Studies On Genetic Relationships Of Pyrous Species And Cultivars Based On Retrotransposons Markers

Pyrus L.(common name pear) naturally distributes across Eurasia. According to their original distribution areas, Pyrus is divided geographically into two groups:the occidental pears and the oriental pears. Owing to the lack of a reproductive barrier among Pyrus species, interspecific hybridization has been thought to be one of the main modes of speciation, which makes it difficult to classify the Pyrus species. The phylogeny of Pyrus has not been well resolved by using AFLP, SSR, chloroplast sequences and nuclear sequences. Recently, the whole genome of pear has been sequenced, which has provided a new research measure to analysize the Phylogeny of Pyrus. In Pyrus,42.4%of the genome was reported to be long terminal repeat (LTR) retrotransposons with high insertion polymorphisms. In this study, we predicted the retrotransposons in Pyrus genome, and developed new retrotransposon-based markers to study the genetic relationship and evoluation of Pyrus species. The main results were listed as follows;1. Bioinformatics methods were used to isolate the Tyl-copia and Ty3-gypsy retrotransposons in pear genome, and a total of1,836full-length LTR retrotransposons were mined. According to the result of cluster analysis, ten families with high copy numbers were identified. We also isolated the reverse transcriptase (RT) to further analysize the feature of retrotransposons. A total of345copia and99gypsy RT sequences were obtained. Sequence alignment showed a high heterogeneity in both copia and gypsy RT sequences. The results of blasting with RNA-Seq data and semi-quantitative RT-PCR proved the expression of RTs in pear organs under the normal growing condition.2. The LTR sequences of retrotransposons and their flanking region were used to develop the retrotransposon-based insertion polymorphisms (RBIP) markers, and196RBIP primers were developed, of which24pairs of primers developed from the Ppcrl retrotransposons were used to analyze genetic diversity among110Pyrus accessions. Our results showed that RBIP markers had high insertion polymorphisms. The genetic structure analysis and the unweighted pair group method with arithmetic mean (UPGMA) dendrogram indicated that all accessions could be divided into oriental and occidental groups. In oriental pears, wild pea pears clustered separately into independent groups. Cultivars of P. ussuriensis Maxim, P. pyrifolia Nakai, and P. pyrifolia Chinese white pear were mingled together. Two P. pyrifolia cultivars,’Puguali’ and ’Yangdanxueli’, showed100%similarity, suggesting that these two cultivars might be synonyms.3. Because of less informative sites of RBIP makers, the genetic relationship within oriental pears was still uncertain. Therefore, we developed retrotransposon based sequence-specific amplification polymorphisms (SSAP) markers, and12primer combinations were used to evaluate genetic diversity of the Asian pears. The result showed2,833fragments were produced, and the average percentage of polymorphic bands for all primer combinations was98.80%. Results of a population structure analysis indicated that all Asian pear species originated from five primitive genepools. Four genepools corresponded to four Asian species: P. betulaefolia, P. pashia, P. pyrifolia, and P. ussuriensis. The remaining genepool was from occidental pears. Nearly all Asian pear accessions were composed with more than two genepools, suggesting that Asian pears experienced hybridization events. Based on these findings, four primary Asian speices were identified:P. betulaefolia, P. pashia, P. pyrifolia, and P. ussuriensis. The other Asian speices were of hybrid origin. P. calleryana, which has long been treated as primary species, probably originated from the hybridization between P. betulaefolia and P. pashia. Pyrus xerophila might originate through the hybridization involving in P. pashia, P. ussuriensis, and the occidental pears. Pyrus phaeocarpa originated from the hybridization of P. betulaefolia, P. pyrifolia, and P. ussuriensis. The hybrid origins of P. hopeiensis involved P. betulaefolia and wild P. ussuriensis. Pyrus hondoensis had a close relationship with P. ussuriensis and originated from hybridization with local species, most probably P. dimorphophylla.4. The results based on SSAP markers showed that the genepool compositon of Chinese sand pear and Chinese white pear was almost same, suggesting their similar genetic background. In clustering tree, they did not form a discrete group, but were intermingled. Our result supported that the Chinese white pear was treated as an ecotype of the Chinese sand pear. The geographic distribution of Pyrus indicated the gene introgression occurred from P. pashia to P. pyrifolia in southwestern China and Japan, while in northern China the gene introgression happened from P. ussuriensis to P. pyrifolia Chinses white pear. The result of STRUCTURE showed there was almost no difference between the Japanese pear, Chinese white pear and the Chinese sand pear, but most Japanese pears clustered separately from Chinese accessions of the P. pyrifolia in dendrogram. The possible reason was that primitive germplasm of the Japanese pear was introduced from ancient China, and then, Japanese pear cultivars developed independently and became specific because of geographical isolation with Chinese pear. The cultivars of P. ussuriensis should be involved in the hybridization between P. pyrifolia and the wild P. ussuriensis. These interspecific hybridization events caused the poor resolution of classification of Pyrus species.