Identification of genes related to salt stress tolerance using intron-length polymorphic markers, association mapping and virus-induced gene silencing in cotton

Intron length polymorphisms (ILPs), a type of gene-based functional marker, could themselves be related to the particular traits. Here, we developed a genome-wide cotton ILPs based on orthologs annotation from two sequenced diploid species, A-genome Gossypium arboreum and D-genome G. raimondii. We identified 10,180 putative ILP markers from 5,021 orthologous genes. Among these, 535 ILP markers from 9 gene families related to stress were selected for experimental verification. Polymorphic rates were 72.71% between G. arboreum and G. raimondii and 36.45% between G. hirsutum acc. TM-1 and G. barbadense cv. Hai7124. Furthermore, 14 polymorphic ILP markers were detected in 264 G. hirsutum accessions. Coupled with previous simple sequence repeats (SSRs) evaluations and salt tolerance assays from the same individuals, we found a total of 25 marker-trait associations involved in nine ILPs. The nine genes, temporally named as C1 to C9, showed the various expressions in different organs and tissues, and five genes (C3, C4, C5, C7 and C9) were significantly upregulated after salt treatment. We verified that the five genes play important roles in salt tolerance. Particularly, silencing of C4 (encodes WRKY DNA-binding protein) and C9 (encodes Mitogen-activated protein kinase) can significantly enhance cotton susceptibility to salt stress.