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[1]苏亚春,凌辉,王恒波,等.甘蔗SCoT-PCR反应体系优化与多态性引物筛选及应用[J].应用与环境生物学报,2012,18(05):810-818.[doi:10.3724/SP.J.1145.2012.00810]
 SU Yachun,LIN Hui,WANG Hengbo,et al.Optimization of SCoT-PCR Reaction System, and Screening and Utilization of Polymorphic Primers in Sugarcane[J].Chinese Journal of Applied & Environmental Biology,2012,18(05):810-818.[doi:10.3724/SP.J.1145.2012.00810]
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甘蔗SCoT-PCR反应体系优化与多态性引物筛选及应用()
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《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
18卷
期数:
2012年05期
页码:
810-818
栏目:
技术与方法
出版日期:
2012-10-25

文章信息/Info

Title:
Optimization of SCoT-PCR Reaction System, and Screening and Utilization of Polymorphic Primers in Sugarcane
作者:
苏亚春凌辉王恒波阙友雄吴期滨陈珊珊许莉萍
(福建农林大学农业部甘蔗生物学与遗传育种重点实验室 福州 350002)
Author(s):
SU Yachun LIN Hui WANG Hengbo QUE Youxiong WU Qibin CHEN Shanshan XU Liping
(Key Laboratory of Sugarcane Biology and Genetic Breeding of Ministry of Agriculture, Fujian Agriculture & Forestry University, Fuzhou 350002, China)
关键词:
甘蔗SCoT标记体系优化引物筛选多态性能力
Keywords:
sugarcane (Saccharum officinarum) ScoT marker system optimization primers screening polymorphic ability
分类号:
S566.103
DOI:
10.3724/SP.J.1145.2012.00810
文献标志码:
A
摘要:
目标起始密码子多态性(Start condon targeted polymorphism,SCoT)分子标记是一种新型的标记,结合了ISSR标记和RAPD标记的优点. 本研究针对SCoT-PCR反应体系的影响因素,以甘蔗叶片DNA为材料,在单因子实验的基础上,采用L16(45)正交实验设计,进一步探讨了模板DNA、Mg2+、dNTPs、引物及Taq酶等5个因素对甘蔗SCoT-PCR扩增效果的影响,建立了甘蔗SCoT-PCR的优化反应体系. 25 μL PCR反应混合液中,含50 ng DNA模板、2.0 μL 10×Ex Taq Buffer(Mg2+ Plus)、0.625 U Ex Taq酶,dNTP和引物的终浓度分别为0.22 mmol/L和0.9 μmol/L. 以我国种植面积最大的栽培品种新台糖22号为模板,应用优化体系,对40条SCoT标记引物进行测试,筛选出16条有效扩增的引物,且均为多态性引物,其GC含量在50%~67%之间. 该体系的稳定性和SCoT标记引物的扩增能力,通过基于随机选择的4条引物对9份具有地理来源和遗传背景不同的甘蔗种质进行标记分析来验证,结果共扩增出84条带,其中多态性条带占82.14%,平均单条引物可扩增出21条. 研究结果为在甘蔗上进一步开发和应用功能性SCoT标记奠定了基础. 图9 表5 参23
Abstract:
SCoT (Start codon targeted polymorphism) incorporates the advantages of both ISSR and RAPD, such as simple operation, low cost, simple design of primers, good reproducibility and universality and high polymorphism of the amplification. In this paper, five impact factors of template, Mg2+, dNTPs, primer and DNA polymerase, which were the components of PCR reaction system and affected the products of SCoT-PCR, were determined by the methods of single factor experiments. Based on the results of the single factor experiments, L16(45) orthogonal design was applied for further testing. Accordingly, a SCoT-PCR system suitable for sugarcane was established, of which the total 25 μL reaction system contained 50 ng/μL template DNA, 2.0 μL 10×Ex Taq Buffer (Mg2+ Plus), 0.625 U Ex Taq DNA polymerase, and the final concentrations of dNTPs and primer were 0.22 mmol/L and 0.9 μmol/L, respectively. Furthermore, taking DNA of variety ROC22 with top cultivated area in mainland China as the template, 16 primers were screened out from 40 tested primers based on the ability of polymorphic amplification in the above optimized system. The G+C contents were between 50% and 67%. In addition, in order to testify the stability and the amplification ability of the primers, four out of 16 primers were randomly selected for testing on 9 sugarcane germplasms with different geographical sources and different genetic background. 84 bands which including 82.14% of polymorphic bands were amplified with an average of 21 bands detected by each primer. This study provided the basis for the in-depth development and application of the functional SCoT marker in sugarcane research. Fig 9, Tab 5, Ref 23

参考文献/References:

1 Chen RK (陈如凯). Theory and Practice of Modern Sugarcane Breeding. Beijing: China Agricultural Press (北京: 中国农业科学院), 2003. 1~2
2 Liao JQ (廖江雄). Research and prospect in molecular marker of complex polyploid plants of sugarcane. Sugarcane (甘蔗), 1998, 5 (2): 10~15
3 Collard BCY, Mackil DJ. Start-codon targeted (SCoT) polymorphism: a simple, novel DNA marker technique for generating gene-targeted markers in plants. Plant Mol Biol Rep, 2009, 27: 86~93
4 Xiong FQ (熊发前), Jiang Q (蒋菁), Zhong RC (钟瑞春), Han ZQ (韩柱强), He LQ (贺梁琼), Li Z (李忠), Zhuang WJ (庄伟建). Tang RH (唐荣华). Application of SCoT molecular marker in genus Arachis. Acta Agron Sin (作物学报), 2010, 36 (12): 2055~2061
5 Chen H (陈虎), He XH (何新华), Luo C (罗聪), Zhu JH (朱建华), Li F (李峰). Analysis on the genetic diversity of 24 longan (Dimocarpus longan) accessions by SCoT markers. Acta Hortic Sin (园艺学报), 2010, 37 (10): 1651~1654
6 Xiong FQ (熊发前), Tang RH (唐荣华), Chen ZL (陈忠良), Pan LH (潘玲华), Zhuang WJ (庄伟建). SCOT: A novel gene targeted marker technique based on the translation start codon. Mol Plant Breed (分子植物育种), 2009, 7 (3): 635~638
7 Carroll B, Mclntyre L, Berding N. Final report-an assessment of the application of DNA markers to studies of genetic diversity and marker assisted selection in sugarcane. BSES Publication, 1999. 1~12
8 Lao FY (劳方业), Liu R (刘睿), He HY (何慧怡), Deng HH (邓海华), Li QW (李奇伟), Chen ZH (陈仲华), Chen JW (陈健文), Fu C (符成), Qi YW (齐永文), Zhang CM (张垂明). Genetic diversity analysis of sugarcane parents with AFLP in China. Genomics & Genom Appl Biol (基因组学与应用生物学), 2009, 28 (3): 503~508
9 Li M (李鸣), Liang CX (梁朝旭), Fang WK (方位宽), Liang J (梁俊), Li YR (李杨瑞,), Tan YM (谭裕模), He WZ (何为中). AFLP molecular marker technology and its application in sugarcane genetics and breeding. Sugar Crops China (中国糖料), 2006 (1): 43~46
10 Nair NV, Selvi A, Sreenivasan TV, Pushpalatha KN. Molecular diversity in Indian sugarcane cultivars as revealed by randomly amplified DNA polymorphisms. Euphytica, 2002, 127 (2): 219~225
11 Xu LP (许莉萍), Chen RK (陈如凯). Identification of RAPD marker linked to smut resistance gene in sugarcane. Chin J Appl Environ Biol (应用与环境生物学报), 2004, 10 (3): 263~267
12 Cordeiro GM, Pan YB, Henry RJ. Sugarcane microsatellites for the assessment of genetic diversity in sugarcane germplasm. Plant Sci, 2003, 165 (1): 181~189
13 Yu AL (俞爱丽), Zhang MQ (张木清), Chen RK (陈如凯). Applicability of inter-simple sequence repeat polymorphisms in sugarcane and its related genera as DNA markers. J Fujian Agric For Univ (福建农林大学学报), 2002, 31 (4): 484~489
14 Que YX (阙友雄), Chen TS (陈天生), Xu LP (许莉萍). Genetic diversity of important sugarcane germplasm on TRAP marker. J Agric Biotechnol (农业生物技术学报), 2009 (3): 496~503
15 Kwok S, Kellogg DE, McKinney N, Spasic D, Goda D, Levenson C. Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies. Nucleic Acid Res, 1990, 18 (4): 999~1005
16 Jiang F (蒋锋), Liu PF (刘鹏飞), Du SZ (杜世州), Wang XM (王晓明). Research progress on crop QTL papping method. J Anhui Agric Sci (安徽农业科学), 2009, 37 (26): 12496~12497, 12508
17 Ming DX (明道绪). The Field Experiment and Statistics Analysis. Beijing: Science Press (北京: 科学出版社), 2005. 294
18 Wang SQ (王水琦), Lin YX (林一心), Ye XL (叶小丽). Molecular markers and its application in sugarcane breeding. Sugarcane Canesugar (甘蔗糖业), 2003 (4): 21~25
19 Chen H (陈辉), Fan YH (范源洪), Cai Q (蔡青). RAPD molecular marker technology and its application in sugarcane breeding. Sugar Crops China (中国糖料), 2004 (1): 46~48
20 Zhu YF (朱岩芳), Zhu SJ (祝水金), Li YP (李永平), Ma WG (马文广), Zheng JY (郑钧晔), Hu J (胡晋). Application of ISSR molecular marker in plant germplasm research. Seed (种子), 2010, 29 (2): 55~59
21 Liu J (刘静). The development and application of AFLP. Shandong Agric Sci (山东农业科学), 2010 (5): 10~14
22 Zhang JC (张君诚), Song YH (宋育红), Zhu Y (朱勇), Zhang HY (张杭颖), Zhong Y (钟扬). AFLP analysis of genetic diversity and population structure of Huperzia serrata (Thunb. ex Murray) Trev. var. longipetiolata (Spring) H. M. Chang. Chin J Appl Environ Biol (应用与环境生物学报), 2011, 17 (1): 18~23
23 Cheng XL (陈香玲), Li YR (李杨瑞), Yang LT (杨丽涛), Wu JM (吴建明), Xiong FQ (熊发前), Luo C (罗聪). Comparison of two electrophoresis methods in cDNA-SCoT gene differential expression. Biothchnol Bull (生物技术通报), 2010 (10): 93~95

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备注/Memo

备注/Memo:
现代农业产业技术体系建设专项资金(No. CARS-20)资助
更新日期/Last Update: 2012-10-26