蚕豆SSR-PCR体系建立及优化
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摘要
为了建立适宜蚕豆的SSR-PCR反应体系,用于蚕豆的SSR分子标记研究。以青海12号、透心绿为材料,通过正交设计L16(45)对蚕豆SSR-PCR反应体系中的Taq酶,Mg~(2+),dNTPs,引物和模板DNA 5个因素的4个水平进行优化试验。结果表明:各因素不同水平对PCR反应均有影响,各因素对PCR扩增结果的影响大小分别为:Taq酶>dNTPs>模板DNA>引物>Mg2+。最终筛选出蚕豆最佳SSR-PCR反应体系(20μL):1U Taq酶,0.75 mmol/L d NTPs,100 ng模板DNA,2μmol/L引物和1.5 mmol/L Mg~(2+)。
The variety of Qinghai No.12 and Touxinlv were chosen as experimental materials in order to establish the appropriate SSR-PCR system for analyzing SSR molecular marker of faba bean. The SSR-PCR system of faba bean were optimized by using the orthogonal design L_(16)( 4~5) for Taq DNA polymerase,Mg~(2+),dNTPs,primer and DNA template. The results showed that different levels of concentration of various were significantly affected the results of PCR. And the effect size is Taq DNA polymerase > d NTPs > DNA template > primer > Mg~(2+). Finally,an optimal 20μL volumes reaction system of faba bean consisted of 1U Taq DNA polymerase,0. 75 mmol/L d NTPs,100 ng DNA template,2μmol/L primer and 1. 5 mmol/L Mg~(2+).
The variety of Qinghai No.12 and Touxinlv were chosen as experimental materials in order to establish the appropriate SSR-PCR system for analyzing SSR molecular marker of faba bean. The SSR-PCR system of faba bean were optimized by using the orthogonal design L_(16)( 4~5) for Taq DNA polymerase,Mg~(2+),dNTPs,primer and DNA template. The results showed that different levels of concentration of various were significantly affected the results of PCR. And the effect size is Taq DNA polymerase > d NTPs > DNA template > primer > Mg~(2+). Finally,an optimal 20μL volumes reaction system of faba bean consisted of 1U Taq DNA polymerase,0. 75 mmol/L d NTPs,100 ng DNA template,2μmol/L primer and 1. 5 mmol/L Mg~(2+).
引文
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[9]闫伟,姚丹,张洋,等.大豆SSR-PCR反应体系优化[J].河南农业科学,2010(8):36-39.
[10]龙萄,黄春琼,刘国道.正交设计优化狗牙根SSR-PCR反应体系[J].基因组学与应用生物学,2016,35(1):198-205.
[11]王志勇,郭海林,刘建秀.正交设计优化狗牙根SSR-PCR反应体系[J].分子植物育种,2007,5(6):201-206.
[12]帕提古丽·麦麦提敏,张延辉,李培英,等.狗牙根EST-SSR反应体系优化及其遗传多样性初步分析[J].新疆农业大学学报,2014,37(2):112-118.
[2]韩梅,马晓彤,曹卫东,等.青海蚕豆根瘤菌的系统发育与多样性研究[J].青海大学学报(自然科学版),2015,33(5):5-9.
[3]MOORE S S,SARGEANT L L,KING T J,et al.The conservation of dinucleotide microsatellites among mammalian genomes allows the use of heterologous PCR primer pairs in closely related species[J].Genomics,1991,10(3):654-660.
[4]罗冉,吴委林,张旸,等.SSR分子标记在作物遗传育种中的应用[J].基因组学与应用生物学,2010,29(1):137-143.
[5]杨迪菲,秦智伟,王桂玲,等.黄瓜SSR-PCR反应体系的优化[J].东北农业大学学报,2006,37(5):619-623.
[6]李亚利,陈书霞,孟焕文,等.利用正交设计优化黄瓜的SSR-PCR反应体系[J].西北农业学报,2008,17(3):280-284.
[7]侯万伟,刘玉皎.蚕豆RAPD反应体系的建立与优化[J].西南农业学报,2011,24(1):194-196.
[8]刘玉皎,李萍,张小田.β-巯基乙醇和PVP对蚕豆DNA质量的影响[J].湖北农业学报,2008,47(3):248-250.
[9]闫伟,姚丹,张洋,等.大豆SSR-PCR反应体系优化[J].河南农业科学,2010(8):36-39.
[10]龙萄,黄春琼,刘国道.正交设计优化狗牙根SSR-PCR反应体系[J].基因组学与应用生物学,2016,35(1):198-205.
[11]王志勇,郭海林,刘建秀.正交设计优化狗牙根SSR-PCR反应体系[J].分子植物育种,2007,5(6):201-206.
[12]帕提古丽·麦麦提敏,张延辉,李培英,等.狗牙根EST-SSR反应体系优化及其遗传多样性初步分析[J].新疆农业大学学报,2014,37(2):112-118.