Submitted to: Theoretical and Applied Genetics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/25/2014
Publication Date: 10/1/2014
Citation: Thyssen, G.N., Fang, D.D., Turley, R.B., Florane, C.B., Li, P., Naoumkina, M.A. 2014. Next generation genetic mapping of the Ligon-lintless-2 (Li2) locus in upland cotton (Gossypium hirsutum L.). Theoretical and Applied Genetics. 127:2183-2192.
Interpretive Summary: The identification of the genes that control fiber elongation and development is important to cotton breeders and will enable the development of superior cotton varieties. Although the genome of cultivated cotton (Gossypium hirsutum) has not yet been sequenced, we were able to use the genome of a closely related plant (Gossypium raimondii) and next generation sequencing technologies to identify, in a streamlined fashion, a candidate gene that controls fiber elongation. First, we sequenced RNA from elongating fiber cells from the short fiber mutant Ligon-lintless 2 (Li2) and its wild-type relative DP5690. We identified differences in the DNA between Li2 and DP5690 that would result in differences in proteins. We found a cluster of such mutations that we confirmed by two additional approaches: deep sequencing of DNA and traditional genetic mapping using a new type of genetic marker that we developed for this study. Our resulting genetic interval contains only one gene, an aquaporin, which is highly expressed in wild-type fibers and is significantly under-expressed in elongating Li2 fiber cells. The discovery of this gene will be immediately important to cotton breeders, and our improved methods will be useful to researchers who study the genetics of crop plants.
Technical Abstract: Next generation sequencing offers new ways to identify the genetic mechanisms that underlie mutant phenotypes. The release of a reference diploid Gossypium raimondii (D5) genome and bioinformatics tools to sort tetraploid reads into subgenomes has brought cotton genetic mapping into the genomics era. We used multiple high throughput sequencing approaches to identify the relevant region of reference sequence and identify single nucleotide polymorphisms (SNPs) near the short fiber mutant Ligon-lintless 2 (Li2) gene locus. First, we performed RNAseq on 8 days post anthesis (DPA) fiber cells from the Li2 mutant and its wild-type near isogenic line (NIL) Gossypium hirsutum cv. DP5690. We aligned sequence reads to the D5 genome, sorted the reads into A and D subgenomes with PolyCat and called SNPs with InterSNP. We then identified SNPs that would result in non-synonymous substitutions 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. Next, we sequenced total DNA from pools of F2 plants, using a super bulked segregant analysis sequencing (sBSAseq) approach. The sBSAseq predicted 82 non-synonymous SNPs among 3494 SNPs in a 3-Mb region that includes the RNAseq peak. We designed subgenome specific SNP markers and tested them in an F2 population of 1733 individuals to construct a genetic map. Our resulting genetic interval contains only one gene, an aquaporin, which is highly expressed in wild-type fibers and is significantly under-expressed in elongating Li2 fiber cells.