Phenotypic evaluation
After 28 days of inoculation, all plants of the parent Y4 and the four differential cultivars, including Litter Marvel, Darkskin Perfection, New Era, and New Season, displayed wilt and dead. Conversely, each seedling of the parent SJ1 and the three differential cultivars, including WSU 23, WSU 28, and WSU 31 remained viable and showed no discernible disease symptoms (Fig. 1A). Resistance reactions of seven differential cultivars confirmed race 5 identity of Fop isolate PF22b.
Within the combined mapping population of 589 F2 individuals, a total of 445 individuals were resistant (R) and 144 were susceptible (S), and the observed segregation ratio of R:S fit the expected 3:1 ratio (χ2 = 0.10, p = 0.76) (Table 1). For each F2 populations, the observed segregation ratio was consisted with the expected 3:1 ratio as well (Table 1). The results indicated Fop resistance in SJ1 was controlled by a single dominant gene, and named FwS1.
Furthermore, the DW4 showed resistance to PF22b with symptoms similar to SJ1, while Y3, Y25, DDR11 was susceptible with the same symptoms as Y4 (Fig. 1B-D). A total of 63 and 53 F2 individuals were identified as resistant, and 14 and 20 as susceptible in the SJ1 × Y3 and SJ1 × Y25 population, respectively (Table S1, Table S2), while in the DDR11 × D4 population, 51 individuals were resistant and 19 were susceptible (Table S3). The observed segregation ratio of R:S in the three populations were also the expected 3:1 ratio. For 200 pea accessions, 134 accessions exhibited high resistance or resistance to isolate PF22b, 29 accessions displayed intermediate and another 37 accessions were susceptible or high susceptible, and a collection of 155 resistant and 55 susceptible individual plants were obtained from the identified pea accessions and differential cultivars for subsequent haplotype analysis (Table S4).
Reads mapping and BSA-seq analysis
Leveraging the Illumina platform, approximately 45.29, 45.72, 95.63, and 95.36 Gb clean base data were generated for the Y4, SJ1, Susceptible-bulk, and Resistant-bulk, respectively (Table 2). Upon aligning these clean base data to the reference genome Caméor v1a, 300 million, 312 million, 611 million, and 635 million paired-end 150-bp clean reads were obtained from the Y4, SJ1, Susceptible-bulk, and Resistant-bulk, resulting in mean depths of 13.89×, 14.03×, 26.35×, and 26.81×, and the 4× genome coverage of 74.75%, 74.67%, 81.25%, and 81.54%, respectively (Table 2).
Table 2
Whole-genome resequencing based on Illumina sequencing of parents and bulks
Genotype sample
|
Clean base (Gb)
|
Number of clean reads
|
Mapping rate (%)
|
Mean depth (×)
|
4 × genome coverage rate (%)
|
Y4
|
45.29
|
300,171,969
|
99.42
|
13.89
|
74.75
|
Shijiadacaiwan 1
|
45.72
|
302,904,242
|
99.38
|
14.03
|
74.67
|
Susceptible-bulk
|
95.63
|
611,740,352
|
99.46
|
26.35
|
81.25
|
Resistant-bulk
|
95.36
|
635,739,658
|
99.61
|
26.81
|
81.54
|
After mapping reads and filtering SNPs and InDels, a total of 4,119,468 reliable SNPs and InDels were identified across the seven chromosomes (Table S5). In this study, the InDels were treated as SNPs for calculating and analyzing the SNP-index. The analysis of SNP-index and ΔSNP-index of each SNPs in the Resistant-bulk and Susceptible-bulk revealed that a 10.4 Mb genomic region (1,169,564 − 11,563,817) at the top of chr6LG2 was significantly associated with pea resistance to Fop (Fig. 2A). The SNP-index at the 10.4 Mb genomic region in Resistant-bulk exhibited a considerably higher value, while it was notably lower in Susceptible-bulk, and the ΔSNP-index was significantly higher than the random segregation scenario (Fig. 2A). Thus, this genomic region is preliminary designated as the candidate region for the gene FwS1.
Validation of candidate region and fine mapping of FwS1
In order to validate the precision of the 10.4 Mb candidate genomic region harboring FwS1, as pinpointed through WGRS and BSA-seq analysis, and to refine the target region, 23 candidate SNPs in this interval to were chosen to design KASP markers for sequential genotyping F2 populations (Table S7). A total of 17 tested KASP markers were firstly genotyped 68 individuals of Y4 × SJ1 - A F2 population (Fig. 2B, Fig S1, Fig S2A). Nine markers A015910, A015911, A016031, A015982, A016180, A016182, A016183, A015912, and A015985 displayed consistent linkage to FwS1, and the interval harboring FwS1 was narrowed down to a 1.63 Mb region bordered by markers A015910 and A015985 (Fig. 2B).
