A Long‐Read Transcriptome Assembly of Cotton (Gossypium hirsutum L.) and Intraspecific Single Nucleotide Polymorphism Discovery

Abstract

Upland cotton (Gossypium hirsutum L.) has a narrow germplasm base, which constrains marker development and hampers intraspecific breeding. A pressing need exists for high-throughput single nucleotide polymorphism (SNP) markers that can be readily applied to germplasm in breeding and breeding-related research programs. Despite progress made in developing new sequencing technologies during the past decade, the cost of sequencing remains substantial when one is dealing with numerous samples and large genomes. Several strategies have been proposed to lower the cost of sequencing for multiple genotypes of large-genome species like cotton, such as transcriptome sequencing and reduced-representation DNA sequencing. This paper reports the development of a transcriptome assembly of the inbred line Texas Marker-1 (TM-1), a genetic standard for cotton, its usefulness as a reference for RNA sequencing (RNA-seq)-based SNP identification, and the availability of transcriptome sequences of four other cotton cultivars. An assembly of TM-1 was made using Roche 454 transcriptome reads combined with an assembly of all available public expressed sequence tag (EST) sequences of TM-1. The TM-1 assembly consists of 72,450 contigs with a total of 70 million bp. Functional predictions of the transcripts were estimated by alignment to selected protein databases. Transcriptome sequences of the five lines, including TM-1, were obtained using an Illumina Genome Analyzer-II, and the short reads were mapped to the TM-1 assembly to discover SNPs among the five lines. We identified >14,000 unfiltered allelic SNPs, of which ~3,700 SNPs were retained for assay development after applying several rigorous filters. This paper reports availability of the reference transcriptome assembly and shows its utility in developing intraspecific SNP markers in upland cotton.

SNP‐Based MAS in Cotton under Depressed‐Recombination for Renlon–Flanking Recombinants: Results and Inferences on Wide‐Cross Breeding Strategies

“…The ability to extract DNA nondestructively from seed allowed for greater temporal flexibility, and the MAS with SNPs greatly reduced the numbers of seed that had to be planted. These kinds of MAS based on targeted SNP activities seem certain to become increasingly common in cotton breeding and related research, given recent strides in SNP discovery (Ashrafi et al, 2015; Hulse‐Kemp et al, 2014, 2015a; Islam et al, 2015; Wang and Chen et al, 2015), development of a high‐quality high‐density SNP array (Hulse‐Kemp et al, 2015b), some progress in validated genotyping by sequencing (Gore et al, 2014), G. hirsutum genome sequence assembly (Li et al, 2015; Zhang et al, 2015), and large‐scale genome‐wide SNP mapping (Hulse‐Kemp et al, 2015b; Wang et al, 2015). The ability to conduct large‐scale seed‐based MAS will revolutionize the science, scope and efficacy of wide‐cross cotton breeding; these trends will foster the assimilation of much more germplasm diversity into mainstream cotton breeding.…”

Insights into Drought Stress Signaling in Plants and the Molecular Genetic Basis of Cotton Drought Tolerance

“…Whole-genome (2.5 Gb) SNPs have been developed in allotetraploid cotton with advances in NGS techniques and in silico methods. In cotton, development of the SNP63K array containing 17,954 and 45,104 putative interspecific and intraspecific SNP marker assays, respectively, is valuable [170]. This provides a high-throughput genotyping platform, a basis and standard tool for genetic analyses of stress-related, agronomically, and economically important traits in cotton.…”

“…Transcriptome profiling is an important tool that to extract information through sequence data to gain knowledge on various gene functions and pathways. Recently, the whole-genome transcriptome was reported which provides information about expressed sequence tag (EST) assemblies of TM-1 inbred line (Gossypium hirsutum), and serves as a reference genome for all RNA-sequence-based SNP studies [170]. Transcriptome libraries of Gossypium barbadense for stress-related traits such as drought, salt, heat, cold, and phosphorus, were also standardized as a reference to identify novel stress-related genes [172].…”

Recent insights into cotton functional genomics: progress and future perspectives

“…The rising efficacy of NGS technique and advanced in silico methods has permitted the development of single nucleotide polymorphisms (SNPs) at the whole-genome level, even for the 2.5-Gb genome of allotetraploid cotton. In cotton, SNP63K has been developed that contains assays for 45 104 and 17 954 putative intraspecific and interspecific SNP markers (Ashrafi et al, 2015). This initial effort for developing SNP63K array of cotton provides a standard high-throughput genotyping tool and a base for the genetic analysis of economically and agronomically important traits.…”