Molecular Phylogeny And Evolution Of Avena L.

The genus Avena L. in the tribe Aveneae, subfamily Pooideae, Poaceae, consists of 29 species, including 6 genome constitutions (A, C, AB, AC, CC, AACCDD). As an important gene pool for improving cultivated oats and related species, this genus occupies a most important status in cereal plants. However, the species in this genus are often overlapping in ditribution and morphologically so similar that it is very difficult to identify them with certainy if only using gross-morphological charecters. Difference in genome constitution and ploidy level add even greater complexity. Such situation has undoubtedly restricted the effective utilization of the the valuable genetic resources in the genus. In additon, the incomplete knowledge on the diploid donors of the polyploid members in the genus has made the situation become more complicated. Indeed, the origin of the allopolyploid members in the genus has long been a controversial matter. In this study, based on PCR-RFLP and ccSSR analysis of plasmon, the plastid matK gene and the trnL-F region, the nuclear ribosomal internal transcribed spacers (ITS) and 5S rRNA gene, and the intron region of low-copy LEAFY gene, the phylogenetic relationships of the species and the origin of the allotetraploid members were discussed. In additon, based on the former results, primers specific for genome were designed and used to identify the different genomes. The main results are summarized as follows:1. Plasmon genetic varitions of 96 accessions including 25 Avena taxa and 1 outgroup were investigated by using PCR-RFLP markers. Twelve plasmon universal primers produced 203 bands, only 39 out of which were polymorphic (19.2%), indicating that it is not suitable for identify the different genome, species or accessions of Avena. Thus, the consensus chloroplast simple sequence repeat (ccSSR) makers and plastid matK gene and the trnL-F region were used to study the genetic polymorphisms of the Avena plasmon.2. Consensus chloroplast simple sequence repeat (ccSSR) makers were used to assess the genetic variation and genetic relationships of 80 accessions from 25 taxa of the genus Avena. A total of 51 alleles were detected at the 16 ccSSR loci. Among these ccSSR loci, the highest polymorphism information content (PIC) value was 0.754. The mean genetic similarity index among the 80 Avena accessions was 0.545. To assess the usefulness of ccSSRs in separating and distinguishing between haplome (genome) groups, we used ordination by canonical discriminant analysis and classificatory discriminant analysis. The analysis of genetic similarity showed that diploid species with the A haplome were more diverse than other species. Among the species with the C haplome, A. clauda was more diverse than A. eriantha and A. ventricosa. In the cluster analysis, we found that the Avena accessions with the same genomes and/or belonging to the same species had the tendency to cluster together. As for the maternal donors of polyploid species based on this maternally inherited marker, A. strigosa served as the maternal donor of some Avena polyploidy species such as A. sativa, A. sterilis and A. occidentalis from Morocco. A. fatua is genetically distinct from other hexaploid Avena species, and A. damascena might be the A genome donor of A. fatua. A. lusitanica served as the maternal parents during the polyploid formation of the AACC tetraploids and some AACCDD hexaploids. These results suggested that different diploid species were the putative A haplome donors of the tetraploid and hexaploid species. The C genome species A. eriantha and A. ventricosa are largely differentiated from the Avena species containing the A, or B, or D haplomes, whereas A. clauda from different accessions were found to be scattered within different groups, and might play an important role in the phylogeny of Avena.3. The maternally inherited matK and trnL-F sequences revealed the separate A genome diploid species as maternal parents of the different polyploid species. One of the A_SA_S donor diploid species, A. wiestii, fell together with most hexaploid species, A. sativa, A. sterilis and A. occidentalis, with the two AACC tetraploids, A. maroccana and A. murphy, and with one of the AABB genome tetraploid, A. agadiriana on the chloroplast gene trees. Another diploid species, A. damascena carrying the A_dA_d genome, always fell together with the hexaploid A. fatua. Three AABB tetraploids, A. abyssinica, A. vaviloviana and A. barbata, were close to the AA genome diploid A. hirtula in a subclade on the tree. Therefore, the maternal donor was