五叶草莓超低温保存及遗传稳定性分析

【目的】为了找出野生种质资源五叶草莓(Fragaria pentapylla)的超低温保存方法和分析超低温保存后五叶草莓的遗传信息稳定性,【方法】运用玻璃化法对五叶草莓离体茎尖超低温保存技术进行了研究,并采用AFLP和MSAP技术对其遗传稳定性作了探讨。【结果】结果表明,不同的低温锻炼时间、预培养时间、预处理时间、玻璃化溶液(PVS2)处理时间和液氮保存时间对五叶草莓超低温保存后成活率的影响各不相同,经优化后,低温锻炼3周,预培养3 d,预处理30 min,PVS2处理60 min时,超低温保存的最高成活率可达79.7%,液氮处理时间对成活率影响不明显;运用AFLP技术对超低温后成活生长120 d的材料及对照材料基因组DNA进行了分析,超低温前后带型一致,未发现明显差异片段;进一步用MSAP技术分析了超低温保存后成活的材料和对照材料DNA甲基化水平差异,结果表明超低温保存后五叶草莓DNA甲基化与去甲基化分别为5.70%和12.43%。【结论】玻璃化法超低温保存技术可以有效的保存五叶草莓种质资源,超低保存前后的五叶草莓基因组DNA没有发生多态性变化,但DNA甲基化水平降低。

Cryopreservation of Fragaria pentapylla and analysis of genetic stability

【Objective】Objective of the study was to investigate the method of the cryopreservation of Fragaria pentapylla and to analyze genetic stability of the samples.【Method】The cryopreservation technology of the F.pentapylla shoot tip in vitro was studied by vitrification early,then genetic stability of the samples was analyzed by Amplification Fragment Length Polymorphism(AFLP) and Methylation Sensitive Amplified Polymorphism(MSAP).【Result】Cold acclimation,pre-culture,pre-treatment,PVS2 treatment,and liquid nitrogen preservation time had different effects on Fragaria pentapylla's survival rate after its cryopreservation.The highest survival rate of shoot tips could reach to 79.7%,but first of all,shoot tips must be acclimated for 21 days,then pre-cultured for 3 days,and pre-treated for 30 mins,ultimately,treated with PVS2 for 60 mins.Genomic DNA of the materials had been analyzed by AFLP after cryopreservation for 120 days,and there was not any obvious differences in banding patterns of DNA fragments between survival material and control material.And then the differences of DNA methylation between survival material and control material were analyzed by MSAP.Results showed that the methylation and demethylation rate of F.pentapylla DNA were 5.70% and 12.43%,respectively.【Conclusion】This study demonstrates that cryopreservation is an efficient method to keep the F.pentapylla resource.Polymorphism changes had not been found in the genomic DNA between survival material and control material,but the DNA methylation level had dropped.