GbPP2C80 Interacts with GbWAKL14 to Negatively Co-Regulate Resistance to Fusarium and Verticillium wilt via MPK3 and ROS Signaling in Sea Island Cotton.

Fusarium wilt (FW) is widespread in global cotton production, but the mechanism underlying FW resistance in superior-fiber-quality Sea Island cotton is unclear. This study reveals that FW resistance has been the target of genetic improvement of Sea Island cotton in China since the 2010s. The key nonsynonymous single nucleotide polymorphism (SNP, T/C) of gene Gbar_D03G001670 encoding protein phosphatase 2C 80 (PP2C80) results in an amino acid shift (L/S), which is significantly associated with FW resistance of Sea Island cotton. Silencing GbPP2C80 increases FW resistance in Sea Island cotton, whereas overexpressing GbPP2C80 reduces FW resistance in Arabidopsis. GbPP2C80 and GbWAKL14 exist synergistically in Sea Island cotton accessions with haplotype forms "susceptible-susceptible" (TA) and "resistant-resistant" (CC), and interact with each other. CRISPR/Cas9-mediated knockout of GbWAKL14 enhances FW and Verticillium wilt (VW) resistance in upland cotton and overexpression of GbWAKL14 and GbPP2C80 weakens FW and VW resistance in Arabidopsis. GbPP2C80 and GbWAKL14 respond to FW and VW by modulating reactive oxygen species (ROS) content via affecting MPK3 expression. In summary, two tandem genes on chromosome D03, GbPP2C80, and GbWAKL14, functions as cooperative negative regulators in cotton wilt disease defense, providing novel genetic resources and molecular markers for the development of resistant cotton cultivars.

Yield-related quantitative trait loci identification and lint percentage hereditary dissection under salt stress in upland cotton.

Salinity is frequently mentioned as a major constraint in worldwide agricultural production. Lint percentage (LP) is a crucial yield-component in cotton lint production. While the genetic factors affect cotton yield in saline soils are still unclear. Here, we employed a recombinant inbred line population in upland cotton (Gossypium hirsutum L.) and investigated the effects of salt stress on five yield and yield component traits, including seed cotton yield per plant, lint yield per plant, boll number per plant, boll weight, and LP. Between three datasets of salt stress (E1), normal growth (E2), and the difference values dataset of salt stress and normal conditions (D-value), 87, 82, and 55 quantitative trait loci (QTL) were detectable, respectively. In total, five QTL (qLY-Chr6-2, qBNP-Chr4-1, qBNP-Chr12-1, qBNP-Chr15-5, qLP-Chr19-2) detected in both in E1 and D-value were salt related QTL, and three stable QTL (qLP-Chr5-3, qLP-Chr13-1, qBW-Chr5-5) were detected both in E1 and E2 across 3 years. Silencing of nine genes within a stable QTL (qLP-Chr5-3) highly expressed in fiber developmental stages increased LP and decreased fiber length (FL), indicating that multiple minor-effect genes clustered on Chromosome 5 regulate LP and FL. Additionally, the difference in LP caused by Gh_A05G3226 is mainly in transcription level rather than in the sequence difference. Moreover, silencing of salt related gene (GhDAAT) within qBNP-Chr4-1 decreased salt tolerance in cotton. Our findings shed light on the regulatory mechanisms underlining cotton salt tolerance and fiber initiation.

Identification of fertility restoration candidate genes from a restorer line R186 for Gossypium harknessii cytoplasmic male sterile cotton.

BACKGROUND: The utilization of heterosis based on three-line system is an effective strategy in crop breeding. However, cloning and mechanism elucidation of restorer genes for cytoplasmic male sterility (CMS) in upland cotton have yet been realized. RESULTS: This research is based on CMS line 2074A with the cytoplasm from Gossypium harknessii (D2-2) and restorer line R186. The offspring of 2074A × R186 were used to conduct genetic analysis. The fertility mechanism of 2074A can be speculated to be governed by multiple genes, since neither the single gene model nor the double genes model could be used. The bulked segregant analysis (BSA) for (2074A × R186) F2 determined the genetic interval of restorer genes on a region of 4.30 Mb on chromosome D05 that contains 77 annotated genes. Four genes were identified as candidates for fertility restoration using the RNA-seq data of 2074A, 2074B, and R186. There are a number of large effect variants in the four genes between 2074A and R186 that could cause amino acid changes. Evolutionary analysis and identity analysis revealed that GH_D05G3183, GH_D05G3384, and GH_D05G3490 have high identity with their homologs in D2-2, respectively. Tissue differential expression analysis revealed that the genes GH_D05G3183, GH_D05G3384, and GH_D05G3490 were highly expressed in the buds of the line R186. The predicted results demonstrated that GH_D05G3183, GH_D05G3384 and GH_D05G3490 might interact with GH_A02G1295 to regulate orf610a in mitochondria. CONCLUSION: Our study uncovered candidate genes for fertility restoration in the restorer line R186 and predicted the possible mechanism for restoring the male fertility in 2074A. This research provided valuable insight into the nucleoplasmic interactions.

