杉木木材形成功能基因内SSR标记的开发及应用
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摘要
开发一套基于杉木(Cunninghamia lanceolata)木材形成相关功能基因的SSR标记,可为后续木材材质性状的分子标记辅助育种提供重要工具。本研究对参与杉木木材形成的10个基因序列进行SSR位点和引物设计,应用毛细管电泳检测引物的有效性和多态性,使用TAASEL(trait analysis by association,evolution and linkage)软件进行SSR位点组合间连锁不平衡(linkage disequilibrium, LD)及其与木材密度的关联分析。在与木材形成相关的7个功能基因中共检测到15个SSR位点。碱基重复形式有二碱基、三碱基、四碱基与六碱基,其中三碱基重复位点所占比例为46.7%。依据SSR位点两侧的保守序列设计了14对PCR扩增引物,其中有7对引物的扩增结果呈现多态性。结合已有的表达序列标签(expressed sequence tag, EST)-SSR数据进行LD分析,检测到644个SSR位点组合皆存在一定程度的LD(D'≠0),其中D'值大于0.5的组合有183个。关联分析发现来自杉木纤维素合成酶基因(C. lanceolata cellulose synthase 3,ClCesA3) 5'非翻译区(5'untranslated region, 5'UTR)的CLSSR4位点与杉木木材密度极显著关联(P<0.01),该位点对性状变异的解释率为26.07%,且相应等位变异A201和A205对木材密度分别表现为增效和减效效应。本研究共设计筛选出了7对功能基因SSR多态性引物,其中来源于ClCesA3基因5'UTR的SSR位点与杉木木材密度显著关联,为分子标记辅助杉木木材性状改良提供了重要的标记和途径。
To provide an important tool for molecular marker-assisted breeding of wood quality traits, a new nuclear genome SSR marker was developed from functional genes related to Cunninghamia lanceolata wood formation. The SSR locus was detected through analyzing the sequences of 10 genes involved in the formation of C. lanceolata. The availability and polymorphism of the designed SSR primers were examined by capillary electrophoresis. Linkage disequilibrium of pairwise loci and the association analysis between developed SSR loci and wood density were performed by using TASSEL(trait analysis by association, evolution and linkage)GLM(general linear model) program. A total of 15 SSR loci were identified in 7 functional genes. There were di-, tri-, tetra-and hexa-nucleotide repeats, with trinucleotide SSRs being the most frequent,accounting for46.7% of the total loci. Fourteen pairs of PCR amplification primers were designed according to conserved sequences on both sides of SSR loci. Among them, 7 pairs of primers showed polymorphism. A total of 644 SSR loci combinations were found to have a certain degree of linkage disequilibrium(D'≠ 0), with D' values of183 combinations greater than 0.5. The association analysis revealed that the CLSSR4 locus from the 5'untranslated region(5' UTR) of ClC esA 3(C. lanceolata cellulose synthase 3) was extremely significantly associated with wood density(P<0.01). The wood density variance explained by this locus was 26.07%, and the corresponding alleles of A201 and A205 showed the positive and negative phenotypic effects, respectively.In this study, seven polymorphic SSR primer pairs were developed from genomic sequence of functional genes,and a SSR locus derived from the 5' UTR of ClC esA 3 gene was significantly associated with wood density of C.lanceolata. These results will provide an important tool for molecular marker-assisted improvement of C.lanceolata wood quality.
To provide an important tool for molecular marker-assisted breeding of wood quality traits, a new nuclear genome SSR marker was developed from functional genes related to Cunninghamia lanceolata wood formation. The SSR locus was detected through analyzing the sequences of 10 genes involved in the formation of C. lanceolata. The availability and polymorphism of the designed SSR primers were examined by capillary electrophoresis. Linkage disequilibrium of pairwise loci and the association analysis between developed SSR loci and wood density were performed by using TASSEL(trait analysis by association, evolution and linkage)GLM(general linear model) program. A total of 15 SSR loci were identified in 7 functional genes. There were di-, tri-, tetra-and hexa-nucleotide repeats, with trinucleotide SSRs being the most frequent,accounting for46.7% of the total loci. Fourteen pairs of PCR amplification primers were designed according to conserved sequences on both sides of SSR loci. Among them, 7 pairs of primers showed polymorphism. A total of 644 SSR loci combinations were found to have a certain degree of linkage disequilibrium(D'≠ 0), with D' values of183 combinations greater than 0.5. The association analysis revealed that the CLSSR4 locus from the 5'untranslated region(5' UTR) of ClC esA 3(C. lanceolata cellulose synthase 3) was extremely significantly associated with wood density(P<0.01). The wood density variance explained by this locus was 26.07%, and the corresponding alleles of A201 and A205 showed the positive and negative phenotypic effects, respectively.In this study, seven polymorphic SSR primer pairs were developed from genomic sequence of functional genes,and a SSR locus derived from the 5' UTR of ClC esA 3 gene was significantly associated with wood density of C.lanceolata. These results will provide an important tool for molecular marker-assisted improvement of C.lanceolata wood quality.
