Genome-wide association study reveals novel SNPs and genes in Gossypium hirsutum underlying Aphis gossypii resistance.

KEY MESSAGE: A. gossypii resistance showed great variability in G. hirsutum varieties. One hundred and seventy-six SNPs associated with A. gossypii resistance were identified using GWAS. Four candidate resistance genes were functionally validated. Aphis gossypii is an economically important sap-feeding pest and is widely distributed in the world's cotton-producing regions. Identification of cotton genotypes and developing cultivars with improved A. gossypii resistance (AGR) is essential and desirable for sustainable agriculture. In the present study, A. gossypii was offered no choice but to propagate on 200 Gossypium hirsutum accessions. A relative aphid reproduction index (RARI) was used to evaluate the AGR, which showed large variability in cotton accessions and was classified into 6 grades. A significantly positive correlation was found between AGR and Verticillium wilt resistance. A total of 176 SNPs significantly associated with the RARI were identified using GWAS. Of these, 21 SNPs could be repeatedly detected in three replicates. Cleaved amplified polymorphic sequence, a restriction digestion-based genotyping assay, was developed using SNP1 with the highest observed -log10(P-value). Four genes within the 650 kb region of SNP1 were further identified, including GhRem (remorin-like), GhLAF1 (long after far-red light 1), GhCFIm25 (pre-mRNA cleavage factor Im 25 kDa subunit) and GhPMEI (plant invertase/pectin methylesterase inhibitor superfamily protein). The aphid infection could induce their expression and showed a significant difference between resistant and susceptible cotton varieties. Silencing of GhRem, GhLAF1 or GhCFIm25 could significantly increase aphid reproduction on cotton seedlings. Silencing of GhRem significantly reduced callose deposition, which is reasonably believed to be the cause for the higher AGR. Our results provide insights into understanding the genetic regulation of AGR in cotton and suggest candidate germplasms, SNPs and genes for developing cultivars with improved AGR.

Transcriptome, Ectopic Expression and Genetic Population Analysis Identify Candidate Genes for Fiber Quality Improvement in Cotton.

Comparative transcriptome analysis of fiber tissues between Gossypium barbadense and Gossypium hirsutum could reveal the molecular mechanisms underlying high-quality fiber formation and identify candidate genes for fiber quality improvement. In this study, 759 genes were found to be strongly upregulated at the elongation stage in G. barbadense, which showed four distinct expression patterns (I-IV). Among them, the 346 genes of group IV stood out in terms of the potential to promote fiber elongation, in which we finally identified 42 elongation-related candidate genes by comparative transcriptome analysis between G. barbadense and G. hirsutum. Subsequently, we overexpressed GbAAR3 and GbTWS1, two of the 42 candidate genes, in Arabidopsis plants and validated their roles in promoting cell elongation. At the secondary cell wall (SCW) biosynthesis stage, 2275 genes were upregulated and exhibited five different expression profiles (I-V) in G. barbadense. We highlighted the critical roles of the 647 genes of group IV in SCW biosynthesis and further picked out 48 SCW biosynthesis-related candidate genes by comparative transcriptome analysis. SNP molecular markers were then successfully developed to distinguish the SCW biosynthesis-related candidate genes from their G. hirsutum orthologs, and the genotyping and phenotyping of a BC3F5 population proved their potential in improving fiber strength and micronaire. Our results contribute to the better understanding of the fiber quality differences between G. barbadense and G. hirsutum and provide novel alternative genes for fiber quality improvement.

Tissue-specific expression of GhnsLTPs identified via GWAS sophisticatedly coordinates disease and insect resistance by regulating metabolic flux redirection in cotton.

Cotton (Gossypium hirsutum) is constantly attacked by pathogens and insects. The most efficient control strategy is to develop resistant varieties using broad-spectrum gene resources. Several resistance loci harboured by superior varieties have been identified through genome-wide association studies. However, the key genes and/or loci have not been functionally identified. In this study, we identified a locus significantly associated with Verticillium wilt (VW) resistance, and within a 145.5-kb linkage disequilibrium, two non-specific lipid transfer protein genes (named GhnsLTPsA10) were highly expressed under Verticillium pathogen stress. The expression of GhnsLTPsA10 significantly increased in roots upon Verticillium dahliae stress but significantly decreased in leaves under insect attack. Furthermore, GhnsLTPsA10 played antagonistic roles in positively regulating VW and Fusarium wilt resistance and negatively mediating aphid and bollworm resistance in transgenic Arabidopsis and silenced cotton. By combining transcriptomic, histological and physiological analyses, we determined that GhnsLTPsA10-mediated phenylpropanoid metabolism further affected the balance of the downstream metabolic flux of flavonoid and lignin biosynthesis. The divergent expression of GhnsLTPsA10 in roots and leaves coordinated resistance of cotton against fungal pathogens and insects via the redirection of metabolic flux. In addition, GhnsLTPsA10 contributed to reactive oxygen species accumulation. Therefore, in this study, we elucidated the novel function of GhnsLTP and the molecular association between disease resistance and insect resistance, balanced by GhnsLTPsA10. This broadens our knowledge of the biological function of GhnsLTPsA10 in crops and provides a useful locus for genetic improvement of cotton.

