桉树DNA自动提取及木素相关QTL定位

发布时间：2018-09-03 11:22

【摘要】：人工林为工业生产提供高质量的木质生物素原料,同时也作为重要的固碳库。近年来,随着杨树、桉树等树种全基因组测序的完成和高通量测序技术的发展,树木的分子生物学研究进入了高通量的基因组研究阶段,并取得了一些成果。对于样品量大的林木群体基因组学和全基因组关联研究等,自动化、高通量的实验操作尤为必要。因此,本研究重点针对树木分子生物学研究中DNA提取这一基础环节,优化和建立了一种树木叶片DNA的自动提取方法,并将其成功应用于桉树杂种子代的父本分析中。并且,由于桉树是全球范围内热带和亚热带地区广泛种植的用材树种,本研究还对其进行了材性性状中木素含量和愈创木基比紫丁香基的比例(Gyringyl-to-suaiacyl ratio,G/S)的QTL定位研究。主要研究内容如下:(1)树木叶片DNA自动提取方法的优化和建立。以桉树4种/杂种的叶片为材料,比较了5种磁珠试剂盒在KingFisher Flex核酸纯化系统上自动提取的DNA得率和纯度,初步选出了2种较好的试剂盒。比较了KingFisher Flex上不同的洗涤速度,最优为Medium。初选的2种试剂盒所提的DNA均可有效用于PCR扩增,表明DNA纯度均较好,确定得率最高的为最优试剂盒。对比手动的改良CTAB法(DNA得率19.0μg/g,OD260/OD280为1.97,OD260/OD230为2.03),本文优化的方法可显著提高DNA得率(103.6μg/g)且DNA纯度相似(OD260/OD280为1.93,OD260/OD230为1.89)。优化的方法可有效用于马占相思(Acacia mangium)、银中杨(Populus alba×Po.berolinensis)、降香黄檀(Dalbergia odorifera)、思茅松(Pinus kesiya var.langbianensis)、马尾松(Pi.massoniana)和短枝木麻黄(Casuarina equisetifolia)叶片的DNA提取。这对树木分子生物学研究的高通量DNA提取有重要的应用价值,对非木本植物的DNA提取亦可提供有益的方法参考。(2)DNA自动提取方法在桉树杂交子代的父本分析中的应用。参试材料为包含1株母本、2株待确定父本和93株子代的桉树群体,参试标记为具多态性的16个EST-SSR标记。在95%的置信水平下,成功鉴定出92株子代的父本。其中,87株子代的父本为目的父本,5株子代的父本为外源花粉污染。由此,证明了本研究所构建的DNA自动提取方法能成功应用于桉树基于EST-SSR标记的基因分型实验中,为该方法进一步推广应用到其他林木树种的分子生物学研究中奠定基础。(3)桉树木素含量和G/S相关QTL定位。参试材料为课题前期构建的尾叶桉和细叶桉杂交F1无性系群体。QTL定位的遗传图谱为课题前期构建,所需标记分型数据为图谱构建所用标记对该参试群体的分型数据。在母本尾叶桉和父本细叶桉的遗传图谱上,共检测到4个与木素含量和G/S相关的QTLs,LOD值为3.8-20.6,表型变异解释率为5.1%-23.1%。
[Abstract]:Artificial forests provide high-quality lignin feedstock for industrial production and also serve as important carbon sequestration reservoirs. In recent years, with the complete genome sequencing of poplar, eucalyptus and other species and the development of high-throughput sequencing technology, the molecular biology of trees has entered the stage of high-throughput genome research, and some achievements have been made. It is necessary to automate and high-throughput experimental operation for large sample size forest population genomics and whole genome association research. Therefore, aiming at the basic link of DNA extraction in tree molecular biology, an automatic extraction method of DNA from tree leaves was optimized and established, and it was successfully applied to the paternal analysis of eucalyptus hybrids. Since eucalyptus is a widely planted timber species in tropical and subtropical regions worldwide, the QTL mapping of lignin content and ratio of guaiacyl to lilac (Gyringyl-to-suaiacyl ratio,G/S) in wood properties was also studied. The main contents are as follows: (1) Optimization and establishment of automatic DNA extraction method for tree leaves. Using the leaves of 4 species / hybrids of eucalyptus as materials, the yield and purity of DNA extracted automatically from 5 magnetic bead kits in KingFisher Flex nucleic acid purification system were compared, and two better kits were preliminarily selected. The different washing speed on KingFisher Flex is compared. The optimal washing speed is Medium.. The DNA from the two primary kits could be effectively used for PCR amplification, which indicated that the purity of DNA was good, and the best kit was the one with the highest yield. Compared with the manual modified CTAB method (the yield of DNA was 19.0 渭 g / g OD260 / OD280 was 1.97 / OD260 / OD230 was 2.03), the optimized method could significantly improve the yield of DNA (103.6 渭 g / g) and the purity of DNA was similar (OD260/OD280 = 1.93 渭 g / OD230 = 1.89). The optimized method can be used to extract the leaves of (Populus alba 脳 Po.berolinensis, (Pinus kesiya var.langbianensis, Pi.massoniana and (Casuarina equisetifolia) of Acacia equisetifolia (Acacia mangium), (Dalbergia odorifera), (Pinus kesiya var.langbianensis), P. massoniana (Pi.massoniana) and Casuarina equisetifolia (Casuarina equisetifolia). It has important application value for high-throughput DNA extraction in tree molecular biology, and also provides a useful reference for DNA extraction from non-woody plants. (2) the application of DNA automatic extraction method in the paternal analysis of eucalyptus hybrid progeny. The eucalyptus population consisted of 1 female parent and 2 female parents and 93 progenies were selected. The markers were 16 polymorphic EST-SSR markers. At 95% confidence level, 92 male progenies were successfully identified. Among them, the male parent of the 87 progenies was the purpose. The male parent of the 5 progenies of the male parent was exogenous pollen pollution. Therefore, it is proved that the automatic DNA extraction method constructed in this paper can be successfully applied to the genotyping experiment of eucalyptus based on EST-SSR markers. It lays a foundation for the further application of this method in the study of molecular biology of other tree species. (3) Eucalyptus lignin content and G / S correlation QTL mapping. The genetic map of F1 clone population of Eucalyptus urophylla and Eucalyptus grandis was constructed. On the genetic map of female eucalyptus and male eucalyptus, four QTLs,LOD values related to lignin content and G / S were found to be 3.8-20.6.The phenotypic variation interpretation rate was 5.1-23.1.
【学位授予单位】：中国林业科学研究院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S792.39

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