结缕草SSR反应体系的正交优化(英文)

doi:10.11733/j.issn.1007-0435.2008.01.018

›› 2008, Vol. 16 ›› Issue (1): 89-93,102.DOI: 10.11733/j.issn.1007-0435.2008.01.018

结缕草SSR反应体系的正交优化(英文)

桂金山, 王赟文, 方程

中国农业大学草地研究所, 农业部草地植被恢复与重建重点开放实验室, 北京, 100094

收稿日期:2007-07-26 修回日期:2007-12-12 出版日期:2008-02-15 发布日期:2008-02-15
通讯作者: 方程, E-mail:Fangcheng@cau.edu.cn
作者简介:桂金山(1982- ),男,湖南张家界人,硕士研究生,研究方向为牧草生物技术,E-mail:guijinshan2008@126.com
基金资助:
国家自然科学基金项目(30500354)

Optimization of the SSR Reaction System for Zoysiagrass Using Orthogonal Design

GUI Jin-shan, WANG Yun-wen, FANG Cheng

Institute of Grassland Science, China Agricultural University, Ministry of Agriculture Key Laboratory of GrasslandVegetation Restoration and Reconstruction, Beijing, 100094, China

Received:2007-07-26 Revised:2007-12-12 Online:2008-02-15 Published:2008-02-15

摘要/Abstract

摘要： 为了快速获得结缕草(Zoysia japonica)SSR反应体系,采用5因素(模板DNA,Mg²⁺,dNTP,Taq酶和引物)4水平正交设计筛选合适的结缕草SSR体系,并通过随机挑选3对Tm相近的引物对该优化体系进行验证。结果表明:正交设计可应用于SSR-PCR反应体系的优化,20μl最佳PCR反应体系中包括2μl10×buffer、1.0 U Taq酶、0.2mM引物、100ng模板DNA、0.2mMdNTP、2.0 mM Mg⁺;对结缕草进行梯度退火实验,其最佳退火温度为56-58℃;可行扩增程序是:94℃预变性4min、进行35个循环的94℃变性30s、56-58℃退火40s,72℃延伸1min;72℃延伸10min,4℃保存;该最适反应体系的建立,为今后结缕草SSR分析奠定了坚实基础。

关键词: 结缕草, SSR标记, 反应体系, 正交设计, 优化

Abstract: In order to obtainthe opti mal SSR-PCRsystem for zoysiagrass(Zoysiajaponica),an experi mental design of orthog-onal diagramL16(45) was employed to evaluate five factors (template DNA,Mg²⁺,dNTP,Taq DNA polymerase,and primer) at four different levels.Three randomly selected SSR primers (RM84,RM220,RM531) with resembled Tmwere used to vali-date the reaction system.The results indicate that orthogonal design could be used to optimize the SSR-PCR system;the optimal systemof terminal volume 20μl consisted of 2μl 10×buffer,1.0 U Taq DNA polymerase,0.2mM primers,100ng template DNA,0.2 mM dNTP,and 2.0mM Mg⁺.The optimal annealing temperature was determined to be 56-58℃by gradient PCR;the suitable thermal cycling conditions with initial melting at 94℃ for 4min,followed by 35 cycles at 94℃ for 30 s,56 -58℃ for 40s,72℃ for 60 s;then keep the reaction mixture at 4℃ after a final extension step of 72℃ for 10 min.The optimized system would be effective as a solid foundation for zoysiagrass SSR analysis.

Key words: Zoysia japonicaSteud., SSR markers, Reaction system, Orthogonal design, Opti mization

中图分类号:

Q943

桂金山, 王赟文, 方程. 结缕草SSR反应体系的正交优化(英文)[J]. , 2008, 16(1): 89-93,102.

GUI Jin-shan, WANG Yun-wen, FANG Cheng. Optimization of the SSR Reaction System for Zoysiagrass Using Orthogonal Design[J]. , 2008, 16(1): 89-93,102.

