Genentic Diversity Studies Of Asian Common Wild Rice(Oryza Rufipogon Griff.) And Primary Functional Analysis Of Its2Specific Genes

Cultivated rice (Oryza sativa L.) is a major source of nutrition for more than half the global population. Asian common wild rice(Oryza rufipogon Griff.) has long been believed to be the direct ancestor of Asian cultivated rice. It serves as a valuable gene pool for the improvement of cultivated rice because of its many extraordinary traits, therefore, a better understanding of the genetic diversity of Asian common wild rice will be beneficial for the efficient use of the Oryza gene pool and will certainly have significant impact on rice production. In the present study,12different common wild rice distributed from18.73N to28.14N in China were studied, and six indica cultivars, four japonica cultivars were used as controls. A more comprehensive comparison of nine highly variable cpDNA regions and471SSR markers on chromosome4were performed to elucidate the genetic diversity of common wild rice.The results will deepen our understanding of the genetic diversity of common wild rice and ultimately accelerate the efficient use of the extraordinary genes of common wild rice for the genetic improvement of cultivated rice. Meanwhile, the cpDNA genetic diversity of17different cytoplasm male sterile (CMS) lines was also studied in this research. The main results are as follows:1. CpDNA genetic diversity analysis of common wild rice and cultivated rice The chloroplast DNA (cpDNA) polymorphism among18cultivated rice and12Asian common wild rice were analyzed by comparing the polymorphic sites in nine highly variable regions. A total of144polymorphic bases were detected. O. rufipogon from different distributions, with117polymorphic bases, showed rich genetic diversity. The131bases in13sites were identified with indica/japonica characteristics, as they showed differences between the indica and japonica subspecies at these sites. The javanica strains and japonica shared similar bases at these131polymorphic sites, suggesting that javanica has a close relationship with japonica. Using the amplified fragment length analysis of ORF100and ORF29-TrnCGCA can simply and rapidly identify the indica/japonica types of O. sativa cytoplasm. Based on the results of the length analyses of the ORF100and the ORF29-TrnCGCA fragments, the O. rufipogon strains can also be classified into indica/japonica subgroups, which indicate that differences in indica and japonica also exist in the cpDNA genome of the O. rufipogon strains. However, these differences showed certain primitiveness and incompleteness, as an O. rufipogon line may show different indica/japonica attributes at different sites. Consequently, the O. rufipogon cannot be simply classified into the indica/japonica types according one or several molecular markers. Our data support the hypothesis that Asian cultivated rice, indica and japonica, separately evolved from different Asian common wild rice strains, which have different indica-japonica differentiation trends.2. CpDNA genetic diversity analysis of cytoplasm male sterile (CMS) lines The cpDNA genetic diversity of different types of CMS lines was elucidated by comparing the polymorphic sites in nine highly variable regions, taking cultivated rice and common wild rice as controls. According to the cpDNA polymorphism, the17CMS lines were divided into three cytoplasmic types:CMS-WA (wild-abortive), CMS-HL (Honglian) and CMS-BT (Boro II). So-called CMS-Yinshui, CMS-GA and CMS-K types had the same sequences with CMS-WA lines, so they were redefined as CMS-WA cytoplasmic type. Each CMS type had specific bases:CMS-WA lines uniquely had a "GTTGAG" sequence at position220-225of rps16intron, CMS-HL lines contained a specific "G" base at position595of rps16intron, and CMS-BT lines were distinguishable by their indica specific sequences "GCTT" in the ccsA gene under a japonica cpDNA background. All of these specific sequences can be used as DNA fingerprinting for them. On the other hand, it was proved that some genetic variations occurred in cpDNA of CMS lines. However, further study is needed to reveal the correlationship between these variations and pollen sterility.3. Genetic diversity analysis on chromosome4of common wild rice and cultivated rice471pairs of SSR markers were used in this study to explore the genetic diversity on chromosome4in common wild rice and cultivated rice. O. rufipogon from different distributions showed rich genetic diversity which is much higher than that of cultivated rice. And there also exists some allelic genes that cannot be found in cultivated rice, which indicated that common wild rice contains many extraordinary genes used for genetic improvement of cultivated rice.141indica-japonica characteristic SSR markers were identified, and O. rufipogon strains showed different indica-japonica differentiation at these sites. So it proved again that there exists an indica-japonica differentiation trend in genome of the O. rufipogon strains. The constructed dendrogram of these materials also showed this result. Thus, these results provided evidence for dual origin of cultivated rice on the level of molecular genetics. However, javanica rices in this study showed different indica-jaopnica differentiations, which was inconsistent with the results showed by using cpDNA sequences. So it is reasonable to classified javanica in to japonica subspecies based on the cpDNA sequences characteristic of them, while it also seems have a lag to stand on to regard javanica as an independent subspecies of cultivated rice according to the complex analysis results of IGS and ITS sequences (studied by the other researcher of our lab) and chromosome4.4. Identification of specific genes in common wild rice and the functional analysis of these genes Two common wild rice specific genes located on chromosome4were identified, cloned and transferred into cultivated rice in this study. And the chlorophyll content and proline of one transgenic line were higer than wild type, which suggested this common wild rice specific gene might be related to the stress tolerance of plant. According to the primary results of abiotic stress treatment of this transgenic line, it was suuggetted that this gene might play a role in high salt tolerance of plant. Generally, common wild rice showed rich genentic diversity in both cpDNA and chromosome4. And there also exist indica-japonica differentiation trend in common wild rice which supported the dual origin of cultivated rice.2common wild rice specific genes were also indentified and one of them was suggested plays a role in high salt tolerance of plant in this study. All these results will deepen our understanding of genetic diversity of common wild rice and provide therotical support for genetic improvement of cultivated rice. Meanwhile, according to the cpDNA genetic diversity,17different CMS lines were classified in to3types:CMS-WA, CMS-HL and CMS-BT, and a corresponding DNA fingerprinting system for them was preliminarily established.