南方灵芝ITS、EF1-α和RPB2序列多态性研究（英文）
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摘要
内转录间隔区(ITS)、延伸因子1-α(EF1-α)与RNA聚合酶II第二大亚基(RPB2)是真菌系统学研究中常用的基因片段。已有研究发现,在同一个体内ITS也可能存在差异,但EF1-α和RPB2是否也存在同样的现象却鲜有报道。本研究通过克隆测序,分析了南方灵芝Ganodermaaustrale的ITS、EF1-α和RPB2在同一个体中的序列差异,并探讨这种序列差异是否会影响分子鉴定的准确性。结果表明,在南方灵芝供试样品中,ITS、EF1-α和RPB2序列都有可能存在个体内变异,但个体内变异程度不尽相同。基于供试样品和克隆测序结果发现,ITS序列在同一个体内的变异最高可达2.3%,而RPB2序列在同一个体内的差异仅0.5%,EF1-α序列在同一个体内序列差异也可高达1.8%。ITS和EF1-α序列在同一个体内的差异可能超过了灵芝属部分物种间的差异,对于部分灵芝属物种来说直接利用克隆序列进行分子鉴定或系统发育分析可能存在一些问题。
The internal transcribed spacer(ITS), translation elongation factor 1-α(EF1-α) and second largest subunit of RNA polymerase Ⅱ(RPB2) are commonly used for taxonomic or phylogenetic studies of fungi. Intragenomic variation of ITS has been detected in many fungal groups but few cases on EF1-α and RPB2 have been reported. In this study, the intragenomic variation of ITS, EF1-α and RPB2 in Ganoderma australe was detected using PCR cloning to determine whether intragenomic variation affects accuracy of molecular identification. The results showed that intragenomic variation of ITS, EF1-α and RPB2 was present in different specimens and their heterogeneity level was different. Heterogeneity level of the same gene also varied between individuals. Based on the examined samples and cloned sequences, the intragenomic variation of ITS was up to 2.3%, while that of RPB2 only 0.5%. Intragenomic variation of EF1-α could reach 1.8%. The highest intragenomic variation of ITS and EF1-α might exceed the interspecific difference, and therefore the use of these sequences for phylogenetic analyses and molecular identification of Ganoderma species could be problematic.
The internal transcribed spacer(ITS), translation elongation factor 1-α(EF1-α) and second largest subunit of RNA polymerase Ⅱ(RPB2) are commonly used for taxonomic or phylogenetic studies of fungi. Intragenomic variation of ITS has been detected in many fungal groups but few cases on EF1-α and RPB2 have been reported. In this study, the intragenomic variation of ITS, EF1-α and RPB2 in Ganoderma australe was detected using PCR cloning to determine whether intragenomic variation affects accuracy of molecular identification. The results showed that intragenomic variation of ITS, EF1-α and RPB2 was present in different specimens and their heterogeneity level was different. Heterogeneity level of the same gene also varied between individuals. Based on the examined samples and cloned sequences, the intragenomic variation of ITS was up to 2.3%, while that of RPB2 only 0.5%. Intragenomic variation of EF1-α could reach 1.8%. The highest intragenomic variation of ITS and EF1-α might exceed the interspecific difference, and therefore the use of these sequences for phylogenetic analyses and molecular identification of Ganoderma species could be problematic.
引文
Belbahri L,McLeod A,Paul B,Calmin G,Moralejo E,Spies CFJ,Botha WJ,Clemente A,Descale E,Sanchez-Hernandez E,Lefort F,2008.Intraspecific and within-isolate sequence variation in the ITS rRNA gene region of Pythium mercuriale sp.nov.(Pythiaceae).FEMS Microbiology Letters,284:17-27
Cao Y,Wu SH,Dai YC,2012.Species clarification of the prize medicinal Ganoderma mushroom“Lingzhi”.Fungal Diversity,56:49-62
Hasegawa E,Ota Y,Hattori T,Kikuchi T,2010.Sequence-based identification of Japanese Armillaria species using the elongation factor-1 alpha gene.Mycologia,102:898-910
He XL,Li Q,Peng WH,Zhou J,Cao XL,Wang D,Huang ZQ,Tan W,Li Y,Gan BC,2017.Intra-and inter-isolate variation of ribosomal and protein-coding genes in Pleurotus:implications for molecular identification and phylogeny on fungal groups.BMC Microbiology,17:139
Jargalmaa S,Eimes JA,Park MS,Park JY,Oh SY,Lim YW,2017.Taxonomic evaluation of selected Ganoderma species and database sequence validation.Peer J,5:e3596
