云南重楼ITS序列单核苷酸多态性与甾体皂苷特征的相关性分析

Correlation Analysis Between Single Nucleotide Polymorphism of ITS Sequences and Profile of Steroidal Saponins Composition of Paris polyphylla var.yunnanensis

为了解云南重楼(Paris polyphylla var. yunnanensis) ITS序列的单核苷酸多态性(SNP)特征与其甾体皂苷构成特征的相关性,并探讨其对药材质量稳定性的影响,利用MegAlign软件对37份云南重楼样本的ITS序列进行对比,根据SNP位点进行分型;采用HPLC法测定7种甾体皂苷成分(重楼皂苷Ⅰ、Ⅱ、Ⅴ、Ⅵ、Ⅶ、H和薯蓣皂苷);用SPSS 25.0软件和SIMCA-P15.0软件对各基因型甾体皂苷构成特征进行统计分析。结果表明,云南重楼ITS序列存在40个双等位多态性位点,其中碱基转换32个,碱基颠换8个,根据SNP特征可划分为2类基因型YN-Ⅰ和YN-Ⅱ。6种甾体皂苷(重楼皂苷Ⅰ、Ⅱ、Ⅵ、Ⅶ、H、薯蓣皂苷)在云南重楼广泛分布,而重楼皂苷Ⅴ稀少。YN-Ⅰ和YN-Ⅱ的药典指标成分总含量分别为1.070%和0.93%,样本合格率分别为68.42%和77.78%;YN-Ⅰ的甾体皂苷总含量为1.65%,YN-Ⅱ为1.32%。方差分析表明,2类基因型的甾体皂苷特征存在一定程度的区别,但药典指标成分无显著差异,药材应该具有等效性。聚类分析和主成分分析表明,YN-Ⅰ的离散度更高,YN-Ⅱ的聚集度更好,表明YN-Ⅱ的药材质量更加稳定,植株个体的甾体皂苷合成和累积差异更小,YN-Ⅱ的SNP特征对云南重楼良种选育的分子遗传标记筛选具有重要意义。

In order to understand the correlation between characters of single nucleotide polymorphism(SNP) of internal transcribed spacer(ITS) sequences and composition of steroidal saponins of Paris polyphylla var.yunnanensis, the effect of SNP on the quality stability of medicinal materials, the ITS sequences of 37 samples were compared by MegAlign, and then classified according to SNP loci. In addition, 7 steroidal saponins(polyphyllin Ⅰ, Ⅱ, Ⅴ, Ⅵ, Ⅶ, H, dioscin) in samples were determined by HPLC-DAD. The steroidal saponin composition characteristics of each genotype were statistical analysis by SPSS 25.0 & SIMCA-P 15.0. The results showed that there were 40 double-allelic polymorphic sites in ITS sequences of P. polyphylla var. yunnanensis,including 32 base conversion sites and 8 base inversion sites. According to SNP characteristics, 37 samples were divided into two genotypes: YN-Ⅰ and YN-Ⅱ. Six kinds of steroidal saponins(polyphyllin Ⅰ, Ⅱ, Ⅵ, Ⅶ, H, dioscin)were widely distributed in samples, while polyphyllin Ⅴ was relatively rare. The mean total content of pharmacopoeia index components in YN-Ⅰ and YN-Ⅱ were 1.070% and 0.93%, which the sample pass rate were 68.42% and 77.78%, respectively. The mean total content of 7 steroidal saponins were 1.65% and 1.32%,respectively. ANOVA analysis showed that there was significant differentiation of steroidal saponins profile between two genotypes, while there was no significant difference in the index components of pharmacopoeia, so the medicinal value of two genotypes should be equivalent. Cluster analysis(CA) and principal component analysis(PCA) showed the dispersion of YN-Ⅰ was higher than that of YN-Ⅱ, indicating the quality of medicinal materials of YN-Ⅱ was more stable and the individual differences in steroidal saponins synthesis and accumulation in YN-Ⅱ was smaller than YN-Ⅰ. Therefore, the SNP characteristics of YN-Ⅱ were of great significance for the screen of molecular genetic markers in the breeding of Paris polyphylla var. yunnanensis.