Genome-wide Association Study And Candidate Gene Mining Of Alkalinity Tolerance In Japonica Rice At The Seedling Stage

Saline-alkali stress is one of the main factors limiting food production worldwide.As one of the most important food crops in the world,rice is an important guarantee for a country’s food security.However,due to the continuous deterioration of saline-alkali soil,the area of cultivated land has decreased year by year,and the growth and yield of rice have been seriously affected.Therefore,studying the salt and alkalinity tolerance of rice and cultivating salt and alkalinity-tolerant rice varieties have important practical significance for improving the utilization rate of saline-alkali land and ensuring stable production of rice.Based on the differences of main anions,we can divide the salinity-alkalinity stress into two kinds:salt stress(NaCl and Na2SO4)and alkalinity stress(NaHCO3and Na2CO3).Alkalinity stress is more harmful to plants than neutral salt stress in many aspects.So far,the excavation and utilization of alkalinity tolerance genes of rice have lagged behind salt stress research._Moreover,most of the related research of alkalinity tolerance of rice is in the primary stage of QTL mapping.In this study,the natural population consisting of 295 japonica rice varieties was subjected to 14.62× re-sequencing genotyping.Based on the criteria of having less than 20%missing data and a minor allele frequency(MAF)greater than 5%in the selected population,a total of 788,396 SNPs were selected for GWAS.We assessed the alkalinity tolerance of rice at the seedling stage and performed a genome-wide association study(GWAS).We used the score of alkalinity tolerance(SAT),the concentrations of Na+and K in the shoots(SNC and SKC,respectively),the concentrations of Na+ and K+ in the roots(RNC and RKC,respectively),the Na+/K ratio of shoots and roots(SNK and RNK,respectively),relative shoots and roots fresh weight(SFW and RFW,respectively),relative shoots and roots dry weight(SDW and RDW,respectively),relative root number(RRN),relative root length(RRL)and relative seedling height(RSH)as indices to assess alkalinity tolerance at the seedling stage in rice.In addition,transcriptome sequencing analysis was performed on the alkali-tolerant rice "WD20342" and the alkali-sensitive rice "Caidao" under alkalinity stress and control conditions,and further screening and analysis of candidate genes.In order to provide a theoretical basis for excavating new genes for rice alkalinity tolerance and improving rice varieties through molecular marker-assisted selection.The main results were as follows:1.The statistical analysis of 14 alkalinity tolerance related traits in japonica rice at the seedling stage showed that the alkalinity tolerance related traits in this study were normally distributed.Among them,SAT is distributed between 1-9,and the population variation coefficient is 45.17%.This result shown that the natural population selected in this study shows a wide diversity in alkalinity tolerance.2.The combined results of population structure(Q),principal component analysis(PCA)and the neighbour-joining(NJ)tree divided the japonica rice panel into three subgroups,and these varieties did not show an extremely differentiated population structure.The analysis of kinship(K)shows that the genetic relationship between individuals is far away,which is more suitable for GWAS.3.The linkage disequilibrium(LD)analysis of the indica population in this study showed that the LD distance at the genome-wide level was approximately 109.77 kb,consistent with previous studies.The LD distance of different chromosomes is significantly different.The LD distance of chromosome 11 is the smallest,about 13.79 kb.The LD distance of chromosome 5 is the largest,about 415.77 kb.4.Using the mixed linear model(MLM)of TASSLE 5.0 software,38 SNP loci significantly associated with rice alkalinity tolerance were detected at P<6.34×10-8,which were distributed in 30 QTL intervals.Among them,salt-tolerant genes with known functions can be retrieved at internal or adjacent positions of 12 QTLs.5.A total of 8,332 differentially expressed genes(DEGs)related to alkalinity stress were detected by transcriptome sequencing analysis of "WD20342" and "Caidao" under alkaline stress and control conditions.The results of GO enrichment analysis indicated that some of the DEGs were involved in stimulation,stress response and ion transmembrane transport.The results of KEGG pathway enrichment analysis indicated that DEG was mainly involved in the plant hormone signal transduction and biosynthesis of secondary metabolites.6.This study was further analyzed for a QTL that was significantly associated with SAT,SNC and SNK detected on chromosome 3.Combined with transcriptome sequencing and GO enrichment analysis,candidate genes were initially screened,and 12 candidate genes related to alkalinity tolerance were obtained.By using qRT-PCR analysis,two genes differentially expressed between alkali-tolerant and alkali-sensitive varieties and between alkalinity stress and control were detected,which were LOC_Os03g26210 and LOC Os03g26430,respectively.7.The sequence analysis of LOC Os03g26210 showed that the alkali-tolerant varieties contained one deletion in the starting position of 5’ untranslated region(UTR)(ATG start codon 193-199bp upstream)with a total length of 7 bp compared with the alkali-sensitive varieties.A previous study indicated that LOC_Os03g26210 is a negative regulator of the Fe-deficiency response in rice and was named OsIR03.8.Furthermore,we designed an insertion-deletion(Indel)marker using the 7-bp deletion sequence to genotype 126 japonica rice varieties(60 tolerant and 66 sensitive varieties).The results showed that 85.00%(51 of 60)of the tolerant varieties(SAT≤3)had the tolerant allele,and 83.33%(55 of 66)of the sensitive varieties(SAT>6)had the sensitive allele.