【作者】 田露申；

【导师】 牛应泽；

【作者基本信息】 四川农业大学 ， 作物遗传育种， 2011， 博士

【摘要】 油菜是世界重要油料作物之一,在我国种植面积和总产均居世界首位,也是我国重要的食用油来源。油菜白花具有重要的理论研究价值和实践应用前景。本研究以甘蓝型油菜异源白花品系HW243为材料,围绕白花性状展开遗传研究。主要研究结果如下：1.油菜花色观察方法的比较以HW243为母本,黄花品系(品种)HZ21-1和中油821为父本,通过杂交配置出两个杂交组合(HW243×HZ21-1和HW243×中油821)的P1、P2、F1、B1、B2及F2群体。使用各个世代群体内的单株来研究比较目测法、数码扫描法和色素比色法调查花色是否准确可靠。研究发现：(1)使用数码扫描仪扫描得到油菜花瓣颜色的RGB值,对不分离世代群体样本分析时发现,RGB值中R和G值变化幅度很小,B值变化幅度很大,白花的B值最大(>200),黄花的B值很小(<10),乳白花居于之间,且B值的变化与田间花色的变化高度一致。将B值选为数码扫描法调查花色的指标；(2)用无水乙醇提取花瓣中的色素在200-600nm波长范围进行紫外-可见光光谱扫描。发现在439nm处的吸光度值变化与田间花色变化高度一致,黄花的吸光度值很大(>1.4),乳白花较小,白花的吸光度值最小(<0.1)。将439nm处的吸光度值选为色素比色法调查花色的指标；(3)使用RGB值、吸光度值和目测法调查两个杂交组合(HW243×HZ21-1和HW243×中油821)的B1、B2及F2群体的花色。结果显示三种方法具有较一致的观察结果,扫描法和色素法能够减少人为主观等因素造成的误差,可以取代目测法实现对油菜花色的调查。2.甘蓝型油菜异源白花性状的遗传模式分析使用吸光度值和B值在2年对2组合(HW243×HZ21-1、HW243×中油821)的P1、P2、F1、B1、B2、F2共6个世代的油菜花色变异进行了调查。分析发现,花色既表现出主基因分离的特征,又表现出微效多基因效应的特点。分离群体B1、B2和F2中吸光度值和B值显示出一定的连续分布,其中B1群体呈单峰分布,B2和F2群体中均呈现双峰分布。使用植物数量遗传体系的主基因+多基因模型进一步分析,两种调查方法的结果一致表明,甘蓝型油菜异源白花性状符合2对加性-显性-上位性主基因+加性-显性-上位性多基因遗传模型(E-0),花色性状以主基因遗传效应为主,多基因的作用相对较小；其中主基因加性、显性和上位性效应均具有重要的作用。在F2、B1和B2群体中主基因遗传率(46.55%-98.14%)一致高于多基因遗传率(0.32%-46.9%),表明在甘蓝型油菜异源白花性状的遗传变异中主基因作用大于多基因的作用。3.甘蓝型油菜分子图谱的构建和异源白花性状的QTL定位以杂交组合(HW243×HZ21-1)的F2为作图群体,采用B值调查F2群体内单株的花色,使用SSR、RAPD和SRAP分子标记构建遗传连锁图谱,并对花色性状进行QTL定位。结果构建得到2102cM的连锁遗传图谱,含有217个标记位点,标记间平均距离为9.69cM。QTL扫描发现花色存在2个QTL位点,即qPE4(Nal 2-B06—ME25-EM20-3)和qPE6(Nal 0-H06—ME27-EM26-2),2个QTL位点共解释表型变异为27.45%。同时还检测到3个上位性QTL位点,3个QTL位点,共解释表型变异为13.33%。因此,花色性状是以加性效应为遗传主效应,但上位性也存在一定的遗传作用,不可忽略。4.甘蓝型油菜类胡萝卜素代谢相关基因的克隆与分析使用RACE技术克隆分离出甘蓝型油菜类胡萝卜素代谢途径相关酶基因BnPSY(HQ260432,HQ260433)、BnPDS(HM989806,HM989807,HM989808,HM989809)、BnZDS (HQ260435, HQ260436)、BnLYCE (HM212502, HM212503)、BnLYCB (HM989810)、BnBCH(HM212504, HM212505)、BnZEP (GU361616, GU561839)、BnNCED3 (HQ260434)、BnCCDl (HQ260430, HQ260431)。使用实时荧光定量PCR技术分析了各个基因在不同组织部位及不同花色中的表达,结果显示,黄花花瓣中各个基因的表达均要高于白花花瓣；F1花瓣中除BnZEP和BnCCD1基因的表达略高于黄花外,其他基因的表达均低于黄花,但所有基因的表达均高于白花花瓣。分析HW243不同发育阶段花瓣中各个基因的表达显示,开花过程中花色的变化可能主要是由BnPSY、BnPDS、BnZDS、BnLYCB、BnLYCE、BnNCED3和BnCCD1等基因共同调控的结果。更多还原

