毛白杨遗传连锁图谱的构建及重要性状的分子标记.doc

1 毛白杨遗传连锁图谱的构建及重要性状的分子标记毛白杨遗传连锁图谱的构建及重要性状的分子标记 作者 张德强 作者 张德强指导教师 张志毅教授 指导教师 张志毅教授 摘要摘要 毛白杨 Populus tomentosa Carr 是我国特有的乡土杨树 属杨柳科 Salicaceae 杨属 Populus L 白杨派 Section Leuce Duby 树种 主要分布在我国黄 淮海流域约 100 万平方公里的范围内 在我国北方 尤其是在黄河中下游林业生产和 生态环境建设中占有重要地位 构建毛白杨遗传连锁图谱并进行数量性状位点 Quantitative Trait Loci QTLs 和质量性状位点分析 将对毛白杨物理图谱的构建 基因的图位克隆和分子标记辅助早期选择等研究具有重要的理论意义和潜在的实践意 义 本研究建立了适合于毛白杨特点的遗传作图分离群体 运用扩增性片断长度多态 性 Amplified Fragment Length Polymorphisms AFLPs 标记技术和拟测交作图策略 构建了第一张毛白杨及其杂种毛新杨的 AFLP 遗传连锁图谱 结合作图群体的表型资 料 对毛新杨 毛白杨 F1杂种的几个性状进行 QTLs 定位 并利用混合分群分析法 Bulked Segregant Analysis BSA 与 AFLP 技术相结合 对毛白杨种子胚根发育生根 基因进行了分子标记 得到如下主要结论 1 以毛白杨 毛新杨及银腺杨为材料 利用切枝水培和种子组织培养技术相结 合建立了用于毛白杨遗传作图的三个较大的分离群体 均超过 500 株 对三个作图群 体的亲本进行 AFLP 标记多态性检测和对子代进行田间表型性状调查后 选取了 DNA 多态性高 生长性状 适应性和木材化学成分含量均有较大差异的毛新杨无性系 TB01 毛白杨无性系 LM50 的子代分离群体作为构建第一张毛白杨遗传连锁图的作图 群体 2 选用 AFLP 标记技术结合拟测交作图策略首次构建了我国特有白杨派乡土树 种毛白杨及其杂种毛新杨的 AFLP 遗传连锁图 51 对 AFLP 引物组合在作图亲本间共 检测到 808 个多态性位点 在 P 2 0 时 对于材积量 分枝角 分枝数 叶长 叶宽 叶面积 叶柄长 叶脉数目 春季顶芽展叶时间 木材纤维素和木质素 含量共 11 个性状检测到 50 个 QTLs 4 当 LOD 值 2 0 时 利用区间作图法共检测到 18 个 QTLs 与主干材积量 分 枝角和分枝数性状相连锁 每一性状可检测到 4 8 个 QTLs 每一 QTL 可解释表型变 异的 7 2 12 8 而对于主干材积量性状共检测到 4 个 QTLs 它们分别位于遗传连 锁图的 TLG1 TLG6 TLG8 和 TBLG2 上 每一 QTL 可解释表型变异的 8 0 11 0 8 2 和 8 6 相应每一 QTL 的加性效应值分别为 0 936 m3 0 946 m3 1 198 m3和 0 950 m3 其中位于 TLG1 TLG6 TLG8 上的 QTLs 的增效等位基因源于亲本毛白杨无性系 LM50 位于 TBLG2 上的 QTLs 为亲本毛新杨 无性系 TB01 的等位基因 这些 QTLs 总和可解释表型变异的 35 8 对于分枝角性 状 利用区间作图共检测出 6 个 QTLs 分别位于 TLG2 TLG5 TLG7 TLG10 TLG16 和 TBLG14 上 每一 QTL 可解释表型变异的 7 4 12 8 共解释 表型变异的 57 7 其中位于 TLG7 TLG10 和 TLG16 上的 QTLs 对分枝角有增效 影响 其加性效应值分别为 5 08 6 31 和 4 37 它们均源于亲本毛白杨无 性系 LM50 对分枝数性状共检测出 8 个 QTLs 其中 7 个对分枝数有增效影响 其加 性效应值分别为 8 76 个 9 43 个 8 06 个 9 87 个 8 76 个 8 89 个和 8 16 个 每一 QTL 可解释表型变异的 7 0 9 2 所有 QTLs 总和可解释表型变异 的 61 7 5 对控制叶片表型性状如叶长 叶宽 叶面积和叶柄长以及春季顶芽展叶时间 共 5 个性状进行了 QTL 分析 利用区间作图软件共检测到控制叶片表型性状的 QTLs14 个 位于毛白杨和毛新杨遗传连锁图的 9 个连锁群上 而对于春季顶芽展叶时 3 间共检测到 3 个 QTLs 分别位于 3 个不同的连锁群上 在检测到的 17 个 QTLs 中 每一 QTL 可解释表型变异的 7 6 15 8 此外 发现控制叶长 叶宽和叶面积等相 关性状的 QTLs 位于相同的基因组区域 这些 QTLs 主要位于毛白杨遗传连锁图的 TLG2 和 TLG11 上以及毛新杨连锁图的 TBLG1 上 据控制叶片表型和春季萌芽时间 的 QTLs 所处的基因组区域 可推测叶片表型和春季萌芽时间这两类性状是由各自相 应的基因控制 6 在杨树中 首次利用区间作图检测到了控制杨树木材化学成分含量的多个 QTLs 对于木材纤维素含量 在 LOD 值 2 0 条件下 共检测到 8 个 QTLs 每一 QTL 可解释表型变异的 8 7 14 6 分别位于毛白杨遗传连锁图的 TLG1 TLG5 TLG12 TLG13 TLG16 和 TLG18 以及毛新杨遗传连锁图的 TBLG11 和 TBLG14 上 共解释表变异的 83 7 其中位于 TLG1 TLG5 TLG18 TBLG11 和 TBLG14 上的 QTLs 对木材纤维素含量有增效影响 其加性效应值分别为 1 48 1 60 1 83 2 41 和 1 52 在相同条件下 利用区间作图 共检测到 6 个 QTLs 与木材木质素含量相连锁 每一 QTL 可解释表型变异的 8 5 10 0 其中来自毛白杨的 QTLs 有 2 个 分别位于毛白杨遗传连锁图的 TLG2 和 TLG15 上 其加性效应值分别为 1 52 和 1 64 可分别解释表型变异的 9 2 和 10 0 而来自毛新杨的 QTLs 有 4 个 分别位于毛新杨遗传连锁图的 TBLG3 TBLG5 TBLG6 和 TBLG8 上 每一 QTL 可解释表型变异的 8 5 9 8 所有 QTLs 总和可解释木质素含量的 54 9 7 利用 AFLP 标记技术与 BSA 法相结合检测到了控制毛白杨种子胚根发育生根 性状的分子标记 以毛白杨无性系 5082 为母本 截叶毛白杨为父本 切枝水培控制授 粉杂交 共得种子 3 193 粒 消毒后将种子点播在 1 2 MS 培养基上 25 光照培养 15 天后统计发芽和生根情况 发芽率为 48 74 生根幼苗 1 179 棵 不生根幼苗 376 棵 生根幼苗 不生根幼苗符合 3 1 2 0 557 2 0 05 3 84 这初步表明毛白杨 种子胚根发育属质量性状 并推测该性状受一对完全显性基因或几个紧密连锁的基因 控制 在此基础上 利用 AFLP 标记技术与 BSA 法相结合检测到了控制毛白杨种子胚 根发育生根性状的两个 