Project description:The role of microRNAs (miRNAs) during cotton fiber development remains unclear. Here, a total of 54 miRNAs belonging to 39 families were selected to characterize miRNA regulatory mechanism in eight different fiber development stages in upland cotton cv BM-1. Among 54 miRNAs, 18 miRNAs were involved in cotton fiber initiation and eight miRNAs were related to fiber elongation and secondary wall biosynthesis. Additionally, 3,576 protein-coding genes were candidate target genes of these miRNAs, which are potentially involved in cotton fiber development. We also investigated the regulatory network of miRNAs and corresponding targets in fiber initiation and elongation, and secondary wall formation. Our Gene Ontology-based term classification and KEGG-based pathway enrichment analyses showed that the miRNA targets covered 220 biological processes, 67 molecular functions, 45 cellular components, and 10 KEGG pathways. Three of ten KEGG pathways were involved in lignan synthesis, cell elongation, and fatty acid biosynthesis, all of which have important roles in fiber development. Overall, our study shows the potential regulatory roles of miRNAs in cotton fiber development and the importance of miRNAs in regulating different cell types. This is helpful to design miRNA-based biotechnology for improving fiber quality and yield.
Project description:Fiber quality is an important economic index and a major breeding goal in cotton, but direct phenotypic selection is often hindered due to environmental influences and linkage with yield traits. A genome-wide association study (GWAS) is a powerful tool to identify genes associated with phenotypic traits. In this study, we identified fiber quality genes in upland cotton (Gossypium hirsutum L.) using GWAS based on a high-density CottonSNP80K array and multiple environment tests. A total of 30 and 23 significant single nucleotide polymorphisms (SNPs) associated with five fiber quality traits were identified across the 408 cotton accessions in six environments and the best linear unbiased predictions, respectively. Among these SNPs, seven loci were the same, and 128 candidate genes were predicted in a 1-Mb region (±500 kb of the peak SNP). Furthermore, two major genome regions (GR1 and GR2) associated with multiple fiber qualities in multiple environments on chromosomes A07 and A13 were identified, and within them, 22 candidate genes were annotated. Of these, 11 genes were expressed [log2(1 + FPKM)>1] in the fiber development stages (5, 10, 20, and 25 dpa) using RNA-Seq. This study provides fundamental insight relevant to identification of genes associated with fiber quality and will accelerate future efforts toward improving fiber quality of upland cotton.
Project description:BACKGROUND: Cotton fiber development undergoes rapid and dynamic changes in a single cell type, from fiber initiation, elongation, primary and secondary wall biosynthesis, to fiber maturation. Previous studies showed that cotton genes encoding putative MYB transcription factors and phytohormone responsive factors were induced during early stages of ovule and fiber development. Many of these factors are targets of microRNAs (miRNAs) that mediate target gene regulation by mRNA degradation or translational repression. RESULTS: Here we sequenced and analyzed over 4 million small RNAs derived from fiber and non-fiber tissues in cotton. The 24-nucleotide small interfering RNAs (siRNAs) were more abundant and highly enriched in ovules and fiber-bearing ovules relative to leaves. A total of 31 miRNA families, including 27 conserved, 4 novel miRNA families and a candidate-novel miRNA, were identified in at least one of the cotton tissues examined. Among 32 miRNA precursors representing 19 unique miRNA families identified, 7 were previously reported, and 25 new miRNA precursors were found in this study. Sequencing, miRNA microarray, and small RNA blot analyses showed a trend of repression of miRNAs, including novel miRNAs, during ovule and fiber development, which correlated with upregulation of several target genes tested. Moreover, 223 targets of cotton miRNAs were predicted from the expressed sequence tags derived from cotton tissues, including ovules and fibers. The cotton miRNAs examined triggered cleavage in the predicted sites of the putative cotton targets in ovules and fibers. CONCLUSIONS: Enrichment of siRNAs in ovules and fibers suggests active small RNA metabolism and chromatin modifications during fiber development, whereas general repression of miRNAs in fibers correlates with upregulation of a dozen validated miRNA targets encoding transcription and phytohormone response factors, including the genes found to be highly expressed in cotton fibers. Rapid and dynamic changes in siRNAs and miRNAs may contribute to ovule and fiber development in allotetraploid cotton.
