Characterization And Functional Analysis Of Major QTL For Seed Dormancy And Germination In Rice

Seed dormancy affects seed germination,seedling establishment,seed quality and grain yield.However,the genetic and molecular mechanisms underlying seed dormancy and germination in rice（Oryza sativa L.）remain unclear.Here,two populations（CSSLs and BILs）,both developed from the common parents japonica cultivar Nipponbare and indica cultivar 9311,were used to conduct linkage mapping for seed dormancy.In addition,a genome wide association study of seed dormancy in a rice core collection of 252 accessions was performed.A number of QTLs for seed dormancy were detected in the three populations,and one of the major loci for seed dormancy was cloned.The main results are as follows:1.Genotyping of 122 9311/NIP CSSLs using a RICE6 K chip generated a total of 3383 high-quality single nucleotide polymorphisms（SNPs）.These SNPs were then used as markers for bin-map construction.A total of 387 bins with an average length of 800 kb were generated.For BILs,a total of 49,890 high-quality SNPs were identified by using a genotyping-by-sequencing strategy.A bin-map was generated with 3235 bins in 400 BILs,and the bin lengths ranged from 30 kb to 3.0 Mb with an average of 115 kb in the BILs.2.QTL analysis of four germination（dormancy）parameters with bin-maps in the CSSLs and BILs was performed using a linear ridge regression method.A total of 12 and 27 QTL regions for seed dormancy were detected in CSSLs and BILs,respectively.Among these regions,eight major loci were detected in both two populations,and four loci were commonly identified for multiple germination parameters associated with seed dormancy in both populations.3.Two major loci（3.1 and 3.5）exhibited the most significant effects on seed dormancy in CSSLs and BILs.3.1 and 3.5 were individually validated in the near-isogenic lines that contain the target QTL,with the NIP alleles increasing seed dormancy.The effects of 3.1 and 3.5 on seed dormancy were further confirmed in a CSSL-derived F2 population.Furthermore,the NIP alleles of both 3.1 and 3.5 were sensitive to abscisic acid and exhibited a significant epistatic interaction to increase seed dormancy.4.Genotyping of 252 rice accessions by using a RICE50 K chip generated a total of 43,386 high-quality SNPs.The linkage disequilibrium decay values for all 252 rice accessions,two subpopulations of japonica and indica were approximately 130 kb,250 kb and 200 kb,respectively.Genome-wide association analysis in 252 rice accessions was performed for four germination parameters.A total of 22 loci for seed dormancy were detected in the rice collection.Among them,DOM3.5 located nearby 28 Mb on chromosome 3 was colocalized in the same region of 3.5,which had the most significant effect for multiple parameters in the rice collection.DOM3.5 was finely mapped into a 38 kb region using progeny testing of recombinants from a CSSL-derived F2 population that were developed from Nipponbare and Zhenshan 97 B.Sequence and expression analyses in the two parents revealed that LOC_Os03g49400 was the most possible putative gene for DOM3.5.5.Complementary transgenic experiments confirmed that LOC_Os03g49400 is the gene associated with seed dormancy.LOC_Os03g49400 is an ortholog of ethylene insensitive gene 2 in Arabidopsis,which encodes an ethylene insensitive protein.LOC_Os03g4940 is named as Seed Dormancy and Germination 3（OsSDG3）.CRISPR/Cas9 induced OsSDG3 mutants in the NILNIP background showed the mutants significantly promoted seed germination and increased germination uniformity compared with the wild type.These results indicate that the OsSDG3 NIP alleles contribute to high seed dormancy.RNAi transgenic test supported that the OsSDG3 NIP positively regulated seed dormancy at transcription level.6.Eight haplotypes of OsSDG3 were identified in the rice collection,exhibiting significant differences in seed dormancy.Complementary transgenic experiments of OsSDG3SNP122 A to the tropical japonica AZUCENA（containing OsSDG3SNP122C）confirmed that OsSDG3SNP122 A accounted for higher seed dormancy,suggesting that the SNP（+122A>C）in coding region is a functional locus for seed dormancy.7.The NILs that contain various OsSDG3 alleles exhibited significant differences in seed dormancy.The contents of endogenous l-aminocyclopropane-l-carboxylic acid（ACC）and abscisic acid（ABA）in the embryo of NIL-OsSDG3 NIP was significantly higher that those in NIL-OsSDG3^ZS97.Expression analyses of the ABA biosynthesis and signaling genes revealed that OsSDG3 may modulate several genes in the ethylene and ABA pathways to increase seed dormancy.