Five reference genes, 18S, EF1alpha, alpha-Tubulin, Ubiquitin and Actin, from Salvia miltiorrhiza were analyzed as internal controls for gene expression profiling assay using quantitative real-time polymerase chain reaction (qRT-PCR). The five candidate genes were measured for their transcriptional level in seven tissues, including roots, stems, leaves, sepals, petals, stamens and pistils. Then they were ranked by the GeNorm tool. The results showed that Actin and Ubiquitin were the most stable whereas EF1alpha and 18S did not favor normalization of qRT-PCR results in these tissues. Expression levels of the SmDXR gene were studied in parallel, with Actin and Ubiquitin both or each as reference in the seven tissues, and varying relative quantifications of the SmDXR gene in seven tissues. This study indicated that selection of the most stable genes plays an important role in gene expression profiling assays.

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.

Soil salinization is one of the major problems in global agricultural production. Cotton is a pioneer crop with regard to salt stress tolerance, and can be used for saline-alkali land improvement. The large-scale detection of salt tolerance traits in cotton accessions, and the identification of elite quantitative trait loci (QTLs)/genes for salt-tolerance have been very important in salt tolerance breeding. Here, 43 advanced salt-tolerant and 31 highly salt-sensitive cultivars were detected by analyzing ten salt tolerance related traits in 304 upland cotton cultivars. Among them, 11 advanced salt-tolerance and eight highly salt-sensitive cultivars were consistent with previously reported results. Association analysis of ten salt-tolerance related traits and 145 SSRs was performed, and a total of 95 significant associations were detected; 17, 41, and 37 of which were associated with germinative index, seedling stage physiological index, and four seedling stage biochemical indexes, respectively. Of these associations, 20 SSR loci were simultaneously associated with two or more traits. Furthermore, we detected 117 elite alleles associated with salt-tolerance traits, 4 of which were reported previously. Among these loci, 44 (37.60%) were rare alleles with a frequency of less than 5%, 6 only existed in advanced salt-tolerant cultivars, and 2 only in highly salt-sensitive cultivars. As a result, 13 advanced salt-tolerant cultivars were selected to assemble the optimal cross combinations by computer simulation for the development of salt-tolerant accessions. This study lays solid foundations for further improvements in cotton salt-tolerance by referencing elite germplasms, alleles associated with salt-tolerance traits, and optimal crosses.

Background: Salinity is one of the most significant environmental factors limiting the productivity of cotton. However, the key genetic components responsible for the reduction in cotton yield in saline-alkali soils are still unclear. Results: Here, we evaluated three main components of lint yield, single boll weight (SBW), lint percentage (LP) and boll number per plant (BNPP), across 316 G. hirsutum accessions under four salt conditions over two years. Phenotypic analysis indicated that LP was unchanged under different salt conditions, however BNPP decreased significantly and SBW increased slightly under high salt conditions. Based on 57,413 high-quality single nucleotide polymorphisms (SNPs) and genome-wide association study (GWAS) analysis, a total of 42, 91 and 25 stable quantitative trait loci (QTLs) were identified for SBW, LP and BNPP, respectively. Phenotypic and QTL analysis suggested that there was little correlation among the three traits. For LP, 8 stable QTLs were detected simultaneously in four different salt conditions, while fewer repeated QTLs for SBW or BNPP were identified. Gene Ontology (GO) analysis indicated that their regulatory mechanisms were also quite different. Via transcriptome profile data, we detected that 10 genes from the 8 stable LP QTLs were predominantly expressed during fiber development. Further, haplotype analyses found that a MYB gene (GhMYB103), with the two SNP variations in cis-regulatory and coding regions, was significantly correlated with lint percentage, implying a crucial role in lint yield. We also identified that 40 candidate genes from BNPP QTLs were salt-inducible. Genes related to carbohydrate metabolism and cell structure maintenance were rich in plants grown in high salt conditions, while genes related to ion transport were active in plants grown in low salt conditions, implying different regulatory mechanisms for BNPP at high and low salt conditions. Conclusions: This study provides a foundation for elucidating cotton salt tolerance mechanisms and contributes gene resources for developing upland cotton varieties with high yields and salt stress tolerance.