【作者】 陈发波；

【导师】 杨克诚；

【作者基本信息】 四川农业大学 ， 作物遗传育种， 2010， 博士

【摘要】 茎瘤芥(Brassica juncea var. tumida Tsen et Lee)是双子叶植物,十字花科,芸苔属,芥菜种的变种。作为中国特有作物资源,仅在长江流域种植,其加工产品“涪陵榨菜”蜚声海内外,与欧洲甜酸甘兰、日本腌菜并称世界三大名腌菜。本研究以从全国收集的133份茎瘤芥、7份父本系及8份不育系和72份茎瘤芥及其近缘植物为供试材料,采用表型分析和SSR分子标记对133份种质材料进行遗传多样性分析,选用的7个父本系8个不育系按不完全双列杂交组配56个组合,并分析茎瘤芥主要性状配合力、瘤茎产量杂种优势以及瘤茎产量杂种优势与亲本间遗传距离的相关性,通过单拷贝核Chs基因序列分析茎瘤芥及其近缘植物的系统发育关系,对茎瘤芥的起源进化进行初步探讨。主要结果如下：(1)表型变异系数分析表明,调查的21个农艺、经济性状除裂叶对数、沟间深度、茎叶比和瘤茎产量的变异系数较大外,其余性状材料间的变异系数均在一个较小的范围内。(2)表型性状主成分分析表明,可用前8个主成分(累计贡献率达87.41%)概括21个性状的总信息量,并以此对供试的133份材料进行综合评价,结合各主成分表现较为明显性状的分析,可以看出各份茎瘤芥材料都具有相应的特点,在茎瘤芥种质保存及育种中可以有针对性地进行利用。(3)表型性状系统聚类分析表明,茎瘤芥材料间的遗传距离变幅为0.01-7.77,平均值为2.50,供试材料间遗传距离变幅较小,平均值较小,且有88.93%的遗传距离小于4.00。当遗传距离为2.21时,可将供试材料分成8类。所聚类型较少,A81.20%供试材料集中在第3、6和8类,茎瘤芥种质间遗传差异较小,遗传基础相对狭窄。(4)从600对引物中筛选出81对扩增条带清晰,具明显多态性的共同引物。81对引物共扩增到810个条带,每对引物扩增到5-16个条带,平均10.0个,其中多态性条带724个,占89.38%。81对引物共检测到2846个基因型,每对引物检测到6-52个基因型,平均35.14个,其中有效基因型998.32个,平均12.32个,有效基因型比例为35.08%。Shannon多态性指数变幅为0.52-3.72,平均值为2.74。材料间的遗传相似系数变幅为0.77-0.91,平均值为0.85,遗传相似系数值在0.85-0.91的分布较多,占65.99%。当相似系数为0.83时,可将供试材料分成6类,所聚类型较少,且有90.23%的材料集中在第5和6类。进一步说明茎瘤芥种质间相似程度较高,遗传差异较小,遗传基础相对狭窄。(5)基于SSR分子标记的主成分分析表明,前3个主成分所能解释的遗传变异分别为85.47%、0.67%和0.61%,可将供试材料分为6类,第1类包括50和86,第2类包括8、11和64,第3类包括14、15和65,第4类包括9、26、99和125,其余材料被分到第5和6类,且多数材料在第5和6类,同样说明茎瘤芥间遗传差异较小。(6)根据地理来源将供试茎瘤芥材料分成长江上游、长江中游和长江下游3个类群,基于表型和SSR标记的分析均表明,3个类群遗传多样性水平从高到低的顺序为：长江中游>长江上游>长江下游。基于SSR分子标记数据的主成分分析还表明,长江中游茎瘤芥基本位居133份茎瘤芥种质特征根分布3维图的中心区域。据此,推测长江中游的重庆市可能是茎瘤芥的起源地或遗传多样性中心,在其传播过程中主要是以重庆市为中心,沿长江流域向其上游或下游传播。(7)配合力分析表明,同一性状不同亲本间及同一亲本不同性状间一般配合力存在较大差异。瘤茎产量一般配合力相对效应值变幅为-29.88-13.48,其中显著或极显著正向效应值5个,占33.33%,父本系60和156一般配合力正向效应值达极显著水平,不育系133.2A、118-3A和145-1A一般配合力正向效应值达显著水平。瘤茎产量特殊配合力相对效应值变幅为-35.86-39.93,其中显著或极显著正向效应值24个,占42.86%。瘤茎产量一般配合力和特殊配合力效应共同作用强优势组合,强优势组合一般具有较好的特殊配合力,或双亲具有较好的一般配合力,或二者兼备。在茎瘤芥杂种优势利用中,既要重视一般配合力选择,更要重视特殊配合力选择。(8)瘤茎产量平均超亲优势为6.24%,变幅为-46.345-77.22%,正向优势组合占64.29%,说明茎瘤芥亲本系间瘤茎产量存在较明显的杂种优势。利用父本系19和60,不育系140-5A、122-3A和133-2A组配出瘤茎产量高杂交组合的几率较大。相关分析表明,瘤茎产量的超亲优势与特殊配合力间相关系数达极显著水平,且相关系数较大,说明可用特殊配合力效应预测瘤茎产量杂种优势。瘤茎产量、超亲优势、特殊配合力效应与亲本间表型遗传距离和SSR标记的遗传距离相关系数不显著,且相关系数较小,不能用表型遗传距离和SSR标记遗传距离来预测瘤茎产量杂种优势和特殊配合力。(9)利用单拷贝核Chs基因序列对茎瘤芥及其近缘植物系统关系的分析表明,可将117个Chs基因序列分成11个分支。网状结构分析表明,茎瘤芥及其近缘植物间不仅存在树状的进化关系,还大量存在非树状的进化史,在茎瘤芥及其近缘植物植物进化史上存在较多的网状进化事件。网状支系分析表明,白菜可能是茎瘤芥A基因组亲本的供体,黑芥可能是茎瘤芥B基因组亲本的供体,茎瘤芥可能是由白菜和黑芥杂交后产生的某种芥菜发生自然突变再经人工驯化选择产生的,但具体是由哪一种芥菜变种进化而来还需要做进一步的研究。更多还原

【Abstract】 Tuber mustard (Brassica juncea var.tumida Tsen et Lee), a variation in the species Brassica juncea belonging to the genus Brassica in the Cruciferae family, is an agriculturally and economically important crop merely cultivated along the Yangtze river in China. Fuling hot pickled tuber mustard, a product of mustard tuber, as well as European pickled vegetable and Japanese pickles, are hailed as the three most famous pickled vegetables in the world. In present study analysis of phenotypic characters and SSR molecular markers were made to invetigate the genetic diversity in 133 tuber mustard accessions. Fifteen tuber mustard inbred lines were used to detected their genetic diversity by SSR markers, among them, seven inbred lines (male) and eight sterile male inbred lines (female) were crossed to get 56 hybrids. The heterosis and combining ability were analyzed among these hybrids. And the low-copy nuclear Chs gene sequences of seventy-two brassica accessions were used to estimate the phylogenetic relationships among tuber mustard and its related genera. The main results are as follows:(1) The results showed that the variation coefficients of the twenty-one phenotypic characters changed in a small range except the number of bilobed leaf, depth of inter-nodule and stem weight per plant.