Liao DQ,1999.Concerted evolution:molecular mechanism and biological implications.The American Journal of Human Genetics,64:24-30
Lindner DL,Banik MT,2011.Intragenomic variation in the ITSrDNA region obscures phylogenetic relationships and inflates estimates of operational taxonomic units in genus Laetiporus.Mycologia,103:731-740
Matheny PB,2005.Improving phylogenetic inference of mushrooms with RPB1 and RPB2 nucleotide sequences(Inocybe;Agaricales).Molecular Phylogenetics and Evolution,35:1-20
Naidoo K,Steenkamp ET,Coetzee MPA,Wingfield MJ,Wingfield BD,2013.Concerted evolution in the ribosomal RNA cistron.PLoS One,8:e59355
Okabe I,Arakawa M,Matsumoto N,2001.ITS polymorphism within a single strain of Sclerotium rolfsii.Mycoscience,42:107-113
Pannecoucque J,H?fte M,2009.Detection of rDNA ITSpolymorphism in Rhizoctonia solani AG 2-1 isolates.Mycologia,101:26-33
Paterson RR,2006.Ganoderma-a therapeutic fungal biofactory.Phytochemistry,67:1985-2001
Rehner SA,Buckley E,2005.A Beauveria phylogeny inferred from nuclear ITS and EF1-a sequences:evidence for cryptic diversification and links to Cordyceps teleomorphs.Mycologia,97:84-98
Ryvarden L,Gilbertson RL,1993.European polypores.Synopsis Fungorum,6:1-387
Simon UK,Weiss M,2008.Intragenomic variation of fungal ribosomal genes is higher than previously thought.Molecular Biology and Evolution,25:2251-2254
Tamura K,Stecher G,Peterson D,Filipski A,Kumar S,2013.MEGA6:molecular evolutionary genetics analysis version6.0.Molecular Biology and Evolution,30:2725-2729
Tang JM,Toe L,Back C,Unnasch TR,1996.Intra-specific heterogeneity of the rDNA internal transcribed spacer in the Simulium damnosum(Diptera:Simuliidae)complex.Molecular Biology and Evolution,13:244-252
Vydryakova GA,Van DT,Shoukouhi P,Psurtseva NV,Bissett J,2012.Intergenomic and intragenomic ITS sequence heterogeneity in Neonothopanus nambi(Agaricales)from Vietnam.Mycology,3:89-99
Waalwijk C,Koning JRA,Baayen RP,Gams W,1996.Discordant groupings of Fusarium spp.from sections Elegans,Liseola and Dlaminia based on ribosomal ITS1and ITS2 sequences.Mycologia,88:361-368
Wang DM,Yao YJ,2005.Intrastrain internal transcribed spacer heterogeneity in Ganoderma species.Canadian Journal of Microbiology,51:113-121
White TJ,Bruns T,Lee S,Taylor J,1990.Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics.In:Innis MA,Gelfand DH,Sninsky JJ,White TJ(eds.)PCR protocols:a guide to methods and applications.Academic Press,San Diego.315-322
Woo PCY,Leung SY,To KKW,Chan JFW,Ngan AHY,Cheng VCC,Lau SKP,Yuen KY,2010.Internal transcribed spacer region sequence heterogeneity in Rhizopus microsporus:implications for molecular diagnosis in clinical microbiology laboratories.Journal of Clinical Microbiology,48:208-214
Xing JH,Song J,Decock C,Cui BK,2016.Morphological characters and phylogenetic analysis reveal a new species within the Ganoderma lucidum complex from South Africa.Phytotaxa,266:115-124
Xing JH,Sun YF,Han YL,Cui BK,Dai YC,2018.Morphological and molecular identification of two new Ganoderma species on Casuarina equisetifolia from China.Mycokeys,34:93-108
Zhao Y,Tsang C,Xiao M,Cheng J,Xu Y,Lau SKP,Woo PCY,2015.Intra-genomic internal transcribed spacer region sequence heterogeneity and molecular diagnosis in clinical microbiology.International Journal of Molecular Sciences,16:25067-25079
Zhou LW,Cao Y,Wu SH,Vlasák J,Li DW,Li MJ,Dai YC,2015.Global diversity of the Ganoderma lucidum complex(Ganodermataceae,Polyporales)inferred from morphology and multilocus phylogeny.Phytochemistry,114:7-15
Cao Y,Wu SH,Dai YC,2012.Species clarification of the prize medicinal Ganoderma mushroom“Lingzhi”.Fungal Diversity,56:49-62
Hasegawa E,Ota Y,Hattori T,Kikuchi T,2010.Sequence-based identification of Japanese Armillaria species using the elongation factor-1 alpha gene.Mycologia,102:898-910
