云南草果SSR-PCR反应体系的优化及引物的筛选
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摘要
草果为药食两用的草本植物,近年来在市场上供不应求,但关于草果居群的分子鉴定及种质资源的研究极为稀少。为使云南草果产业更加健康和可持续的发展,本实验对云南草果进行了SSR-PCR反应体系的优化及引物的筛选,为研究草果遗传多样性提供了依据。实验采用L16(43)正交试验设计,对PCR反应体系中的3个因素(2×Taq PCR Starmix,引物, DNA模板)进行优化,并利用单因素分析法,对PCR退火温度、PCR扩增产物点样量进行实验与分析。最终确定5个因素的最佳水平,即DNA模板(50 ng/μL) 2μL,2×Taq PCR StartMix 6μL,正反引物(10μmol/L)各1.5μL,dd H2O补足至20μL (即加入ddH2O 9μL),PCR产物点样量可以选择在1.5～2μL之间。从48对SSR引物中筛选出了7对扩增结果好的引物。7个SSR引物可用于草果资源的遗传多样性研究,为分子技术研究提供了科学依据。
Amomum tsao-ko is a herbaceous herb which used for medicine and food. In recent years, it has been in short supply on the market. However, the molecular identification and germplasm resources of Amomum tsao-ko population are extremely rare. In order to make the Yunnan Amomum tsao-ko industry more healthy and sustainable development, the optimization of SSR-PCR reaction system and screening of primers were carried out in this experiment, which provided the basis for the study of the genetic diversity of Amomum tsao-ko. Experiments and analysis were performed on 3 factors including 2×Taq PCR Starmix, primers, and DNA template in the PCR reaction using L16(43) orthogonal test design, single factor PCR annealing temperature, and the amount of PCR amplification product. The best level of factors. Finally, the best level of 5 factors was determined. DNA(50 ng/μL) 2 μL,2 ×Taq PCR StartMix 6 μL, forward primer and reverse primer(10 μmol/L) each 1.5 μL, and fill up to 20 μL with ddH2O(add ddH2O9 μL). 7 pairs of primers which have good amplification results were screened out from 48 pairs of SSR primers. 7 pairs of SSR primers can be used to explore genetic diversity of Amomum tsao-ko resources, this study provid the basis with for molecular technology.
Amomum tsao-ko is a herbaceous herb which used for medicine and food. In recent years, it has been in short supply on the market. However, the molecular identification and germplasm resources of Amomum tsao-ko population are extremely rare. In order to make the Yunnan Amomum tsao-ko industry more healthy and sustainable development, the optimization of SSR-PCR reaction system and screening of primers were carried out in this experiment, which provided the basis for the study of the genetic diversity of Amomum tsao-ko. Experiments and analysis were performed on 3 factors including 2×Taq PCR Starmix, primers, and DNA template in the PCR reaction using L16(43) orthogonal test design, single factor PCR annealing temperature, and the amount of PCR amplification product. The best level of factors. Finally, the best level of 5 factors was determined. DNA(50 ng/μL) 2 μL,2 ×Taq PCR StartMix 6 μL, forward primer and reverse primer(10 μmol/L) each 1.5 μL, and fill up to 20 μL with ddH2O(add ddH2O9 μL). 7 pairs of primers which have good amplification results were screened out from 48 pairs of SSR primers. 7 pairs of SSR primers can be used to explore genetic diversity of Amomum tsao-ko resources, this study provid the basis with for molecular technology.
