Non-functional GoFLA19s are responsible for the male sterility caused by hybrid breakdown in cotton (Gossypium spp.).

Hybrid breakdown (HB) functions as a common reproductive barrier and reduces hybrid fitness in many species, including cotton. However, the related genes and the underlying genetic mechanisms of HB in cotton remain unknown. Here, we found that the photosensitive genetic male sterile line CCRI9106 was a hybrid progeny of Gossypium hirsutum and Gossypium barbadense and probably a product of HB. Fine mapping with F2 s (CCRI9106 × G. hirsutum/G. barbadense lines) identified a pair of male sterility genes GoFLA19s (encoding fasciclin-like arabinogalactan family protein) located on chromosomes A12 and D12. Crucial variations occurring in the fasciclin-like domain and the arabinogalactan protein domain were predicted to cause the non-functionalization of GbFLA19-D and GhFLA19-A. CRISPR/Cas9-mediated knockout assay confirmed the effects of GhFLA19s on male sterility. Sequence alignment analyses showed that variations in GbFLA19-D and GhFLA19-A likely occurred after the formation of allotetraploid cotton species. GoFLA19s are specifically expressed in anthers and contribute to tapetal development, exine assembly, intine formation, and pollen grain maturation. RNA-sequencing and quantitative reverse transcriptase-polymerase chain reaction analyses illustrated that genes related to these biological processes were significantly downregulated in the mutant. Our research on male sterility genes, GoFLA19s, improves the understanding of the molecular characteristics and evolutionary significance of HB in interspecific hybrid breeding.

A comprehensive analysis of cotton VQ gene superfamily reveals their potential and extensive roles in regulating cotton abiotic stress.

BACKGROUND: Valine-glutamine (VQ) motif-containing proteins play important roles in plant growth, development and abiotic stress response. For many plant species, the VQ genes have been identified and their functions have been described. However, little is known about the origin, evolution, and functions (and underlying mechanisms) of the VQ family genes in cotton. RESULTS: In this study, we comprehensively analyzed the characteristics of 268 VQ genes from four Gossypium genomes and found that the VQ proteins evolved into 10 clades, and each clade had a similar structural and conservative motif. The expansion of the VQ gene was mainly through segmental duplication, followed by dispersal. Expression analysis revealed that many GhVQs might play important roles in response to salt and drought stress, and GhVQ18 and GhVQ84 were highly expressed under PEG and salt stress. Further analysis showed that GhVQs were co-expressed with GhWRKY transcription factors (TFs), and microRNAs (miRNAs) could hybridize to their cis-regulatory elements. CONCLUSIONS: The results in this study broaden our understanding of the VQ gene family in plants, and the analysis of the structure, conserved elements, and expression patterns of the VQs provide a solid foundation for exploring their specific functions in cotton responding to abiotic stresses. Our study provides significant insight into the potential functions of VQ genes in cotton.

Fine mapping and molecular characterization of the virescent gene vsp in Upland cotton (Gossypium hirsutum).

KEY MESSAGE: The vsp gene was fine mapped to a 353.7-kb region, and a 201-bp deletion that affected chloroplast development and chlorophyll biosynthesis was found in the candidate gene GhPUR4. Virescent mutations can be used as marker traits in heterosis breeding and can also be used to research chloroplast development, chlorophyll biosynthesis and photosynthesis mechanisms. Here, we obtained a light-sensitive virescent mutant, vsp, that has reduced chlorophyll (Chl) content and abnormal chloroplast development. Then, the virescent space (vsp) gene in the vsp mutant was preliminarily mapped to a 38.32-Mb region of chromosome D04 using a high-density SNP genetic map with a total length of 5384.33 cM and 4472 bin markers. Furthermore, the vsp gene was narrowed down to a 353.7-kb region that contains 15 candidate genes using 484 virescent individuals from an F2 population. Sequence analysis of genes in this region showed that a 201-bp deletion was present in the Gh_D04G1108 (GhPUR4) gene in the vsp mutant. The 201-bp deletion of Gh_D04G1108 caused the deletion of 67 AAs in the GhPUR4 protein. Virus-induced gene silencing (VIGS) of GhPUR4 in normal plants caused reduced GhPUR4 gene expression levels, reduced Chl content, abnormal chloroplast development and virescent true leaves. This study could help us unravel the function of GhPUR4 in chloroplast development and Chl biosynthesis at the early developmental stages of the true leaves in cotton, which could promote the research and application of virescent mutations in cotton heterosis breeding.

Fine mapping and candidate gene analysis of the virescent gene v 1 in Upland cotton (Gossypium hirsutum).

