GhGASA14 regulates the flowering time of upland cotton in response to GA3.

KEY MESSAGE: The silencing of GhGASA14 and the identification of superior allelic variation in its coding region indicate that GhGASA14 may positively regulate flowering and the response to GA3. Gibberellic acid-stimulated Arabidopsis (GASA), a member of the gibberellin-regulated short amino acid family, has been extensively investigated in several plant species and found to be critical for plant growth and development. However, research on this topic in cotton has been limited. In this study, we identified 38 GhGASAs that were dispersed across 18 chromosomes in upland cotton, and all of these genes had a GASA core domain. Transcriptome expression patterns and qRT-PCR results revealed that GhGASA9 and GhGASA14 exhibited upregulated expression not only in the floral organs but also in the leaves of early-maturing cultivars. The two genes were functionally characterized by virus-induced gene silencing (VIGS), and the budding and flowering times after silencing the target genes were later than those of the control (TRV:00). Compared with that in the water-treated group (MOCK), the flowering period of the different fruiting branches in the GA3-treated group was more concentrated. Interestingly, allelic variation was detected in the coding sequence of GhGASA14 between early-maturing and late-maturing accessions, and the frequency of this favorable allele was greater in high-latitude cotton cultivars than in low-latitude ones. Additionally, a significant linear relationship was observed between the expression level of GhGASA14 and flowering time among the 12 upland cotton accessions. Taken together, these results indicated that GhGASA14 may positively regulate flowering time and respond to GA3. These findings could lead to the use of valuable genetic resources for breeding early-maturing cotton cultivars in the future.

The Silencing of GhPIP5K2 and GhPIP5K22 Weakens Abiotic Stress Tolerance in Upland Cotton (Gossypium hirsutum).

Phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks), essential enzymes in the phosphatidylinositol signaling pathway, are crucial for the abiotic stress responses and the overall growth and development of plants. However, the GhPIP5Ks had not been systematically studied, and their function in upland cotton was unknown. This study identified a total of 28 GhPIP5Ks, and determined their chromosomal locations, gene structures, protein motifs and cis-acting elements via bioinformatics analysis. A quantitative real-time PCR (qRT‒PCR) analysis showed that most GhPIP5Ks were upregulated under different stresses. A virus-induced gene silencing (VIGS) assay indicated that the superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities were significantly decreased, while malondialdehyde (MDA) content were significantly increased in GhPIP5K2- and GhPIP5K22-silenced upland cotton plants under abiotic stress. Furthermore, the expression of the stress marker genes GhHSFB2A, GhHSFB2B, GhDREB2A, GhDREB2C, GhRD20-1, GhRD29A, GhBIN2, GhCBL3, GhNHX1, GhPP2C, GhCBF1, GhSnRK2.6 and GhCIPK6 was significantly decreased in the silenced plants after exposure to stress. These results revealed that the silencing of GhPIP5K2 and GhPIP5K22 weakened the tolerance to abiotic stresses. These discoveries provide a foundation for further inquiry into the actions of the GhPIP5K gene family in regulating the response and resistance mechanisms of cotton to abiotic stresses.

Genome-Wide Identification of the GhANN Gene Family and Functional Validation of GhANN11 and GhANN4 under Abiotic Stress.

Annexins (ANNs) are a structurally conserved protein family present in almost all plants. In the present study, 27 GhANNs were identified in cotton and were unevenly distributed across 14 chromosomes. Transcriptome data and RT-qPCR results revealed that multiple GhANNs respond to at least two abiotic stresses. Similarly, the expression levels of GhANN4 and GhANN11 were significantly upregulated under heat, cold, and drought stress. Using virus-induced gene silencing (VIGS), functional characterization of GhANN4 and GhANN11 revealed that, compared with those of the controls, the leaf wilting of GhANN4-silenced plants was more obvious, and the activities of catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) were lower under NaCl and PEG stress. Moreover, the expression of stress marker genes (GhCBL3, GhDREB2A, GhDREB2C, GhPP2C, GhRD20-2, GhCIPK6, GhNHX1, GhRD20-1, GhSOS1, GhSOS2 and GhSnRK2.6) was significantly downregulated in GhANN4-silenced plants after stress. Under cold stress, the growth of the GHANN11-silenced plants was significantly weaker than that of the control plants, and the activities of POD, SOD, and CAT were also lower. However, compared with those of the control, the elasticity and orthostatic activity of the GhANN11-silenced plants were greater; the POD, SOD, and CAT activities were higher; and the GhDREB2C, GhHSP, and GhSOS2 expression levels were greater under heat stress. These results suggest that different GhANN family members respond differently to different types of abiotic stress.

