Allelic Variation And Epistatic Analysis Of Rice Heading Date Genes Based On SSSLs

Heading date in rice（Oryza sativa L.）is a complex quantitative trait,which directly determines the planting district and season,and the goal of rice breeding,thus plays an important role for rice varieties producing and introducing.In this paper,we used single segment substitution lines（SSSLs）to detect allelic variations and estimate various epistatic components,then revealed the genetic and flowering mechanism on heading date genes.The aims are to find different alleles on heading date for the molecular pyramiding breeding,and to enrich the genetic and mechanism on controlling flowering in rice.The main results are as follows:1.There were more than one allelic variations being found on the seven heading date genes.Three alleles among Hd1,Ehd1,Hd3a,RFT1 and DTH8,and four alleles of OsMADS50,and two alleles in OsGI were identified,respectively.Among these alleles,the Hd1-2,Hd1-3,Ehd1-2,Ehd1-3,Hd3a-2,RFT1-2,OsMADS50-3 and OsGI-2 were new allelic variations,which we found at first time.Meanwhile,several new functional nucleotide polymorphisms（FNPs）were revealed.The differences of heading date were according to sequence variations of coding region among Hd1,Ehd1,Hd3a,RFT1,DTH8and OsGI,while heading date variations might be corresponding to sequence variations of the first intron and promoter region in OsMADS50.Interestingly,RNA levels of different alleles in DTH8 had no connection with their flowering times,whereas,expression levels among Hd1,Ehd1,Hd3a,RFT1,OsMADS50 and OsGI were proportional to flowering time,which higher expression levels were correlated with earlier flowering under different conditions.In addition,we also investigated these variations at 23 ancestors by the data from high-throughput sequencing,to build a solid foundation for further screening rice germplasm resource.2.Seven heading date genes showed difference on the sensitivity to temperature and day-length.Based on their character of sensitivity to temperature and day-length,they were divided into three types:photoperiod-sensitive type（Hd3a）,temperature-sensitive type（Ehd1,RFT1,OsMADS50 and OsGI）,and both photoperiod and temperature sensitive type（Hd1 and DTH8）.3.Significant epistatic interaction were generally detected between different heading date genes and pyramiding effect among different alleles were inconsistent.The significant additive effect proved that these SSSLs indeed harbored different heading date genes which were sensitive to different environment conditions.The pyramiding effects of OsMADS50and Ehd1 promoted heading date,while pyramiding effects of DTH8 delayed flowering,and the pyramiding effects showed different among alleles.21 polymers on heading date were detected with significant epistatic effects with the ratios of 88.89%,and the ratios of58.13%were significant epistatic effects on 20 epistatic components from F₂population.It was found that the heading date tend to early,when combined early heading date gene with late heading date gene.It was a solution for late material breeding improvement.The diverse pyramiding effects on different heading date alleles laid on a solid foundation for further molecular breeding by design on maturity to different environment condition.4.The protein interaction was detected between Hd1 and Hd3a,which interaction intensity was related to the Zinc finger domain of Hd1 N-end but not CCT domain.Meanwhile,we also found that the interaction was exist between Hd1 and RFT1.However,the intensity was rarely weak,even no interaction,as the mutation on E105K position of RFT1.Full length Hd1 had the transcriptional activity and its active domain was in the C-end of Hd1 protein without any connection with Zinc finger domain.5.Seven genes form a network to control rice flowering in this study.By the analysis of expression level and epistatic interaction,it was illustrated that Hd1 was as a promoter under long day and low temperature conditions and as an inhibitor under short day and high temperature conditions.Under different photoperiod and temperature conditions,OsMADS50,Ehd1,Hd3a and RFT1 both as promoter to regulate rice flowering by OsMADS50-Ehd1-Hd3a/RFT1-OsMADS14/OsMADS15 pathway.Contrarily,DTH8 was as an inhibitor downgrading the expression of Ehd1 by a direct or indirect way（inhibiting OSFLF1/OsMADS50）,and had strongly interaction between Hd3a and DTH8,which enhanced photosensitivity to delay heading date strongly.Interestingly,two new heading date pathways by OsGI were found:one way is mainly by OsGI-Hd1-RFT1 under low temperature conditions;another is mainly by OsGI-（OsMADS50/Ghd7）-Ehd1-Hd3a under high temperature conditions.In addition,using the analysis of protein interaction,the interaction were found between Hd1 and Hd3a,RFT1.Hd1 actived the transcription of Hd3a/RFT1 in transcription level,also interacting with Hd3a/RFT1 protein in post-transcription level,assisting or blocking the expression of the function of Hd3a/RFT1.Visibly,the network of rice heading date was complex.By the analysis of expression,epestical interaction and protein interaction can accurate and whole reveal the position and function of these heading date genes at the network.Overall,in this study,based on SSSL materials,we excavated several new allelic variations of heading date genes,cleared their positions and functions on heading date regulation network,confirmed the importance and universality of espestatic interaction between heading date genes,and found their new protein interaction relationships.These results made the regulatory mechanism of rice flowering more clear and built a solid foundation for further rice molecular breeding by design for flowering under multi-environments.Moreover,the methods in this paper also provided a reference for other crops or traits of allelic and espestatic research.