Unravelling cucumber resistance to several viruses via genome-wide association studies highlighted resistance hotspots and new QTLs

Abstract

The mapping and introduction of sustainable resistance to viruses in crops is a major challenge in modern breeding, especially regarding vegetables. We hence assembled a panel of cucumber elite lines and landraces from different horticultural groups for testing with six virus species. We mapped 18 quantitative trait loci (QTL) with a multiloci genome wide association studies (GWAS), some of which have already been described in the literature. We detected two resistance hotspots, one on chromosome 5 for resistance to the cucumber mosaic virus (CMV), cucumber vein yellowing virus (CVYV), cucumber green mottle mosaic virus (CGMMV) and watermelon mosaic virus (WMV), colocalizing with the RDR1 gene, and another on chromosome 6 for resistance to the zucchini yellowing mosaic virus (ZYMV) and papaya ringspot virus (PRSV) close to the putative VPS4 gene location. We observed clear structuring of resistance among horticultural groups due to plant virus coevolution and modern breeding which have impacted linkage disequilibrium (LD) in resistance QTLs. The inclusion of genetic structure in GWAS models enhanced the GWAS accuracy in this study. The dissection of resistance hotspots by local LD and haplotype construction helped gain insight into the panel's resistance introduction history. ZYMV and CMV resistance were both introduced from different donors in the panel, resulting in multiple resistant haplotypes at same locus for ZYMV, and in multiple resistant QTLs for CMV.

Figure 1

Organization of the 256 cucumber accessions across nine genetic groups A. Each color represents the horticultural group (when known) of the accession. B. Barplot representing the percentage attribution of 256 cucumber accessions to nine genetic groups. Accessions were rarely attributed 100% to one genetic group and accessions considered as admixed resulted from crosses between breeding programs. Values were extracted with the snmf R package. Genetic groups were consistent with the horticultural groups. C. Boxplot representing the phenotypic variance in the diversity panel for each virus. Accessions were assigned to a genetic group when the contribution of this group was above 0.6, but if no group reached this threshold the accession was considered as admixed (in black). The number of accessions per group is indicated in brackets.

Figure 2

The phenotypic variance distribution among i/ fixed effects: both the genetic structure (grey) and top SNPs from previous MLMM steps (blue) ii/ random effect (genetic, green) and iii/ error (red). The white dashed line indicates significant SNPs using Bonferroni correction. MLMM for CGMMV and WMV respectively stopped at the 14th and 18th steps because no further phenotypic variance explained by the genetic remained after the previous step.

Figure 3

A Miami plot consisting of two mirrored Manhattan plots corresponding to two different GWAS. The two significance thresholds are represented by the two dashed lines. These Miami plots were built from p-values collected from the first KQ9 MLMM step (without marker correction). The 18 SNPs selected in one of the MLMM steps were represented by a red triangle, their positions as well as the step in which they were selected are labelled. A. CMV (pink) and CVYV (blue) shared a common peak and a common top SNP on chromosome 5, CMV had two other isolated QTLs on chromosomes 2 and 6. The peak on chr7 was finally not selected during the MLMM steps as it disappeared as it explained the same phenotypic variance as the peak on chr6 B. CGMMV (yellow) and WMV (purple) shared the same peak on chromosome 5 but had different top SNPs. CGMMV presented another QTL on chromosome 2 when tested independently from the top SNP on chromosome 5, even with the lowest threshold. C. ZYMV (grey) and PRSV (green) shared two common peaks, i.e. a large one on chromosome 6, with the same top SNPs, and a small one on chromosome 1 that was not selected in further MLMM steps.

Figure 4

The hotspot on chr 5 A. Representation of the LD between the WMV top SNP and all other SNPs at the hotspot on chr5. The X-axis represents the physical distance from the WMV top SNP, while the Y-axis represents the r² corrected by the structure between SNPs and the top SNP. Each dot represents an SNP which is colored according the p-value in the first step of MLMM GWAS for WMV. Dots are grey when the SNP was not tested by GWAS due to missing data. The dot shape represents the top SNP of the QTL or top SNPs from other resistance QTLs. The QTL interval is delineated by the red dashed line. B. Genes located in the QTL, candidate genes in red (at least one SNP in LD with the top SNP in a coding area), potential candidate genes in orange (at least one SNP in LD with the top SNP in a non-coding area), with other genes in grey or blue. The first line represented these categories for broad spectrum resistance to CMV and CVYV and below for broad-spectrum resistance to CGMMV and WMV.

Figure 5

Genetic organization of the QTL_CMVCH2 (26 793 SNPs between 7.7 Mb and 9.6 Mb) in relation with the genetic structure and level of resistance A. Heatmap of the local kinship B. Genetic group of accessions (see color code Figure 1). C. Resistance level to CMV 1-resistant, 9-susceptible. D. Haplotype of each accession. Each SNP is colored according to its allelic state: in blue when the SNP is homozygous as the reference genome CCL, purple for alternative homozygous, pink for heterozygous. The red cross represents the location of the top SNP.

Figure 6

Genetic organization of the hotspot on chr 6 (25 825 SNPs between 9.4 Mb and 12.8 Mb) in relation with the genetic structure and level of resistance A. Heatmap of the local kinship B. Genetic group of accessions (see color code Figure 1). C. Resistance level to ZYMV 1-resistant, 9-susceptible. D. Haplotype of each accession. . Each SNP is colored regarding its allelic state: in blue when the SNP is homozygous as the reference genome CCL, purple for alternative homozygous, pink for heterozygous. The red cross represents the location of the top SNP.