發(fā)布時(shí)間：2018-09-17 07:55

【摘要】：長(zhǎng)白落葉松是我國(guó)林區(qū)主要的速生造林樹(shù)種之一。具有重要的生態(tài)與經(jīng)濟(jì)價(jià)值。但其在造紙生產(chǎn)過(guò)程中,排出含有木質(zhì)素等化學(xué)物品,對(duì)生態(tài)環(huán)境的破壞也不可小視。因此,高纖維素、低木質(zhì)素的生物合成對(duì)于紙漿材樹(shù)種的生態(tài)和生產(chǎn)效益至關(guān)重要。本研究以11個(gè)種源264個(gè)長(zhǎng)白落葉松單株為研究對(duì)象,通過(guò)對(duì)與木質(zhì)素和纖維相關(guān)的3個(gè)重要基因的單核苷酸多態(tài)性分析,篩選出與材性性狀關(guān)聯(lián)顯著的SNP位點(diǎn),找到具有育種價(jià)值的功能SNP位點(diǎn),為長(zhǎng)白落葉松木材材性性狀分子標(biāo)記輔助育種提供理論依據(jù)。主要研究結(jié)果如下：1.已有分析研究表明CesAl、Csl D3基因與纖維素的合成相關(guān)、C3H基因與木質(zhì)素的合成相關(guān)。通過(guò)對(duì)長(zhǎng)白落葉松轉(zhuǎn)錄組測(cè)序與在NCBI數(shù)據(jù)庫(kù)中進(jìn)行檢索,利用Primer5.0軟件設(shè)計(jì)引物,以實(shí)驗(yàn)采集的264個(gè)長(zhǎng)白落葉松個(gè)體的DNA為模板擴(kuò)增基因片段克隆測(cè)序。3個(gè)材性相關(guān)基因克隆約2343bp序列中共發(fā)現(xiàn)82個(gè)常見(jiàn)SNP位點(diǎn),編碼區(qū)出現(xiàn)64個(gè),非編碼區(qū)出現(xiàn)18個(gè),其中同義突變68個(gè),非同義突變14個(gè)。轉(zhuǎn)換類(lèi)型A-G 22個(gè),T-C 24個(gè)；顛換類(lèi)型A-C 6個(gè),T-G 11個(gè),A-T 9個(gè),G-C 10個(gè)。2.利用Dnasp5.0軟件對(duì)長(zhǎng)白落葉松CesAl、Csl D3、C3H基因進(jìn)行核苷酸多態(tài)性分析,基因平均核苷酸多態(tài)性θW和πT為0.00547和0.01154,總體來(lái)說(shuō)核苷酸多樣性程度較高。0W值變化為0.00102-0.01121,；πT值變化為0.00211-0.02281,Csl D3基因核苷酸多樣性水平最高,C3H基因最低。3.中性檢測(cè)結(jié)果,CesAl和Csl D3基因的Tajima's D, Fu and Li F*和Fu Fs的值均為顯著性負(fù)值,均出現(xiàn)負(fù)值,說(shuō)明這些基因中存在很多低頻率突變。C3H基因Tajima's D、Fu and Li F*和Fu Fs值均為正值。4.利用Haploview4.2軟件,對(duì)SNP位點(diǎn)進(jìn)行連鎖不平衡,CesAl基因SNP之間的連鎖程度較高。相對(duì)CesAl基因來(lái)說(shuō),Csl D3基因和C3H基因SNP之間的連鎖程度較低。CesAl和Csl D3基因?qū)2值的回歸分析結(jié)果為SNP連鎖不平衡程度隨核苷酸序列長(zhǎng)度逐漸減弱,但減緩很慢,而C3H的連鎖不平衡程度在50bp左右,r2下降至0.20,表明其連鎖程度比CesAl和Csl D3基因低很多。三個(gè)基因的連鎖不平衡(LD)分析,表明長(zhǎng)白落葉松適合采用候選基因進(jìn)行關(guān)聯(lián)分析和功能SNP標(biāo)記的開(kāi)發(fā)。5.SNP的篩選與分析。采用單個(gè)SNP位點(diǎn)和單倍型兩種方法對(duì)SNP位點(diǎn)與纖維素和木質(zhì)素進(jìn)行關(guān)聯(lián)分析。在CesAl基因中三個(gè)SNP (SNP4、SNP19、SNP20)和纖維素含量顯著相關(guān)聯(lián),SNP4位于內(nèi)含子上,為非編碼區(qū)突變,SNP19位于外顯子上,為同義突變,SNP20位于外顯子上,為同義突變。在Csl D3基因中有一個(gè)重要SNP (SNP44)和纖維素含量極顯著相關(guān)聯(lián),位于外顯子上,為非同義突變A＞C突變導(dǎo)致賴(lài)氨酸(Lys)變?yōu)樘於彼岚被?Asp)。在C3H基因中有兩個(gè)重要SNP (SNP1、SNP2)與木質(zhì)素含量顯著相關(guān)聯(lián),位于內(nèi)含子上,非編碼區(qū)突變。多重比較分析結(jié)果表明CesAl基因SNP4基因型為AA的長(zhǎng)白落葉松具有較高的纖維素；SNP19基因型為T(mén)T的長(zhǎng)白落葉松具有較高的纖維素；SNP20基因型為GG的長(zhǎng)白落葉松具有較高的纖維素。Csl D3基因SNP44基因型為CC的長(zhǎng)白落葉松具有較高的纖維素。C3H基因SNP1的TT基因型具有較低木質(zhì)素；SNP2基因型為CC的長(zhǎng)白落葉松具有較低的木質(zhì)素。
[Abstract]:Larix olgensis is one of the main fast-growing afforestation tree species in China. It has important ecological and economic value. However, in the process of papermaking, it discharges chemicals such as lignin, which can damage the ecological environment. In this study, 264 Larix olgensis individual plants from 11 provenances were selected as the research object. Through single nucleotide polymorphism analysis of three important genes related to lignin and fiber, SNP loci with significant correlation with wood properties were screened out, and functional SNP