Genome-wide association study reveals the genetic basis of yield- and quality-related traits in wheat.

BACKGROUND: Identifying the loci and dissecting the genetic architecture underlying wheat yield- and quality-related traits are essential for wheat breeding. A genome-wide association study was conducted using a high-density 90 K SNP array to analyze the yield- and quality-related traits of 543 bread wheat varieties. RESULTS: A total of 11,140 polymorphic SNPs were distributed on 21 chromosomes, including 270 significant SNPs associated with 25 yield- and quality-related traits. Additionally, 638 putative candidate genes were detected near the significant SNPs based on BLUP data, including three (TraesCS7A01G482000, TraesCS4B01G343700, and TraesCS6B01G295400) related to spikelet number per spike, diameter of the first internode, and grain volume. The three candidate genes were further analyzed using stage- and tissue- specific gene expression data derived from an RNA-seq analysis. These genes are promising candidates for enhancing yield- and quality-related traits in wheat. CONCLUSIONS: The results of this study provide a new insight to understand the genetic basis of wheat yield and quality. Furthermore, the markers detected in this study may be applicable for marker-assisted selection in wheat breeding programs.

Evolution, expression and functional analysis of cultivated allotetraploid cotton DIR genes.

BACKGROUND: Dirigent (DIR) proteins mediate regioselectivity and stereoselectivity during lignan biosynthesis and are also involved in lignin, gossypol and pterocarpan biosynthesis. This gene family plays a vital role in enhancing stress resistance and in secondary cell-wall development, but systematical understanding is lacking in cotton. RESULTS: In this study, 107 GbDIRs and 107 GhDIRs were identified in Gossypium barbadense and Gossypium hirsutum, respectively. Most of these genes have a classical gene structure without intron and encode proteins containing a signal peptide. Phylogenetic analysis showed that cotton DIR genes were classified into four distinct subfamilies (a, b/d, e, and f). Of these groups, DIR-a and DIR-e were evolutionarily conserved, and segmental and tandem duplications contributed equally to their formation. In contrast, DIR-b/d mainly expanded by recent tandem duplications, accompanying with a number of gene clusters. With the rapid evolution, DIR-b/d-III was a Gossypium-specific clade involved in atropselective synthesis of gossypol. RNA-seq data highlighted GhDIRs in response to Verticillium dahliae infection and suggested that DIR gene family could confer Verticillium wilt resistance. We also identified candidate DIR genes related to fiber development in G. barbadense and G. hirsutum and revealed their differential expression. To further determine the involvement of DIR genes in fiber development, we overexpressed a fiber length-related gene GbDIR78 in Arabidopsis and validated its function in trichomes and hypocotyls. CONCLUSIONS: These findings contribute novel insights towards the evolution of DIR gene family and provide valuable information for further understanding the roles of DIR genes in cotton fiber development as well as in stress responses.

A high-density genetic map and multiple environmental tests reveal novel quantitative trait loci and candidate genes for fibre quality and yield in cotton.

KEY MESSAGE: A high-density linkage map of an intraspecific RIL population was constructed using 6187 bins to identify QTLs for fibre quality- and yield-related traits in upland cotton by whole-genome resequencing. Good fibre quality and high yield are important production goals in cotton (Gossypium hirsutum L.), which is a leading natural fibre crop worldwide. However, a greater understanding of the genetic variants underlying fibre quality- and yield-related traits is still required. In this study, a large-scale population including 588 F7 recombinant inbred lines, derived from an intraspecific cross between the upland cotton cv. Nongdamian13, which exhibits high quality, and Nongda601, which exhibits a high yield, was genotyped by using 232,946 polymorphic single-nucleotide polymorphisms obtained via a whole-genome resequencing strategy with 4.3-fold genome coverage. We constructed a high-density bin linkage map containing 6187 bin markers spanning 4478.98 cM with an average distance of 0.72 cM. We identified 58 individual quantitative trait loci (QTLs) and 25 QTL clusters harbouring 94 QTLs, and 119 previously undescribed QTLs controlling 13 fibre quality and yield traits across eight environments. Importantly, the QTL counts for fibre quality in the Dt subgenome were more than two times that in the At subgenome, and chromosome D02 harboured the greatest number of QTLs and clusters. Furthermore, we discovered 24 stable QTLs for fibre quality and 12 stable QTLs for yield traits. Four novel major stable QTLs related to fibre length, fibre strength and lint percentage, and seven previously unreported candidate genes with significantly differential expression between the two parents were identified and validated by RNA-seq. Our research provides valuable information for improving the fibre quality and yield in cotton breeding.

SEP-like genes of Gossypium hirsutum promote flowering via targeting different loci in a concentration-dependent manner.