To furtherly reduce the candidate region of the FwS1 gene, a larger segregating population composed of 589 F2 individuals was genotyped using nine aforementioned full linkage markers, and the FwS1 was fine-mapped to a 91.4 kb region flanked by markers A015982 and A015912, with complete co-segregation with markers A016180, A016182, and A016183, revealing the absence of recombination events (Fig. 2C). The 91.4 kb region contains 23 genes according to the annotation of Caméor reference genome (Fig. 2D). For confirming putative genes, 10 additional KASP markers were developed using the non-candidate SNPs near the co-segregating markers (Table S6, Table S7), and six new KASP markers, A016299, A016557, A016558, A016559, A016561, and A016563, linked to FwS1 were employed to refine the candidate region (Fig. S1). The 589 F2 individuals could be classified into 10 major haplotypes by the 11 markers (Fig. 2E). The individuals containing SNPs corresponding to markers A016180, A016182, A016183, A016561, and A016563 consistently exhibited resistance to Fop, while individuals lacking these five SNPs displayed a susceptible phenotype (Fig. 2E). This result suggested the gene FwS1 was situated between markers A016299 and A015912, co-segregating with markers A016180, A016182, A016183, A016561, and A016563.
Haplotype analyses of FwS1
In order to ascertain SNPs associated with the resistance in the gene FwS1 and discover pea accessions containing the FwS1 unique haplotype, the eight co-segregated or linked KASP markers with the gene FwS1 were employed to detect the individuals of 207 pea accessions with a definite resistance phenotype (Fig. S2B). Twelve haplotypes were identified among 207 accessions, and a total of 88 accessions carrying marker A016180 displayed a resistant phenotype, whereas 55 accessions lacking marker A016180 exhibited a susceptible phenotype (Fig. 2F, Table S4). Furthermore, 64 accessions lacking marker A016180 remained a resistant phenotype, suggesting the presence of other Fop resistant genes in them (Fig. 2F, Table S4). The results showed that the pea accessions with resistant phenotype and carrying SNP marker A016180 genotype of “T/T” or “T/C” could harbor the gene FwS1, while the accessions with susceptible or resistant genotype with “C/C” SNP genotype were lack of Fop resistance gene or possess other resistance genes. Some accessions containing other seven makers exhibited resistance or susceptible to Fop, suggesting that these SNPs were unrelated to the resistance gene FwS1.
Consequently, only the SNP corresponding to KASP marker A016180 displayed exclusive correlation with the resistance gene FwS1. In accordance with the Caméor reference genome, the SNP, positioned at 2,600,848 bp in chr6LG2 or at 721 bp in Psat6g003960, was a “T/C” substitution that caused the amino acid change from Aspartic (Asp) to Asparagine (Asn) (Fig. 2G, Fig. 3). The annotation of Caméor reference genome suggested that Psat6g003960 was a potential disease resistance gene containing an NB-ARC domain.
Specificity validation of the marker A016180 for FwS1
The Y3 and Y25 was selected from SJ1 and shared a similar genetic background with SJ1 and Y4, while DW4 and DDR11 were dry pea cultivars having large genetic difference with SJ1. Haplotype analysis showed Y3, Y25 and DW4 could carry the gene FwS1. In order to demonstrate the marker A016180 is specific for the gene FwS1, three F2 populations derived from the crosses of SJ1 × Y3, SJ1 × Y25, and DDR11 × DW 4 were employed for mapping Fop resistance gene. As expected, co-segregated markers with the crosses of SJ1 × Y4, including the marker A016180, were also co-segregated with the crosses of SJ1 × Y3, SJ1 × Y25, and DDR11 × DW 4, respectively (Fig. 4A-C). The results also indicated that the Fop resistance gene in Y3, Y25 and DW4 was the gene FwS1. The marker A016180 could efficiently and accurately identify the individuals harboring the FwS1 gene within the three populations. The genotype of the resistant individuals was “T/T” or “T/C”, and the susceptible individuals was “C/C” (Table S1, S2, S3). This finding further confirmed the marker A016180 was specific for identification of the gene FwS1 in different genetic background.
Integrating the findings of haplotype analysis and mapping Fop resistance gene in the crosses of SJ1 × Y3, SJ1 × Y25, and DDR11 × DW 4, the KASP marker A016180 could be used as a diagnostic marker for precise identification of the gene FwS1 in diverse pea populations and accessions and MAS for resistance breeding.
The association between the resistance gene FwS1 and Fwf
The candidate gene Psat6g003960 of FwS1 is situated within the potential interval of gene Fwf (Okubara et al. 2002). To test the relationship between gene FwS1 and Fwf, the markers utilized for constructing the genetic linkage map of Fwf were used to map the gene FwS1 with the SJ1 × Y4, SJ1 × Y3, SJ1 × Y25, and DDR11 × DW4. However, none of the constructing markers exhibited polymorphisms across these populations.
Based on the Pulse Crop Database (https://www.pulsedb.org/), the adjacent markers of these constructing markers in other genetic linkage map were identified and employed for screening across the aforementioned four populations. It is found that a gene marker RNApol2, closely linked to the constructing marker T03_650 of Fwf (0.8 cM) in a new pea consensus map (Bordat et al. 2011), exhibited polymorphism in the F2 population derived from the cross of DDR11 × DW 4 and was mapped to the gene FwS1 region with a distance of 0.7 cM (Fig. 4). Therefore, we speculated that the genes FwS1 and Fwf were closely linked or identical. Subsequently, the FwS1 candidate gene Psat6g003960 from line 74SN3B, carrying Fwf, was cloned and compared with that from SJ1. The Psat6g003960 sequence of 74SN3B shared 100% homology with that of SJ1 (Fig. 3), which suggested that the FwS1 and Fwf may represent the same Fusarium wilt resistance gene. However, further confirmation through additional allelic tests is warranted.