Fine Mapping And RNA-seq Analysis Of A Novel Seed Dormancy Gene QSD7-1 In Rice(oryza Sativa L.)

Seed dormancy is an important agronomic trait that is closely associated with the pre-harvest and seed storage,and obviously affects yield and quality in rice.Therefore,it is of importance to investgate the genetic base of seed dormancy in order to develop rice varieties with moderate seed dormancy and make seed production and storage safety.In this study,SSSL W22S and the receptor parent W0 were used to construct a F₂ segregation population and screen secondary SSSLs for further fine mapping of qSD7-1,and RNA-Seq analysis was also carried out and the candidate related to seed dormancy at qSD7-1 locus was identified.The main results are as follows:1.A F₃ population were developed by self-pollination of the selected plants from the F₂ population of“W22S/W0”.The recombinant F₃ plants were identified and eight key homozygous recombinants were obtained in the F₄ generation.Substitution mapping with homozygous recombinants showed that qSD7-1 was narrowed down to a 48.9 kb fragment between markers LCB14 and LCB5.There were 9 ORFs in the target region.2.Quantitative real-time PCR indicated that ORF1,ORF3,ORF6,ORF8 and ORF9expressed in various tissues,especially in panicles.All of them were expressed at higher levels in SSSL W22S than that in the receptor parent W0 at the late panicle development stage.There were significant differences in expressions of ORF1,ORF3and ORF8 between W22S and W0 in leaf and branch,while expressions of ORF6 and ORF9 were relatively low or hardly detected in the tissues expressed.Therefore,ORF1,ORF3,ORF6,ORF8 and ORF9 were probably the target candidate genes with different expression patterns.3.Sequence analysis showed that there were no differences in the sequences of all ORFs except ORF1 and ORF6 between W0 and W22S.Compared with W0,the sequence of ORF1 in W22S had four changes,including a AT deletion at 317-318 bp in the first intron and single base mutations at the second,eighth and ninth exons,which did not changed the amino acid,while that of ORF6 existed single base difference from C to T at coding region,which made the amino acid changed from arginine to tryptophan.Tryptophan is an important precursor of auxin synthesis,and ORF6 contains an F-Box domain,which play a vital role in signal transduction of plant hormones?especially in GA andABA?and the process of sugar metabolism,etc.At the same time,genetic effect indicated that the C/T mutation locus was highly correlated with seed dormancy.Therefore,ORF6 was considered to be the target candidate gene.4.The allelic variation of qSD7-1 was investigated by using four SSSLs from different donor.Three alleles qSD7-1¹,qSD7-1² and qSD7-1³,were identified at qSD7-1 locus,seed dormancy of which showed the following trend:qSD7-1¹>qSD7-1²>qSD7-1³.5.RNA-Seq analysis manifested that there were 59 differential expressed genes.Among of them,14 were up regulated in expression and the others were down regulated.Gene annotation demonstrated that these genes mainly involved in the metabolism of linoleic acid,starch and sucrose metabolism,and galactose metabolism,glycine,serine and threonine metabolism,arginine and proline metabolism,glycolysis pathway and other secondary metabolites biosynthesis.This suggested that regulation of seed dormancy of qSD7-1 seemed to be associated with secondary metabolites such as sugars,amino acids.In the study,a novel seed dormancy gene,was fine mapped on chromosome seven,and the candidate genes were identified through sequencing and quantitative expression.Three alleles with different genetic effect were identified at qSD7-1 locus,genetic basis of rice seed dormancy was further clearified.These laid a foundation for qSD7-1 gene clone and molecular design breeding in future.