(2) The principal component analysis of the twenty-one phenotypic characters showed that the first eight principal components can explain the genetic variation with 87.41% which can summarize the total information of the twenty-one phenotypic characters. From the first eight principal of the 133 tuber mustard accessions and the characteristic of each accessions, we can see that each tuber mustard accession has its characteristics correspondingly which could be used in the conservation and breeding of tuber mustard.(3) The results of system cluster analysis of 21 phenotypic characters showed that the genetic distances among the 133 tuber mustard accessions ranged from 0.01 to 7.77, with an average of 2.50. It was indicated that the genetic distance among the accessions raged in a small degree and the mean among them was small with 69.5% of all genetic distances were less than 4.00. Cluster analysis indicated that all the tuber mustard accession could be divided into eight Clusters. So litter Clusters were obtained and Cluster 3,6 and 8 included 81.20% of all accessions. It was indicated that the genetic distance was small and the genetic diversity was narrow among the 133 mustard tuber accessions.(4) Eighty-one pairs of SSR primers from 600 pairs of SSR primers in Brassica produced stable amplified bands and 810 bands were detected among the accessions with 724 polymorphic bands, accounting for 89.38%. The average number of bands per locus was 10.0 with a range from 5 to 16. The observed number of alleles was 2846 with 35.08% was effective number of alleles. The average number of observed number of alleles per locus was 35.14 with a range from 6 to 52. The values of Shannon’s information index for each SSR locus varied from 0.52 to 3.72 with an average of 2.74. The coefficients of genetic similarity in the SSR marker patterns among the 133 accessions ranged from 0.77 to 0.91, with an average of 0.85. There were 5793 pairs of genetic similarity, in which 65.99% were 0.85 to 0.91. The cluster analysis showed that the accessions could be classified into six clusters when the genetic similarity was 0.83, with 90.23% of the accessions included in Cluster 5 and Cluster 6. It was also indicated that the similarity was high and the genetic diversity was narrow among the 133 mustard tuber accessions.(5) Principal component analysis was carried on based on the SSR data. The result showed that the first three principal components can explain the genetic variation with 85.47%,0.67% and 0.61%, and the 133 accessions could be divided into six Clusters according to the nearest phylogenetic relationship. Cluster 1 included the accessions 50 and 86. Cluster 2 included the accessions 8,11 and 64. Cluster 3 included the accessions 14,15 and 65. Cluster 4 included the accessions 9,26,99 and 125. The remains were included in Cluster 5 and 6. It was also indicated that the genetic diversity was narrow among the 133 mustard tuber accessions.