Fine Mapping Of The Heading Date Gene Dth2Involved In Photoperiod Sensitivity And Map-based Cloning Of The LTG1Gene Controlling Low Temperature Growth In Rice(Oryza Sativa L.)

Heading date (HD) is a key determinant for adaptation to different cultivation areas and cropping seasons in rice. Therefore, high-yielding potential combined with moderate heading date is a major target in rice breeding. Heading date is mainly affected by three factors, i.e. duration of the basic vegetative growth (BVG), photoperiod-sensitivity (PS) and temperature-sensitivity (TS). Diversification of heading date resulted from the complexity of the genetic mechanism of HD provides abundant resources for rice breeding in different habitats. However, it also synchronously enhances difficulty in breeding of rice cultivars with optimum growth duration. Therefore, identification, isolation as well as map-based cloning of novel genes/QTLs will be helpful for disclosing the molecular mechanism of flowering regulation pathway and provide guidance for breeding in rice.In this study, by using a set of chromosome segment substitution lines (CSSL), we successfully isolated a photoperiod sensitivity QTL dth2and a temperature sensitivity gene LTG1which controls low temperature growth. Then we mainly focused on LTG1and analyzed the function of LTG1.Our results suggested that LTG1is a domestication-related gene and the substitution of artificial selection in the Itgl region during domestication led to the transition from low temperature sensitive to insensitive. On the other hand, we found that LTG1controls yield when planted in a low temperature duration condition. Moreover, LTG1also plays a very important role in stabilizing rice HD, which is crucial for rice breeding. Taken together, the main founding are as follows:1. QTL analysis of heading date in rice and fine mapping of a photoperiod sensitivity allele, dth2, located on chromosome2In this study, the genetic basis of HD in rice was dissected into three quantitative trait loci (QTL, denoted dth2, dth6, and dth8) using71F7recombinant inbred lines derived from a single cross between Asominori (Japonica) and IR24(Indica). These three QTLs were located on chromosome2,6and8, respectively. The loci of these three QTLs overlap with previously reported heading date QTL Hd7, Hdl and Hd5, respectively. As Hdl and Hd5had been cloned and Hd7was only roughly mapped to about20cM region between markers C1221and C1408, we selected dth2for fine mapping. The genetic effect of dth2was first confirmed in the chromosome segment substitution line23(CSSL23) through its extremely late heading and transgressive phenotype. Then the QTL was remapped using the CSSL23/Asominori BC4F2population. The QTL dth3, corresponding to the previously reported hd8, was also detected in this population. The LOD score was13.12and the phenotypic variance explained was14.91%. Two nearly isogenic lines (NILs), NIL (dth2) and NIL (dth3), were developed from the BC4F2population using a marker-assisted selection strategy. Further analysis showed that dth2and dth3were independently involved in photoperiod sensitivity, resulting in the transgressive phenotype of CSSL23. Finally, based on the segregation of the HD phenotype and molecular marker genotype in NIL (dth2)/Asominori F2, F3, and F4populations, dth2was dissected into a single recessive gene located in a56.8-kb genomic region with13predicted open reading frames (ORFs). We speculated that ORF8was the candidate gene. This study provides a basis for map-based cloning of the dth2gene and guidance for controlling HD in rice breeding.2. Map-based cloning and functional analysis of the LTG1gene controlling low temperature growth in riceIn this study, by using CSSLs, we found a line CSSL13which was more sensitive to low temperature condition than Asominori. Further, we mapped this gene (named as LTG1) between markers RM3762and RM263by using the secondary segregation population. Then we constructed the near isogenic line (NIL) which harbored a very narrow target segment. By using this NIL and the advanced segregation population, LTG1was delimited to a25.4kb region located between markers dcapsl and ind14and co-segregated with markers L264and dcaps2. According to the genome annotation by TIGR, this region has four putative ORFs. Based on the sequence polymorphism beween the two parents and expression information, we speculate the ORF1and ORF3were the most probable target genes. We then confirmed that ORF3was the authentic target gene by genetic transformation experiments. Subsequently, Blast analysis demonstrated that ORF3encodes a putative casein kinase I subfamily protein. In order to clarify the potential reason of the phenotype differences between the two parents, we measured and compared the kinase activity of the LTG1protein encoded by the two parents under different temperature conditions. Moreover, we also analyzed the expression differences under different temperature conditions. Our results suggested that proteins encoded by the LTG1and Itgl allele may target different substrates to produce the phenotype differences under low temperature condition. The decreased expression of Itgl in NIL (Itgl) under low temperature condition may further enhance the low temperature sensitivity of Itgl. In addition, the LTG1protein was localized in the nuclear and cytoplasm and this subcellular location pattern did not change with the variation of the temperature. On the other hand, RT-PCR, real-time PCR and GUS staining analysis indicated that LTG1was a constitutively expressed gene. By using the microarray and real-time PCR analysis, we found that LTG1may participate in the ABA regulation pathway and HD pathway. LTG1may locate upstream of the HD genes Hd3a and RFT1. Finally, we also analyzed the geographical distribution and the temperature sensitivity variation of rice cultivars with different haplotypes of the LTG1gene. The potential breeding value of LTG1with regard to controlling yield and stabilizing HD was discussed.