Molecular Phylogeny Of The A-genome Species Of Oryza, With A Reference To Origin Of The Asian Cultivated Rice

The genus Oryza L. in the Poaceae consists of more than 20 wild species and two cultivated species, O. sativa and O. glaberrima, and is distributed across tropical and sub-tropical area. It is represented cytogenetically by 10 genome groups (i.e. the A-, B-, C-, BC-, CD-, E-, F-, G-, HJ-and HK-genomes). The A-genome group, which contains the cultivated rice, has the widest geographic distribution and is the most recently diverged lineage within the genus Oryza. This genome group includes seven or eight diploid species based on different taxonomical treatments. Because of the closest affinity with the cultivated rice, wild species with the A-genome serve an important gene pool for genetic improvment of the cultivated rice. During the past decades, a wealth of data from morphological and cytological and molecular markers has significantly enhanced our understanding of classification and evolution of the A-genome species. However, many problems regarding the phylogenetic relationships and evolutionary history of this group are still unsolved due to the similar morphology and overlapping distribution of the species. In this study, we reconstructed the phylogeny and estimated the divergence time among A-genome species in Oryza using phylogenetic analysis of intron sequences of four nuclear genes and MITE insertions. In addition, we investigated the population genetics and phylogeography of O. rufipogon and O. nivara as well as the origin of the Asian cultivated rice based on cloning and sequencing of population samples. The main results are summarized as follows: 1. Phylogeny of the A-genome species Based on the search in the whole genome database of O. sativa, we selected and sequenced the intron of four nuclear genes, including Adh1 and other three anonymous genes. The separate and combined analyses the intron sequences by both maximum parsimony and Bayesian MCMC methods indicated that (1) O. meridionalis from Australia was the basal lineage of A-genome species; (2) The two subspecies of the Asian cultivated rice, O. sativa ssp. indica and O. sativa ssp. japonica, showed closer affinity with different Asian wild accessions, supporting the hypothesis of the polyphyletic origins of the Asian cultivated rice; (3) O. rufipogon and O. nivara mixed with each other in the phylogenetic trees, indicating their close genetic relationships; (4) One of the African wild species, O. barthii was the immediate progenitor of the African cultivated O. glaberrima, while O. longistaminata, another wild species from Africa, showed a distant relationship with O. glaberrima and O. barthii. These three species from Africa did not form a monophyly on the combined or individual phylogenetic trees; (5) Based on the molecular clock approach, we estimated that the divergence of A-genome group occurred approximately 2.0 million years ago (MYA), while the Africa cultivated rice and Asian cultivated rice, two subspecies (indica and japonica) of the Asian cultivated rice separated roughly 0.7 and 0.4 MYA, respectively. In addition, comparisons between the intron sequences and other commonly used nrITS and matK demonstrated that intron sequences of nuclear genes were verypowerful and informative for phylogenetic study of the closely related species. 2. Population genetics of O. rufipogon / O. nivara and origin of the Asian cultivated rice Whether O. rufipogon and O. nivara should be combined into a signle or partitioned into two independent species has long been a subject of controversy for decades. In this study, we sequenced introns and 5'-UTRs regions of four nuclear genes (Waxy, LHS,CatA and one unknown gene) by cloning and sequencing of population samples of O. rufipogon and O. nivara. The results indicated that: (1) High levels of nucleotide polymorphisms in the two species were detected with the average estimated nucleotide diversity being 0.011 ( π ) and 0.014 (θw). (2) No distinct genetic differentiation was observed between O. rufipogon and O. nivara. Abundant shared polymorphisms and no fixed difference at these four loci suggested the two species might remain in a large population in history, which supported the previous treatment of the two species into two ecotypes of a single species. (3) The geneaological analysis revealed that the two subspecies of the Asian cultivated rice were closer to different wild accessions than to each other, in agreement with the polyphyletic hypotheses of origin of O. sativa. 3. Utility of transposons in studies of population genetics and phylogeny Considering the lack of molecular markers with sufficient phylogenetically information or intraspecific variation in studies of plant phylogeography, we explored the utility of the fast-evolving transposable elements based on the allele genealogical analysis in O. rufipogon and O. nivara. Our results demonstrated that: (1) Much more nucleotide variation in transposon regions than in other regions of sequences was detected on both the species and the population levels. (2) High nucleotide divergence was found in both the outcrossing O. rufipogon and the inbreeding O. nivara, suggesting that mating system was not related directly with the level of genetic diversity. (3) Transposon insertion diversity is a useful marker for the inference of phylogenetic relationships. Presence of three out of seven transposons at three unlinked genes on different chromosomes further testified that O. meridionalis was the most basal lineage of the A-genome group. Additionally, a survey of three thansposon-like deletions in the choloroplast genomes of the tribe Oryzeae revealed that two of the three deletions were related to the material origin of allotetraploid with BC genome and genus Leersia.