基于RAD测序技术的黄绿卷毛菇(Floccularia luteovirens)微卫星引物的开发
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摘要
黄绿卷毛菇(Floccularia luteovirens)是广泛分布于青藏高原高寒草甸的一种重要的可食用的菌根真菌。先前的研究利用EST-SSR引物已经对该物种的遗传多样性及群落结构进行了研究,但研究表明EST-SSR位点多态性较低,因此想要进一步探讨形成现有遗传分布格局的机制(该物种小尺度空间范围内基株的密度及大小)需要重新开发具有更高多态性的基于核基因的微卫星位点。本研究利用RAD测序技术使用直接测序分析的方法重新开发了12对具有多态性的引物。测序结果去接头拼装后共获得46 036条重叠群。在这些序列当中共扫描到342条含有重复单元的序列,在这当中70.47%为三碱基重复(241),8.19%为二碱基重复(28)。随机选取48对检测,12对具有多态性且测序结果良好,基于3个居群63个个体的遗传多样性研究显示,所有样品中共获得60个单倍型,每个位点的单倍型数量介于2 (GSSR26L)至9 (GSSR7L,GSSR11L)之间。Gst值介于-0.03(GSSR46L)至0.28(GSSR6L)之间、Fst值介于-0.03(GSSR33L)至0.42(GSSR6L)之间。在引物GSSR47L扩增测序中发现,其扩增出的片段不仅具有重复单元数量的变化,重复单元本身也存在突变的现象,这是之前使用聚丙烯凝胶电泳所检测不到的。本研究获得的微卫星引物将为接下来小尺度空间下研究黄绿卷毛菇基株的密度、大小及动态变化提供有力的支持。
Floccularia luteovirens(Physalacriaceae) is an important edible mycorrhizal fungus mushroom, which is widely distributed on alpine meadows of the Qinghai-Tibet Plateau. Expressed sequence tag-simple sequence repeat(EST-SSR) markers have been used to investigate its genetic diversity and community structure in the previous studies,but the results showed that the polymorphism of EST-SSR locus is low. Therefore, in order to further explore the mechanism that led to its current genotype distribution pattern(the density and size of the genets in the small scale space of the species), we need to redevelop the nuclear gene based microsatellite sites with higher polymorphism. In this study,12 pairs of primers with polymorphism were re-developed by direct sequencing analysis using RAD sequencing technology.After Illumina pair-end sequencing and assembled to yield A total of 46 036 contigs were obtained after reassembling the sequences. In these sequences, a total of 342 SSRs were detected, of which 70.47% were tri-nucleotide repeats(241)and 8.19% were bi-nucleotide repeats(28). Of the 48 pairs randomly selected,, twelve were polymorphic and sequenced well. Genetic diversity studies based on 63 individuals from 3 populations showed that 60 haplotypes were obtained in all samples, with number of haplotypes ranging from 2(GSSR26 L) to 9(GSSR7 L, GSSR11 L) per locus. Gstvalue ranged from-0.03(GSSR46 L) to 0.28(GSSR6 L) and Fstvalue ranged from-0.03(GSSR33 L) to 0.42(GSSR6 L). In the amplified sequences of primer GSSR47 L, it was found that the amplified fragment not only had the change of the number of repeat units, but also the repeat units themselves had mutations, which could not be detected by polyacrylamidegel electrophoresis before. The microsatellite markers obtained in this study would provide a powerful support for the study of the density, size and dynamic changes of the genets of Floccularia luteovirens in small scale space.
Floccularia luteovirens(Physalacriaceae) is an important edible mycorrhizal fungus mushroom, which is widely distributed on alpine meadows of the Qinghai-Tibet Plateau. Expressed sequence tag-simple sequence repeat(EST-SSR) markers have been used to investigate its genetic diversity and community structure in the previous studies,but the results showed that the polymorphism of EST-SSR locus is low. Therefore, in order to further explore the mechanism that led to its current genotype distribution pattern(the density and size of the genets in the small scale space of the species), we need to redevelop the nuclear gene based microsatellite sites with higher polymorphism. In this study,12 pairs of primers with polymorphism were re-developed by direct sequencing analysis using RAD sequencing technology.After Illumina pair-end sequencing and assembled to yield A total of 46 036 contigs were obtained after reassembling the sequences. In these sequences, a total of 342 SSRs were detected, of which 70.47% were tri-nucleotide repeats(241)and 8.19% were bi-nucleotide repeats(28). Of the 48 pairs randomly selected,, twelve were polymorphic and sequenced well. Genetic diversity studies based on 63 individuals from 3 populations showed that 60 haplotypes were obtained in all samples, with number of haplotypes ranging from 2(GSSR26 L) to 9(GSSR7 L, GSSR11 L) per locus. Gstvalue ranged from-0.03(GSSR46 L) to 0.28(GSSR6 L) and Fstvalue ranged from-0.03(GSSR33 L) to 0.42(GSSR6 L). In the amplified sequences of primer GSSR47 L, it was found that the amplified fragment not only had the change of the number of repeat units, but also the repeat units themselves had mutations, which could not be detected by polyacrylamidegel electrophoresis before. The microsatellite markers obtained in this study would provide a powerful support for the study of the density, size and dynamic changes of the genets of Floccularia luteovirens in small scale space.
