几种森林大型真菌纯培养菌种的RAPD及ITS分子标记鉴定

doi:10.11707/j.1001-7488.20071216

林业科学 ›› 2007, Vol. 43 ›› Issue (12): 94-100.doi: 10.11707/j.1001-7488.20071216

几种森林大型真菌纯培养菌种的RAPD及ITS分子标记鉴定

李海波¹ 吴学谦^1,2 魏海龙^1,2 付立忠^1,2 吴庆其^1,2

1.浙江省林业科学研究院,杭州310023；2.浙江益圣菌物发展有限公司,丽水323000

收稿日期:2006-06-14 修回日期:1900-01-01 出版日期:2007-12-25 发布日期:2007-12-25

Identification of Pure Culture for Macrofungi with RAPD and ITS Molecular Markers

Li Haibo¹ Wu Xueqian^1,2 Wei Hailong¹ Fu Lizhong^1,2 Wu Qingqi^1,2

1. Zhejiang Forestry Academy Hangzhou 310023; 2. Lishui Edible Fungal Reasearch and Development Center Lishui 323000

Received:2006-06-14 Revised:1900-01-01 Online:2007-12-25 Published:2007-12-25

摘要/Abstract

摘要：

以采自野外的4对外生菌根菌和1对木腐菌的子实体及其各自的组织分离菌株作为研究材料,运用RAPD分子标记来分析组织分离菌株与子实体间的亲缘关系,通过PCR产物克隆测序比较二者的ITS碱基序列差异,最终对组织分离菌株是否为其纯培养菌种作出判定。RAPD分析结果表明,彩色豆马勃、白乳菇以及缘盖牛肝菌的子实体及其组织分离菌株分别在0.949、0.953以及0.969的Dice相似系数水平上聚为一类,而正红菇、香杯伞的子实体及其分离菌株仅在0.04和0.08的Dice相似系数水平上聚为一类。ITS序列测定分析结果显示,彩色豆马勃、白乳菇以及缘盖牛肝菌子实体与其各自组织分离菌株的ITS片段在长度和碱基序列上完全一致,而正红菇、香杯伞子实体与其各自分离菌株具有数量和长度均不同的ITS片段。RAPD分析结果与ITS分析结果相互支持,表明彩色豆马勃、白乳菇以及缘盖牛肝菌的组织分离菌株为其纯培养菌种,而正红菇、香杯伞的组织分离菌株并非其纯培养菌种,RAPD和ITS二种分子标记的结合运用可以更加高效准确地鉴定出外生菌根菌组织分离菌株是否为其纯培养菌种。ITS分析结果还提示正红菇、香杯伞子实体内可能存在多种伴生菌。

关键词: 大型真菌, 外生菌根菌, 纯培养菌种, 亲菌鉴定, ITS, RAPD

Abstract:

5 pair samples, including 4 ectomycorrhizal mushrooms and their isolates from tissue isolation, 1 saprotrophic fungi fruitibody and its isolate from tissue isolation, which were collected in Lishui area, in Zhejiang Province, were selected in this study. To determine wheather the isolates from tissue isolation were their own pure cultures or not, RAPD molecular marker was used to analyse the genetic relationships among 5 fuitibodies and their own isolates, and the differences of the sequences of ITS among these fruitbodies and their own isolates were compared by clone sequencing of PCR products. The results of RAPD analysis revealed that the fuitibodies and their corresponding isolates of Pisolithus tinctorius, Lactarius piperatus and Boletus appendiculatus can be clustered into one group at the similarity coefficient level of 0.949, 0.953 and 0.969 respectively, but for Russula vinosa and Clitocybe odora, they were clustered into one group only at the similarity coefficient level of 0.04 and 0.08 respectively. The sequence comparing of ITS revealed that the fuitibodies and their corresponding isolates of P. tinctorius, L. piperatus and B. appendiculatus shared the same size and completely homologous sequence respectively, but for R. vinosa and C. odora, they were remarkably different not only at the size but also at the numbers of the ITS band among the fuitibodies and their corresponding isolate. The results of RAPD analysis, supported by the results of ITS analysis, indicates that the isolates of P. tinctorius, L. piperatus and B. appendiculatus were the pure cultures of their fuitibodies respectively, but it is not true for the R. vinosa and C. odora. In addition, the results of ITS analysis also revealed some unnamed companion fungi or bacteria lived in the fuitibodies of R. vinosa and C. odora. In conclusion, the present study suggests that RAPD, combined with ITS analysis could be used to identify the pure cultures of ectomycorrhizal fungi more effectively and accurately.

Key words: macrofungi, ectomycorrhizal fungi, pure culture, DNA identification, ITS, RAPD

李海波吴学谦;魏海龙;付立忠;吴庆其;. 几种森林大型真菌纯培养菌种的RAPD及ITS分子标记鉴定[J]. 林业科学, 2007, 43(12): 94-100.

Li Haibo Wu Xueqian;Wei Hailong Fu Lizhong;Wu Qingqi;. Identification of Pure Culture for Macrofungi with RAPD and ITS Molecular Markers[J]. Scientia Silvae Sinicae, 2007, 43(12): 94-100.

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几种森林大型真菌纯培养菌种的RAPD及ITS分子标记鉴定

Identification of Pure Culture for Macrofungi with RAPD and ITS Molecular Markers

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