GhAP1-D3 positively regulates flowering time and early maturity with no yield and fiber quality penalties in upland cotton.

Flowering time (FTi) is a major factor determining how quickly cotton plants reach maturity. Early maturity greatly affects lint yield and fiber quality and is crucial for mechanical harvesting of cotton in northwestern China. Yet, few quantitative trait loci (QTLs) or genes regulating early maturity have been reported in cotton, and the underlying regulatory mechanisms are largely unknown. In this study, we characterized 152, 68, and 101 loci that were significantly associated with the three key early maturity traits-FTi, flower and boll period (FBP) and whole growth period (WGP), respectively, via four genome-wide association study methods in upland cotton (Gossypium hirsutum). We focused on one major early maturity-related genomic region containing three single nucleotide polymorphisms on chromosome D03, and determined that GhAP1-D3, a gene homologous to Arabidopsis thaliana APETALA1 (AP1), is the causal locus in this region. Transgenic plants overexpressing GhAP1-D3 showed significantly early flowering and early maturity without penalties for yield and fiber quality compared to wild-type (WT) plants. By contrast, the mutant lines of GhAP1-D3 generated by genome editing displayed markedly later flowering than the WT. GhAP1-D3 interacted with GhSOC1 (SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1), a pivotal regulator of FTi, both in vitro and in vivo. Changes in GhAP1-D3 transcript levels clearly affected the expression of multiple key flowering regulatory genes. Additionally, DNA hypomethylation and high levels of H3K9ac affected strong expression of GhAP1-D3 in early-maturing cotton cultivars. We propose that epigenetic modifications modulate GhAP1-D3 expression to positively regulate FTi in cotton through interaction of the encoded GhAP1 with GhSOC1 and affecting the transcription of multiple flowering-related genes. These findings may also lay a foundation for breeding early-maturing cotton varieties in the future.

An RTM-GWAS procedure reveals the QTL alleles and candidate genes for three yield-related traits in upland cotton.

BACKGROUND: Cotton (Gossypium spp.) fiber yield is one of the key target traits, and improved fiber yield has always been thought of as an important objective in the breeding programs and production. Although some studies had been reported for the understanding of genetic bases for cotton yield-related traits, the detected quantitative trait loci (QTL) for the traits is still very limited. To uncover the whole-genome QTL controlling three yield-related traits in upland cotton (Gossypium hirsutum L.), phenotypic traits were investigated under four planting environments and 9244 single-nucleotide polymorphism linkage disequilibrium block (SNPLDB) markers were developed in an association panel consisting of 315 accessions. RESULTS: A total of 53, 70 and 68 significant SNPLDB loci associated with boll number (BN), boll weight (BW) and lint percentage (LP), were respectively detected through a restricted two-stage multi-locus multi-allele genome-wide association study (RTM-GWAS) procedure in multiple environments. The haplotype/allele effects of the significant SNPLDB loci were estimated and the QTL-allele matrices were organized for offering the abbreviated genetic composition of the population. Among the significant SNPLDB loci, six of them were simultaneously identified in two or more single planting environments and were thought of as the stable SNPLDB loci. Additionally, a total of 115 genes were annotated in the nearby regions of the six stable SNPLDB loci, and 16 common potential candidate genes controlling target traits of them were predicted by two RNA-seq data. One of 16 genes (GH_D06G2161) was mainly expressed in the early ovule-development stages, and the stable SNPLDB locus (LDB_19_62926589) was mapped in its promoter region. CONCLUSION: This study identified the QTL alleles and candidate genes that could provide important insights into the genetic basis of yield-related traits in upland cotton and might facilitate breeding cotton varieties with high yield.