参考文献

[1] Duble R L.Turfgrasses:their management and use in the southern zone[M]. College Station,TX:Texas A&M University Press,1996
[2] Forbes I Jr.Chromosome numbers and hybrids in Zoysia[J]. Agron.J.,1952,44:147-151
[3] Arumuganathan K,Tallury S P,Fraser M L.Nuclear DNA content of thirteen turfgrass species by flow cytometry[J]. Crop Sci.,1999,39:1518-1521
[4] White R H,Engelke M C,Anderson S J.Zoysiagrass water relations[J]. Crop Sci.,2001,41:133-138
[5] Brede D.Turfgrass maintenance reduction handbook:sports,lawns,and golf[M]. Chelsea,MI:Ann Arbor Press,2000
[6] Ge Y X,Norton T,Wang Z Y.Transgenic zoysiagrass (Zoysia japonica) plants obtained by Agrobacterium-mediated transformation[J]. Plant Cell Rep.,2006,25:792-798
[7] Spangenberg G,Wang Z Y,Potrykus I.Biotechnology in forage and turf grass improvement[M]. Berlin:Springer,1998
[8] Wang Z Y,Hopkins A,Mian R.Forage and turf grass biotechnology[J]. Crit.Rev.Plant Sci.,2001,20:573-619
[9] Cai H W,Maiko I,Nana Y.Isolation,characterization and mapping of simple sequence repeat markers in zoysiagrass (Zoysia spp.)[J]. Theor.Appl.Genet.,2005,112:158-166
[10] Zietkiewicz E,Rafalski A,Labuda D.Genome fingerprinting by simple sequence repeat (SSR)-anchored polymerase chain reaction amplification[J]. Genomics,1994,20:176-183
[11] Yaish M W F,Pérez de la Vega M.Isolation of (GA)n microsatellite sequences and description of a predicted MADS-box sequence isolated from common bean (Phaseolus vulgaris)[J]. Genet.Mol.Biol.,2003,26:337-342
[12] Morgante M,Olivieri A M.PCR-amplified microsatellites as markers in plant genetics[J]. Plant J.,1993,3:175-182
[13] 魏臻武,符昕,耿小丽,等.苜蓿遗传多样性和亲缘关系的SSR和ISSR分析[J]. 草地学报,2007,15(2):118-123
[14] 毛培胜,张涛,杨青川.紫花苜蓿品种鉴定的RAPD分子标记技术研究[J]. 草地学报,2007,15(2):124-128
[15] 沙伟,滕兆岩,倪红伟.正交设计优化星星草ISSR-PCR反应体系研究[J]. 中国草地学报,2006,28(6):52-55
[16] Gu X Y,Kianian S F,Hareland G A.Genetic analysis of adaptive syndromes interacted with seed dormancy in weedy rice (Oryza sativa)[J]. Theor.Appl.Genet.,2005,110:1108-1118
[17] Gu X Y,Kianian S F,Foley M E.Dormancy Genes From Weedy Rice Respond Divergently to Seed Development Environments[J]. Genetics,2006,172:1199-1211
[18] Wan J M,Cao Y J,Wang C M.Quantitative trait loci associated with seed dormancy in rice[J]. Crop Sci.,2005,45:712-716
[19] Murray M G,Thompson W F.The isolation of high molecular weight plant DNA[J]. Nucleic Acids Res.,1980,8:4321-4325
[20] Dieffenbach C W,Dveksler G S.PCR Primer:a laboratory manual[M]. New York:Cold Spring Harbor Laboratory Press,1995.23-26
[21] Birren B,Eric D,Green E D.Analysing DNA:a laboratory manual[M]. New York:Cold Spring Harbor Laboratory Press,1997.264-266

结缕草SSR反应体系的正交优化(英文)

Optimization of the SSR Reaction System for Zoysiagrass Using Orthogonal Design

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	王子玥, 刘曼, 刘凌云, 常智慧. 苇状羊茅EST-SSR标记在草甸羊茅和多年生黑麦草的通用分析[J]. 草地学报, 2021, 29(9): 1900-1908.
[2]	叶刚, 张笑笑, 陈静波, 李建建, 李玲, 刘建秀, 郭爱桂, 郭海林. 结缕草属植物杂交后代形态特征和SRAP分子标记鉴定[J]. 草地学报, 2021, 29(7): 1406-1415.
[3]	陈志祥, 罗小燕, 李拴林, 吴如月, 王文强, 丁西朋. 基于SSR标记的木豆种质资源遗传多样性与群体结构分析[J]. 草地学报, 2021, 29(5): 904-911.
[4]	许正, 徐倩, 刘明稀, 向佐湘. 除草剂‘禾耐斯’对‘兰引3号’结缕草交播草坪春季转换的影响[J]. 草地学报, 2021, 29(3): 603-609.
[5]	单旭东, 高娅楠, 张桐瑞, 程锦, 许立新. 保温和浇水处理对结缕草冬季胁迫的缓解作用[J]. 草地学报, 2021, 29(1): 202-210.
[6]	黄春琼, 张子雄, 王文强, 刘国道. 柱花草花粉不育基因连锁的SCAR标记研究[J]. 草地学报, 2020, 28(5): 1478-1483.
[7]	罗建章, 李双铭, 向佐湘, 胡龙兴. 浦绿对高尔夫球道结缕草草坪耐荫性的影响[J]. 草地学报, 2020, 28(3): 743-749.
[8]	滕珂, 檀鹏辉, 范希峰, 岳跃森, 姜红岩, 武菊英. 日本结缕草衰老叶片全长cDNA文库构建及酵母单杂交筛选验证[J]. 草地学报, 2019, 27(6): 1486-1493.
[9]	李双铭, 徐庆国, 杨勇, 刘雪勇, 罗建章, 向佐湘, 胡龙兴. 低温胁迫对结缕草抗氧化酶活性和脂肪酸含量的影响[J]. 草地学报, 2019, 27(4): 906-912.
[10]	彭雪, 程俊康, 辛国荣. AMF与坪床基质对“兰引3号”结缕草草坪磷元素有效性及其流失的影响[J]. 草地学报, 2019, 27(3): 553-559.
[11]	孙金金, 焦婷, 李亚娟, 王芳, 汪鹏斌, 鱼小军. 高寒区草地-家畜优化配置研究——以青海省玛沁县1牧户为例[J]. 草地学报, 2019, 27(3): 728-735.
[12]	李统华, 冯中红, 杨成德. 高寒草甸牧草内生解淀粉芽胞杆菌261MY6发酵工艺优化[J]. 草地学报, 2019, 27(2): 503-509.
[13]	刘晶, 赵方媛, 李冬梅, 李雪, 田新会, 杜文华. 饲用型小黑麦遗传图谱构建及草产量相关性状QTLs初步定位[J]. 草地学报, 2019, 27(1): 219-226.
[14]	肖晓琳, 王凯, 张兵, 刘建秀. 沟叶结缕草蛋白质二硫键异构酶(ZmPDIs)基因家族耐盐功能分析[J]. 草地学报, 2018, 26(5): 1181-1189.
[15]	王贵珍, 花立民, 杨思维, 赵海忠. 高寒草甸家庭牧场管理模型的初步开发[J]. 草地学报, 2018, 26(4): 991-1003.