斑痣盘菌科的物种多样性和齿裂菌属等的RAPD分子标记
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摘要
本文以世界较先进的分类系统为原则,从形态解剖、个体发育、生态习性、地理分布等表型性状方面对斑痣盘菌科中一些种进行较系统的分类研究。共查明隶属于齿裂菌属(Coccomyces)、皮下盘菌属(Hypoderma)、散斑壳属(Lophodermium)、湿皮盘菌属(Hypohelion)4属的8个种,其中包括4个新种,即栗齿裂菌(Coccomycescastaneae)、杉生皮下盘菌(Hypoderma cunninghamiicota)、清风藤散斑壳(Lophodermium sabiae)、络石散斑壳(L.trachelosperi);并且对4个国内已知种进行了补充研究,追加了这些种的新寄主和地理新分布。
以组织分离法对采自天堂寨国家森林公园的斑痣盘菌活材料进行分离培养,共获得隶属于齿裂菌属和皮下盘菌属的17号分离株,接菌于MA培养基上,定期观察、记载各菌株的菌落形状、大小、色泽、结构、气生菌丝和潜生菌丝状况、生长速度、分泌色素与否以及是否产生分生孢子等特征。对悬钩子皮下盘菌(Hypoderma rubi)各菌株,以培养性状为依据,对其进行种下阶元分类,共划分出8个生物(培养)型。
分别对21个皮下盘菌属(Hypoderma)分离株和12个齿裂菌属(Coccomyces)菌株进行了DNA提取,RAPD-PCR扩增体系的优化,并对提取的DNA进行RAPD-PCR扩增反应。因RAPD技术的稳定性和重复性相对较差,所以有必要寻找一个适合本试验的RAPD-PCR反应体系,以提高其稳定性与重复性。笔者从引物用量、模板用量、Mg~(2+)用量、dNTPs用量、Taq酶用量、退火温度及时间、延伸时间、循环次数等方面对齿裂菌属和皮下盘菌属两属的RAPD反应体系进行了优化,得到结果是:25μl反应体系中,ddH_2O 16.3μl,10×Buffer 2.5μl,Mg~(2+)(25mmol/L)2μl,dNTPs(10mmol/L)2μl,引物(12.5μmol/L)1μl,Taq酶(5U/μl)0.2μl,模板(100ng/μl)1μl。扩增程序为:95℃预变性5min,50次循环:94℃变性60s,36℃退火100s,72℃延伸140s;最后一次循环后在72℃延伸10 min。
筛选出14个随机引物,使用优化的RAPD-PCR反应参数及程序对齿裂菌属12个菌株的DNA进行扩增,共扩增出140个位点,其中多态性位点数为133个,占总数的95%。扩增位点最多的是引物S11,有20个位点数;S404引物次之;扩增位点数最少的则是引物S7和S220,只有3个位点数。扩增出的片断长度大多在250-2000bp之间。采用UPGMA方法构建聚类树状图,通过表型性状分类和RAPD分析结果比较,二者能较好地统一起来。
按优化的RAPD-PCR反应体系对皮下盘菌21个菌株的DNA进行扩增,14个随机引物共扩增出132个位点,其中多态性位点数为120个,占总数的91%。扩增位点最多的是引物S130,有17个位点数;引物$298和$33为次;扩增位点数最少的是引物S7,仅有4个位点数。扩增片断长度也是位于250-2000bp之间。使用UPGMA法构建聚类树状图,在遗传距离4.10处,21个菌株被分成7类,该结果与菌株形态解剖学特征、寄主范围、采集地基本一致。
Some important members of 8 species of 4 genera,which were Coccomyces, Hypoderma,Lophodermium and Hypohelion of Rytismataceae were systematically studied from morphology,ontogeny,ecology and distribution and so on according to the more advanced taxonomic system.Among them,Coccomyces castaneae sp.nov.,Hypoderma cunninghamiicola sp.nov.,Lophodermium sabiae sp.nov.and Lophodermiun trachelospesperi sp.nov.were new species,and 4 known species of China were supplied and studied in new hosts and new geography distribution.