loci with breeding value were identified as wood properties of Larix olgensis. The main results are as follows: 1. Previous studies have shown that CesAl and Csl D3 genes are related to cellulose synthesis and C3H genes are related to lignin synthesis. DNA of 264 individuals of Larix olgensis was cloned and sequenced as template amplified gene fragments. A total of 82 common SNP loci were found in the 2343 BP sequence of 3 clones of wood-related genes, 64 in coding regions and 18 in non-coding regions, including 68 synonymous mutations, 14 non-synonymous mutations, 22 in conversion type A-G, 24 in T-C, 6 in transversion type A-C, and 6 in T-C. The nucleotide polymorphisms of CesAl, Csl D3 and C3H genes in Larix gmelinii were analyzed by Dnasp5.0 software. The average nucleotide polymorphisms of theta W and PI T were 0.00547 and 0.01154. The nucleotide diversity of Larix gmelinii was high. The variation of PI T value was 0.00102-0.01121, 0.00211-0.02281 and 0.02281, respectively. The values of Tajima's D, Fu and Li F * and Fu F s of CesAl and CSl D3 genes were significantly negative, indicating that there were many low frequency mutations in these genes. The values of Tajima's D, Fu and Li F * and Fu F s of C3H genes were all positive. Compared with CesAl gene, the linkage between Csl D3 gene and C3H gene was lower. The regression analysis of R2 value between CesAl and Csl D3 gene showed that the degree of SNP linkage disequilibrium decreased gradually with the length of nucleotide sequence, but slowed down very slowly. The disequilibrium degree was about 50 BP and R2 decreased to 0.20, indicating that the linkage degree of the three genes was much lower than that of CesAl and Csl D3. The linkage disequilibrium (LD) analysis of the three genes indicated that the candidates were suitable for association analysis and the development of functional SNP markers. 5. SNP screening and analysis. Three SNPs (SNP4, SNP19, SNP20) in the CesAl gene were significantly associated with cellulose content. SNP4 was located in the intron and mutated in the non-coding region. SNP19 was located in the exon and was synonymous with cellulose and lignin. There was an important SNP in the Csl D3 gene. There are two important SNPs (SNP1, SNP2) in the C3H gene that are significantly associated with lignin content, located on the intron and mutated in the non-coding region. Larix olgensis with SNP4 genotype AA had higher cellulose content; Larix olgensis with SNP19 genotype TT had higher cellulose content; Larix olgensis with SNP20 genotype GG had higher cellulose content. Larix olgensis with Csl D3 genotype SNP44 genotype CC had higher cellulose content. Larix olgensis with lower lignin and SNP2 genotype CC has lower lignin.
【學(xué)位授予單位】：東北林業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：S791.22

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