【作者】 王艳惠；

【导师】 牛应泽；

【作者基本信息】 四川农业大学 ， 作物遗传育种， 2009， 博士

【摘要】 人工合成甘蓝型油菜是创建甘蓝型油菜新种质,获得更多重要农艺经济性状的遗传资源,利用人工合成种作为桥梁将一些近缘物种的有利性状导入甘蓝型油菜,可拓宽油菜的遗传基础并对其农艺及经济性状进行改良。四川农业大学油菜研究中心通过人工合成甘蓝型油菜创造出了一群遗传变异丰富新材料,并从中选育出了一些优良的特异材料,如特长角、高千粒重、多分枝以及大角大粒等优良农艺性状的材料。本研究对人工合成甘蓝型油菜获得的特长角油菜材料H218的角果长度进行了遗传分析,探讨油菜角果长度的遗传变异规律,进一步丰富油菜的理论基础,改良现有油菜的角果长度服务于生产,并从分子生物学方面,采用基因组原位杂交、DNA甲基化等技术分析了特长角形成的可能因素。其主要研究结果如下：1、在特长角“H218”的F2群体中,全果长、果身长、果喙长均呈连续正态分布,显示为多基因控制的数量性状,全果长最少受5对基因控制,果身长最少受6对基因控制,果喙长最少受2对基因控制。特长角H218的全果长、果身长的广义遗传力较高而狭义遗传力相对较低,且狭义遗传力均大于广义和狭义遗传力之差,表明基因的加性效应远比非加性效应对表型变异的贡献更大。果喙长的广义遗传力、狭义遗传力均较低,表明他们能够遗传的部分所占比例较低。对F2群体进行相关分析表明,全果长与果身长、果喙长、每果粒数之间,果身长与果喙长、每果粒数之间均呈极显著正相关,表明选择长角果性状可间接增加角果的有效长度和每果粒数。2、对H218特长角系进行三重测交分析,全果长、果身长和果喙长三性状的I、I1、I2的χ2值均达到显著,表明三性状存在显著的上位性效应。全果长、果身长和果喙长三性状的和式、差式、和差式三种方差家系间均达极显著水平,并且三性状的i型互作、j和l型互作也达极显著水平,说明这三性状是由这三种基因效应共同作用的结果,其遗传均符合加性-显性-上位性模型,三性状的i型互作的显‘著性均大于j型和l型互作的显著性,表明长角果性状通过不断的自交是可以固定的,但也不能忽视j型和l型互作的变异。3、采用世代均数法对特长角H218进行遗传模型检验和基因效应分析的结果,全果长、果身长、果喙长三性状不符合简单的加性-显性遗传模型,符合加性-显性-上位性模型,这与三重测交的结果一致。全果长、果身长和果喙长三性状均为[d]+[j]>[h]+[l],表明角果长度性状在F1代难以出现超亲杂种优势。全果长、果身长的[d]>[i]+[j]+[l],基因加性效应远比上位性效应对表型变异的贡献更大,但上位性效应不可忽视,果喙长的[d]+[h]>[i]+[j]+[l],基因的加性、显性效应远比上位性效应对表型变异的贡献更大,表明角果长度性状通过不断的自交进行轮回选择,可以积累较多的有利基因。4、用主基因+多基因模型分析特长角系H218的遗传特性表明,全果长、果身长均以C模型为最适遗传模型,即受加性-显性-上位性多基因系统控制,未检测到主基因效应,表明全果长、果身长可通过轮回选择集中增效基因或通过聚合回交转移这些多基因。B1、F2的多基因遗传率相对较高,B2的多基因遗传率低,遗传参数因遗传背景不同而异。果喙长的遗传模型、遗传参数因遗传背景不同而异。5、对特长角H218进行基因效应对总遗传变异的贡献分析表明,全果长、果身长、果喙长三性状的加性效应平方和大于显性效应平方和,角果长度性状的加性遗传变异占世代间总变异的80%以上,加性效应是世代间遗传变异的主导因素,显性效应、上位性效应对总遗传变异的贡献率也起一定的作用。表明角果长度性状通过轮回选择是可在一个自交系中积累较多的有利基因,但也不能忽视显性效应与上位性效应的作用。6、用540对SSR引物对特长角H218和普通长度角果中油821的基因组DNA进行筛选,结果有115对引物可以在两亲本间扩增出差异带谱,用有差异的115对引物在F2的长、短角果基因池之间筛选出3对有差异的引物,即A05、J35、N05,表明这3对SSR引物与角果长度性状存在连锁。经软件分析,A05、J35、N05三标记间无连锁关系,采用单标记分析法进行了t检验,A05、N05两标记可能与控制角果长的QTL存在连锁关系,而J35与控制角果长的QTL不存在连锁关系。6、对特长角系及其后代的DNA甲基化进行(MSAP法)分析结果表明,长角果的总甲基化率低于普通长度角果品系的总甲基化率,长角果的全甲基化率低于普通长度角果品系的全甲基化率,长角果的半甲基化率低于普通长度角果品系的半甲基化率,即长角果的甲基化水平低于普通角果。特长角果的去甲基化和次甲基化程度高于普通角果,普通角果的全、半甲基化和过或超甲基化程度高于长角果。相对较低的甲基化水平以及去甲基化程度的提高有可能是特长角果形成的原因之一。7、采用GISH分析方法对特长角变异系及其后代的染色体差异进行分析结果表明,以白菜型油菜基因组作探针,用不同来源的甘蓝型油菜作封阻DNA,特长角H218都表现出了其与白菜型油菜的同源性高于普通角果与白菜型油菜的同源性,即H218的特长角性状可能与所用白菜型油菜提供的新特异基因有关；用白花芥蓝基因组作探针,用不同来源的甘蓝型油菜DNA作封阻,特长角H218都表现出了其与白花芥蓝的同源性高于普通角果与白花芥蓝的同源性,这表明H218的特长角表现可能又与白花芥蓝的特异基因导入有关。综合分析认为,人工合成系H218的特长角表现,还可能与白花芥蓝特异基因与白菜型油菜特异基因的互作有关。更多还原

【Abstract】 Artificially resynthesized Brassica napus lines are the genetic resources of creating new germplasm, as well as obtaining more significant agronomic and economic characteristics of Brassica napus L.. With the help of resynthesis technologies, we can transform some beneficial traits of relative species into Brassica napus L., accordingly, the hereditary basis of rapeseed can be expanded, and the agronomic and economic characteristics of rapeseed can be modificated. A population of new rapeseed materials which are rich in genetic variation has been created by resynthesis technologies in Rapeseed Research Center of Sichuan Agricultural University. A lot of excellent metamaterials were selected from the population, such as specially-long pod Variant Line, high 1 000-grain weight line, multiple-branch line, and big pod, big seed line. In this