无柄灵芝遗传多样性的SRAP、ITS、TEF1-α和LSU分析
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摘要
【目的】研究来自我国不同地区的45株无柄灵芝菌株的遗传多样性。【方法】利用ITS、TEF1-α和LSU多基因分析及SRAP分子标记两种方法,对供试无柄灵芝菌株进行聚类分析和遗传多样性研究。【结果】筛选出8对SRAP引物共扩增出95条条带,其中具有多态性条带79条,平均多态性比例为82.4%,多态性信息含量(PIC)变幅在0.28-0.43,平均为0.38。ITS、TEF1-α和LSU多基因序列分析结果显示,同一地域的部分菌株聚在一起,亲缘关系较近,而地域相隔较远的部分菌株也聚在同一个进化支上,其亲缘关系也很近,这与SRAP聚类分析结果相吻合。【结论】无柄灵芝菌株遗传多样性较为丰富,其遗传相似性与地理分布存在一定的相关性,ITS、TEF1-α、LSU基因及多基因分析更适合无柄灵芝分类鉴定,而SRAP分子标记更适合于无柄灵芝遗传多样性分析。
[Objective] Genetic diversity and relationship of 45 Ganoderma resinaceum strains from different areas in China were analyzed. [Methods] We studied clustering analysis and genetic diversity of these isolates using multi-gene analysis with ITS, TEF1-α, LSU sequences and SRAP technique. [Results] 8 pairs of primers on 45 strains were selected and amplificated using PCR-SRAP system, and 95 fragments were amplified, the number of polymorphic bands was 79, the average polymorphic rate was 82.4%. The PIC(polymorphism information content) value of these markers varied from 0.28 to 0.43, averaging 0.38. The results of multi-gene analysis with ITS, TEF1-α, LSU sequences and SRAP technique showed that part of the strains from the same area were clustered with each other, their kinship was close, and some strains with large gaps about the geographical distribution were also clustered in the same phylogenetic tree branch, their genetic relationship may also be relatively close, the result above was consistent with SRAP clustering analysis. [Conclusion] The genetic diversity of 45 strains was relatively abundant, and it had some correlation between genetic similarity and geographical distribution. ITS, TEF1-α and LSU sequences analysis and multi-gene analysis were more useful for classification and identification of Ganoderma resinaceum, and SRAP technique was more suitable for the genetic diversity analysis of Ganoderma resinaceum.
[Objective] Genetic diversity and relationship of 45 Ganoderma resinaceum strains from different areas in China were analyzed. [Methods] We studied clustering analysis and genetic diversity of these isolates using multi-gene analysis with ITS, TEF1-α, LSU sequences and SRAP technique. [Results] 8 pairs of primers on 45 strains were selected and amplificated using PCR-SRAP system, and 95 fragments were amplified, the number of polymorphic bands was 79, the average polymorphic rate was 82.4%. The PIC(polymorphism information content) value of these markers varied from 0.28 to 0.43, averaging 0.38. The results of multi-gene analysis with ITS, TEF1-α, LSU sequences and SRAP technique showed that part of the strains from the same area were clustered with each other, their kinship was close, and some strains with large gaps about the geographical distribution were also clustered in the same phylogenetic tree branch, their genetic relationship may also be relatively close, the result above was consistent with SRAP clustering analysis. [Conclusion] The genetic diversity of 45 strains was relatively abundant, and it had some correlation between genetic similarity and geographical distribution. ITS, TEF1-α and LSU sequences analysis and multi-gene analysis were more useful for classification and identification of Ganoderma resinaceum, and SRAP technique was more suitable for the genetic diversity analysis of Ganoderma resinaceum.
