Juan M. Osorno

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

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

Developing market class specific InDel markers from next generation sequence data in Phaseolus vulgaris L.

Next generation sequence data provides valuable information and tools for genetic and genomic research and offers new insights useful for marker development. This data is useful for the design of accurate and user-friendly molecular tools. Common bean (Phaseolus vulgaris L.) is a diverse crop in which separate domestication events happened in each gene pool followed by race and market class diversification that has resulted in different morphological characteristics in each commercial market class. This has led to essentially independent breeding programs within each market class which in turn has resulted in limited within market class sequence variation. Sequence data from selected genotypes of five bean market classes (pinto, black, navy, and light and dark red kidney) were used to develop InDel-based markers specific to each market class. Design of the InDel markers was conducted through a combination of assembly, alignment and primer design software using 1.6× to 5.1× coverage of Illumina GAII sequence data for each of the selected genotypes. The procedure we developed for primer design is fast, accurate, less error prone, and higher throughput than when they are designed manually. All InDel markers are easy to run and score with no need for PCR optimization. A total of 2687 InDel markers distributed across the genome were developed. To highlight their usefulness, they were employed to construct a phylogenetic tree and a genetic map, showing that InDel markers are reliable, simple, and accurate.

SNP Assay Development for Linkage Map Construction, Anchoring Whole-Genome Sequence, and Other Genetic and Genomic Applications in Common Bean

A total of 992,682 single-nucleotide polymorphisms (SNPs) was identified as ideal for Illumina Infinium II BeadChip design after sequencing a diverse set of 17 common bean (Phaseolus vulgaris L) varieties with the aid of next-generation sequencing technology. From these, two BeadChips each with >5000 SNPs were designed. The BARCBean6K_1 BeadChip was selected for the purpose of optimizing polymorphism among market classes and, when possible, SNPs were targeted to sequence scaffolds in the Phaseolus vulgaris 14× genome assembly with sequence lengths >10 kb. The BARCBean6K_2 BeadChip was designed with the objective of anchoring additional scaffolds and to facilitate orientation of large scaffolds. Analysis of 267 F2 plants from a cross of varieties Stampede × Red Hawk with the two BeadChips resulted in linkage maps with a total of 7040 markers including 7015 SNPs. With the linkage map, a total of 432.3 Mb of sequence from 2766 scaffolds was anchored to create the Phaseolus vulgaris v1.0 assembly, which accounted for approximately 89% of the 487 Mb of available sequence scaffolds of the Phaseolus vulgaris v0.9 assembly. A core set of 6000 SNPs (BARCBean6K_3 BeadChip) with high genotyping quality and polymorphism was selected based on the genotyping of 365 dry bean and 134 snap bean accessions with the BARCBean6K_1 and BARCBean6K_2 BeadChips. The BARCBean6K_3 BeadChip is a useful tool for genetics and genomics research and it is widely used by breeders and geneticists in the United States and abroad.

Genome-Wide Association Study Identifies Candidate Loci Underlying Agronomic Traits in a Middle American Diversity Panel of Common Bean

Common bean (Phaseolus vulgaris L.) breeding programs aim to improve both agronomic and seed characteristics traits. However, the genetic architecture of the many traits that affect common bean production are not completely understood. Genome‐wide association studies (GWAS) provide an experimental approach to identify genomic regions where important candidate genes are located. A panel of 280 modern bean genotypes from race Mesoamerica, referred to as the Middle American Diversity Panel (MDP), were grown in four US locations, and a GWAS using >150,000 single‐nucleotide polymorphisms (SNPs) (minor allele frequency [MAF] ≥ 5%) was conducted for six agronomic traits. The degree of inter‐ and intrachromosomal linkage disequilibrium (LD) was estimated after accounting for population structure and relatedness. The LD varied between chromosomes for the entire MDP and among race Mesoamerica and Durango–Jalisco genotypes within the panel. The LD patterns reflected the breeding history of common bean. Genome‐wide association studies led to the discovery of new and known genomic regions affecting the agronomic traits at the entire population, race, and location levels. We observed strong colocalized signals in a narrow genomic interval for three interrelated traits: growth habit, lodging, and canopy height. Overall, this study detected ∼30 candidate genes based on a priori and candidate gene search strategies centered on the 100‐kb region surrounding a significant SNP. These results provide a framework from which further research can begin to understand the actual genes controlling important agronomic production traits in common bean.

