Large genomic introgression blocks of Phaseolus parvifolius Freytag bean into the common bean enhance the crossability between tepary and common beans.

The production of the common bean (Phaseolus vulgaris L.), one of the most important sources of protein and minerals and one of the most consumed grain legumes globally, is highly affected by heat and drought constraints. In contrast, the tepary bean (Phaseolus acutifolius A. Gray), a common bean-related species, is adapted to hot and dry climates. Hybridization to introduce complex traits from the tepary bean into the common bean has been challenging, as embryo rescue is required. In this study, we report three novel interspecific lines that were obtained by crossing lines from prior common bean × tepary bean hybridization with Phaseolus parvifolius Freytag in order to increase the male gametic diversity to facilitate interspecific crosses. These interspecific lines enhanced the crossability of the common bean and tepary bean species while avoiding the embryo rescue process. Crossing these three interspecific lines with tepary beans resulted in 12-fold more hybrid plants than crossing traditional common beans with tepary beans. Whole-genome sequencing analysis of these three interspecific lines shows large introgressions of genomic regions corresponding to P. parvifolius on chromosomes that presumably contribute to reproductive barriers between both species. The development of these lines opens up the possibility of increasing the introgression of desirable tepary bean traits into the common bean to address constraints driven by climate change.

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.

Genetic mapping for agronomic traits in a MAGIC population of common bean (Phaseolus vulgaris L.) under drought conditions.

BACKGROUND: Common bean is an important staple crop in the tropics of Africa, Asia and the Americas. Particularly smallholder farmers rely on bean as a source for calories, protein and micronutrients. Drought is a major production constraint for common bean, a situation that will be aggravated with current climate change scenarios. In this context, new tools designed to understand the genetic basis governing the phenotypic responses to abiotic stress are required to improve transfer of desirable traits into cultivated beans. RESULTS: A multiparent advanced generation intercross (MAGIC) population of common bean was generated from eight Mesoamerican breeding lines representing the phenotypic and genotypic diversity of the CIAT Mesoamerican breeding program. This population was assessed under drought conditions in two field trials for yield, 100 seed weight, iron and zinc accumulation, phenology and pod harvest index. Transgressive segregation was observed for most of these traits. Yield was positively correlated with yield components and pod harvest index (PHI), and negative correlations were found with phenology traits and micromineral contents. Founder haplotypes in the population were identified using Genotyping by Sequencing (GBS). No major population structure was observed in the population. Whole Genome Sequencing (WGS) data from the founder lines was used to impute genotyping data for GWAS. Genetic mapping was carried out with two methods, using association mapping with GWAS, and linkage mapping with haplotype-based interval screening. Thirteen high confidence QTL were identified using both methods and several QTL hotspots were found controlling multiple traits. A major QTL hotspot located on chromosome Pv01 for phenology traits and yield was identified. Further hotspots affecting several traits were observed on chromosomes Pv03 and Pv08. A major QTL for seed Fe content was contributed by MIB778, the founder line with highest micromineral accumulation. Based on imputed WGS data, candidate genes are reported for the identified major QTL, and sequence changes were identified that could cause the phenotypic variation. CONCLUSIONS: This work demonstrates the importance of this common bean MAGIC population for genetic mapping of agronomic traits, to identify trait associations for molecular breeding tool design and as a new genetic resource for the bean research community.

Fine-mapping of angular leaf spot resistance gene Phg-2 in common bean and development of molecular breeding tools.

KEY MESSAGE: The Common Bean Angular Leaf Spot Resistance Gene Phg-2 was fine-mapped to a 409-Kbp region, and molecular markers for breeders were developed and validated in field experiments. Common bean (Phaseolus vulgaris L.) is an important food legume in Latin America, Asia and Africa. It is an important source of protein, carbohydrates and micro-minerals, particularly for smallholder farmers. Common bean productivity is affected by angular leaf spot (ALS) disease caused by the pathogenic fungus Pseudocercospora griseola, resulting in significant yield losses, particularly in low-input smallholder farming systems in the tropics. The ALS resistance gene Phg-2, which was found in several highly resistant common bean genotypes, was investigated in crosses between Mesoamerican pre-breeding lines and elite Andean breeding lines. Next-generation sequencing (NGS) data sets were used to design new SNP-based molecular markers. The Phg-2 locus was confirmed to be the major locus providing ALS resistance in these crosses. The locus was fine-mapped to a 409-Kbp region on chromosome 8. Two clusters of highly related LRR genes were identified in this region, which are the best candidate genes for Phg-2. Molecular markers were identified that are closely linked to the Phg-2 resistance gene and also highly specific to the donor germplasm. Marker-assisted selection (MAS) was used to introgress the Phg-2 resistance locus into Andean breeding germplasm using MAB lines. The usefulness of molecular markers in MAS was confirmed in several field evaluations in complex breeding crosses, under inoculation with different ALS pathotypes. This project demonstrates that NGS data are a powerful tool for the characterization of genetic loci and can be applied in the development of breeding tools.

Analyses of African common bean (Phaseolus vulgaris L.) germplasm using a SNP fingerprinting platform: diversity, quality control and molecular breeding.

