【作者】 范小宁；

【导师】 张盛周； 姚小华；

【作者基本信息】 安徽师范大学 ， 细胞生物学， 2011， 硕士

【摘要】 油茶是我国重要的木本油料本土树种,综合利用价值较高,为了更好的开发利用其资源为人类社会所服务,与此息息相关的工作之一是培育油茶优良品种,其良种培育以选择育种为主。在长期的栽培生产过程中,部分天然杂交种表现出明显的杂种优势,预示着杂交育种是油茶选择育种的一个具有巨大潜力的途径。为了深入探讨油茶杂交子代的遗传多样性,为油茶杂交育种提供指导和理论支持。本文针对油茶控制授粉家系七个共127份实验材料,采用SRAP和SSR两种分子标记的方法,分别对实验材料进行遗传位点的扩增,通过软件POPGENE 3.2和NTSYS 2.10对其扩增位点的统计结果进行计算分析,探讨家系内和家系间的遗传多样性丰富度。两种标记的实验结果分析均得到家系内的遗传多样性大于家系间的遗传多样性,此实验结果为油茶优良新品种的选择提供了更为有价值的理论指导信息,家系内个体水平上选育出油茶优良品种具有较大潜质;同时,从分子水平上对家系内的杂交后代进行了聚类分析,并探索各家系之间的亲缘关系,为确立油茶品种类群的划分提供理论和遗传上的依据。采用SRAP分子标记时,从105对SRAP引物中筛选出9对扩增稳定、重复性好、多态性高的引物,利用优化的SRAP-PCR反应体系对油茶杂交子代进行位点扩增。共检测到片段大小在100-700 bp之间的位点191个,其中多态性比率高达100%,平均每对引物扩增位点21.2个。利用POPGENE 3.2和NTSYS 2.10软件对统计结果进行分析处理显示:Nei基因多样性指数（H）在0.3405-0.3664之间,Shannon信息指数（I）在0.5146-0.5450之间;家系内的遗传多样性（Hs）为0.3547,家系间的遗传多样性（Dst）为0.0200,表明总的遗传变异中家系内占94.67%,家系间仅占5.33%,证实了家系内存在丰富的遗传多样性,并且远大于家系间的丰富度。油茶杂交子代个体间存在着丰富的遗传变异,说明从家系内筛选综合性状优良的油茶品种存在可行性。油茶SSR-PCR反应体系的优化研究,实验应用改良CTAB法提取油茶基因组DNA,采用L₁₆（4⁵）正交设计试验,对影响油茶SSR- PCR反应体系的5因素(Taq聚合酶浓度、模板浓度、dNTPs浓度、引物浓度、Mg²⁺浓度)在4水平上进行筛选,PCR统计结果经分析软件DPS处理,此实验还对其退火温度进行了摸索,确立了油茶SSR-PCR反应的优化体系:总体积为20μL,Taq聚合酶量为1.0 U·20μL^-1,模板浓度75 ng·20μL^-1,dNTPs浓度为0.15 mmol·L^-1,引物浓度为0.4μmol·L^-1,Mg²⁺浓度为1.5 mmol·L^-1,退火温为50℃。在此优化体系的基础上,从61对SSR引物中筛选出10对多态性高、扩增稳定、重复性好的引物,对127份实验材料的遗传变异位点进行了扩增。通过统计分析及其软件计算处理,探讨了油茶家系内和家系间遗传多样性的丰富度,为油茶优良品种的选育提供分子层面的理论指导信息。10对引物组合共检测到60个位点,位点的大小分布在100-280bp之间,其中多态性位点60个,平均每对引物6.0个,多态性比率为100%。利用POPGENE3.2和NTSYS2.10软件对统计结果进行分析显示:Nei基因多样性指数（H）在0.3279-0.3883之间,Shannon信息指数（I）在0.4864-0.5573之间;家系内的遗传多样性（Hs）为0.3641,家系间的遗传多样性（Dst）为0.0329,表明总的遗传变异中家系内占89.44%,家系间仅占10.56%,家系内存在丰富的遗传多样性,并且大于家系间的丰富度。油茶杂交子代个体间存在着丰富的遗传变异,家系内的遗传丰富度大于家系间,为油茶的选择育苗提供了理论方向,说明从中筛选综合性状优良的油茶品种存在可行性。据此推测,在通过杂交育种技术培育油茶新品种工作中,应以杂交后代个体选择为主,优良家系选择对油茶产量提升的潜力相对较小。更多还原

