【作者】 齐晓花；

【导师】 张明方；

【作者基本信息】 浙江大学 ， 蔬菜学， 2009， 博士

【摘要】 芥菜（Brassica juncea Tsen et Lee）属十字花科芸薹属作物,具有十分重要的经济价值。Brassica juncea在欧亚大陆长期繁衍过程中,不但在分化出了黄籽芥菜（印度型油用芥菜）,而且以中国为演化中心分化出丰富多彩的菜用芥菜。芥菜类蔬菜在漫长的历史演变过程中由原始矮小的根、茎、叶、薹等均不发达的野生类型经过自然选择和人工选择逐渐发展到今天的根、茎、叶、薹等形态和产品器官十分发达且变异类型非常丰富的群体。其丰富的变异为研究芥菜类蔬菜的遗传多样性及系统进化提供了理想材料。此外,芥菜品质性状和重要农艺性状的遗传特性对芥菜品种选育及良种繁育等影响甚大,因此,本论文以中国特有的芥菜类蔬菜为材料,针对芥菜类蔬菜的起源、系统进化以及重要农艺性状及品质性状的遗传规律进行分析。主要的研究内容和结论如下:（1）利用基因组原位杂交（genomic in situ hybridization,GISH）来鉴定芥菜的原始亲本并对中国芥菜和印度芥菜的染色体组进行比较。以B.rapa和B.nigra的总基因组为探针,其中B.rapa（A）基因组探针和B.nigra（B）基因组探针分别与B.juncea的20和16条染色体杂交并且信号十分清晰。这就再次证实了B.rapa和B.nigra是异源四倍体中国芥菜和印度芥菜的原始亲本并且两套基因组的同源性很低。A基因组（绿色荧光）信号比较集中的位于染色体的着丝粒附近,我们推测是因为整个芥菜的基因组较小,重复的基因组家族含量相对也较少,因此集中分布于着丝粒周围。而B基因组（红色荧光）信号并没有完全集中在着丝点周围,而是以着丝点为中心向染色体臂方向有明显扩散,这说明芥菜中的来自于B基因组的重复DNA家族在芥菜染色体上以着丝粒为中心向染色体臂区域扩散分布的。A、B基因组不同的分布模式说明两套基因组芥菜进化过程中有着不同的进化途径,结合前人的细胞学和分子研究说明A、B基因组的差异不仅仅是量上的差别。由此推测B基因组是相对较早的从原始十字花科基因组中分化出来的。此外,利用GISH技术获得中国芥菜和印度芥菜荧光图象信号强弱几乎一致,这说明两者的分化时间差别不大,因此我们推测两者在进化上是几乎平行的。（2）利用核糖体DNA中的内转录间隔区（internal transcribed spacers,ITS）序列变异对中国芥菜进行系统进化分析和遗传多样性分析。以16个形态差异明显的不同变种中国芥菜材料与芥菜的原始亲本芸薹和黑芥为研究材料,通过对其ITS序列变异进行分析表明,中国芥菜可明显的区分为两组,一组是包括‘中熟芥菜’（分蘖芥）,‘浙桐三号’（茎芥）,‘黑叶忝菜’（薹芥）和‘广饶芥末’（籽芥菜）的黑芥分支,序列长度均为622 bp。另外的12个材料与芸薹的三个亚种构成芸薹分支,序列长度均为608 bp。中国芥菜从ITS序列上明显的分成A芸薹、B黑芥两组,这说明中国芥菜的进化过程由于不同的亲本重组方式而发生了不同方向的协同进化。据此我们推测芸薹类型的中国芥菜杂种在形成过程中不断的与芸薹类亲本回交,而黑芥分支的中国芥菜在杂种形成中和黑芥发生反复的回交,最后渐渐分别形成稳定的以A、B基因组为主的芥菜类型。（3）利用两个叶绿体非编码区,trnL和trnL-F序列变异对芥菜杂种形成的母本遗传进行分析,该序列在芸薹属种内高度保守,但是在黑芥和芜菁之间存在核苷酸突变,芥菜与芜菁（芸薹）的同源性很高,说明芥菜的母本是芸薹而父本为黑芥。（4）利用AFLP（Amplified Fragment Length Polymorphism）分子标记对中国芥菜的16个变种材料以及3个芸薹种材料和1个黑芥材料进行系统分类分析。中国芥菜16个材料形成了几个小的分支,其中‘特大棒菜’是和其他芥菜的分歧最大的一支,相似系数为0.67。其次是籽芥菜‘广饶芥末’明显区分与另外14个类型的芥菜形成一个独立的分支。其他的14个芥菜类型分成两个分支,第一组包括两个根芥菜类型,第二组包括其余的12个芥菜类型。从AFLP分子标记聚类分析的结果看,和传统的形态分类有很大的分歧。这些结果表明芥菜在经过长期的栽培和驯化后,表型已经发生了很大的变化,单单以形态进行分类已经不能反应整个芥菜家族的亲缘关系和进化历史。（4）针对中国芥菜产量相关的主要农艺性状以及品质性状的遗传规律进行了分析。以根、茎、叶、薹四个变种的芥菜品种为亲本进行双列杂交获得F₁和F₂代杂交组合,以四个亲本、F₁和F₂代组合为材料,于2005年在浙江大学农场和绍兴农科院两个试验点进行了随机区组试验。调查了株高、分蘖数、叶数、最大叶片长度、最大叶片宽度、最大叶片重、单株根重、单株重、株幅9个农艺性状,并取样测定了芥菜叶片中的总氨基酸、维生素C、可溶性糖、可溶性蛋白质、黑芥子硫苷酸钾和总硫代葡萄糖苷含量。采用加性-显性-加加上位性与环境互作的遗传模型对上述性状进行遗传和相关分析。获得了如下主要结果。除了株高和最大叶宽两个性状主要受基因环境互作效应影响外,其他农艺性状主要受遗传效应的影响。在总的遗传效应中,加性效应是控制分蘖数、叶片数、最大叶宽、最大叶重的主要效应;显性效应是控制最大叶重,最大叶长、根重和株高的主要效应;加加上位性效应是控制株幅的主要遗传效应。在基因环境互作效应中,最大叶宽,最大叶重,根重主要受到加性与环境互作效应的影响,单株重、最大叶长、株高和株幅主要受显性环境互作效应影响;此外,加加上位性与环境互作效应是控制中国芥菜分蘖数和叶片数的主要效应。对农艺性状杂种优势进行分析,分蘖数、叶片数、最大叶宽、叶重性状表现正向的平均优势和超亲优势;株高、单株重、最大叶长、株幅和根重性状表现正向的平均优势和超亲优势。不同试点的基因环境互作杂种优势表现不同,这说明不同环境中杂种优势有波动性,因此应该在特定的环境下对杂交组合进行选择。品质性状的遗传分析结果表明,维生素C和总蛋白含量主要受加性效应和加性环境互作效应控制;总糖、氨基酸、黑芥子硫苷酸钾和总硫代葡萄糖苷含量主要受显性和显性与环境互作效应的影响;对品质性状的杂种优势分析结果表明,品质性状的平均杂种优势和超亲杂种优势多数不显著或者为负值;互作效应平均优势随着试点和性状不同而表现不同,但超亲优势均表现为负值或不显著,因此利用杂种优势改良品质性状比较困难。更多还原

【Abstract】 Mustard crop（Brassica juncea（L.） Czern.）,belonging to the genus Brassica in the Cruciferae family,is an agriculturally and economically important crop widely cultivated in Asia and Europe.In India,B.juncea is used as oil-bearing crop and has been studied extensively.China is considered the original region of varietal differentiation,with the highest level of differentiation around Sichuan san Zhejiang.Chinese vegetable mustard has been increasingly attractive to biologist and plant breeders largely due to its higher diversification and economically important values.According to the U-triangle,B.rapa（2n=20,AA） and B. nigra（2n=16,BB） were hybridized naturally to synthesize the present species of B.juncea （2n=36,AABB）.Although most studies on the species of U-triangle have confirmed the diploid origins of amphidiploid