Delong Wang

The MAPKKK gene family in Gossypium raimondii: genome-wide identification, classification and expression analysis.

Mitogen-activated protein kinase (MAPK) cascades are conserved signal transduction pathways in all eukaryotic organisms. MAPKKKs (MAPK kinase kinases) operate at the top levels of these cascades. Recently, this family of genes has been systematically investigated in Arabidopsis, rice and maize, but has not yet been characterized in cotton. In this study, we identified 78 putative MAPKKK genes in the genome of the diploid cotton, Gossypium raimondii. They were classified into three subfamilies, of which 12 were ZIK, 22 were MEKK and 44 were Raf. The ZIK and MEKK genes displayed a scattered genomic distribution across 11 of the 13 chromosomes, whereas Raf genes were distributed across the entire genome. Their conserved patterns observed for introns and additional domains were consistent with the evolutionary relationships inferred from the phylogenetic analysis within subfamily. Transcriptome sequencing data were used to investigate their transcript profiles in mature leaves, 0 day and 3 days post-anthesis (DPA) ovules. Sixty MAPKKK genes were expressed, of which 41 were strongly expressed in mature leaves. Twelve MAPKKK genes were more highly expressed in 3-DPA ovules than in 0-DPA ovules. Our results provide a foundation for future evolutionary and functional characterizations of MAPKKK genes in cotton and probably other Gossypium plants.

Targeted mutagenesis in cotton ( Gossypium hirsutum L.) using the CRISPR/Cas9 system

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.

Genome-Wide Analysis of Long Noncoding RNAs and Their Responses to Drought Stress in Cotton (Gossypium hirsutum L.)

Recent researches on long noncoding RNAs (lncRNAs) have expanded our horizon of gene regulation and the cellular complexity. However, the number, characteristics and expression patterns of lncRNAs remain poorly characterized and how these lncRNAs biogenesis are regulated in response to drought stress in cotton are still largely unclear. In the study, using a reproducibility-based RNA-sequencing and bioinformatics strategy to analyze the lncRNAs of 9 samples under three different environment stresses (control, drought stress and re-watering, three replications), we totally identified 10,820 lncRNAs of high-confidence through five strict steps filtration, of which 9,989 were lincRNAs, 153 were inronic lncRNAs, 678 were anti-sense lncRNAs. Coding function analysis showed 6,470 lncRNAs may have the ability to code proteins. Small RNAs precursor analysis revealed that 196 lncRNAs may be the precursors to small RNAs, most of which (35.7%, 70) were miRNAs. Expression patterns analysis showed that most of lncRNAs were expressed at a low level and most inronic lncRNAs (75.95%) had a consistent expression pattern with their adjacent protein-coding genes. Further analysis of transcriptome data uncovered that lncRNAs XLOC_063105 and XLOC_115463 probably function in regulating two adjacent coding genes CotAD_37096 and CotAD_12502, respectively. Investigations of the content of plant hormones and proteomics analysis under drought stress also complemented the prediction. We analyzed the characteristics and the expression patterns of lncRNAs under drought stress and re-watering treatment, and found lncRNAs may be likely to involve in regulating plant hormones pathway in response to drought stress.

Genome-wide Identification and analysis of the stress-resistance function of the TPS (Trehalose-6-Phosphate Synthase) gene family in cotton

