Converting restriction fragment length polymorphism to single‐strand conformation polymorphism markers and its application in the fine mapping of a trichome gene in cotton

Abstract

A well‐characterized and systematically organized collection of genetic markers is crucial in the study of any crop species. It is the basis of map‐based gene cloning and crop improvements through marker‐assisted selections. Single‐strand conformation polymorphism (SSCP) has been a robust way of discovering new polymorphisms in marker development without the requirement of sequencing. Here, we report the first approach of applying SSCP marker discovery methods in the genetic map construction and gene mapping of cotton species. A total of 80 restriction fragment length polymorphism (RFLP) markers were selected from a region on published cotton genetic maps around the T1 gene related to cotton trichome. Among the 80 RFLPs, 28 showed polymorphisms through SSCP, showing a polymorphic rate of approximately 35%, which is much higher than that of simple sequence repeat (SSR) markers in the same region (7.8%). By integrating these newly generated SSCP markers, a detailed genetic map was reconstructed around this region using an F2 population derived from a cross between Gossypium arboreum and G. herboceum. The reconstructed region comprises 22 SSCP markers, eight SSR markers and the T1 gene, spanning 21.6 cM. The marker order of the new map agrees well with published reference RFLP maps. The above results suggest that SSCP method can be applied very efficiently and reliably to the marker development of cotton genomes. It will prove to be even more valuable and robust after the public release of cotton whole‐genome sequences.

The Hairless Stem Phenotype of Cotton (Gossypium barbadense) Is Linked to aCopia-Like Retrotransposon Insertion in aHomeodomain-Leucine ZipperGene (HD1)

“…This trait cosegregates with the loss-of-fiber mutant sma-4 (Desai et al 2008;He et al 2013). Recently, map-based cloning of sma-4 was conducted by our research group using a large-scale high-resolution finemapping population containing 2500+ individuals, which suggested strongly that the underlying causative gene for the sma-4 phenotype is HD1 on chromosome LG A03.…”

“…Genetic marker candidates that are potentially linked to trichome phenotypes were chosen from both published A genome (He et al 2013) and tetraploid genome mapping studies (Wright et al 1999;Rong et al 2007). Candidate RFLP markers were screened for polymorphisms between the parents of the mapping population in this study (T586 and Pima S6), as reported by He et al (2013).…”

“…Candidate RFLP markers were screened for polymorphisms between the parents of the mapping population in this study (T586 and Pima S6), as reported by He et al (2013). In addition, the genome sequences of G. raimondii (Paterson et al 2012), spanning 5624.28 kb ranging from 26,943 to 5,651,218 nt [trichome gene (T1) region] of chromosome 10 determined by synteny between genetic maps (Wright et al 1999;Rong et al 2007;Desai et al 2008) and a physical map (Paterson et al 2012), were used to develop additional DNA markers.…”

“…In addition, the genome sequences of G. raimondii (Paterson et al 2012), spanning 5624.28 kb ranging from 26,943 to 5,651,218 nt [trichome gene (T1) region] of chromosome 10 determined by synteny between genetic maps (Wright et al 1999;Rong et al 2007;Desai et al 2008) and a physical map (Paterson et al 2012), were used to develop additional DNA markers. Primer design and PCR and SSCP procedures were reported previously (He et al 2013). In brief, primer design for SSCP marker development was based on the following criteria: the length of the amplified fragments should be 250-600 bp and cover the introns.…”

“…In brief, primer design for SSCP marker development was based on the following criteria: the length of the amplified fragments should be 250-600 bp and cover the introns. PCR products were first checked on 1% agarose gels, and those showing one to two clear bands for both genotypes were used for SSCP employing the protocol described by Lee et al (1992) with some modifications (He et al 2013).…”