梨（Pyrus L.）AFLP反应体系的建立及在品种鉴定中的应用
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摘要
AFLP(扩增片段长度多态性)是一种新型的分子标记技术，目前已广泛应用于分子生物学及动植物育种等领域的研究。尽管前人利用分子标记技术对梨也进行了一些研究，但利用AFLP技术进行研究的报道很少。故本研究对影响AFLP技术体系的多方面因素进行了探讨，同时对梨属44个品种(包括6组芽变)进行了DNA水平上的研究，探讨AFLP技术在梨品种鉴定中的应用；通过对梨属部分种质进行鉴定和亲缘关系分析，解决如苹果梨等分类存在争议的品种的归属问题，以期为梨的高密度分子遗传图谱的构建、目标基因的定位和核心种质的确立提供参考。主要研究结果如下：
1．对影响梨AFLP指纹分析的多个因素进行了实验与摸索，最后建立了一套梨AFLP指纹分析的最适体系。该体系中各优化因素为：①模板DNA的浓度为150ng／μL，模板DNA用量为450ng；②酶切体系中，MseⅠ和EcoRⅠ各加入3Units，使用MseⅠ Buffer，反应时间为5h；③连接酶最适用量为1.5Units，反应时间为10h；④连接产物最适稀释倍数为5倍；⑤预扩增产物最适稀释倍数为5倍。
2．对64个引物组合进行筛选，选出10个引物组合对44个梨品种进行扩增，共扩增出371个位点，平均每对引物扩增37.1个位点，其中多态性位点306个，占总扩增位点数的82.5％。
3．初步建立了梨属44个品种的AFLP指纹。利用10个引物组合对44个供试品种进行扩增，其中M-CTT／E-ACG能将44个供试品种(包括6组芽变)全部区分开，充分显示了AFLP鉴别率高的优越性。
4．共有19个品种扩增出特有标记，占供试类型的48.2％。其中属于西洋梨系统的4个品种，分别为红巴梨、保利阿斯卡、朱丽比恩和茄梨；3个来源于新疆梨的品种，分别为库尔勒香梨、早熟句句和黄酸梨；另外还有黄金梨、贵妃、中梨1号等12个品种。
5．对供试44个梨品种AFLP数据结果进行了聚类分析。结果表明，白梨系统与砂梨系统品种首先聚在一起，然后是秋子梨系统，最后与西洋梨系统聚在一起。
6．探讨了苹果梨的分类地位。苹果梨与白梨系统的鸭梨欧氏距离最近为7.416，而与砂梨系统的新星为7.746，与秋子梨系统的小香水为8.062，与西洋梨系统的红茄欧氏距离为9.849。从聚类树状图中可以看出，苹果梨首先与金花4号聚在一起，再与其杂种后代苹香聚在一起，认为将苹果梨归为白梨系统比较适宜。
AFLP marker is a new kind of technique, which is widely applied in the molecular biology, high mammal and plants in recent years. The fingerprinting analysis based on AFLP was carried out in 44 different pear cultivars, including six pairs of sports. This study aimed at discussing the feasibility of AFLP applying on Pyrus germplasm, which could find out the ownership of some cultivars, such as Pingguli pear and so on. The main results were as follows:
1 Some factors affecting establishment of AFLP technological system have been tested and analyzed again and again, and at last a most effective system was established. Some improved factors were as follows: (1) the concentration of template DNA was ISOng/^L, the template DNA dosage was 450ng; (2) in the digestion system, each of Msel and EcoRI was input SUnits, Msel Buffer was used and reaction time was 5 hours; (3) the most available ligase dosage was 1.5U, the reaction time was 10 hours; (4) the products of ligation was diluted to 5 times for preamplification; (5) the products of preamplification reaction was diluted to 5 times for selective amplification.
2 10 pairs of primers selected from 64 primer combinations were identified to be high polymorphic and were applied on AFLP amplification in 44 Pyrus plants. 371 loci were gained among which 306 loci were polymorphic. Average locus numbers amplified by per pair of primer were 37.1.
3 The fingerprinting pattern based on AFLP was preliminarily established. The identification efficiency of 10 pairs of primer differed from each other, among which My/Es was able to distinguish 44 pear cultivars.
4 19 forms had specific AFLP markers which were 48.2% of the total.