引文
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[5]Budak H,Shearman RC,Parmaksiz I,et al.Molecular characterization of Buffalograss germplasm using sequence-related amplified polymorphism markers[J].Theoretical and Applied Genetics,2004,108(2):328-334
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[7]Bunyard BA,Chaichuchote S,Nicholson MS,et al.Ribosomal DNA analysis for resolution of genotypic classes of Pleurotus[J].Mycological Research,1996,100(2):143-150
[8]Dunham SM,O’dell TE,Molina R.Analysis of nr DNA sequences and microsatellite allele frequencies reveals a cryptic chanterelle species Cantharellus cascadensis sp.nov.from the American Pacific Northwest[J].Mycological Research,2003,107(10):1163-1177
[9]Smith BJ,Sivasithamparam K.Internal transcribed spacer ribosomal DNA sequence of five species of Ganoderma from Australia[J].Mycological Research,2000,104(8):943-951
[10]Cao Y,Wu SH,Dai YC.Species clarification of the prize medicinal Ganoderma mushroom“Lingzhi”[J].Fungal Diversity,2012,56(1):49-62
[11]Chen GF.Morphological and phylogenetic analysis and detection by fluorescent in situ hybridization of several red tide causative microalgae[D].Qingdao:Doctoral Dissertation of Institute of Oceanology,Chinese Academy of Sciences,2007(in Chinese)陈国福.几种赤潮微藻的形态和系统进化分析及荧光原位杂交检测[D].青岛:中国科学院海洋研究所博士学位论文,2007
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[13]Thorn RG,Moncalvo JM,Reddy CA,et al.Phylogenetic analyses and the distribution of nematophagy support a monophyletic Pleurotaceae within the polyphyletic pleurotoid-lentinoid fungi[J].Mycologia,2000,92(2):241-252
[14]Hibbett DS,Binder M.Evolution of complex fruiting–body morphologies in homobasidiomycetes[J].Proceedings of the Royal Society of London B:Biological Sciences,2002,269(1504):1963-1969
[15]Moncalvo JM,Vilgalys R,Redhead SA,et al.One hundred and seventeen clades of euagarics[J].Molecular Phylogenetics and Evolution,2002,23(3):357-400
[16]Li XL.Phylogenetic position of Pleurotus calyptratus based on nr DNA—LSU and ITS sequences[J].Journal of Beijing Forestry University,2005,27(3):67-71(in Chinese)李雪玲.贝盖侧耳的系统发育地位—基于nr DNA-LSU和ITS序列分析的研究[J].北京林业大学学报,2005,27(3):67-71
[17]Lin RB,Zhang H,Chen JC,et al.SRAP analysis of Agaricus blazei Murill cultivars[J].Chinese Journal of Tropical crops,2012,33(3):472-474(in Chinese)林戎斌,张慧,陈济琛,等.姬松茸种质资源多样性SRAP分析[J].热带作物学报,2012,33(3):472-474
[18]Liu N,Liu ZL,Gong GS,et al.Virulence structure of Blumeria graminis f.sp.tritici and its genetic diversity by ISSR and SRAP profiling analyses[J].PLo S One,2015,10(6):e0130881
[19]Zhang X,Li Q,Liu HJ,et al.Application of SRAP and ITS molecular markers in genetic diversity of wild Auricularia auricula in Daxing’anling area[J].Heilongjiang Science,2013,4(4):18-21(in Chinese)张旭,李倩,刘华晶,等.SRAP和ITS分子标记在大兴安岭地区野生黑木耳遗传多样性上的应用[J].黑龙江科学,2013,4(4):18-21
[20]Jia DH,Wang B,Peng WH,et al.Analysis of ITS sequences in 4wild Ganoderma lucidum strains[J].Southwest China Journal of Agricultural Sciences,2012,25(4):1414-1416(in Chinese)贾定洪,王波,彭卫红,等.4个野生灵芝菌株的ITS序列分析[J].西南农业学报,2012,25(4):1414-1416
[21]Moncalvo JM,Wang HH,Hseu RS.Phylogenetic relationships in Ganoderma inferred from the internal transcribed spacers and 25S ribosomal DNA sequences[J].Mycologia,1995,87(2):223-238
[22]Moncalvo JM,Wang HF,Hseu RS.Gene phylogeny of the Ganoderma lucidum complex based on ribosomal DNA sequences.Comparison with traditional taxonomic characters[J].Mycological Research,1995,99(12):1489-1499
[23]Hong SG,Jung HS.Phylogenetic analysis of Ganoderma based on nearly complete mitochondrial small-subunit ribosomal DNA sequences[J].Mycologia,2004,96(4):742-755
[24]Lan J,Xu JT,Wang QY,et al.Electrophoretic studies on estease and peroxidase isozymes in Ganoderma sp.[J].Chinese Pharmaceutical Journal,1998,33(12):714-716(in Chinese)兰进,徐锦堂,王秋颖,等.灵芝过氧化物同工酶和酯酶同工酶的研究[J].中国药学杂志,1998,33(12):714-716
