Upland cotton is a model for polyploid crop domestication and transgenic improvement. Here we sequenced the allotetraploid Gossypium hirsutum L. acc. TM-1 genome by integrating whole-genome shotgun reads, bacterial artificial chromosome (BAC)-end sequences and genotype-by-sequencing genetic maps. We assembled and annotated 32,032 A-subgenome genes and 34,402 D-subgenome genes. Structural rearrangements, gene loss, disrupted genes and sequence divergence were more common in the A subgenome than in the D subgenome, suggesting asymmetric evolution. However, no genome-wide expression dominance was found between the subgenomes. Genomic signatures of selection and domestication are associated with positively selected genes (PSGs) for fiber improvement in the A subgenome and for stress tolerance in the D subgenome. This draft genome sequence provides a resource for engineering superior cotton lines.

High-throughput genotyping platforms play important roles in plant genomic studies. Cotton (Gossypium spp.) is the world’s important natural textile fiber and oil crop. Upland cotton accounts for more than 90% of the world’s cotton production, however, modern upland cotton cultivars have narrow genetic diversity. The amounts of genomic sequencing and re-sequencing data released make it possible to develop a high-quality single nucleotide polymorphism (SNP) array for intraspecific genotyping detection in cotton. Here we report a high-throughput CottonSNP80K array and its utilization in genotyping detection in different cotton accessions. 82,259 SNP markers were selected from the re-sequencing data of 100 cotton cultivars and used to produce the array on the Illumina Infinium platform. 77,774 SNP loci (94.55%) were successfully synthesized on the array. Of them, 77,252 (99.33%) had call rates of >95% in 352 cotton accessions and 59,502 (76.51%) were polymorphic loci. Application tests using 22 cotton accessions with parent/F1 combinations or with similar genetic backgrounds showed that CottonSNP80K array had high genotyping accuracy, good repeatability, and wide applicability. Phylogenetic analysis of 312 cotton cultivars and landraces with wide geographical distribution showed that they could be classified into ten groups, irrelevant of their origins. We found that the different landraces were clustered in different subgroups, indicating that these landraces were major contributors to the development of different breeding populations of modern G. hirsutum cultivars in China. We integrated a total of 54,588 SNPs (MAFs >0.05) associated with 10 salt stress traits into 288 G. hirsutum accessions for genome-wide association studies (GWAS), and eight significant SNPs associated with three salt stress traits were detected. We developed CottonSNP80K array with high polymorphism to distinguish upland cotton accessions. Diverse application tests indicated that the CottonSNP80K play important roles in germplasm genotyping, variety verification, functional genomics studies, and molecular breeding in cotton.

Background: SNPs are the most abundant polymorphism type, and have been explored in many crop genomic studies, including rice and maize. SNP discovery in allotetraploid cotton genomes has lagged behind that of other crops due to their complexity and polyploidy. In this study, genome-wide SNPs are detected systematically using next-generation sequencing and efficient SNP genotyping methods, and used to construct a linkage map and characterize the structural variations in polyploid cotton genomes. Results: We construct an ultra-dense inter-specific genetic map comprising 4,999,048 SNP loci distributed unevenly in 26 allotetraploid cotton linkage groups and covering 4,042 cM. The map is used to order tetraploid cotton genome scaffolds for accurate assembly of G. hirsutum acc. TM-1. Recombination rates and hotspots are identified across the cotton genome by comparing the assembled draft sequence and the genetic map. Using this map, genome rearrangements and centromeric regions are identified in tetraploid cotton by combining information from the publicly-available G. raimondii genome with fluorescent in situ hybridization analysis. Conclusions: We report the genotype-by-sequencing method used to identify millions of SNPs between G. hirsutum and G. barbadense. We construct and use an ultra-dense SNP map to correct sequence mis-assemblies, merge scaffolds into pseudomolecules corresponding to chromosomes, detect genome rearrangements, and identify centromeric regions in allotetraploid cottons. We find that the centromeric retro-element sequence of tetraploid cotton derived from the D subgenome progenitor might have invaded the A subgenome centromeres after allotetrapolyploid formation. This study serves as a valuable genomic resource for genetic research and breeding of cotton.

Cotton is an economically important crop throughout the world and is a pioneer crop in salt stress tolerance research. Investigation of the genetic regulation of salinity tolerance will provide information for salt stress-resistant breeding. Here, we employed next-generation RNA-Seq technology to elucidate the salt-tolerant mechanisms in cotton using the diploid cotton species Gossypium davidsonii which has superior stress tolerance. A total of 4744 and 5337 differentially expressed genes (DEGs) were found to be involved in salt stress tolerance in roots and leaves, respectively. Gene function annotation elucidated salt overly sensitive (SOS) and reactive oxygen species (ROS) signaling pathways. Furthermore, we found that photosynthesis pathways and metabolism play important roles in ion homeostasis and oxidation balance. Moreover, our studies revealed that alternative splicing also contributes to salt-stress responses at the posttranscriptional level, implying its functional role in response to salinity stress. This study not only provides a valuable resource for understanding the genetic control of salt stress in cotton, but also lays a substantial foundation for the genetic improvement of crop resistance to salt stress.

Cotton is an important industrial crop worldwide and upland cotton (Gossypium hirsutum L.) is most widely cultivated in the world. Due to ever-increasing water deficit, drought stress brings a major threat to cotton production. Thus, it is important to reveal the genetic basis under drought stress and develop drought tolerant cotton cultivars. To address this issue, in present study, 319 upland cotton accessions were genotyped by 55,060 single nucleotide polymorphisms (SNPs) from high-density CottonSNP80K array and phenotyped nine drought tolerance related traits. The two datasets were used to identify quantitative trait nucleotides (QTNs) for the above nine traits using multi-locus random-SNP-effect mixed linear model method. As a result, a total of 20 QTNs distributed on 16 chromosomes were found to be significantly associated with six drought tolerance related traits. Of the 1,326 genes around the 20 QTNs, 205 were induced after drought stress treatment, and 46 were further mapped to Gene ontology (GO) term “response to stress.” Taken genome-wide association study (GWAS) analysis, RNA-seq data and qRT-PCR verification, four genes, RD2 encoding a response to desiccation 2 protein, HAT22 encoding a homeobox-leucine zipper protein, PIP2 encoding a plasma membrane intrinsic protein 2, and PP2C encoding a protein phosphatase 2C, were proposed to be potentially important for drought tolerance in cotton. These results will deepen our understanding of the genetic basis of drought stress tolerance in cotton and provide candidate markers to accelerate the development of drought-tolerant cotton cultivars.