【Abstract】 Camellia oleifera is a characteristic arboreal oil tree plant, the comprehensive utilization value is quite high. In order to exploit its resources for human society, one of the related jobs is to cultivate the thoroughbred Camellia oleifera, and the selection breeding plays an important part in the thoroughbred cultivation. In the long-term of the cultivation process, some natural hybrids have showed obvious advantages. It indicates that the hybrid breeding will play an enormous potential part in the thoroughbred cultivation of Camellia oleifera. In order to further explore the genetic diversity of Camellia oleifera for selecting high-quality seeds, in this study for a total of 127 varieties with 7 families. Mainly including SRAP and SSR marks, POPGENE 3.2 and NTSYS 2.10 were used to detect the genetic diversity among 127 Camellia oleifera cultivars of 7 families. Genetic diversity index information by SRAP and SSR marks indicated that plentiful genetic diversity was existed in this 7 families, genetic diversity in the family was more than that among the family genetic diversity. The rich genetic variation was existed in the hybrid offspring camellia oleifera, it’s feasible to choose the superior comprehensive offspring camellia oleifera in the family. The study is expected to provide some technical foundations for the study of the genetic breeding.In this study for a total of 127 varieties of 7 families, SRAP-PCR of Camellia oleifera was applicated in the basis of optimization system. 9 pairs of SRAP primers had been selected from 105 pairs of SRAP primers. POPGENE 3.2 and NTSYS 2.10 were used to detect the genetic diversity among 127 Camellia oleifera cultivars, and total 191 sites were obtained, The size of the sites being between 100 bp and 700 bp, the average bands per SRAP primer pairs being 21.2, and the ratio of polymorphism was 100.00%. Nei index number was from 0.3405 to 0.3664 and Shannon index number was from 0.5146 to 0.5450. It was 0.3547 in the family genetic diversity, and it’s 0.0200 among the family genetic diversity. It’s 94.67% in the family genetic diversity, and it’s 5.33% among the family genetic diversity. Genetic diversity index information indicated that plentiful genetic diversity was existed in this 7 families and genetic diversity in the family was more than that among the family genetic diversity. The rich genetic variation was existed in the hybrid offspring camellia oleifera, it’s feasible to choose the superior comprehensive offspring camellia oleifera in the family.The genomic DNA extracted from 1eaves of Camellia oleifera with an improved method of CTAB was app1ied to optimize the SSR-PCR system. The four levels of five factors(Taq DNA polymerase, DNA, dNTPs, primers, Mg²⁺)in SSR-PCR system were studied by L₁₆（4⁵） orthogonal design. The electrophoresis profiles of SSR-PCR were ana1yzed by software DPS. The annealing temperature of SSR-PCR reaction was also explored. A suitable SSR-PCR system （20μl） was established as: 1.0 U·20μL^-1U Taq DNA po1ymerase, 75ng·20μL^-1, 0.15 mmol·L^-1 dNTPs, 0.4μmol·L^-1 primers, 1.5 mmol·L^-1Mg²⁺, and annealing temperature at 50℃. The optimization SSR-PCR system is expected to provide some technical foundations for the study of the genetic diversity of Camellia oleifera Resources. In this study for a total of 127 varieties with 7 families, SSR-PCR of Camellia oleifera was applicated in the basis of optimization system. 10 pairs of SSR primers had been selected from 61 pairs of SSR primers. POPGENE 3.2 and NTSYS 2.10 were used to detect the genetic diversity among 127 Camellia oleifera cultivars, and total 60 sites were obtained, The size of the sites being between 100 bp and 280 bp, the average bands per SSR primer pairs being 6.0, and the ratio of polymorphism was 100%. Nei index number was from 0.3279 to 0.3833 and Shannon index number was from 0.4864 to 0.5573. It was 0.3641 in the family genetic diversity, and it’s 0.0329 among the family genetic diversity. It’s 89.44% in the family genetic diversity, and it’s 10.56% among the family genetic diversity.Genetic diversity index information indicated that plentiful genetic diversity was existed in this 7 families and genetic diversity in the family was more than that among the family genetic diversity. The rich genetic variation was existed in the hybrid offspring Camellia oleifera, it’s feasible to choose the superior comprehensive offspring camellia in the family, and it’s high potential to select superior Camellia oleifera species in the family.更多还原