Project description:Abiotic stress is a major environmental factor that limits cotton growth and yield, moreover, this problem has become more and more serious recently, as multiple stresses often occur simultaneously due to the global climate change and environmental pollution. In this study, we sought to identify genes involved in diverse stresses including abscisic acid (ABA), cold, drought, salinity and alkalinity by comparative microarray analysis. Our result showed that 5790, 3067, 5608, 778 and 6148 transcripts, were differentially expressed in cotton seedlings under treatment of ABA (1 ?M ABA), cold (4°C), drought (200 mM mannitol), salinity (200 mM NaCl) and alkalinity (pH=11) respectively. Among the induced or suppressed genes, 126 transcripts were shared by all of the five kinds of abiotic stresses, with 64 up-regulated and 62 down-regulated. These common members are grouped as stress signal transduction, transcription factors (TFs), stress response/defense proteins, metabolism, transport facilitation, as well as cell wall/structure, according to the function annotation. We also noticed that large proportion of significant differentially expressed genes specifically regulated in response to different stress. Nine of the common transcripts of multiple stresses were selected for further validation with quantitative real time RT-PCR (qRT-PCR). Furthermore, several well characterized TF families, for example, WRKY, MYB, NAC, AP2/ERF and zinc finger were shown to be involved in different stresses. As an original report using comparative microarray to analyze transcriptome of cotton under five abiotic stresses, valuable information about functional genes and related pathways of anti-stress, and/or stress tolerance in cotton seedlings was unveiled in our result. Besides this, some important common factors were focused for detailed identification and characterization. According to our analysis, it suggested that there was crosstalk of responsive genes or pathways to multiple abiotic or even biotic stresses, in cotton. These candidate genes will be worthy of functional study under diverse stresses.
Project description:Cotton leaf curl Allahabad virus (CLCuAV) belongs to genus Begomovirus, family Geminiviridae. It has single stranded monopartite DNA genome transmitted by whitefly (Bemisia tabaci). MicroRNAs (miRNAs) belong to class of endogeneous small RNAs which suppress expression of genes following cleavage or translational inhibition of target messenger RNAs. They are demonstrated to be involved in a number of plant processes such as, development, biotic and abiotic stresses. Employing in silico approach, high scoring miRNA-target pairs satisfying rules of minimum free energy and maximum complementarity were selected to investigate if they possess the potential to bind the genome CLCuAV. Our results revealed that miRNA species viz., ghr-miR2950 can target all the viral genes, ghr-miR408 targets overlapping transcripts of AC1 and AC2 genes; while ghr-miR394 and ghr-miR395a and miR395d could bind overlapping transcripts of AC1 and AC4 genes. This is the first report of prediction of cotton miRNAs which have the potential to target CLCuAV genes including AC1 and AC4, involved in viral replication and gene silencing suppression, respectively.
Project description:The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 system has been widely used for genome editing in various plants because of its simplicity, high efficiency and design flexibility. However, to our knowledge, there is no report on the application of CRISPR/Cas9-mediated targeted mutagenesis in cotton. Here, we report the genome editing and targeted mutagenesis in upland cotton (Gossypium hirsutum L., hereafter cotton) using the CRISPR/Cas9 system. We designed two guide RNAs to target distinct sites of the cotton Cloroplastos alterados 1 (GhCLA1) and vacuolar H+-pyrophosphatase (GhVP) genes. Mutations in these two genes were detected in cotton protoplasts. Most of the mutations were nucleotide substitutions, with one nucleotide insertion and one substitution found in GhCLA1 and one deletion found in GhVP in cotton protoplasts. Subsequently, the two vectors were transformed into cotton shoot apexes through Agrobacterium-mediated transformation, resulting in efficient target gene editing. Most of the mutations were nucleotide deletions, and the mutation efficiencies were 47.6-81.8% in transgenic cotton plants. Evaluation using restriction-enzyme-PCR assay and sequence analysis detected no off-target mutations. Our results indicated that the CRISPR/Cas9 system was an efficient and specific tool for targeted mutagenesis of the cotton genome.
