【作者】 耿建峰；

【导师】 侯喜林；

【作者基本信息】 南京农业大学 ， 蔬菜学， 2007， 博士

【摘要】 不结球白菜（Brassica campestris ssp．chinensis）原产于我国，是东亚地区重要的蔬菜作物之一，在日常生活中占有重要地位。不结球白菜遗传图谱的构建及重要农艺性状的QTL定位将为其遗传育种及分子标记辅助选择提供有益的参考。本研究重点做了以下工作：对不结球白菜游离小孢子培养的关键技术进行了单因素和多因素分析。单因素研究结果表明：基因型之间的小孢子胚诱导率差异显著，54个基因型根据小孢子胚诱导率的高低可分为4类；对供体材料低温处理小孢子胚诱导率差异不大；高温诱导在12～60h范围内差异不大；NAA和6-BA的添加对小孢子胚诱导率差异不大，浓度过大时小孢子胚诱导率反而降低；活性炭的有无和浓度大小对小孢子胚诱导率影响极大。通过基因型、NAA、6-BA、活性炭四因素分析结果表明：基因型之间差异显著，活性炭不同浓度之间差异显著；不同基因型与活性炭不同浓度之间的互作差异显著；其它的互作差异均不显著。利用从F₁代杂交种‘暑绿’中得到的112个双单倍体（doubled haploid，DH）株系构成的群体作为作图群体，应用SRAP、SSR、RAPD和ISSR四种标记来构建遗传连锁图谱，通过Mapmaker3.0／EXP软件分析，得到1张不结球白菜分子遗传图谱，图谱总长度1 116.9 cM，平均图距6.0 cM，共包括14个连锁群，186个多态性分子标记，包括114个SRAP、33个SSR、24个RAPD和15个ISSR标记。每个连锁群上的标记数在4～27个之间，连锁群的长度在30.3～165.8 cM的范围内，平均图距在3.4～11.1cM之间。利用已构建的包括186个分子标记的不结球白菜遗传连锁图谱，采用复合区间作图法（CIM），对维生素C、可溶性糖、可溶性蛋白、粗纤维和干物质含量以及叶片、叶柄重比值6个品质性状进行了QTL定位和遗传效应分析，共检测到控制可溶性蛋白的QTL 2个，控制干物质的QTL 3个，控制叶片、叶柄重比的QTL 4个，但是未得到控制维生素C、可溶性糖和粗纤维的QTL。以包括186个分子标记的不结球白菜遗传连锁图谱为框架，采用复合区间作图法（CIM），对这2个耐寒相关性状进行了QTL分析。结果检测到4个控制相对电导率的QTL；得到控制冷害指数的QTL 7个。同时对两种方法的QTL定位结果进行了比较分析，并对QTL的解释变异百分率及加性效应进行了分析。以由186个分子标记构成的不结球白菜分子遗传图谱为框架，分8个时期对株高、开展度和最大叶叶型指数3个形态学性状进行了动态QTL定位。结果在8个时期检测到控制株高的非条件QTL8个，分布于4个连锁群上。在这些QTL有1个QTL在各个时期均被检测到，其余的在某1个或几个时期被检测到，这些QTL解释变异百分率全部大于10％；除第1期外，在6个时期检测到控制株高条件QTL 12个，这些QTL分别分布于6个连锁群上，都是非条件OTL检测时未检测到的，这些QTL的解释变异百分率大于10％的有8个。在8个时期共检测到控制开展度的非条件QTL 17个，分布于8个连锁群上，这些非条件QTL，没有1个在8个时期都被检测到，只在某1个或几个时期被检测到，这17个非条件QTL解释变异百分率大于10％的有15个；除第1期外，分别在4个时期检测到控制开展度的条件QTL 10个，这些条件QTL分别分布于5个连锁群上，其解释变异百分率大于10％的有6个。在8个不同时期检测到控制叶型指数的非条件QTL 21个，分布于7个连锁群上，其中有1个在8个时期均被检测到，其余的在某1个或几个时期被检测到，这些控制叶型指数的非条件QTL解释变异百分率大于10％的有14个；除了第1期以外，分别在4个时期新检测到控制叶型指数的条件QTL总共5个，这些QTL分别分布于5个连锁群上，这5个条件QTL解释变异百分率大于10％的有4个。更多还原

