桦剥管菌SRAP分子标记反应体系优化与遗传多样性分析
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摘要
桦剥管菌Piptoporus betulinus作为一种褐腐真菌,是白桦木材的专性腐朽菌,在工业污染物降解和医药领域中起着重要作用。以桦剥管菌为材料,在SRAP-PCR体系优化的基础上,对来自东北主要林区(带岭、塔河、帽儿山以及敦化)不同种群的桦剥管菌进行遗传多样性分析,为桦剥管菌多态性鉴定以及进一步利用提供理论依据。结果表明:用单因素法及均匀设计法优化后的SRAP-PCR体系为10×Buffer 2.0μL、Primer各8μmol/L、dNTP 16 mmol/L、Taq polymerase 2.5 U、DNA 12 ng、Mg~(2+)45 mmol/L,总体积20μL;利用优化的反应体系筛选出15对引物,并扩增出239条清晰的条带,其中237条为多态性条带,多态性比率为99.16%;采用POPGENE软件分析,4个种群的平均有效等位基因平均数、平均Nei’s多样性指数、平均Shannon多样性指数分别为1.499 5、0.303 0、0.465 5;系统聚类结果显示,15个菌株分成了两个类群,这一结果表明来自同一地点菌株的遗传背景较为相似,揭示了桦剥管菌菌株间遗传特性与地理分布间的相互关系。
Piptoporus betulinus, as a kind of brown rot fungus specially decaying the birch wood, plays a vital role in degradation of industrial pollutants and medicine fields. Therefore, P. betulinus from different populations of the northeast China(Dailing, Tahe, Mao'er Mountain and Dunhua) was concerned as research material based on optimizing of SRAP reaction system. The purpose is to make the polymorphism identification among the populations and providing theoretical basis for further utilization. The results showed that the optimal reaction system was 20 μL total volume including 10×Buffer 2.0 μL, each primer 8 μmol/L, d NTP 16 mmol/L, Taq polymerase 2.5 U, DNA 10 ng, Mg~(2+) 45 mmol/L. Fifteen primer combinations were screened using optimal reaction, and produced 239 clear bands, of which 237 were polymorphic bands with 99.16% polymorphism. Using POPGENE software, the mean number of effective alleles, Nei's gene diversity and Shannon's information index were 1.499 5, 0.303 0 and 0.465 5, respectively. All the samples were divided into two groups by cluster analysis, suggesting that genetic background in the fungal strains from the same population was relatively similar and revealing the relationship between the geographical distribution and genetic characteristics of the species.
Piptoporus betulinus, as a kind of brown rot fungus specially decaying the birch wood, plays a vital role in degradation of industrial pollutants and medicine fields. Therefore, P. betulinus from different populations of the northeast China(Dailing, Tahe, Mao'er Mountain and Dunhua) was concerned as research material based on optimizing of SRAP reaction system. The purpose is to make the polymorphism identification among the populations and providing theoretical basis for further utilization. The results showed that the optimal reaction system was 20 μL total volume including 10×Buffer 2.0 μL, each primer 8 μmol/L, d NTP 16 mmol/L, Taq polymerase 2.5 U, DNA 10 ng, Mg~(2+) 45 mmol/L. Fifteen primer combinations were screened using optimal reaction, and produced 239 clear bands, of which 237 were polymorphic bands with 99.16% polymorphism. Using POPGENE software, the mean number of effective alleles, Nei's gene diversity and Shannon's information index were 1.499 5, 0.303 0 and 0.465 5, respectively. All the samples were divided into two groups by cluster analysis, suggesting that genetic background in the fungal strains from the same population was relatively similar and revealing the relationship between the geographical distribution and genetic characteristics of the species.
引文
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[27]Ferriol M,PicóB,Nuez F.Genetic diversity of a germplasm collection of Cucurbita pepo using SRAP and AFLP marker[J].Theoretical and Applied Genetics,2003,107(2):271-282.
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[29]Ma D,Yang G,Mu L,et al.Application of SRAP in the genetic diversity of Tricholoma matsutake in northeastern China[J].African Journal of Biotechnology,2010,38(9):6244-6250.
[2]Deswal D,Gupta R,Nandal P,et al.Fungal pretreatment improves amenability of lignocellulosic material for its saccharification to sugars[J].Carbohydrate Polymers,2014,99(1):264-269.
[3]Fissore A,Carrasco L,Reyes P,et al.Evaluation of a combined brown rot decay-chemical delignification process as a pretreatment for bioethanol production from Pinus radiata wood chips[J].Journal of Industrial Microbiology&Biotechnology,2010,37(9):893-900.