Project description:Dwarf cottons are more resistant to damage from wind and rain and associated with stable, increased yields, and also desirable source for breeding the machine harvest varieties. In an effort to uncover the transcripts and miRNA networks involved in plant height, the transcriptome and small RNA sequencing were performed based on dwarf mutant Ari1327 (A1), tall-culm mutant Ari3697 (A3) and wild type Ari971 (A9) in Gossypium hirsutum.The stem apexes of wild-type upland cotton (Ari971) and its dwarf mutant (Ari1327) and tall-culm mutant (Ari3697) at the fifth true leaf stage were extracted for RNA, respectively. Transcriptome and small RNA libraries were constructed and subjected to next generation sequencing.The transcriptome sequencing analysis showed that the enriched pathways of top 3 differentially expressed genes (DEGs) were categorized as carotenoid biosynthesis, plant-pathogen interaction and plant hormone signal transduction in both A1-A9 and A3-A9. The ABA and IAA related factors were differentially expressed in the mutants. Importantly, we found the lower expressed SAUR and elevated expressed GH3, and ABA related genes such as NCED and PP2C maybe relate to reduced growth of the plant height in Ari1327 which was consistent with the higher auxin and ABA content in this mutant. Furthermore, miRNA160 targeted to the auxin response factor (ARF) and miRNA166 (gma-miR166u and gma-miR166h-3p) targeted to ABA responsive element binding factor were related to the mutation in cotton. We have noticed that the cell growth related factors (smg7 targeted by gra-miR482 and 6 novel miRNAs and pectate-lyases targeted by osa-miR159f), the redox reactions related factors (Cytochrome P450 targeted by miR172) and MYB genes targeted by miR828, miR858 and miR159 were also involved in plant height of the cotton mutants. A total of 226 conserved miRNAs representing 32 known miRNA families were obtained, and 38 novel miRNAs corresponding to 23 unique RNA sequences were identified. Total 531 targets for 211 conserved miRNAs were obtained. Using PAREsnip, 27 and 29 miRNA/target conserved interactions were validated in A1-A9 and A3-A9, respectively. Furthermore, miRNA160, miRNA858 and miRNA172 were validated to be up-regulated in A1-A9 but down-regulated in A3-A9, whereas miRNA159 showed the opposite regulation.This comprehensive interaction of the transcriptome and miRNA at tall-culm and dwarf mutant led to the discovery of regulatory mechanisms in plant height. It also provides the basis for in depth analyses of dwarf mutant genes for further breeding of dwarf cotton.
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.
Project description:The single-celled cotton (Gossypium hirsutum) fiber provides an excellent model to investigate how human selection affects phenotypic evolution. To gain insight into the evolutionary genomics of cotton domestication, we conducted comparative transcriptome profiling of developing cotton fibers using RNA-Seq. Analysis of single-celled fiber transcriptomes from four wild and five domesticated accessions from two developmental time points revealed that at least one-third and likely one-half of the genes in the genome are expressed at any one stage during cotton fiber development. Among these, ~5,000 genes are differentially expressed during primary and secondary cell wall synthesis between wild and domesticated cottons, with a biased distribution among chromosomes. Transcriptome data implicate a number of biological processes affected by human selection, and suggest that the domestication process has prolonged the duration of fiber elongation in modern cultivated forms. Functional analysis suggested that wild cottons allocate greater resources to stress response pathways, while domestication led to reprogrammed resource allocation toward increased fiber growth, possibly through modulating stress-response networks. This first global transcriptomic analysis using multiple accessions of wild and domesticated cottons is an important step toward a more comprehensive systems perspective on cotton fiber evolution. The understanding that human selection over the past 5,000+ years has dramatically re-wired the cotton fiber transcriptome sets the stage for a deeper understanding of the genetic architecture underlying cotton fiber synthesis and phenotypic evolution.
Project description:MicroRNAs (miRNAs) are endogenous non-coding ~21 nucleotide RNAs that regulate gene expression at the transcriptional and post-transcriptional levels in plants and animals. They play an important role in development, abiotic stress, and pathogen responses. miRNAs with their targets have been widely studied in model plants, but limited knowledge is available on the small RNA population of cotton (Gossypium hirsutum)-an important economic crop, and global identification of related targets through degradome sequencing has not been developed previously. In this study, small RNAs and their targets were identified during cotton somatic embryogenesis (SE) through high-throughput small RNA and degradome sequencing, comparing seedling hypocotyl and embryogenic callus (EC) of G. hirsutum YZ1. A total of 36 known miRNA families were found to be differentially expressed, of which 19 miRNA families were represented by 29 precursors. Twenty-five novel miRNAs were identified. A total of 234 transcripts in EC and 322 transcripts in control (CK) were found to be the targets of 23 and 30 known miRNA families, respectively, and 16 transcripts were targeted by eight novel miRNAs. Interestingly, four trans-acting small interfering RNAs (tas3-siRNAs) were also found in degradome libraries, three of which perfectly matched their precursors. Several targets were further validated via RNA ligase-mediated rapid amplification of 5' cDNA ends (RLM 5'-RACE). The profiling of the miRNAs and their target genes provides new information on the miRNAs network during cotton SE.