species,many questions remain concerning how the amphidiploid species evolved from their parental diploids.My research was focus on the phylogenetic analysis of B.juncea and genetic analysis of important characters on Chinese vegetable mustard.The main contents and results were as follows:（1） We applied genomic in situ hybridization（GISH） in this study that has allowed assignment of B.juncea chromosomes to the A or B genomes.The simultaneous application of B.nigra（red） and B.rapa（green） total genomic DNA probes allowed unambiguous discrimination of all 16 and 20 chromosomes of the genomes B and A,respectively.This demonstrates that despite their evolutionary proximity,a majority of repetitive DNA families from these two genomes do not share any significant level of homology.A detailed comparison of different A and B genomes revealed that the hybridization pattern of the B genome nuclear DNA differs from that of A genome.In the chromosomes of the AABB genome of B.juncea,although the GISH signals are localized predominantly in the pericentromeric regions,clear extensions of the signals towards the intercalary parts of the arm can be seen.In contrast,in the A genome,the signals are restricted almost exclusively to the pericentromeric regions;this may suggest that some differences in the distribution of repetitive DNA between the B genome and A genome.The results of most other cytogenetic （Harrison and Heslop-Harrison,1995） and molecular（Song et al.,1988;Warwick and Black, 1993） studies clearly suggest that these differences are not only quantitative but also qualitative.This result also supported the hypothesis that the B genome was the first to diverge from the common ancestral Brassica genome（Quiros,1995）.（2） Sequence variation of nuclear internal transcribed spacer regions of ribosomal DNA （ITS1,5.8S rRNA and ITS2） from Chinese vegetable mustards（AB-genome） and its putative parents Brassica rapa（the A-genome） and Brassica nigra（the B-genome） were used to investigate the molecular phylogeny and the probable evolutional pattern of this amphidiploid species that uniquely formed in China.Totally,sixteen accessions of Chinese vegetable mustard those covering nearly all the diverse variations were included in this study, and together with three accessions of B.rapa and one accession of B.nigra.The results disclosed two strongly supported clades,one containing four accessions of vegetable mustard which have closer relationship with B-genome species "B.nigra" lineage and the other containing twelve accessions of B.juncea and three A-genome accessions.This classification was in disagreement with the evidence from chloroplast DNA,mitochondrial DNA,nuclear DNA restriction fragment length polymorphism（RFLP）,which suggested that B.juncea was closely related to the A-genome type.For the incongruence,we speculated that the B.juncea crops derived from Chinese have evolved through different recombined events of the diploid morphutypes and evolved unidirectional concerted evolution.The traditional phenotypic classification of B.juncea was not wholly supported by ITS results,and hence the phylogenetic relationships among these subspecies need to be reconsidered on molecular level.