The living materials of fungi of Rytismataceae from Tiantangzhai National Forest Park were separated and cultivated by tissue isolation techniques.Sixteen strains of Coccomyces spp.and Hypoderma rubi were obtained.The strains were cultured on MA culture medium, and then regularly observed and noted the shape,size,luster,structure,mycelium,the rate of growth,and if which produce pigment and conidiomata.The strains of Hypoderma rubi were classified to 8 biology(culture)types based on the characteristics of culture.
The DNA of 12 Coccomyces strains and 21Hypoderma ones were respectively extracted.The RAPD-PCR reaction system was optimized,and the DNA from intraspecies and interspecies were amplified.It is necessary to find a RAPD reaction system suitable for this experiment in order to promote the stability and repetition because of their more lacking stability and repetition.The reaction system of Coccomyces and Hypoderma were optimized in the quantity of primers,template,Mg~(2+),dNTPs,Taq DNA polymerase and annealing temperature,annealing time,elongation and cycle numbers.The optimal RAPD-PCR reaction system included 16.3μl ddH_2O,2.5μl 10×Buffer,2μl Mg~(2+) (25mmol/L),2μl dNTPs(10mmol/L),1μl(100ng/μl)template,0.2μl Taq DNA polymerase (5U/μl),1μl random primers(12.5μmol/L)in the system of 25μl.The amplification process as follow:95℃beforehand denaturalization for 5 minutes,50 cycles;94℃denaturalization for one minute,36℃annealing for 100 seconds,72℃elongation for 140 seconds,after the last circulation,then 72℃elongation for 10 minutes.
The DAN of 12 strains of Coccomyces were amplified with 14 collected random primers by the optimal process,140 fragments were obtained,133 polymorphic ones among,accounting for 95%.The most sites,20 ones,were amplified by Primer S11,the second ones were Primer S404,the least,three ones,were amplified by Primer S7 and Primer S220.The length of most amplified fragments ranged from 250bp to 2000bp.The phenotype was well consistent with the results of RAPD based on the clustering tree map which were established by the method of UPGMA.
The DAN of 21 strains of Hypoderma were amplified with 14 collected random primers using the reaction process,132 fragments were obtained,120 polymorphic ones among them,accounting for 91%.The most sites,17 ones,were amplified by S130,the second ones were 16.Four sites were the least,amplified by Primers S7.The length of most fragments ranged from 250bp to 2000bp,too.From the clustering tree map which were established by the method of UPGMA,21 strains were classified into 7 sorts at the genetic distance of 4.10,the results was basically consistent with the phenotype,host and location.
以组织分离法对采自天堂寨国家森林公园的斑痣盘菌活材料进行分离培养,共获得隶属于齿裂菌属和皮下盘菌属的17号分离株,接菌于MA培养基上,定期观察、记载各菌株的菌落形状、大小、色泽、结构、气生菌丝和潜生菌丝状况、生长速度、分泌色素与否以及是否产生分生孢子等特征。对悬钩子皮下盘菌(Hypoderma rubi)各菌株,以培养性状为依据,对其进行种下阶元分类,共划分出8个生物(培养)型。
分别对21个皮下盘菌属(Hypoderma)分离株和12个齿裂菌属(Coccomyces)菌株进行了DNA提取,RAPD-PCR扩增体系的优化,并对提取的DNA进行RAPD-PCR扩增反应。因RAPD技术的稳定性和重复性相对较差,所以有必要寻找一个适合本试验的RAPD-PCR反应体系,以提高其稳定性与重复性。笔者从引物用量、模板用量、Mg~(2+)用量、dNTPs用量、Taq酶用量、退火温度及时间、延伸时间、循环次数等方面对齿裂菌属和皮下盘菌属两属的RAPD反应体系进行了优化,得到结果是:25μl反应体系中,ddH_2O 16.3μl,10×Buffer 2.5μl,Mg~(2+)(25mmol/L)2μl,dNTPs(10mmol/L)2μl,引物(12.5μmol/L)1μl,Taq酶(5U/μl)0.2μl,模板(100ng/μl)1μl。扩增程序为:95℃预变性5min,50次循环:94℃变性60s,36℃退火100s,72℃延伸140s;最后一次循环后在72℃延伸10 min。
筛选出14个随机引物,使用优化的RAPD-PCR反应参数及程序对齿裂菌属12个菌株的DNA进行扩增,共扩增出140个位点,其中多态性位点数为133个,占总数的95%。扩增位点最多的是引物S11,有20个位点数;S404引物次之;扩增位点数最少的则是引物S7和S220,只有3个位点数。扩增出的片断长度大多在250-2000bp之间。采用UPGMA方法构建聚类树状图,通过表型性状分类和RAPD分析结果比较,二者能较好地统一起来。