Identification of candidate genes involved in salt stress response at germination and seedling stages by QTL mapping in upland cotton.

Salinity is a major abiotic stress at critical stages of seed germination and seedling establishment. Germination rate (GR) and field emergence rate (FER) are the key traits that determine the basic number of plants stand under field conditions. To explore molecular mechanisms in upland cotton under salt stress, a population of 177 recombinant inbred lines, and their parents were evaluated for seed germination traits (GP, germination potential; GR; FW, fresh weight; DW, dry weight; GL, germinal length) and seedling traits (FER; SH, seedling height; NL, number of main stem leaves) in 2016-2018. Based on the linkage map contained 2,859 single nucleotide polymorphism and simple sequence repeat markers, traits under salt stress (E1) and normal conditions (E2), and in the converted relative index (R-value) dataset of 3 years' trials were used to map quantitative trait loci (QTL). A total of 3 QTL and 2 clusters were detected as salt-tolerant QTL. Three QTL (qGR-Chr4-3, qFER-Chr12-3, and qFER-Chr15-1) were detected under salt stress conditions and R-value dataset, which explained variance of phenotype 9.62-13.67%, and 4.2-4.72%, 4.75-8.96%, respectively. Two clusters (Loci-Chr4-2 and Loci-Chr5-4) harboring the QTL for 4 germination traits (GR, FER, GL, and NL) and 6 seedling traits (GR, FER, DW, FW, SH, and NL) were detected related under salt stress. A total of 691 genes were found in the candidate QTL or clusters. Among them, 4 genes (Gh_A04G1106, Gh_A05G3246, Gh_A05G3177, and Gh_A05G3266) showed expression differences between salt-sensitive and -tolerant lines under salt stress conditions, and were assigned as candidate genes in response to salt stress. The consistent salt-tolerance QTL identified in both germination and seedling stages will facilitate novel insights into effective utilization of cotton genetic resources.

Genomic and GWAS analyses demonstrate phylogenomic relationships of Gossypium barbadense in China and selection for fibre length, lint percentage and Fusarium wilt resistance.

Sea Island cotton (Gossypium barbadense) is the source of the world's finest fibre quality cotton, yet relatively little is understood about genetic variations among diverse germplasms, genes underlying important traits and the effects of pedigree selection. Here, we resequenced 336 G. barbadense accessions and identified 16 million SNPs. Phylogenetic and population structure analyses revealed two major gene pools and a third admixed subgroup derived from geographical dissemination and interbreeding. We conducted a genome-wide association study (GWAS) of 15 traits including fibre quality, yield, disease resistance, maturity and plant architecture. The highest number of associated loci was for fibre quality, followed by disease resistance and yield. Using gene expression analyses and VIGS transgenic experiments, we confirmed the roles of five candidate genes regulating four key traits, that is disease resistance, fibre length, fibre strength and lint percentage. Geographical and temporal considerations demonstrated selection for the superior fibre quality (fibre length and fibre strength), and high lint percentage in improving G. barbadense in China. Pedigree selection breeding increased Fusarium wilt disease resistance and separately improved fibre quality and yield. Our work provides a foundation for understanding genomic variation and selective breeding of Sea Island cotton.

QTL controlling fiber quality traits under salt stress in upland cotton (Gossypium hirsutum L.).

KEY MESSAGE: QTL for fiber quality traits under salt stress discerned candidate genes controlling fatty acid metabolism. Salinity stress seriously affects plant growth and limits agricultural productivity of crop plants. To dissect the genetic basis of response to salinity stress, a recombinant inbred line population was developed to compare fiber quality in upland cotton (Gossypium hirsutum L.) under salt stress and normal conditions. Based on three datasets of (1) salt stress, (2) normal growth, and (3) the difference value between salt stress and normal conditions, 51, 70, and 53 QTL were mapped, respectively. Three QTL for fiber length (FL) (qFL-Chr1-1, qFL-Chr5-5, and qFL-Chr24-4) were detected under both salt and normal conditions and explained 4.26%, 9.38%, and 3.87% of average phenotypic variation, respectively. Seven genes within intervals of two stable QTL (qFL-Chr1-1 and qFL-Chr5-5) were highly expressed in lines with extreme long fiber. A total of 35 QTL clusters comprised of 107 QTL were located on 18 chromosomes and exhibited pleiotropic effects. Thereinto, two clusters were responsible for improving five fiber quality traits, and 6 influenced FL and fiber strength (FS). The QTL with positive effect for fiber length exhibited active effects on fatty acid synthesis and elongation, but the ones with negative effect played passive roles on fatty acid degradation under salt stress.