The young leaves of virescent mutants are yellowish and gradually turn green as the plants reach maturity. Understanding the genetic basis of virescent mutants can aid research of the regulatory mechanisms underlying chloroplast development and chlorophyll biosynthesis, as well as contribute to the application of virescent traits in crop breeding. In this study, fine mapping was employed, and a recessive gene (v 1) from a virescent mutant of Upland cotton was narrowed to an 84.1-Kb region containing ten candidate genes. The GhChlI gene encodes the cotton Mg-chelatase I subunit (CHLI) and was identified as the candidate gene for the virescent mutation using gene annotation. BLAST analysis showed that the GhChlI gene has two copies, Gh_A10G0282 and Gh_D10G0283. Sequence analysis indicated that the coding region (CDS) of GhChlI is 1269 bp in length, with three predicted exons and one non-synonymous nucleotide mutation (G1082A) in the third exon of Gh_D10G0283, with an amino acid (AA) substitution of arginine (R) to lysine (K). GhChlI-silenced TM-1 plants exhibited a lower GhChlI expression level, a lower chlorophyll content, and the virescent phenotype. Analysis of upstream regulatory elements and expression levels of GhChlI showed that the expression quantity of GhChlI may be normal, and with the development of the true leaf, the increase in the Gh_A10G0282 dosage may partially make up for the deficiency of Gh_D10G0283 in the v 1 mutant. Phylogenetic analysis and sequence alignment revealed that the protein sequence encoded by the third exon of GhChlI is highly conserved across diverse plant species, in which AA substitutions among the completely conserved residues frequently result in changes in leaf color in various species. These results suggest that the mutation (G1082A) within the GhChlI gene may cause a functional defect of the GhCHLI subunit and thus the virescent phenotype in the v1 mutant. The GhChlI mutation not only provides a tool for understanding the associations of CHLI protein function and the chlorophyll biosynthesis pathway but also has implications for cotton breeding.

Identification of favorable SNP alleles and candidate genes for traits related to early maturity via GWAS in upland cotton.

BACKGROUND: Early maturity is one of the most important and complex agronomic traits in upland cotton (Gossypium hirsutum L). To dissect the genetic architecture of this agronomically important trait, a population consisting of 355 upland cotton germplasm accessions was genotyped using the specific-locus amplified fragment sequencing (SLAF-seq) approach, of which a subset of 185 lines representative of the diversity among the accessions was phenotypically characterized for six early maturity traits in four environments. A genome-wide association study (GWAS) was conducted using the generalized linear model (GLM) and mixed linear model (MLM). RESULTS: A total of 81,675 SNPs in 355 upland cotton accessions were discovered using SLAF-seq and were subsequently used in GWAS. Thirteen significant associations between eight SNP loci and five early maturity traits were successfully identified using the GLM and MLM; two of the 13 associations were common between the models. By computing phenotypic effect values for the associations detected at each locus, 11 highly favorable SNP alleles were identified for five early maturity traits. Moreover, dosage pyramiding effects of the highly favorable SNP alleles and significant linear correlations between the numbers of highly favorable alleles and the phenotypic values of the target traits were identified. Most importantly, a major locus (rs13562854) on chromosome Dt3 and a potential candidate gene (CotAD_01947) for early maturity were detected. CONCLUSIONS: This study identified highly favorable SNP alleles and candidate genes associated with early maturity traits in upland cotton. The results demonstrate that GWAS is a powerful tool for dissecting complex traits and identifying candidate genes. The highly favorable SNP alleles and candidate genes for early maturity traits identified in this study should be show high potential for improvement of early maturity in future cotton breeding programs.

Identification and Expression Analysis of CAMTA Genes in Tea Plant Reveal Their Complex Regulatory Role in Stress Responses.

Calmodulin-binding transcription activators (CAMTAs) are evolutionarily conserved transcription factors and have multi-functions in plant development and stress response. However, identification and functional analysis of tea plant (Camellia sinensis) CAMTA genes (CsCAMTAs) are still lacking. Here, five CsCAMTAs were identified from tea plant genomic database. Their gene structures were similar except CsCAMTA2, and protein domains were conserved. Phylogenetic relationship classified the CsCAMTAs into three groups, CsCAMTA2 was in group I, and CsCAMTA1, 3 and CsCAMTA4, 5 were, respectively, in groups II and III. Analysis showed that stress and phytohormone response-related cis-elements were distributed in the promoters of CsCAMTA genes. Expression analysis showed that CsCAMTAs were differentially expressed in different organs and under various stress treatments of tea plants. Three-hundred and four hundred-one positive co-expressed genes of CsCAMTAs were identified under cold and drought, respectively. CsCAMTAs and their co-expressed genes constituted five independent co-expression networks. KEGG enrichment analysis of CsCAMTAs and the co-expressed genes revealed that hormone regulation, transcriptional regulation, and protein processing-related pathways were enriched under cold treatment, while pathways like hormone metabolism, lipid metabolism, and carbon metabolism were enriched under drought treatment. Protein interaction network analysis suggested that CsCAMTAs could bind (G/A/C)CGCG(C/G/T) or (A/C)CGTGT cis element in the target gene promoters, and transcriptional regulation might be the main way of CsCAMTA-mediated functional regulation. The study establishes a foundation for further function studies of CsCAMTA genes in stress response.