Background Trehalose (a-D-glucopyranosyl a-D-glucopyranoside) is a nonreducing disaccharide and is widely distributed in bacteria, fungi, algae, plants and invertebrates. In the study, the identification of trehalose-6-phosphate synthase (TPS) genes stress-related in cotton, and the genetic structure analysis and molecular evolution analysis of TPSs were conducted with bioinformatics methods, which could lay a foundation for further research of TPS functions in cotton. Results The genome information of Gossypium raimondii (group D), G. arboreum L. (group A), and G. hirsutum L. (group AD) was used in the study. Fifty-three TPSs were identified comprising 15 genes in group D, 14 in group A, and 24 in group AD. Bioinformatics methods were used to analyze the genetic structure and molecular evolution of TPSs. Real-time PCR analysis was performed to investigate the expression patterns of gene family members. All TPS family members in cotton can be divided into two subfamilies: Class I and Class II. The similarity of the TPS sequence is high within the same species and close within their family relatives. The genetic structures of two TPS subfamily members are different, with more introns and a more complicated gene structure in Class I. There is a TPS domain(Glyco transf_20) at the N-terminal in all TPS family members and a TPP domain(Trehalose_PPase) at the C-terminal in all except GrTPS6, GhTPS4, and GhTPS9. All Class II members contain a UDP-forming domain. The responses to environmental stresses showed that stresses could induce the expression of TPSs but the expression patterns vary with different stresses. Conclusions The distribution of TPSs varies with different species but is relatively uniform on chromosomes. Genetic structure varies with different gene members, and expression levels vary with different stresses and exhibit tissue specificity. The upregulated genes in upland cotton TM-1 is significantly more than that in G. raimondii and G. arboreum L. Shixiya 1. Electronic supplementary material The online version of this article (doi:10.1186/s12863-016-0360-y) contains supplementary material, which is available to authorized users.BackgroundTrehalose (a-D-glucopyranosyl a-D-glucopyranoside) is a nonreducing disaccharide and is widely distributed in bacteria, fungi, algae, plants and invertebrates. In the study, the identification of trehalose-6-phosphate synthase (TPS) genes stress-related in cotton, and the genetic structure analysis and molecular evolution analysis of TPSs were conducted with bioinformatics methods, which could lay a foundation for further research of TPS functions in cotton.ResultsThe genome information of Gossypium raimondii (group D), G. arboreum L. (group A), and G. hirsutum L. (group AD) was used in the study. Fifty-three TPSs were identified comprising 15 genes in group D, 14 in group A, and 24 in group AD. Bioinformatics methods were used to analyze the genetic structure and molecular evolution of TPSs. Real-time PCR analysis was performed to investigate the expression patterns of gene family members. All TPS family members in cotton can be divided into two subfamilies: Class I and Class II. The similarity of the TPS sequence is high within the same species and close within their family relatives. The genetic structures of two TPS subfamily members are different, with more introns and a more complicated gene structure in Class I. There is a TPS domain(Glyco transf_20) at the N-terminal in all TPS family members and a TPP domain(Trehalose_PPase) at the C-terminal in all except GrTPS6, GhTPS4, and GhTPS9. All Class II members contain a UDP-forming domain. The responses to environmental stresses showed that stresses could induce the expression of TPSs but the expression patterns vary with different stresses.ConclusionsThe distribution of TPSs varies with different species but is relatively uniform on chromosomes. Genetic structure varies with different gene members, and expression levels vary with different stresses and exhibit tissue specificity. The upregulated genes in upland cotton TM-1 is significantly more than that in G. raimondii and G. arboreum L. Shixiya 1.

Single-base resolution methylomes of upland cotton ( Gossypium hirsutum L.) reveal epigenome modifications in response to drought stress

BackgroundDNA methylation, with a cryptic role in genome stability, gene transcription and expression, is involved in the drought response process in plants, but the complex regulatory mechanism is still largely unknown.ResultsHere, we performed whole-genome bisulfite sequencing (WGBS) and identified long non-coding RNAs on cotton leaves under drought stress and re-watering treatments. We obtained 31,223 and 30,997 differentially methylated regions (representing 2.48% of the genome) after drought stress and re-watering treatments, respectively. Our data also showed that three sequence contexts, including mCpG, mCHG, mCHH, all presented a hyper-methylation pattern under drought stress and were nearly restored to normal levels after the re-watering treatment. Among all the methylation variations, asymmetric CHH methylation was the most consistent with external environments, suggesting that methylation/demethylation in a CHH context may constitute a novel epigenetic modification in response to drought stress. Combined with the targets of long non-coding RNAs, we found that long non-coding RNAs may mediate variations in methylation patterns by splicing into microRNAs. Furthermore, the many hormone-related genes with methylation variations suggested that plant hormones might be a potential mechanism in the drought response.ConclusionsFuture crop-improvement strategies may benefit by taking into account not only the DNA genetic variations in cotton varieties but also the epigenetic modifications of the genome.

