【作者】 赵云雷；

【导师】 喻树迅； 张献龙；

【作者基本信息】 华中农业大学 ， 作物遗传育种学， 2007， 博士

【摘要】 利用杂种优势可以提高作物产量，节约土地资源，并创造了巨大的经济和社会效益，是遗传学应用于实践最成功的范例之一。对杂种优势机理的研究已将近一个世纪，然而在许多方面仍然是一个谜。棉花是世界上最重要的纺织作物，在世界经济、政治、以及社会事务中起了很重要的作用。杂交棉在中国和印度已经得到广泛的商业化利用，并研究了许多年。然而到目前为止，关于棉花杂种优势的分子方面的研究报道却很少。本研究以不同优势棉花杂交组合为材料，系统研究了它们在营养生长、产量及品质性状以及根系性状上的杂种优势，然后进一步分析了不同发育阶段棉花杂交种和亲本间的DNA甲基化差异以及基因表达差异，最后将基因差异表达模式与产量性状的表现及杂种优势进行了相关分析，分别从表观遗传调控和功能基因转录水平对杂种优势的分子机理进行了探讨。其实验结果和主要结论如下：1．以同一母本与两个不同的父本杂交获得两个杂交组合，经田间比较试验，结果表明，组合石远345×CRI41是一个优势比较高的杂交组合，其优势不仅表现在营养生长和一些产量相关性状上，而且还表现在根系性状上；而组合石远345×SGK321是一个在地上部的优势相对比较低的组合，但是在某些根系性状上却存在比较高的杂种优势。2．为探讨DNA甲基化与杂种优势的关系，采用甲基化敏感的扩增多态性技术（MSAP），以杂交组合石远345×CRI41和石远345×SGK321为材料，分析了花期阶段这两个杂交组合中杂交种和双亲中的胞嘧啶甲基化水平。结果表明棉花基因组中至少有20％的CCGG位点发生胞嘧啶甲基化。3．依据特定位点的胞嘧啶甲基化从亲本向杂交种的遗传及其变异，我们比较了杂交种与亲本之间的胞嘧啶甲基化模式。研究结果表明杂交种与其双亲相比，发生了广泛的胞嘧啶甲基化变化，这种变化包括超甲基化和去甲基化同时还包括甲基化类型的潜在转变（如从外部胞嘧啶甲基化到内部胞嘧啶甲基化的转变或从内部胞嘧啶甲基化到外部胞嘧啶甲基化的转变）；对于表现出杂交种相对于亲本的甲基化水平降低的类型，在高优势组合石远345／CRI41（Hybrid A）中的去甲基化位点数高于在低优势组合石远345／SGK321（Hybrid B）中的去甲基化位点数。尽管有一类表现为在杂交种中的甲基化水平升高，但是这些超甲基化位点仅仅占两个杂交组合中总多态性位点数的很少一部分。4．通过对三个亲本系和两个杂交种在苗期阶段和花期阶段的5′-CCGG位点的胞嘧啶甲基化进行总体分析，我们发现花期阶段比苗期阶段有更多的位点发生去甲基化。说明棉花中的胞嘧啶甲基化具有组织特异性。5．存在于低拷贝基因组区域的胞嘧啶甲基化变化可以被Southern杂交证实。对杂交种相对于亲本表现甲基化变化的位点进行测序表明同功能已知的基因同源，包括转录调控、信号识别、富含亮氨酸重复、钙离子通道、类PDR的ABC转移、同工酶、脱羧酶、磷酸酶以及ATP酶等。6．以高优势组合石远345×CRI41和低优势组合石远345×SGK321的叶片组织为材料，用mRNA差异显示方法分析了两个杂交组合的杂交种与其亲本在苗期阶段、蕾期阶段、花期阶段和铃期阶段的基因表达差异，结果表明两个棉花杂交种与其亲本间在四个阶段均存在显著的基因表达差异，我们既观察到量上的表达差异也观察到质上的表达差异，而主要的基因表达差异发生在质的水平，包括：（ⅰ）双亲共沉默型，即该条带在双亲都有，而F₁中没有（BPnF₁）；（ⅱ）单亲表达沉默型，即该条带仅出现亲本之一，而在另一亲本和F₁中没有出现（UPnF₁）；（ⅲ）杂种特异表达型，即该条带仅在F₁中出现，在双亲中均不出现（UF₁nP）；（ⅳ）单亲表达一致型，即该条带在双亲之一和杂种中出现，而在另一亲本中不出现（UPF₁）。7．进一步分析了质的差异表达模式在不同阶段的变化，结果表明差异模式的数量在不同阶段是变化的，而花期阶段的差异模式的数量要相对高于其它阶段，暗示了花期阶段的基因差异表达在杂交种的产量形成和优势表现上起了关键的作用。同时在花期阶段，UF₁nP和UPF₁模式（分别代表杂种特异表达或超显性表达和显性表达）在高优势杂交种中的数量要比低优势杂交种高得多，这表明在花期阶段更高水平的这两种模式可能与杂种优势具有正向的相关关系。8．最后以一套双列杂交组合（24个F₁，10个亲本）在盛花期的叶片为材料，用差异显示法进一步分析了四种质的差异模式的数量。以差异模式的数量为变量，与棉花5个产量性状（子棉产量、皮棉产量、铃数、铃重和衣分）的杂种表现、杂种优势进行相关分析的结果表明：单亲基因表达一致模式（UPF₁）与杂种产量性状中单位面积铃数呈显著正相关，但与杂种优势的相关却未达到显著水平，说明此模式有利于产量形成，但对杂种优势贡献不大；而杂种特异表达模式（UF₁nP）在差异表达模式中所占比例处于中等，但与产量性状杂种优势（皮棉产量优势）达到显著正相关，这在某种程度上与基于超显性假说的预测相一致；另外单亲表达沉默模式（UPnF₁）与多数产量性状杂种优势呈负相关，并且与单位面积铃数相关达显著水平，暗示了亲本基因在杂交种中的抑制不利于杂交种表现杂种优势。更多还原

