Study On The Mechanism Of Salt-tolerance In Cotton Based On The Transcriptome Sequencing And SNP Array Analysis

Cotton is an economically important crop throughout the world,and also is a pioneer crop in salt stress tolerance research.Nevertheless,in recent years,due to the adjustment of cultivated area and climate change,the demand for cotton adversity resistance is higher and higher.Based on the advantage of salt tolerance of cotton,the study on the salt tolerance can contribute to the salt tolerance mechanism research and the breeding of salt tolerant varieties of crops.In present study,based on the transcriptional sequencing and SNP microarray technologies,the mechanism of salt tolerance in cotton was studied at the transcriptome,spliceosome and genome levels.The main results are as follows:1.The diploid cotton species Gossypium davidsonii which has superior stress tolerance was selected for salt-tolerant mechanisms study.Plant was treated with 200 mM NaCl,when seedlings cultured to containing two simple leaves and one heart-shaped leaf.The roots and leaves at five salt treatment periods were selected for transcriptome analysis by next generation sequencing technology.Compared to the released Gossypium raimondii genome sequence,43,219 genes were identified in G.davidsonii,including 6,173 novel genes.Compared to the control,a total of 4,744 and 5,337 differentially expressed genes(DEGs)were found to be involved in salt stress tolerance in roots and leaves,respectively.Gene function annotation showed that these differentially expressed genes mainly involved in plant stress response,metabolism and photosynthesis.According to the number of differentially expressed genes in different tissues and periods,the expression of these salt stress responsive genes had obvious space-time specificity.Based on the differences in gene expression and functional classification at different time periods after salt treatment,the process of salt stress response in cotton was divided into two parts,namely,early stress stage(12-24 h)and late stage(48-144 h).The roots responded to stress strongly at the early stage,and the related genes such as osmotic regulators,ion transport,antioxidant enzymes and a large number of transcription factors were differentially expressed at this stage.At the late stage,the number of differentially expressed genes decreased gradually,and there were no significant changes in physiological processes compared with conventional treatments.In leaves,a few genes related to osmotic stress were differentially expressed at the early stage.As the stress time went on,the expression of genes such as ion transport and redox activities became active.At the late stage,a large number of genes differentially expressed,including not only stress responsive genes,but also genes related to plant growth and development,such as hormones,carbohydrates,Oils and fats.In addition,a large number of genes related to the process of photosynthesis were down regulated at this stage,suggesting that energy metabolism and normal growth and development of plants were seriously affected under long-term salt stress.Pathway analysis showed that the key genes in the SOS and ROS pathways were very active under salt induction.The SOS pathway plays an important role in the maintenance of ion homeostasis.At present,most studies have shown that the plant mainly achieve the efflux of intracellular Na+ according to the SOS3-SOS2-SOS1 pathway.However,the differential expression of SOS3 was not detected in this study,but a SCaBP8 with the same function as SOS3 was highly expressed under salt stress.Therefore,cotton may be mainly controlled by SCaBP8-SOS2-SOS1 signaling pathways to regulate efflux of Na+ and achieve ion homeostasis.In addition,we found that a large number of K+transport related genes were differentially expressed under salt stress,such as HKT1,KUP1,and KUP2.This result suggests that active K+transport plays an important role in maintaining K+/Na+ ion ratios and reducing Na+ toxicity in cotton.Therefore,in maintaining ion balance,cotton mainly through two pathways,Na+efflux and K+accumulation.In addition,the ROS pathwayROS pathway also plays an important role in salt stress response.Under salt stress,accumulation of ROS can cause serious damage to plant cell components.2.Based on the study at the transcription level,we further studied the alternative splicing(AS)at post transcriptional in G.davidsonii under salt stress.The number of AS events between the tissues and between the stresses were counted.It was found that 5,069 were common in roots and leaves,and 5,035 and 5,893 events were specific in roots and leaves,respectively.In addition.8,271 were common in control and treatment group,and 3,618 and 4,108 were specific in control and treatment group,respectively.The analysis revealed that a total of 14,172 AS events were identified in 