Epigenetic mechanisms of salt tolerance and heterosis in Upland cotton (Gossypium hirsutum L.) revealed by methylation-sensitive amplified polymorphism analysis

In order to explore the molecular mechanism of salt tolerance and heterosis in cotton, a methylation-sensitive amplification polymorphism method based on capillary electrophoresis was used to analyze DNA methylation level in a cotton hybrid CCRI 29 and its two parents. The major results and conclusions are as follows: Firstly, salt tolerance test showed that CCRI 29 had higher salt-tolerance level than its both parents. The global DNA methylation level in CCRI 29 under salt treatment significantly increased, whereas the two parents did not change significantly between salt treatment and control. All kinds of variation of DNA methylation happened in cotton under salt treatment, and hypermethylation happened at a significantly higher rate than that of hypomethylation in CCRI 29 but not in its two parents. The results suggested that the increase of global DNA methylation level in cotton genome and also different methylation types played an important role in tolerance to salt treatment in cotton. Secondly, both hypomethylation and hypermethylation happened to different genes and some genes maintained the same methylation level after salt stress, which meant that complex gene expression alterations occurred when responding to salt stress in cotton, indicating the complicated characteristics of roles that specific genes played in salt tolerance. Thirdly, although most cytosine methylation sites in hybrid CCRI 29 shared the same status as that of at least one of the parents, the site number of hypomethylation is significantly higher than that of hypermethylation in CCRI 29 compared to parents under both control and salt stress, indicating that demethylation could be the mechanism to explain heterosis in cotton hybrid.

GhSOS1, a plasma membrane Na+/H+ antiporter gene from upland cotton, enhances salt tolerance in transgenic Arabidopsis thaliana

Upland cotton (Gossypium hirsutum L.), an important source of natural fiber, can tolerate relatively high salinity and drought stresses. In the present study, a plasma membrane Na+/H+ antiporter gene, GhSOS1, was cloned from a salt-tolerant genotype of G. hirsutum, Zhong 9807. The expression level of GhSOS1 in cotton roots was significantly upregulated in the presence of high concentrations of NaCl (200 mM), while its transcript abundance was increased when exposed to low temperature and drought stresses. Localization analysis using onion epidermal cells showed that the GhSOS1 protein was localized to the plasma membrane. The overexpression of GhSOS1 in Arabidopsis enhanced tolerance to salt stress, as indicated by a lower MDA content and decreased Na+/K+ ratio in transgenic plants. Moreover, the transcript levels of stress-related genes were significantly higher in GhSOS1 overexpression lines than in wild-type plants under salt treatment. Hence, GhSOS1 may be a potential target gene for enhancing salt tolerance in transgenic plants.

Mining and Analysis of SNP in Response to Salinity Stress in Upland Cotton (Gossypium hirsutum L.).

Salinity stress is a major abiotic factor that affects crop output, and as a pioneer crop in saline and alkaline land, salt tolerance study of cotton is particularly important. In our experiment, four salt-tolerance varieties with different salt tolerance indexes including CRI35 (65.04%), Kanghuanwei164 (56.19%), Zhong9807 (55.20%) and CRI44 (50.50%), as well as four salt-sensitive cotton varieties including Hengmian3 (48.21%), GK50 (40.20%), Xinyan96-48 (34.90%), ZhongS9612 (24.80%) were used as the materials. These materials were divided into salt-tolerant group (ST) and salt-sensitive group (SS). Illumina Cotton SNP 70K Chip was used to detect SNP in different cotton varieties. SNPv (SNP variation of the same seedling pre- and after- salt stress) in different varieties were screened; polymorphic SNP and SNPr (SNP related to salt tolerance) were obtained. Annotation and analysis of these SNPs showed that (1) the induction efficiency of salinity stress on SNPv of cotton materials with different salt tolerance index was different, in which the induction efficiency on salt-sensitive materials was significantly higher than that on salt-tolerant materials. The induction of salt stress on SNPv was obviously biased. (2) SNPv induced by salt stress may be related to the methylation changes under salt stress. (3) SNPr may influence salt tolerance of plants by affecting the expression of salt-tolerance related genes.

