百山祖冷杉SSR体系的建立及人工辅助授粉子代的初步鉴定

An SSR reaction system for Abies beshanzuensis progeny with artificial pollination and progeny identification

应用十六烷基三甲基溴化铵(CTAB)法提取百山祖冷杉Abies beshanzuensis叶片DNA,建立百山祖冷杉简单重复序列(SSR)反应体系。在体系建立过程中,采用单因素法分别对影响聚合酶链反应(PCR)的因素进行分析。结果表明:在20.00μL反应体系中,模板DNA用量、镁离子(Mg2+)浓度、三磷酸鸟嘌呤脱氧核苷酸(dNTPs)浓度、引物浓度、Taq DNA聚合酶用量分别是60 ng,3.75 mmol·L-1,0.15 mmol·L-1,0.40μmol·L-1,16.67 nkat(1.0 U)时结果最好。随机挑取其中1对引物扩增的多态性片段进行克隆测序,序列经比对后得到的一致性值达到94%,表明其他杉科植物的SSR引物可以在百山祖冷杉中应用。通过引物筛选,得到15对引物可在百山祖冷杉中扩增得到多态性条带,其中4对引物扩增得到的部分片段在百山祖冷杉和日本冷杉Abies firma间有明显区别。这些结果表明,利用SSR分子标记技术能够用于百山祖冷杉人工辅助授粉子代的初步鉴定。

To establish a Simple Sequence Repeat with Polymerase Chain Reaction（SSR-PCR） system for Abies beshanzuensis and to optimize the factors which affect the PCR reaction,the cetyl-trimethyl-ammonium bromide（CTAB） method was used to extract DNA from its leaves.Then,to evaluate the universality of the primers,the amplified product of one pair of primers were cloned and sequenced.Finally,primer pairs with good amplification and diversity were selected.Results showed that the optimal PCR reaction system was 60 ng template DNA,3.75 mmol·L-1 Mg2＋,0.15 mmol·L-1 dNTPs,0.4 μmol·L-1 primers,and 16.67 nkat（1.0 U） Taq polymerase in a volume of 20 μL.Blast results also showed that the identity value reached 94%.Then,15 pairs of primers with good amplification and diversity were obtained with four that could produce differing DNA fragments between Abies beshanzuensis and Abies firma.Identity value results indicated that SSR primers from other Taxodiaceae species could be used with Abies beshanzuensis;additionally the SSR-PCR technique was applicable to preliminary identification of progeny from artificial pollination.