[4]Hastrup A,Iii F,Lebow P,et al.Enzymatic oxalic acid regulation correlated with wood degradation in four brown-rot fungi[J].International Biodeterioration&Biodegradation,2012,75(3):109-114.
[5]Li G,Quiros C.Sequence-related amplified polymorphism(SRAP),a new marker system based on a simple pcr reaction:is application to mapping and gene tagging in Brassica[J].Theoretical Applied Genetics,2001,103(2-3):455-461.
[6]Lin X,Lou Y,Zhang Y,et al.Identification of Genetic Diversity Among Cultivars of Phyllostachys violascens Using ISSR,SRAP and AFLP Markers[J].Botanical Review,2011,77(3):223-232.
[7]Lu J,Zhao H,Suo N,et al.Genetic linkage maps of Dendrobium moniliforme and D.officinale based on EST-SSR,SRAP,ISSR and RAPD markers[J].Scientia Horticulturae,2012,137(4):1-10.
[8]逯晓萍,刘丹丹,王树彦,等.高丹草遗传效应与杂种表现预测模型[J].作物学报,2014,40(3):466-475.
[9]Sun S,Gao W,Lin S,et al.Analysis of genetic diversity in Ganoderma population with a novel molecular marker SRAP[J].Applied Microbiology&Biotechnology,2006,72(3):537-543.
[10]Yu M,Ma B,Luo X,et al.Molecular diversity of Auricularia polytricha revealed by inter-simple sequence repeat and sequence-related amplified polymorphism markers[J].Current Microbiology,2008,56(3):240-245.
[11]Yin Y,Liu Y,Li H,et al.Genetic diversity of Pleurotus pulmonarius revealed by RAPD,ISSR,and SRAP fingerprinting[J].Current Microbiology,2014,68(3):397-403.
[12]訾晓雪,曹宇,闫绍鹏,等.3种白腐菌木质纤维素酶活性及酶相关基因的TRAP标记遗传多态性[J].林业科学,2015,(6):111-118.
[13]王桂娥,晁群芳,梁建芳,等.改良CTAB法提取新疆一枝蒿干叶基因组DNA[J].中国实验方剂学杂志,2015,21(12):19-22.
[14]吕世翔.三种木腐菌木材降解相关酶以及相关基因TRAP标记的变异[D].哈尔滨:东北林业大学,2010.
[15]曹宇.海绵状白腐菌木质纤维素酶活性及其基因的遗传变异研究[D].哈尔滨:东北林业大学,2013.
[16]王玉英,吕世翔,王秋玉.3种木腐菌木质纤维素相关酶基因的TRAP标记体系优化与遗传变异初探[J].林业科技,2012,37(2):17-20.
[17]盛文涛,周劲松,汤泳萍,等.芦笋SRAP反应体系优化及引物筛选[J].分子植物育种,2010,8(3):612-618.
[18]王成树,李增智.分子数据的遗传多样性分析方法[J].安徽农业大学学报,2002,29(1):90-94.
[19]王兵,王晓春.均匀设计直观分析法优化PCR条件[J].检验医学,2007,22(5):620-622.
[20]方开泰.均匀设计及其应用(Ⅲ)[J].数理统计与管理,1994(3):52-55.
[21]常双双,王承南,王森,等.5种丛枝菌根真菌对君迁子幼苗光合生长的影响[J].经济林研究,2016,34(2):79-85.
[22]余贵湘,段忠俊,卢靖,等.施肥和覆盖对澳洲坚果344和OC生长和产量的影响[J].经济林研究,2016,34(4):73-79.
[23]牛广俊,陈清英,朱思,等.用层次分析法多指标评价优选金花茶超声提取工艺[J].经济林研究,2015,33(1):119-122.
[24]胡薇,黄儒珠.均匀设计优化建兰ISSR-PCR体系[J].生命科学研究,2007,11(1):58-63.
[25]孙晋科.巴旦杏RAPD-PCR反应体系的正交优化[J].新疆农业科学,2007,44(5):715-719.
[26]郭丽琴,卫尊征,张金凤,等.均匀设计优化杨属的SRAP—PCR反应体系[J].北京林业大学学报,2010,32(2):34-38.
[27]Ferriol M,PicóB,Nuez F.Genetic diversity of a germplasm collection of Cucurbita pepo using SRAP and AFLP marker[J].Theoretical and Applied Genetics,2003,107(2):271-282.
[28]傅常娥.桦褐孔菌菌株遗传多样性的SRAP和RSAP分析[D].延边:延边大学,2014.
[29]Ma D,Yang G,Mu L,et al.Application of SRAP in the genetic diversity of Tricholoma matsutake in northeastern China[J].African Journal of Biotechnology,2010,38(9):6244-6250.