Comparative Transcriptome Analysis of Resistant and Susceptible Common Bean Genotypes in Response to Soybean Cyst Nematode Infection

Soybean cyst nematode (SCN; Heterodera glycines Ichinohe) reproduces on the roots of common bean (Phaseolus vulgaris L.) and can cause reductions in plant growth and seed yield. The molecular changes in common bean roots caused by SCN infection are unknown. Identification of genetic factors associated with SCN resistance could help in development of improved bean varieties with high SCN resistance. Gene expression profiling was conducted on common bean roots infected by SCN HG type 0 using next generation RNA sequencing technology. Two pinto bean genotypes, PI533561 and GTS-900, resistant and susceptible to SCN infection, respectively, were used as RNA sources eight days post inoculation. Total reads generated ranged between ~ 3.2 and 5.7 million per library and were mapped to the common bean reference genome. Approximately 70–90% of filtered RNA-seq reads uniquely mapped to the reference genome. In the inoculated roots of resistant genotype PI533561, a total of 353 genes were differentially expressed with 154 up-regulated genes and 199 down-regulated genes when compared to the transcriptome of non- inoculated roots. On the other hand, 990 genes were differentially expressed in SCN-inoculated roots of susceptible genotype GTS-900 with 406 up-regulated and 584 down-regulated genes when compared to non-inoculated roots. Genes encoding nucleotide-binding site leucine-rich repeat resistance (NLR) proteins, WRKY transcription factors, pathogenesis-related (PR) proteins and heat shock proteins involved in diverse biological processes were differentially expressed in both resistant and susceptible genotypes. Overall, suppression of the photosystem was observed in both the responses. Furthermore, RNA-seq results were validated through quantitative real time PCR. This is the first report describing genes/transcripts involved in SCN-common bean interaction and the results will have important implications for further characterization of SCN resistance genes in common bean.

Two endornaviruses show differential infection patterns between gene pools of Phaseolus vulgaris

We investigated the occurrence of two plant endornaviruses, Phaseolus vulgaris endornavirus 1 and Phaseolus vulgaris endornavirus 2, in breeding lines, cultivars, landraces, and wild genotypes of common bean (Phaseolus vulgaris) collected from the two centers of common bean domestication: Mesoamerica and the Andes. The two endornaviruses were detected in many genotypes of Mesoamerican origin but rarely in genotypes of Andean origin. The results suggest that these two endornaviruses were introduced into the Mesoamerican modern genotypes during common bean domestication and provide more evidence for the existence of two divergent gene pools of common bean.

Genetic Architecture of Flooding Tolerance in the Dry Bean Middle-American Diversity Panel

Flooding is a devastating abiotic stress that endangers crop production in the twenty-first century. Because of the severe susceptibility of common bean (Phaseolus vulgaris L.) to flooding, an understanding of the genetic architecture and physiological responses of this crop will set the stage for further improvement. However, challenging phenotyping methods hinder a large-scale genetic study of flooding tolerance in common bean and other economically important crops. A greenhouse phenotyping protocol was developed to evaluate the flooding conditions at early stages. The Middle-American diversity panel (n = 272) of common bean was developed to capture most of the diversity exits in North American germplasm. This panel was evaluated for seven traits under both flooded and non-flooded conditions at two early developmental stages. A subset of contrasting genotypes was further evaluated in the field to assess the relationship between greenhouse and field data under flooding condition. A genome-wide association study using ~150 K SNPs was performed to discover genomic regions associated with multiple physiological responses. The results indicate a significant strong correlation (r > 0.77) between greenhouse and field data, highlighting the reliability of greenhouse phenotyping method. Black and small red beans were the least affected by excess water at germination stage. At the seedling stage, pinto and great northern genotypes were the most tolerant. Root weight reduction due to flooding was greatest in pink and small red cultivars. Flooding reduced the chlorophyll content to the greatest extent in the navy bean cultivars compared with other market classes. Races of Durango/Jalisco and Mesoamerica were separated by both genotypic and phenotypic data indicating the potential effect of eco-geographical variations. Furthermore, several loci were identified that potentially represent the antagonistic pleiotropy. The GWAS analysis revealed peaks at Pv08/1.6 Mb and Pv02/41 Mb that are associated with root weight and germination rate, respectively. These regions are syntenic with two QTL reported in soybean (Glycine max L.) that contribute to flooding tolerance, suggesting a conserved evolutionary pathway involved in flooding tolerance for these related legumes.

