黑木耳菌株的分子鉴定与遗传多样性_英文_

黑木耳菌株的分子鉴定与遗传多样性_英文_ () 文章编号 : 1001 - 3717 200104 - 0297 - 05 Discrimination and Genetic Diversity of Ξ A u ricul a ri a a u ricul a stra ins 1 1 2 2 YAN Pei - She ng,J IAN G J i a - Hui,LUO Xin - Cha ng, ZHOU Qi ( 1 . Instit ute of Applied Mycology , L AC , L aiyang 265200 ,China ; 2 . State Key L abo rato ry )of Agro - Micro biology , Cent ral China Agricult ural U niversit y , Wuhan , 430070 , China Abstract : The st rains of A u ricul a ri a au ricul a were discriminated and t heir genetic diversity was evaluated us2 ing rDNA - R FL P and RA PD. I TS rDNA - R FL P showed t hat eleven A . au ricul a st rains possessed t he same agarose elect rop horetic pat terns rest ricted by four different rest rictio n enzymes. The Hae ? and Ms p ? re2 st rictio n gave t he species - specific pat terns , and could be used in species identificatio n. RA PD analysis showed t hat all eleven st rains could be discriminated by different rando m p rimers. The genetic diversity derived f ro m polymorp hic RA PD mar kers suggested t hat t he genetic similarity coefficient amo ng A. auricula was t he lowest , t he average was 0 . 48 , t herefore , t he pop ulatio ns of A . au ricul a st rains were highly diverse. In general , t he PCR and RA PD technologies p rovide a powerf ul tool to discriminate t he A . au ricul a and to evaluate t heir ge2 netic diversity. Key words : Identificatio n ; Diversity ; I TS ; R FL P ; RA PD ; J elly mushroo m CLC number : Q939 . 5 Document code : A 黑木耳菌株的分子鉴定与遗传多样性 1 1 2 2 闫培生,蒋家慧,罗信昌,周启 ( )1 . 莱阳农学院应用真菌研究室 ,山东 莱阳 265200 ; 2 . 华中农业大学 农业部农业微生物重点实验室 摘要 : 利用 rDNA - R FL P 和 RA PD 对黑木耳菌株进行了鉴定和遗传从样 性 评 估 。结 果 明 , I TS 的 Hae ?和 Ms p ?酶切电泳图谱可用于黑木耳种的快速鉴定 ,筛选的 RA PD 引物与标记可用于黑木耳菌 株的快速鉴定 ; 供试黑木耳菌株的 DNA 相似系数是已的食用菌中相似系数最低的种类 ,表明黑木 耳菌株拥有丰富的遗传多样性 。 关键词 : 分子鉴定 ; 遗传多样性 ; 胶质食用菌 中图分类号 : Q939 . 5 中图分类号 : A China is t he first co unt ry to cultivate A u ricul a ri a mushroo m artificially in t he wo rld. It was nearly abo ut t welve hundreds years. Because of t he wide and lo ng time cultivatio n , t he genetic diversi2 ( ) t y of A u ricul a ri a a u ricul a might be decreased Yang , 1996. In o rder to utilize t he reso urces of A u2 Ξ 收稿日期 : 2001 - 07 - 12 ( ) 作者简介 : 闫培生 1964 - ,男 ,山东栖霞人 ,博士 ,教授 ,主要从事微生物学 、应用真菌学和资源微生物及其学的教学和 科研工作 。 ricul a ri a a u ricul a effectively in breeding and p ro ductio n , we firstly used t he PCR technology to dis2 criminate and evaluate t he ger m reso urces diversit y f ro m rDNA and total DNA levels. 1 Mat e ri al a nd Met ho d 1 . 1 Stra ins Eleven A . a u ricul a st rains f ro m different geo grap hical locatio ns were used as follow s : A01 f ro m Changsha of Hunan Province ; A02 f ro m Bao kang of Hubei Province ; A03 , A04 and A10 f ro m Wuhan of Hubei Province ; A07 f ro m Fangxian of Hubei Province ; A08 f ro m Nanzhang of Hubei Province ; A05 f ro m U rumuqi of Xinjiang Province ; A06 f ro m Changchun of J ilin Province , A09 f ro m Bao ding of (Hebei Province , and A11 f ro m Taiyuan of Xianxi Province . St rains of A . cor nea f ro m Fuzho u of ) () Fujian Provinceand A . pol y t richa f ro m Changsha of Hunan Provincewere used as o ut gro up . All t he cult ures are depo sited in and available f ro m t he Instit ute of Applied Mycology , L aiyang Agricult ur2 al U niversit y. 1 . 2 Extract ion of D NA and PCR React ion It was difficult to isolate DNA f ro m A . a u ricul a st rains because t heir mycelia co ntained a high quantit y of polysaccharides t hat were not easy to eliminate . Af ter co mparing several met ho ds , we mo dified t he C TAB met ho d slightly and got t he fine DNA f ro m liquid cult ures of mycelia fo r PCR ( ) Yan et al . , 1999. ( ) Inter nal Transcribed Spacer I TSof ribo so mal DNA was cho sen fo r PCR amplificatio n . Primers ( ) used were I TS1/ I TS4 . The PCR reactio n was perfo r med o n t he t her mal cycler SABC , Chinaby t he following parameters : initial denat uratio n at 92 ?fo r 5 min , t hen 40 cycles of 92 ?fo r 1 min , 55 ? fo r 1 min , 72 ?fo r 2 min , and final extensio n at 72 ?fo r 10 min . The amplified I TS f ragment s were subjected to rest rictio n by selected fo ur o r five - base rest rictio n enzymes. Ten - base rando m p rimers ( ) were used fo r geno mic DNA amplificatio n by RA PD Sango ng p ro duct s , Shanghai. The RA PD reac2 tio n was t he same as in PCR reactio n excep t t hat t he annealing was at 35 ?fo r 1 min . μThe amplified DNA was elect rop ho resed in 2 . 