He XL,Li Q,Peng WH,Zhou J,Cao XL,Wang D,Huang ZQ,Tan W,Li Y,Gan BC,2017.Intra-and inter-isolate variation of ribosomal and protein-coding genes in Pleurotus:implications for molecular identification and phylogeny on fungal groups.BMC Microbiology,17:139
Jargalmaa S,Eimes JA,Park MS,Park JY,Oh SY,Lim YW,2017.Taxonomic evaluation of selected Ganoderma species and database sequence validation.Peer J,5:e3596
Liao DQ,1999.Concerted evolution:molecular mechanism and biological implications.The American Journal of Human Genetics,64:24-30
Lindner DL,Banik MT,2011.Intragenomic variation in the ITSrDNA region obscures phylogenetic relationships and inflates estimates of operational taxonomic units in genus Laetiporus.Mycologia,103:731-740
Matheny PB,2005.Improving phylogenetic inference of mushrooms with RPB1 and RPB2 nucleotide sequences(Inocybe;Agaricales).Molecular Phylogenetics and Evolution,35:1-20
Naidoo K,Steenkamp ET,Coetzee MPA,Wingfield MJ,Wingfield BD,2013.Concerted evolution in the ribosomal RNA cistron.PLoS One,8:e59355
Okabe I,Arakawa M,Matsumoto N,2001.ITS polymorphism within a single strain of Sclerotium rolfsii.Mycoscience,42:107-113
Pannecoucque J,H?fte M,2009.Detection of rDNA ITSpolymorphism in Rhizoctonia solani AG 2-1 isolates.Mycologia,101:26-33
Paterson RR,2006.Ganoderma-a therapeutic fungal biofactory.Phytochemistry,67:1985-2001
Rehner SA,Buckley E,2005.A Beauveria phylogeny inferred from nuclear ITS and EF1-a sequences:evidence for cryptic diversification and links to Cordyceps teleomorphs.Mycologia,97:84-98
Ryvarden L,Gilbertson RL,1993.European polypores.Synopsis Fungorum,6:1-387
Simon UK,Weiss M,2008.Intragenomic variation of fungal ribosomal genes is higher than previously thought.Molecular Biology and Evolution,25:2251-2254
Tamura K,Stecher G,Peterson D,Filipski A,Kumar S,2013.MEGA6:molecular evolutionary genetics analysis version6.0.Molecular Biology and Evolution,30:2725-2729
Tang JM,Toe L,Back C,Unnasch TR,1996.Intra-specific heterogeneity of the rDNA internal transcribed spacer in the Simulium damnosum(Diptera:Simuliidae)complex.Molecular Biology and Evolution,13:244-252
Vydryakova GA,Van DT,Shoukouhi P,Psurtseva NV,Bissett J,2012.Intergenomic and intragenomic ITS sequence heterogeneity in Neonothopanus nambi(Agaricales)from Vietnam.Mycology,3:89-99
Waalwijk C,Koning JRA,Baayen RP,Gams W,1996.Discordant groupings of Fusarium spp.from sections Elegans,Liseola and Dlaminia based on ribosomal ITS1and ITS2 sequences.Mycologia,88:361-368
Wang DM,Yao YJ,2005.Intrastrain internal transcribed spacer heterogeneity in Ganoderma species.Canadian Journal of Microbiology,51:113-121
White TJ,Bruns T,Lee S,Taylor J,1990.Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics.In:Innis MA,Gelfand DH,Sninsky JJ,White TJ(eds.)PCR protocols:a guide to methods and applications.Academic Press,San Diego.315-322
Woo PCY,Leung SY,To KKW,Chan JFW,Ngan AHY,Cheng VCC,Lau SKP,Yuen KY,2010.Internal transcribed spacer region sequence heterogeneity in Rhizopus microsporus:implications for molecular diagnosis in clinical microbiology laboratories.Journal of Clinical Microbiology,48:208-214
Xing JH,Song J,Decock C,Cui BK,2016.Morphological characters and phylogenetic analysis reveal a new species within the Ganoderma lucidum complex from South Africa.Phytotaxa,266:115-124
Xing JH,Sun YF,Han YL,Cui BK,Dai YC,2018.Morphological and molecular identification of two new Ganoderma species on Casuarina equisetifolia from China.Mycokeys,34:93-108
Zhao Y,Tsang C,Xiao M,Cheng J,Xu Y,Lau SKP,Woo PCY,2015.Intra-genomic internal transcribed spacer region sequence heterogeneity and molecular diagnosis in clinical microbiology.International Journal of Molecular Sciences,16:25067-25079
Zhou LW,Cao Y,Wu SH,Vlasák J,Li DW,Li MJ,Dai YC,2015.Global diversity of the Ganoderma lucidum complex(Ganodermataceae,Polyporales)inferred from morphology and multilocus phylogeny.Phytochemistry,114:7-15