Genome-Wide Association Mapping For Seed Dormancy And Molecular Cloning And Characterization Of OsDOG1,A Rice Seed Dormancy Candidate Gene

Seed dormancy is an adaptive trait employed by flowering plants to avoid harsh environmental conditions for the continuity of their next generations. In cereal crops, moderate seed dormancy could help prevent pre-harvest sprouting and improve grain yield and quality. In order to dissect the genetic basis of seed dormancy in rice, we performed a genome wide association study (GWAS) for seed germination percentage (GP) in freshly harvested seeds (FHS) and after-ripened seeds (ARS) in 350 worldwide accessions that were characterized with strong population structure of indica, japonica and Aus subpopulations. Meanwhile we tested the Oryza sativa dormancy candidate gene (OsDOG1) from a dormant Minghui63 rice variety (indica) by transforming it into a non-dormant Nipponbare rice variety (japonica). The main results are as below.1. The germination tests revealed that Aus and indica Ⅱ (ⅠndⅡ) rice had stronger seed dormancy than indica Ⅰ (ⅠndⅠ) and japonica rice in FHS. Majority of Aus and a few IndⅡ varieties maintained dormancy in the ARS with majority of IndⅠ and japonica varieties completely breaking their dormancy.2. Association analysis revealed 16 loci significantly associated with GP in FHS and 38 in ARS. Three out of the 38 loci detected in ARS were also detected in FHS and 13 of the ARS loci were detected near previously mapped dormancy QTL.3. In FHS, three of the association loci were located within 100 kb around previously cloned GA/IAA inactivation genes such as GA2ox3, EUII and GH3-2 and one near dormancy gene, Sdr4.4. In ARS an association signal was detected near ABA signaling gene AB15. No similar association peaks were detected among the sub-populations in FHS and only one association peak was detected in both indica and japonica populations in ARS.5. Sdr4 and GA2OX3 haplotype analysis showed that Aus and indica Ⅱ (IndⅡ) varieties had stronger dormancy alleles whereas indica I (IndI) and japonica had weak or non-dormancy alleles.6. The germination test results from the positive OsDOGl transgenic plants showed lower germination rates as compared to the negative transgenic plants. It was however noticed that Nipponbare (control) had unexpected lowest germination rates compared to both positive and negative transgenic plants. The ABA content measurements among Nipponbare, the negative transgenic and positive transgenic plants of OsDOGl showed no significant difference.7. The expression levels of genes involved in ABA biosynthesis pathway were significantly increased in the positive transgenic plants than in the negative controls. It was however, difficult to conclude whether OsDOG1 was conferring dormancy since Nipponbare was expected to have higher germination rates but instead lower germination rates were experienced.Conclusion:The association study and haplotype analysis together, indicate an involvement of independent genes and alleles contributing towards regulation and natural variation of seed dormancy among the rice sub-populations. It is hard to claim whether OsDOG1 functionally regulates seed dormancy. We therefore, intend to change the recipient rice variety in order to ascertain the functionality of OsDOG1 in conferring dormancy.