0 % agaro se gel at 5v/ cm , stained wit h 0 . 5g/ L EB , and p hotograp hed under U V light . 1 . 3 Data treatment Sizes of t he RA PD f ragment s were calculated acco rding to t he sof t ware package GEL . Fo r each st rain , a data reco rd was co nst ructed in w hich each of a particular molecular weight , as generated by () () each p rimer , was rep resented as eit her being p resent 1o r absent 0. A binary mat rix co mbined all t he data reco rds fo r all eleven st rains used in t his st udy f ro m all ten p rimers. The mat rix was t hen used ( ) as inp ut fo r t he S IMQ UAL mo dule of t he N TS YS - pc sof t ware package Ro hlf , 1990wit h t he simi2 larit y coefficient set to Dice . Clustering was perfo r med wit h t he SA HN feat ure of t he p rogram by t he ( ) unweighted pair - gro up met ho d wit h arit hmetic average U P GMA. 2 Re sult s a nd Di scus sio n 2 . 1 Ribosomal D NA a mpl if icat ion and RFL P Only o ne unique f ragment was amplified f ro m I TS - rDNA amo ng eleven st rains. This suggested ( ) t hat no co ntaminatio n occurred. The amplified f ragment size was 650 bp Fig. 1. The f ragment was subjected to rest rict by fo ur rest rictio n enzyme of Hi nf ?, T aq ?, Hae ? and Ms p ?. I TS - R FL P showed t hat t he elect rop ho retic pat ter ns fo r eleven st rains rest ricted by t he same enzyme were exactly t he same . This suggested t hat A . a u ricul a was mo re co nserved amo ng t he regio ns of I TS. The elect rop ho retic pat ter ns fo r Hae ?and M s p ? rest rictio n were stable and specific fo r A . a u ric2 ( ) A . a r2 Therefo re , t hey co uld be used in species identificatio n fo r ul a species Fig. 2 and Yan , 1998. icul a quickly and accurately. Fig. 1 Ampl if ied ITS - rD NA f ragments f rom Fig. 2 ITS - RFL P electrophoretic pattern restricted by Hae ? three A u ricul a ri a species λ) (1 . molecular size markers DNA digested by Hi n d ? and EcoR ?;1. A . cornea ; 2. A . pol yt richa ; 3 . cont rol ; 4 . A . au ricul a A05 ; λ()5 . ismolecular size markers DNA digested wit h Hi n d ? 2 . A . cornea ; 3 . A . pol yt richa ; 4 - 5. A . au ricul a A05 and A08 2 . 2 RAPD a mpl if icat ion Of t went y - six rando m p rimers tested , ten gave t he clear and stable elet rop ho retic pat ter ns amo ng eleven ( ) A . a u ricul a st rains Fig. 3 . The pat ter ns showed not o nly t he species stabilit y but also t he st rain differ2 ence as fo r different p rimers. Fo r example , p rimer S4 co uld amplified 1,4 DNA bands ranged f ro m 0 . 75 , 2 . 54 kb amo ng eleven st rains , and all t hese st rains po ssessed t he 2 . 54 kb band wit h very high bright , L ane 1 2 3 4 5 6 7 8 9 10 11 12 w hich co uld o nly be amplified f ro m a u ricul a Fig. 3 RAPD patterns of eleven A . A . a u ricul a st rains , but not f ro m stra ins a mpl if ied by primer S5 L ane 1 to lane 12 rep resent s A01 , A02 , A03 , A04 , A05 , A06 ,M ,A07 to A11 , ( ot her Auricularia species Yan , (λ)L ane M is a molecular size mar ker DNA digested by Hi n d ?and EcoR ? ) 1998. However , amo ng amplified DNA bands , o nly 12 % were shared by eleven A . a u ricul a st rains. Primer S15 gave t he mo st poly2 mo rp hic elect rop ho retic pat ter ns w hich amplified 3 - 12 DNA bands ranged f ro m 0 . 42 - 3 . 04 kb a2 mo ng eleven st rains ; each st rain had it s ow n pat ter n . Therefo re , different st rains co uld be differentiat2 ed by different p rimers. 