Genetic dissection of stem WSC accumulation and remobilization in wheat (Triticum aestivum L.) under terminal drought stress.

BACKGROUND: The accumulation and remobilization of stem water soluble carbohydrates (WSC) are determinant physiological traits highly influencing yield potential in wheat against drought stress. However, knowledge gains of the genetic control are still limited. A hexaploid wheat population of 120 recombinant inbred lines were developed to identify quantitative trait loci (QTLs) and to dissect the genetic basis underlying eight traits related to stem WSC under drought stress (DS) and well-watered (WW) conditions across three environments. RESULTS: Analysis of variance (ANOVA) revealed larger environmental and genotypic effects on stem WSC-related traits, indicating moderate heritabilities of 0.51-0.72. A total of 95 additive and 88 pairs of epistatic QTLs were identified with significant additive and epistatic effects, as well as QTL× water environmental interaction (QEI) effects. Most of additive QTLs and additive QEIs associated with drought-stressed environments functioned genetic effects promoting pre-anthesis WSC levels and stem WSC remobilization to developing grains. Compared to other genetic components, both genetic effects were performed exclusive contributions to phenotypic variations in stem WSC-related traits. Nineteen QTL clusters were identified on chromosomes 1B, 2A, 2B, 2D, 3B, 4B, 5A, 6A, 6B and 7A, suggestive of the genetic linkage or pleiotropy. Thirteen additive QTLs were detectable repeatedly across two of the three water environments, indicating features of stable expressions. Some loci were consistent with those reported early and were further discussed. CONCLUSION: Stem WSC-related traits were inherited predominantly by additive and QEI effects with a moderate heritability. QTL cluster regions were suggestive of tight linkage or pleiotropy in the inheritance of these traits. Some stable and common loci, as well as closely linked molecular markers, had great potential in marker-assisted selection to improve stem WSC-related traits in wheat, especially under drought-stressed environments.

Genome-wide association study identified genetic variations and candidate genes for plant architecture component traits in Chinese upland cotton.

KEY MESSAGE: Thirty significant associations between 22 SNPs and five plant architecture component traits in Chinese upland cotton were identified via GWAS. Four peak SNP loci located on chromosome D03 were simultaneously associated with more plant architecture component traits. A candidate gene, Gh_D03G0922, might be responsible for plant height in upland cotton. A compact plant architecture is increasingly required for mechanized harvesting processes in China. Therefore, cotton plant architecture is an important trait, and its components, such as plant height, fruit branch length and fruit branch angle, affect the suitability of a cultivar for mechanized harvesting. To determine the genetic basis of cotton plant architecture, a genome-wide association study (GWAS) was performed using a panel composed of 355 accessions and 93,250 single nucleotide polymorphisms (SNPs) identified using the specific-locus amplified fragment sequencing method. Thirty significant associations between 22 SNPs and five plant architecture component traits were identified via GWAS. Most importantly, four peak SNP loci located on chromosome D03 were simultaneously associated with more plant architecture component traits, and these SNPs were harbored in one linkage disequilibrium block. Furthermore, 21 candidate genes for plant architecture were predicted in a 0.95-Mb region including the four peak SNPs. One of these genes (Gh_D03G0922) was near the significant SNP D03_31584163 (8.40 kb), and its Arabidopsis homologs contain MADS-box domains that might be involved in plant growth and development. qRT-PCR showed that the expression of Gh_D03G0922 was upregulated in the apical buds and young leaves of the short and compact cotton varieties, and virus-induced gene silencing (VIGS) proved that the silenced plants exhibited increased PH. These results indicate that Gh_D03G0922 is likely the candidate gene for PH in cotton. The genetic variations and candidate genes identified in this study lay a foundation for cultivating moderately short and compact varieties in future Chinese cotton-breeding programs.

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.

Genome-wide identification and functional analysis of the TIFY gene family in response to drought in cotton.

