Rapid identification of a candidate gene related to fiber strength using a superior chromosome segment substitution line from Gossypium hirsutum ×Gossypium barbadense via bulked segregant RNA-sequencing

Abstract

Gossypium is the most widely cultivated commercial crop producing natural fiber around the world, and fiber strength principally determined during the secondary wall thickening period is a critical trait for fiber quality. Based on the developed BC 5 F 3:5 CSSLs (chromosome segment substitution lines) from G. hisutum CCRI36 × G. barbadense Hai1, the superior MBI9915 was chosen to construct the secondary segregated population BC 7 F 2 with its recurrent parent CCRI36, which was subjected to Bulk segregant RNA-sequencing (BSR-seq) for rapid identification of candidate genes related to fiber strength. Four fiber-transcriptome libraries were separately constructed and sequenced, including two parents (CCRI36 and MBI9915) and two extreme pools at 20 DPA (days post anathesis). Through multiple comparisons, 3742 DEGs (differentially expressed genes) and 3252 DEGs were separately identified between two parents and between two extreme pools, while 536 DEGs were overlapped between parent and extreme pool groups. A total of 831high-probability SNPs (single nucleotide polymorphism) were identified relevant to fiber strength between two extreme pools through allelic-polymorphism comparison in mRNA sequences, and 18 correlated regions with 1981 annotation genes were finally screened by linkage analysis with SNP-index method, of which including only 12 common genes differentially expressed both between two parents and two pools. Interesting, there was one correlated region consistent with the previous study with the same parents on chromosome A07 with 13-14 Mb, and one common DEG ( Gh_A07G0837 ) in the candidate region was identified in both parents and extreme pools, which has been reported to be involved in fiber strength development through regulating reactive oxygen species (ROS) activity. The reliability of BSR-seq results was validated by the quantitative real-time PCR (qRT-PCR) experiments on 5 DEGs at 20 DPA. This study focuses on bulked segregant analysis of the extreme pools from segregation population developed by superior CSSL and its recurrent parent, indicating that BSR-seq can be efficiently applied on rapid identification for candidate genes related to the significant quantitative traits, which provides valuable contributions for comprehension of fiber strength formation in cotton.