Phylogenetic Analysis Of Pear And Huyou Based On SSR, AFLP And ITS Markers

In this thesis, different principles, characteristics and their applications of RFLP,RAPD,SSR,AFLP and ITS markers were reviewed. The phylogenetic relationship of Pyrus spp and huyou were analyzed by using SSR,AFLP and ITS markers.Simple Sequence Repeat (SSR) markers were used to assess genetic diversity and relationship of Pyrus cultivars native mainly to East Asia. A total of 168 putative alleleswere generated from six primer-pairs BGA35,KU10,BGT23b,NH004a,NH011b and NH015a. All the SSR markers showed a high level of genetic polymorphism with a mean of 28 putative alleles per locus and the heterozygosity of 0.631.The Dice's similarity coefficient between cultivars ranged from 0.02 to 0.98and occidental pears generally had low affinities to Asian pears. Thirteen groups were generated from all the accessions by UPGMA clusters analysis. Chinese sand pears consisted of 4 groups with Chinese white pears and Japanese pears, of which Chinese sand pears occurred in all 4 groups, presenting a large genetic diversity, Chinese white pears were included in 2 groups, and Japanese pears only fell into two groups. Some Japanese pear cultivars had high affinities to Chinese sand pear cultivars. These results support the previous viewpoints of Chinese white pears as a variety or an ecotype of Chinese sand pears (P. pyrifolia var.sinensis (Lindley) Y.Teng et K.Tanabe) and progenitor of Japanese pears coming from China. Cultivars of P. ussuriensis Maxim were clustered together with one clone of P. hondoensis Nakai et Kikuchi. Cultivars of P. commusis L and other Occidental species formed three different groups and were distant from most Asian pears,except for P. betulaefolia Bge. A further research had been carried out with AFLP markers. The six primer combinations (EcoRI-AAG/MseI-CAT, EcoRI-ACT/MseI-CTT, EcoRI-AAC/Mse I-CAG, EcoRI-AAC/MseI -CTA, EcoRI-AAC/MseI-CAA, EcoRI-AAG/MseI-CTC) could successfully differentiate all the materials and generated 649 fragments of which 575fragments were polymorphic. Dendrogram analysis clustered the 100 Pyrus spp accessions into 15 groups. Chinese sand pears scattered in 7 groups mingled with Chinese white pears,which suggested a close relationship between them. Some Japanese cultivars in group 2 were clustered with Chinese sand pear 'Puguali' from Zhejiang province. The Japanese pears 'Hakataao' and 'Konpeito' were also clustered with Chinese sand pears 'Yingzhiqing' and 'Kuixingmake' from Jiangsu province and Zhejiang province, respectively. This finding further supported the close relationship between Japanese pears and Chinese sand pears. Both studies with SSR and AFLP markers suggest a common origin for Chinese sand pears, Chinese white pears and Japanese pears.In order to unravel the mystery of the origin of huyou {Citrus changshanensis K. S. Chen et C. X. Fu), all citrus existed before huyou in Qingshi Town of Changshan County where huyou originated were evaluated for the possibility of being parents of huyou based on ITS sequences. Sequencing of 6 individual 'Changhuyihao' recombinat plasmids revealed 2 different ITS sequences. Sequence variation was observed in 8 sites (62nd, 63rd, 67th, 74th, 99th, 129th, 218th, 220th) in ITS1 region, while no variation was found in ITS2 region as well as 5. 8 S,partial 18 S or partial 26 S region. The lengths of ITS 15.8 S and ITS2 regions for huyou are 250,164,and 228 bp,respectively. The existence of two different ITS1 sequences,designated as ITS II and ITS III in this study, supported the hybrid origin of huyou. Applying other ITS1 sequences,sour orange and pummelo were identified to be the parents of huyou. In all previous studies,pummelo was suggested to be a parent but sour orange was never evaluated. Sweet orange,which was supposed to be the other parent of huyou in previous studies, can be excluded because none of the three ITS1 sequences are identical to either of ITS 1 sequences of huyou. Sour orange as a parent can also well explain the strong freezing resistance of huyou,since sour orange is much more resistant to freezing than huyou,pummelo and sweet orange.