Jasmonates control many aspects of plant biological processes. They are important for regulating plant responses to various biotic and abiotic stresses, including drought, which is one of the most serious threats to sustainable agricultural production. However, little is known regarding how jasmonate ZIM-domain (JAZ) proteins mediate jasmonic acid signals to improve stress tolerance in cotton. This represents the first comprehensive comparative study of TIFY transcription factors in both diploid A, D and tetraploid AD cotton species. In this study, we identified 21 TIFY family members in the genome of Gossypium arboretum, 28 members from Gossypium raimondii and 50 TIFY genes in Gossypium hirsutum. The phylogenetic analyses indicated the TIFY gene family could be divided into the following four subfamilies: TIFY, PPD, ZML, and JAZ subfamilies. The cotton TIFY genes have expanded through tandem duplications and segmental duplications compared with other plant species. Gene expression profile revealed temporal and tissue specificities for TIFY genes under simulated drought conditions in Gossypium arboretum. The JAZ subfamily members were the most highly expressed genes, suggesting that they have a vital role in responses to drought stress. Over-expression of GaJAZ5 gene decreased water loss, stomatal openings, and the accumulation of H2O2 in Arabidopsis thaliana. Additionally, the results of drought tolerance assays suggested that this subfamily might be involved in increasing drought tolerance. Our study provides new data regarding the genome-wide analysis of TIFY gene families and their important roles in drought tolerance in cotton species. These data may form the basis of future studies regarding the relationship between drought and jasmonic acid.

GhBRX.1, GhBRX.2, and GhBRX4.3 improve resistance to salt and cold stress in upland cotton.

Introduction: Abiotic stress during growth readily reduces cotton crop yield. The different survival tactics of plants include the activation of numerous stress response genes, such as BREVIS RADIX (BRX). Methods: In this study, the BRX gene family of upland cotton was identified and analyzed by bioinformatics method, three salt-tolerant and cold-resistant GhBRX genes were screened. The expression of GhBRX.1, GhBRX.2 and GhBRXL4.3 in upland cotton was silenced by virus-induced gene silencing (VIGS) technique. The physiological and biochemical indexes of plants and the expression of related stress-response genes were detected before and after gene silencing. The effects of GhBRX.1, GhBRX.2 and GhBRXL4.3 on salt and cold resistance of upland cotton were further verified. Results and discussion: We discovered 12, 6, and 6 BRX genes in Gossypium hirsutum, Gossypium raimondii and Gossypium arboreum, respectively. Chromosomal localization indicated that the retention and loss of GhBRX genes on homologous chromosomes did not have a clear preference for the subgenomes. Collinearity analysis suggested that segmental duplications were the main force for BRX gene amplification. The upland cotton genes GhBRX.1, GhBRX.2 and GhBRXL4.3 are highly expressed in roots, and GhBRXL4.3 is also strongly expressed in the pistil. Transcriptome data and qRT‒PCR validation showed that abiotic stress strongly induced GhBRX.1, GhBRX.2 and GhBRXL4.3. Under salt stress and low-temperature stress conditions, the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) and the content of soluble sugar and chlorophyll decreased in GhBRX.1-, GhBRX.2- and GhBRXL4.3-silenced cotton plants compared with those in the control (TRV: 00). Moreover, GhBRX.1-, GhBRX.2- and GhBRXL4.3-silenced cotton plants exhibited greater malondialdehyde (MDA) levels than did the control plants. Moreover, the expression of stress marker genes (GhSOS1, GhSOS2, GhNHX1, GhCIPK6, GhBIN2, GhSnRK2.6, GhHDT4D, GhCBF1 and GhPP2C) decreased significantly in the three target genes of silenced plants following exposure to stress. These results imply that the GhBRX.1, GhBRX.2 and GhBRXL4.3 genes may be regulators of salt stress and low-temperature stress responses in upland cotton.

Genome-wide investigation and expression profiles of the NPF gene family provide insight into the abiotic stress resistance of Gossypium hirsutum.

