【作者】 陈新；

【导师】 孟金陵；

【作者基本信息】 华中农业大学 ， 作物遗传育种学， 2008， 博士

【摘要】 甘蓝型油菜(Brassica napus,A~nA~nC~nC~n2n=38)是一种重要的油料作物和生物能源载体,它起源于欧洲地中海地区,由白菜型油菜(Brassica.rapa,A~rA~r2n=20)和甘蓝(Brassica oleracea,C~oC~o2n=18)天然杂交,自然加倍而来。作为一种栽培作物,随着产量潜力的不断提升和品质性状的改良,其栽培面积和地域不断扩大,遍布亚洲,欧洲,北美和澳洲。然而,由于其较短的栽培历史和高强度的现代育种,使其遗传基础相对狭窄。育种工作者们试图通过各种途径来丰富其遗传基础,其中通过种间杂交渗入外源基因组的遗传成份来合成甘蓝型油菜就是一种行之有效的方法。事实上,在合成的甘蓝型油菜中已经发现一些新的遗传变异,如染色体重组、DNA序列丢失、DNA甲基化水平变化和基因表达的改变等。杂种优势在农业上被广泛地利用来提作物的高籽粒产量,而且利用杂种优势的作物数量在不断增加。在许多作物中的研究表明亲本和杂种间的基因差异表达是理解杂种优势分子基础的重要方法,如小麦,水稻和玉米。孙逢吉1943年在中国第一次报道了芸苔属物种间杂种的生物学产量杂种优势。随后的许多研究都对油菜籽粒产量的杂种优势程度进行了评价。近年来,又发现了新型甘蓝型油菜和常规甘蓝型油菜间存在着较强的杂种优势,进一步的研究表明其中一些从外源基因组渗入甘蓝型油菜的等位基因对油菜籽粒产量的增加有利。为了深入理解新型甘蓝型油菜及其亲本,新型甘蓝型油菜和甘蓝型油菜组合间基因表达谱的差异对杂种优势的影响,主要进行了以下6个方面的研究:1.利用5个含有白菜型油菜A~r基因组成分或埃塞俄比亚芥C~c基因组成分的新型甘蓝型油菜作母本,5个甘蓝型油菜品种作父本,按NCⅡ设计配制杂交组合得到25个亚基因组间杂种。这25个杂交种的平均中亲优势和超亲优势分别为33.3%和17.0%。其中7个杂种的中亲优势大于40%,10个杂种的超亲优势大于20%。2.选择3个新型甘蓝型油菜以及合成它们的原始亲本共计9份材料为研究对象,利用cDNA-AFLP基因表达差异显示技术分析新型甘蓝型油菜和它们的原始亲本间的一组基因表达谱,32对引物组合获得1167个转录获得片段(Transcript-derivedFragment,TDF)。通过分析表达谱发现,有超过60%的TDF在新型甘蓝型油菜及其甘蓝型油菜亲本间存在差异表达。3.从25个亚基因组间杂交组合中,选择了9个组合,同样利用cDNA-AFLP基因表达差异显示技术分析亚基因组间杂种和甘蓝型油菜亲本间的另一组基因表达谱,32对引物组合获得总计为1231个的TDF。通过对该组表达谱的分析,发现TDF只在亲本之一中出现的表达模式在7个不同的表达模式中占主导地位,另外显性、超显性和负显性表达模式次之,加性效应最少。4.通过分析单个差异表达的TDF与杂种产量表现,中亲优势和超亲优势的相关性,发现同一TDF对相同性状的不同数据类型,效应方向是一致的,但对不同的性状的相同数据类型,效应方向则不定。对于不同数据类型的分析发现:杂种和亲本的表现,TDF为负效应的数量多于正效应的,负效应TDF占58.1%;其它两类,则是正效应的数量多于负效应的,中亲优势的正效应TDF占54.8%,超亲优势的正效应TDF占75.4%。5.回收,克隆和测序了近40个差异表达的TDFs,并对其中20个TDFs的序列进行深入电子拼接分析。首先在NCBI和TAIR网站上搜索同源序列,下载同源序列,然后使用Bioedit软件构建contig,并设计引物验证预测结果。另外,根据对cDNA-AFLP的实验操作经验累积和理解的加深,本研究提出了一个比较可行的cDNA-AFLP改进方案。6.将杂种优势相关TDF转化为分子标记运用到QTL作图研究中验证所得TDF与产量性状相关QTL的相关性。7个杂种优势相关TDF定位在TN分子标记遗传连锁图的12个连锁群上。QTL扫描之后发现其中4个TDF转化的7个标记落于TN群体23个籽粒产量相关性状QTL的置信区间,预示着这些TDF确实可影响油菜的籽粒产量。更多还原