引文
Bu rc hhardt K.M.,Rivera Y.,Baldwin T.,Vanearden D.,and Kretzer A.M.,2011,Analysis of genet size and local gene flow in the ectomycorrhizal basidiomycete Suillus spraguei(synonym S.pictus),Mycologia,103(4):722-730
Cao Y.,Zhang Y.,Yu Z.,Liu C.,Tang X.,Long Y.,He X.,Wang P.,and Xu J.,2013,Structure,gene flow,and recombination among geographic populations of a russula virescens ally from southwestern China,PLo S One,8(9):e73174
Cromack K.,Sollins P.,Graustein W.C.,Speidel K.,Todd A.W.,Spycher G.,Li C.Y.,and Todd R.L.,1979,Calcium oxalate accumulation and soil weathering in mats of the hypogeous fungus Hysterangium crassum,Soil Biolgy And Biochemstry,11(5):463-468
Doyle J.J.,1987,A rapid DNA isolation procedure from small quantities of fresh leaf tissues,Phytochemistry,19(1):11-15
Eujayl I.,Sorrells M.E.,Baum M.,Wolters P.,and Powell W.,2002,Isolation of EST-derived microsatellite markers for genotyping the A and B genomes of wheat,Theor.Appl.Genet.,104(2-3):399-407
Gryta H.,Carriconde F.,Charcosset J.Y.,Jargeat P.,and Gardes M.,2006,Population dynamics of the ectomycorrhizal fungal species Tricholoma populinum and Tricholoma scalpturatum associated with black poplar under differing environmental conditions,Environmental Microbiology,8(5):773-786
Guidot A.,Debaud J.C.,and Marmeisse R.,2001,Correspondence between genet diversity and spatial distribution of above-and below-ground populations of the ectomycorrhizal fungus Hebeloma cylindrosporum,Molecular Ecology,10(5):1121-1131
Gutierrez M.V.,Patto M.C.V.,Huguet T.,Cubero J.I.,Moreno M.T.,and Torres A.M.,2005,Cross-species amplification of Medicago truncatula microsatellites across three major pulse crops,Theor.Appl.Genet.110(7):1210-1217
Hortal S.,Trocha L.K.,Murat C.,Chybicki I.J.,Buee M.,Trojankiewicz M.,Burczyk J.,and Martin F.,2012,Beech roots are simultaneously colonized by multiple genets of the ectomycorrhizal fungus Laccaria amethystina clustered in two genetic groups,Mol.Ecol.,21(9):2116-2129
Horton T.R.,and Bruns T.D.,1998,Multiple-host fungi are the most frequent and abundant ectomycorrhizal types in a mixed stand of Douglas fir(Pseudotsuga menziesii)and bishop pine(Pinus muricata),The New Phytologist,139(2):331-339
Iotti M.,Lancellotti E.,Hall I.,and Zambonelli A.,2010,The ectomycorrhizal community in natural Tuber borchii grounds,Fems Microbiology Ecology,72(2):250-260
Lian C.,Narimatsu M.,Nara K.,and Hogetsu T.,2006,Tricholoma matsutake in a natural Pinus densiflora forest:correspondence between above-and below-ground genets,association with multiple host trees and alteration of existing ectomycorrhizal communities,New Phytologist,171(4):825-836
Molinier V.,Murat C.,Frochot H.,Wipf D.,and Splivallo R.,2015,Fine-scale spatial genetic structure analysis of the black truffle Tuber aestivum and its link to aroma variability,Environmental Microbiology,17(8):3039-3050
O'Hanlon R.,2012,Below-ground ectomycorrhizal communities:the effect of small scale spatial and short term temporal variation,Symbiosis,57(2):57-71
Roy M.,Dubois M.P.,Proffit M.,Vincenot L.,Desmarais E.,and Selosse M.A.,2008,Evidence from population genetics that the ectomycorrhizal basidiomycete Laccaria amethystina is an actual multihost symbiont,Mol.Ecol.,17(12):2825-2838
