Field Screen and Genotyping of Phaseolus vulgaris against Two Begomoviruses in Georgia, USA

Abstract

The production and quality of Phaseolus vulgaris (snap bean) have been negatively impacted by leaf crumple disease caused by two whitefly-transmitted begomoviruses: cucurbit leaf crumple virus (CuLCrV) and sida golden mosaic Florida virus (SiGMFV), which often appear as a mixed infection in Georgia. Host resistance is the most economical management strategy against whitefly-transmitted viruses. Currently, information is not available with respect to resistance to these two viruses in commercial cultivars. In two field seasons (2018 and 2019), we screened Phaseolus spp. genotypes (n = 84 in 2018; n = 80 in 2019; most of the genotypes were common in both years with a few exceptions) for resistance against CuLCrV and/or SiGMFV. We also included two commonly grown Lima bean (Phaseolus lunatus) varieties in our field screening. Twenty Phaseolus spp. genotypes with high to moderate-levels of resistance (disease severity ranging from 5%-50%) to CuLCrV and/or SiGMFV were identified. Twenty-one Phaseolus spp. genotypes were found to be highly susceptible with a disease severity of ≥66%. Furthermore, based on the greenhouse evaluation with two genotypes-each (two susceptible and two resistant; identified in field screen) exposed to viruliferous whiteflies infected with CuLCrV and SiGMFV, we observed that the susceptible genotypes accumulated higher copy numbers of both viruses and displayed severe crumple severity compared to the resistant genotypes, indicating that resistance might potentially be against the virus complex rather than against the whiteflies. Adult whitefly counts differed significantly among Phaseolus genotypes in both years. The whole genome of these Phaseolus spp. [snap bean (n = 82); Lima bean (n = 2)] genotypes was sequenced and genetic variability among them was identified. Over 900 giga-base (Gb) of filtered data were generated and >88% of the resulting data were mapped to the reference genome, and SNP and Indel variants in Phaseolus spp. genotypes were obtained. A total of 645,729 SNPs and 68,713 Indels, including 30,169 insertions and 38,543 deletions, were identified, which were distributed in 11 chromosomes with chromosome 02 harboring the maximum number of variants. This phenotypic and genotypic information will be helpful in genome-wide association studies that will aid in identifying the genetic basis of resistance to these begomoviruses in Phaseolus spp.

Keywords: cucurbit leaf crumple virus; lima beans; sida golden mosaic Florida virus; snap beans; whitefly.

Figure 1

Symptoms of leaf crumple on P. vulgaris plants under field conditions. A non-infected P. vulgaris (A) and an infected plant (B) with severe stunting, leaf crumpling and distortion, and leaf mosaic symptoms.

Figure 2

Accumulation of CuLCrV and SiGMFV and leaf crumple severity in P. vulgaris genotypes in greenhouse (susceptible vs. resistant; identified in field screen). Data points represent the mean copy numbers of SiGMFV (A) and CuLCrV (B) in two susceptible (Top crop and Gold mine) and two resistant genotypes (Prevail and Sybaris) in two independent experiments. Bars indicate standard error of the mean. Means with similar letter are not significantly different according to least significant difference (LSD) at p = 0.05 level. Response of two susceptible [Top crop (C) and Gold mine (D)] and two resistant [Prevail (E) and Sybaris (F)] genotypes to mixed infection (CuLCrV and SiGMFV).

Figure 3

Mean number of whitefly adults counted on different snap bean genotypes in 2018 (A) and 2019 (B). Bars with standard errors represent the average number of whiteflies present on each plant of the genotype. Whiteflies adults were enumerated on the three top leaves of the plant. Y-axis is shown in a logarithmic scale. Bars with different letters are significantly different from one another (p < 0.05).

Figure 4

Circos plot to show the density (no. of variants/kb) of SNPs, insertions and deletions in bins of 100 Kb on 11 chromosomes of Phaseolus vulgaris. The outermost track (A) denotes the physical distance on each of the eleven chromosomes at 5 Mb break-point. Track (B) denotes the chromosome numbers. Track (C) shows the area plot of SNP density. Track (D) represent the line plot of insertion density and Track E represent the scatter plot of deletion density.

Figure 5

Genome-wide distribution of SNPs and InDels. Chromosome-wise distribution of SNPs and InDels (A). Chromosome-wise distribution of indels ranging from 2 bp to >50 bp (B). Chromosome-wise distribution of indels ranging from 2 bp to >50 bp (C).

Figure 6

Annotation of single nucleotide polymorphisms (SNPs) and InDels. Distribution of SNPs (A) and InDels (B) in different regions of genome. SNPs in exonic regions are classified into synonymous, non-synonymous, stop-loss and stop-gain categories (A). Indels in exonic regions are classified into frameshift deletion, frameshift insertion, nonframeshift deletion, and stop-gain and stop-loss categories (B).