Genome Fingerprinting Of Pear Cultivars By ISSR

ISSR marker is a new kind of technique that developed in recent years, and used in the research of high mammal and plants in relative fields. The fingerprinting analysis based on 1SSR-PCR was carried out in 52 different pear cultivars. This study aimed at establishing ISSR fingerprinting pattern and clearing genetic relationship so that can offer some reference for efficiently using these pear cultivars and molecular marker-assisted, breeding The main results were as follows:1 The optimum reaction system of ISSR-PCR and program for cultivar pear were established. In a total volume of 20uL, containing template DNA 40ng, 1.0U Taq-DNA polymerase,0.2umol·L^-1ISSR primer,200u mol·L^-1,dNTP,Buffer(Tris-Hcl(pH8.3)10m mol·L^-1,Kcl 50 mmol·L^-1 ,Mgcl₂ 1.5 mmol·L^-1). Amplified procedure was programmed for 5 minutes at 94℃, at annealing temperature(52℃) for 60s,extension 120s at 72℃ ;39 cycles : 60 seconds at 94℃, at annealing temperature(52℃) for 60s,extension 120s at 72℃ and then a final extension 72℃ for 10 min after the last cycle and then keep at 4℃ .Amplified products were detected by electrophoresis in 1.4% agarose gel (containing 1 ug·mL^-1EB).2 11 ISSR primers selected from 100 primers were polymorphic in pear cultivar and were used in ISSR-PCR of 52 pear cultivars. 193 loci were gained among which 182 loci were polymorphic. Average locus number amplified by per primer and length of locus were 18 and 280～3000bp respectively.3 The fingerprinting pattern based on ISSR were preliminarily established. The identification efficiency of 11 primers differed with each other. The identification efficency of primer UBC810 and UBC845,each of them was able to distinguish 52 pear cultivars.4 Total 6 forms which were respectively Guyot, Piaoli Aikunqieke, Kuerlexiangli, Shenbuzhi, and Jingbaili, had specific ISSR markers which were 11.5% .5 According to the result of ISSR-PCR, Clustering analyses on the 52 pear cultivars were carried out,The phylogenetic relationship of four cultivar system and the relationshipof Pingguoli and Xinjiangli by Ward's method with Euclidean were discussed.(1) 25 pear cultivars selected which were characteristics on four cultivar systemed , were clustered, these pear cultivars from the same cultivar system clustered together at first, that is the same pattern by morphology.(2) Accordining to the results of the ISSR statistics analysis, the relationship of the 52 pear cultivars could be studied accurately. Pingguoli pear clustered together at first with Ya-li ,Bai-zhimuyang and Chili which belongs to Pyrus bretschneider Rehd.so Pingguoli has neareast genetic relationship with Pyrus bretschneider Rehd..(3) In virtue of ISSR technique, the genetic relationship of Xinjiangli by Ward's method with Euclidean were discussed.Pyrus sinkiangensis Yu can be divided many clusters.Hongnahe clustered together with Pyrus bretschneider Rehd..Ku-er-lie Xiangli originated from Xinjiang clustered with those cultivars from the same origin., and then clustered with Flemish Beauty belonging to Pyrus communis . so the result indicate that Pyrus sinkiangensis YU has both Pyrus bretschneider Rehd.and Pyrus communis.6. The amplification products based on ISSR proved that ISSR markers was an effective marker type characterized by rapidness, cheap cost and high polymorphisms and be suitable for use in fingerprinting analysis and cultivar identification of pear cultivars.