桑树多倍体育种材料遗传背景的AFLP分析

【作者】 王卓伟；

【导师】 余茂德；

【作者基本信息】 西南农业大学 ， 特种经济动物饲养， 2001， 硕士

【摘要】 在桑树品种选育中，由于人工三倍体桑在经济性状和农艺性状上比二倍体桑品种和自然三倍体桑更为优良，已成为国内外桑树品种选育的重要途径。本研究利用DNA多态性检出率较强的AFLP（Amplified Fragment Length Polymorphism）分子标记技术，即扩增片段长度多态性，从DNA分子水平对19份人工三倍体桑育种材料，其中14份四倍体材料（通过秋水仙素人工化学诱变而成），5份二倍体材料，进行遗传背景分析。构建了这些人工多倍体桑育种材料的指纹图，得到19份材料的UPGMA聚类图。根据各材料间的遗传距离及相似系数和前人研究结果，本研究以结合线法认为遗传相似系数0.7500可作为选择杂交亲本的标准。在19个供试材料中，二倍体桑材料与四倍体桑材料间的相似系数小于该值，认为是亲缘关系较远的种间关系，杂交后代可能产生较强的杂交优势，适于用作人工三倍体桑育种的杂交亲本。从19份桑育种材料70个杂交组合中，选出16个杂交组合，认为适于用作人工三倍体桑育种亲本间杂交组合。 本研究还利用AFLP技术对二倍体桑与人工诱导（秋水仙素）的同源四倍体桑之间的遗传差异进行了分析。根据三个桑种5组二倍体桑与同源四倍体桑共10个材料的DNA多态性及遗传距离分析，二倍体桑与同源四倍体桑的遗传差异小于种间遗传差异，大于种内同倍性单株间遗传差异，在种内变异水平上。由此认为二倍体桑经秋水仙素诱导成同源四倍体的同时，其遗传结构即DNA分子序列也发生了一定改变，变化程度因材料的不同而有差异。 对于AFLP分子标记技术及RAPD（Random Amplified polymorphic DNA）分子标记技术应用于桑树遗传育种研究，经与前人用RAPD方法得出的结果进行比较分析，在亲缘关系较近的材料间两种方法的实验结果有较高的一致性。结合AFLP和RAPD两种方法各自的特点，认为AFLP更适宜于有较高亲缘关系材料间的遗传分析。 在DNA提取过程中，根据桑叶自身特点，探索出一种提取高质量桑叶总DNA的方法，即利用CTAB（Cetyltrimethyle Ammonium Bromide，十六烷基三乙基溴化铵）缓冲液，附加一定量PVP（Polyvinylpyrrolidone，聚乙烯吡咯烷酮），采用平衡酚-氯仿-异戊醇-核糖核酸酶法抽提，不仅可较彻底去除蛋白质，RNA，并发现PVP能有效去除桑叶中高含量的多糖，使所提取DNA能满足AFLP实验要求，保证实验结果可靠性。更多还原

【Abstract】 Mulberry artificial triploid breeding is becoming the focus in mulberry breeding because the economic characters and the agronomic characters of artificial triploid mulberry are excellent than that of diploid mulberry and natural triploid mulberry. Genetic background of 19 materials used for artificial triploid breeding, which including 14 tetraploid materials and 5 diploid rnatercials, are analyzed by the AFLP (Amplified Fragment Length Polymorphism) from which the fingerprint and the UPGNIA dendrogram of the materials are abtained . According to the results, if the genetic coefficient between diploid material and tetraploid material is less than 0.7500. which is the identification of mulberry species according to the former study and this study, the two materials can be used for hibridization .16 hibrid combinations selected from 19 assayed materials can be used for mulberry artificial triploid breeding. The genetic diversity between diploid and homologous tetraploid chemically induced by colchicines are analyzed. The result indicates that the genetic diversity between diploid and homologous tetraploid is less than that of interspecies, more than that of?individuals, at the level of? intraspecies. It can be conclLtded that the DNA molecular sequence of homologous tctraploid materials changes compares with diploid according to the results. As to the study in which AFLP and RAPD are used for mulberry research, the results by two means are consistant when the bloodrelationship of materials is close, AFLP is more appropriate to materials whose bloodreltionship is close through comparison and analyzation. A means is explored through which high quality DNA are extracted, that is CTAB buffer plus I 3~ PVP. with the method of phenol-chloroform-isopentamol-endoribonucleases. The protein and RNA are eliminated completely. and it is found that PVP has the function to eliminate polyhexose. The DNA extracted is fit for AFLP.更多还原