【Abstract】 Brassica napus（AACC,2n=38） is an amphidiploid species that originated from spontaneous hybridization between B.rapa（AA） and B.oleracea（CC）.The role of cultivated B.napus as a commercial oil crop in Asia,Europe,North America and Australia,has progressively increased due to better production potential and improvement in seed quality.However,the short cultivation history and intensive breeding of this species has led to a comparatively narrow genetic base.Considerable efforts have been made to enrich the genetic diversity of B.napus by the introgression of genomic components of its related species or by developing synthetic B.napus lines by hybridization between B.rapa and B.oleracea.Several interesting changes have been observed in synthetic B.napus,such as chromosomal rearrangements,deletions of DNA sequences,variations at DNA methylation loci and the alteration of gene expression patterns.Heterosis has been widely used in agriculture to increase the seed yield,and it has been applied to a large number of crops.Research has revealed that analysis of the differences in gene expression profiles between the hybrids and their parents is an important method for elucidating the molecular basis of heterosis in crops such as wheat, riceand maize.These results are consistent with the hypothesis that multiple molecular mechanisms contribute to heterosis.In Brassica,heterosis was first reported in biomass for Sun in China.Subsequently many studies have estimated the extent of heterosis for seed yield.Divergent evolution and isolation have led to differentiation within the same genome among different species.To distinguish different subgenomes in Brassica,the genomes of the three diploid species B.rapa,B.nigra and B.oleracea were designated as A^r,Bⁿ and C^o while the genomes of the three amphidiploid species B.napus,B.juncea and B.carinata were designated as AⁿCⁿ,A^jC^j and B^cC^c,respectively.A new-type B. napus,A^r/nA^r/nCⁿCⁿ or A^r/nA^r/nC^c/nC^c/n,was created by interspecific hybridization and artificial selection.A hybrid(A^r/nAⁿCⁿCⁿ or A^r/nAⁿC^c/nCⁿ) was developed by crossing the new-type B.napus to B.napus and strong heterosis was observed in the hybrids.This typeof heterosis was considered as intersubgenomic heterosis and its existence showed that some alleles that were derived from related species could favourably contribute to increasing the seed yield of rapeseed. In this study,we compared the gene expression profiles of new-type B.napus lines with those of hybrids derived from crosses between new-type B.napus lines and B.napus cultivars,six objectives are addressed in this paper:1.Twenty-five hybrids developed between five lines of new-typed B.napus,two with A^r introgression from B.rapa and three with A^r and C^c introgression from both B. rapa and B.carinata as female parents and five cultivars of natural B.napus as male parents to hybridize each other.The average value of mid-parent heterosis（MPH） and high-parent heterosis（HPH） in hybrids was 33.3%and 17.0%,respectively.2.Three new-typed B.napus lines and their parents were chose,and cDNA-AFLP differential display technique was applied to obtain the first set of gene expression profiles between new-typed B.napus and their original parents.With 33 primer combination,we detected 1167 differential expressed TDFs.Over 50%transcript-derived fragment（TDF） altered their display pattern between new-typed B.napus and its parental natural B.napus.3.From 25 combinations,we chose nine combinations to obtain the second set of expression profiles between hybrids and their two B.napus parents by cDNA-AFLP differential display technique.With 32 primer combination,we detected 1210 differentially displayed TDFs.The type N₁⁺H^-N^- and N_t^-H^-N⁺,accounting for 51.4%of 1210 detected TDFs,were predominance among seven different patterns,followed by the dominant patterns and overdominant pattern.4.Through correlation analysis between presence or absence of single differential TDF and three types of data（HP,MPH,HPH） for six investigated agronomic traits,we found that the same differential TDF had consistent effect direction for same trait in three type heterosis data.However,for different traits,the effect direction was indefinite.For hybris performance,the negative effect TDFs were more than positive effect TDFs,they accounted for 58.1%;for MPH and HPH,the positive effect bands were much more,they accounted for 54.8%and 72.8%,respectively.5.Reused,cloned and sequenced 20 heterosis associated TDFs.We applied correlated internet servers and software to analyze these sequences information: downloading homologous sequences and assembling them into contigs.Then, EuGene’Hom was used to predict the functions of these genes with these contigs,and designed primers to confirm the assembled sequences.Based on the experience of manipulation and deeper understanding of cDNA-AFLP,we put forward a project to improve cDNA-AFLP technique. 6.Transformed some heterosis-associated TDFs into molecular marker to validate the relationship with QTLs for the yield related traits.Seven heterosis-associated TDFs were mapped in TN linkage map by 12 SSR and TRAP markers.After QTL analysis,7 markers of them developed from 4 TDFs located in the confident intervals of 23 QTL for seed yield and yield related traits in TN population.更多还原