【作者】 王潇然；

【导师】 刘宝；

【作者基本信息】 东北师范大学 ， 遗传学， 2013， 博士

【摘要】 组织培养可以诱发基于遗传和表观遗传变异的可遗传的表型变异,这种现象被称为体细胞克隆变异(somaconal variation)。尽管已经有大量工作对组织培养诱导的基因组变异的分子机制以进行了探索研究,但对于水稻纯系,杂交种和多倍体对组织培养的应答是否存在差异以及差异程度的研究却鲜有报道。本文研究了两种水稻纯系(不同亚种),二者正反交杂交种F1代和相应多倍体在组织培养条件下发生的遗传变异和表观遗传变异。我们利用扩增片段长度多态性(AFLP)和甲基化敏感扩增多态性(MSAP)两种分子标记技术检测了六个基因型的愈伤组织和再生苗的遗传变异和DNA甲基化变异,发现总体上遗传变异频率高于表观遗传变异频率。对于两种类型的变异,各个基因型之间存在着显著差异,但仅有一种杂交种(N/9)的遗传变异与其他各个基因型表现出极其显著的差异。我们发现两个杂交种之间的遗传变异的差异大于两个纯系亚种之间的差异。两个多倍体之间的遗传变异频率亦存在差异,但差异程度也低于两杂交种之间的差异。对DNA修复和DNA甲基化相关基因的表达水平检测证实,愈伤组织和再生苗中均发生了基因表达变化,部分检测基因的表达变化与遗传变异或表观遗传变异存在相关性。本文研究结果表明：水稻体细胞克隆变异中,遗传变异是主要变异类型并伴随表观遗传变异的发生；组织培养导致DNA修复和DNA甲基化相关基因的表达变化,这种变化与遗传变异和表观遗传变异存在相关性；组织培养诱发的遗传和表观遗传变异特点在纯系、杂交种和多倍体种存在本质差异；正反交杂交种之间体细胞克隆变异频率存在显著差异。更多还原

【Abstract】 Genetic and epigenetic alterations can be invoked by plant tissue culture, which may result in heritable changes in phenotypes, a phenomenon collectively termed somaclonal variation. Although extensive studies have been conducted on the molecular nature and spectrum of tissue culture-induced genomic alterations, the issue of whether and to what extent distinct plant genotypes, e.g., pure-lines, hybrids and polyploids, may respond differentially to the tissue culture condition remains poorly understood.We investigated tissue culture-induced genetic and epigenetic alterations in a set of rice genotypes including two pure-lines (different subspecies), a pair of reciprocal Fl hybrids parented by the two pure-lines, and a pair of reciprocal tetraploids resulted from the hybrids. Using two molecular markers, amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP), both genetic and DNA methylation alterations were detected in calli and regenerants from all six genotypes, but genetic alteration is more prominent than epigenetic alteration. Significant genotypic difference was observed in frequencies of both types of alterations, but only genetic alteration showed distinctive features among the three types of genomes, with one hybrid (N/9) being exceptionally labile. Surprisingly, difference in genetic alteration frequencies between the pair of reciprocal F1hybrids is much greater than that between the two pure-line subspecies. Difference also exists in the pair of reciprocal tetraploids, but is to a less extent than that between the hybrids. The steady-state transcript abundance of genes involved in DNA repair and DNA methylation was significantly altered in both calli and regenerants, and some of which were correlated with the genetic and/or epigenetic alterations.Out results document that genetic alteration is the major cause of somaclonal variation in rice, which is concomitant epigenetic alteration. Perturbed expression by tissue culture of genes encoding for enzymes involved in DNA repair and DNA methylation is associated with both genetic and epigenetic alterations. There exist fundamental differences among distinct types of genotypes, pure-lines, hybrids and tetraploids, in propensities of generating both genetic and epigenetic alterations under the tissue culture condition. Unexpectedly, parent-of-origin has a conspicuous effect on the alterationfrequencies.更多还原