【作者】 宋丰顺；

【导师】 朱立武；

【作者基本信息】 安徽农业大学 ， 果树学， 2004， 硕士

【摘要】 安徽砀山地区梨资源丰富，砀山酥梨在世界水果市场占据重要地位。由于梨遗传背景复杂，种内变化大，加上种间杂交，使砀山地区梨在长期的进化和栽培中形成了丰富的资源类型。对砀山地区梨种质资源进行研究，以达到合理利用这些资源，对加快梨育种进程有重要意义。本试验调查并收集了砀山地区梨24个资源类型，进行RAPD研究，主要结果如下：1、以CTAB法和SDS法提取了幼叶、成熟叶、韧皮部和芽中的DNA，通过纯度、颜色、产率、电泳，扩增结果等比较，认为CTAB法更适合梨基因组DNA提取。用幼叶提取的DNA，质量好，产率高；成熟叶用改良CTAB法提取也可得到适于RAPD分析的DNA；韧皮部和芽用上述两种方法提取，DNA产率均较低。2、用正交设计筛选出适合梨树的RAPD反应参数为：在25μl反应体系中，模板DNA 60ng、引物4pmol、dNTPs 5×10-9mol、10×PCR Buffer 2.0μl、MgCl2 5×10-8mol、Taq酶1.0U。3、运用6个引物的特异带构建出的梨DNA指纹图谱，可区分14个生物型中的9个。用8个引物的带型组合构建的指纹图谱，可将这14个资源类型全部区分。4、从60个随机引物中筛选出40个表现稳定的引物进行PCR扩增。砀山地区梨种质资源聚类分析表明，当阀值为68.6~70.4时，全部生物型可分为5类。第一类：砀山酥梨及其变异、鸭梨、雪花梨、莱阳慈梨和紫酥，主要是白梨；第二类：新高、丰水、二十世纪、皇冠和苍溪梨，除皇冠外，都为沙梨；库尔勒香梨和红香酥聚为一类；伏酥和豆梨各为一类。5、逐步判别分析显示，Fisher判别函数值只与引物S54 1500bp，S58 1375bp，S58 947bp和S1461831bp 4个位点有关，判别函数为：Y1= -27X1+18X2-18X3+18X4-19.609；Y2=9X1-3.60×10-15X2+3.597×10-15X3-3.60×10-15X4-5.209；Y3=31.5X1-13.5X2+22.5X3-13.5X4-19.609；Y4= 31.5X1-4.5X2+40.5X3-22.5X4-35.359。6、砀山酥梨的8个变异类型，用60个随机引物进行RAPD分析，与对照相比，变化的条带为：良梨早酥29条，锈酥10条，细皮酥6条，扁酥4条，黄花酥3条，棱酥2条，王酥1条，八怪酥无变化带。更多还原

【Abstract】 There are abundant pear resources in Dangshan County, Anhui province. The yield of Dangshan su pear holds the great share in the world pear market. Many new types of pear (Pyrus.) have been forming in the long history of pear evolution because of the complex genetic background and a lot variation from mutation and cross between species. So it will be very significant to study and reasonably utilize the germplasm resources for accelerating the program of pear breeding.The RAPD analysis was undertaken with 24 biotypes, which were cultivated in Dangshan. The main results were as follows:1. Two kinds of method—CTAB and SDS—were applied to extract the genomic DNAs of young leave, mature leave, phloem and dormant bud. The results showed that the CTAB method was better than SDS method based on the compare of DNA purity, color, yield, amplification etc. The DNAs extracted from young leave by two methods all had good quality and yield. The DNA from mature leave extracted only by improved CTAB method could be suitable for RAPD analysis. The yield of DNAs was very low, which were extracted from phloem and dormant bud by either SDS or CTAB method.2. The parameters in RAPD reaction system were determined by orthogonal experiment: In the system of 25μl, the quantity of template DNA is 60ng; primer 4pmol; dNTPs 5×10-9mol, 10×PCR Buffer 2.0μl; MgCl2 5×10-8mol; Taq polymerase 1.0U.3. The DNA fingerprint constructed by the special bands of 6 arbitrary primers could identify 9 biotypes of 14 ones. The Fingerprint constructed by the patterns of 8 arbitrary primers could completely discriminate whole biotypes studied.4. The genomic DNAs of 22 biotypes were amplified by RAPD analysis using the 40 steady arbitrary primers screened from 60 ones. The result of cluster analysis showed that all studied biotypes could be divided into five groups in the threshold of similarity coefficient from 68.6 to 70.4. The first group was mainly Pyrus bretschneideri, including Dangshan su pear and its mutants, Yali, Xuehuali, Laiyangcili. The second group was all P. pyrifolia except for Huangguanli, including: Niitaka, Kosui, Nichisaki, and Cangxili. The third group was composed of Kuerle pear and Hongxiangsu. Fusu and Pyrus betulaefolia formed a group respectively.5. The result of stepwise discriminant analysis showed that the Fisher functions were only relative with 4 loci—S54 1500bp，S58 1375bp，S58 947bp and S1461 831bp. The Fisher equations were as follows: <WP=7>Y 1= -27X1+18X2-18X3+18X4-19.609；Y2 = 9X1-3.60×10-15X2+3.597×10-15X3-3.60×10-15X4-5.209；Y3 = 31.5X1-13.5X2+22.5X3-13.5X4-19.609；Y4 = 31.5X1-4.5X2+40.5X3-22.5X4-35.3596. Comparing with contrasted cultivar, eight mutants of Dangshan su pear were analyzed by RAPD analysis with 60 arbitrary primers. The result showed that Baguaisu had no changing band, Lianglizaosu had 29 changing bands, Xiusu 10, Xipisu 6, Biansu 4, Hanghuaxu 3, Lengsu 2, and Wangsu 1.更多还原