利用PCR與RFLP技術分析台灣產南方靈芝兩雜交不孕性群之遺傳變異

Genetic Diversity of Two Intersterility Groups of Ganoderma australe in Taiwan

10.29812/BBNTNU.199512.0006

葉增勇(Zeng-Yung Yeh)；張君玉(Chung-Yuh Chang)；李桂楨(Guey-Jen Lee-Chen)

南方靈芝 ； 雜交不孕性群 ； 核醣體DNA ； 遺傳變異 ； 聚合酵素鏈反應 ； 限制片段長度多型性 ； Ganoderma austral ； Intersterility group ； Ribosomal DNA ； Genetic diversity ； PCR ； RFLP

師大生物學報

30卷2期（1995 / 12 / 01）

117 - 124

繁體中文

本報告在以聚合酵素鏈反應（PCR）及限制片段長度多型性（RFLP）等分子生物的技術，探討台灣產南方靈芝Ganoderma australe (Fr.) Pat種內兩個「雜交不孕性群」（intersterility group）之遺傳變異，同時加入美國產的G. australe FL-01作為比較研究。另以台灣產的新日本靈芝G. neo-japonicum Imaz. CCRC 36049當做本研究之外群（out group）。以互補於酵母菌rRNA基因的引子與DNA聚合酵素，將2.16-Kb的17S rRNA基因片段及1.85-Kb的25S rRNA基因5端片段選殖出來。依據限制酵素AvaII、HhaI、HinfI切割PCR產物及多聚丙烯醯膠體電泳，分析各靈芝菌種的限制表限型（restriction phenotype）：台灣產南方靈芝的兩個雜交不孕性群呈現遺傳上的變異，可以AvaII及HinfI的限制表現型區別；美國產的南方靈芝部分rDNA重覆單位（rDNA repeat）出現序列異質性（sequence heterogeneity）。以限制酵素部分切割（partial digest）及南方墨點法（Southern blot）建築的限制酵素圖譜（restriction map），呈現各靈芝菌種保守和變異的限制酵素切點。將這些酵素切點利用PAUP軟體繪出親緣關係樹狀圖，結果顯示台灣產南方靈芝兩群已明顯分化。

The purpose of this study was to investigate genetic diversity of two intersterility groups of Ganodnma australe (Fr.) Pat. in Taiwan. One culture of G. australe FL-01 from Florida was also employed for comparative studies. G. neo-japonicum Imaz. CCRC 36049 was served as the out group in parsimony analysis. Polymerase chain reaction (PCR) and restriction fragment length polymorphisms (RFLPs) molecular approaches were applied to assess these genetic problems. By using conserved primers complementary to rRNA genes of Saccharomyces cerevisiae, a 2.16-kb A fragment containing 17S rRNA gene and a 1.85-kb B fragment containing 5' half of 25S rRNA gene were efficiently amplified from ribosomal DNA (rDNA) repeats of these fungi. Electrophoresis of HhaI, AvaII or HinfI digested PCR products produced restriction phenotypes (fingerprints). The two intersterility groups of G. australe in Taiwan can be differentiated by Ava II and HinfI restriction phenotypes. In addition, minor sequence heterogeneity of rDNA repeats within G. australe FL-01 isolate was observed. Detail mapping of restriction sites within rDNA locus by partial digestion and Southern hybridization reveals conserved and variable restriction sites for each species or group. Phylogenetic structure was constructed from restriction sites by PAUP program. Our data suggest the two groups of G. australe in Taiwan are genetically separated. G. neo-japonicum CCRC 36049 is shown to be genetically distinct from group 1 of G. australe in Taiwan.