| 钟礼义，应正河，刘新锐，陈体强.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[J].广东农业科学,2022,49(2):9-15 |
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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 WuzhiNo. 2 Based on Ribosomal DNA ITS and ITS2 Sequence |
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| DOI：10.16768/j.issn.1004-874X.2022.02.002 |
| 中文关键词: 紫芝 武芝 2 号 紫芝 S2 rDNA ITS/ITS2 序列分析 |
| 英文关键词: Ganoderma sinense Wuzhi No. 2 Zizhi S2 ribosomal DNA ITS/ITS2 molecular identification |
| 基金项目:福建省财政专项 - 福建省农业科学院创新团队建设项目（CXTD2021-2-14，STIT2017-2-8） |
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| 中文摘要: |
| 【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。 |
| 英文摘要: |
| 【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. |
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