RNAseq Analysis of Endornavirus-Infected vs. Endornavirus-Free Common Bean (Phaseolus vulgaris) Cultivar Black Turtle Soup

Common bean (Phaseolus vulgaris L.) is the most important grain legume for direct human consumption worldwide and represents a rich source of protein, vitamins, minerals, and fiber (Broughton et al., 2003). The recent sequencing of the common bean genome, together with the availability of genomic and transcriptomic data have provided useful information to common bean breeders that will help in the development of genotypes with desirable characteristics (Schmutz et al., 2014; Vlasova et al., 2016). Endornaviruses are persistent viruses with a non-encapsidated RNA genome that ranges from 9.8 to 17.6 kb, infect plants, fungi, and oomycetes, are transmitted only via gametes, and do not cause apparent symptoms (Stielow et al., 2011; Fukuhara and Gibbs, 2012). Although endornaviruses have been reported in several economically important plant species, little is known about the effect they have on their hosts. One of the major obstacles to study their effect to the host is the lack of a transmission method. In plants, endornaviruses do not move from cell to cell and spread only during cell division. Recently, Khankhum et al. (2015) reported that most common bean genotypes of Mesoamerican origin are double-infected with Phaseolus vulgaris endornavirus 1 (PvEV1) and Phaseolus endornavirus 2 (PvEV2); in contrast, genotypes of Andean origin are often endornavirus-free. Black Turtle Soup (BTS), a cultivar of Mesoamerican origin has been reported to be double-infected by these two endornaviruses (Okada et al., 2013). A BTS endornavirus-free selection (BTS−), obtained from an endornavirus-infected BTS (BTS+) seed lot has been reported by Okada et al. (2013). To establish the bases for future research on the role that endornaviruses play in the common bean plant, and the effect these viruses have on the host gene expression, we conducted RNAseq on two BTS lines: one endornavirus-infected and the other endornavirus-free.

Common Bean Genomics and Its Applications in Breeding Programs

Because of its nutritional value, easiness of cultivation, and cultural preference in many cases, common bean (Phaseolus vulgaris L.) is the most important grain legume in the human diet worldwide. Recent genomic evidence suggest that common bean originated in Central America and confirms the two centers of domestication previously characterized (Mesoamerican and Andean), with well-defined races within each gene pool. Total world production of dry bean from the 10 year period 1961–1970 increased 65 % to 169 million MT in the period 2001–2010. The main challenge now is how to apply these genomic tools into breeding programs for increased efficiency. Applications go from marker-assisted breeding to tracking of F1 crosses, and even DNA fingerprinting, among others. More recently, the development of thousands of single nucleotide polymorphisms (SNP) markers and the completion of the bean genome sequence have opened numerous opportunities for fine mapping and gene characterization. The exploitation of linkage disequilibrium through association mapping allows for rapid identification of important genomic regions associated with traits of economic importance without the need of creating bi-parental populations for this goal. The following sections will describe specific examples of applications of these genomic tools into breeding programs and illustrate some of the possible future directions some of these technologies may follow.

Genetic Analysis of Flooding Tolerance in an Andean Diversity Panel of Dry Bean (Phaseolus vulgaris L.)

Climate change models predict temporal and spatial shifts in precipitation resulting in more frequent incidents of flooding, particularly in regions with poor soil drainage. In these flooding conditions, crop losses are inevitable due to exposure of plants to hypoxia and the spread of root rot diseases. Improving the tolerance of bean cultivars to flooding is crucial to minimize crop losses. In this experiment, we evaluated the phenotypic responses of 277 genotypes from the Andean Diversity Panel to flooding at germination and seedling stages. A randomized complete block design, with a split plot arrangement, was employed for phenotyping germination rate, total weight, shoot weight, root weight, hypocotyl length, SPAD index, adventitious root rate, and survival score. A subset of genotypes (n = 20) were further evaluated under field conditions to assess correlations between field and greenhouse data and to identify the most tolerant genotypes. A genome-wide association study (GWAS) was performed using ~203 K SNP markers to understand the genetic architecture of flooding tolerance in this panel. Survival scores between field and greenhouse data were significantly correlated (r = 0.55, P = 0.01). Subsequently, a subset of the most tolerant and susceptible genotypes were evaluated under pathogenic Pythium spp. pressure. This experiment revealed a potential link between flooding tolerance and Pythium spp. resistance. Several tolerant genotypes were identified that could be used as donor parents in breeding pipelines, especially ADP-429 and ADP-604. Based on the population structure analysis, a subpopulation consisting of 20 genotypes from the Middle American gene pool was detected that also possessed the highest root weight, hypocotyl length, and adventitious root development under flooding conditions. Genomic regions associated with flooding tolerance were identified including a region on Pv08/3.2 Mb, which is associated with germination rate and resides in vicinity of SnRK1.1, a central gene involved in response of plants to hypoxia. Furthermore, a QTL at Pv07/4.7 Mb was detected that controls survival score of seedlings under flooding conditions. The association of these QTL with the survivability traits including germination rate and survival score, indicates that these loci can be used in marker-assisted selection breeding to improve flooding tolerance in the Andean germplasm.