| Soil salinity is the accumulation of mineral salts in soil water and considered as major limiting factor for cotton yield and fiber quality.Cotton(Gossypium hirsutum L.)is regarded as the foremost natural fiber crop worldwide,with guesstimated production of ～115 million bales.The economic impact of the cotton industry is about ～$500 billion per year on the globe.In China,it has significant importance to increase the income of farmers and satisfy the needs of development of national economy.Seed germination is the beginning of the seed growing,which plays a decisive role in the whole life cycle of the plant.More than 6.7% of China’s arable land is saline.Cotton is salt tolerance compared to other cultivated crops,but the salt content of more than 0.3% still inhibited cotton seed germination,and then influence the seedling rate,becoming the limiting factor of cotton planting in saline alkali soil.In recent years,due to the changing climatic conditions,increased soil salinization,cotton production is relatively concentrated in the saline areas of eastern coastal and northwest inland,causing harmful effects on the development of cotton industry.Our study used China’s cotton variety Sicot 73 as research plant material and analyzed germination at different salt concentrations.We determined the germination rate at 0.6% and 0.8% of Na Cl and studied the dynamic changes of gene expression during seed germination by RNA-Seq.Based on the transcriptome data and bioinformatics analysis,we obtain the following conclusions:1)Germination test showed that when the external salt concentration of 0.0%,0.2%,0.4%,0.6%,0.8%,and 1.0%,the germination rate of seeds was: 100.00%,83.33%,73.33%,66.67%,46.67%,and 10.00% respectively.When the salt concentration was higher than 1.0%,cotton seeds could not germinate.According to the results of experiment,we selected 0.6% and 0.8% Na Cl concentration for the following study.2)RNA-Seq received a total of 114,139,977 clean reads,and more than 87%of them can map to the G.hirsutum L.,with the standard of both log2>1 or log2<1(fold change>2)and the adjusted FDR ≤ 0.001 in the comparisons,we identified 26,650 differentially expressed genes(DEGs).3)The results showed that the number of DEGs was gradually increased with the extension of salt treatment duration,and the DEGs of 0.8% Na Cl were significantly higher than that of 0.6% Na Cl under same duration.Using the rigorous cutoff as mentioned,we identified 914 and 1529 DEGs in the samples of YR4-6 and YR4-8 compared with YR4-0,625 and 1146 DEGs of YR8-6 and YR8-8,and 10649 and 15529 DEGs of YR24-6 and YR24-8 respectively.To explore the DEGs in a time series way,we also performed sequential pairwise comparisons among 4h,8h and 24 h in three times course.In control,there were 1483 DEGs between YR1-0 and YR4-0 or 1603 DEGs between YR4-0 and YR8-0,while 13625 DEGs between YR8-0 and YR24-0.In the salt concentration of 0.6% and 0.8%,315,1613,4604 and 869,942,3885 DEGs were identified within the same comparison of periods.We illustrated different time points in the event of physiological change need of gene regulation of different types and numbers and cotton seed germination process of different concentrations of salt stress contain same or different expression regulations.4)Transcriptomic studies have unveiled the role of several salt-responsive transcription factors and genes,either up-regulated or down-regulated in cotton.Among all these DEGs,there were 1053 transcription factors(TFs)belongs to 74 families according to Plant Transcription Factor Database,as ERF(138),b HLH(103),WRKY(93),NAC(66),ARF(51),GATA(48),MYB(44),HSF(36),Trihelix(35)et al.These transcription factors showed different distribution at different time points,such as 4h,the main TFs belongs to ERF(38),WRKY(28),GATA(12);8h,are WRKY(16),HSF(12),ERF(11);and 24 h for ERF(90),b HLH(63),WRKY(59);indicating the seed germination in different periods needs different transcription factors and many transcription factors have relatively specific regulatory functions.Many members of transcription factors are reported in plant stress germination or growth for regulation or response,and further depicted the role of TFs in stress conditions during seedgermination and important biological functions.5)According to the characteristics of gene expression,we can divide the differentially expressed genes into 30 clusters by K-mean analysis.We further found genes in cluster 7,8,and 10 were predominantly enriched in transcription,DNA repair,RNA processing and ubiquitin-dependent protein catabolic process;genes in cluster 21 were predominantly enriched in signal transduction;genes in cluster 1,6,12,and 14 included carbohydrate metabolic process,transport and microtubule-based movement;genes in clusters 4 and 5 response to stress;clusters 15,20,24 and 28 enriched in translation process;clusters 2 and 13 related to protein phosphorylation.6)Pathway analysis showed that the three time points after the germination of 4h,8h and 24 h,there were 54 DEGs involved in glucose metabolism at the concentration of 0.6% Na Cl,168 DEGs enriched in glutathione metabolism biosynthesis of secondary metabolites and protein processing at 0.8%Na Cl and 17 DEGs at both conditions.7)Comparing the genes in the A-and D-sub genome,there were 12990 DEGs belongs to A-sub genome,including 509 transcription factors and these genes mainly involved in ribosome,carbohydrate metabolism and protein biological processes;13384 DEGs belongs to D-sub genome,including 534 transcription factors and their mainly biological effects were similar with A-sub genome.Moreover,there were 8960 DEGs of Asub genome and 9008 DEGs of D-sub genome considered as homologous genes.The results showed that cotton A-sub genome and Dsub genome do not have visible difference in salt tolerance of cotton.In summary,using genome-wide transcriptome analysis in real-time,we obtain a series of differentially expressed genes under salt stress on seed germination.We validated homologous genes response induced by salt stress,also get more salt stress response genes.Real time transcriptome analysis provides us with a comprehensive understanding of the transcriptional regulation of cotton seed germination,and also provides more genetic resources for further study on seed germination under stress conditions.We also highlighted the potential areas for future investigation to elucidate the key transcription factors for effective breeding of salt-tolerant cotton genotypes.These results have some reference valuefor cotton genetic engineering and resistance breeding. |
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