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.
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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-017-3681-y) contains supplementary material, which is available to authorized users.
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.
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