试栽羊肚菌分子系统分析
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摘要
[目的]为了弄清陕西汉中试栽羊肚菌的种类归属。[方法]收集到汉中地区试栽羊肚菌子实体19个样品,采用CTAB法提取其基因组DNA,使用通用引物PCR扩增5个基因序列(ITS、LSU、EF1-α、RPB1和RPB2)保守区片段,用1. 0%的琼脂糖凝胶电泳对PCR产物进行检测,并对PCR产物送样测序。依据测序结果构建系统发育树,结合系统发育树进行物种归属鉴定。[结果]获取了所有样品的5个基因序列保守区片段,通过测序比对19个样品5个基因序列保守区片段并进行序列联合,对总长度3 025 bp的保守区序列分析并构建系统发育树,依据系统发育树鉴定出汉中试栽羊肚菌分别为六妹羊肚菌(Morchella sextelata)、梯棱羊肚菌(M. importuna)、隐形羊肚菌(M. cryptica)和北方羊肚菌(M. septentrionalis)。[结论]采用多基因联合分析法弄清楚汉中试栽羊肚菌的种类归属。
[Objective]The classification of planting morel species in Hanzhong of Shaanxi province was identified.[Method]A total of 19 samples of morels fruiting bodies were collected from the Hanzhong area and the genomic DNA was extracted by CTAB method. Polymerase chain reaction( PCR) was used to amplify nuclear ribosomal internal transcribed spacer( ITS),domains D1 and D2 of the Ribosomal 28 S large subunit( LSU),translation elongation factor 1-alpha( EF1-α),RNA polymeraseⅡ largest( RPB1) and second largest subunit( RPB2) using universal primers. Five conserved region gene fragments were detected by 1. 0% agarose gel electrophoresis,and the PCR products were sent for sequencing.[Result]Phylogenetic tree was constructed based on the sequencing results,and species identification was performed in combination with phylogenetic tree. As a result,ITS,LSU,EF1-α,RPB1 and RPB2 sequences of all species were obtained. Through sequencing and comparison,every samples had 3 025 bp conserved region. Phylogenetic tree was constructed by the joint analysis of 3 025 bp sequences. The morels were identified as Morchella sextelata,M. importuna,M. cryptica,and M. septentrionalis by the phylogenetic tree. [Conclusion] The species of test cultivation morels were preliminarily ascertained in Hanzhong area of Shaanxi province.
[Objective]The classification of planting morel species in Hanzhong of Shaanxi province was identified.[Method]A total of 19 samples of morels fruiting bodies were collected from the Hanzhong area and the genomic DNA was extracted by CTAB method. Polymerase chain reaction( PCR) was used to amplify nuclear ribosomal internal transcribed spacer( ITS),domains D1 and D2 of the Ribosomal 28 S large subunit( LSU),translation elongation factor 1-alpha( EF1-α),RNA polymeraseⅡ largest( RPB1) and second largest subunit( RPB2) using universal primers. Five conserved region gene fragments were detected by 1. 0% agarose gel electrophoresis,and the PCR products were sent for sequencing.[Result]Phylogenetic tree was constructed based on the sequencing results,and species identification was performed in combination with phylogenetic tree. As a result,ITS,LSU,EF1-α,RPB1 and RPB2 sequences of all species were obtained. Through sequencing and comparison,every samples had 3 025 bp conserved region. Phylogenetic tree was constructed by the joint analysis of 3 025 bp sequences. The morels were identified as Morchella sextelata,M. importuna,M. cryptica,and M. septentrionalis by the phylogenetic tree. [Conclusion] The species of test cultivation morels were preliminarily ascertained in Hanzhong area of Shaanxi province.
引文
[1]肖江勇,何秀霞,王丹,等.ELISA鉴别条件下的野生羊肚菌菌种分离方法[J].中国食用菌,2015,34(3):11-14.
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[3]顾可飞,周昌艳,邵毅,等.云南省野生牛肝菌与羊肚菌营养成分分析[J].食品研究与开发,2017,38(17):129-133.
[4]龚燕川,陈影,唐杰,等.梯棱羊肚菌M83和M101菌株培养特性差异[J].中国食用菌,2016,35(4):7-11.
[5]张能,赵苗,谢敬宜,等.梯棱羊肚菌Morchella importuna对重金属离子的耐受性研究[J].菌物学报,2017,36(3):367-375.