引文
胡梦霄.2016.基于SSR标记的杉木重要性状关联分析研究[D].硕士学位论文,浙江农林大学,导师:童再康,pp.31-45.(Hu MX.2016.Association analysis of important traits based on SSR markers in Cunninghamia lanceolata[D].Thesis for M.S.,Zhejiang A&F University,Supervisor:Tong Z K,pp.31-45.)
黄海燕,杜红岩,刘攀峰.2013.基于杜仲转录组序列的SSR分子标记的开发[J].林业科学,49(5):176-181.(Huang HY,Du HY,Liu PF.2013.Development of SSR molecular markers based on transcriptome sequencing of Eucommia ulmoides[J].Scientia Silvae Sinicae,49(5):176-181.)
刘琼瑶.2015.杉木NAC基因的克隆、表达与SNP分析[D].硕士学位论文,浙江农林大学,导师:黄华宏,pp.10-28.(Liu QY.2015.Isolation,expression and single nucleotide polymorphisms analysis of NAC genes in Cunninghamia lanceolata[D].Thesis for M.S.,Zhejiang A&F University,Supervisor:Huang H H,pp.10-28.)
宋志姣,翁启杰,周长品,等.2016.细叶桉(Eucalyptus tereticornis)早期生长的SSR标记关联分析[J].分子植物育种,14(1):195-203.(Song Z J,Weng Q J,Zhou C P,et al2016.SSR Markers associated with early growth in Eucalyptus tereticornis[J].Molecular Plant Breeding,14(10):173-180)
王保垒,王博文,陈清清,等.2011.杨树抗逆转录因子基因内SSR标记的开发[J].林业科学,47(8):67-74.(Wang B L,Wang B W,Chen Q Q,et al.2011.Identification of SSR loci from transcription factor genes expressed under abiotic stresses in Populus[J].Scientia Silvae Sinicae,47(8):67-74.)
文自翔,赵团结,郑永战,等.2008.中国栽培和野生大豆农艺及品质性状与SSR标记的关联分析Ⅱ.优异等位变异的发掘[J].作物学报,34(8):1339-1349.(Wen Z XZhao T J,Zheng Y Z,et al.2008.Association analysis of agronomic and quality traits with SSR markers in Glycine max and Glycine soja in China:Ⅱ.Exploration of Elite Alleles[J].Acta Agronomica Sinica.34(8):1339-1349.)
文亚峰,韩文军,周宏,等.2015.杉木转录组SSR挖掘及EST-SSR标记规模化开发[J].林业科学,51(11):40-49.(Wen Y F,Han W J,Zhou H,et al.2015.SSR mining and development of EST-SSR markers for Cunninghamia lanceolata based on transcriptome sequences[J]Scientia Silvae Sinicae,51(11):40-49.)
张圣,黄华宏,林二培,等.2013.杉木与台湾杉EST-SSR标记的开发与应用[J].林业科学,49(10):173-180(Zhang S,Huang H H,Lin E P,et al.2013.Development and application of EST-SSR markers for Cunninghamia lanceolata and Taiwania cryptomerioides[J].Scientia Silvae Sinicae,49(10):173-180.)
Cordeiro G M,Casu R,Mcintyre C L,et al.2001.Microsatellite markers from sugarcane(Saccharum spp.)ESTs cross transferable to erianthus and sorghum[J].Plan Science,160(6):1115-1123.
Dillon S K,Nolan M,Li W,et al.2010.Allelic variation in cell wall candidate genes affecting solid wood properties in natural populations and land races of Pinus radiate[J].Genetics,185:1477-1487.
Du Q,Gong C,Pan W,et al.2013.Development and application of microsatellites in candidate genes related to wood properties in the Chinese White Poplar(Populus tomentosa Carr.)[J].DNA Research,20(1):31-44.
Du Q,Pan W,Xu B,et al.2012.Polymorphic simple sequence repeat(SSR)loci within cellulose synthase(PtoCesA)genes are associated with growth and wood properties in Populus tomentosa[J].New Phytologist,197(3):763-776.
