GBS Mapping and Analysis of Genes Conserved between Gossypium tomentosum and Gossypium hirsutum Cotton Cultivars that Respond to Drought Stress at the Seedling Stage of the BC?F? Generation.
ABSTRACT: Cotton production is on the decline due to ever-changing environmental conditions. Drought and salinity stress contribute to over 30% of total loss in cotton production, the situation has worsened more due to the narrow genetic base of the cultivated upland cotton. The genetic diversity of upland cotton has been eroded over the years due to intense selection and inbreeding. To break the bottleneck, the wild cotton progenitors offer unique traits which can be introgressed into the cultivated cotton, thereby improving their performance. In this research, we developed a BC?F? population between wild male parent, G. tomentosum as the donor, known for its high tolerance to drought and the elite female parent, G. hirsutum as the recurrent parent, which is high yielding but sensitive to drought stress. The population was genotyped through the genotyping by sequencing (GBS) method, in which 10,888 single-nucleotide polymorphism (SNP) s were generated and used to construct a genetic map. The map spanned 4191.3 cM, with average marker distance of 0.3849 cM. The map size of the two sub genomes had a narrow range, 2149 cM and 2042.3 cM for At and Dt_sub genomes respectively. A total of 66,434 genes were mined, with 32,032 (48.2%) and 34,402 (51.8%) genes being obtained within the At and Dt_sub genomes respectively. Pkinase (PF00069) was found to be the dominant domain, with 1069 genes. Analysis of the main sub family, serine threonine protein kinases through gene ontology (GO), cis element and miRNA targets analysis revealed that most of the genes were involved in various functions aimed at enhancing abiotic stress tolerance. Further analysis of the RNA sequence data and qRT-PCR validation revealed 16 putative genes, which were highly up regulated under drought stress condition, and were found to be targeted by ghr-miR169a and ghr-miR164, previously associated with NAC(NAM, ATAF1/2 and CUC2) and myeloblastosis (MYB), the top rank drought stress tolerance genes. These genes can be exploited further to aid in development of more drought tolerant cotton genotypes.
Project description:BACKGROUND:Late embryogenesis abundant (LEA) proteins are large groups of hydrophilic proteins with major role in drought and other abiotic stresses tolerance in plants. In-depth study and characterization of LEA protein families have been carried out in other plants, but not in upland cotton. The main aim of this research work was to characterize the late embryogenesis abundant (LEA) protein families and to carry out gene expression analysis to determine their potential role in drought stress tolerance in upland cotton. Increased cotton production in the face of declining precipitation and availability of fresh water for agriculture use is the focus for breeders, cotton being the backbone of textile industries and a cash crop for many countries globally. RESULTS:In this work, a total of 242, 136 and 142 LEA genes were identified in G. hirsutum, G. arboreum and G. raimondii respectively. The identified genes were classified into eight groups based on their conserved domain and phylogenetic tree analysis. LEA 2 were the most abundant, this could be attributed to their hydrophobic character. Upland cotton LEA genes have fewer introns and are distributed in all chromosomes. Majority of the duplicated LEA genes were segmental. Syntenic analysis showed that greater percentages of LEA genes are conserved. Segmental gene duplication played a key role in the expansion of LEA genes. Sixty three miRNAs were found to target 89 genes, such as miR164, ghr-miR394 among others. Gene ontology analysis revealed that LEA genes are involved in desiccation and defense responses. Almost all the LEA genes in their promoters contained ABRE, MBS, W-Box and TAC-elements, functionally known to be involved in drought stress and other stress responses. Majority of the LEA genes were involved in secretory pathways. Expression profile analysis indicated that most of the LEA genes were highly expressed in drought tolerant cultivars Gossypium tomentosum as opposed to drought susceptible, G. hirsutum. The tolerant genotypes have a greater ability to modulate genes under drought stress than the more susceptible upland cotton cultivars. CONCLUSION:The finding provides comprehensive information on LEA genes in upland cotton, G. hirsutum and possible function in plants under drought stress.