Membrane transporters encoded by NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER (NPF) genes, which play crucial roles in plant growth, development and resistance to various stresses, are involved in the transport of nitrate (NO3 -) and peptides. In several plant species, NPF genes are involved in the resistance to abiotic stresses; however, whether the whole NPF gene family in cotton contributes to this resistance has not been systematically investigated. Here, 201 genes encoding NPF proteins with a peptide transporter (PTR) domain were confirmed in three different Gossypium species, namely, Gossypium hirsutum, Gossypium arboreum and Gossypium raimondii. The NPF proteins in these three Gossypium species and Arabidopsis thaliana were classified into three different subfamilies via phylogenetic analysis. Among the genes that encode these proteins, most GhNPF genes in the same subfamily contained similar gene structures and conserved domains. Predictions of the promoters of these genes revealed that the cis-acting elements included phytohormone- and light-responsive elements, indicating that some of these genes might be expressed in response to abiotic stress. Furthermore, 52 common potential candidate genes in 98 GhNPFs were predicted to exhibit specific spatiotemporal expression patterns in different tissues based on two RNA sequencing (RNA-seq) datasets. Finally, the gene expression profiles of abiotic stress indicated that 31 GhNPF genes were upregulated in at least one treatment period. Under abiotic stress for 12 and 24 h, the expression of GhNPF8 was upregulated upon cold treatment but downregulated with heat treatment, salt treatment and drought treatment. Furthermore, the expression of genes GhNPF8, GhNPF54 and GhNPF43 peaked at 6 h after heat and salt treatment. These results indicated that these genes exhibit underlying characteristics related to responses to abiotic stress. The verification of NPFs and analysis of their expression profiles in different tissues and in response to different abiotic stresses of cotton provide a basis for further studying the relationship between abiotic stress resistance and nitrogen (N) transport in cotton, as well as identifying candidate genes to facilitate their functional identification.

Nucleotide Evolution, Domestication Selection, and Genetic Relationships of Chloroplast Genomes in the Economically Important Crop Genus Gossypium.

Gossypium hirsutum (upland cotton) is one of the most economically important crops worldwide, which has experienced the long terms of evolution and domestication process from wild species to cultivated accessions. However, nucleotide evolution, domestication selection, and the genetic relationship of cotton species remain largely to be studied. In this study, we used chloroplast genome sequences to determine the evolutionary rate, domestication selection, and genetic relationships of 72 cotton genotypes (36 cultivated cotton accessions, seven semi-wild races of G. hirsutum, and 29 wild species). Evolutionary analysis showed that the cultivated tetraploid cotton genotypes clustered into a single clade, which also formed a larger lineage with the semi-wild races. Substitution rate analysis demonstrated that the rates of nucleotide substitution and indel variation were higher for the wild species than the semi-wild and cultivated tetraploid lineages. Selection pressure analysis showed that the wild species might have experienced greater selection pressure, whereas the cultivated cotton genotypes underwent artificial and domestication selection. Population clustering analysis indicated that the cultivated cotton accessions and semi-wild races have existed the obviously genetic differentiation. The nucleotide diversity was higher in the semi-wild races compared with the cultivated genotypes. In addition, genetic introgression and gene flow occurred between the cultivated tetraploid cotton and semi-wild genotypes, but mainly via historical rather than contemporary gene flow. These results provide novel molecular mechanisms insights into the evolution and domestication of economically important crop cotton species.

TaABC1, a member of the activity of bc1 complex protein kinase family from common wheat, confers enhanced tolerance to abiotic stresses in Arabidopsis.

Abiotic stresses such as drought, salinity, and low temperature have drastic effects on plant growth and development. However, the molecular mechanisms regulating biochemical and physiological changes in response to stresses are not well understood. Protein kinases are major signal transduction factors among the reported molecular mechanisms mediating acclimation to environmental changes. Protein kinase ABC1 (activity of bc(1) complex) is involved in regulating coenzyme Q biosynthesis in mitochondria in yeast (Saccharomyces cersvisiae), and in balancing oxidative stress in chloroplasts in Arabidopsis thaliana. In the current study, TaABC1 (Triticum aestivum L. activity of bc(1) complex) protein kinase was localized to the cell membrane, cytoplasm, and nucleus. The effects of overexpressing TaABC1 in transgenic Arabidopsis plants on responses to drought, salt, and cold stress were further investigated. Transgenic Arabidopsis overexpressing the TaABC1 protein showed lower water loss and higher osmotic potential, photochemistry efficiency, and chlorophyll content, while cell membrane stability and controlled reactive oxygen species homeostasis were maintained. In addition, overexpression of TaABC1 increased the expression of stress-responsive genes, such as DREB1A, DREB2A, RD29A, ABF3, KIN1, CBF1, LEA, and P5CS, detected by real-time PCR analysis. The results suggest that TaABC1 overexpression enhances drought, salt, and cold stress tolerance in Arabidopsis, and imply that TaABC1 may act as a regulatory factor involved in a multiple stress response pathways.

