Fine Mapping And Candidate Gene Analysis Of Low Temperature Germinability QTL QLTGR3-1 In Rice(Oryza Sativa L.)

Temperature is a key factor for seed germination,to affect planting area sowing time and sowing method of crop.Low temperature can lead to low germination rate,delayed germination,unorderly seedling growth,low seedling vigor,and ultimately reduce crop yield.Therefore,the study of the genetic and molecular mechanism of low temperature germinability（LTG）in rice is very necessary,will provide genetic resources and theoretical basis for the cultivation of new rice varieties of resistance to low temperature germination.In our laboratory,208 single-segment substitution lines（SSSLs）with HJX74 as background were used to identify LTG QTL,and a stable expression of major QTL,qLTGR3-1 is located in 319 kb substitution segment on chromosome 3.Based on the previous research,we used segregation population to fine mapping and candidate gene analysis of qLTGR3-1 by BSA（bulk sequencing analysis）,map-based cloning and molecular biology method.First,BSA was carried out by using the F₂segregation population,and nearly 1.15million mutation sites were identified,including more than 980,000 SNP sites and more than 160,000 Indel sites.There were 45 SNP sense mutations in interval,which were 43SNP non-synonymous mutations and 2 frame shift mutations,causing 21 genes variants.SNP peaks were detected in Chr3:2800001-9600001 on the front of chromosome 3.Then,molecular markers were designed to detect recombinant based on the mutation sites by BSA analysis identification.25 secondary SSSLs with different length segments were identified from the four seasons of 2018-2019.The results showed that 6 SSSLs had stable low-temperature germination phenotype in four consecutive seasons,and 5 SSSLs had overlapping segments.Therefore,the qLTGR3-1 was located to 6.7 kb region between maker M4316-M6341 by substitution mapping.To determine the target genes,the Rice Genome Annotation Project database was used to annotate the target fragment,and found one annotation gene,OsMST4.Furthermore,the sequence sequence alignment of candidate genes showed the sequence difference in the promoter region,intron region and 3’UTR region,but no difference in the gene coding region was found.CDS sequence alignment further verified unchanged in deduced amino acid sequences.Quantitative analysis showed that the relative expression of OsMST4 was abundant on the 5th and 7th day of seeds germination,and the expression level of OsMST4 was significantly higher than in resistance to low temperature germination of ZRQ10 comparison with HJX74.Therefore,we could speculate OsMST4 is the target gene of qLTGR3-1.OsMST4 gene encodes a monosaccharide transporter（MST）,which is a member of the MST family,located on the cell membrane.Using the Rice e FP website to predict the spatiotemporal expression patterns of OsMST4 gene,showed that the OsMST4 gene was expressed ubiquitously in all tissues and organs of rice and at different stage of development.The expression level of the OsMST4 gradually decreased with the maturity of seeds,and was expressed substantively in the late heading stage and early seedling growth stage.The analysis of main agronomic traits showed that the OsMST4 gene might not be related to plant height,grain type and 1000 grain weight.Therefore,the gene OsMST4 can be used to improve the rice varieties with-not resistant to low temperature but with other excellent characters.In this study,the major QTL,qLTGR3-1,was map-based cloned by combining BSA sequencing with the traditional location method.OsMST4 was speculated as the objective gene of qLTGR3-1,which is the first reported MST gene related to LTG in rice,which laid a foundation for further verification of gene function.The results will further improve the genetic regulation mechanism of LTG of rice and lay a foundation for the cultivation of new rice varieties with excellent low-temperature germination resistance.