Rozen S.,and Skaletsky H.,2000,Primer3 on the WWW for general users and for biologist programmers,Methods Mol.Biol.,132:365-386
Taylor A.F.,and Alexander I.,2005,The ectomycorrhizal symbiosis:life in the real world,Mycologist,19(3):102-112
Thiel T.,Michalek W.,Varshney R.K.,and Graner A.,2003,Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley(Hordeum vulgare L.),Tag.Theoretical and Applied Genetics.Theoretische Und Angewandte Genetik,106(3):411-422
Wadud M.A.,Nara K.,Lian C.,Ishida T.A.,and Hogetsu T.,2014,Genet dynamics and ecological functions of the pioneer ectomycorrhizal fungi Laccaria amethystina and Laccaria laccata in a volcanic desert on Mount Fuji,Mycorrhiza,24(7):551-563
Xie Z.L.,Zhao L.Z.,Li Y.,Lei J.Q.,and Zhang F.M.,2016,The correlation of geographic distribution and ecological environment of endemic species Floccularia luteovirens on Qinghai-Tibet Plateau,Shengtai Xuebao(Acta Ecologica Sinica),36(10):2851-2857(谢占玲,赵联正,李椰,雷洁琼,张凤梅,2016,青藏高原特有种黄绿卷毛菇的地理分布与生态环境的相关性,生态学报,36(10):2851-2857)
Xing R.,Gao Q.B.,Zhang F.Q.,Fu P.C.,Wang J.L.,Yan H.Y.,and Chen S.L.,2017,Genetic variation and phylogenetic relationships of the ectomycorrhizal Floccularia luteovirens on the Qinghai-Tibet Plateau,Journal of Microbiology,55(8):600-606
Xing R.,Gao Q.B.,Zhang F.Q.,Li Y.H.,Fu P.C.,Zhang J.H.,Wang J.L.,and Chen S.L.,2014,Isolation and characterization of 27 polymorphic microsatellite markers in Armillaria luteo-virens(Physalacriaceae),Weishengwu Xuebao(Acta Microbiologica Sinica),54(9):1045-1052(邢睿,高庆波,张发起,李印虎,付鹏程,张金华,王久利,陈世龙,2014,基于454测序技术的青藏高原黄绿蜜环菌微卫星引物的开发,微生物学报,54(9):1045-1052)
Zerbino D.R.,and Birney E.,2008,Velvet:algorithms for de novo short read assembly using de Bruijn graphs,Genome Research,18(5):821-829
Cao Y.,Zhang Y.,Yu Z.,Liu C.,Tang X.,Long Y.,He X.,Wang P.,and Xu J.,2013,Structure,gene flow,and recombination among geographic populations of a russula virescens ally from southwestern China,PLo S One,8(9):e73174
Cromack K.,Sollins P.,Graustein W.C.,Speidel K.,Todd A.W.,Spycher G.,Li C.Y.,and Todd R.L.,1979,Calcium oxalate accumulation and soil weathering in mats of the hypogeous fungus Hysterangium crassum,Soil Biolgy And Biochemstry,11(5):463-468
Doyle J.J.,1987,A rapid DNA isolation procedure from small quantities of fresh leaf tissues,Phytochemistry,19(1):11-15
Eujayl I.,Sorrells M.E.,Baum M.,Wolters P.,and Powell W.,2002,Isolation of EST-derived microsatellite markers for genotyping the A and B genomes of wheat,Theor.Appl.Genet.,104(2-3):399-407
Gryta H.,Carriconde F.,Charcosset J.Y.,Jargeat P.,and Gardes M.,2006,Population dynamics of the ectomycorrhizal fungal species Tricholoma populinum and Tricholoma scalpturatum associated with black poplar under differing environmental conditions,Environmental Microbiology,8(5):773-786
Guidot A.,Debaud J.C.,and Marmeisse R.,2001,Correspondence between genet diversity and spatial distribution of above-and below-ground populations of the ectomycorrhizal fungus Hebeloma cylindrosporum,Molecular Ecology,10(5):1121-1131
Gutierrez M.V.,Patto M.C.V.,Huguet T.,Cubero J.I.,Moreno M.T.,and Torres A.M.,2005,Cross-species amplification of Medicago truncatula microsatellites across three major pulse crops,Theor.Appl.Genet.110(7):1210-1217