5 On the base of AFLP data abtained from the study, clustering analysis on the 44 materials of pear were carried out, and the tree diagrams were established by ward's method, the relationships within different systems of Pyrus were discussed.
6 The existent demurral on systematic status of Pingguoli pear was discussed, Pingguli pear clustered together with Jinhuasihao pear which belonged to Pyrus bretschneideri, so we considered that Pingguoli pear should belong to white-pear system.
1．对影响梨AFLP指纹分析的多个因素进行了实验与摸索，最后建立了一套梨AFLP指纹分析的最适体系。该体系中各优化因素为：①模板DNA的浓度为150ng／μL，模板DNA用量为450ng；②酶切体系中，MseⅠ和EcoRⅠ各加入3Units，使用MseⅠ Buffer，反应时间为5h；③连接酶最适用量为1.5Units，反应时间为10h；④连接产物最适稀释倍数为5倍；⑤预扩增产物最适稀释倍数为5倍。
2．对64个引物组合进行筛选，选出10个引物组合对44个梨品种进行扩增，共扩增出371个位点，平均每对引物扩增37.1个位点，其中多态性位点306个，占总扩增位点数的82.5％。
3．初步建立了梨属44个品种的AFLP指纹。利用10个引物组合对44个供试品种进行扩增，其中M-CTT／E-ACG能将44个供试品种(包括6组芽变)全部区分开，充分显示了AFLP鉴别率高的优越性。
4．共有19个品种扩增出特有标记，占供试类型的48.2％。其中属于西洋梨系统的4个品种，分别为红巴梨、保利阿斯卡、朱丽比恩和茄梨；3个来源于新疆梨的品种，分别为库尔勒香梨、早熟句句和黄酸梨；另外还有黄金梨、贵妃、中梨1号等12个品种。
5．对供试44个梨品种AFLP数据结果进行了聚类分析。结果表明，白梨系统与砂梨系统品种首先聚在一起，然后是秋子梨系统，最后与西洋梨系统聚在一起。
6．探讨了苹果梨的分类地位。苹果梨与白梨系统的鸭梨欧氏距离最近为7.416，而与砂梨系统的新星为7.746，与秋子梨系统的小香水为8.062，与西洋梨系统的红茄欧氏距离为9.849。从聚类树状图中可以看出，苹果梨首先与金花4号聚在一起，再与其杂种后代苹香聚在一起，认为将苹果梨归为白梨系统比较适宜。
AFLP marker is a new kind of technique, which is widely applied in the molecular biology, high mammal and plants in recent years. The fingerprinting analysis based on AFLP was carried out in 44 different pear cultivars, including six pairs of sports. This study aimed at discussing the feasibility of AFLP applying on Pyrus germplasm, which could find out the ownership of some cultivars, such as Pingguli pear and so on. The main results were as follows:
1 Some factors affecting establishment of AFLP technological system have been tested and analyzed again and again, and at last a most effective system was established. Some improved factors were as follows: (1) the concentration of template DNA was ISOng/^L, the template DNA dosage was 450ng; (2) in the digestion system, each of Msel and EcoRI was input SUnits, Msel Buffer was used and reaction time was 5 hours; (3) the most available ligase dosage was 1.5U, the reaction time was 10 hours; (4) the products of ligation was diluted to 5 times for preamplification; (5) the products of preamplification reaction was diluted to 5 times for selective amplification.
2 10 pairs of primers selected from 64 primer combinations were identified to be high polymorphic and were applied on AFLP amplification in 44 Pyrus plants. 371 loci were gained among which 306 loci were polymorphic. Average locus numbers amplified by per pair of primer were 37.1.
3 The fingerprinting pattern based on AFLP was preliminarily established. The identification efficiency of 10 pairs of primer differed from each other, among which My/Es was able to distinguish 44 pear cultivars.
4 19 forms had specific AFLP markers which were 48.2% of the total.
5 On the base of AFLP data abtained from the study, clustering analysis on the 44 materials of pear were carried out, and the tree diagrams were established by ward's method, the relationships within different systems of Pyrus were discussed.
6 The existent demurral on systematic status of Pingguoli pear was discussed, Pingguli pear clustered together with Jinhuasihao pear which belonged to Pyrus bretschneideri, so we considered that Pingguoli pear should belong to white-pear system.
引文
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