Isolation and Single Nucleotide Polymorphisms Analysis of Cellulose Synthase Gene Fragment in Larix kaempferi

YI Min; ZHANG Shou-gong; XIE Yun-hui; SUN Xiao-mei

Volume 28 Issue 3

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Isolation and Single Nucleotide Polymorphisms Analysis of Cellulose Synthase Gene Fragment in Larix kaempferi

1.
State Key Laboratory of Tree Genetics and Breeding, Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091, China

Received Date: 2014-05-26

Abstract

Objective Cellulose synthase (CesA) plays a key role in the biosynthesis pathway of plant cellulose,which controls the quality and yield of wood fiber. This work aims at cloning the homologous genes cellulose-related from Larix kaempferi, and further analyzing the nucleotide diversity and linkage disequilibrium, which may provide important genetic foundation associated with LkCesA gene and gene-assisted breeding of new germplasms with desirable wood fiber traits in L.kaempferi.Method According to the expressed sequence tags (ESTs) of cellulose synthase from L.kaempferi transcriptome database, a cDNA fragment encoding CesA was isolated from L. kaempferi by gene-specific PCR amplification. The genomic sequences of LkCesA in 40 individuals were cloned and sequenced, then the single nucleotide polymorphisms (SNPs) diversity and linkage disequilibrium of LkCesA were analyzed using DnaSP5.0 software.Result The CesA fragment was 1 209 bp in length with a partial open reading frame (ORF, 1 053 bp) which would be capable to encode a predicted protein of 350 AA. The sequence indicated that the deduced amino acids shared 95.4% identity with PtCesA2 from Pinus taeda. A total of 83 SNPs was detected, and the frequency and diversity of SNPs (πT) were 1/21 bp and 0.006 05, respectively. There were 69 SNPs belonging to transition type and 24 belonging to transversion type, including 19 common SNPs and 64 rare SNPs. In total, 54 SNPs were detected in the coding regions of LkCesA, among which 20 were synonymous mutation and 34 were missense mutation. The results of linkage disequilibrium analysis showed that the LD declined rapidly with the nucleotide length.Conclusion The results of this study indicate that LkCesA is a member of CesA gene family. The linkage disequilibrium declined rapidly within the gene regions of LkCesA suggests that the gene could be used as the candidate gene for LD mapping, which contribute to the directional cultivation and wood quality improvement in Japanese larch. In addition, many common SNPs were detected in LkCesA which could be used in further LD mapping.
- Larix kaempferi,
- cellulose synthase,
- gene cloning,
- single nucleotide polymorphism

