Phenotypic data genetic analysis
The hilum-eye types in parents, F1, and RILs are presented in Fig.1 and the segregation data in different classes in Table 1. As evident, the hilum-eye type of seeds in GEC was ‘Small eye’; in IT98K-476-8 was ‘Watson eye type’; and in F1 was self-colored, indicating a complementary gene action. The segregation ratio of the seed hilum-eye type in the RILs was 23 Self: 52 Watson: 27 Small: 44 Ring: 24 Large hilum-eye type and all had brown color as expected since both parents had brown color. The data fitted well to a three gene model with two alleles (W-w, R-r, and S-s) giving different phenotypes based on their interactions. Thus, all the plants with dominant homozygotes at all three loci (WWSSRR) were Self hilum-eye type; the genotypes WWSSrr, WWssRR, and WWssrr exhibited Watson hilum-eye type; the genotypes wwSSRR exhibited Small hilum-eye type; the genotype wwssRR exhibited Large hilum-eye type, and the genotype wwssrr exhibited Ring hilum-eye type (Table 1). All the three genes were inherited independently, and the observed data fitted well to the expected segregation as confirmed by the Chi-square test. The total calculated Chi-square value of 3.381 is less than Chi-square table value of 9.49 for 4 degrees of freedom at 0.05 alpha level (95% probability), thus confirming the three gene segregation model (Table 1).
We mapped QTLs for the hilum-eye type of seeds. We identified three major QTLs for hilum-eye phenotype (Fig.2, Table 2), defined qHilum7.1, qHilum9.1,and qHilum10.1. SNP markers, SNP6683 and SNP32139, with a LOD of 37.4, flanked qHilum7.1 and its LOD score peak was positioned at the 57 cM position, within the physical region of 18.9 to 20.2 Mbp on Chr7. This QTL explained the highest phenotypic variation (38.43%) of the cowpea seed hilum-eye type. Parent GEC, which had Small-type hilum-eye type, donated the alleles for hilum-eye color in this QTL. qHilum9.1 was mapped at the 52 cM position, within the physical region of 30.5 to 30.8 Mbp on Chr9 and flanked by SNP3899 and SNP26097, with a LOD score of 18.1. This QTL explained 13.6% of the seed hilum-eye type phenotype. Parent IT98K-476-8 contributed the alleles for hilum-eye color for this QTL (Table 2). qHilum10.1 was mapped at the 5 cM position, within the physical region of 38.4 to 38.9 Mbp on Chr10, with a LOD score of 28.5. It was flanked by SNP32261 and SNP17347 and explained 25.2% of the seed hilum-eye type phenotype. Parent IT98K-476-8, with Watson-type hilum-eye color, contributed the alleles of this QTL.
Determination of the seed hilum-eye type genes and their corresponding QTLs
Both genetic analysis and QTL mapping consistently showed that three major genes: R, W, and S, or three QTLs: qHilum 7.1, qHilum 9.1, and qHilum 10.1, controlled the seed hilum-eye type variation in the RIL mapping population. Nevertheless, what was the relationship between the three genes and three QTLs? To determine their correspondence, we extracted the genotypes of the SNPs immediately flanking each QTL and their corresponding seed hilum-eye type phenotypes, and conducted association analysis between the QTL flanking SNPs and seed hilum-eye types by Chi-square Test. We found that both SNP 6683 and SNP 32139 flanking qHilum 7.1 were associated with the Ring hilum-eye type seeds (P < 0.0001), while none of the SNPs flanking either qHilum 9.1 or qHilum 10.1 was associated with the Ring hilum-eye type seeds (P > 0.05). Similarly, both SNP 3899 and SNP 26097 were associated with the Watson hilum-eye type seeds (P < 0.0001), and both SNP 32261 and SNP 17347 were associated with the Small hilum-eye type seeds (P < 0.0001). These results indicated that qHilum7.1 corresponded to the R gene; qHilum9.1 corresponded to the W gene; and qHilum10.1 corresponded to the S gene (Table 2).
Physical location of the SNP markers flanking the mapped three QTLs was identified using BLAST tool available at phytozome.jgi.doe.gov/pz/portal.html using IT98K-499-35 reference genome (Supp. Table S1). Genes within the mapped QTLs qHilum7.1 (Supp. Table S2), qHilum9.1 (Supp. Table S3),and qHilum10.1 (Supp. Table S4) were mined using tools available at https://mines.legumeinfo.org/cowpeamine/begin.do (Lonardi et al. 2019). Number of genes discovered under QTL qHilum7.1, qHilum9.1, qHilum10.1 were 53, 23, and 43, respectively. QTL qHilum10.1 harbors eight genes, close to SNP32261, produce iron binding protein and two other genes in the QTL – Vigun10g165300 and Vigun10g165400, are myb transcription factors (chromatin binding protein) may play role in iron deficiency resistant (Supp. Table S2, S3, and S4).