Two Aquaporin Genes, GhPIP2;7 and GhTIP2;1, Positively Regulate the Tolerance of Upland Cotton to Salt and Osmotic Stresses.

Aquaporins (AQPs) facilitate the transport of water and small molecules across intrinsic membranes and play a critical role in abiotic stresses. In this study, 111, 54, and 56 candidate AQP genes were identified in Gossypium hirsutum (AD1), Gossypium arboreum (A2), and Gossypium raimondii (D5), respectively, and were further classified into five subfamilies, namely, plasma intrinsic protein (PIP), tonoplast intrinsic protein (TIP), nodulin 26-like intrinsic protein (NIP), small basic intrinsic protein (SIP), and uncategorized X intrinsic protein (XIP). Transcriptome analysis and quantitative real-time PCR (qRT-PCR) revealed some high-expression GhPIPs and GhTIPs (PIP and TIP genes in G. hirsutum, respectively) in drought and salt stresses. GhPIP2;7-silenced plants decreased in the chlorophyll content, superoxide dismutase (SOD) activity, and peroxidase (POD) activity comparing the mock control (empty-vector) under 400 mM NaCl treatment, which indicated a positive regulatory role of GhPIP2;7 in salt tolerance of cotton. The GhTIP2;1-silenced cotton plants were more sensitive to osmotic stress. GhTIP2;1-overexpressed plants exhibited less accumulation of H2O2 and malondialdehyde but higher proline content under osmotic stress. In summary, our study elucidates the positive regulatory roles of two GhAQPs (GhPIP2;7 and GhTIP2;1) in salt and osmotic stress responses, respectively, and provides a new gene resource for future research.

GhCIPK6a increases salt tolerance in transgenic upland cotton by involving in ROS scavenging and MAPK signaling pathways.

BACKGROUND: Salt stress is one of the most damaging abiotic stresses in production of Upland cotton (Gossypium hirsutum). Upland cotton is defined as a medium salt-tolerant crop. Salinity hinders root development, shoots growth, and reduces the fiber quality. RESULTS: Our previous study verified a GhCIPK6a gene response to salt stress in G. hirsutum. The homologs of GhCIPK6a were analyzed in A2 (G. arboreum), D5 (G. raimondii), and AD1 (G. hirsutum) genomes. GhCIPK6a localized to the vacuole and cell membrane. The GhCBL1-GhCIPK6a and GhCBL8-GhCIPK6a complexes localized to the nucleus and cytomembrane. Overexpression of GhCIPK6a enhanced expression levels of co-expressed genes induced by salt stress, which scavenged ROS and involved in MAPK signaling pathways verified by RNA-seq analysis. Water absorption capacity and cell membrane stability of seeds from GhCIPK6a overexpressed lines was higher than that of wild-type seeds during imbibed germination stage. The seed germination rates and seedling field emergence percentages of GhCIPK6a overexpressed lines were higher than that of control line under salt stress. Moreover, overexpressing of GhCIPK6a in cotton increased lint percentage, and fiber length uniformity under salt stress. CONCLUSIONS: We verified the function of GhCIPK6a by transformation and RNA-seq analysis. GhCIPK6a overexpressed lines exhibited higher tolerance to abiotic stresses, which functioned by involving in ROS scavenging and MAPK pathways. Therefore, GhCIPK6a has the potential for cotton breeding to improve stress-tolerance.

Evolution of PEPC gene family in Gossypium reveals functional diversification and GhPEPC genes responding to abiotic stresses.

Phosphoenolpyruvate carboxylase (PEPC) family genes play important roles in regulating plant growth and abiotic stress response. Based on the sequenced Gossypium genomes, we performed comprehensive analysis of PEPC homolog genes in cotton, which six, six, eleven and ten PEPC genes were identified in Gossypium arboreum (A2), G. raimondii (D5), G. hirsutum (AD1) and G. barbadense (AD2), respectively. These genes were divided into six subgroups: PEPC-i, PEPC-ii, PEPC-iii, PEPC-iv, PEPC-v and PEPC-vi; PEPC genes in each subgroup displayed conserved gene structure and motifs. Segmental duplication and whole genome duplication (WGD) events yielded the expansion of PEPC genes. Expression assays showed that the duplicated PEPC genes displayed diverse expression patterns, indicating that they experienced functional divergence. Of which, genes in PEPC-iv subgroup played crucial role for substrate distribution in cottonseed. Cis-elements, putative miRNAs and expression analyses showed that GhPEPC homologs might respond to abiotic stresses, expression levels of GhPEPC1 and GhPEPC2/GhPEPC2D genes were larger induced than other GhPEPC genes under cold, heat, salt, and drought stresses, indicating the crucial roles in abiotic stresses response. Present study serves new information to decipher the evolution and function of PEPC genes in Gossypium.