Elkassaby Y A.2004.Single-copy,species-transferable microsatellite markers developed from loblolly pine ESTs[J].Theoretical&Applied Genetics,109(2):361-369.
Ellis J R,Burke J M.2007.EST-SSRs as a resource for population genetic analyses[J].Heredity,99(2):125-132.
Ferr?o L F V,Caixeta E T,Pena G,et al.2015.New EST-SSR markers of Coffea arabica:Transferability and application to studies of molecular characterization and genetic mapping[J].Molecular Breeding,35(1):31.
Huang H H,Xu L L,Tong Z K,et al.2012.De novo characterization of the Chinese fir(Cunninghamia lanceolata)transcriptome and analysis of candidate genes involved in cellulose and lignin biosynthesis[J].BMC Genomics.13:648.
Iglesias A R,Kindlund E,Tammi M,et al.2004.Some microsatellites may act as novel polymorphic cis-regulatory elements through transcription factor binding[J].Gene,341:149-165.
Kalia R K,Rai M K,Kalia S,et al.2011.Microsatellite markers:An overview of the recent progress in plants[J].Euphytica,177(3):309-334.
Li Y C,Korol A B,Fahima T,et al.2004.Microsatellites within genes:Structure,function,and evolution[J].Molecular Biology and Evolution,21(6):991-1007.
Li Y,Wilcox P,Telfer E,et al.2016.Association of single nucleotide polymorphisms with form traits in three New Zealand populations of radiata pine in the presence of genotype by environment interactions[J].Tree Genetics&Genomes,12(4):1-12.
Neale D,Kremer A.2011.Forest tree genomics:Growing resources and applications[J].Nature Reviews Genetics,12(2):111-122.
Scott K D,Eggler P,Seaton G,et al.2000.Analysis of SSRs derived from grape ESTs[J].Theoretical&Applied Genetics,100(5):723-726.
Song Q J,Marek L F,Shoemaker R C,et al.2004.A new integrated genetic linkage map of the soybean[J].Theoretical&Applied Genetics,109(1):122-8.
Stewart C N,Via L E.1993.A rapid CTAB DNA isolation technique useful for RAPD fingerprinting and other PCR applications[J].Biotechniques,14(5):748-750.
Tuskan G A,Gunter L E,Yang Z K.2004.Characterization of microsatellites revealed by genomic sequencing of Populus trichocarpa[J].Canadian Journal of Forest Research.34(1):85-93.
Wei Z,Du Q,Zhang J,et al.2013.Genetic diversity and population structure in Chinese indigenous Poplar(Populus simonii)populations using microsatellite markers[J].Plant Molecular Biology Reporter,31(3):620-632.
黄海燕,杜红岩,刘攀峰.2013.基于杜仲转录组序列的SSR分子标记的开发[J].林业科学,49(5):176-181.(Huang HY,Du HY,Liu PF.2013.Development of SSR molecular markers based on transcriptome sequencing of Eucommia ulmoides[J].Scientia Silvae Sinicae,49(5):176-181.)
刘琼瑶.2015.杉木NAC基因的克隆、表达与SNP分析[D].硕士学位论文,浙江农林大学,导师:黄华宏,pp.10-28.(Liu QY.2015.Isolation,expression and single nucleotide polymorphisms analysis of NAC genes in Cunninghamia lanceolata[D].Thesis for M.S.,Zhejiang A&F University,Supervisor:Huang H H,pp.10-28.)
宋志姣,翁启杰,周长品,等.2016.细叶桉(Eucalyptus tereticornis)早期生长的SSR标记关联分析[J].分子植物育种,14(1):195-203.(Song Z J,Weng Q J,Zhou C P,et al2016.SSR Markers associated with early growth in Eucalyptus tereticornis[J].Molecular Plant Breeding,14(10):173-180)
王保垒,王博文,陈清清,等.2011.杨树抗逆转录因子基因内SSR标记的开发[J].林业科学,47(8):67-74.(Wang B L,Wang B W,Chen Q Q,et al.2011.Identification of SSR loci from transcription factor genes expressed under abiotic stresses in Populus[J].Scientia Silvae Sinicae,47(8):67-74.)
文自翔,赵团结,郑永战,等.2008.中国栽培和野生大豆农艺及品质性状与SSR标记的关联分析Ⅱ.优异等位变异的发掘[J].作物学报,34(8):1339-1349.(Wen Z XZhao T J,Zheng Y Z,et al.2008.Association analysis of agronomic and quality traits with SSR markers in Glycine max and Glycine soja in China:Ⅱ.Exploration of Elite Alleles[J].Acta Agronomica Sinica.34(8):1339-1349.)