References

[1]	Delmer D P. Cellulose biosynthesis: exciting times for a difficult field of study[J].Annu Rev Plant Physiol Plant Mol Biol, 1999, 50:245-276
[2]	Fengel D, Wegener G. Wood: chemistry, ultrastructure, reactions[M].Berlin:Walter de Gruyter, 1984, 26-65.
[3]	王军辉, 张守攻, 石淑兰, 等.日本落叶松纸浆材造纸性能的研究[J].北京林业大学学报, 2004, 26(5):71-74.
[4]	孙晓梅, 张守攻, 李时元, 等.日本落叶松纸浆材优良家系多性状联合选[J].林业科学, 2005, 41(4):48-54.
[5]	Kimura S, Laosinchai W, Itoh T. Immunogold labeling of rosste terminal cellulose synthesizing complexes in the vascular plant Vigna angularis[J].Plant Cell, 1999, 11:2075-2085.
[6]	Pear J R, Kawagoe Y, Schreckengost W E, et al. Higher plants contain homologs of the bacterial celA genes encoding the catalytic subunit of cellulose synthase[J]. Proceedings of the National Academy of Sciences, USA, 1996, 93:12637-12642.
[7]	Richmond T A, Somerville C R. The cellulose synthase superfamily[J].Plant Physiology, 2000, 124:495-498.
[8]	Appenzeller L, Doblin M, Barreiro R, et al. Cellulose synthesis in maize: isolation and expression analysis of the cellulose synthase(CesA) gene family[J].Cellulose, 2004, 11, 287-299.
[9]	Tanaka K, Murata K, Yamazaki M, et al. Three distinct rice cellulose synthase catalytic subunit genes required for cellulose synthesis in the secondary wall[J].Plant Physiology, 2003, 133, 73-83.
[10]	Wu L, Joshi C P, Chiang V L.A xylem-specific cellulose synthase gene from aspen(Populus tremuloides) is responsive to mechanical stress[J].Plant Journal, 2000, 22:495-502.
[11]	Soraya D, Mats L, Lars A, et al. The genome sequence of black cottonwood(Populus trichocarpa) reveals 18 conserved cellulose synthase(CesA) genes[J].Planta, 2005, 221(5):739-746.
[12]	Suzuki S, Li L, Sun Y H, et al. The cellulose synthase gene superfamily and biochemical functions of xylem-specific cellulose synthase-like genes in Populus trichocarpa[J].Plant Physiology, 2006, 142:1233-1245.
[13]	Krauskopf E, Harris P J, Putterill J. The cellulose synthase gene PrCESA10 is involved in cellulose biosynthesis in developing tracheids of the gymnosperm Pinus radiata[J].Gene, 2005, 350(2):107-116.
[14]	Nairn C J, Haselkorn T. Three loblolly pine CesA genes expressed in developing xylem are orthologous to secondary cell wall CesA 敧?杮敥湳攠?晦甠湡据瑧楩潯湳孰?嵲??敛瑊桝漮摎獥??潰汨??楯潬汯???ぴ???日???ㄠ㈱???????戹爱?嬮监?嵲￣?漱渵穝???日??泶敾竺??憘狊瓺??????湂旍稘?卢‐????牛獄潝種?????犗漚矑湦—??刬?′?攱洳?攼瑢?愾汛???攠浊???乩??猠敐焬甠敂湨捡敮?癡慲物椠慓琬椠潒湡?慪湡摮?獐攬氠攼捥瑭椾潥湴?潡晬?琯慥杭￣献椠湇来汮敯?湩畣捳氠敯潦琠楣摥敬?灵潬汯祳浥漠牢灩桯楳獹浮獴?慥瑳?捳愠湩摮椠摰慯瑰敬?杲敳湛敊獝?晎潥牷?摐牨潹畴杯桬瑯?獩瑳牴攬猠猲‰爰攴猬瀠漱渶猴攺?椵渳??攱洮?偢楲渾畛猱?瑝愠敒摩慣????敤洠?嬠?崬??敯湭敥瑲楶捩獬???ぃ???ㄠ???ㄠ??????????扳特?孴??嵳??癳潵牰湥祲欠?噡??卬楹牛癊楝???????????楩歯歬漬渠攲渰‰????攲洴?攴琹‵愭水??攮洼??￣?漱眸?渠畒捩汣敨潭瑯楮摤攠?搮椠癈敩牧獨楥瑲礠?慬瑡?瑴栠散?灬慬汵??汳潥挠畳獹?楴湨?瑳桥敳?睊楝搮敇汥祮?摭楥猠瑂物楯扬甬琠攲搰‰?攬洠?倨椴温町猳‰猰礱氭瘳攰猰琶爮椼獢??敛洱?孝?嵨???漠汎攱挬甠汈愐牬??楉漮泥漬朽秶?慂滍擊??瘼潥汭甾琴楃潌渼???????????????ㄧ???J].林业科学研究, 2013, (专刊):018-024.
[15]	Krutovsky K V, Neale D B. Nucleotide diversity and linkage disequilibrium in cold-hardiness and wood quality-related candidate genes in Douglas fir[J].Genetics, 2005, 171:2029-2041.
[16]	Du Q, Pan W, Tian J, et al. The UDP-glucuronate decarboxylase gene family in Populus: structure, expression, and association genetics[J].PLoS One, 2013, 8: e60880.
[17]	Semerikov V L, Semerikova S A, Polezhaeva M A. Nucleotide diversity and linkage disequilibrium of adaptive significant genes in Larix (Pinaceae)[J].Russian Journal of Genetics, 2013, 49:915-923.
[18]	Namroud M C, Guillet-Claude C, Mackay J, et al. Molecular evolution of regulatory genes in spruces from different species and continents: heterogeneous patterns of linkage disequilibrium and selection but correlated recent demographic changes[J].J Mol Evol, 2010, 70:371-386.
[19]	Zhang D, Xu B, Yang X, et al. The sucrose synthase gene family in Populus: structure, expression, and evolution[J].Tree Genetics & Genomes, 2010, 7:443-456.
[20]	Kado T, Yoshimaru H, Tsumura Y, et al. DNA variation in a conifer, Cryptomeria japonica (Copressaceae sensu lato)[J].Genetics, 2003, 164:1547-1599.
[21]	Dvornyk V, Sirviö A, Mikkonen M, et al. Low nucleotide diversity at the pal1 locus in the widely distributed Pinus sylvestris[J]. Molecular Biology and Evolution, 2002, 19:179-188.
[22]	Whitt S R, Buckler E B.Using natural allelic diversity to evaluat