【Abstract】 Non-heading Chinese cabbage {Brassica campestris ssp. chinensis) is originated from China, which is one of the important vegetables in eastern Asia and play an important role in daily menu. Construction of genetic map and QTL mapping will provide an available reference to genetic breeding and molecular marker assisted selection in non-heading Chinese cabbage. The results are shown as follows:Several key factors affecting isolated microspore culture in non-heading Chinese cabbage were studied by mono-factor and multi-factor experimental designs. The results of mono-factor experiments were shown as follows: there were significant differences in the frequency of microspore-induced embryos among 54 genotypes, whose genotypes were classified into four levels; for the low temperature treatment, no significant differences had been found; there were no significant differences under high temperature when the incubation time was within 12～60 h; the addition of NAA and the 6-BA in NLN medium had no significant influence on the frequency of microspore-induced embryos, and if the concentration was excessive, the frequency would decrease; the presence and concentration of activated charcoal had a great influence on the frequency of microspore-induced embryos. Moreover, as indicated by analysis results of genotypes, NAA, 6-BA and activated charcoal concentrations, there were significant differences among different genotypes and activated charcoal concentrations respectively as well as interaction of these two factors, while other interactions remained non-significant.A genetic linkage map of non-heading Chinese cabbage was constructed, which was based on a DH （doubled haploid） population with 112 individuals derived from a F₁ hybrid variety ’Shulv’. Four kinds of markers of SRAP, SSR, RAPD and ISSR were adopted and 186 polymorphic markers including 114 SRAP, 33 SSR, 24 RAPD and 15 ISSR markers were integrated into the resulted map using Mapmaker 3.0 version. This map consisted of 14 linkage groups, covering 1 116.9 cM with a mean marker interval of 6.0 cM. Various linkage groups were featured by 4-27 maker numbers, 30.3-165.8 cM length and 3.4-11.1 cM mean marker interval.By using composite interval mapping （CIM） method, a molecular genetic map with 186 markers was adopted to map and analyze QTL for the quality-related traits including vitamin C, soluble sugar, soluble protein, coarse fibrin, dry weight in leaves and weight ratio between the leaf and the leafstalk in non-heading Chinese cabbage. Two QTL were detected for soluble protein, three QTL for dry weight and four QTL for weight ratio between the leaf and the leafstalk, while no QTL were detected for controlling vitamin C, soluble sugar and coarse fibrin.Based on composite interval mapping （CIM） method., a genetic linkage map with 186 molecular markers was employed in mapping QTL for two cold-tolerant traits in non-heading Chinese cabbage Four QTL controlling electric conductance were detected in LG1, while seven QTL of cold damage index were detected in three LGs. The results of QTL mapping of relative electric conductance and cold damage index were compared and analyzed, as well as the explained variance and the additive effect of these QTL were estimated.A genetic linkage map with 186 molecular markers was adopted in dynamic mapping QTL for morphological traits including the plant height, plant width and the leaf index at eight different growth stages in non-heading Chinese cabbage. A total of eight unconditional QTL for the plant height were mapped in four linkage groups at eight growth stages. As revealed by results, only one unconditional QTL for plant height was detected at all eight stages, others were detected at one or several stages respectively. The explained variance of each unconditional QTL was more than 10%. Twelve conditional QTL for the plant height were detected in six linkage groups at six stages except the first stage. These conditional QTL did not emerge in detecting the unconditional QTL. The explained variance of eight in twelve conditional QTL was more than 10%. For plant diameter, a total of seventeen unconditional QTL were detected in eight linkage groups at eight growth stages. Among these unconditional QTL, none was detected at all growth stages, just at one or several stages respectively. The explained variance of single QTL was more than 10% except two of them. Ten conditional QTL for plant diameter were detected in five linkage groups at four stages except the first stage. The explained variance of six in ten conditional QTL was more than 10%. For the leaf index, a total of twenty-one unconditional QTL was detected in seven linkage groups at eight growth stages. Just one of them was detected at all eight stages, others were detected at one or several stages respectively. The explained variance of fourteen in twenty-one unconditional QTL was more than 10%. Five new conditional QTL for the leaf index were detected in five linkage groups at four stages expect the first stage. The explained variance of four in five conditional QTL was more than 10%.更多还原