按优化的RAPD-PCR反应体系对皮下盘菌21个菌株的DNA进行扩增,14个随机引物共扩增出132个位点,其中多态性位点数为120个,占总数的91%。扩增位点最多的是引物S130,有17个位点数;引物$298和$33为次;扩增位点数最少的是引物S7,仅有4个位点数。扩增片断长度也是位于250-2000bp之间。使用UPGMA法构建聚类树状图,在遗传距离4.10处,21个菌株被分成7类,该结果与菌株形态解剖学特征、寄主范围、采集地基本一致。
Some important members of 8 species of 4 genera,which were Coccomyces, Hypoderma,Lophodermium and Hypohelion of Rytismataceae were systematically studied from morphology,ontogeny,ecology and distribution and so on according to the more advanced taxonomic system.Among them,Coccomyces castaneae sp.nov.,Hypoderma cunninghamiicola sp.nov.,Lophodermium sabiae sp.nov.and Lophodermiun trachelospesperi sp.nov.were new species,and 4 known species of China were supplied and studied in new hosts and new geography distribution.
The living materials of fungi of Rytismataceae from Tiantangzhai National Forest Park were separated and cultivated by tissue isolation techniques.Sixteen strains of Coccomyces spp.and Hypoderma rubi were obtained.The strains were cultured on MA culture medium, and then regularly observed and noted the shape,size,luster,structure,mycelium,the rate of growth,and if which produce pigment and conidiomata.The strains of Hypoderma rubi were classified to 8 biology(culture)types based on the characteristics of culture.
The DNA of 12 Coccomyces strains and 21Hypoderma ones were respectively extracted.The RAPD-PCR reaction system was optimized,and the DNA from intraspecies and interspecies were amplified.It is necessary to find a RAPD reaction system suitable for this experiment in order to promote the stability and repetition because of their more lacking stability and repetition.The reaction system of Coccomyces and Hypoderma were optimized in the quantity of primers,template,Mg~(2+),dNTPs,Taq DNA polymerase and annealing temperature,annealing time,elongation and cycle numbers.The optimal RAPD-PCR reaction system included 16.3μl ddH_2O,2.5μl 10×Buffer,2μl Mg~(2+) (25mmol/L),2μl dNTPs(10mmol/L),1μl(100ng/μl)template,0.2μl Taq DNA polymerase (5U/μl),1μl random primers(12.5μmol/L)in the system of 25μl.The amplification process as follow:95℃beforehand denaturalization for 5 minutes,50 cycles;94℃denaturalization for one minute,36℃annealing for 100 seconds,72℃elongation for 140 seconds,after the last circulation,then 72℃elongation for 10 minutes.
The DAN of 12 strains of Coccomyces were amplified with 14 collected random primers by the optimal process,140 fragments were obtained,133 polymorphic ones among,accounting for 95%.The most sites,20 ones,were amplified by Primer S11,the second ones were Primer S404,the least,three ones,were amplified by Primer S7 and Primer S220.The length of most amplified fragments ranged from 250bp to 2000bp.The phenotype was well consistent with the results of RAPD based on the clustering tree map which were established by the method of UPGMA.
The DAN of 21 strains of Hypoderma were amplified with 14 collected random primers using the reaction process,132 fragments were obtained,120 polymorphic ones among them,accounting for 91%.The most sites,17 ones,were amplified by S130,the second ones were 16.Four sites were the least,amplified by Primers S7.The length of most fragments ranged from 250bp to 2000bp,too.From the clustering tree map which were established by the method of UPGMA,21 strains were classified into 7 sorts at the genetic distance of 4.10,the results was basically consistent with the phenotype,host and location.
引文
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