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Detection of DNA methylation changes during seed germination in rapeseed (Brassica napus)

Chinese Science Bulletin 2006年第2期51卷 182-190页

作者： lu guangyuan wu xiaoming chen biyun gao guizhen xu kun li xiangzhi Key Laboratory of Oil Crops Genetic Improvement of the Ministry of Agriculture Oil Crops Research Institute CAAS Wuhan 430062 China

DNA methylation is known to play a crucial role in regulating plant development and or- gan or tissue differentiation. In this study, we as- sessed the extent and pattern of cytosine methylation during rapeseed (Brassica napus L.) seed germina- tion, and compared the methylation level of various tissues in seedling, using the techniques of methyla- tion-sensitive amplified polymorphism (MSAP) and HPLC separation and quantification of nucleosides. In all, 484 bands, each representing a recognition site cleaved by either or both of the isoschizomers, were amplified by 12 pairs of selective primers in DNA ob- tained from dry seeds. A total of 76 sites were found to be differentially digested by the isoschizomers, indicating that approximately 15.7% of 5′-CCGG-3′ sites in the genome were cytosine methylated. Four classes of patterns were observed in a comparative assay of cytosine methylation in the dry and germi- nating seeds; a small number of hypermethylation events occurred at 5′-CCGG-3′ sites in germinating seeds compared with dry seeds, while many more hypomethylation events were detected after seed germination. Differences in DNA methylation level in various tissues were also detected; radicel was less methylated than hypocotyl and cotyledon. These observations were further confirmed by HPLC analy- sis. In addition, sequencing of eleven differentially methylated fragments and the subsequent blast search revealed that cytosine methylated 5′-CCGG- 3′ sequences were equally distributed between cod- ing and non-coding regions. These results clearly demonstrate the power of MSAP technique for large-scale DNA methylation detection in rapeseed genome, and the complexity of DNA methylationchange during seed germination. DNA Hypomethyla- tion going with seed germination appears to be a necessary step toward transcriptional activation in gene expression, and may well contribute to the de- velopmental gene regulation.

关键词：油菜籽芸苔属 DNA甲基化作用种子萌芽 MSAP HPLC

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Evaluation of Genetic and Epigenetic Modification in Rapeseed(Brassica napus)Induced by Salt Stress

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Journal of Integrative Plant Biology 2007年第11期49卷 1599-1607页

作者： guangyuan lu xiaoming wu biyun chen guizhen gao kun xu Brassica Germplasm Division Oil Crops Research Institute Chinese Academy of Agricultural Science Wuhan 430062 China

Salinity is an important limiting environmental factor for rapeseed production worldwide. In this study, we assessed the extent and pattern of DNA damages caused by salt stress in rapeseed plants. Amplified fragment length polymorphism （AFLP） analysis revealed dose-related increases in sequence alterations in plantlets exposed to 10-1000 mmol/L sodium chloride. In addition, individual plantlets exposed to the same salt concentration showed different AFLP and selected region amplified polymorphism banding patterns. These observations suggested that DNA mutation in response to salt stress was random in the genome and the effect was dose-dependant. DNA methylation changes in response to salt stress were also evaluated by methylation sensitive amplified polymorphism （MSAP）. Three types of MSAP bands were recovered. Type Ⅰ bands were observed with both isoschizomers Hpa Ⅱ and Msp Ⅰ, while type Ⅱ and type Ⅲ bands were observed only with Hpa Ⅱ and Msp Ⅰ, respectively. Extensive changes in types of MSAP bands after NaCI treatments were observed, including appearance and disappearance of type Ⅰ, Ⅱ and Ⅲ bands, as well as exchanges between either type Ⅰand type Ⅱ or type Ⅰ and type Ⅲ bands. An increase of 0.2-17.6% cytosine methylated CCGG sites were detected in plantlets exposed to 10- 200 mmol/L salt compared to the control, and these changes included both de novo methylation and demethylation events. Nine methylation related fragments were also recovered and sequenced, and one sharing a high sequence homology with the ethylene responsive element binding factor was identified. These results demonstrated clear DNA genetic and epigenetic alterations in planUets as a response to salt stress, and these changes may suggest a mechanism for plants adaptation under salt stress.

关键词： Brassica napus DNA-methylation genetic variation methylation sensitive amplified polymorphism salt stress.

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国家冬油菜区试新品种的SSR指纹图谱分析

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分子植物育种 2008年第4期6卷 709-716页

作者：陈碧云伍晓明张冬晓刘凤兰陆光远许鲲高桂珍中国农业科学院油料作物研究所武汉 430062 农业部全国农业技术推广服务中心北京 100026

以2006-2007年度全国冬油菜区试的89份参试品种为材料,运用筛选确定的25对多态性好、带纹清晰的SSR引物对这些品种进行分子标记分析,共获得106个多态性片段,平均每对引物4.2个,多态性比率平均达到77.7%,PIC平均值为0.7991,利用106个多... 详细信息

以2006-2007年度全国冬油菜区试的89份参试品种为材料,运用筛选确定的25对多态性好、带纹清晰的SSR引物对这些品种进行分子标记分析,共获得106个多态性片段,平均每对引物4.2个,多态性比率平均达到77.7%,PIC平均值为0.7991,利用106个多态性片段构建了这些品种的SSR指纹图谱。遗传聚类和主成分分析结果显示,在相似系数为0.95时,89份区试品种完全区分开;同一单位育成的品种的遗传距离较近,不同地区或单位育成的品种间遗传距离较大,揭示的遗传结构与品种系谱来源相吻合;除了少数品种外,大部分品种都具有很高的特异性。对不同类型及不同区片的品种(品系)遗传多样性指数分析,结果显示,杂交种的多样性水平要明显高于常规种;长江中游区品种(品系)遗传多样性指数最高,长江上游区和长江下游区次之,黄淮区最低。

关键词：油菜区域试验 SSR DNA指纹

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