Project description:Drought stress massively restricts plant growth and the yield of crops. Reducing the deleterious effects of drought is necessary for agricultural industry. The plant-specific NAC (NAM, ATAF1/2 and CUC2) transcription factors (TFs) are widely involved in the regulation of plant development and stress response. One of the NAC TF, JUNGBRUNNEN1 (JUB1), has been reported to involve in drought resistance in Arabidopsis. However, little is known of how the JUB1 gene respond to drought stress in cotton. In the present study, we cloned GhJUB1L1, a homologous gene of JUB1 in upland cotton. GhJUB1L1 is preferentially expressed in stem and leaf and could be induced by drought stress. GhJUB1L1 protein localizes to the cell nucleus, and the transcription activation region of which is located in the C-terminal region. Silencing GhJUB1L1 gene via VIGS () reduced cotton drought tolerance, and retarded secondary cell wall (SCW) development. Additionally, the expression of some drought stress-related genes and SCW synthesis-related genes were altered in the GhJUB1L1 silencing plants. Collectively, our findings indicate that GhJUB1L1 may act as a positive regulator in response to drought stress and SCW development in cotton. Our results enriched the roles of NAC TFs in cotton drought tolerance and laid a foundation for the cultivation of transgenic cotton with higher drought tolerance.
Project description: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.
Project description:Late embryogenesis abundant (LEA) proteins play key roles in plant drought tolerance. In this study, 157, 85 and 89 candidate LEA2 proteins were identified in G. hirsutum, G. arboreum and G. raimondii respectively. LEA2 genes were classified into 6 groups, designated as group 1 to 6. Phylogenetic tree analysis revealed orthologous gene pairs within the cotton genome. The cotton specific LEA2 motifs identified were E, R and D in addition to Y, K and S motifs. The genes were distributed on all chromosomes. LEA2s were found to be highly enriched in non-polar, aliphatic amino acid residues, with leucine being the highest, 9.1% in proportion. The miRNA, ghr-miR827a/b/c/d and ghr-miR164 targeted many genes are known to be drought stress responsive. Various stress-responsive regulatory elements, ABA-responsive element (ABRE), Drought-responsive Element (DRE/CRT), MYBS and low-temperature-responsive element (LTRE) were detected. Most genes were highly expressed in leaves and roots, being the primary organs greatly affected by water deficit. The expression levels were much higher in G. tomentosum as opposed to G. hirsutum The tolerant genotype had higher capacity to induce more of LEA2 genes. Over expression of the transformed gene Cot_AD24498 showed that the LEA2 genes are involved in promoting root growth and in turn confers drought stress tolerance. We therefore infer that Cot_AD24498, CotAD_20020, CotAD_21924 and CotAD_59405 could be the candidate genes with profound functions under drought stress in upland cotton among the LEA2 genes. The transformed Arabidopsis plants showed higher tolerance levels to drought stress compared to the wild types. There was significant increase in antioxidants, catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) accumulation, increased root length and significant reduction in oxidants, Hydrogen peroxide (H2O2) and malondialdehyde (MDA) concentrations in the leaves of transformed lines under drought stress condition. This study provides comprehensive analysis of LEA2 proteins in cotton thus forms primary foundation for breeders to utilize these genes in developing drought tolerant genotypes.
Project description:BACKGROUND:Mitogen-activated protein kinase kinase kinases (MAPKKKs) are significant components in the MAPK signal pathway and play essential roles in regulating plants against drought stress. To explore MAPKKK gene family functioning in cotton response and resistance to drought stress, we conducted a systematic analysis of GhMAPKKKs. RESULTS:In this study, 157 nonredundant GhMAPKKKs (including 87 RAFs, 46 MEKKs and 24 ZIKs) were identified in cotton (Gossypium hirsutum). These GhMAPKKK genes are unevenly distributed on 26 chromosomes, and segmental duplication is the major way for the enlargement of MAPKKK family. Furthermore, members within the same subfamily share a similar gene structure and motif composition. A lot of cis-elements relevant to plant growth and response to stresses are distributed in promoter regions of GhMAPKKKs. Additionally, these GhMAPKKKs show differential expression patterns in cotton tissues. The transcription levels of most genes were markedly altered in cotton under heat, cold and PEG treatments, while the expressions of some GhMAPKKKs were induced in cotton under drought stress. Among these drought-induced genes, we selected GhRAF4 and GhMEKK12 for further functional characterization by virus-induced gene silencing (VIGS) method. The experimental results indicated that the gene-silenced cotton displayed decreased tolerance to drought stress. Malondialdehyde (MDA) content was higher, but proline accumulation, relative leaf water content and activities of superoxide dismutase (SOD) and peroxidase (POD) were lower in the gene-silenced cotton, compared with those in the controls, under drought stress. CONCLUSION:Collectively, a systematic survey of gene structure, chromosomal location, motif composition and evolutionary relationship of MAPKKKs were performed in upland cotton (Gossypium hirsutum). The following expression and functional study showed that some of them take important parts in cotton drought tolerance. Thus, the data presented here may provide a foundation for further investigating the roles of GhMAPKKKs in cotton response and resistance to drought stress.