Phylogenetic Analysis of the Membrane Attack Complex/Perforin Domain-Containing Proteins in Gossypium and the Role of GhMACPF26 in Cotton Under Cold Stress.

The membrane attack complex/perforin (MACPF) domain-containing proteins are involved in the various developmental processes and in responding to diverse abiotic stress. The function and regulatory network of the MACPF genes are rarely reported in Gossypium spp. We study the detailed identification and partial functional verification of the members of the MACPF family. Totally, 100 putative MACPF proteins containing complete MACPF domain were identified from the four cotton species. They were classified into three phylogenetic groups and underwent multifold pressure indicating that selection produced new functional differentiation. Cotton MACPF gene family members expanded mainly through the whole-genome duplication (WGD)/segmental followed by the dispersed. Expression and cis-acting elements analysis revealed that MACPFs play a role in resistance to abiotic stresses, and some selected GhMACPFs were able to respond to the PEG and cold stresses. Co-expression analysis showed that GhMACPFs might interact with valine-glutamine (VQ), WRKY, and Apetala 2 (AP2)/ethylene responsive factor (ERF) domain-containing genes under cold stress. In addition, silencing endogenous GhMACPF26 in cotton by the virus-induced gene silencing (VIGS) method indicated that GhMACPF26 negatively regulates cold tolerance. Our data provided a comprehensive phylogenetic evolutionary view of Gossypium MACPFs. The MACPFs may work together with multiple transcriptional factors and play roles in acclimation to abiotic stress, especially cold stress in cotton.

Detection of Favorable QTL Alleles and Candidate Genes for Lint Percentage by GWAS in Chinese Upland Cotton.

Improving cotton yield is a major breeding goal for Chinese upland cotton. Lint percentage is an important yield component and a critical economic index for cotton cultivars, and raising the lint percentage has a close relationship to improving cotton lint yield. To investigate the genetic architecture of lint percentage, a diversity panel consisting of 355 upland cotton accessions was grown, and the lint percentage was measured in four different environments. Genotyping was performed with specific-locus amplified fragment sequencing (SLAF-seq). Twelve single-nucleotide polymorphisms (SNPs) associated with lint percentage were detected via a genome-wide association study (GWAS), in which five SNP loci distributed on chromosomes At3 (A02) and At4 (A08) and contained two major-effect QTLs, which were detected in the best linear unbiased predictions (BLUPs) and in more than three environments simultaneously. Furthermore, favorable haplotypes (FHs) of two major-effect QTLs and 47 putative candidate genes in the two linkage disequilibrium (LD) blocks of these associated loci were identified. The expression levels of these putative candidate genes were estimated using RNA-seq data from ten upland cotton tissues. We found that Gh_A02G1268 was very highly expressed during the early fiber development stage, whereas the gene was poorly expressed in the seed. These results implied that Gh_A02G1268 may determine the lint percentage by regulating seed and fiber development. The favorable QTL alleles and candidate genes for lint percentage identified in this study will have high potential for improving lint yield in future Chinese cotton breeding programs.

Two genomic regions associated with fiber quality traits in Chinese upland cotton under apparent breeding selection.

Fiber quality is one of the most important agronomic traits of cotton, and understanding the genetic basis of its target traits will accelerate improvements to cotton fiber quality. In this study, a panel comprising 355 upland cotton accessions was used to perform genome-wide association studies (GWASs) of five fiber quality traits in four environments. A total of 16, 10 and 7 SNPs were associated with fiber length (FL), fiber strength (FS) and fiber uniformity (FU), respectively, based on the mixed linear model (MLM). Most importantly, two major genomic regions (MGR1 and MGR2) on chromosome Dt7 and four potential candidate genes for FL were identified. Analyzing the geographical distribution of favorable haplotypes (FHs) among these lines revealed that two favorable haplotype frequencies (FHFs) were higher in accessions from low-latitude regions than in accessions from high-latitude regions. However, the genetic diversity of lines from the low-latitude regions was lower than the diversity of lines from the high-latitude regions in China. Furthermore, the FHFs differed among cultivars developed during different breeding periods. These results indicate that FHs have undergone artificial selection during upland cotton breeding in recent decades in China and provide a foundation for the further improvement of fiber quality traits.