Working group session:
Structural Genomics
Presentation type:
poster
Authors:
Thyssen, Gregory; Naoumkina, Marina; Fang, David
Presenter:
Fang, David
Correspondent:
Fang, David
Abstract:
Next generation sequencing technologies offer new ways to identify the genetic mechanisms that underlie mutant phenotypes. The release of a reference diploid Gossypium ramondii (D5) genome and bioinformatics tools to sort tetraploid reads into subgenomes has finally brought cotton into the genomics era. We used multiple high throughput sequencing approaches to identify the relevant region of reference sequence and identify SNPs near the Ligon-lintless short fiber Li2 gene. First, we performed RNAseq on 8-DPA fiber cells from Li2 and its nearly isogenic line (NIL) DP5690. We aligned reads to the reference D5 genome, sorted the reads into At and Dt subgenomes with PolyCat and called SNPs with InterSNP. We further filtered these SNPs and then identified SNPs that would result in a non-synonymous substitution to amino acid sequences of annotated genes. This final step allowed us to identify a 1-Mb region with 24 non-synonymous SNPs, representing the introgressed region that differentiates Li2 from its NIL and contains the causative mutation. Since these SNPs were confined to genes that were expressed in both mutant and wild type fiber, we also sequenced total DNA from pools of segregating F2 plants, in a super bulked segregant analysis sequencing (sBSAseq) approach. The sBSAseq predicted 82 non-synonymous SNPs among 3400 SNPs in a 3-Mb region that includes the RNAseq peak. We used all types of SNPs identified in our sequencing data to design subgenomic specific SNP markers which we tested in a F2 population of 1700 individuals to construct a genetic map. Our resulting genetic interval contains only one gene, which is significantly under-expressed in developing Li2 fiber cells.