican, which contribute to the high diversity of growth type, pod and seed characteristics. The climbing growth habit is associated with increased days to flowering (DF), seed iron concentration (SdFe), nitrogen fixation, and yield. However, breeding efforts in climbing beans have been limited and independent from bush type beans. To advance climbing bean breeding, we carried out genome-wide association studies and genomic predictions using 1,869 common bean lines belonging to five breeding panels representing both gene pools and all growth types. The phenotypic data were collected from 17 field trials and were complemented with 16 previously published trials. Overall, 38 significant marker-trait associations were identified for growth habit, 14 for DF, 13 for 100 seed weight, three for SdFe, and one for yield. Except for DF, the results suggest a common genetic basis for traits across all panels and growth types. Seven QTL associated with growth habits were confirmed from earlier studies and four plausible candidate genes for SdFe and 100 seed weight were newly identified. Furthermore, the genomic prediction accuracy for SdFe and yield in climbing beans improved up to 8.8% when bush-type bean lines were included in the training population. In conclusion, a large population from different gene pools and growth types across multiple breeding panels increased the power of genomic analyses and provides a solid and diverse germplasm base for genetic improvement of common bean.

The Common Bean V Gene Encodes Flavonoid 3'5' Hydroxylase: A Major Mutational Target for Flavonoid Diversity in Angiosperms.

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.

Coding Mutations in Vacuolar Protein-Sorting 4 AAA+ ATPase Endosomal Sorting Complexes Required for Transport Protein Homologs Underlie bc-2 and New bc-4 Gene Conferring Resistance to Bean Common Mosaic Virus in Common Bean.

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.

Genome-Wide Association Mapping of bc-1 and bc-u Reveals Candidate Genes and New Adjustments to the Host-Pathogen Interaction for Resistance to Bean Common Mosaic Necrosis Virus in Common Bean.

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 F3 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.

NAC Candidate Gene Marker for bgm-1 and Interaction With QTL for Resistance to Bean Golden Yellow Mosaic Virus in Common Bean.

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.