【作者】 王万兴；

【导师】 刘玉梅；

【作者基本信息】 中国农业科学院 ， 蔬菜学， 2013， 博士

【摘要】 结球甘蓝（Brassica oleracea L. var. capitata），别名圆白菜、卷心菜、包菜等，属十字花科（Brassiceae）芸薹属（Brassica）的一年生或二年生草本植物。结球甘蓝具有品质好、适应性强、产量高、耐储运等特点，已成为国内外普遍栽培的一种重要蔬菜作物，在我国蔬菜的周年供应中起到重要作用。本研究基于结球甘蓝全基因组测序项目，利用全基因组de novo和重测序数据开发出SSR、SNP和InDel标记；利用不同生态型的结球甘蓝高代纯合自交系01-88和02-12杂交获得F1，通过游离小孢子培养获得DH群体，利用该DH群体构建甘蓝高密度遗传连锁图谱，该图谱作为参考图谱服务于基因组的组装，同时为功能基因克隆及基因组比较研究提供技术支持，并在探寻十字花科芸薹属作物的起源，特别是在通过比较图谱阐述基因组组成和进化关系中起到重要作用；针对未锚定到基因组且片段较大的Scaffolds设计InDel标记，并将其补充组装到甘蓝基因组上，进一步充实甘蓝基因组信息；通过对甘蓝主要农艺性状进行遗传和相关性分析以及基因定位，结合转录组测序信息对控制相关性状的基因进行预测，获得更多重要农艺性状完整的遗传信息，为结球甘蓝分子标记辅助育种及相关基因的克隆打下理论基础；筛选与甘蓝显性雄性核不育基因紧密连锁的分子标记，建立一种高效甘蓝显性雄性不育基因的鉴定方法；本研究对提高结球甘蓝的育种水平，提高选择效率具有重要的理论意义和实用价值。本研究获得的主要成果如下：（1）选用不同生态型的结球甘蓝高代纯合自交系01-88与02-12杂交获得F1，通过游离小孢子培养技术构建了含有189个基因型的DH群体。（2）甘蓝基因组中共检测到233844个SSR位点，共包含6种类型的重复单元，其中单核苷酸重复单元MNRs（163621，69.97%）数目最多，最少为六核苷酸重复单元HNRs（311，0.13%）。利用这些位点信息通过Primer3.0软件共设计并合成SSR引物3378对；通过比对de novo测序及重测序数据，共检测到SNP位点1026766个，针对含有单碱基变异位点的序列，应用SNAPER软件共设计并合成SNP引物2200对。（3）利用双亲01-88和02-12对8479对引物进行筛选，最终筛选出有多态性的引物1274对，多态性比例为14.99%；将有多态性的引物用于DH群体检验，利用Joinmap4.0软件构建一张包含1227对分子标记（602SSRs；625SNPs）、覆盖基因组总长度为1197.9cM，平均遗传距离和物理距离分别为0.98cM和503.3Kb的结球甘蓝高密度遗传连锁图谱。该图谱是目前最饱和的甘蓝类作物参考遗传图谱。（4）针对71个未锚定到基因组上，且长度大于200kb的Scaffolds设计了172对InDel标记，通过双亲筛选有84对InDel标记具有多态性。利用该84对多态性标记补充锚定了43个Scaffolds，物理长度总计为22.6Mb。结合补充锚定的Scaffolds，目前甘蓝全基因组组装已达到562.6Mb，组装比率从原来的85.0%上升到89.3%。（5）采用植物数量性状主基因+多基因混合遗传模型对5个与甘蓝叶球相关（叶球重、叶球纵径、中心柱长、叶球颜色、外短缩茎长）和6个与植株相关（外叶数、开展度、株高、外叶长、外叶宽、外叶颜色）的农艺性状进行遗传和相关性分析。DH群体分析结果表明，11个主要农艺性状均为数量性状，除外叶宽和外叶颜色性状为多基因控制外，其余9个农艺性状均由2对主基因+多基因控制。主基因遗传率范围为19.56%（开展度）~79.45%（叶球重）。（6）应用IBM SPSS Statistics19软件偏相关分析的Pearson方法，对甘蓝11个主要农艺性状相关性进行分析。结果表明叶球颜色与外叶颜色，叶球纵径与中心柱长等成对性状呈显著正相关；叶球重与外短缩茎长，叶球纵径与外叶数等成对性状呈显著负相关；叶球纵径与外叶长，外短缩茎长与开展度等成对性状呈不显著相关。（7）应用MapQTL4.0软件，采用IM和MQM作图法，根据甘蓝高密度遗传连锁图谱和DH群体农艺性状表型数据，共定位了控制甘蓝11个主要农艺性状的39个QTLs。其中，叶球纵径、外短缩茎长、外叶数和开展度性状定位的QTL数量最多为5个；中心柱长、叶球颜色和外叶宽性状定位的QTL数量最少为2个。定位的39个QTLs在9条连锁群上均有分布，其中有些QTLs呈现重合和连锁分布。（8）采用BSA方法，从含有425个单株的育性分离群体中随机挑选20株可育株和不育株的DNA组建2对可育池和不育池。利用分子标记技术，在2对育性池中共筛选得到3对与不育基因CDMs399-3连锁的SSR分子标记。其中最近的双侧翼标记为scaffold38115和scaffold13994的遗传距离分别为8.3和15.3cM。（9）通过对构建甘蓝DH群体的双亲进行转录组分析，将得到的差异表达基因与主要农艺性状的QTLs相比较分析，共筛选得到控制8个主要农艺性状的10个候选基因，其中有8个候选基因与生长素和细胞分裂素合成、代谢和运输相关，2个候选基因与叶绿素和类胡萝卜素合成、代谢相关。更多还原