[6]秦小波,时小东,张国珍,等.梯棱羊肚菌菌丝培养条件研究及观察分析[J].食用菌,2016,38(3):8-10.
[7]朱斗锡.羊肚菌人工栽培研究进展[J].中国食用菌,2008,27(4):3-5.
[8]Ower R.Notes on the development of the morel ascocarp:Morchella esculenta[J].Mycologia,1982,74(1):142-144.
[9]陈诚,李强,黄文丽,等.羊肚菌白霉病发生对土壤真菌群落结构的影响[J].微生物学通报,2017,44(11):2652-2659.
[10]Kerry O'Donnell,Alejandro P.Rooney,Gary L.Mills,et al.Phylogeny and historical biogeography of true morels(Morchella)reveals an early cretaceous origin and high continental endemism and provincialism in the Holarctic[J].Fungal Genetics&Biology,2011,48(3):252-265.
[11]武冬梅,许文涛,谢宗铭,等.新疆野生羊肚菌物种多样性研究[J].食品工业科技,2015,36(2):167-172.
[12]刘伟,蔡英丽,张亚,等.我国羊肚菌人工栽培快速发展的关键技术解析[J].食药用菌,2018,26(3):142-147.
[13]Voitk A,Beug MW,Kerry O'Donnell,et al.Two new species of true morels from Newfoundland and Labrador:cosmopolitan Morchella eohespera and parochial M.laurentiana[J].Mycologia,2016,108(1):1-28.
[14]Hatra Ta?kn,Saadet Büyükalaca,Hasan Hüseyin Dogˇan,et al.Amultigene molecular phylogenetic assessment of true morels(Morchella)in Turkey[J].Fungal Genetics&Biology,2010,47(8):672-682.
[15]杜习慧,赵琪,杨祝良,等.羊肚菌的多样性、演化历史及栽培研究进展[J].菌物学报,2014,33(2):183-197.
[16]何培新,刘伟,蔡英丽,等.我国人工栽培和野生黑色羊肚菌的菌种鉴定及系统发育分析[J].郑州轻工业学院学报:自然科学版,2015,30(3-4):26-29.
[17]Kuo M,Dewsbury D R,O'Donnell K,et al.Taxonomic revision of true morels(Morchella)in Canada and the United States[J].Mycologia,2012,104(5):1159-1177.
[18]Richard F,Bellanger J M,Clowez P,et al.True morels(Morchella,Pezizales)of Europe and North America:evolutionary relationships inferred from multilocus data and a unified taxonomy[J].Mycologia,2015,107(2):359-382.
[19]易铭,梁嘉俊,史建,等.采用EF-1α序列分析法对苜蓿根腐病病原菌-锐顶镰刀菌的鉴定[J].草业学报,2017,26(02):61-68.
[20]Du X H,Zhao Q,O'Donnell K,et al.Multigene molecular phylogenetics reveals true morels(Morchella)are especially speciesrich in China[J].Fungal Genetics&Biology,2012,49(6):455-469.
[21]Yatsiuk I,Saar I,Kalamees K,et al.Epitypification of Morchella steppicola(Morchellaceae,Pezizales),a morphologically,phylogenetically and biogeographically distinct member of the Esculenta Clade from central Eurasia[J].Phytotaxa,2016,284(1):31-40.
[22]Richard F,Bellanger J M,Clowez P,et al.True morels(Morchella,Pezizales)of Europe and North America:evolutionary relationships inferred from multilocus data and a unified taxonomy[J].Mycologia,2015,107(2):359-382.
[23]Chai H,Chen L,Chen W,et al.Characterization of mating-type idiomorphs suggests that Morchella importuna,Mel-20,and M.sextelata,are heterothallic[J].Mycological Progress,2017,16(12):1-10.
[24]Loizides M,Bellanger JM,Clowez P,et al.Combined phylogenetic and morphological studies of true morels(Pezizales,Ascomycota)in Cyprus reveal significant diversity,including Morchella arbutiphila and M.disparilis spp.nov.[J].Mycological Progress,2016,15(39):DOI:10.1007/s11557-016-1180-1.
[2]林晓民,李振岐,侯军,等.中国菌物[M].北京:中国农业出版社,2007:107-108.
[3]顾可飞,周昌艳,邵毅,等.云南省野生牛肝菌与羊肚菌营养成分分析[J].食品研究与开发,2017,38(17):129-133.