AFLP 标记 为此 选用了 78 对 AFLP 引物组合对生根池 15 株生根幼苗 和无根池 15 株无根幼苗 筛选了大小为 40 bp 650 bp 的 5 600 个多 态性片段 平均每对引物可扩增出 72 个片断 经统计分析后发现有 2 个标记 4 EEAG MAAT 492 和 EEAG MCCA 502 在生根池中有约 492 bp 和 502 bp 大小的片断而在 相应位点的无根池中未出现这两个片断 因此初步认为这两个标记与该性状相连锁 为了进一步检测这两个标记与控制胚根发育生根基因位点的遗传距离 我们随机各选 取了 49 株生根幼苗 DNA 和不生根幼苗 DNA 共对 98 个单株进行 AFLP 标记和种子胚 根发育基因位点间遗传距离的连锁分析 经对 98 个单株 DNA 进行 AFLP 检测后 对 于引物组合 EEAG MAAT仅发现一个重组个体 而对于引物组合 EEAG MCCA发现了两个 重组个体 因此 可以认为这两个标记 EEAG MAAT 492 和 EEAG MCCA 502 与毛白杨种 子胚根发育基因位点紧密连锁 且这两个标记与该基因位点的遗传距离分别为 1 02 1 02 cM 和 2 04 2 04 cM 以上结果为毛白杨基因组结构和功能的研究奠定了基础 对分子标记辅助毛白杨 重要性状的早期选择 基因的图位克隆等提供了理论依据 同时对于进一步开展毛白 杨这些重要性状的遗传机制研究具有重要意义 将对毛白杨进行分子遗传改良产生重 要的影响 关键词 关键词 毛白杨 扩增性片断长度多态性 Amplified Fragment Length Polymorphisms AFLPs 遗传连锁图谱 分子标记 数量性状位点 Quantitative Trait Loci QTLs 分析 胚根发育 5 Genetic Linkage Map Construction and Molecular Markers of Some Important Traits in Populus tomentosa Carr Zhang Deqiang Directed by Prof Zhang Zhiyi ABSTRACT Populus tomentosa Carr is a native species in section Leuce in China It is mainly distributed in the vast area of northern China and occupies about one million km2 It has played a key role in forest production and ecological environment construction along the Yellow River The construction of genetic linkage map and analysis of quantitative and qualitative traits are of theoretical and practical importance to physical map construction map based cloning and marker assisted selection The first AFLP genetic linkage maps based on P tomentosa P bolleana P tomentosa employing the Pseudo test cross strategy were constructed and the traits of interest were mapped Additionally two AFLP markers tightly linked to the embryonic root controlling gene in P tomentosa were identified The major results and conclusions are described as follows 1 Three large segregant mapping populations 500 individuals have been established by combining the controlled hybridization and seed culture in vitro using the material of P tomentosa P tomentosa P bolleana or P alba P glandulossa The optimizing one with the highest diversity on DNA level and phenotypic traits including growth traits adaptability and wood chemical properties were identified to be used for the construction of the first genetic linkage map in P tomentosa P bolleana clone TB01 P tomentosa clone LM50 2 The AFLP genetic linkage maps of an elite clone of LM50 P tomentosa and its hybrid clone TB01 P tomentosa P bolleana were constructed with AFLP markers in the Pseudo test cross mapping strategy Fifty one pairs of AFLP primers generated 808 6 polymorphic fragments among which 655 segregated in a 1 1 ratio 81 1 corresponding to DNA polymorphisms heterozygous in one parent and a null in the other P 1 0 At LOD 2 0 however a total of 50 QTLs associated with 11 traits stem volume sylleptic branch angle sylleptic branch numbers leafblade length leafblade width leaf area leaf petiole length leaf vein numbers spring bud flushing day cellulose content and lignin content were detected 4 QTL analysis