鉴别紫芝菌株的PCR引物筛选及其序列比对验证

Selection and Sequence Alignment of PCR Primers for Identifying Zizhi Strain

【目的】紫芝S2(品种名:武芝2号)是近年来在福建及周边省份推广应用的紫芝栽培新品种,为了避免与遗传背景不同的栽培菌株混杂而建立有效的分子标记。【方法】采用PCR扩增筛选条带清晰稳定、呈现多态性的引物,根据菌株间UPMGA聚类构建系统发生树,通过遗传距离确定主要栽培菌株间的亲缘关系,并与菌株间的拮抗反应结果相映证,进而与‘紫芝S2’基因组序列进行比对验证。【结果】筛选得到能清晰且稳定地扩增出特异性或多态性条带的2个RAPD-PCR引物和3个ISSR-PCR引物,比对发现这5个引物短序列与‘紫芝S2’基因组序列完全匹配上的位点与数目不同。【结论】基于紫芝S2基因组序列比对,确认其中3个引物(ISSR13、 S1326和S1506)可以有效地用于紫芝栽培菌株鉴定。

【Objective】 The molecular markers of the cultivated strain of Ganoderma sinense, Zizhi S2(aka Wu-Zhi No. 2),recently popularized in Fujian and surrounding provinces were studied to facilitate the authentication of the medicinal fungus.【 Method】 Relevant primers of Zizhi S2 showing clear and stable bands and polymorphism were screened using PCR.Phylogenetic tree of UPMGA clustering analysis on the verified authentic cultivars was constructed to determine their relationship by genetic distance as well as antagonistic reaction. Subsequently, sequences of the selected primers were blasted on the genome of Zizhi S2 to validate the methodology. 【Result】 There were 2 RAPD-PCR and 3 ISSR-PCR primers found to clearly and stably amplify the specific or polymorphic bands. However, the sites and numbers on the scaffolds of the Zizhi S2 genome that matched the sequences of the 5 primers were not same. 【 Conclusion】 It was confirmed that three primers(ISSR13, S1326, and S1506)could be effectively used for identification of Zizhi cultivated strains based on sequences alignment with the genome of G. sinense strain Zizhi S2.