长白落叶松材相关基因单核苷酸多态性研究及关联分析

发布时间：2018-09-17 07:55

【摘要】：长白落叶松是我国林区主要的速生造林树种之一。具有重要的生态与经济价值。但其在造纸生产过程中,排出含有木质素等化学物品,对生态环境的破坏也不可小视。因此,高纤维素、低木质素的生物合成对于纸浆材树种的生态和生产效益至关重要。本研究以11个种源264个长白落叶松单株为研究对象,通过对与木质素和纤维相关的3个重要基因的单核苷酸多态性分析,筛选出与材性性状关联显著的SNP位点,找到具有育种价值的功能SNP位点,为长白落叶松木材材性性状分子标记辅助育种提供理论依据。主要研究结果如下：1.已有分析研究表明CesAl、Csl D3基因与纤维素的合成相关、C3H基因与木质素的合成相关。通过对长白落叶松转录组测序与在NCBI数据库中进行检索,利用Primer5.0软件设计引物,以实验采集的264个长白落叶松个体的DNA为模板扩增基因片段克隆测序。3个材性相关基因克隆约2343bp序列中共发现82个常见SNP位点,编码区出现64个,非编码区出现18个,其中同义突变68个,非同义突变14个。转换类型A-G 22个,T-C 24个；颠换类型A-C 6个,T-G 11个,A-T 9个,G-C 10个。2.利用Dnasp5.0软件对长白落叶松CesAl、Csl D3、C3H基因进行核苷酸多态性分析,基因平均核苷酸多态性θW和πT为0.00547和0.01154,总体来说核苷酸多样性程度较高。0W值变化为0.00102-0.01121,；πT值变化为0.00211-0.02281,Csl D3基因核苷酸多样性水平最高,C3H基因最低。3.中性检测结果,CesAl和Csl D3基因的Tajima's D, Fu and Li F*和Fu Fs的值均为显著性负值,均出现负值,说明这些基因中存在很多低频率突变。C3H基因Tajima's D、Fu and Li F*和Fu Fs值均为正值。4.利用Haploview4.2软件,对SNP位点进行连锁不平衡,CesAl基因SNP之间的连锁程度较高。相对CesAl基因来说,Csl D3基因和C3H基因SNP之间的连锁程度较低。CesAl和Csl D3基因对r2值的回归分析结果为SNP连锁不平衡程度随核苷酸序列长度逐渐减弱,但减缓很慢,而C3H的连锁不平衡程度在50bp左右,r2下降至0.20,表明其连锁程度比CesAl和Csl D3基因低很多。三个基因的连锁不平衡(LD)分析,表明长白落叶松适合采用候选基因进行关联分析和功能SNP标记的开发。5.SNP的筛选与分析。采用单个SNP位点和单倍型两种方法对SNP位点与纤维素和木质素进行关联分析。在CesAl基因中三个SNP (SNP4、SNP19、SNP20)和纤维素含量显著相关联,SNP4位于内含子上,为非编码区突变,SNP19位于外显子上,为同义突变,SNP20位于外显子上,为同义突变。在Csl D3基因中有一个重要SNP (SNP44)和纤维素含量极显著相关联,位于外显子上,为非同义突变A＞C突变导致赖氨酸(Lys)变为天冬氨酸氨基酸(Asp)。在C3H基因中有两个重要SNP (SNP1、SNP2)与木质素含量显著相关联,位于内含子上,非编码区突变。多重比较分析结果表明CesAl基因SNP4基因型为AA的长白落叶松具有较高的纤维素；SNP19基因型为TT的长白落叶松具有较高的纤维素；SNP20基因型为GG的长白落叶松具有较高的纤维素。Csl D3基因SNP44基因型为CC的长白落叶松具有较高的纤维素。C3H基因SNP1的TT基因型具有较低木质素；SNP2基因型为CC的长白落叶松具有较低的木质素。
[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.
【学位授予单位】：东北林业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S791.22

【参考文献】