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通讯作者: 陈斌, bchen63@163.com

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沈阳化工大学材料科学与工程学院沈阳 110142

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Isolation and Single Nucleotide Polymorphisms Analysis of Cellulose Synthase Gene Fragment in Larix kaempferi

1. State Key Laboratory of Tree Genetics and Breeding, Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091, China

Received Date: 2014-05-26

Abstract: Objective Cellulose synthase (CesA) plays a key role in the biosynthesis pathway of plant cellulose,which controls the quality and yield of wood fiber. This work aims at cloning the homologous genes cellulose-related from Larix kaempferi, and further analyzing the nucleotide diversity and linkage disequilibrium, which may provide important genetic foundation associated with LkCesA gene and gene-assisted breeding of new germplasms with desirable wood fiber traits in L.kaempferi.Method According to the expressed sequence tags (ESTs) of cellulose synthase from L.kaempferi transcriptome database, a cDNA fragment encoding CesA was isolated from L. kaempferi by gene-specific PCR amplification. The genomic sequences of LkCesA in 40 individuals were cloned and sequenced, then the single nucleotide polymorphisms (SNPs) diversity and linkage disequilibrium of LkCesA were analyzed using DnaSP5.0 software.Result The CesA fragment was 1 209 bp in length with a partial open reading frame (ORF, 1 053 bp) which would be capable to encode a predicted protein of 350 AA. The sequence indicated that the deduced amino acids shared 95.4% identity with PtCesA2 from Pinus taeda. A total of 83 SNPs was detected, and the frequency and diversity of SNPs (πT) were 1/21 bp and 0.006 05, respectively. There were 69 SNPs belonging to transition type and 24 belonging to transversion type, including 19 common SNPs and 64 rare SNPs. In total, 54 SNPs were detected in the coding regions of LkCesA, among which 20 were synonymous mutation and 34 were missense mutation. The results of linkage disequilibrium analysis showed that the LD declined rapidly with the nucleotide length.Conclusion The results of this study indicate that LkCesA is a member of CesA gene family. The linkage disequilibrium declined rapidly within the gene regions of LkCesA suggests that the gene could be used as the candidate gene for LD mapping, which contribute to the directional cultivation and wood quality improvement in Japanese larch. In addition, many common SNPs were detected in LkCesA which could be used in further LD mapping.

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Reference (22)