Common bean (Phaseolus vulgaris L.) is an important staple crop for smallholder farmers, particularly in Eastern and Southern Africa. To support common bean breeding and seed dissemination, a high throughput SNP genotyping platform with 1500 established SNP assays has been developed at a genotyping service provider which allows breeders without their own genotyping infrastructure to outsource such service. A set of 708 genotypes mainly composed of germplasm from African breeders and CIAT breeding program were assembled and genotyped with over 800 SNPs. Diversity analysis revealed that both Mesoamerican and Andean gene pools are in use, with an emphasis on large seeded Andean genotypes, which represents the known regional preferences. The analysis of genetic similarities among germplasm entries revealed duplicated lines with different names as well as distinct SNP patterns in identically named samples. Overall, a worrying number of inconsistencies was identified in this data set of very diverse origins. This exemplifies the necessity to develop and use a cost-effective fingerprinting platform to ensure germplasm purity for research, sharing and seed dissemination. The genetic data also allows to visualize introgressions, to identify heterozygous regions to evaluate hybridization success and to employ marker-assisted selection. This study presents a new resource for the common bean community, a SNP genotyping platform, a large SNP data set and a number of applications on how to utilize this information to improve the efficiency and quality of seed handling activities, breeding, and seed dissemination through molecular tools.

Resequencing of Common Bean Identifies Regions of Inter-Gene Pool Introgression and Provides Comprehensive Resources for Molecular Breeding.

Common bean ( L.) is the most important grain legume for human consumption and is a major nutrition source in the tropics. Because bean production is reduced by both abiotic and biotic constraints, current breeding efforts are focused on the development of improved varieties with tolerance to these stresses. We characterized materials from different breeding programs spanning three continents to understand their sequence diversity and advance the development of molecular breeding tools. For this, 37 varieties belonging to , (A. Gray), and L. were sequenced by whole-genome sequencing, identifying more than 40 million genomic variants. Evaluation of nuclear DNA content and analysis of copy number variation revealed important differences in genomic content not only between and the two other domesticated species, but also within , affecting hundreds of protein-coding genomic regions. A large number of inter-gene pool introgressions were identified. Furthermore, interspecific introgressions for disease resistance in breeding lines were mapped. Evaluation of newly developed single nucleotide polymorphism markers within previously discovered quantitative trait loci for common bacterial blight and angular leaf spot provides improved specificity to tag sources of resistance to these diseases. We expect that this dataset will provide a deeper molecular understanding of breeding germplasm and deliver molecular tools for germplasm development, aiming to increase the efficiency of bean breeding programs.

Bioinformatic analysis of genotype by sequencing (GBS) data with NGSEP.

BACKGROUND: Therecent development and availability of different genotype by sequencing (GBS) protocols provided a cost-effective approach to perform high-resolution genomic analysis of entire populations in different species. The central component of all these protocols is the digestion of the initial DNA with known restriction enzymes, to generate sequencing fragments at predictable and reproducible sites. This allows to genotype thousands of genetic markers on populations with hundreds of individuals. Because GBS protocols achieve parallel genotyping through high throughput sequencing (HTS), every GBS protocol must include a bioinformatics pipeline for analysis of HTS data. Our bioinformatics group recently developed the Next Generation Sequencing Eclipse Plugin (NGSEP) for accurate, efficient, and user-friendly analysis of HTS data. RESULTS: Here we present the latest functionalities implemented in NGSEP in the context of the analysis of GBS data. We implemented a one step wizard to perform parallel read alignment, variants identification and genotyping from HTS reads sequenced from entire populations. We added different filters for variants, samples and genotype calls as well as calculation of summary statistics overall and per sample, and diversity statistics per site. NGSEP includes a module to translate genotype calls to some of the most widely used input formats for integration with several tools to perform downstream analyses such as population structure analysis, construction of genetic maps, genetic mapping of complex traits and phenotype prediction for genomic selection. We assessed the accuracy of NGSEP on two highly heterozygous F1 cassava populations and on an inbred common bean population, and we showed that NGSEP provides similar or better accuracy compared to other widely used software packages for variants detection such as GATK, Samtools and Tassel. CONCLUSIONS: NGSEP is a powerful, accurate and efficient bioinformatics software tool for analysis of HTS data, and also one of the best bioinformatic packages to facilitate the analysis and to maximize the genomic variability information that can be obtained from GBS experiments for population genomics.

Fine-mapping of a major QTL controlling angular leaf spot resistance in common bean (Phaseolus vulgaris L.).

KEY MESSAGE: A major QTL for angular leaf spot resistance in the common bean accession G5686 was fine-mapped to a region containing 36 candidate genes. Markers have been developed for marker-assisted selection. Common bean (Phaseolus vulgaris L.) is an important grain legume and an essential protein source for human nutrition in developing countries. Angular leaf spot (ALS) caused by the pathogen Pseudocercospora griseola (Sacc.) Crous and U. Braun is responsible for severe yield losses of up to 80%. Breeding for resistant cultivars is the most ecological and economical means to control ALS and is particularly important for yield stability in low-input agriculture. Here, we report on a fine-mapping approach of a major quantitative trait locus (QTL) ALS4.1(GS, UC) for ALS resistance in a mapping population derived from the resistant genotype G5686 and the susceptible cultivar Sprite. 180 F3 individuals of the mapping population were evaluated for ALS resistance and genotyped with 22 markers distributed over 11 genome regions colocating with previously reported QTL for ALS resistance. Multiple QTL analysis identified three QTL regions, including one major QTL on chromosome Pv04 at 43.7 Mbp explaining over 75% of the observed variation for ALS resistance. Additional evaluation of 153 F4, 89 BC1F2 and 139 F4/F5/BC1F3 descendants with markers in the region of the major QTL delimited the region to 418 kbp harboring 36 candidate genes. Among these, 11 serine/threonine protein kinases arranged in a repetitive array constitute promising candidate genes for controlling ALS resistance. Single nucleotide polymorphism markers cosegregating with the major QTL for ALS resistance have been developed and constitute the basis for marker-assisted introgression of ALS resistance into advanced breeding germplasm of common bean.