[25]Zhao MW,Chen MJ,Wang N,et al.Study on genetic relationship among some commercial strains of Ganoderma[J].Journal of Nanjing Agricultural University,2003,26(3):60-63(in Chinese)赵明文,陈明杰,王南,等.灵芝生产用种的亲缘关系研究[J].南京农业大学学报,2003,26(3):60-63
[26]Tang CH,Zhang JS,Chen MJ,et al.Preliminary study on genetic diversity among ten strains of Ganoderma[J].Journal of Nanjing Agricultural University,2005,28(2):133-136(in Chinese)唐传红,张劲松,陈明杰,等.灵芝属菌株遗传多样性的初步研究[J].南京农业大学学报,2005,28(2):133-136
[27]Zheng LY,Jia DH,Fei XF,et al.An assessment of the genetic diversity within Ganoderma strains with AFLP and ITS PCR-RFLP[J].Microbiological Research,2009,164(3):312-321
[28]Zhang HY,Fu LZ,Wu XQ,et al.Identification of protoplast fusion hybrids of Ganoderma lucidum by SRAP analysis[J].Acta Edulis Fungi,2009,16(4):5-9(in Chinese)张红玉,付立忠,吴学谦,等.灵芝原生质体融合子的SRAP分子标记鉴定[J].食用菌学报,2009,16(4):5-9
[29]Steyaert RL.Species of Ganoderma and related genera mainly of the Bogor and Leiden herbaria[J].Persoonia-Molecular Phylogeny and Evolution of Fungi,1972,7(1):55-118
[30]Gilbertson RL,Ryvarden L.European Polypores[M].Oslo:Synopsis Fungorum,1993,6:743
[31]Hseu RS,Wang HH,Wang HF,et al.Differentiation and grouping of isolates of the Ganoderma lucidum complex by random amplified polymorphic DNA-PCR compared with grouping on the basis of internal transcribed spacer sequences[J].Applied and Environmental Microbiology,1996,62(4):1354-1363
[2]Al-Fatimi M,Wurster M,Kreisel H,et al.Antimicrobial,cytotoxic and antioxidant activity of selected basidiomycetes from Yemen[J].Die Pharmazie-An International Journal of Pharmaceutical Sciences,2005,60(10):776-780
[3]Li P,Zhan X,Que QM,et al.Genetic diversity and population structure of Toona Ciliata Roem.Based on sequence-related amplified polymorphism(SRAP)markers[J].Forests,2015,6(4):1094-1106
[4]Li G,Quiros CF.Sequence-related amplified polymorphism(SRAP),a new marker system based on a simple PCR reaction:its application to mapping and gene tagging in Brassica[J].Theoretical and Applied Genetics,2001,103(2/3):455-461
[5]Budak H,Shearman RC,Parmaksiz I,et al.Molecular characterization of Buffalograss germplasm using sequence-related amplified polymorphism markers[J].Theoretical and Applied Genetics,2004,108(2):328-334
[6]Sun SJ,Gao W,Lin SQ,et al.Analysis of genetic diversity in Ganoderma population with a novel molecular marker SRAP[J].Applied Microbiology and Biotechnology,2006,72(3):537-543
[7]Bunyard BA,Chaichuchote S,Nicholson MS,et al.Ribosomal DNA analysis for resolution of genotypic classes of Pleurotus[J].Mycological Research,1996,100(2):143-150
[8]Dunham SM,O’dell TE,Molina R.Analysis of nr DNA sequences and microsatellite allele frequencies reveals a cryptic chanterelle species Cantharellus cascadensis sp.nov.from the American Pacific Northwest[J].Mycological Research,2003,107(10):1163-1177
[9]Smith BJ,Sivasithamparam K.Internal transcribed spacer ribosomal DNA sequence of five species of Ganoderma from Australia[J].Mycological Research,2000,104(8):943-951
[10]Cao Y,Wu SH,Dai YC.Species clarification of the prize medicinal Ganoderma mushroom“Lingzhi”[J].Fungal Diversity,2012,56(1):49-62
[11]Chen GF.Morphological and phylogenetic analysis and detection by fluorescent in situ hybridization of several red tide causative microalgae[D].Qingdao:Doctoral Dissertation of Institute of Oceanology,Chinese Academy of Sciences,2007(in Chinese)陈国福.几种赤潮微藻的形态和系统进化分析及荧光原位杂交检测[D].青岛:中国科学院海洋研究所博士学位论文,2007
[12]Vilgalys R,Moncalvo JM,Liou S R,et al.Recent advances in molecular systematics of the genus Pleurotus[A]//Royse,DJ.Mushroom Biology and Mushroom Products[M].University Park,PA,USA:Pennsylvania State University,1996:91-101