引文
Guo X.,Ni Z.X.,Luo J.Z.,and Xu L.A.,2017,Optimization of SSR-PCR reaction system and primer screening of Eucalyptus,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(2):626-632(郭鑫,倪州献,罗建中,徐立安,2017,桉树SSR-PCR体系优化及引物筛选应用,分子植物育种,15(2):626-632)
Hu Y.,Li Y.,Huang Y.Z.,and Liu P.W.,2016,Genetic diversity evaluation of sugarcane varieties using SSR and RAPD markers,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(9):2494-2503(胡杨,李赟,黄有总,刘平武,2016,利用SSR与RAPD分子标记评估甘蔗品种的遗传多样性,基因组学与应用生物学,35(9):2494-2503)
Sun J.,Yan T.X.,Tu Y.Q.,Le M.W.,Rao Y.L.,Lei G.,Yan X.W.and Zhou H.Y.,2015,Orthogonal optimization and verification of RSAP-PCR reaction system in sesame(Sesamum indicum L.),Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(10):2200-2209(孙建,颜廷献涂玉琴,乐美旺,饶月亮,雷刚,颜小文,周红英,2015,芝麻RSAP-PCR反应体系的正交优化与验证,基因组学与应用生物学,35(10):2200-2209)
Yang N.,Su W.M.,Jin Y.H.,and Wang J.,2016,Establishmen of SSR-PCR reaction system and primers screening for Curcama kwangsinesis S.G.Lee C.F.Liang,Hubei Nongye Kexue(Hubei Agricultural Sciences),55(23):6270-6275(杨妮,苏伟敏,靳雅慧,王建,2016,广西莪术SSR-PCR反应体系优化和引物筛选研究,湖北农业科学,55(23):6270-6275)
Zhang L.,Ding X.P.,Yan L.L.,Hu S.N.,and Bai C.J.,2014,Optimization of SSR-PCR system and primers screening of 8species of Stylosanthe,Caoye Kexue(Pratacultural Science),31(2):232-242(张龙,丁西朋,严琳玲,胡珊娜,白昌军,2014,8种柱花草属牧草SSR-PCR反应体系优化和引物筛选,草业科学,31(2):232-242)
Zhang L.F.,and Fang Y.M.,2018,Optimization and primer delection of SSR-PCR system on Parrotia subaequalis,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),16(16):5340-5346(张丽芳,方炎明,2018,银缕梅SSR-PCR反应体系的优化及引物筛选,分子植物育种,16(16):5340-5346)
Hu Y.,Li Y.,Huang Y.Z.,and Liu P.W.,2016,Genetic diversity evaluation of sugarcane varieties using SSR and RAPD markers,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(9):2494-2503(胡杨,李赟,黄有总,刘平武,2016,利用SSR与RAPD分子标记评估甘蔗品种的遗传多样性,基因组学与应用生物学,35(9):2494-2503)
Sun J.,Yan T.X.,Tu Y.Q.,Le M.W.,Rao Y.L.,Lei G.,Yan X.W.and Zhou H.Y.,2015,Orthogonal optimization and verification of RSAP-PCR reaction system in sesame(Sesamum indicum L.),Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(10):2200-2209(孙建,颜廷献涂玉琴,乐美旺,饶月亮,雷刚,颜小文,周红英,2015,芝麻RSAP-PCR反应体系的正交优化与验证,基因组学与应用生物学,35(10):2200-2209)
Yang N.,Su W.M.,Jin Y.H.,and Wang J.,2016,Establishmen of SSR-PCR reaction system and primers screening for Curcama kwangsinesis S.G.Lee C.F.Liang,Hubei Nongye Kexue(Hubei Agricultural Sciences),55(23):6270-6275(杨妮,苏伟敏,靳雅慧,王建,2016,广西莪术SSR-PCR反应体系优化和引物筛选研究,湖北农业科学,55(23):6270-6275)
Zhang L.,Ding X.P.,Yan L.L.,Hu S.N.,and Bai C.J.,2014,Optimization of SSR-PCR system and primers screening of 8species of Stylosanthe,Caoye Kexue(Pratacultural Science),31(2):232-242(张龙,丁西朋,严琳玲,胡珊娜,白昌军,2014,8种柱花草属牧草SSR-PCR反应体系优化和引物筛选,草业科学,31(2):232-242)
Zhang L.F.,and Fang Y.M.,2018,Optimization and primer delection of SSR-PCR system on Parrotia subaequalis,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),16(16):5340-5346(张丽芳,方炎明,2018,银缕梅SSR-PCR反应体系的优化及引物筛选,分子植物育种,16(16):5340-5346)