[1]	Delmer D P. Cellulose biosynthesis: exciting times for a difficult field of study[J].Annu Rev Plant Physiol Plant Mol Biol, 1999, 50:245-276
[2]	Fengel D, Wegener G. Wood: chemistry, ultrastructure, reactions[M].Berlin:Walter de Gruyter, 1984, 26-65.
[3]	王军辉, 张守攻, 石淑兰, 等.日本落叶松纸浆材造纸性能的研究[J].北京林业大学学报, 2004, 26(5):71-74.
[4]	孙晓梅, 张守攻, 李时元, 等.日本落叶松纸浆材优良家系多性状联合选[J].林业科学, 2005, 41(4):48-54.
[5]	Kimura S, Laosinchai W, Itoh T. Immunogold labeling of rosste terminal cellulose synthesizing complexes in the vascular plant Vigna angularis[J].Plant Cell, 1999, 11:2075-2085.
[6]	Pear J R, Kawagoe Y, Schreckengost W E, et al. Higher plants contain homologs of the bacterial celA genes encoding the catalytic subunit of cellulose synthase[J]. Proceedings of the National Academy of Sciences, USA, 1996, 93:12637-12642.
[7]	Richmond T A, Somerville C R. The cellulose synthase superfamily[J].Plant Physiology, 2000, 124:495-498.
[8]	Appenzeller L, Doblin M, Barreiro R, et al. Cellulose synthesis in maize: isolation and expression analysis of the cellulose synthase(CesA) gene family[J].Cellulose, 2004, 11, 287-299.
[9]	Tanaka K, Murata K, Yamazaki M, et al. Three distinct rice cellulose synthase catalytic subunit genes required for cellulose synthesis in the secondary wall[J].Plant Physiology, 2003, 133, 73-83.
[10]	Wu L, Joshi C P, Chiang V L.A xylem-specific cellulose synthase gene from aspen(Populus tremuloides) is responsive to mechanical stress[J].Plant Journal, 2000, 22:495-502.
[11]	Soraya D, Mats L, Lars A, et al. The genome sequence of black cottonwood(Populus trichocarpa) reveals 18 conserved cellulose synthase(CesA) genes[J].Planta, 2005, 221(5):739-746.
[12]	Suzuki S, Li L, Sun Y H, et al. The cellulose synthase gene superfamily and biochemical functions of xylem-specific cellulose synthase-like genes in Populus trichocarpa[J].Plant Physiology, 2006, 142:1233-1245.
[13]	Krauskopf E, Harris P J, Putterill J. The cellulose synthase gene PrCESA10 is involved in cellulose biosynthesis in developing tracheids of the gymnosperm Pinus radiata[J].Gene, 2005, 350(2):107-116.
[14]	Nairn C J, Haselkorn T. Three loblolly pine CesA genes expressed in developing xylem are orthologous to secondary cell wall CesA 敧?杮敥湳攠?晦甠湡据瑧楩潯湳孰?嵲??敛瑊桝漮摎獥??潰汨??楯潬汯???ぴ???日???ㄠ㈱???????戹爱?嬮监?嵲￣?漱渵穝???日??泶敾竺??憘狊瓺??????湂旍稘?卢‐????牛獄潝種?????犗漚矑湦—??刬?′?攱洳?攼瑢?愾汛???攠浊???乩??猠敐焬甠敂湨捡敮?癡慲物椠慓琬椠潒湡?慪湡摮?獐攬氠攼捥瑭椾潥湴?潡晬?琯慥杭￣献椠湇来汮敯?湩畣捳氠敯潦琠楣摥敬?灵潬汯祳浥漠牢灩桯楳獹浮獴?慥瑳?捳愠湩摮椠摰慯瑰敬?杲敳湛敊獝?晎潥牷?摐牨潹畴杯桬瑯?獩瑳牴攬猠猲‰爰攴猬瀠漱渶猴攺?椵渳??攱洮?偢楲渾畛猱?瑝愠敒摩慣????敤洠?嬠?崬??敯湭敥瑲楶捩獬???ぃ???ㄠ???ㄠ??????????扳特?孴??嵳??癳潵牰湥祲欠?噡??卬楹牛癊楝???????????楩歯歬漬渠攲渰‰????攲洴?攴琹‵愭水??攮洼??￣?漱眸?渠畒捩汣敨潭瑯楮摤攠?搮椠癈敩牧獨楥瑲礠?慬瑡?瑴栠散?灬慬汵??汳潥挠畳獹?楴湨?瑳桥敳?睊楝搮敇汥祮?摭楥猠瑂物楯扬甬琠攲搰‰?攬洠?倨椴温町猳‰猰礱氭瘳攰猰琶爮椼獢??敛洱?孝?嵨???漠汎攱挬甠汈愐牬??楉漮泥漬朽秶?慂滍擊??瘼潥汭甾琴楃潌渼???????????????ㄧ???J].林业科学研究, 2013, (专刊):018-024.
[15]	Krutovsky K V, Neale D B. Nucleotide diversity and linkage disequilibrium in cold-hardiness and wood quality-related candidate genes in Douglas fir[J].Genetics, 2005, 171:2029-2041.
[16]	Du Q, Pan W, Tian J, et al. The UDP-glucuronate decarboxylase gene family in Populus: structure, expression, and association genetics[J].PLoS One, 2013, 8: e60880.
[17]	Semerikov V L, Semerikova S A, Polezhaeva M A. Nucleotide diversity and linkage disequilibrium of adaptive significant genes in Larix (Pinaceae)[J].Russian Journal of Genetics, 2013, 49:915-923.
[18]	Namroud M C, Guillet-Claude C, Mackay J, et al. Molecular evolution of regulatory genes in spruces from different species and continents: heterogeneous patterns of linkage disequilibrium and selection but correlated recent demographic changes[J].J Mol Evol, 2010, 70:371-386.
[19]	Zhang D, Xu B, Yang X, et al. The sucrose synthase gene family in Populus: structure, expression, and evolution[J].Tree Genetics & Genomes, 2010, 7:443-456.
[20]	Kado T, Yoshimaru H, Tsumura Y, et al. DNA variation in a conifer, Cryptomeria japonica (Copressaceae sensu lato)[J].Genetics, 2003, 164:1547-1599.
[21]	Dvornyk V, Sirviö A, Mikkonen M, et al. Low nucleotide diversity at the pal1 locus in the widely distributed Pinus sylvestris[J]. Molecular Biology and Evolution, 2002, 19:179-188.
[22]	Whitt S R, Buckler E B.Using natural allelic diversity to evaluat

Isolation and Single Nucleotide Polymorphisms Analysis of Cellulose Synthase Gene Fragment in Larix kaempferi

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Isolation and Single Nucleotide Polymorphisms Analysis of Cellulose Synthase Gene Fragment in Larix kaempferi

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