[13]Thorn RG,Moncalvo JM,Reddy CA,et al.Phylogenetic analyses and the distribution of nematophagy support a monophyletic Pleurotaceae within the polyphyletic pleurotoid-lentinoid fungi[J].Mycologia,2000,92(2):241-252
[14]Hibbett DS,Binder M.Evolution of complex fruiting–body morphologies in homobasidiomycetes[J].Proceedings of the Royal Society of London B:Biological Sciences,2002,269(1504):1963-1969
[15]Moncalvo JM,Vilgalys R,Redhead SA,et al.One hundred and seventeen clades of euagarics[J].Molecular Phylogenetics and Evolution,2002,23(3):357-400
[16]Li XL.Phylogenetic position of Pleurotus calyptratus based on nr DNA—LSU and ITS sequences[J].Journal of Beijing Forestry University,2005,27(3):67-71(in Chinese)李雪玲.贝盖侧耳的系统发育地位—基于nr DNA-LSU和ITS序列分析的研究[J].北京林业大学学报,2005,27(3):67-71
[17]Lin RB,Zhang H,Chen JC,et al.SRAP analysis of Agaricus blazei Murill cultivars[J].Chinese Journal of Tropical crops,2012,33(3):472-474(in Chinese)林戎斌,张慧,陈济琛,等.姬松茸种质资源多样性SRAP分析[J].热带作物学报,2012,33(3):472-474
[18]Liu N,Liu ZL,Gong GS,et al.Virulence structure of Blumeria graminis f.sp.tritici and its genetic diversity by ISSR and SRAP profiling analyses[J].PLo S One,2015,10(6):e0130881
[19]Zhang X,Li Q,Liu HJ,et al.Application of SRAP and ITS molecular markers in genetic diversity of wild Auricularia auricula in Daxing’anling area[J].Heilongjiang Science,2013,4(4):18-21(in Chinese)张旭,李倩,刘华晶,等.SRAP和ITS分子标记在大兴安岭地区野生黑木耳遗传多样性上的应用[J].黑龙江科学,2013,4(4):18-21
[20]Jia DH,Wang B,Peng WH,et al.Analysis of ITS sequences in 4wild Ganoderma lucidum strains[J].Southwest China Journal of Agricultural Sciences,2012,25(4):1414-1416(in Chinese)贾定洪,王波,彭卫红,等.4个野生灵芝菌株的ITS序列分析[J].西南农业学报,2012,25(4):1414-1416
[21]Moncalvo JM,Wang HH,Hseu RS.Phylogenetic relationships in Ganoderma inferred from the internal transcribed spacers and 25S ribosomal DNA sequences[J].Mycologia,1995,87(2):223-238
[22]Moncalvo JM,Wang HF,Hseu RS.Gene phylogeny of the Ganoderma lucidum complex based on ribosomal DNA sequences.Comparison with traditional taxonomic characters[J].Mycological Research,1995,99(12):1489-1499
[23]Hong SG,Jung HS.Phylogenetic analysis of Ganoderma based on nearly complete mitochondrial small-subunit ribosomal DNA sequences[J].Mycologia,2004,96(4):742-755
[24]Lan J,Xu JT,Wang QY,et al.Electrophoretic studies on estease and peroxidase isozymes in Ganoderma sp.[J].Chinese Pharmaceutical Journal,1998,33(12):714-716(in Chinese)兰进,徐锦堂,王秋颖,等.灵芝过氧化物同工酶和酯酶同工酶的研究[J].中国药学杂志,1998,33(12):714-716
[25]Zhao MW,Chen MJ,Wang N,et al.Study on genetic relationship among some commercial strains of Ganoderma[J].Journal of Nanjing Agricultural University,2003,26(3):60-63(in Chinese)赵明文,陈明杰,王南,等.灵芝生产用种的亲缘关系研究[J].南京农业大学学报,2003,26(3):60-63
[26]Tang CH,Zhang JS,Chen MJ,et al.Preliminary study on genetic diversity among ten strains of Ganoderma[J].Journal of Nanjing Agricultural University,2005,28(2):133-136(in Chinese)唐传红,张劲松,陈明杰,等.灵芝属菌株遗传多样性的初步研究[J].南京农业大学学报,2005,28(2):133-136
[27]Zheng LY,Jia DH,Fei XF,et al.An assessment of the genetic diversity within Ganoderma strains with AFLP and ITS PCR-RFLP[J].Microbiological Research,2009,164(3):312-321
[28]Zhang HY,Fu LZ,Wu XQ,et al.Identification of protoplast fusion hybrids of Ganoderma lucidum by SRAP analysis[J].Acta Edulis Fungi,2009,16(4):5-9(in Chinese)张红玉,付立忠,吴学谦,等.灵芝原生质体融合子的SRAP分子标记鉴定[J].食用菌学报,2009,16(4):5-9
[29]Steyaert RL.Species of Ganoderma and related genera mainly of the Bogor and Leiden herbaria[J].Persoonia-Molecular Phylogeny and Evolution of Fungi,1972,7(1):55-118
[30]Gilbertson RL,Ryvarden L.European Polypores[M].Oslo:Synopsis Fungorum,1993,6:743
[31]Hseu RS,Wang HH,Wang HF,et al.Differentiation and grouping of isolates of the Ganoderma lucidum complex by random amplified polymorphic DNA-PCR compared with grouping on the basis of internal transcribed spacer sequences[J].Applied and Environmental Microbiology,1996,62(4):1354-1363