DOI: 10.25338/b8k03w
title: Towards the introgression of PvPdh1 for increased resistance to pod shattering in common bean

description: Some varieties of common bean (Phaseolus vulgaris L.) suffer from pod
 shattering, which can severely reduce yields, especially in arid
 conditions. The PvPdh1 locus on chromosome Pv03 has recently been
 described as a major locus controlling pod shattering in common bean and
 could be used to mitigate pod shattering in the future. Despite this, the
 role of a possible second locus on chromosome Pv08 remains unclear and
 patterns of dominance and epistasis between alleles of these genes have
 not been resolved. This information will be vital for efficient selection
 to decrease pod shattering. Further, the genetic diversity around the
 PvPdh1 gene has not yet been thoroughly explored and there are not yet
 genetic screens that can be used to evaluate pod shattering in segregating
 populations. Here, we have developed a recombinant inbred population to
 determine the roles of genes implicated in pod shattering and evaluate the
 patterns of dominance among the relevant alleles. Our results suggest that
 a PvPdh1 allele reduces pod valve twisting, and its dominance varies by
 phenotyping method. This allele is the only genetic variant that provides
 environmentally stable and widespread resistance to pod shattering in
 Middle American common beans grown for grain. Further analyses identified
 a selective sweep around PvPdh1 with greater nucleotide diversity in
 individuals with the ancestral, shattering-susceptible allele. Finally, we
 developed simple, effective CAPS markers to facilitate the introgression
 of PvPdh1 into new varieties of common bean. These genetic resources will
 be critical for improving the aridity resilience of a major global staple.
description: Three populations were
 evaluated in this study; a biparental population and two diversity panels,
 which represent each of the two domestication events of common bean. The
 biparental population was developed to study two shattering-related QTLs,
 their patterns of dominance, and their interactions. Cultivars ‘Mayflower’
 (ecogeographic race Mesoamerica, Kelly et al. 1989) and ‘Bill Z’ (race
 Durango, Wood et al. 1989) showed total resistance to pod shattering when
 field-grown in Davis in 2017 (n=27, n=19, respectively). These varieties
 were among the most distantly related accessions in the MDP, with neither
 showing any evidence of admixture between ecogeographic races (Moghaddam
 et al. 2016; Parker et al. 2020). Mayflower is a navy bean type (white,
 small-seeded), which possesses a SNP allele on Pv08 that is weakly
 associated with resistance to pod shattering in race Mesoamerica. Bill Z
 is a pinto bean type and has a SNP variant on Pv03 associated with strong
 <i>PvPdh1-</i>mediated shattering resistance common in race
 Durango. The population can therefore be used to determine if a reduction
 in pod shattering was independently selected in each of these
 ecogeographic races. An F<sub>3</sub> population of 138
 individuals was developed by hybridization between these cultivars. Each
 F<sub>3</sub> individual was descended from a distinct
 F<sub>2</sub> plant, and all of the F<sub>2</sub>s
 were the progeny of a single F<sub>1</sub> developed by
 cross-pollinating Mayflower and Bill Z. This 138-member Mayflower x Bill Z
 (MxB) population was used to validate the possible alleles on Pv03 and
 Pv08 and test any patterns of dominance and epistasis between the
 loci. The two
 diversity panels were grown to evaluate the degree of pod shattering
 across diverse accessions of common bean. In 2016, 98 members of major
 market classes in the Andean Diversity Panel (ADP, Cichy et al. 2015) were
 field-grown in Davis, California, to evaluate each variety’s
 susceptibility to pod shattering. In 2017, 278 varieties of the BeanCAP
 Middle American Diversity Panel (MDP, Moghaddam et al. 2016) were
 similarly field-grown in Davis to evaluate pod shattering. At maturity, a
 sample of pods (mean n = 30) was harvested from each variety.
 Mature pods of all
 phenotyped varieties were harvested and then exposed to seven days of
 desiccation at 65°C and a further seven days of re-equilibration to room
 temperature. The desiccation conditions for all varieties were identical
 and desiccation was conducted using the same drying chamber. The
 proportion of pods dehiscing in this treatment was recorded, along with
 the market class of each variety. For evaluation of pod twists in the MxB
 population, all non-shattering pods were fractured by hand and then all
 pods were subjected to the desiccation treatment and re-equilibration
 again. The number of twists was counted for ten pods of each genotype,
 with “1” indicating a complete 360° rotation of the valve. DNA was extracted from young
 trifoliate leaves (approximately 1cm in length) of the greenhouse-grown
 biparental MxB F<sub>3</sub> generation, using a modified CTAB
 protocol (adapted from Allen et al. 2006). DNA was quantified with a
 NanoDrop spectrophotometer and genotyped using the BARCBean6K_3 BeadChip
 (Song et al. 2015), yielding 5398 initial SNPs. SNPs that were missing or
 heterozygous in either parent, or identical between the parents, were
 filtered from further analysis. The remaining SNPs were arranged into a
 linkage map using the ASMap R package (Taylor et al. 2017). SNPs that did
 not map to one of the 11 major linkage groups were removed, leaving 1794
 SNPs for QTL mapping. QTL mapping was conducted using the expectation
 maximization method (Lander and Botstein 1989) in R/qtl (Broman et al.
 2003). Phenotypes for QTL mapping were generated by harvesting all the
 pods from each greenhouse-grown F<sub>3</sub> plant (mean n=27
 pods/plant), then subjecting them to seven days at 65°C and seven further
 days of re-equilibration to room temperature. Pods that had fractured to
 the tip of the beak due to this treatment were counted as shattered, while
 those with no opening or only fissuring along the sutures were considered
 non-shattering. The percentage of pods that shattered in this treatment
 were used for QTL mapping. The maximum LOD score of 1000 randomized
 analyses of the data was used as a significance threshold. To test
 dominance, F<sub>3</sub> individuals were subset by genotype
 at highly significant SNPs, and comparisons were made between groups by
 t-test. Next, the 43 SNPs within 100kb of
 <i>PvPdh1</i> in the MDP data set were analyzed to identify
 patterns of selection and diversity around the gene. To simplify and
 visualize the data, principal component analysis was performed on the SNPs
 using R. Sequence variation was converted to integer values and the
 imputePCA() function of the missMDA package was used to impute missing
 data (Josse and Husson 2016). The genotype data were also sorted to
 identify unique haplotypes within the populations. The degree of
 similarity between the PCA and haplotype diversity was then compared.
 Individuals with missing data for SNPs distinguishing the haplotypes or
 haplotype clusters were not shown in plots, and not numbered in plots as
 they could not be unambiguously placed within any haplotype
 group. The SNPs
 tightly linked to <i>PvPdh1</i> in the MDP data set were then
 screened for other positions that could be useful for conversion to
 additional CAPS markers. The SNP closest to <i>PvPdh1 </i>in
 this data set, at Pv03 position 49,132,438 (accession G19833 genome v2.1),
 is distinguishable by <i>Eco</i>RI and is highly correlated
 with pod shattering. Unlike the <i>Taq</i>II-based CAPS
 marker, the allele cleaved by <i>Eco</i>RI is the
 shattering-resistant variant, reducing the risk of falsely identifying a
 susceptible individual as resistant due to technical errors in digestion.
 The SNP distinguished by <i>Eco</i>RI is separated from the
 <i>PvPdh1 </i>causal polymorphism by less than 7kb.