Genetic Analysis Of Cold Tolerance At The Germination And Booting Stages In Rice By Association Mapping, Cloning And Functional Verification Of Cold Tolerance Genes At Booting Stage

Low temperature is a major limiting factor for agricultural productivity and quality in temperate areas for Asian cultivated rice.During the growth stage,Low temperature impairs seed germination,causes dead rice and reduces spikelet fertility.Rice is sensitive to low temperature especially at booting stage.Low temperature causes significant crop losses by reducing spikelet fertility.The objective of this study was to identify and analyze QTL(s)and germplasm with cold tolerance derived from core germplasm collection by genome-wide SSR marker association analysis.Here,we report the identification of two genes responsible for cold tolerance at booting stage then confirmed the result by improvement in sensitive cultivar.1.Using mini-core collection of 174 Chinese rice germplasm accessions and 273 SSR markers,we investigated the cold tolerance(CT)at the germination and booting stages along with their underlying genetic bases by association mapping.Two distinct populations,corresponding to subspecies indica and japonica showed evident differences in CT and its genetic basis.Both indica and japonica were sensitive to cold stress at both growth stages.However,japonica was more tolerant than indica at both stages as measured by seedling survival rate and seed setting.There was a low correlation for CT between the germination and booting stages.Fifty one quantitative trait loci(QTLs)for CT were identified on all 12 chromosomes;22 detected at germination stage and 33 at the booting stage.Eight QTLs were associated with at least two indicators.About 46%of QTLs represented new loci.The only QTL shared between indica and japonica for the same indicator was qLTSSvR6-2 for SSvR.This implied a complex of CT in rice.According to the relative genotypic effect(RGE)of each genotype for each QTL,we detected 18 positive genotypes and 21 negative genotypes in indica,and 19 positive genotypes and 24 negative genotypes in japonica.In general,the negative effects were much stronger than the positive effects in both subspecies.Markers for QTLs with positive effect in one subspecies were proven to be effective for selection of CT in that subspecies,but not in the other subspecies.QTL with strong negative effects on CT should be avoided during MAS breeding for favorable QTL at other loci.2.Another study was about fine mapping of a QTL qCTB4-1 for cold tolerance that was identified on chromosomes 4 derived from a population.Using a cold-tolerant 1913 developed by backcrossing KMXBG,reported to be cold-tolerant rice variety at the booting stage,as donor,with a cold-sensitive Japanese commercial japonica variety,Towada.The QTL have been mapped on a 130-kb region containing 4 putative genes.The study cloned a gene that encoded LRR-protein kinase.And this study identified the function of the gene.i.Sequence analyses indicated the seven polymorphisms between KMXBG and Towada in the coding regions but only three SNPs cause amino acid changes.The result implies that qCTB4-1-1 cannot show cold tolerance at germination stage.We evaluated the effects of the transgenes on cold tolerance based on differences in mean spikelet fertility between T2 plants carrying the transgene andTowada with cool water irrigation(19-C-7d,)or cool air(17-C-7d)at booting stage and cool water irrigation(19-C-14d,)at young spikelet differentiation stage.The results indicated that the LRR-protein kinase gene confers cold tolerance.Cold tolerance is associated with greater spikelet fertility and the transgenic plants had greater spikelet fertility than Towada.LRR-protein kinase gene keeps more primary branches and decreases production loss of the transgenic plants at young spikelet differentiation stage.LRR-protein kinase keeps whole anther,anther activity and spikelet fertility.The results show that qCTB4-1-1 code LRR-protein kinase governs cold tolerance at reproduction stage.ii.qCTB4-1-1codes a membrane protein.After cold treatment,the relative electric conductivity and MDA contents in T2 plants carrying the transgene as compared to Towada were reduced,while the proline contents were enhanced.qCTB4-1-1 expression was observed in young leaf at seedling stage,and it was mainly observed in root,stem and panicles at booting stage.Low temperature induces the expression of qCTB4-1-1,and the qCTB4-1-1 expression also impacts the expression of cor-genes.iii.We analyzed the single haplotype of 65 cultivars.We just find four cultivars are tolerant with same genotype as KMXBG,but no one has same genotype as Towada.The results show that the SNP located at 424-bp(C>T)is associate with spikelet fertility.3.The current study was also focused to identify the qCTB4-1-2 that codes UDP-glucose:sterol glucosyltransferase.Here we created transgenic plants that exhibited the cold tolerance at the booting stage.Cold tolerance is associated with greater spikelet fertility and the transgenic plants had greater spikelet fertility and production than Towada.qCTB4-1-1 was expressed in mature root,stem and panicles.qCTB4-1-2 was identified as a locus responsible for membrane and nucleus proteins.