（3） trnL intron and trnL-F were highly conserved among B.juncea species,however, nucleotide mutation points were found between B.rapa and B.nigra species.Informative sites in these two cpDNA regions povided evidence of the maternal origin of B.juncea.It was found that the materal parent of B.juncea could be B.rapa because the B.juncea were closely related to the accession of B.rapa （4） Amplified fragment length polymorphism（AFLP） analysis was used to evaluate the genetic relationships and diversities of Chinese vegetable mustards.The cluster analysis showed that the vegetable mustards could be grouped into two main groups and some minor rami,which was partially in accordance with the traditional classification that based on different edible organs of vegetable mustards.The incongruity between morphological and molecular classification might be attributed to the high selection pressure during domestication of Chinese vegetable mustards,producing some accessions with similar genetic backgrounds evolving into abundant morphological variations.The great diversification among Chinese vegetable mustards not only provides an excellent object for molecular evolution research of B.juncea but also is of great value for widening the genetic basis of breeding programs and breeding materials selection.Besides,our study also indicates that AFLP are informative and can provide significant insights for genetic diversity research in B.juncea.（5） Genetic effects and genotype by environment（GE） interaction effects for some important agronomic traits and qualitative traits of Chinese vegetable mustard were analyzed by using a genetic model including additive,dominance,additive×additive effects and their interaction effects with environments.Four different variations of Chinese vegetable mustard as parental lines and their F_1s,F_2s were evaluated in fields of two locations.It was revealed that the agronomic traits of Chinese vegetable mustard were mainly controlled by genetic effects except plant weight（PW） and leaf weight（LW） were observed to be more affected by GE interaction effects.Among the genetic effects,additive effects took the main proportion for tillering number（TN）,leaf number（LN）,leaf breadth （LB） and LW;dominance effects were the main components of PW,leaf length（LL）,root weight（RW） and plant height（PH）;additive×additive effects were the main components of plant breadth（PB）.Among the GE interaction effects,LB,LW and RW were mainly affected by additive interaction effects and PW,LL,PH and PB were mainly controlled by dominance interaction effects.Besides,additive×additive interaction was the main factor which controlled TN and LN of Chinese vegetable mustard.For heterosis analyses,TN,LN, LB and LW of Chinese vegetable mustard showed positive H_PM and negative H_PB.The other traits showed positive H_PM and H_PB.Heterosis arising from GE interaction was found to varying degree for different locations.It was shown that genetic heterosis and GE interaction effects were important factors for agronomic traits in Chinese vegetable mustard.For quality traits,it was revealed that Vitamin C content and total protein content were mainly controlled by additive effects and additive environment interaction effects.Sugar content,amino acid content,sinigrin content and total glucosinolate content were mainly controlled by dominance and dominance environment interaction effects.For heterosis analyses,sinigrin content and total glucosinolate content of Chinese vegetable mustard showed positive H_PM and negative H_PB The sugar content and total protein content showed negative H_PM and H_PB.Vitamin C content showed negative H_PB although no H_PM.Amino acid content of Chinese vegetable mustard showed positive H_PM（F₁） and negative H_PM（F₂） and H_PB.更多还原