study, in order to study the formation reasons of long-pod rapeseed, quantitative genetic analysis of specially-long pod resynthesized Brassica napus line H218 was investigated by the methods of molecular biology technologies such as GISH(Genomic in Situ Hybridization) and DNA methylation. The main results of the research are as the follows:1. The broad-sense heritability of whole-fruit length and fruit length are relatively higher than narrow-sense heritability in specially-long pod Brassica napus line H218, and narrow-sense heritability are significantly higher than the difference between broad-sense heritability and narrow-sense heritability, indicating that the contribution of additive effects to phenotype is much higher than non-additive effects. The broad-sense heritability and narrow-sense heritability of the length of beak are both lower, indicating that the ratio of heredity is lower. The whole-fruit length is controlled by at least five genes, the fruit length is controlled by at least six genes, the length of beak is controlled by at least two genes. In F2 population, the whole-fruit length, fruit length and the beak length are normal distribution, demonstrate that they are quantitative genetic traits controlled by multiple genes. The correlation analysis on F2 population indicated that they are significant positive between whole-fruit length and fruit length, beak length, the grains on each pod, fruit length and beak length, the grains on each pod. It means that select whole-fruit length can improve fruit length indirectly, selecting long pod trait can improve the grains on each pod indirectly.2. Genetic effects were analysed and genetic models were tested by the method of generation means analysis, the results showed that the whole-fruit length, the fruit length and beak length are not conform to simple additive-dominant-epistasis genetic model, but conform to [d]+[i]>[h]+[l], which suggests that the three traits are mainly behaved as additive effects, and it is hard for them to behave heterobeltiosis in F2 generation. The result of [d]+[h]>[i]+[j]+[l] suggests that the additive effects, dominant effects contribute more to phenotype variance than epistasis effects. The additive effects [d] of the three traits, the interaction effects between additive effects and additive effects [i] are showed as significant and positive, which proved that the genes of all the three traits are joint probability distribution. As far as the three traits are concerned, the sum of squares of additive effects are higher than that of the dominant effects, in different hybridized combination, the contribution of gene effects to total genetic variance are similarly, additive genetic variance contribute more than 80% to total variance, and additive is the main factor to genetic variance between generations. Dominant effects, epistasis effects also contribute to total genetic variance.3. TTC analysis was carried out on specially-long pod Brassica napus line H218, when it comes to the three traits, theχ2 value of I, I1,I2 were significant, which proved that there exist significant epistasis effects. Between the families, the sum, the