【作者】 王超；

【导师】 路丙社；

【作者基本信息】 河北农业大学 ， 林木遗传育种， 2010， 硕士

【摘要】 黄连木（Pistacia chinesis Bunge.）和阿月浑子（Pistacia vera L）是黄连木属重要的生物质能源和经济树种。本文采用形态学观察和DNA分子标记技术对不同种源的黄连木群体和引进的阿月浑子品种进行了遗传多样性和亲缘关系分析,以期为黄连木优良种质和阿月浑子优良品种的筛选提供科学依据,主要研究结果如下:1.采用形态学观察和生理测定的方法,对不同种源黄连木种子的横纵径比、千粒重等形态学指标和发芽率、发芽势等生理指标进行了分析比较,结果表明,不同种源黄连木种子的横纵径比、千粒重、发芽率、发芽势等指标均存在显著或极显著差异。种子千粒重为37.56⁵6.20 g ,横纵径比为0.55⁰.75 ;发芽率为15.25%⁵4.38%,发芽势在20.55%⁵4.38%之间。武安种子的千粒重为56.20 g,明显大于其它种源的种子,最小的为安徽种子。林州种源发芽率（54.38%）最高。其次是四川、房山、武安和房山,最低的是绛县。2.利用UPGMA聚类分析,依据黄连木种子表型性状进行了分类研究。参试6个种源可分为两个组群:第一组群包括安徽滁州、四川康定、北京房山3个群体,其中四川康定与北京房山群体种子表型特征基本相同;第二组群包括山西绛县、河南林州、河北武安3个群体,其中山西绛县与河南林州群体种子表型特征较为相近。3. DNA提取方法:用预处液(3%可溶性PVP,2%β-巯基乙醇,100mmol/L^-1 EDTA)和CTAB提取缓冲液（2%β-巯基乙醇、水溶性PVP和4%CTAB）提取阿月浑子和黄连木基因组DNA,其OD₂₆₀/OD₂₈₀值在1.78¹.9之间,可用于PCR扩增。4.利用正交试验设计L₁₆（4⁴）,对模板DNA、Taq DNA聚合酶、引物浓度、dNTP浓度进行了筛选研究,建立了适宜黄连木的RAMP反应体系。20μL PCR反应体系为:1×buffer﹢0.8μL dNTP(250μmolL^-1)﹢0.8μL引物﹢0.8μL Taq酶。5.不同种源黄连木的RAMP分析:13对引物组合所产生的115条DNA扩增片段中,有109条具有多态性,多态位点百分率（PPB）为94.78%;Shannon多样性指数I=0.5541,Nei’s多样性指数H=0.38030,物种总基因多样性（Ht）为0.2963;种群内基因多样性（Hs）为0.3803,群体间基因分化系数（Gst）为0.2209,表明22.09%的变异存在于种群间,群体内的变异占了总变异的77.91%;基因流（Nm）为1.7633,显示种群间的基因交流顺畅。UPGMA聚类分析表明,以遗传距离0.16为阈值,可以分为两类:武安、林州、房山、滁州、绛县居群聚为一类;康定居群独自聚为一类。5.不同阿月浑子品种亲缘关系分析:用筛选的21个随机引物对13个品种阿月浑子DNA进行PCR扩增,共扩增出137个位点,其中多态位点122个,多态位点比率占89.05%,平均每个引物扩增多态性DNA的带数为5.81条。品种间遗传距离在0.2015⁰.5163之间,表明各品种间具有一定的遗传差异。UPGMA聚类分析表明,13个阿月浑子品种在遗传距离0.40处可划分为3个类群,基本反映了阿月浑子品种间的遗传多样性。更多还原

【Abstract】 Pistacia chinesis Bunge. and pistachio (Pistacia vera L) are important energy and economic trees, this article from the seed morphological markers and DNA markers analysis of genetic diversity of Pistacia chinesis Bunge.and Pistacia vera L. genetic relationship between sub-species, and has achieved initial results.In this paper, the length to width ratio, 1000 seeds’mass, germinating rate, germinationcharacters of seeds which originated from six different geographic of Pistacia chinensis Bunge were studied. The result showed that the range of the mass was 37.56 g~56.20 g, the range of length and width ratio of the seeds from 0.55~0.75, and the range of germinating energy from 20.25% to 54.38%, the germinating tendency from 15.25% to 54.38%. The Pistacia chinensis Bunge seeds from different geographic including vertical and length to width ratio, 1000 seeds’mass, germinating rate arrived significant or extremely significant. The WuAn 1000 seeds’mass was 56.20 g, significantly higher than other. the smallest seed was Anhui. Lin states provenance The germination rate of LinZhou was (54.38%) it is Max. Followed by Sichuan, Fangshan, Wu Ann, the lowest was JiangXian.Pistacia chinesis Bunge. seed phenotype based on UPGMA clustering index is divided into two groups: the first group including Chuzhou, Kangding, Fangshan. The Kangding seed phenotypes similar with Fangshan; the second one including Jiangxian, Linzhou,Wuan. The seed phenotypes of Jiangxian state are more similar to Linzhou.With a pretreatment solution (3% soluble PVP, 2%β-mercaptoethanol, 100 mmol / L-1 EDTA) and CTAB extraction buffer (2%β-mercaptoethanol, water-soluble PVP and 4% CTAB) extraction of pistachio and Pistacia chinensis Bunge genomic DNA, ultraviolet spectrophotometry agent test results OD260/OD280 values were between 1.78 to 1.9, can be a template for PCR amplification of DNA.Using orthogonal design L16(44)to investigate four factors(TaqDNA polymerase, dNTP, primer, DNA) at four levels respectively. The RAMP reaction system suitable for Pistacia chinesis Bunge.was established, that was, 20μl amplification reactions system containing 1×PCR buffer, 250μl mol/L-1.dNTPs, 0.8μL primer, 10 ng template DNA, 0.8μL TaqDNA polymerase.The genetic diversity of Pistacia chinesis Bunge. was analyzed using random amplified microsatellite polymorphism (RAMP) markers. The results indicated that there was relatively high genetic diversitv in china. 115 bands were amplified from 6 populations in different habitats with 13 reliable primers, 109 bands were polymorphic, The polymorphic percentage was 94.78%; The Shannon information index I was 0.5541 and Nei’s index H was 0.38030. The total gene diversity Ht was 0.2963, and the gene diversity within populations Hs was 0.3803. The Gst was 0.2209, 77.91% of genetic variation existed within populations while 22.09% of genetic variation existed among populations, and the gene flow was 1.7633. There was a strong gene flow among the populations, So the genetic drift would not cause the genetic differentiation.In order to analyze Pistacia vera L. genetic relationship, random amplified polymorphic DNA (RAPD) was used to study the classification and identification of 13 pistachio cultivars which includs 12 exotic varities and one native selects of Xinjiang. Twenty-one 10 bp primers that selected from eighty arbitrary primers were applied for amplifying the Pistachio DNA. Total 137 bands were produced, among which 122 bands (89.05%) were polymorphic. Genetic distances among the cultivars were estimated based on the amount of band sharing and ranged from 0.2015~0.5163, it is indicated that there are genetic differences among the cultivars. UPGMA cluster analysis showed that 13 pistachio varieties can be divided into three groups in the genetic distance of 0.40 level: The first category includes eleven varieties, the second category is the‘Xinjiang selection’and the third category is‘Mateur’varieties.更多还原