Study On Pear (Pyrus) Germplasm Resources Cultivated In Dangshan Anhui Province By AFLP Technique

There are abundant pear germplasm resources in Dangshan County, Anhui province. The yield of Dangshan su pear held the great share in the world pear market. Many new types of pear (Prus.) have been forming in the long history of pear evolution because of the complex genetic background and a lot variation from mutation and hybridization between species. So it would be very significant to study and utilize the germplasm resources for accelerating the program of pear breeding.AFLP (amplified fragment length polymorphism) was a new DNA molecular marker technique with high efficiency, promptitude, stability and accuracy. The system suitable for pear genomic DNA analysis was established according to systematical study of the main factors involved in AFLP technique system. The digestion of genomic DNA was implemented by using of two-step digestion with the two restriction enzymes. 500ng genomic DNA was digested with 3 units of EcoRI and MseI respectively in a reaction total volume of 25μL. Double strainded adaptor was legated to the restriction fragments at 16℃overnight (8-12h). The digested-legated DNA fragment was diluted to 10 times with ddH2O and used in pre-amplification. The products of which were diluted to 10 times with ddH2O and used in selective amplification. The PCR reaction products with the same volume loading buffer denature at 95℃for 5min,which were isolated in 6% denatured polyacrylamide gel on stable power(70w) and then stained by silver nitrate.The 27 biotypes of pear, which cultivated in Dangshan country, were analyzed in the established AFLP technique system to study genetic diversity and mutant's identification. The abundance bands of polymorphic DNA were obtained by amplication with three pairs of selective primer. There were more than 50 bands in each channel of electrophoresis. The size of amplified fragment was 30bp-600bp, and the number of which was 180 bands reliable in AFLP techenique system, of which the 166 bands were polymorphic accounting for 92.2% of total bands in the genus. The polymorphism amplified with EcoRⅠ-3/MseⅠ-1 was the highest, up to 100%, and which with EcoRⅠ-2/MseⅠ-3 was the lowest, 89.7%. There were no the same genetic biotype found in the test. The bands of biotypes that had close relative relationship had little similarity, on the country, had larger similarity.The result of cluster analysis showed that all studied biotypes could be divided into six groups at threshold of 39.2-40.1 in coefficient of squared euclidean distance. The first group includes Dangshan su pear and its 8 mutants, intraspecies hybrid, local cultivars. The second group includes Xuehuali, Yali, Laiyangcili, Zisu, Zaomeisu, Kosui, Niitaka, where P. bretschneideri Rehd and P.pyrifolia Naka were clustered into one group. The third group included Kuerle and Hongxiangsu. Nanguoli, Hongbali and Pyrus betulaefolia Bunge formed a group resepectively.Dangshan su pear and its eight mutants were analyzed by AFLP techenique with three pairs of selective primers. The result showed that 7 bands altered in Baguaisu, 10 in Lianglizaosu, 9 in Xiusu, 6 in Youpisu, 15 in Xipisu, 5 in wangsu, 8 in Lengsu, 5 in Biansu, comparing with the stock plant.