紫芝栽培新品种武芝2号基于rDNA-ITS和ITS2的分子鉴定与分析

Molecular Identification and Analysis of New Cultivar Wuzhi No.2 Based on Ribosomal DNA ITS and ITS2 Sequence

【目的】探讨利用rDNA-ITS及其ITS2二级结构进行紫芝栽培新品种武芝2号(原名紫芝S2)的分子鉴定。【方法】通过第一代和第三代测序技术分别获得武芝2号的ITS序列,针对灵芝属(Ganoderma spp.)相关种类的ITS序列构建系统进化树进行分子分类鉴定,采用rDNA-ITS及相关引物序列进行BLAST比对分析,在线预测ITS2二级结构。【结果】Sanger法测序得到的武芝2号ITS序列(MG282563.1,649 bp),与GenBank中5个紫芝ITS序列具有高相似度(99.53%~100%),并且与已知4个紫芝菌株或实物标本在灵芝属系统进化树上聚为一个分支,同属一个种群。已知2个紫芝特异性引物短序列(GS1_22bp、GS2_22bp)出现在武芝2号ITS序列的119~140 bp正向和588~567 bp反向位置;在武芝2号基因组Scaffold No.49上rDNA中找到4个串联重复的ITS区域,与Sanger法测序得到的单个ITS序列高度一致;在ITS2序列二级结构上,武芝2号与ITS2数据库中紫芝Ganoderma sinense的3个螺旋区结构相同,仅螺旋IV区间夹角不同。【结论】基于ITS和ITS2分子鉴定,确认了武芝2号与已知紫芝G.sinense同属一个种群,在ITS2二级结构螺旋IV区存在夹角差异;此外,验证了2个已知紫芝特异性扩增引物的有效性。

【Objective】The study was carried out to explore the molecular identification of Wuzhi No. 2(original name: Zizhi S2), a new cultivar of Ganoderma sp., by rDNA-ITS sequence analysis and ITS2 secondary structure model.【Method】The sequences of Wuzhi No. 2 were acquired by the first and third generation sequencing techniques respectively, and the phylogenetic tree was constructed based on ITS sequences of related species of genus Ganoderma for molecular classification and identification, the rDNA-ITS and related primer sequences were aligned by BLAST with the whole genome, and the secondary structure of ITS2 was predicted online.【Result】Sanger sequencing showed that the ITS sequence of Ganoderma sp. strain Wuzhi No. 2(GenBank: MG282563.1, 649 bp) displayed high similarity(99.53%-100%)with five known Ganoderma sinense strains in the GenBank, while Wuzhi No. 2 and four known strains or voucher specimens of G. sinense(strain) were clustered into a same branch on the Ganoderma spp. phylogenetic tree. Two known short sequences of specific primers(GS_22bp and GS2_22bp) appeared in the forward position of 119-140 bp and the reverse position of 588-567 bp of ITS sequence of Wuzhi No. 2, respectively. Four tandem repeat ITS regions were found from the rDNA on Scaffold No. 49 of Wuzhi No. 2 genome, which was highly consistent with the single ITS sequence by Sanger method. In terms of the secondary structure of ITS2 sequence, three helix regions in Wuzhi No. 2 and Ganoderma sinense were the same, but only the included angle in Helix IV region was different.【Conclusion】Based on ITS/ITS2 molecular identification, it was confirmed that the new cultivar Wuzhi No. 2 and the known strains of Ganoderma sinense belonged to the same population, there were angle differences in the Helix IV region of secondary structure of ITS2, and the effectiveness of two known specific amplification primers were also verified.