Genetic Analysis Of Seedling Stage Salinity Tolerance In Indica Rice (Oryza Sativa L.)

Soil salinity is a serious menace in rice production threatening global food security.Understanding the molecular responses of rice under salt stress is beneficial in developing highly salt-tolerant cultivars.Detection of quantitative trait loci(QTLs)/genes conferring salt tolerance is therefore a crucial step in breeding.This research provides essential genetic resource directly relevant in further understanding the complex network of salt tolerance in rice.The specific objectives are:i)to quickly identify novel and stable QTLs useful for breeding by combining QTL-seq and linkage analysis using bi-parental population;ii)to compare the genetic mapping power and resolution of multi-parent advanced generation intercross(MAGIC)population via different association mapping models;and iii)to characterize and dissect the gene underlying SNC4–conferring differential shoot Na⁺content in indica rice cultivars–identified from MAGIC population.The first study was conducted using 625 and 192 F₂ lines derived by intercrossing the salt-tolerant landrace from Bangladesh(Bhera Dawa)and salt-sensitive cultivar(IR36).Meanwhile,the second study was conducted using 385 MAGIC recombinant inbred lines(RILs)derived from intercrossing eight elite rice cultivars–namely,Feddearroz50,Sanhuangzhan No.2,PSBRc82,IR 4630-22-2-5-1-3,IR 45427-2B-2-2B-1-1,IR 84196-12-32(Sambha Mahsuri+Sub1),IR 77298-14-1-2-10 and IR 77186-122-2-2-3.Salinity tolerance screening in both populations was carried out by applying 180 m M analytic grade Na Cl to the modified Yoshida's hydroponic solution when seedlings reached 3rd leaf stage.Salt tolerance of the F₂ mapping population were assessed using two morphological traits,the salt injury score based on IRRI standard evaluation system(SES)and days of seedling survival(DSS).Meanwhile,MAGIC RILs were assessed using physiological traits related to salt tolerance–including,shoot Na⁺content(SNC),shoot K⁺content(SKC),root Na⁺content(RNC)and root K⁺content(RKC).QTL-seq and linkage mapping were combined to detect loci controlling salt tolerance in the F₂mapping population.A total of 10 QTLs on chromosomes 1,3,7,11 and 12 were detected via QTL-seq.Eight of these QTLs–namely,q ST1,q ST3.2,q ST11.1,q ST11.2,q ST11.3,q ST12.1,q ST12.2 and q ST12.3–had positive significant?SNP index which suggests that the salt tolerance alleles in these regions were contributed by the tolerant parent Bhera Dawa.In contrast,the two other QTLs,q ST3.1 and q ST7,had salt tolerance alleles contributed by the salt-sensitive parent IR36 as evident by negative significant?SNP index.Meanwhile,only three QTLs were detected in the population via linkage analysis implementing inclusive composite interval mapping.The low mapping resolution via linkage analysis was possibly due to the low marker coverage used.The q ST1(37.24-39.29 Mb)and q DSS1(37.29-40.40 Mb)detected from QTL-seq and linkage analysis,respectively,were located in the same genomic region.Using the sequence data generated from QTL-seq,we analyzed the non-synonymous SNP variants and identified 45 potential candidate genes.Some of these genes are members calmodulin-dependent protein kinases(CDPKs),erythromycin encoding proteins and transcription factors(TFs),such as zinc finger(ZF)and no apical meristem(NAM)genes.All these candidate genes have not yet been reported for their role on salinity tolerance in rice.It is also worth noting that the Sal Tol/SKC1 region was not detected in both mapping methods conducted.Hence,Bhera Dawa confers salt tolerance mechanism different from Nona Bokra and Pokkali.Therefore,fine mapping the q ST1(37.24-39.29 Mb)and q DSS1(37.29-40.40 Mb)is needed to narrow down the region and clone novel salt tolerance gene directly useful for breeding.The use of MAGIC population has been proven effective in identifying major loci controlling complex traits in rice.Using 385 MAGIC RILs,we compared the statistical mapping power and resolution of multi-parent linkage analysis implementing inclusive composite interval mapping(ICIM)method,single locus(SL)genome-wide association study(GWAS)and multi-locus(ML)GWAS.A total of 29QTLs were detected from ICIM,of which four and 11 QTLs overlapped in SL-GWAS and ML-GWAS,respectively.These QTLs revealed small to large phenotypic variances ranging from 2.51 to 20.09%.These results demonstrated that the higher mapping power and resolution of MAGIC population was fully exploited using linkage analysis than GWAS-based models.A total of 18 minor effect QTLs were missed out from the GWAS result.It has been previously reported that GWAS has low power in detecting loci with relatively small genetic variation since it depends on the“common phenotype-common genotype”hypothesis.Meanwhile,linkage mapping method captured many small effect loci as it included genetic background control into the model.This therefore increases the power of QTL detection while reducing false positive rates.Natural variation for salt tolerance exists in rice,mainly ought to their differential shoot Na⁺content(SNC).Assessing the MAGIC RILs for physiological traits related to salt tolerance detected two major loci,q SNC1 and q SNC4,controlling SNC.The causal gene underlying q SNC1 was the previously cloned SKC1,whereas,causal gene underlying q SNC4 is still unknown.Hence,we conducted further analysis within the q SNC4 region.Linkage disequilibrium(LD)analysis of q SNC4 region revealed 25 LD blocks.The LD block 17 spanned?63.57 kb and harbors the Os HAK1 gene,the only transporter gene found in the region.Although HAK transporter genes are known mediators of K⁺transport in plants,previous studies have reported that some HAK genes confer Na⁺transporting activity.To further confirm if q SNC4is encoded by Os HAK1,the MAGIC lines and its parents were sequenced for Os HAK1 region.Sequence analysis detected one non-synonymous variant(G₁₆₈₉to C₁₆₈₉)in the first exon of Os HAK1.This non-synonymous variant results to Ser₄₀to Thr₄₀amino acid substitution.Moreover,sequencing the MAGIC population for Os HAK1 subdivided it into two groups-the Os HAK1-Hap1 and Os HAK1-Hap2haplotypes.The Os HAK1-Hap1 and Os HAK1-Hap2 haplotypes are low-and high-Na⁺isoforms,respectively.Os HAK1 haplotype analysis in the 3K resequenced panel revealed that the Ser₄₀to Thr₄₀amino acid substitution is responsible for differential SNC in indica cultivars but not japonica.These results indicate that Os HAK1 has a role in shoot Na⁺exclusion mechanism,apart from the previously cloned Os HKT1;5/SKC1.However,further study is needed to provide full evidence on the Na⁺-transporting activity of Os HAK1.Taken together,our study provides a useful genetic information in further understanding the complex mechanism of salt tolerance in rice.