Screening Of Cassava Salt Tolerent Germplasms&Transcriptome Analysis Of Salt-tolerant Germplasm SC8 Salt Stress

Soil salinization is the main environmental factor that limits crop growth and productivity.It is necessary to develop and utilize saline soil.The screen of salt-tolerant germplasms and the exploration of its salt-tolerant mechanism are important to develop and utilizate the saline soil.Cassava（Manihot esculenta Crantz）is a plant of Euphorbiaceae and Manihot.The roots are rich in starch and it is an important raw material for industrial starch and alcohol processing.Cassava has strong adaptability of drought and barren tolerances,which lays a foundation for its popularization and planting on saline land.In this study,28 cassava germplasms were used as experimental materials to analyze their agronomic traits by cluster analysis,membership function analysis and correlation analysis.9 typical cassava germplasms（salt tolerance,moderate salt-tolerance and salt sensitivity）were screened out and their physiological and biochemical indices were determined.The physiological changes of cassava under salt stress were investigated.The leaves of the salt tolerant germplasm SC8 at different stages of salt stress were used for further transcriptome sequence;and the key genes and metabolic pathways in response to salt stress were analyzed.The results were as follows:1.Analysis of agronomic traits in the fieldBy analyzing the salt tolerance indices of 7 agronomic traits of the 28 tested cassava germplasms,we find that all the salt tolerance indices of relative plant height,relative stem diameter,relative leaf number,relative survival rate,relative tuber setting rate per plant,relative yield per plant and relative dry matter rate were lesser than I.The 28 germplasms were divided into 3 categories by cluster analysis,which include 6 salt tolerant germplasms（account up to 21.4%）:Luoyong 90,F03P,Thailand,SC 124,Luoyong 9,SC8;13 moderate salt-tolerant germplasms（account up to 46.4%）:Guire No.3,CH16 cold resistance,F274 cold resistance,GR911,KU50,COL173,F297,Q10,2B,F200,F124 cold resistance,CH20 cold resistance,SC205;9 salt-sensitive germplasms（account up to 32.2%）:F17312,18R,27-6,11K,Guire 5,HB60,BRA755,BRA755-1,F128.2.Analysis of physiological and biochemical indicators11 physiological and biochemical indices of the 9 typical cassava germplasms（salt tolerance,moderate salt-tolerant and salt sensitivity）were analyzed,it was found that with the increase of stress time,the contents of hydrogen peroxide,oxygen-,relative conductivity,CAT and proline of 9 cassava germplasms were increased continuously.the POD content was increased at first,followed a decrease,and then increased again.The relative water content was decreased continuously;and the chlorophyll content was increased first and then decreased.The malondialdehyde（MDA）content in some germplasms showed a decreasing trend in the early stressed stage,and then was increased..The contents of SOD and soluble sugar were increased first and then decreased in the salt-sensitive germplasms,but were increased continuously in the salt-tolerant and moderate salt-tolerant germplasms.The results of subordinate function analysis showed that the order of salt tolerance of 9 germplasms was SC8>F03P>Luoyong 9>Q10>SC205>F297>Guire 5>27-6>BRA755-1.The correlation results showed that the correlation coefficient between proline content and the average membership function was the highest one,which the absolute value of correlation coefficient was up to 0.973.The correlation coefficients between MDA and O2-with the average membership function were the secondary higher ones,which the absolute value of correlation coefficient was up to 0.94,0.896,respectively.The absolute correlation coefficients of H2O2,SOD,relative water content,POD,relative conductivity,and solubility,sugar,chlorophyll content,CAT content with the