difference, the sum and difference of the three traits showed as extremely significant differences, and the interaction models of i, j,l are all extremely significant differences, which suggest that three different of genes cooperate to each other, and result in three different traits, it also conform to additive-dominant-epistasis genetic model.4. Analysis especially-long pod Brassica napus line H218 with the model of main gene-multiple genes, C model is the best for whole-fruit length and fruit length, it also means that they are controlled by additive-dominant-epistasis multiple genes, no main gene effects was founded, it suggest that collect enhancin gene by recurrent selection, or transform these multiple genes by convergent backcross method can effect the whole-fruit length and fruit length. Heritability of multiple genes in B1, F2 population are relatively higher than B2 population, and genetic parameters are different based on hereditary background difference. The genetic model and genetic parameters of beak length are different based on different hereditary background.5.540 pairs of SSR primers are screened between especially-long pod H218 and Zhongyou 821,115 pairs of which showed difference, make use of the 115 pairs to screen between long pod gene pool and short pod gene pool in F2 population,3 pairs of primers showed difference, that is A05、J35、N05,which suggest that the three pairs of primers linked with pod length trait. Through software analysis, there are no linkage relationship among the three markers, t test was carried out by single marker analysis, and the result suggest that it is possible that markers A05 and N05 linked with QTL of length controlled, but not for J35.6. The total methylation ratio of long pod is lower than normal pod, the methylation ratio of long pod is lower than normal pod, the half methylation ratio of long pod is lower than normal pod, that is to say, the methylation ratio of long pod is lower than that of normal pod. Demethylation and submethylation level of especially-long pod rapeseed are higher than normal pod length rapeseed, total methylation, half methylation, super methylation level of normal pod length are higher than that of especially-long pod.7. Use genome DNA of B campestris L. as probe, chromosome of different Brassica napus L. as blocking DNA, especially-long pod rapeseed H218 performed higher homology with Brassica campestris L. than normal pod, which means the eapecially-long pod may has something to do with the transformation of special genes from Brassica campestris L.. Use genome DNA of Brassica oleracea var.alboglabra as probe, chromosome of different Brassica napus L. as blocking DNA, especially-long pod H218 performed higher homology with Brassica oleracea var.alboglabra than normal pod, which means the eapecially-long pod trait may has something to do with the transformation of special genes from Brassica oleracea var.alboglabra. In resynthesized Brassica napus line H218, special genes from Brassica campestris L. and Brassica oleracea var.alboglabra interact with each other, then formed especially-long pod, and can inherit stably to the next generation.更多还原