[4]龚燕川,陈影,唐杰,等.梯棱羊肚菌M83和M101菌株培养特性差异[J].中国食用菌,2016,35(4):7-11.
[5]张能,赵苗,谢敬宜,等.梯棱羊肚菌Morchella importuna对重金属离子的耐受性研究[J].菌物学报,2017,36(3):367-375.
[6]秦小波,时小东,张国珍,等.梯棱羊肚菌菌丝培养条件研究及观察分析[J].食用菌,2016,38(3):8-10.
[7]朱斗锡.羊肚菌人工栽培研究进展[J].中国食用菌,2008,27(4):3-5.
[8]Ower R.Notes on the development of the morel ascocarp:Morchella esculenta[J].Mycologia,1982,74(1):142-144.
[9]陈诚,李强,黄文丽,等.羊肚菌白霉病发生对土壤真菌群落结构的影响[J].微生物学通报,2017,44(11):2652-2659.
[10]Kerry O'Donnell,Alejandro P.Rooney,Gary L.Mills,et al.Phylogeny and historical biogeography of true morels(Morchella)reveals an early cretaceous origin and high continental endemism and provincialism in the Holarctic[J].Fungal Genetics&Biology,2011,48(3):252-265.
[11]武冬梅,许文涛,谢宗铭,等.新疆野生羊肚菌物种多样性研究[J].食品工业科技,2015,36(2):167-172.
[12]刘伟,蔡英丽,张亚,等.我国羊肚菌人工栽培快速发展的关键技术解析[J].食药用菌,2018,26(3):142-147.
[13]Voitk A,Beug MW,Kerry O'Donnell,et al.Two new species of true morels from Newfoundland and Labrador:cosmopolitan Morchella eohespera and parochial M.laurentiana[J].Mycologia,2016,108(1):1-28.
[14]Hatra Ta?kn,Saadet Büyükalaca,Hasan Hüseyin Dogˇan,et al.Amultigene molecular phylogenetic assessment of true morels(Morchella)in Turkey[J].Fungal Genetics&Biology,2010,47(8):672-682.
[15]杜习慧,赵琪,杨祝良,等.羊肚菌的多样性、演化历史及栽培研究进展[J].菌物学报,2014,33(2):183-197.
[16]何培新,刘伟,蔡英丽,等.我国人工栽培和野生黑色羊肚菌的菌种鉴定及系统发育分析[J].郑州轻工业学院学报:自然科学版,2015,30(3-4):26-29.
[17]Kuo M,Dewsbury D R,O'Donnell K,et al.Taxonomic revision of true morels(Morchella)in Canada and the United States[J].Mycologia,2012,104(5):1159-1177.
[18]Richard F,Bellanger J M,Clowez P,et al.True morels(Morchella,Pezizales)of Europe and North America:evolutionary relationships inferred from multilocus data and a unified taxonomy[J].Mycologia,2015,107(2):359-382.
[19]易铭,梁嘉俊,史建,等.采用EF-1α序列分析法对苜蓿根腐病病原菌-锐顶镰刀菌的鉴定[J].草业学报,2017,26(02):61-68.
[20]Du X H,Zhao Q,O'Donnell K,et al.Multigene molecular phylogenetics reveals true morels(Morchella)are especially speciesrich in China[J].Fungal Genetics&Biology,2012,49(6):455-469.
[21]Yatsiuk I,Saar I,Kalamees K,et al.Epitypification of Morchella steppicola(Morchellaceae,Pezizales),a morphologically,phylogenetically and biogeographically distinct member of the Esculenta Clade from central Eurasia[J].Phytotaxa,2016,284(1):31-40.
[22]Richard F,Bellanger J M,Clowez P,et al.True morels(Morchella,Pezizales)of Europe and North America:evolutionary relationships inferred from multilocus data and a unified taxonomy[J].Mycologia,2015,107(2):359-382.
[23]Chai H,Chen L,Chen W,et al.Characterization of mating-type idiomorphs suggests that Morchella importuna,Mel-20,and M.sextelata,are heterothallic[J].Mycological Progress,2017,16(12):1-10.
[24]Loizides M,Bellanger JM,Clowez P,et al.Combined phylogenetic and morphological studies of true morels(Pezizales,Ascomycota)in Cyprus reveal significant diversity,including Morchella arbutiphila and M.disparilis spp.nov.[J].Mycological Progress,2016,15(39):DOI:10.1007/s11557-016-1180-1.