【摘要】：人工林為工業(yè)生產(chǎn)提供高質(zhì)量的木質(zhì)生物素原料,同時(shí)也作為重要的固碳庫(kù)。近年來(lái),隨著楊樹(shù)、桉樹(shù)等樹(shù)種全基因組測(cè)序的完成和高通量測(cè)序技術(shù)的發(fā)展,樹(shù)木的分子生物學(xué)研究進(jìn)入了高通量的基因組研究階段,并取得了一些成果。對(duì)于樣品量大的林木群體基因組學(xué)和全基因組關(guān)聯(lián)研究等,自動(dòng)化、高通量的實(shí)驗(yàn)操作尤為必要。因此,本研究重點(diǎn)針對(duì)樹(shù)木分子生物學(xué)研究中DNA提取這一基礎(chǔ)環(huán)節(jié),優(yōu)化和建立了一種樹(shù)木葉片DNA的自動(dòng)提取方法,并將其成功應(yīng)用于桉樹(shù)雜種子代的父本分析中。并且,由于桉樹(shù)是全球范圍內(nèi)熱帶和亞熱帶地區(qū)廣泛種植的用材樹(shù)種,本研究還對(duì)其進(jìn)行了材性性狀中木素含量和愈創(chuàng)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?jī)?nèi)容如下:(1)樹(shù)木葉片DNA自動(dòng)提取方法的優(yōu)化和建立。以桉樹(shù)4種/雜種的葉片為材料,比較了5種磁珠試劑盒在KingFisher Flex核酸純化系統(tǒng)上自動(dòng)提取的DNA得率和純度,初步選出了2種較好的試劑盒。比較了KingFisher Flex上不同的洗滌速度,最優(yōu)為Medium。初選的2種試劑盒所提的DNA均可有效用于PCR擴(kuò)增,表明DNA純度均較好,確定得率最高的為最優(yōu)試劑盒。對(duì)比手動(dòng)的改良CTAB法(DNA得率19.0μg/g,OD260/OD280為1.97,OD260/OD230為2.03),本文優(yōu)化的方法可顯著提高DNA得率(103.6μg/g)且DNA純度相似(OD260/OD280為1.93,OD260/OD230為1.89)。優(yōu)化的方法可有效用于馬占相思(Acacia mangium)、銀中楊(Populus alba×Po.berolinensis)、降香黃檀(Dalbergia odorifera)、思茅松(Pinus kesiya var.langbianensis)、馬尾松(Pi.massoniana)和短枝木麻黃(Casuarina equisetifolia)葉片的DNA提取。這對(duì)樹(shù)木分子生物學(xué)研究的高通量DNA提取有重要的應(yīng)用價(jià)值,對(duì)非木本植物的DNA提取亦可提供有益的方法參考。(2)DNA自動(dòng)提取方法在桉樹(shù)雜交子代的父本分析中的應(yīng)用。參試材料為包含1株母本、2株待確定父本和93株子代的桉樹(shù)群體,參試標(biāo)記為具多態(tài)性的16個(gè)EST-SSR標(biāo)記。在95%的置信水平下,成功鑒定出92株子代的父本。其中,87株子代的父本為目的父本,5株子代的父本為外源花粉污染。由此,證明了本研究所構(gòu)建的DNA自動(dòng)提取方法能成功應(yīng)用于桉樹(shù)基于EST-SSR標(biāo)記的基因分型實(shí)驗(yàn)中,為該方法進(jìn)一步推廣應(yīng)用到其他林木樹(shù)種的分子生物學(xué)研究中奠定基礎(chǔ)。(3)桉樹(shù)木素含量和G/S相關(guān)QTL定位。參試材料為課題前期構(gòu)建的尾葉桉和細(xì)葉桉雜交F1無(wú)性系群體。QTL定位的遺傳圖譜為課題前期構(gòu)建,所需標(biāo)記分型數(shù)據(jù)為圖譜構(gòu)建所用標(biāo)記對(duì)該參試群體的分型數(shù)據(jù)。在母本尾葉桉和父本細(xì)葉桉的遺傳圖譜上,共檢測(cè)到4個(gè)與木素含量和G/S相關(guān)的QTLs,LOD值為3.8-20.6,表型變異解釋率為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.
【學(xué)位授予單位】：中國(guó)林業(yè)科學(xué)研究院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S792.39

【參考文獻(xiàn)】

相關(guān)期刊論文 前8條

1 巴華杰;馬駿;劉亞楠;朱愛(ài)華;林子清;;4種固相顆粒吸附法提取濾紙血痕DNA效果的比較[J];中國(guó)法醫(yī)學(xué)雜志;2016年04期

2 王占軍;李嬌嬌;張家效;徐楠楠;歐祖蘭;徐忠東;何子昂;沈夏;陳延松;;黃山木蘭葉片基因組DNA提取方法的比較研究[J];分子植物育種;2016年03期

3 楊海峰;王敏杰;趙樹(shù)堂;唐芳;盧孟柱;;利用擬南芥基因芯片和突變體對(duì)木材形成相關(guān)基因的初步分析[J];林業(yè)科學(xué);2011年12期

4 黃真池;余秋梅;歐陽(yáng)樂(lè)軍;曾富華;;一種木本植物RNA和DNA的簡(jiǎn)捷提取方法[J];湖北農(nóng)業(yè)科學(xué);2010年04期

5 饒紅欣;Briony Patterson;Brad Potts;Réne Vaillancourt;;藍(lán)桉樹(shù)木園異型雜交及花粉污染的微衛(wèi)星分子標(biāo)記研究(英文)[J];Journal of Forestry Research;2008年02期

6 甘四明;李梅;李發(fā)根;吳坤明;吳菊英;盧國(guó)桓;白嘉雨;;尾葉桉×細(xì)葉桉雜種無(wú)性系扦插生根和生長(zhǎng)性狀的研究[J];林業(yè)科學(xué)研究;2006年02期

7 郭寶生,張輝,耿軍義,張香云;改良SDS法快速提取小樣品量棉花總DNA及其純化[J];棉花學(xué)報(bào);2005年05期

8 張博,張露,諸葛強(qiáng),王明庥,黃敏仁;一種高效的樹(shù)木總DNA提取方法[J];南京林業(yè)大學(xué)學(xué)報(bào)(自然科學(xué)版);2004年01期

相關(guān)會(huì)議論文 前1條

1 郭嫻;張寒霜;趙俊麗;王永強(qiáng);;高鹽低pH值法提取棉花基因組DNA研究[A];中國(guó)棉花學(xué)會(huì)2011年年會(huì)論文匯編[C];2011年

相關(guān)博士學(xué)位論文 前1條

1 于曉麗;桉樹(shù)高密度遺傳圖譜構(gòu)建及生長(zhǎng)和材性相關(guān)QTL解析[D];中國(guó)林業(yè)科學(xué)研究院;2015年

，

本文編號(hào)：2219837