【Abstract】 Exploitation of heterosis is one of the major example of genetics that could increase the yield of crops, save the land, have made the significant contribution to the world. The efforts for exploring the mechanism of heterosis have lasted for nearly one centure. However, many aspects remain unknown. As the most important textile crop and the world’s second most important oilseed crop, cotton plays a vital role in the economic, political, and social affairs of the world. Hybrid cotton has been widely used commercially and studied for many years in India and China. However, until now, there has been no report on the molecular aspects of cotton heterosis because of its allopolyploidy and relatively large genome size. In this paper, we study the heterosis of growth, yield-related traits, quality traits and root traits in different cotton crosses, then we investigated both the differences in cytosine methylation patterns among two cotton hybrids and their parental lines by using the methylation-sensitive amplified polymorphism （MSAP） method and the differential gene expression between the two hybrids and their parents by using the differential display technique, finally we analyzed the correlation between differential expression patterns and heterosis at full opening flower stage. This work was carried out for dissecting the molecular basis of heterosi on epigenetic regulation level and on gene differential expression level, respectively. The main results are as follows:1. One female parent crossed with two male parent, resulting in a highly heterotic hybrid, 345xCRI41 （Hybrid A）, which shows high heterosis in growth, yield-related traits and root traits, and a low heterotic hybrid, 345×SGK321 （Hybrid B）, which only shows heterosis in some root traits.2. To investigate the relation between DNA methylation and heterosis, we investigated the cytosine methylation level in cotton heterotic hybrid/nonheterotic hybrids and their parental lines at flowering stage by using the methylation-sensitive amplified polymorphism （MSAP） method. The results showed that about 20% of the 5’-CCGG sites in cotton genome at the flowering stage were methylated.3. According to the inheritance and alteration of cytosine methylation from parent to hybrid, the cytosine methylation patterns between hybrids and their parents were compared. Our results showed that extensive cytosine methylation alterations including hyper-and demethylation as well as the potential conversion of methylation types （from external cytosine to internal cytosine or vice visa） occurred in the hybrid compared with the parents; For the group showing a decreased level of methylation in the hybrid compared with the parents, the demethylation loci that occurred in cross 345/ CRI41 （Hybrid A） were greater than those in 345/SGK321 （Hybrid B）. Another group showed an increased level of methylation in the hybrid, but these hyper-methylation loci account for only a small portion of the total polymorphism loci in both hybrids.4. On the basis of the whole analysis of methylation status at 5’-CCGG sites in the three parental lines and the two hybrids at the seedling stage and the flowering stage, we found that more loci were demethylated at the flowering stage than at the seedling stage.5. The altered methylation patterns at low-copy genomic regions can be confirmed by DNA gel blot analysis. The loci that underwent methylation alterations in the hybrid compared with the parents were sequenced and found to be homologous to functionally characterized genes, including transcription regulator, signal recognition, leucine-rich repeat, calcium channel, PDR-like ABC transporter, decarboxylase, phosphatase, isomerase and ATPase.6. Using differential display technique, we analyzed the gene differential expression in leaves between cotton heterotic hybrid （345×CRI41）/non-heterotic hybrid（345×SGK321） and their parents at the seedling, squaring, flowering and boll-forming stages, respectively. The results indicated that there existed significant differences of gene expression between cotton hybrids and their parents during the four stages, whatever these hybrids were heterotic ones or non-heterotic ones. Both quantitative and qualitative differences were observed. The major differences of gene expression occurred in the qualitative level for each stage. The qualitative differences include: （ⅰ） bands observed in both parents but not in F₁ hybrid （BPnF₁）; （ⅱ） bands occurring in either of the parents but not in F₁ hybrid （UPnF₁）; （ⅲ） bands present only in F₁ hybrid but not in either of the parents（UF₁nP）; （ⅳ） bands detected in either of the parents and F₁ hybrid（UPF₁）.7. The amount of differential patterns was changeable for each stage and it was relatively higher at the flowering stage than those at other stages, which implies that the differential gene expression at this stage plays a crucial role in yield forming and heterosis performance of hybrid.. The amount of UF₁nP and UPF₁ patterns in highly heterotic hybrid was obviously higher than that in non-heterotic hybrid at the flowering stage. This indicated that higher percentage of these two patterns at this stage may be positively related to the observed heterosis at the later stage.8. The correlation between differential expression patterns and heterosis was estimated in a cotton diallel cross involving 24 hybrids and ten parents at full opening flower stage. The results showed that hybrid-specific expression pattern had a significant positive correlation with heterosis and silenced expression pattern was significantly negative correlation with heterosis at full opening flower stage.更多还原