Mapping Cucumber Vein Yellowing Virus Resistance in Cucumber (Cucumis sativus L.) by Using BSA-seq Analysis

Abstract

Cucumber vein yellowing virus (CVYV) causes severe yield losses in cucurbit crops across Mediterranean countries. The control of this virus is based on cultural practices to prevent the presence of its vector (Bemisia tabaci) and breeding for natural resistance, which requires the identification of the loci involved and the development of molecular markers for linkage analysis. In this work, we mapped a monogenic locus for resistance to CVYV in cucumber by using a Bulked Segregant Analysis (BSA) strategy coupled with whole-genome resequencing. We phenotyped 135 F₃ families from a segregating population between a susceptible pickling cucumber and a resistant Long Dutch type cucumber for CVYV resistance. Phenotypic analysis determined the monogenic and incomplete dominance inheritance of the resistance. We named the locus CsCvy-1. For mapping this locus, 15 resistant and 15 susceptible homozygous F₂ individuals were selected for whole genome resequencing. By using a customized bioinformatics pipeline, we identified a unique region in chromosome 5 associated to resistance to CVYV, explaining more than 80% of the variability. The resequencing data provided us with additional SNP markers to decrease the interval of CsCvy-1 to 625 kb, containing 24 annotated genes. Markers flanking CsCvy-1 in a 5.3 cM interval were developed for marker-assisted selection (MAS) in breeding programs and will be useful for the identification of the target gene in future studies.

Keywords: BSA-seq; Cucumber vein yellowing virus; breeding; cucumber; mapping; marker-assisted selection; resistance.

Figure 1

Phenotyping plants of the susceptible (P_S) and resistant (P_R) parental lines, and their F₁, for CVYV susceptibility. Symptoms were scored and virus accumulation was measured at 9, 16, 23 and 30 days post-inoculation (dpi) (A) Symptoms in plants at 9 dpi. Symptoms could be assigned unequivocally to one of the following categories: (0) No symptoms; (1) mild chlorotic mottling in young but fully expanded leaves in interveinal petiole-proximal leaf areas; (2) similar to (1) plus vein yellowing evident in fully expanded leaves and incipient in young developing leaves; (3) obvious vein yellowing in all leaves, including young developing leaves, chlorotic mosaics in fully expanded leaves and overall plant growth reduction. (B) Virus accumulation was measured by quantitative dot-blot hybridization on total plant RNA extracts from basal, intermediate and apical leaves, as indicated for each graph. Three leaf discs (8 mm) were taken per leaf sampled. Virus accumulation in F₁ plants was intermediate between the susceptible and resistant parental lines in basal and intermediate leaves at 16, 23 and 30 dpi, respectively; an asterisk marks statistically significant differences in Kruskal-Wallis tests (P < 0.027). A minimum of 9 plants were used per treatment. Symptom category (as in (A)) is indicated for each line on the right side of each graph for each time period after inoculation.

Figure 2

(A) Genetic linkage of SNP markers and the resistance locus CsCvy-1 in Chr05. Genetic distance is expressed as centiMorgans (cM). The CsCvy-1 gene is flanked by two markers in an interval of 5.3 cM. (B) Major QTL detected on Chr05 corresponding to CsCvy-1 locus.

Figure 3

SNP-index and Δ(SNP-index) distribution (blue line). SNP-index for R-bulk (A) and S-bulk (B) was calculated using sliding windows of 4.7 Mbp in length with a step measuring 10 kb. The corresponding Δ(SNP-index) (C) was calculated as the difference SNP-index R-bulk – SNP-index S-bulk. Regions of statistical significance are detected as those that surpass the threshold of 0.95 (orange line) or 0.99 (green line) confidence intervals.

Figure 4

Scheme of fine mapping of the CsCvy-1 locus. (A) DeltaSNP graph on Chr05 with the statistical confidence interval of 0.95 (orange line) or 0.99 (green line) (B) Physical map of Chr05 with all the markers used for mapping the CsCvy-1 locus: preliminary mapping (black), flanking markers (magenta), fine mapping markers (blue). Distance is expressed in Mb on the bottom of the bar. (C) Genotyping result of recombinant individuals with the markers covering CsCvy-1 locus. Alleles are represented in three colors: black for homozygous P_R allele, dark grey for heterozygous P_R/P_S, and light grey for homozygous P_S allele.