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Next generation genetic mapping of the Ligon-lintless-2 (Li 2) locus in upland cotton (Gossypium hirsutum L.)

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Abstract

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Mapping-by-sequencing and novel subgenome-specific SNP markers were used to fine map the Ligon-lintless 2 ( Li 2 ) short-fiber gene in tetraploid cotton. These methodologies will accelerate gene identification in polyploid species.

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 (Li 2) gene locus. First, we performed RNAseq on 8-day post-anthesis (DPA) fiber cells from the Li 2 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 step allowed us to identify a 1-Mb region with 24 non-synonymous SNPs, representing the introgressed region that differentiates Li 2 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 3,494 SNPs in a 3-Mb region that includes the region identified by RNAseq. We designed subgenome-specific SNP markers and tested them in an F2 population of 1,733 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 Li 2 fiber cells.

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Acknowledgments

This project was financially supported by the USDA-ARS CRIS project # 6435-21000-0017D and Cotton Incorporated project # 12-210. We greatly appreciate the contributions of Dr. Xianliang Song in our group. We also appreciate Mrs. Sheron Simpson and Dr. Brian Scheffler at the Genomics and Bioinformatics Research Unit, USDA-ARS at Stoneville, MS, for their support in SSR marker analysis. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture that is an equal opportunity provider and employer.

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Cotton Fiber Bioscience Research Unit, USDA-ARS-SRRC, 1100 Robert E. Lee Blvd, New Orleans, LA, 70124, USA

    Gregory N. Thyssen, David D. Fang, Christopher Florane, Ping Li & Marina Naoumkina

  2. Crop Genetics Research Unit, USDA-ARS, 141 Experiment Station Road, Stoneville, MS, 38776, USA

    David D. Fang & Rickie B. Turley

Authors
  1. Gregory N. Thyssen
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  2. David D. Fang
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  3. Rickie B. Turley
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  4. Christopher Florane
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  5. Ping Li
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  6. Marina Naoumkina
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Correspondence to Marina Naoumkina.

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Communicated by Christiane Gebhardt.

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Thyssen, G.N., Fang, D.D., Turley, R.B. et al. Next generation genetic mapping of the Ligon-lintless-2 (Li 2) locus in upland cotton (Gossypium hirsutum L.). Theor Appl Genet 127, 2183–2192 (2014). https://doi.org/10.1007/s00122-014-2372-1

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  • DOI: https://doi.org/10.1007/s00122-014-2372-1

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