2 . 3 Genet ic similarity and cl uster analysis Of 133 DNA bands amplified by 10 rando m p rimers amo ng eleven A . a u ricul a st rains , 117 bands were polymo rp hic. The genetic similarit y analysis showed t hat t he genetic similarit y coefficient (ranged f ro m 0 . 22 to 0 . 72 bet ween t wo st rains. The lowest was 0 . 22 bet ween A04 f ro m Wuhan of ) () Hubei p rovinceand A11 f ro m Taiyuan of Xianxi p rovince, and t he highest was 0 . 72 bet ween A02 ) () ( ) (f ro m Bao kang of Hubei p rovinceand A05 f ro m U rumuqi of Xinjiang p rovinceTable 1. The av2 ( erage was 0 . 48 w hich was t he lowest co mpared wit h ot her edible mushroo ms Zhan et al . , 1997 ; ) Zhang et al . , 1996 ; Zhan et al . , 1998 ;. This suggested t hat t he genetic diversit y of A . a u ricul a in China was very plentif ul . Ta ble 1 Matrix of similarity coeff ic ient of a mpl if ied D NA f ragments by RAPD within A . a u r icul a St rains A01 A02 A03 A04 A05 A06 A07 A08 A09 A10 A11 A01 1 . 00 A02 0 . 44 1 . 00 A03 0 . 48 0 . 51 1 . 00 A04 0 . 41 0 . 38 0 . 53 1 . 00 A05 0 . 48 0 . 72 0 . 59 0 . 39 1 . 00 A06 0 . 45 0 . 41 0 . 56 0 . 44 0 . 45 1 . 00 A07 0 . 58 0 . 51 0 . 70 0 . 39 0 . 54 0 . 56 1 . 00 A08 0 . 68 0 . 46 0 . 52 0 . 38 0 . 50 0 . 52 0 . 67 1 . 00 A09 0 . 41 0 . 42 0 . 51 0 . 65 0 . 39 0 . 47 0 . 53 0 . 46 1 . 00 A10 0 . 51 0 . 43 0 . 46 0 . 46 0 . 46 0 . 50 0 . 59 0 . 53 0 . 44 1 . 00 A11 0 . 45 0 . 40 0 . 45 0 . 22 0 . 41 0 . 45 0 . 50 0 . 45 0 . 36 0 . 48 1 . 00 Fig. 4 Dendrogra m of the eleven stra ins of A . a u ricul a derived f rom RAPD by UPGMA Cluster analysis showed t hat at abo ut 0 . 50 similarity coefficient level , all eleven st rains co uld be ( ) gro uped into fo ur classes Fig. 4. The first class included st rains A01 , A03 , A06 , A07 , A08 and A10 ; t he seco nd included st rains A02 , and A05 ; t he t hird included o nly st rain A11 ; t he fo urt h includ2 ed st rains A04 and A09 . The cluster result s showed t hat st rains f ro m different geograp hic o rigins co uld be gro uped into t he same class , w hereas st rains f ro m t he same geograp hic o rigins co uld be gro uped into different classes. This suggested t hat t he genetic diversit y of A . a u ricul a st rains be not related to t heir geograp hic o rigins to so me extent . There might be t wo reaso ns. One was related to t he breeding p rac2 tice and alien st rain int ro ductio n . The ot her was t hat t he criteria of geo grap hic o rigins and mo rp hologi2 cal characteristic used in st rain genetic diversit y evaluatio n were not accurate because t hey might not fit ( ) t he genot ype Zhang et al . ,1997. Therefo re , we co uld utilize t he genetic reso urces ratio nally and ac2 curately o nly w hen t he genetic diversit y was elucidated at DNA molecular levels. Ref erences : 1 YAN Pei - Shen g. Molecular identificatio n and p hylogeny of t he genus Auricularia M . PhD. Thesis , Huazho n g Agricult ural U niversit y , Wuhan , China ,1998 . 2 YAN Pei - Shen g , L UO Xin - Chang and ZHOU Qi . RFL P analysis of amplified nuclear ribo so mal DNA in t he genus Auricularia ( ) J . M yco systema , 1999 , 18 2: 206 - 213 . 3 YAN G Xin - Mei . The cultivatio n of edible f un gi in China M . Bei jing : China Agricult ural Press. 1996 ,167 - 180 . 4 ZHAN Cai - Xin , Zhu L anbao and Yan g Xinmei . Evaluatio n of t he ger m reso urces of Flamulina velutipesJ . Acta Edulis Fun gi , ( ) 1997 ,4 1: 1 - 7 . 5 ZHAN Cai - Xin , Zhan g Yinfang. DNA polymo rp hism of cultivated Agaricus bispo rus st rains in China J . Acta Edulis Fun gi ,1998 , ( ) 5 1: 1 - 7 . 6 ZHAN G Shu - Tin g , Lin Fangcan . Mushroo m genetics and BreedingM . Bei jing : China Agricult ural Press , 1997 ,214 . 7 ZHAN G Yin - Fan g , Pan Yingjie . Genetic relatio nship s of cultivated L entinula edo des st rains in China J . J . of A g ricul t u ral ( ) B iotech nology , 1996 , 4 2: 141 - 146 .