Identification And Analysis Of Salt Tolerance Germplasms And Related Genes In Wild Emmer Wheat

As the ancestor of domesticated wheat,wild emmer wheat(Triticum dicoccoides)has many good characteristics such as big grain size,large spike lengh,tolerant to stress and disease etc.Wild emmer holds the genetic potentiality for improvement of wheat yield,quality and stress tolerance such as drought and salt.MicroRNAs(miRNAs)are a class of endogenous small noncoding RNAs which regulate diverse molecular and biochemical processes at post-transcription level in plants.Transcriptome sequencing(RNA-seq)provide effective way for identification of plant stress related genes and analysis their expression pattern in genome wide scale.With the development of high-throughput sequencing and bioinformatics analysis,the constantly published whole genome sequencing data of crops provide new resource for comparative genomics analysis,which could help to understand t evolutionary rates and structural features of the sequences.The evaluation of salt tolerance in wild emmer germplasms was firstly screened and several salt tolerant genotypes were identified.Subsequently,the high throughput sequencing was applied to identify salt responsive genes and miRNAs in wild emmer.The comparison analysis of the expression and regulation patterns between salt tolerance and sensitive genotypes was carried out combined with the omics data and physiological and biochemical indexes.Lastly,the comparative genomic analysis in wild emmer and cultivated emmer was performed.The main research contents and results of this study are as follows:(1)To comprehensively evaluate the salt tolerance of wild emmer wheat,a total of 30traits including growth-,physiological-and photosynthetic related as well as K~+and Na~+content of 30 wild emmer and 14 durum wheat accessions were systematically investigated and compared between normal and saline conditions.Salt tolerance index(STI)based on multiple regression analysis of these traits was calculated and 5 wild emmer accessions showed high salt tolerance,which could be used as valuable resource for wheat salt tolerance improvement.Furthermore,wild emmer genotypes showed wider trait performance variation compared to durum wheat,indicating the higher genetic diversity in wild emmer wheat.Then,shoot Na~+content,shoot K~+/Na~+ratio,root length and root surface area were identified as suitable index for salt tolerance evaluation.Na~+exclusion mechanism was found to be played an important role in response to salt stress in emmer wheat.(2)To obtain more information on miRNAs in wild emmer,we systematically investigated and characterized the salinity-responsive miRNAs using deep sequencing technology in salt tolerance germplasm GilbourB5.A total of 88 conserved and 124 novel miRNAs were identified,of which,50 were proved to be salinity responsive miRNAs with 32significantly up-regulated and 18 down-regulated.Ttd-miR172b,Ttd-mi R1120a as well as Ttd-mi393a were the most significantly differently expressed.Targets of these miRNAs were computationally predicted,then GO and KEGG analysis found that the targets of salinity responsive miRNAs enriched in transcription factors,stress-response metabolic related proteins.Furthermore,the salt and drought responsive miRNAs in wild emmer and the relate species were compared.MiRNAs such as miR156,mi R171 and miR396 etc.were shown as both drought and salt responsive mi RNAs in almost all of the five species Some stress-responsive miRNAs showed species-specific,such as miR1432,miR474 etc.in wild emmer and miR3170,mi R529 etc.in bread wheat.Finally,we investigated the expression profiles of 7 mi RNAs between salt tolerant and sensitive genotypes under different stages(0 h,3 h,6 h,12 h,24 h)and salt concentration(150 mM,250 mM)and found that they played critical roles in salinity tolerance in wild emmer.Our results systematically identified the salinity-responsive mi RNAs in wild emmer,which not only enrich the miRNA resource but also lay the foundation for further study on the biological functions and evolution of mi RNAs in wild wheat and beyond.(3)Based on the transcriptome sequencing(RNA-seq)data,we performed systematic compariative analysis between salt tolerant line(GilbourB5)and salt sensitive line(GilbourA9)on the scope of gene expression and regulation.A total of 83 and 1618 salt responsive genes were identified of in GilboaB5 and GilboaA9.The 36 common salt responsive genes were enriched in a variety of processes such as stress response in response to salt stress,oxidoreductase activity,intracellular ion balance(cellular iron ion homeostasis)etc.Compared with the salt tolerant genotype,the gene expression ratio,root traits,photosynthetic traits and oxidative stress of salt sensitive genotype were more susceptible to salt stress based on a series of analysis,including the number of genes,gene expression patterns,gene function diversity,physiological and biochemical characteristics.In addition,the RNA-seq data was adopted to analyze the RNA editing and alternative splicing events in response to salt stress.For GilboaB5 and GilboaA9,738 and 778 RNA editing sites,as well as 290 and 670alternative splicing events were identified,respectively.This research revealed the transcriptional and post transcriptional regulation pattern of wild emmer response to salt stress,which provides important resource for further research.(4)Based on the genome data of wild emmer and related species,we we conducted the comparative genome analysis of wild emmer genome.We identified the pairwise orthologs between wheat sub-genomes.The identified of positively selected genes(PSGs)between A-sub genome and B sub-genome was 439,and the number between wild emmer and durum as well as T.urartu were 619(we_A_vs_durum_A),654(we_B_vs_durum_B)and 1407(we_A_vs_urartu).We further calculated the rates of nonsynonymous substitution(dN)and the rates of synonymous substitution(dS)then compared the gene features,expression patterns,codon bias,promoter characteristics and evolutionary rates between PSGs and NSGs.The genome of emmer wheat composed two sub-genomes and the characteristics and evolutionary patterns of positively selected genes(PSGs)and negatively selected genes(NSGs)between the sub-genomes and their donors are poorly understood.The results show that PSGs have higher protein evolutionary rates,lower synonymous substitution rates,shorter gene length,fewer exons,higher functionary specificity.These data suggest that PSGs and NSGs differ not only under selective pressure(dN/dS),but also in their structural,evolutionary and functional properties,indicating that selective modes may serve as a determinant factor for measuring evolutionary rates,gene compactness and expression patterns in wheat genomes.The results provide not only a new insight for large-scale comparative genomic analysis,but also a better understanding the evolutionary events wild emmer wheat and related species.