Genetic Analysis Of QTL-HAN2 In Cold Tolerance At Seedling Stage In Rice

Temperature is an important environmental factor that regulates the growth and development of plants and determines the geographical distribution.Low temperature stress can affect the growth and development of plants,and even death in severe cases.As one of the most important staple food crops,rice is sensitive to low temperature due to its tropical and subtropical origin.Temperate japonica rice is an ecotype that acquires low temperature tolerance during the domestication process and adapts to temperate environment.Therefore,analyzing the genetic basis of low-temperature adaptability of temperate japonica rice can not only lay a theoretical support for low-temperature tolerance rice breeding,but also provide genetic resources.In this study,genetic populations were constructed from cold-sensitive indica rice varieties and cold-tolerant temperate japonica rice varieties,and QTL analysis was performed.Finally,a cold-tolerant QTL-HAN2 at the seedling stage of rice was finely mapped and its candidate genes were functionally verified.The main results of this study are as follows:(1)In the genetic populations,a QTL for cold tolerance in rice was located within the 8.9Mb region of chromosome 11,named HAN2.Through genetic analysis,the LOD value of HAN2 is 9.5,and its positional confidence interval is the molecular marker M1-M11(Chr.11:8.8Mb-9.5Mb).The additive effect value and the dominant effect value of HAN2 is 18.7% or-7.0%,respectively,and they explained about 26.8% of the total variability of the resolved phenotype.The japonica rice genotype of this QTL is cold-tolerant alleles,while the indica rice genotype is cold-sensitive alleles.The indica rice genotype is partially dominant to the japonica rice genotype.(2)Through further fine-mapping,it is positioned to the 28.5Kb area and contains three ORFs.Through expression analysis,we found that ORF1 can be induced by low temperature to reduce expression,while ORF2 and ORF3 are not induced by low temperature.Within ORF1,there are abundant natural mutations in its promoter,5’UTR,coding region,and 3’UTR region,which may affect the transcription level of the gene or the function of the encoded protein.(3)Primary functional analysis showed that ORF1,the candidate gene of HAN2,was mainly expressed in vegetative organs,especially in leaf and sheath.Through transient expression in tobacco leaf,we found that ORF1 is localized both in the cytoplasm and nucleus.Through the overexpression and gene knockout of ORF1,it was determined that ORF1 could negatively regulate the cold tolerance of rice seedling.Moreover,the knockout of the HAN2 candidate gene ORF1,can increase the cold tolerance of seedling but did not affect the grain yield of plants under normal temperature conditions.Therefore,this QTL can enhance the cold tolerance of rice,but does not affect the yield of rice.