【Abstract】 Rapeseed (Brassica napus L.) is one of the main oil crops widely grown in the word, as well as in China. It is an important source of vegetable oils in China with the largest planting area and the largest production. White flower character in rapeseed is rare and has important research and breeding values. In this paper, the inheritance of white flower character in a Brassica napus line, HW243, was studied by a series of methods, including different ways of observation, quantitative genetic analysis, QTL analysis and real-time quantitative PCR. The main results were summarized as the following:1. The three different methods for observation of rapeseed flower colorsWhite flower line’HW243’was crossed to two yellow flower B.napus lines,’HZ21-1’ and’Zhongyou821’, i.e.,’HW243×HZ21-1’and’HW243×Zhongyou821’. Six generations (Pi, P2, F1, RF1, F2, B1, B2) for each of the two combinations were prepared to investigate the variations in flower colors in Brassica napus L. The colors of flower in the six generations were observed with three different methods:naked eye observation and classification, spectrophotometry of extracted pigments, and digital scanning of petals. The following importamt results were discovered:(1) With the digital scanning method, rapeseed flower colors could be characterized by the RGB values of the petals, which were obtained with a reading software. The results showed that the parameters R and G varied to a very little extent, but the parameter B showed a large range of variation, among the different generations. The variations in B values were highly consistent with the variation in flower colour observed by naked eyes. In present study, the B values of flower petals were used to analize the genetic characteristics of the flower colors in rapeseed. (2) The flower pigments were extracted with absolute alcohol, and the extracted solution was examinated with a visible/ultra-violet spectrophotometer at variable wavelengths. It was observed that the absorbance value at wavelength 439nm showed the largest difference among P1, P2 and F1, with yellow, white and milky flower colors, respectively. The absorbance values of the extracted pigments at wavelength 439nm were used for the systematic analysis in later experiments. (3) The variations in flower color in the six generations of the two crosses could be evaluated by B values, absorbance values and naked eye grading. It was shown that the three methods generated highly consistent observation results. The digital scanning and the pigment extraction methods, however, could reduce the subjective observation errors, and could be used to replace’naked eye’ observation of flower colour in Brassica napus L.2. Genetic analysis of the Allogenous white flower color in Brassica napusThe flower colors in the six generations (P1, P2, F1, B1, B2 and F2) of the two crosses (HW243×HZ21-1, HW243×Zhongyou 821) were analyzed using the B values and the absorbance values. It was found that Allogenous white flower color in Brassica napus L. was controlled by both major genes and polygene genes. The B values and the absorbance values in the segregating generations showed continuously distribution. One single peak was distributed in B1 population, and bimodal distributed in B1 and F2. The genetic modes of flower color were analyzed with a mixed model for major genes plus polygenes. The results from the both detection methods were consistant. It was shown that Allogenous white flower color in Brassica napus L. was a quantitative trait and its inheritance mode fitted to a mixed genetic model of two major genes with additive-dominant-epistatic effects plus polygenes with additive-dominant-epistatic effects (the E-0 model). The effects of major genes were high and polygenes were relatively small. The effects of additive, dominant and epistatic actions of the two major genes were equally important. The inheritability rates of the two major genes were estimated to be 46.55% to 98.14% in B1, B2 and F2, higher than that of the polygenes, estimated to be 0.32% to 46.9% in B1, B2 and F2 respectively.3. Construction of molecular genetic map and QTL identification of Allogenous white flower color in Brassica napusTaking the F2 generation of HW243 (white petal parent)×HZ21-1 (yellow petal parent) as a mapping population, a genetic linkage map of Brassica napus L. was constructed using molecular markers of SSR, RAPD and SRAP. QTL’s for petal colors were identified. The molecular map included 217 marker loci, covering 2102cM, with an average distance of 9.69cM. Two additive QTL’s were checked out for flower colors in Brassica napus L., i.e., qPE4 (Na12-B06—ME25-EM20-3) and qPE6 (Nal0-H06—ME27-EM26-2). The two QTL’s explained 27.45% of the phenotypic variation. Additionally, three pairs of epistatic QTL’s were detected, which explained 13.33% of the phenotypic variation.4. Cloning and Characterization of genes associated with carotenoid biosynthesis in Brassica napusA group of genes possibly associated with the carotenoid biosynthesis in Brassica napus were cloned with RACE, including BnPSY (HQ260432, HQ260433), BnPDS (HM989806, HM989807, HM989808, HM989809), BnZDS (HQ260435, HQ260436), BnLYCE (HM212502, HM212503), BnLYCB (HM989810), BnBCH (HM212504, HM212505), BnZEP (GU361616, GU561839), BnNCED3 (HQ260434), BnCCDl (HQ260430, HQ260431). The levels of expression of these genes were detected using real-time quantitative PCR in the different tissues from plants of different flower colors. The results showed that the expression levels of all of these genes were higher in the yellow flower petals than in the white flower petals. The expression level of most of these genes was a little bit lower in flower petals of F1 than in the yellow flower petal. But reverse situations were observed for the genes BnZEP and BnCCD1. The results also showed that the expression levels of these genes were, to some extent, variable during the developmental period of the white flower petals in HW243, which was consistant with the change in flower colour from pale yellow in the earlier stage to full white in the later stage. The changes in the white flower color seemed to be due to the co-regulation of BnPSY, BnPDS, BnZDS, BnLYCB, BnLYCE, BnNCED3 and BnCCD1 gene.更多还原