【Abstract】 Cabbage (Brassica oleracea L. var. capitata) is a major crop with the characteristics of high quality,high yield and resistance storage, which is widely cultivated in the world. It plays an important role invegetables all-year supply in China.This study is based on the B. oleracea Genome Sequence Project which was launched in2009,using next generation sequencing technology. A number of SSR, SNP and InDel markers are developedby using the data from the de novo sequence and re-sequence. A DH population derives from F1crossbetween two advanced homozygous inbred lines,01-88and02-12, by microspore culture. Then asaturated genetic map of B. oleracea genome is constructed. This genetic map could be used to orientatesequence Scaffolds from the B. oleracea genome assembly. Some InDel markers are developed by usingthe information of the Scaffolds with long fragments, which are not anchored to the genome. This studyis aim to the supplement assembly of the genome. Combined with the transcriptome sequence, the geneswhich control the main agronomic traits could be predicted, through the genetic, correlative and QTLanalysis. So this study provides the whole genetic information of these traits. The markers are screenedto identify which linked to the male sterile gene, with the purpose of development an efficient malesterile gene identified method. In summary, this study is facilitated to enhance the breeding technologyand selection efficiency.The main conclusions of this study are as listed follows:(1) Two diverse advanced homozygous inbred lines of cabbage,01-88and02-18, were used as theparents to develop a doubled haploid (DH) mapping population containing189lines. The DHpopulation was derived from F1by microspore culture and contained lines with a wide variety ofmorphological traits.(2) A total of233844putative SSR sequences were identified from the cabbage assembled Scaffoldsequences. Six different SSR repeat types were identified, and of these the HNRs (163621,69.97%)were the most abundant and the MNRs were the least abundant (311,0.13%).3378SSR primerpairs were designed using the Primer3.0program. A total of1026766SNPs were detected between01-88and02-12.2200SNP markers were developed by SNAPER program.(3) To construct the map, a total of8497markers were screened with the DNA from the01-88and02-12parental lines.1274markers (14.99%) were polymorphic. The B. oleracea high-densitygenetic linkage map that was constructed includes1227markers in nine linkage groups spanning1197.9cM with an average of0.98cM and503.3kb between two loci. This genetic map is themost saturated in B. oleracea crops.(4) A total of172InDel markers were developed, using the information of long Scaffolds which werenot anchored to the genome.84InDel markers showed the polymorphism. Using thesepolymorphic markers,43Scaffolds with22.6Mb were supplemented anchored to the genome.Now, the size of assembled B. oleracea genome is562.6Mb. And the assembly rate is from85.0% up to89.3%.(5) Using mixed major gene plus inheritance model,5traits related to leaf head (head weight, lengthof stem in head, vertical diameter of head, primary color of head, length of shortening stem) and6traits related to plants (number of outer leaf, plant breadth, plant height, length of outer leaf, widthof outer leaf, primary color of outer leaf) were investigated. All the11main agronomic traits werequantitative. Except the Wol and Pcl traits, the other9traits inheritances showed the models of twomajor genes plus polygenes. The heritability range was from19.56%(Pb) to79.45%(Hw).(6) Using IBM SPSS Statistics19program Pearson method in partial correlation analysis,11mainagronomic traits were analysed. Pch-Pcl and Vdh-Lsh, etc paired traits were significant positivecorrelation. Hw-Ls and Vdh-Nol, etc were significant negative correlation. Vdh-Lol and Ls-Pb, etcwere with no correlation.(7) MapQTL4.0software and IM&MQM methods were employed in QTL mapping. As a result, atotal39QTLs were mapped for11main agronomic traits in B. oleracea. Among them, Vdh, Ls,Nol and Pb traits mapped the most QTLs (5). Lsh, Pch and Wol traits were the least with2QTLs.All the QTLs distributed on all the9linkage groups. Some QTLs showed coincidence and linkagephenomenons.(8) Using BSA method,2male sterile and fertile bulks were construed. A total of3SSR markers wereidentified and linked with CDMS-399-3gene. Respectively, scaffold38115and scaffold13994wereflanking markers with genetic distances of8.3cM and15.3cM.(9) Two parental lines of DH population were analysied by RNA-seq technology. Combined with theQTLs and differentially expressed genes from transcriptomic results. A total10genes wereidentified as candidate genes. Of these genes,8genes were related to the processes of anabolismand transport in auxin and cytokinin. The other2genes were related to the processes of anabolismin chlorophyll and carotenoid.更多还原