Drought Tolerance-related Association Mapping And Natural Varitions Of Two MYB Genes In Cassava (Manihot Esculenta Cranz)

Cassava is one of the most important food crops and energy crops.It is maily grown in tropical and subtropical regions.It is often affected by seasonal drought and has strong tolerance to intermittent drought.Studying the mechanism of cassava drought resistance,it is of positive significance to excavate excellent drought-tolerance alleles to cassava and other crops.In order to further study about cassava drought tolerance,select excellent germplasm materials.Total of 150 casava materials from 13 countries and regions were studied under both normal and drought conditions.We tested the above-ground fresh weight(AGFW),storage root-related traits,and another 6 physilogical and biochemical traits.Then,all of these data were used to evaluate the influence of cassava accessions under water depressed condition and to select excellent materials for drought tolerance according to comprehensive assessements of them.The whole-genome genotype was obtained by using the expressed sequence tag-simple sequence repeats(EST-SSR)marker and SNPs from simplify genomic sequencing.For the screening of drought resistance related gene,we conducted a genome-wide association analysis using the genotype and phenotype under drought stress condition data,and combining with the existing cassava drought stress transcription and i TRAQ protein group data screening of cassava drought-tolerant candidate genes.In addition,our lab is obtained two MYB transcription factors related to drought tolerance of cassava,by omics data and functional study.In this study,the two MYB transcription factor were re-sequenced in their genomic region,combination of cassava genotype drought related phenotypes,analyze natural variation and relationship with the drought phenotype.The main results are as follows:1.Drought stress had significant influence on these phenotypes of cassava germplasm resources.All of cassava accessions were divided into 3 groups(tolerance,moderate and sensitive)according to the drought resistance measurements(D values).As results of further analysis of variance and multiple comparisons,esitamated values of storage root dry matter percentage(SRDMP)and proline in root of water deficit group,DTCs of activity of SOD in leaf,content of SRS in root and content of proline in root were significantly distinct with each other while the three DTCs of traits were duplicated significant difference in both two years water depressed experiments.2.We examined a total of 107 abiotic stress related EST-SSR markers in 134 cassava genotypes and performed drought related markertraits association mapping.As results,we successfully amplified 98 of 107 markers in 97 polymorphic loci and 279 alleles.The genetic coefficient between every two lines was 0.37 on average,ranging from 0.21 to 0.82.We used DTCs of drought tolerance phenotypes of cassava accessions as phenotypes for marker-trait association mapping.We found that 53 markers were significantly associated with these drought-related traits,with a contribution rate for phenotypes variation of 8.60% on average,ranging between 2.66 and 28.09%.Twenty of these 53 associated genes showed differential transcription levels under drought stress when compared to the control conditions in cassava and four were significantly associated to cassava drought stress signal in m RNA and protein level.3.Cassava accessions were genotyped on total of 16,173 high quality SNPs loci and genotypes were used to drought resistance related marker-traits GWAS.Finally,total of 1,990 loci were significantly associated to drought related traits.A number of 256 functional associated SNPs located in 172 genes.Compared with previous omics,we found 42 differential expressed associated genes under normal condition and water deficit condition.4.Functional interaction network analysis was performed with 66 drought responsive associated genes and key genes of target traits related as query gene.Then,total of 29 genes were selected which might be interaction with key genes of target traits.And q RT-PCR was used to vercify the expression patterns of these 29 genes.As results of q RT-PCR,there were 9 genes whose expression pattern was identical with previous omics.Among them,there were 5 pleiotropic genes containg 06G045100,10G078800,12G005000,12G074800 and16G022700.Acting as negative regulating gene,12G005000 was down regulated under water deficit stress while another 4 genes were up-regulated.All of these 5 pleiotropic genes could be used as important candidate genes for the future drought tolerance study in cassava.5.We found that most diversity regions were located in the first intron and the third exon of MeMYB91.All of SNP in exons could be divided into 29 kinds of haplotypes containing seven major haplotypes.These haplotypes belong to 2 clusters respectively one of which was close to the MeMYB91 derived from W14.And Ka/Ks between the two clusters were far bigger than 1.Besides,total of 12 SNPs of MeMYB91 were associated to DTCs of CAT,Proline,SOD,SRS,AGFW,SRN and SRDMP.Total of 21 SNPs of Me MYB26 was extracted in its genomic sequence.There were 5 missense variants and 1 stop gained variant which were significantly associated to DTCs of storage root nuber per plant,AGFW,activities of SOD and CAT,contents of proline and MDA.The results of this study provide a basis for the molecular breeding of cassava drought resistance and high yield,and provide a theoretical basis for studying the mechanism of cassava drought resistance and screening of drought-tolerant genes.