average membership function were 0.884,0.873,0.848,0.845,0.812,0.694,0.633,-0.345,respectively.Thus it preliminarily determines that proline,MDA,O2--could be used as the reference indices for cassava salt tolerance.3.Transcriptome analysis of salt-tolerant germplasm SC8 under salt treatmentThe transcriptomes in leaves of the salt-tolerant germplasm SC8 under salt treatment for 0 h,12 h and 72 h were sequenced.9 cDNA libraries were constructed and 31.2G high-quality base sequences were obtained.The clean bases were up to 3.2 G and the Q20 were up to 99.97%.In SC8₀ h-vs-SC8₁2 h,1591 differentially expressed genes were obtained,among which 689 were up-regulated,902 were down-regulated,and 5791 were differentially expressed.In SC8₀ h-vs-SC8₇2 h,2507 were up-regulated,3284 were down-regulated,and 5893 were differentially expressed.In SC8 12 h-vs-SC8₇2 h,3183 were up-regulated and 2710 were down-regulated.The GO analysis of the differentially expressed genes in SC8₀ h-vs-SC8₁2 h,SC8₀ h-vs-SC8₇2 h,SC8₁2 h-vs-SC8₇2 h showed that there were differences in GO enrichment to be found at three time points,which the plasma membrane components,were especially found in SC8₀ h-vs-SC8₁2 h,which might be related to the index in SC8 in response to salt stress.The folding protein,and the respiratory burst related proteins were especially found in SC8₀ h-vs-SC8₇2 h,which might be related to long-term response to salt stress in SC8.The specific items of immune response,isomerase activity were found in SC8₁2 h-vs-SC8₇2 h,which might be related to protection of SC8 from salt stress damage.KEGG analysis of the diferentially expressed genes of SC8₀ h-vs-SC8₁2 h,SC8₀ h-vs-SC8₇2 h,SC8₁2 h-vs-SC8₇2 h showed that plant hormone signal transduction,phenylalanine metabolism,phenylalanine biosynthesis,starch and sucrose metabolism,and alpha-linolenic acid metabolism were significantly enriched in SC8₀ h-vs-SC8₁2 h,SC8₀ h-vs-SC8₇2 h,among which plant hormone signal transduction,phenylalanine metabolism have been identified to be associated with plant stress tolerance in other plants.With the increase of salt stress time,there was no significant enrichment in SC8₀ h-vs-SC8₁2 h,however,the circadian rhythm,photosynthesis-antenna proteins and so on were significantly enriched in SC8_h-vs-SC8₇2 h and SC8₁2 h-vs-SC8₇2 h.These pathways might be important metabolic pathways for SC8 to respond to salt stress in the long term.The Venn plot was used to analyze the distribution of the differentially expressed genes（DEGs）at three stress time points.The results showed that there were 381,1288 and 1558 DEGs to be specifically expressed at SC8₀ h-vs-SC8₁2 h、SC8 0 h-vs-SC8₇2 h、SC8 12 h-vs-SC8₇2 h,respectively.There were 500,332,3625 DEGs to be overlapped at three time points;and there were 378 DEGs to be shared at SC8₀ h-vs-SC8₁2 h,SC8₀ h-vs-SC8₇2 h,SC8₁2 h-vs-SC8₇2 h.From the trend cluster analysis of these 378 DEGs,the first four types of KEGG Pathway were mainly enriched in the pathways of phenylalanine metabolism,plant hormone signal transduction,cysteine and methionine metabolism,plant circadian rhythm.These pathways have been proved to be related to plant stress resistance.36 differentially expressed genes that are related to salt stress from the above pathways have been identified,which include heat shock protein（HSF）gene family,WRKYs gene family,ABA pathway related kinase CIPKs/SnRK,circadian rhythm related gene LHY,and auxin regulation.Genes SAUR,FLA/SOS,transcription factor ERP/AP2 and so on.4.qRT-PCR verificationThe accuracy of transcriptome data was verified by real-time PCR.12 genes were screened from the 378 differentially expressed genes at three time points for real-time PCR.The results showed that the trend of gene expression in transcriptome and the relative expression in fluorescence quantitative analysis were consistent,indicating that the sequencing results were reliable.