Dynamic QTLs Analysis For Rice Seed Dormancy At Different Developmental Stages And Identification Of Genes Related To Seed Imbibition

Rice(Oryza sativa L.)is the most important food crop in China.With the rapid development of economy,the increase of population and the improvement of people's life,it is important to increase rice yield and improve rice quality in order to meet food's security and market's prosperity.Pre-harvest sprouting of rice is always occurred in the south of China and also in middle-stream and low-stream of Yangtze River.It will reduce seed viability and grain yield as well as grain quality,resulting great economic losses for farmers.Therefore it is becoming more essential to breed varieties with moderate dormancy for rice production and seed industry development in China.Recently,direct seeding rice and other rice cultivation methods in southern China have developed rapidly,it has a high requirement for rice seed vigor.Previous studies have shown that seed priming can effectively improve the seed vigor.However,there is little research on the optimal initiation time for rice seed priming.Thus,it is necessary to develop the biomarker and raise the trigger technology to monitor the seed priming process,and seed priming provides the theoretical basis for the improving seed vigor in rice.In this study,we analyzed the dynamic QTLs for dormancy of rice seeds at different developmental stages using a recombinant inbred line derived from indica and japonica hybrids,and identified the key genes of rice seed imbibition.The main results are as follows:1,One rice population of recombinant inbred lines(RILs),which were derived from 150 families,was used for dynamic QTL analyse of seed dormancy(SD)at the early(4 weeks after heading),middle(5 weeks after heading)and late(6 weeks after heading)development stages.Results showed that the seed dormancy of IR26(indica)was significantly stronger than that of Jiucaiqing(japonica).With the seed development,the dormancy of parents and RILs was gradually decreased.The frequency of seed dormancy was continuous in RILs,and most of them had deep dormancy.Correlation analysis showed that seed germination was significantly and negatively correlated with the days to heading at the early and middle development stages,while seed germination was significantly and positively affected by the temperature during seed developmental stages;the long heading date and the low temperature could lead seeds with deep dormancy.2,Using QTL IciMapping and Network software,the dormancy QTLs of three different developmental stages of seed germination are analyzed.A total of eight additive quantitative trait loci(QTLs)for SD were identified at three development stages.Among them,four,one and three additive QTLs were identified at the early,middle and late development stages,respectively.Epistatic QTLs and QTL-by-development interactions were detected by the joint analysis of multi development phenotypic values,and one additive and two epistatic QTLs were identified.The phenotypic variation of SD explained by each additive,epistatic QTL and QTL × development interaction ranged from 8.0 to 13.5%,0.7 to 3.9%and 1.3 to 2.8%,respectively.One major QTL qSD7.1 for SD was tightly linked to the major QTL qHD7.4 for HD.By comparing chromosomal positions of these additive QTLs with those previously identified,five additive QTLs qSDl.l,qSD2.1,qSD2.2,qSD4.1 and qSD4.2 might represent novel genes.3,The RIL37,RIL55,RIL67 and RIL72 were screened out the best alleles by selecting identified QTLs by RILs.These RILs contain 3 to 7 synergistic alleles controlling seed dormancy,of which RIL3 7 has the most excellent allele.The best three cross combinations for the development of RIL populations were predicted to improve SD.RIL37 × RIL67,RIL55 × RIL72,which can be polymerized with 8 of excellent allele.With selecting excellent lines from RILs population,as well as locating seeds with excellent dormancy sites,the selected RILs and the identified QTLs might be applicable to improve the rice pre-harvest sprouting tolerance by the marker-assisted selection approach.4,Phase ? of seed imbibition is a critical process during seed priming.To identify genes involved in rice seed priming,the altered proteins between the dry and imbibed(24 h)seeds were compared using a two-dimensional gel electrophoresis system in this study.A total of 14 protein spots were identified at the P<0.01 level,and 9 proteins associated with seed imbibition were identified.There are glucose-1-phosphate adenosyltransferase,aminotransferase,starch synthase,heat shock protein,prolamin precursor,FAD-dependent oxidoreductase,phosphoglycerate kinase protein,elongation factor Tu,aminotransferase ?and ? domains.GO analysis showed that these proteins were involved in carbohydrate,biosynthesis,metabolic correlation,signal transduction,storage proteins,stress-related proteins,carbon metabolism,carbon metabolism and amino acid biosynthesis.Meta-analysis showed that most of the genes encoding proteins were expressed in seed filling,maturation,and endosperm tissues.Quantitative real-time PCR analysis showed that the expression of genes associated with stress appeared at the early imbibition stage,while those associated with carbohydrate metabolism,protein synthesis and signalling increased at the late imbibition stage.Three identified proteins(glucose-1-phosphate adenylyltransferase large subunit,aminotransferase and prolamin precursor)had similar transcript and protein expression patterns in embryos.Based on phenotype and gene expression,the optimal stop time for seed priming is 24 h,when these three genes have relatively low expression,followed by significant induction during imbibition in embryos.These three genes are ideal candidate biomarkers for rice seed priming.5,Full-length gene of OsHsp20 was cloned by bioinformatics and PCR.Compared with Hsp20 amino acid sequence,the homology of Hsp20 and monocotyledon was higher than that of dicotyledon.The expression of OsHsp20 gene was the highest in the panicle,followed by the expression level in the node.The transgenic plant was established by using Japonica rice Zhonghua 11 as the receptor,and the germination rate of OsHsp20 RNAi and overexpression transgenic lines was significantly lower and higher than that of wild type,the results showed that the gene could regulate the rice seed Germination rate.