Domestication of cereal crops from wild relatives has reduced seed dormancy, which can be imposed by inherent constraints within the embryo (embryo dormancy) or by restrictive tissues covering the embryo (coat-imposed dormancy). A small region of the genome designated as qSD12 has been isolated from weedy rice and is known to contain one or more genes that control embryo dormancy. Map-based cloning has identified two candidate genes for qSD12. The project aims to determine the function, origin, differentiation, and distribution of the candidates and to identify qSD12 downstream gene networks that regulate the development of embryo dormancy in the rice model system. Isogenic lines for the candidate genes and several approaches, such as complementation, haplotype, and transcriptomic analyses, will be used to achieve these objectives. The project is expected to assign one or two of the candidate genes to the function of embryo dormancy, reveal whether the dormancy gene(s) differentiated before or after the rice domestication, whether cultivated rice species and subspecies originated from different functional mutations, and provide direct knowledge of molecular and physiological pathways through which the naturally occurring gene regulates embryo dormancy. The broader impacts of the project include: 1) practical training for graduate, undergraduate, and high school students and teachers in biology and agricultural programs; and 2) interdisciplinary collaborations to use the dormancy gene for breeding varieties resistant to pre-harvest sprouting and to test the hypothesis that orthologous genes regulate dormancy and germination in other grasses.
Fine mapping and cloning of the SD12 seed dormancy QTL discovered three candidate genes (i.e., SD12a, SD12b & SD12c) in the previous years of this project. Research in the past year was conducted to verify functions of these candidates for seed dormancy and to identify gene networks regulated by the candidates. Three strategies were used to verify the candidate genes. The first was the development of perfect isogenic lines for all of the three loci, including SD12c, to evaluate their seed dormancy under controlled conditions. The second was to the introduction of transgenes for each of the three candidates from Nipponbare (japonica cv.) into the genetic background of the recipient (EM93-1, indica cv.) for the isogenic lines to evaluate their seed dormancy. And the third was the use of purified T-DNA insertion mutation and RNAi silencing lines for SD12a to evaluate its effect on seed dormancy in different genetic backgrounds. All the three approaches confirmed that the SD12 major QTL is a complex of three tightly linked genes and each of them has an effect on seed dormancy. RNA-seq analysis was conducted to identify putative genes regulated by the SD12 seed dormancy genes. About 800 genes were detected in early developing seeds based on genome-wide transcription profiles for the three isogenic lines EM93-1, EM93-1_SD12a, and EM93-1_SD12a+b+c. The most differentially expressed genes are those encoding heat shock proteins, which explained the previous observation that the effect of SD12 on seed dormancy is enhanced by high seed developmental temperatures. In the past year, the resources of this project were used to train a Native Indian American undergraduate, three graduate students and a postdoc researcher, and also used to train six high school teachers from the South Dakota state in a one-week workshop. There has been difficult to clone the full-length cDNA for SD12b, which is annotated as a hypothetic protein. It is also important to know how SD12a, SD12b and SD12c interact with each other to make the complex a major QTL, which is hard to be addressed by the native genes because of their linkage. In addition, haplotype analysis suggested that the SD12 complex may be only present in some wild rice lines collected from the southern part of Thailand. Therefore, research will be conducted to isolate SD12b’s full-length cDNA, to evaluate epistatic effects between/among the three loci, and to determine the similarity in DNA sequence of the SD12 complex between the donor line of functional alleles at SD12a, SD12b and SD12c and the selected wild ancestral lines