Project description:<h4>Background</h4>Plants suffer from various abiotic stresses during their lifetime, of which drought and salt stresses are two main factors limiting crop yield and quality. Previous studies have shown that abscisic acid (ABA) responsive element binding protein (AREB)/ ABRE binding factors (ABFs) in bZIP transcription factors are involved in plant stress response in an ABA-dependent manner. However, little is known about the properties and functions of AREB/ABFs, especially ABF3, in cotton.<h4>Results</h4>Here, we reported the cloning and characterization of GhABF3. Expression of GhABF3 was induced by drought,salt and ABA treatments. Silencing of GhABF3 sensitized cotton to drought and salt stress, which was manifested in decreased cellular antioxidant capacity and chlorophyll content. Overexpression of GhABF3 significantly improved the drought and salinity tolerance of Arabidopsis and cotton. Exogenous expression of GhABF3 resulted in longer root length and less leaf wilting under stress conditions in Arabidopsis thaliana. Overexpressing GhABF3 significantly improved salt tolerance of upland cotton by reducing the degree of cellular oxidation, and enhanced drought tolerance by decreasing leaf water loss rate. The increased expression of GhABF3 up-regulated the transcriptional abundance of downstream ABA-inducible genes under salt stress in Arabidopsis.<h4>Conclusion</h4>In conclusion, our results demonstrated that GhABF3 plays an important role in plant drought and salt tolerance. Manipulation of GhABF3 by biotechnology might be an important strategy to alter the stress resistance of cotton.
Project description:Sucrose non-fermenting-1-related protein kinase 2 (SnRK2) is a plant-specific serine/threonine kinase family involved in the abscisic acid (ABA) signaling pathway and responds to osmotic stress. A genome-wide analysis of this protein family has been conducted previously in some plant species, but little is known about SnRK2 genes in upland cotton (Gossypium hirsutum L.). The recent release of the G. hirsutum genome sequence provides an opportunity to identify and characterize the SnRK2 kinase family in upland cotton.We identified 20 putative SnRK2 sequences in the G. hirsutum genome, designated as GhSnRK2.1 to GhSnRK2.20. All of the sequences encoded hydrophilic proteins. Phylogenetic analysis showed that the GhSnRK2 genes were classifiable into three groups. The chromosomal location and phylogenetic analysis of the cotton SnRK2 genes indicated that segmental duplication likely contributed to the diversification and evolution of the genes. The gene structure and motif composition of the cotton SnRK2 genes were analyzed. Nine exons were conserved in length among all members of the GhSnRK2 family. Although the C-terminus was divergent, seven conserved motifs were present. All GhSnRK2s genes showed expression patterns under abiotic stress based on transcriptome data. The expression profiles of five selected genes were verified in various tissues by quantitative real-time RT-PCR (qRT-PCR). Transcript levels of some family members were up-regulated in response to drought, salinity or ABA treatments, consistent with potential roles in response to abiotic stress.This study is the first comprehensive analysis of SnRK2 genes in upland cotton. Our results provide the fundamental information for the functional dissection of GhSnRK2s and vital availability for the improvement of plant stress tolerance using GhSnRK2s.