Comparative Analysis of Cotton Small RNAs and Their Target Genes in Response to Salt Stress

Small RNAs play an important role in regulating plant responses to abiotic stress. Depending on the method of salt application, whether sudden or gradual, plants may experience either salt shock or salt stress, respectively. In this study, small RNA expression in response to salt shock and long-term salt stress in parallel experiments was described. Cotton small RNA libraries were constructed and sequenced under normal conditions, as well as sudden and gradual salt application. A total of 225 cotton microRNAs (miRNAs) were identified and of these 24 were novel miRNAs. There were 88 and 75 miRNAs with differential expression under the salt shock and long-term salt stress, respectively. Thirty one transcripts were found to be targets of 20 miRNA families. Eight targets showed a negative correlation in expression with their corresponding miRNAs. We also identified two TAS3s with two near-identical 21-nt trans-acting small interfering RNA (tasiRNA)-Auxin Response Factors (ARFs) that coaligned with the phases D7(+) and D8(+) in three Gossypium species. The miR390/tasiRNA-ARFs/ARF4 pathway was identified and showed altered expression under salt stress. The identification of these small RNAs as well as elucidating their functional significance broadens our understanding of post-transcriptional gene regulation in response to salt stress.

Genome-wide identification and expression specificity analysis of the DNA methyltransferase gene family under adversity stresses in cotton

DNA methylation is an important epigenetic mode of genomic DNA modification that is an important part of maintaining epigenetic content and regulating gene expression. DNA methyltransferases (MTases) are the key enzymes in the process of DNA methylation. Thus far, there has been no systematic analysis the DNA MTases found in cotton. In this study, the whole genome of cotton C5-Mtase coding genes was identified and analyzed using a bioinformatics method based on information from the cotton genome. In this study, 51 DNA MTase genes were identified, of which 8 belonged to G. raimondii (group D), 9 belonged to G. arboretum L. (group A), 16 belonged to G. hirsutum L. (group AD1) and 18 belonged to G. barbadebse L. (group AD2). Systematic evolutionary analysis divided the 51 genes into four subfamilies, including 7 MET homologous proteins, 25 CMT homologous proteins, 14 DRM homologous proteins and 5 DNMT2 homologous proteins. Further studies showed that the DNA MTases in cotton were more phylogenetically conserved. The comparison of their protein domains showed that the C-terminal functional domain of the 51 proteins had six conserved motifs involved in methylation modification, indicating that the protein has a basic catalytic methylation function and the difference in the N-terminal regulatory domains of the 51 proteins divided the proteins into four classes, MET, CMT, DRM and DNMT2, in which DNMT2 lacks an N-terminal regulatory domain. Gene expression in cotton is not the same under different stress treatments. Different expression patterns of DNA MTases show the functional diversity of the cotton DNA methyltransferase gene family. VIGS silenced Gossypium hirsutum l. in the cotton seedling of DNMT2 family gene GhDMT6, after stress treatment the growth condition was better than the control. The distribution of DNA MTases varies among cotton species. Different DNA MTase family members have different genetic structures, and the expression level changes with different stresses, showing tissue specificity. Under salt and drought stress, G. hirsutum L. TM-1 increased the number of genes more than G. raimondii and G. arboreum L. Shixiya 1. The resistance of Gossypium hirsutum L.TM-1 to cold, drought and salt stress was increased after the plants were silenced with GhDMT6 gene.