Hortal S.,Trocha L.K.,Murat C.,Chybicki I.J.,Buee M.,Trojankiewicz M.,Burczyk J.,and Martin F.,2012,Beech roots are simultaneously colonized by multiple genets of the ectomycorrhizal fungus Laccaria amethystina clustered in two genetic groups,Mol.Ecol.,21(9):2116-2129
Horton T.R.,and Bruns T.D.,1998,Multiple-host fungi are the most frequent and abundant ectomycorrhizal types in a mixed stand of Douglas fir(Pseudotsuga menziesii)and bishop pine(Pinus muricata),The New Phytologist,139(2):331-339
Iotti M.,Lancellotti E.,Hall I.,and Zambonelli A.,2010,The ectomycorrhizal community in natural Tuber borchii grounds,Fems Microbiology Ecology,72(2):250-260
Lian C.,Narimatsu M.,Nara K.,and Hogetsu T.,2006,Tricholoma matsutake in a natural Pinus densiflora forest:correspondence between above-and below-ground genets,association with multiple host trees and alteration of existing ectomycorrhizal communities,New Phytologist,171(4):825-836
Molinier V.,Murat C.,Frochot H.,Wipf D.,and Splivallo R.,2015,Fine-scale spatial genetic structure analysis of the black truffle Tuber aestivum and its link to aroma variability,Environmental Microbiology,17(8):3039-3050
O'Hanlon R.,2012,Below-ground ectomycorrhizal communities:the effect of small scale spatial and short term temporal variation,Symbiosis,57(2):57-71
Roy M.,Dubois M.P.,Proffit M.,Vincenot L.,Desmarais E.,and Selosse M.A.,2008,Evidence from population genetics that the ectomycorrhizal basidiomycete Laccaria amethystina is an actual multihost symbiont,Mol.Ecol.,17(12):2825-2838
Rozen S.,and Skaletsky H.,2000,Primer3 on the WWW for general users and for biologist programmers,Methods Mol.Biol.,132:365-386
Taylor A.F.,and Alexander I.,2005,The ectomycorrhizal symbiosis:life in the real world,Mycologist,19(3):102-112
Thiel T.,Michalek W.,Varshney R.K.,and Graner A.,2003,Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley(Hordeum vulgare L.),Tag.Theoretical and Applied Genetics.Theoretische Und Angewandte Genetik,106(3):411-422
Wadud M.A.,Nara K.,Lian C.,Ishida T.A.,and Hogetsu T.,2014,Genet dynamics and ecological functions of the pioneer ectomycorrhizal fungi Laccaria amethystina and Laccaria laccata in a volcanic desert on Mount Fuji,Mycorrhiza,24(7):551-563
Xie Z.L.,Zhao L.Z.,Li Y.,Lei J.Q.,and Zhang F.M.,2016,The correlation of geographic distribution and ecological environment of endemic species Floccularia luteovirens on Qinghai-Tibet Plateau,Shengtai Xuebao(Acta Ecologica Sinica),36(10):2851-2857(谢占玲,赵联正,李椰,雷洁琼,张凤梅,2016,青藏高原特有种黄绿卷毛菇的地理分布与生态环境的相关性,生态学报,36(10):2851-2857)
Xing R.,Gao Q.B.,Zhang F.Q.,Fu P.C.,Wang J.L.,Yan H.Y.,and Chen S.L.,2017,Genetic variation and phylogenetic relationships of the ectomycorrhizal Floccularia luteovirens on the Qinghai-Tibet Plateau,Journal of Microbiology,55(8):600-606
Xing R.,Gao Q.B.,Zhang F.Q.,Li Y.H.,Fu P.C.,Zhang J.H.,Wang J.L.,and Chen S.L.,2014,Isolation and characterization of 27 polymorphic microsatellite markers in Armillaria luteo-virens(Physalacriaceae),Weishengwu Xuebao(Acta Microbiologica Sinica),54(9):1045-1052(邢睿,高庆波,张发起,李印虎,付鹏程,张金华,王久利,陈世龙,2014,基于454测序技术的青藏高原黄绿蜜环菌微卫星引物的开发,微生物学报,54(9):1045-1052)
Zerbino D.R.,and Birney E.,2008,Velvet:algorithms for de novo short read assembly using de Bruijn graphs,Genome Research,18(5):821-829