Project description:(1) Background: Upland cotton (Gossypium hirsutum L.) is the most important natural fiber worldwide, and it is extensively planted and plentifully used in the textile industry. Major cotton planting regions are frequently affected by abiotic stress, especially drought stress. Drought resistance is a complex, quantitative trait. A genome-wide association study (GWAS) constitutes an efficient method for dissecting the genetic architecture of complex traits. In this study, the drought resistance of a population of 316 upland cotton accessions was studied via GWAS. (2) Methods: GWAS methodology was employed to identify relationships between molecular markers or candidate genes and phenotypes of interest. (3) Results: A total of 8, 3, and 6 SNPs were associated with the euphylla wilting score (EWS), cotyledon wilting score (CWS), and leaf temperature (LT), respectively, based on a general linear model and a factored spectrally transformed linear mixed model. For these traits, 7 QTLs were found, of which 2 each were located on chromosomes A05, A11, and D03, and of which 1 was located on chromosome A01. Importantly, in the candidate regions WRKY70, GhCIPK6, SnRK2.6, and NET1A, which are involved in the response to abscisic acid (ABA), the mitogen-activated protein kinase (MAPK) signaling pathway and the calcium transduction pathway were identified in upland cotton at the seedling stage under drought stress according to annotation information and linkage disequilibrium (LD) block analysis. Moreover, RNA sequencing analysis showed that WRKY70, GhCIPK6, SnRK2.6, and NET1A were induced by drought stress, and the expression of these genes was significantly different between normal and drought stress conditions. (4) Conclusions: The present study should provide some genomic resources for drought resistance in upland cotton. Moreover, the germplasm of the different phenotypes, the detected SNPs and, the potential candidate genes will be helpful for molecular marker-assisted breeding studies about increased drought resistance in upland cotton.
Project description:The 2OG-Fe(II) oxygenase (RF) family of enzyme proteins can affect bulliform cells and cause leaf curling. However, there are few studies related to this family in cotton, and there has been no systematic analysis of RF genes. Here, we determined 25 RF genes in the complete genome sequence of upland cotton (<i>Gossypium hirsutum L.</i>) and 11 RF genes in the complete genome sequence of <i>Arabidopsis thaliana</i>. Cotton RF proteins can be divided into three categories. Whole genome/fragment and scattered replication events played an important role in the expansion of the RF gene family. qRT-PCR analysis results showed that RF genes respond to drought stress Pairwise comparison results showed that the expression of RF genes in Shi yuan 321 was higher than that in Kui 85-174. Overall, genome-wide identification approach was used to further analyze the related functions of the RF gene family, which may include the response to drought stress, in cotton.<h4>Supplementary information</h4>The online version contains supplementary material available at 10.1007/s12298-021-01065-4.
Project description:Long non-coding RNAs (lncRNAs) represent a class of riboregulators that either directly act in long form or are processed into shorter microRNAs (miRNAs) and small interfering RNAs. Long noncoding RNAs (lncRNAs) are arbitrarily defined as RNA genes larger than 200 nt in length that have no apparent coding potential. lncRNAs have emerged as playing important roles in various biological regulatory processes and are expressed in a more tissue-specific manner than mRNA. Emerging evidence shows that lncRNAs participate in stress-responsive regulation.In this study, in order to develop a comprehensive catalogue of lncRNAs in upland cotton under salt stress, we performed whole-transcriptome strand-specific RNA sequencing for three-leaf stage cotton seedlings treated with salt stress (S_NaCl) and controls (S_CK). In total we identified 1117 unique lncRNAs in this study and 44 differentially expressed RNAs were identified as potential non-coding RNAs. For the differentially expressed lncRNAs that were identified as intergenic lncRNAs (lincRNA), we analysed the gene ontology enrichment of cis targets and found that cis target protein-coding genes were mainly enriched in stress-related categories. Real-time quantitative PCR confirmed that all selected lincRNAs responsive to salt stress. We found lnc_388 was likely as regulator of Gh_A09G1182. And lnc_883 may participate in regulating tolerance to salt stress by modulating the expression of Gh_D03G0339 MS_channel. We then predicted the target mimics for miRNA in Gossypium. six miRNAs were identified, and the result of RT-qPCR with lncRNA and miRNA suggested that lnc_973 and lnc_253 may regulate the expression of ghr-miR399 and ghr-156e as a target mimic under salt stress.We identified 44 lincRNAs that were differentially expressed under salt stress. These lincRNAs may target protein-coding genes via cis-acting regulation. We also discovered that specifically-expressed lincRNAs under salt stress may act as endogenous target mimics for conserved miRNAs. These findings extend the current view on lincRNAs as ubiquitous regulators under stress stress.