Map-based Cloning And Functional Analysis Of The Heading Date Gene Dth6 And Lft1 In Rice(Oryza Sativa L.)

Plants in nature in order to adapt to different latitudes,elevations and different planting environments,resulting in their own unique flowering mechanism to thrive.In the long-term artificial selection,rice cultivars with moderate heading date and stable yield potential were preferred to be adopted in rice breeding.The factors that determine the heading date of rice varieties are basic vegetative growth(BVG),photoperiod-sensitivity(PS)and temperature-sensitivity(TS).Different combinations of basic vegetative growth,photoperiod-sensitivity and temperature-sensitivity result in the diversity of heading date and geographic distribution of different rice varieties,which not only provides abundant resources for breeders but also complicates the research on heading date.Heading date is a typical quantitative trait(QTL),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 suppling guidance in rice breeding.In this study,we performed gene mapping based on a chromosome segment susbstitution line(CSSL)and a late-heading mutant,the main results are as follows:1.Map-based cloning of the heading date QTL locus dth6Previously,we found a photoperiod sensitivity QTL dth6 on chromosome 6 by using a set of Recombinant Inbred Lines(RILs).In order to verify the effect of dth6 locus,CSSL30 line was selected from a set of CSSLs,which contains different fragments from the donor parent IR24 in the same genetic background of the receptor parent Asominori.Under long day(LD)condition,CSSL30 with chromosome segment from IR24 in dth6 region shows earier flowering time than Asominori.By investigating heading date of CSSL30×Asominori F2 population in natural long-day condition in Nanjing,a major QTL was proved in dth6 region,the LOD score was 22.88 and the phenotypic variance explained was 27.8%.In contrast to early flowering in LD condition,CSSL30 delayed flowering in natural short day(NSD)condition compared with Asominori,which indicates that dth6 locus involved in photoperiod sensitivity.Then,we identified recombinant individuals from CSSL30×Asominori F2 population and investigated their heading date of both F2 in NLD condition and the derived F2.3 in NSD condition.Finally,the dth6 locus was restricted within a 480kb region,flanked by molecular marker XLF18 and XLF2.Since Hdl gene was located in this region,we first compared Hdl coding sequence between CSSL30 and Asominori.There were four divergences exist and resulted in amino acid alteration both in conserved B-box domain and CCT domain.No matter in LD or SD condition,Hdl expression was significantly downregulated in CSSL30,consisting with early flowering in LD and later flowering in SD.Newly found Hdl allele may lay the foundation for further analysis of Hdl.2.Identification and map-based gene cloning of a Late Flowering Mutant lftl in Rice(Oryza sativa L.)A Nipponbare T-DNA insertion mutant with late heading date under long-day condition was selected,termed Iftl.Compared to wild type Nipponbare,Iftl delayed heading about 20 days under long-day condition,accompanied by increase of grain length and 1000-grain weight.After investigation in lft1/Nipponbare F2 population,we found that the late heading trait is controlled by a pair of recessive gene and has no co-segregation with T-DNA insertion.Compared with a control Nipponbare/9311 F2 population,extremely late heading individuals were found in lft1/9311 F2 population,from which 58 were chosen for initial gene mapping.Genotyping of these individuals was performed by polymorphism markers developed between Nipponbare and 9311.The gene region was restricted on short arm of chromosome 9,flanked by molecular marker RM219 and RM3 912,a length of 2.94Mb region.In the process of further development of markers,we found that one molecular marker SJ9-32 can be detected in Nipponbare and 9311,but not in the Iftl and 58 extremely late heading individuals.A large missing DNA segment covering SDG724 gene did exist.An allelism test further confirmed that deletion in SDG724 was a causative factor of the late heading in lft1.The qRT-PCR results showed that SDG724 could regulate the expression of OsMADS51 and RFTl.It is feasible to adjust rice heading by modulating the expression level of SDG 724 gene,this will work for rice breeding.