Study on Genomic DNA Extraction Method and AFLP Amplification Reaction System of D. hamiltonii Nees et Arn. ex Munro

doi:10.7668/hbnxb.20102.S1.009

Abstract

Abstract: The study on extraction of DNA with improved CTAB (cetyl-trimethyl-ammonium bromide) method from Dendrocalamus hamiltonii Nees et Arn. ex Munro. It showed that CTAB buffer supplemented with 2% PVP (poly vinyl pyrrolidone) and 2% β-Mercaptoethanal was suitable for the genomic DNA extraction of D. hamiltonii Nees et Arn. ex Munro,with OD₂₆₀ /OD₂₈0value of 1. 7－1. 9,representing high purity of the extracted DNA and the complete elimination of protein,phenol,pigment and RNA. Thus the improved CTAB method was approved suitable for further AFLP analysis of D. hamiltonii. Some key factors affecting DNA extraction,restriction-ligase reaction,preamplification and amplification processes were studied and an optimized AFLP reaction system of D. hamiltonii was established. With this AFLP reaction system,clear DNA electrophoresis fingerprints of D. hamiltonii were obtained, and the reproducibility was high. Eight primer combinations were filtrated from sixty and four pair with Mse I and Pst I,8 primer combinations with stable amplification and rich polymorphism for AFLP were screened,256 DNA bands were amplified by eight pairs of AFLP primers and 40. 90% polymorphic bands per pair of primer were produced on average,which had 32 490 distinct and strong amplification bands with a bright background and 13 289 of them were polymorphic bands from 30 individuals of D. hamiltonii,we can entirely distinguish 30 individuals by 8 primer combinations,which indicates that A FLP markers are useful for genetic analysis of D. hamiltonii.

Key words: Dendrocalamus hamiltonii Nees et Arn. ex Munro, DNA extraction, AFLP, Influential factor

CLC Number:

S795.03

YANG Qing, SU Guang-rong, HAN Lei, WANG Zheng-liang, SUN Qi-xiang, PENG Zhen-hua. Study on Genomic DNA Extraction Method and AFLP Amplification Reaction System of D. hamiltonii Nees et Arn. ex Munro[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2010, 25(S1): 32-37. doi: 10.7668/hbnxb.20102.S1.009.

/ / Recommend / Download Citations

URL: http://www.hbnxb.net/EN/10.7668/hbnxb.20102.S1.009

http://www.hbnxb.net/EN/Y2010/V25/IS1/32

References

[1] 耿平介,王正平.中国植物志第九卷第一分册[M].北京:科学出版社,1996:196-197.
[2] 李延辉.西双版纳高等植物名录[M].昆明:云南民族出版社,1996:205-206.
[3] 李金泽.西双版纳甜龙竹:托起绿色经济新希望[J].生态经济,2003(12):77-79.
[4] Sundriyal M,Sundriyal R C.Wild edible plants of the Sikkim Himalaya:Marketing value addition and implications for management[J].Economic Botany,2004,58(2):300-315.
[5] Bhatt B P,Singh K,Singh Alka.Nutritional values of some commercial edible bamboo species of the North Eastern Himalayan region[J].India Journal of Bamboo and Rattan,2005,4(2):111-124.
[6] 杜凡.云南重要经济竹种特性及其生产中存在问题[J].西南林学院学报,2003,23(2):26-30.
[7] 杨清,苏光荣,许丛恒,等.版纳甜龙竹化学成分与制浆性能研究[J].中华纸业,2007,28(6):83-86.
[8] Doyle J J.DNA protocols for plants-CTAB total DNA isolation[M]//Hewitt G M,Johnston.A Molecular Technique in Taxonomy.Berlin,Springer-Verlag,1991:283-293.
[9] Friar E,Kochea G.Bamboo germplasm screening with nuclear restriction fragment length polymorphisms[J].Theoretical and Applied Genetics,1991,182:697-703.
[10] Smith J J,Scottemig J S,Leadbetter J R.Charaterization of random amplified polymorphic DNA (RAPD) products from Xanthomonas campesris and some comments on the use of BAPD products in phylogenetie analysis[J].Mol Phylogenet Evol,1994,3:135-145.
[11] Becket J.Combined mapping of AFLP markers in barley[J].Mol Gen Genet,1995,249:65-73.
[12] Vos P,Hogers R,Bleeker M,et al.AFLP-a new technique for DNA-fingerprinting[J].Nucleic Acids Res,23:4407-4414.
[13] 邹喻苹,汪小全,雷一丁,等.几种濒危植物及其近缘类群总DNA的提取与鉴定[J].植物学报,1994,36(7):528-533.
[14] Friar E,Kochea G.A study of genetic variation and evolution of PhyHostaehys (Bambusoideae:Poaeeae) using nuclear restriction fragment length polymorphisms[J].Theoretical and Applied Genetics,1994,89:265-270.
[15] 房经贵,刘大钧,章镇.两个芒果品种的AFLP指纹图谱[J].南京农业大学学报,1999,22(2):25-27.
[16] 祝军,王涛,李光晨,等.苹果AFLP分析体系的建立[J].中国农业大学学报,2000,5(3):63-67.
[17] 易干军,于晓英,霍合强,等.香蕉种质资源的AFLP鉴别与分类中DNA模板的制备[J],果树学报,2001,18(6):345-348.
[18] 赵宏,王省芬,张桂寅,等.棉花AFLP技术体系的摸索与建立[J].河北农业大学学报,2002,25(1):1-4.
[19] 高文娜,陈万权,王中康.AFLP双酶切和连接方法探讨[J].植物保护,2003,29(6):35-40.
[20] 候义龙,曹同,蔡丽娜,等.苔藓植物DNA提取方法研究[J].广西植物,2003,23(5):425-428.
[21] 刘勇,孙中海,刘德春,等.柚类种质资源AFLP与SSR遗传多样性分析[J].中国农业科学,2005,38(11):2308-2315.
[22] Zhang L,Huang B B,Kai G Y,Guo M L.Analysis of in transpeeifie variation of Chinese Carthamus tinctorius L.using AFLP marke[J].Acta Pharmaceut Sin,2006,41(1):91-96.
[23] Trevor R H,Stephen A R,Graínne N C,et al.A Comparison of ITS Nuclear rDNA Sequence Data and AFLP Markers for Phylogenetic Studies in Phyllostachys (Bambusoideae,Poaceae)[J].Journal of Plant Research,2000,113(3):259-269.
[24] Koen G,Johan G,Hilde P et al.Somatic embryogenesis from mature Bambusa balcooa Roxburgh as basis for mass production of elite forestry bamboos[J].Plant Cell,Tissue and Organ Culture,2007,91(2):115-123.
[25] 邹喻苹,葛颂,王晓东.系统与进化植物学中的分子标记[M].北京:科学出版社,2001:16-17.
[26] Doyle J J and Doyle J L.A rapid DNA isolation procedure for small quantities of fresh leaf tissue[J].Phytochem Bull Bot So.Amer,1987,19:11-15.
[27] 张汉尧,刘小珍,龚秀会,等.竹子DNA提取方法改良及ITS-RFLP分析的初步研究[J].江西林业科技,2006(3):3-5.

Please choose a citation manager

Content to export