Evolution Of Starch Synthase Gene Family In Medicinal Wild Rice

Nearly a half of the world’s population has been feed on rice grains, or accurately on starch in the grains. Many basic and applied studies that focused on the process and individual gene function of the starch synthase gene family provide important guides on rice breeding and genetic improvement. As a wild relative of rice crop, Oryza officinalis has abundant genetic diversity that can serve as a key genetic resource to improve rice starch quality and yield. Here, based on rice whole genome data, we analyzed the details of the starch synthase gene family in O. sativa, and then using the methods of transcriptome resequencing, PCR amplification and sequencing, and qRT-PCR, we invested member constitutions, sequences differences and expression divergences of the starch synthase genes in O. officinalis. The main results are as follows:1. Using next-generation sequencing technology, we investigated the leaf transcriptome of O. officinalis. Approximately 23 million reads(each read was 100bp) with a total of 2.1×109 bases were produced, and de novo assembly methods constructed 68,132 unigenes. Functional annotations for the unigenes were conducted using sequence similarity comparisons against the NCBI、SWISS、KEGG、COG、GO and InterPro databases.2. By analyzing the whole genome data of O. sativa, we found 11 starch synthase genes(SSI, SSII-1, SSII-2, SSII-3, SSIII-1, SSIII-2, SSIV-1, SSIV-2, SSV, GBSSI, and GBSSII) in rice, and these genes locate in 8 different chromosomes(chr1, 2, 4-8, and 10), and have divergent genes structures. Each gene has 8-20 exons and 1827- 4662 bp’s codon region length.By transcriptome resequencing and data analyzing, 18728 reads were mapped onto above 11 rice starch synthase genes, and after assembly alignment, 8 complete genes(SSI, SSII, SSIII, SSIV, SSV and GBSS) were identified in O. officinalis, and they have high level of sequences similarity(93% at amino acid level) to their homologs in cultivated rice. Further statistical tests based on the ratio of nonsynonymous substitutions to synonymous substitutions showed that all of the 8 genes were undertaken significantly purified selection in both wild and cultivated rices. In addition, 3 rice starch synthase genes(SSII-3, SSIV-1, and GBSSI) failed to match their complete homologs in O. officinalis due to a relatively few numbers of reads. This situation possibly associated with a low expression level of these genes in leaf.3. Based on the amino acid sequences of rice, Arabidopsis, maize, and O. officinalis, we constructed a maximum likelihood tree, and found the starch synthase genes in 4 species were all clustered into different clades in according to different functions. Eight genes in O. officinalis were placed on different clades, suggesting the genes identified in this study being truly starch synthase genes.4. In order to confirm whether the starch synthase genes have different expressions in the same or different organs, we designed mRNA amplified primers based on 8 genes of O. officinalis(SSI, SSII-1, SSII-2, SSIII-1, SSIII-2, SSIV-2, SSV, and GBSSII) and 3 genes of O. sativa(SSII-3, SSIV-1, and GBSSI), and then carried out a series of qRT-PCR comparisons in leaf and seed, respectively. As result, we found that divergent expressions of 11 starch synthase genes in O. officinalis emerged not only in the same organ, but also in different organs. For example, an obviously spatial specific expression of GBSSI was revealed, that the gene had a very low level expression in leaf, but a high level expression in seed.