6,798 genes,indicating that 31.58%of the multiple exon genes underwent AS.In addition,we found that the majority of AS events were different in different ecotypes,different tissues or different stress environments,suggesting that splicing regulation played an important role in gene diversity.Under salt stress,the number of AS events were increased in both roots and leaves,and the increased AS events directly caused an increase in the number of gene isoforms in the two tissues.In order to explore how the DAS events impacted to the functional genes,the Fisher exact test was used to identify the DAS events with significant difference.Finally,1,287 and 1,228 DAS were found in roots and leaves,respectively.Functional analysis of the DAS genes induced by salt stress revealed that most of these genes were related to stress response process in plants.In addition,some processes realted to photosynthetic were enriched in leaves specifically.This result suggests that the DAS genes induced by salt stress are mainly involved in stress response,implying the functional specificity of AS events under salt stress.In addition,we found that the Ca2+ response process was significantly enriched.We hypothesized that the genes in the Ca2+signaling pathway are more susceptible to AS regulation under Salt Stress and these frequently AS genes may play an important role in salt stress response in plants.As a regulator of AS regulation,29 Arabidopsis homologues genes,which encoding SR proteins,were identified in G.davidsonii and 13 of them were differential splicing under salt stress.However,only two genes encoding SR proteins were differential expresiion in G.davidsonii expression profile,implying that SR proteins were more susceptible to AS regulation than transcription regulation.Consistent with most other researches,a large number of splcing factors(SFs),including SR proteins,exist a regulatory and self regulatory process.This regulatory mechanism greatly enriches the diversity of SFs,thereby increasing the diversity of AS events and ultimately leading to the diversity of gene isoforms in plant.Accroding to the heatmap analysis of the DAS events in different tissues and periods,we found that AS events had high spatial and temporal sensitivity.Few AS could be detected continuous differential splicing in both tissues or at long-term period.In addition,in view of the fact that AS regulation is regulated on the basis of transcription products,its effects on gene function are limited by gene transcription abundance.It is the secondary fine-tuning of transcriptional regulation and the partern of regulation is more moderate and diverse than transcriptional regulation.When plants respond to different circumstances,such fine-tuning often does not cause intense harm to plants,but gradually adjusts to gradually adapt to the environment.When plants suffering from different adversities,such fine tuning often does not cause intense damage to plants,but gradually adapts itself to the environment in constant adjustment.Studies have shown that the diversity of AS is increasing in the long evolutionary process of animals or plants.Therefore,we suggest that AS regulation plays an important role in adaptive evolution of plants.3.The mechanism of salt tolerance in plants was also revealed by genome level analysis in present study.Based on the high quality reference sequence of the re-sequencing data from 100 different cultivars of G.hirsutum,we developed a high-density SNP array,which named CottonSNP80K,for high-throughput intraspecific upland cotton genotyping identification.The array contained 77,774 SNP loci,with 45,183(58.10%)and 32,591(41.90%)in the At and Dt subgenome,respectively.Genotyping was performed on 352 cotton samples using CottonSNP80K array.59,502(76.51%)loci showed polymorphism,and 57,071(95.91%)loci showed the minor allele frequency(MAF)greater than 0.05.The quality of the array was assessed by 22 cotton samples.It was proved to be of good genotyping efficiency.And it was suitable for the study of genetic diversity and molecular breeding of upland cotton.In combination with the data related to salt tolerance of Upland Cotton in our previous study,we performed GWAS analysis with 288 upland cotton accessions by using CottonSNP80K array.According to the analysis,8 SNP loci on 4 chromosomes were identified associated with salt stress response.Further,in the linkage regions of these loci,we identified 36 genes related to stimulation responses.According to the gene expression data of G.davidsonii,24 of these 36 genes were differentially expressed under salt stress.Among them,genes such as HB7,FMO1 and NRT2.1,which have been reported involved in salt stress response,exhibit characteristic expression patterns under salt stress.The study provided gene resources for further analysis of salt tolerance mechanism and breeding of resistant variety in cotton.