was performed by the internal mapping approach using an empirical threshold of LOD 2 0 Eighteen putative QTLs associated with the 3 main economic traits stem volume sylleptic branch angle sylleptic branch number were identified based on the two parental genetic maps separately with the number of QTLs for each trait ranged from four to eight The proportion of phenotypic variation explained by each of these QTLs varied from 7 2 to 12 8 As for the stem volume four putative QTLs were identified and they were mapped in the linkage group of TLG1 TLG6 TLG8 and TBLG2 The percentage of 7 trait variation explained by each individual QTL were 8 0 11 0 8 2 and 8 6 with corresponding additive effects were 0 936 0 946 1 198 and 0 950 m3 for the phenotypic value respectively At these four loci the three additive alleles of QTLs located in the linkage group of TLG1 TLG6 and TLG8 originated from the clone LM50 and another one QTL mapped in the linkage group of TBLG2 originated from clone TB01 These four QTLs successfully explained 35 8 of the total phenotypic variation For sylleptic branch angle the six QTLs detected fell into 6 genomic regions corresponding respectively to linkage groups TLG2 TLG5 TLG7 TLG10 TLG16 and TBLG14 The percentage of phenotypic variance explained by each QTL ranged from 7 4 TLG16 to 12 8 TLG10 Total phenotypic variation explained by all six putative QTLs was 57 7 Three of these QTLs originated from clone LM50 had a positive impact on phenotypic values with additive effects were 5 08 6 31 and 4 37 respectively A total of eight genomic regions were found to have effects on sylleptic branch numbers and seven QTLs from both parents clone LM50 and clone TB01 increased phenotypic value The additive effects of alleles associated with SBNTL3 SBNTL6 SBNTL8 SBNTL9 SBNTL14 SBNTL19 and SBNTBL17 were 8 76 9 43 8 06 9 87 8 76 8 89 and 8 16 respectively The proportion of the total variance explained by a single QTL varied between 7 0 TBLG17 and 9 2 TLG9 The total phenotypic variation explained by these seven putative QTLs was 61 7 5 QTLs associated with the leaf morphology traits leafblade length leafblade width leaf area and leaf petiole length and spring bud flush were detected at the threshold of LOD 2 0 using interval mapping approach based on the separate parental linkage maps of clone LM50 and TB01 Fourteen QTLs controlling leaf morphology were identified on nine linkage groups and 3 QTLs affecting spring bud flush were detected on three linkage groups with interval mapping software The phenotypic variance explained by each QTL ranged from 7 6 TBLG14 to 15 8 TLG9 Co localization of QTLs controlling correlated traits such as leafblade length leafblade width and leafblade area were mainly found on linkage groups TLG2 and TLG11 in the genetic map of clone LM50 and on linkage group TBLG1 in the genetic map of clone TB01 Based on the genomic regions of QTLs for leaf morphology and spring bud flush these two traits seem to be controlled