越桔ISSR-PCR反应体系建立与优化
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摘要
以改良CTAB法提取越桔(Vaccinium Linn.)品种薄雾DNA为材料,采用正交设计L16(45)表探讨了Tag酶;Mg~(2+);DNA;dntps及引物浓度5个主要因素对越桔ISSR-PCR扩增反应的影响,通过直观分析和方差分析结合,建立了越桔ISSR-PCR(20μL)最佳反应体系,各组分的浓度为:Tag酶0.75 U,Mg~(2+)1.875 mmol/L,DNA 0.8mmol/L,dntp 0.1mmol/L,引物0.2μmol/L.在此基础上探讨了引物UBC835的最佳退火温度、循环次数和延伸时间,结果分别为54.2℃、35次、60s.选用两个引物对21份越桔资源进行ISSR扩增,结果显示优化的体系稳定性较高.
Based on the genomic DNA extracted from Vaccinium Linn.cultivar Misty with a modified CTAB method,five factors influencing ISSR were optimized in an orthogonal-design experiment.Through intuitive analysis and variance analysis,an optimal ISSR-PCR reaction system was established for this fruit crop,namely 10×buffer 2μL;0.75 U Taq DNA polymerase;1.875 mmol/L Mg~(2+);80ng template DNA;0.1mmol/L dNTPs;and 0.2μmol/L primer.The optimal annealing temperature,number of cycles and extension time of primer UBC835 were investigated,and the results were 54.2 ℃,35 cycles and60s,respectively.Twenty-one Vaccinium accessions were used to test the stability of the optimized reaction system.The results indicated that the optimized reaction system was very stable.
Based on the genomic DNA extracted from Vaccinium Linn.cultivar Misty with a modified CTAB method,five factors influencing ISSR were optimized in an orthogonal-design experiment.Through intuitive analysis and variance analysis,an optimal ISSR-PCR reaction system was established for this fruit crop,namely 10×buffer 2μL;0.75 U Taq DNA polymerase;1.875 mmol/L Mg~(2+);80ng template DNA;0.1mmol/L dNTPs;and 0.2μmol/L primer.The optimal annealing temperature,number of cycles and extension time of primer UBC835 were investigated,and the results were 54.2 ℃,35 cycles and60s,respectively.Twenty-one Vaccinium accessions were used to test the stability of the optimized reaction system.The results indicated that the optimized reaction system was very stable.
引文
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[10]李广平,柴梦颖,张长青.蓝莓ISSR-PCR反应体系建立与优化[J].西北农业学报,2010,19(12):134-137.
[11]张鲁杰,夏秀英,徐娜,等.高效提取越橘成熟组织基因组DNA的方法[J].华北农学报,2008,23(增刊):205-208.
[12]何正文,刘运生,陈立华,等.正交设计直观分析法优化PCR条件[J].湖南医科大学学报,1998,23(4):403-404.
[13]王永清,付燕,杨芩,等.枇杷属植物遗传多样性的ISSR分析[J].林业科学,2014,4(46):201-211.
[14]桂腾琴,孙敏,乔爱民,等.正交设计优化果梅ISSR反应体系[J].果树学报,2009,26(1):108-112.
[15]郑姗,张立杰,谢丽雪,等.蓝莓品种ISSR指纹图谱构建的初步研究[J].福建农业学报,2014,29(12):1198-1201.
[16]郭凌飞,邹明宏,曾辉,等.澳洲坚果ISSR-PCR反应体系的建立与优化[J].林业科学,2008,11(5):37-42.
[17]CARVALHO M,MATOS M,CARNIDE V.Fingerprinting of Vaccinium Corymbosum Cultivars Using DNA of Fruits[J].Horticultural Science,2014,41(4):175-184.
[18]胡甦,王永清,陶炼.三角紫叶酢浆草叶色变异株系的RAPD和ISSR标记初步鉴定[J].园艺学报,2011,38(5):930-938.
[19]曹嵩晓,李娟玲,刘国民.利用单因子和正交设计双重实验方法优化广藿香ISSR-PCR实验体系[J].热带生物学报,2011,3(2):321-325.
[2]罗慧,丁广文,郭启高,等.基于ISSR标记的44份桃种质资源遗传多样性分析[J].西南大学学报(自然科学版),2015,37(5):45-50.
[3]缪恒彬,陈发棣,赵宏波.85个大菊品种遗传关系的ISSR分析[J].园艺学报,2007,31(05):112-120.
[4]王文恩,包满珠,张俊卫,等.狗牙根辐射诱变后代变异植株的形态特征比较和ISSR分析[J].草业科学,2009,6(12):320-328.
[5]李宏平,江曼,石佳,等.越橘“V3”品种四倍体诱导及鉴定研究[J].西南师范大学学报(自然科学版),2013,38(2):90-95.
[6]崔建民,刘红霞,邹荣仟,等.越橘种质资源遗传多样性和亲缘关系研究[J].果树学报,2010,27(3):373-378.
[7]李雪松,赵康,李凌.越橘“Legacy”离体培养及秋水仙素诱变研究初报[J].西南大学学报(自然科学版),2015,37(5):51-57.
[8]於虹,顾姻,贺善安,等.越橘品种的ISSR鉴定及其亲缘关系分析[J].吉林农业大学学报,2009,31(5):512-515.
[9]宋杨,张红军,窦连登.越橘品种资源亲缘关系的ISSR分析[J].中国果树,2014(6):17-20.
[10]李广平,柴梦颖,张长青.蓝莓ISSR-PCR反应体系建立与优化[J].西北农业学报,2010,19(12):134-137.
[11]张鲁杰,夏秀英,徐娜,等.高效提取越橘成熟组织基因组DNA的方法[J].华北农学报,2008,23(增刊):205-208.
[12]何正文,刘运生,陈立华,等.正交设计直观分析法优化PCR条件[J].湖南医科大学学报,1998,23(4):403-404.
[13]王永清,付燕,杨芩,等.枇杷属植物遗传多样性的ISSR分析[J].林业科学,2014,4(46):201-211.
[14]桂腾琴,孙敏,乔爱民,等.正交设计优化果梅ISSR反应体系[J].果树学报,2009,26(1):108-112.
[15]郑姗,张立杰,谢丽雪,等.蓝莓品种ISSR指纹图谱构建的初步研究[J].福建农业学报,2014,29(12):1198-1201.
[16]郭凌飞,邹明宏,曾辉,等.澳洲坚果ISSR-PCR反应体系的建立与优化[J].林业科学,2008,11(5):37-42.
[17]CARVALHO M,MATOS M,CARNIDE V.Fingerprinting of Vaccinium Corymbosum Cultivars Using DNA of Fruits[J].Horticultural Science,2014,41(4):175-184.
[18]胡甦,王永清,陶炼.三角紫叶酢浆草叶色变异株系的RAPD和ISSR标记初步鉴定[J].园艺学报,2011,38(5):930-938.
[19]曹嵩晓,李娟玲,刘国民.利用单因子和正交设计双重实验方法优化广藿香ISSR-PCR实验体系[J].热带生物学报,2011,3(2):321-325.