SEP genes are famous for their function in the morphological novelty of bisexual flowers. Although the diverse functions of SEP genes were reported, only the regulatory mechanisms underlying floral organ development have been addressed. In this study, we identified SEP-like genes in Gossypium and found that SEP3 genes were duplicated in diploid cotton varieties. GhSEP4.1 and GhSEP4.2 were abundantly transcribed in the shoot apical meristem (SAM), but only GhSEP4.2 was expressed in the leaf vasculature. The expression pattern of GhSEPs in floral organs was conserved with that of homologs in Arabidopsis, except for GhSEP2 that was preponderantly expressed in ovules and fibers. The overexpression and silencing of each single GhSEP gene suggested their distinct role in promoting flowering via direct binding to GhAP1 and GhLFY genomic regions. The curly leaf and floral defects in overexpression lines with a higher expression of GhSEP genes revealed the concentration-dependent target gene regulation of GhSEP proteins. Moreover, GhSEP proteins were able to dimerize and interact with flowering time regulators. Together, our results suggest the dominant role of GhSEP4.2 in leaves to promote flowering via GhAP1-A04, and differently accumulated GhSEP proteins in the SAM alternately participate in forming the dynamic tetramer complexes to target at the different loci of GhAP1 and GhLFY to maintain reproductive growth. The regulatory roles of cotton SEP genes reveal their conserved and diversified functions.

Cotton GhSSI2 isoforms from the stearoyl acyl carrier protein fatty acid desaturase family regulate Verticillium wilt resistance.

Lipids are major and essential constituents of plant cells and provide energy for various metabolic processes. However, the function of the lipid signal in defence against Verticillium dahliae, a hemibiotrophic pathogen, remains unknown. Here, we characterized 19 conserved stearoyl-ACP desaturase family proteins from upland cotton (Gossypium hirsutum). We further confirmed that GhSSI2 isoforms, including GhSSI2-A, GhSSI2-B, and GhSSI2-C located on chromosomes A10, D10, and A12, respectively, played a dominant role to the cotton 18:1 (oleic acid) pool. Suppressing the expression of GhSSI2s reduced the 18:1 level, which autoactivated the hypersensitive response (HR) and enhanced cotton Verticillium wilt and Fusarium wilt resistance. We found that low 18:1 levels induced phenylalanine ammonia-lyase-mediated salicylic acid (SA) accumulation and activated a SA-independent defence response in GhSSI2s-silenced cotton, whereas suppressing expression of GhSSI2s affected PDF1.2-dependent jasmonic acid (JA) perception but not the biosynthesis and signalling cascade of JA. Further investigation showed that structurally divergent resistance-related genes and nitric oxide (NO) signal were activated in GhSSI2s-silenced cotton. Taken together, these results indicate that SA-independent defence response, multiple resistance-related proteins, and elevated NO level play an important role in GhSSI2s-regulated Verticillium wilt resistance. These findings broaden our knowledge regarding the lipid signal in disease resistance and provide novel insights into the molecular mechanism of cotton fungal disease resistance.

High-quality genome assembly and resequencing of modern cotton cultivars provide resources for crop improvement.

Cotton produces natural fiber for the textile industry. The genetic effects of genomic structural variations underlying agronomic traits remain unclear. Here, we generate two high-quality genomes of Gossypium hirsutum cv. NDM8 and Gossypium barbadense acc. Pima90, and identify large-scale structural variations in the two species and 1,081 G. hirsutum accessions. The density of structural variations is higher in the D-subgenome than in the A-subgenome, indicating that the D-subgenome undergoes stronger selection during species formation and variety development. Many structural variations in genes and/or regulatory regions potentially influencing agronomic traits were discovered. Of 446 significantly associated structural variations, those for fiber quality and Verticillium wilt resistance are located mainly in the D-subgenome and those for yield mainly in the A-subgenome. Our research provides insight into the role of structural variations in genotype-to-phenotype relationships and their potential utility in crop improvement.

Systematic Analysis of Cotton Non-specific Lipid Transfer Protein Family Revealed a Special Group That Is Involved in Fiber Elongation.

Non-specific lipid transfer proteins (nsLTPs) had been previously isolated from cotton fiber but their functions were unclear so far. Bioinformatic analysis of the tetraploid cotton genome database identified 138 nsLTP genes, falling into the 11 groups as reported previously. Different from Arabidopsis, cacao, and other crops, cotton type XI genes were considerably expanded and diverged earlier on chromosome At11, Dt11, and Dt08. Corresponding to the type XI genes, the type XI proteins (GhLtpXIs) all contained an extra N-terminal cap resulting in larger molecular weight. The research revealed that the expression of type XI genes was dramatically increased in fibers of tetraploid cotton compared with the two diploid progenitors. High-level of GhLtpXIs expression was observed in long-fibered cotton cultivars during fiber elongation. Ectopic expression of GhLtpXIs in Arabidopsis significantly enhanced trichome length, suggesting that GhLtpXIs promoted fiber elongation. Overall, the findings of this research provide insights into phenotypic evolution of Gossypium species and regulatory mechanism of nsLTPs during fiber development. HIGHLIGHT A specific group, type XI nsLTPs, was identified with predominant expression in elongating fibers of Gossypium hirsutum based on evolutionary, transcriptional, and functional analyses.