文亚峰,韩文军,周宏,等.2015.杉木转录组SSR挖掘及EST-SSR标记规模化开发[J].林业科学,51(11):40-49.(Wen Y F,Han W J,Zhou H,et al.2015.SSR mining and development of EST-SSR markers for Cunninghamia lanceolata based on transcriptome sequences[J]Scientia Silvae Sinicae,51(11):40-49.)
张圣,黄华宏,林二培,等.2013.杉木与台湾杉EST-SSR标记的开发与应用[J].林业科学,49(10):173-180(Zhang S,Huang H H,Lin E P,et al.2013.Development and application of EST-SSR markers for Cunninghamia lanceolata and Taiwania cryptomerioides[J].Scientia Silvae Sinicae,49(10):173-180.)
Cordeiro G M,Casu R,Mcintyre C L,et al.2001.Microsatellite markers from sugarcane(Saccharum spp.)ESTs cross transferable to erianthus and sorghum[J].Plan Science,160(6):1115-1123.
Dillon S K,Nolan M,Li W,et al.2010.Allelic variation in cell wall candidate genes affecting solid wood properties in natural populations and land races of Pinus radiate[J].Genetics,185:1477-1487.
Du Q,Gong C,Pan W,et al.2013.Development and application of microsatellites in candidate genes related to wood properties in the Chinese White Poplar(Populus tomentosa Carr.)[J].DNA Research,20(1):31-44.
Du Q,Pan W,Xu B,et al.2012.Polymorphic simple sequence repeat(SSR)loci within cellulose synthase(PtoCesA)genes are associated with growth and wood properties in Populus tomentosa[J].New Phytologist,197(3):763-776.
Elkassaby Y A.2004.Single-copy,species-transferable microsatellite markers developed from loblolly pine ESTs[J].Theoretical&Applied Genetics,109(2):361-369.
Ellis J R,Burke J M.2007.EST-SSRs as a resource for population genetic analyses[J].Heredity,99(2):125-132.
Ferr?o L F V,Caixeta E T,Pena G,et al.2015.New EST-SSR markers of Coffea arabica:Transferability and application to studies of molecular characterization and genetic mapping[J].Molecular Breeding,35(1):31.
Huang H H,Xu L L,Tong Z K,et al.2012.De novo characterization of the Chinese fir(Cunninghamia lanceolata)transcriptome and analysis of candidate genes involved in cellulose and lignin biosynthesis[J].BMC Genomics.13:648.
Iglesias A R,Kindlund E,Tammi M,et al.2004.Some microsatellites may act as novel polymorphic cis-regulatory elements through transcription factor binding[J].Gene,341:149-165.
Kalia R K,Rai M K,Kalia S,et al.2011.Microsatellite markers:An overview of the recent progress in plants[J].Euphytica,177(3):309-334.
Li Y C,Korol A B,Fahima T,et al.2004.Microsatellites within genes:Structure,function,and evolution[J].Molecular Biology and Evolution,21(6):991-1007.
Li Y,Wilcox P,Telfer E,et al.2016.Association of single nucleotide polymorphisms with form traits in three New Zealand populations of radiata pine in the presence of genotype by environment interactions[J].Tree Genetics&Genomes,12(4):1-12.
Neale D,Kremer A.2011.Forest tree genomics:Growing resources and applications[J].Nature Reviews Genetics,12(2):111-122.
Scott K D,Eggler P,Seaton G,et al.2000.Analysis of SSRs derived from grape ESTs[J].Theoretical&Applied Genetics,100(5):723-726.
Song Q J,Marek L F,Shoemaker R C,et al.2004.A new integrated genetic linkage map of the soybean[J].Theoretical&Applied Genetics,109(1):122-8.
Stewart C N,Via L E.1993.A rapid CTAB DNA isolation technique useful for RAPD fingerprinting and other PCR applications[J].Biotechniques,14(5):748-750.
Tuskan G A,Gunter L E,Yang Z K.2004.Characterization of microsatellites revealed by genomic sequencing of Populus trichocarpa[J].Canadian Journal of Forest Research.34(1):85-93.
Wei Z,Du Q,Zhang J,et al.2013.Genetic diversity and population structure in Chinese indigenous Poplar(Populus simonii)populations using microsatellite markers[J].Plant Molecular Biology Reporter,31(3):620-632.