Genetic Dissection For Salt Tolerance At Germination And Seedling Stages Using Association Mapping And Linkage Mapping In Rice

Enormous environmental changes have had a negative impact on food crops in the world;degradation of soil and water quality is increasing the soil salinity day by day.It has been reported that more than 45 Mha of irrigated and 32 Mha of arid agricultural land has been salt affected.Salinity particular has changed the soil texture and cause inhibition of plant growth and yield losses.Rice is highly vulnerable to salinity at germination and seedling stages which ultimately cause yield loss.Most of the agronomic traits are complex traits mostly controlled by number of genes/QTL,which can be dissected by using high throughput analysis to find important QTL and candidate genes.The present study was planned for the genetic dissection for salt tolerance at germination and seedling stages in rice.We have used two different approaches(association mapping and linkage mapping)with high density SNPs(700K)and bins(4568)to find important QTL and candidate genes.In both approaches the populations were screened at germination and seedling stages under salinity.Thirteen(3 and 10)agronomic traits were investigated at germination and seedling stages,FL478(tolerant)and IR29(susceptible)were used as check.In genome wide association mapping,208 natural germplasms of rice were involved;the germplasms panel was selected from core collection of indica rice at International Rice Research Institute(IRRI),Philippines,it belongs to 25 different counties of Asia,Africa and Latin America.SNP sites(395,553)with MAF>0.05% were used for genotyping;these SNPs covering about 372 Mb of the rice genome and average distance between adjacent SNPs was 1Kb.To measure LD decay rate along the chromosome distance and genetic similarity analysis was done using TASSEL5.2.To estimate the effects of replication and genotype,analysis of variance was carried out by SAS PROC GLM.Marker-trait associations were conducted by compressed mixed linear model in the GAPIT with default settings and QTL were selected on the base of P<10 x 105 values and significant adjacent SNP.Haplotype analysis for the candidate genes were carried out using SAS PROC GLM.In the linkage mapping analysis,258 RILs(Minghui 63 x 02428)were involved in study,58936 SNP consisting of 4568 bins in the genome,generated by RAD sequencing were used for genotyping.To estimate the effects of replication and genotype,analysis of variance was carried out by SAS PROC GLM SAS Institute and Ici Mapping were used for QTL mapping and QTL were selected on the basis of LOD>3.In both approaches,RL was found positively correlated with SL and GR at germination stage;RDW and SDW were positively correlated with SSD,KS and KR while negatively correlated with SES,Na S,Na R,Na KS and Na KR at seedling stage.No significant correlation was found between germination and seedling stages under salinity.Association mapping,In PCA analysis 7 PCs were found enough to explain 90% of the variance,LD decay distance in this population was about 100 kb.SNPs(MAF>5%)average heterozygosity was 2.94% and the average PIC was about 29%,reflecting a broad genetic diversity.In Genetic similarity tree,genotypes were divided in three major trees with clusters and subclusters,it was found that each country has its major sub cluster.Eighteen QTL(4 and 14)were identified at germination and seedling stages under salinity.Only two QTL(q SES6 and q SL6)were found having partial overlapping region on chromosome 6(8.3-8.7 Mb)and in 7 important QTL(q Na KS1,q Na S1,q Na KR2,q Na S6,q SDW9 a,q SES9 and q Na KS12)19 candidate genes were identified.11 candidate genes have produced 2-4 haplotypes.Two haplotypes Os01g20160(GA)and Os1g20720(GG)of the candidate genes were found maintaining low Na KS.Haplotype(CA)of the candidate gene Os09g03750 was found maintaining higher SDW.Twelve candidate genes were found having good expression in different tissues.Linkage mapping,twenty five QTL(4 and 21)were identified at germination and seedling stages under salinity.Region of nine identified QTL(q Na KS1,q SES2,q SES3,q SES4,q SES9 a,q KR7,Na KR2,Na KR7 and q RL7)were found reported previously including two QTL(q Na KS1 and q Na KR2)identified in GWAS experiment.Positions of q Na KS1 linked with SKC1 involve in K homeostasis in shoot and position of q Na KR2 linked with RSS1 involve in retarding root length under salinity.Identified QTL explain PVE(3.8 to 11).Seven novels QTL(q SES9 b,q KS3,q Na KS3,q SSD3,q SDW3,q Na KS8 and q Na S7)with PVE more than 8.1 were identified.Novelty of the studyNineteen candidate genes for salt tolerance were identified in 7 important QTL;further investigation of these candidate genes can help to find salt tolerant gene.Two haplotype Os01g20160(GA)and Os1g20720(GG)of the candidate genes involved in maintaining low Na KS and Os09g03750(CA)maintain higher SDW,these haplotypes can help to produce salinity tolerance at seedling stage.Eight genotypes have been identified in association mapping for salinity tolerance that can be used directly in breeding programs.Overlapping region on chromosome 6(8.3-8.7 Mb)for salinity tolerance at germination and seedling stages can be further investigated to find ST gene for both stages.In linkage mapping,seven novels QTL(q SES9 b,q KS3,q Na KS3,q SSD3,q SDW3,Na KS8 and q Na S7)with PVE more than 8.1 were identified.In both experiment position of SKC1 and RSS1 genes were identifiedBoth approaches have confirmed the poor feedback mechanism at seedling for germination under salinity because most of the salt tolerant lines at germination were susceptible at seedling stage.