Complete Chloroplast Genome Sequence of Sonchus brachyotus Helps to Elucidate Evolutionary Relationships with Related Species of Asteraceae.

Sonchus brachyotus DC. possesses both edible and medicinal properties and is widely distributed throughout China. In this study, the complete cp genome of S. brachyotus was sequenced and assembled. The total length of the complete S. brachyotus cp genome was 151,977 bp, including an LSC region of 84,553 bp, SSC region of 18,138 bp, and IR region of 24,643 bp. Sequence analyses revealed that the cp genome encoded 132 genes, including 87 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. The GC content was 37.6%. One hundred mononucleotide microsatellites, 4 dinucleotide microsatellites, 67 trinucleotide microsatellites, 4 tetranucleotide microsatellites, and 1 long repeat were identified. The SSR frequency of the LSC region was significantly greater than that of the IR and SSC regions. In total, 175 SSRs and highly variable regions were recognized as potential cp markers. By analyzing the IR/LSC and IR/SSC boundaries, structural differences between S. brachyotus and 6 other species were detected. According to phylogenetic analyses, S. brachyotus was most closely related to S. arvensis and S. oleraceus. Overall, this study provides complete cp genome resources for S. brachyotus that will be beneficial for identifying potential molecular markers and evolutionary patterns of S. brachyotus and its closely related species.

Abscisic acid associated with key enzymes and genes involving in dynamic flux of water soluble carbohydrates in wheat peduncle under terminal drought stress.

Remobilization of stem water soluble carbohydrates (WSC) can supply crucial carbon resources for grain filling under drought stress, while the regulatory metabolism associated with abscisic acid (ABA) is still limited. Two cultivars, LJ196 (drought-tolerant) and XD18 (drought-prone), were pot-grown under well-watered (WW) and drought-stressed (DS) conditions. Concentrations of WSC components and ABA, and fructan metabolizing enzymes and genes were investigated in peduncle after anthesis. When compared with those under the WW, LJ196 remained higher grain yield and grain-filling rate than XD18 under the DS. During the early period of grain filling (0-14 DAA), DS increased concentrations of total WSC and its components, but thereafter substantially reduced them. The gene expression levels and enzymatic activities of fructan 1-exohydrolases (1-FEH) and fructan 6-exohydrolases (6-FEH) showed similar trends, whereas those of fructan: fructan 1-fructosyltransferase (1-FFT), and sucrose: fructan 6-fructosyltransferase (6-SFT) were depressed and declined over the period of examination. LJ196 still showed higher levels of ABA and fructan metabolizing. The ABA concentration under the DS was positively and significantly correlated with total WSC and fructan concentration, and expression levels of these enzymes and genes as well, with more prominently with those of 6-FEH. Presumably, ABA could enhance fructan hydrolysis by strongly up-regulating the gene expression and enzymatic activity of 6-FEH to accelerate WSC remobilization. However, stem WSC induced by DS could be not fully remobilized to grains, due to its weaker correlation with grain-filling rate and finally indicating lower grain yield. The findings would provide useful information for wheat production under water-deficit environments.

Validation of QTLs for Fiber Quality Introgressed from Gossypium mustelinum by Selective Genotyping.

Gene introgression from wild species has been shown to be a feasible approach for fiber quality improvement in Upland cotton. Previously, we developed an interspecific G. mustelinum × G. hirsutum advanced-backcross population and mapped over one hundred QTL for fiber quality traits. In the current study, a trait-based selective genotyping approach was utilized to prioritize a small subset of introgression lines with high phenotypic values for different fiber quality traits, to simultaneously validate multiple fiber quality QTL in a single experiment. A total of 75 QTL were detected by CIM and/or single-marker analysis, including 11 significant marker-trait associations (P < 0.001) and three putative associations (P < 0.005) also reported in earlier studies. The QTL that have been validated include three each for fiber length, micronaire, and elongation, and one each for fiber strength and uniformity. Collectively, about 10% of the QTL previously reported have been validated here, indicating that selective genotyping has the potential to validate multiple marker-trait associations for different traits, especially those with a moderate to large-effect detected simultaneously in one experimental population. The G. mustelinum alleles contributed to improved fiber quality for all validated loci. The results from this study will lay the foundation for further fine mapping, marker-assisted selection and map-based gene cloning.