an A genome species with several maternal lineages being involved in different polyploid species.4. ITS concerted evolution in the genus of Avena was detected based on the comparison of 169 clone sequences, and phylogenetic implication of such evolution was discussed. Different patterns of concerted evolution are revealed in the allopolyploid members if the genus. First, only maternal ITS copies are found to be reversed within the most hexaploids and all of the AABB and AACC tetraploids, indicating the high degree of homogenization in the ITS sequences of Avena. Second, biparental ITS copies are both found within the A. fatua, which had two types in separate clades, one with A genome and the other with C genome species. It need more consideration when utilizing ITS for phylogenetic reconstruction.5. The molecular diversity of the rDNA sequences (5S rDNA units) in 71 accessions from 26 taxa of Avena was evaluated. The analyses based on 553 sequenced clones, indicated that there were six sequence unit classes, enumerated according to the haplomes (genomes) they putatively represent, namely the Long A1, Long B1, Long M1, Short C1, Short D1 and Short M1 unit classes. The long and short M1 were found in the tetraploid A. macrostachya the only perennial species. The long M1 unit class was closely related to the short C1 unit class, while the short Ml unit class was closely related to the long A1 and the long B1 unit classes. However, the Short D1 unit class was more divergent with the other unit classes. There was only one unit class per haplome in Avena, which was different from haplomes in the Triticeae which often have two haplomes. Most of the sequences captured belong to the long A1 unit class. Sequences identified as the long B1 unit class were found in the tetraploids A. abyssinica and A. vaviloviana, and in the diploids A. atlantica and A. longiglumis. The short C1 unit class was found in the diploid species carrying the C genome i.e. A. clauda, A. eriantha and A. ventricosa, and also found in the diploid A. longiglumis and in the tetraploid A. insularis, A. maroccana and in all the hexaploid species. The short D1 unit class was found in all the hexaploid species and in two clones of A. clauda and A. murphyi. It is noteworthy that, according to previous studies the B genome was found to be present only in tetraploid species and the D genome only in hexaploid species. Unexpectedly we found that various diploid Avena species contained the B1 and D1 units. Thus there might be a clue where to search for the diploids carrying the B and D genomes. The path inferred is that the C genome is more ancient than the A and B genomes, and it is closer to A. macrostachya, the only existing perennial which is presumed to be the most ancestral species in the genus.6. The usefulness of low-copy nuclear sequence markers is becoming increasingly recognised since they frequently outperform ITS and plastid markers, the highly variable second intron region of low-copy gene LEAFY was used to inferring the phylogenetic relationships of Avena species and the origin of the allopolyploids, especially for the orign of the B and D genome which only found in polyploids. The B genome of three AABB tetraploids, A. abyssinica, A. barbata and A. vaviloviana, were from the AA genome diploid A. hirtula, and another B genome copy from A. agadiriana was closed to the AA genome diploid A. damascena. As for the D genome which only present in hexaploids, it maybe originate from CC genome diploids A. clauda and A. eriantha, or from the tetraploid A. murphyi which contained the D genome copy. The C genome of polyploids showed closer relationship with one of the CC genome species A. clauda.7. It is an opporunity to design primers specific for genome based on the 5S rDNA class units which different genomes has specific sequence varations. The specific PCR primers had been developed to identify A and D genome of Avena species, indicating the feasibility and value of the genome specific. According to the phylogenetic study of the Avena genus, the amount of sequence variation in the 5S rRNA gene and LEAFY second intron region seems to be sufficient to allow the development of genome specific primers or RFLP markers for the identification of the Avena genomes. It is necessary to explore more genome specific molecular markers in future in order to develop an approach of PCR or RFLP analysis to identify the Avena genomes quickly and reliably. This can be used to affirm whether all copy types of certain species have been obtained and would greatly facilitate the identification of collected oat germplasm.