by separate genes 8 6 Several QTLs controlling wood chemical components were firstly detected using interval mapping with MAPMAKER QTL in Populus Eight unique QTLs affecting wood cellulose content were detected on linkage group TLG1 TLG5 TLG12 TLG13 TLG18 TBLG9 TBLG11 and TBLG14 respectively The respective putative QTLs accounted for between 8 7 TLG13 and 14 6 TBLG11 of phenotypic variation These eight QTLs account for 83 7 of the total variation observed Of the 8 individual QTLs estimated 5 had a positive impact on wood cellulose content with additive effects were 1 48 TLG1 1 60 TLG5 1 83 TLG18 2 41 TBLG11 and 1 52 TBLG14 respectively For the wood lignin content six putative QTLs were identified They were positioned in linkage group TLG2 and TLG15 on the linkage map of LM50 and in linkage group TBLG3 TBLG6 TBLG8 and TBLG5 on the linkage map of TB01 respectively QTLs for wood lignin content generally explained a slighter lower proportion of the phenotypic variance than those for wood cellulose content ranging from 8 5 TBLG5 to 10 0 TLG15 As a whole these six QTLs explained 54 9 of the additive genetic variation and all but one individually explained less than 10 0 of the total phenotypic variation for the trait analyzed 7 In this study seed radicle development and its association with AFLPs markers were detected based on a segregated progeny population by combining the AFLP technique and BSA strategy The progeny population was generated by intraspecific controlled crossing between a female P tomentosa clone 5082 and a male P tomentosa clone JY A total of 3 193 seeds were obtained and sown on 1 2 Murashige and Skoog medium in vitro The measurements were made at 15 to 20 days after germination The rate of germination is 48 74 for incubated seeds Visual inspection of the germinated seeds showed that 1 179 seedlings had a normal radicle and 376 lacked a root organ The segregation ratio of rooting versus non rooting of seed embryos was observed to be 3 1 in P tomentosa This segregation ratio suggested that seed radicle development character is a qualitative trait and is probably controlled by a single complete dominant gene or a set of tightly linked genes To expedite the identification of the AFLP markers linked to genes involved in radicle development in the post embryo for P tomentosa screening for AFLP polymorphisms was carried out by bulked segregant analysis with bulks pools containing equal amounts of preamplified DNA from 15 normal rooting bulk 1 and 15 lacking visible root organ bulk 2 seedlings respectively 9 A total of 78 AFLP primer pairs were used to test bulks and approximately 5 600 selectively amplified DNA fragments ranging in size from 40 to 650 nucleotides were scored Most primer combinations showed no polymorphic loci between rooting and non rooting bulks However primer pairs E65 M34 EcoR I EAG Mse I AAT and E65 M51 EcoR I EAG Mse I CCA each showed striking differences between these two bulks These two candidate AFLP