Genetic Detection of Lint Percentage Applying Single-Locus and Multi-Locus Genome-Wide Association Studies in Chinese Early-Maturity Upland Cotton.

Upland cotton (Gossypium hirsutum L.) is the most important source of natural fiber in the world. Early-maturity upland cotton varieties are commonly planted in China. Nevertheless, lint yield of early-maturity upland cotton varieties is strikingly lower than that of middle- and late-maturity ones. How to effectively improve lint yield of early maturing cotton, becomes a focus of cotton research. Here, based on 72,792 high-quality single nucleotide polymorphisms of 160 early-maturing upland cotton accessions, we performed genome-wide association studies (GWASs) for lint percentage (LP), one of the most lint-yield component traits, applying one single-locus method and six multi-locus methods. A total of 4 and 45 significant quantitative trait nucleotides (QTNs) were respectively identified to be associated with LP. Interestingly, in two of four planting environments, two of these QTNs (A02_74713290 and A02_75551547) were simultaneously detected via both one single-locus and three or more multi-locus GWAS methods. Among the 42 genes within a genomic region (A02: 74.31-75.95 Mbp) containing the above two peak QTNs, Gh_A02G1269, Gh_A02G1280, and Gh_A02G1295 had the highest expression levels in ovules during seed development from 20 to 25 days post anthesis, whereas Gh_A02G1278 was preferentially expressed in the fibers rather than other organs. These results imply that the four potential candidate genes might be closely related to cotton LP by regulating the proportion of seed weight and fiber yield. The QTNs and potential candidate genes for LP, identified in this study, provide valuable resource for cultivating novel cotton varieties with earliness and high lint yield in the future.

Multi-Locus Genome-Wide Association Studies of Fiber-Quality Related Traits in Chinese Early-Maturity Upland Cotton.

Early-maturity varieties of upland cotton are becoming increasingly important for farmers to improve their economic benefits through double cropping practices and mechanical harvesting production in China. However, fiber qualities of early-maturing varieties are relatively poor compared with those of middle- and late- maturing ones. Therefore, it is crucial for researchers to elucidate the genetic bases controlling fiber-quality related traits in early-maturity cultivars, and to improve synergistically cotton earliness and fiber quality. Here, multi-locus genome-wide association studies (ML-GWAS) were conducted in a panel consisting of 160 early-maturing cotton accessions. Each accession was genotyped by 72,792 high-quality single nucleotide polymorphisms (SNPs) using specific-locus amplified fragment sequencing (SLAF-seq) approach, and fiber quality-related traits under four environmental conditions were measured. Applying at least three ML-GWAS methods, a total of 70 significant quantitative trait nucleotides (QTNs) were identified to be associated with five objective traits, including fiber length (FL), fiber strength (FS), fiber micronaire (FM), fiber uniformity (FU) and fiber elongation (FE). Among these QTNs, D11_21619830, A05_28352019 and D03_34920546 were found to be significantly associated with FL, FS, and FM, respectively, across at least two environments. Among 96 genes located in the three target genomic regions (A05: 27.95 28.75, D03: 34.52 35.32, and D11: 21.22 22.02 Mbp), six genes (Gh_A05G2325, Gh_A05G2329, Gh_A05G2334, Gh_D11G1853, Gh_D11G1876, and Gh_D11G1879) were detected to be highly expressed in fibers relative to other eight tissues by transcriptome sequencing method in 12 cotton tissues. Together, multiple favorable QTN alleles and six candidate key genes were characterized to regulate fiber development in early-maturity cotton. This will lay a solid foundation for breeding novel cotton varieties with earliness and excellent fiber-quality in the future.

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.