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Scientia Silvae Sinicae ›› 2007, Vol. 43 ›› Issue (12): 94-100.doi: 10.11707/j.1001-7488.20071216

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

Li Haibo1 Wu Xueqian1,2 Wei Hailong1 Fu Lizhong1,2 Wu Qingqi1,2   

  1. 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

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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