(6) The accessions of mustard tuber in this study can be divided into up-Yangtze river group, mid-Yangtze river group and down-Yangtze river group based on their geographical distributions. According to analysis of phenotypic characters and SSR data, the order of genetic diversity level in the three groups was mid-Yangtze river group>up-Yangtze river group> down-Yangtze river group. The accessions of the mid-Yangtze river group was in the central region of the 3-D dendrogram by principal component analysis. It was presumed that the origin center or genetic diversity center of tuber mustard was the mid-Yangtze river valley, and the crop was transmited along the Yangtze River to it’s upstream and downstream.(7) The analysis of combining ability indicated that the GCA relative effects among different parents in the same trait and among different traits in the same parent showed great different. The GCA relative effects of stem yield among different parents ranged from-29.88 to 13.48. There were five effects showed significant positive, accounting for 33.33%. Two male inbred lines (60 and 156), and three male sterile inbred lines (133-2A, 118-3A and 145-1A) had significant positive (P< 0.01) GCA relative effect. The SCA relative effects ranged from -35.86 to 39.93 with twenty-four effects were significant, accounting for 42.86%. The heterosis among parents was related to GCA and SCA. The hybrids with strong heterosis had excellent SCA or the parents had excellent GCA, or had both excellent SCA and GCA. During the using of heterrosis in tuber mustard, therefore, it should pay attention to both GCA and SCA.(8) The hight-parent heterosis of stem yield ranged from -46.35% to 77.22% with an average of 6.24%. There were 64.29% hybrids showed positive and significant hight-parent heterosis. It was indicated that there were significant heterosis of stem yield among parents. The results also showed some strong heterosis were existed, it indicated that two male inbred lines (19 and 60) and three female inbred lines (140-5A,122-3A and 133-2A) were the elit inbred lines which can be used to develop strong superiority hybridized hybrids. The correlation coefficient among the stem yield, hight-parent heterosis SCA were big and highly significant, which indicating that the heterosis can be reflected by SCA. The correlation coefficient of genetic distance based on phenotypic characters and SSR data with stem yield, hight-parent heterosis and SCA were small and not significant. It couldn’t use the genetic distance based on phenotypic characters and SSR markers to predict the heterosis and combining ability in tuber mustard.(9) The sequences of the single-copy nuclear gene Chs were used to study the phylogenetic relationships among tuber mustard and its related plants. Sequence analysis indicated that the one hundred and seventeen Chs gene sequences could be classified into 11 branches. The split analysis indicated that there not only existed tree evolution, but also existed reticulate evolution among tuber mustard and its related plants. There were many reticulate evolution events during the evolutionary history of tuber mustard and its related plants. Median-joining(MJ) network analysis indicated that Brassica pekinensis was the parent donor of A genome in tuber mustard, while Brassica nigra was the other parent donor of B genome in tuber mustard. It was presumed that tuber mustard was origin from a natural mutation of Brassica juncea by artificial domestication and selecting, while the certain Brassica juncea was a crossing of Brassica pekinensis and Brassica nigra.更多还原