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1.
基于琼脂糖凝胶电泳的小麦SSR扩增体系优化 总被引：2，自引：0，他引：2
以小麦基因组DNA为试验材料,探索SSR扩增反应中5种反应组分(模板DNA、dNTPs、引物、Mg~(2+)和Taq DNA聚合酶)对SSR扩增结果的影响.研究发现,引物、Mg~(2+)和Taq DNA聚合酶对PCR扩增结果的影响最显著,其次是模板DNA,dNTP对PCR扩增结果的影响不明显.此外,借助该优化的SSR反应体系,对小麦7B染色体上的多对SSR引物进行了多态性筛选.试验结果表明,优化的SSR扩增体系结合高浓度琼脂糖凝胶能够对显性或差异显著的共显性标记进行高效分离. 相似文献
2.
大豆SSR技术反应体系的优化 总被引：3，自引：0，他引：3
以大豆为材料,研究了PCR反应体系的主要成分模板DNA浓度、dNTP浓度、引物浓度、Taq酶浓度及退火温度对大豆SSR扩增结果的影响,探索影响SSR扩增结果的各因素的最佳用量及引物的退火温度。结果表明:在试验设计范围内,DNA浓度和dNTP浓度对扩增影响较大,引物浓度在0.05~0.2μmol/L范围内对扩增影响较小,Taq酶浓度对扩增有一定影响,引物要有其扩增适宜的退火温度。确立了适合大豆SSR分子标记研究的优化体系。最终确定总反应体系为20μL,模板DNA 20 ng,dNTP 200μmol/L,引物0.15μmol/L,Taq酶0.5 U,10×Taq Buffer 1.5 mmol/L,ddH2O补至20μL。 相似文献
3.
以橡胶树品种热研88-13为试材,研究了橡胶树SAM-SSR法中各个过程（酶切、连接、抑制性和预扩增）中各因素对SAM法结果的影响。实验结果表明：5U MseⅠ和5U PstⅠ双酶切1h为最合适酶切浓度、方式和时间; 100~300ng为适宜的DNA模板浓度; 合适的连接温度和时间为16℃连接过夜;连接产物和抑制性PCR产物分别稀释20倍,为抑制性PCR和预扩增PCR的适宜模板浓度。 SAM扩增产物用8%的非变性聚丙烯酰胺凝胶检测,结果显示,扩增条带分布在 200~750bp之间,能够满足建立橡胶树SSR基因组文库的需要。 相似文献
4.
棉花SSR多重PCR技术的初步研究和利用 总被引：3，自引：0，他引：3
简单重复序列(simple sequences repeat,SSR)具有数量丰富、高度多态、共显性等优点,是一种极具实用价值的标记,目前已成为研究种质资源遗传多样性、基因组作图的首选标记.SSR多重PCR扩增法可同时完成多个标记检测,具有高效、经济、快捷的特点,并保持了单一SSR引物扩增的高度敏感性和准确性.本文选取6对SSR引物,配成三组SSR两重PCR反应,并设置五种不同反应体系以探索PCR反应体系对其影响,以单一引物PCR扩增为对照,对用于构建亚洲棉(Gossypium arboreum)遗传图谱所用的亲本、F1及F2群体单株的DNA模板进行多重PCR探索、分析;另外,在扩增产物的电泳检测时,结合银染技术,试验了单一引物PCR扩增产物在同一泳道同时上样和先后分时上样的两重检测的方法,以期对这种方法进行多态性位点检测的效果进行评价.结果表明,即使采用与单一引物PCR相同的反应体系,两重PCR扩增也可获得与之相同的多态性产物;而两重检测法的结果亦显示出与相应的单引物检测到的相应的多态性位点,能准确反映单引物所能达到的检测效果,因而为SSR的多重PCR和扩增后多重检测的技术在高效的遗传图谱构建工作中进一步应用提供实验依据. 相似文献
5.
正交设计优化大豆SSR-PCR反应体系及引物筛选 总被引：5，自引：0，他引：5
以大豆(Glycine max L.)为材料,研究了PCR反应体系的主要成分对大豆SSR扩增结果的影响,并确定影响SSR扩增结果的各因素的最佳用量.以CTAB法提取的大豆叶片DNA为模板,应用L16(44)正交设计对影响大豆SSR-PCR的主要参数进行优化,建立适合大豆SSR-PCR反应的最佳体系.结果表明:各因素不同水平浓度对PCR反应结果均有显著影响.大豆SSR-PCR优化反应体系为:2.0 μL 10×PCR Buffer,30 ng模板DNA,150μmol/L dNTP,0.4 μmol/LSSR引物,1.5 U Taq DNA聚合酶,2.0 mmoL/L Mg2+,加ddH2O至终体积20.0μL.优化的PCR扩增程序为:94℃预变性5 min.94℃变性30 s,50℃退火1 min,72℃延伸1 min,共35个循环,72℃延伸5 min,4℃保存.同时选用200对大豆引物对2份材料进行扩增,筛选出条带清晰,多态性好的引物74对,用于大豆SSR标记的进一步研究. 相似文献
6.
正交设计优化狗牙根SSR-PCR反应体系 总被引：4，自引：0，他引：4
以SDS法提取的狗牙根(Cynodon spp.)叶片DNA为模板,应用正交试验设计,从Mg2 、dNTP、引物和DNA聚合酶4种因素三个水平,对SSR扩增效果进行研究,并通过比较不同浓度模板DNA的浓度对PCR扩增的影响,确立适合狗牙根SSR-PCR反应的最佳体系.结果表明:各因素不同水平浓度对PCR反应结果均有显著影响,狗牙根SSR-PCR优化反应体系为:10×buffer、60 ng模板DNA、Mg2 2.5 mmol/L、dNTP 200 μmol/L、primer 0.8 μmol/L和Taq DNA聚合酶0.5U,总体积10 μL.运用该体系对包括三种10份优良坪用型狗牙根进行了检验,证明该体系稳定可靠.这一优化体系的建立为今后利用SSR标记技术进行狗牙根遗传多样性研究、种质资源鉴定以及亲缘关系分析等提供依据. 相似文献
7.
8.
SSR分子标记已经得到普遍应用,初学者如不能很好地掌握实验操作要点,就难以在较短时间内成功地获得试验结果.本文分别介绍了DNA提取、PCR扩增和电泳检测PCR扩增产物等实验过程中容易被忽视的注意事项,以期为普及SSR分子标记实验技术服务. 相似文献
9.
【目的】针对我国进口棉花原料以及收储棉花的品种产地溯源监控技术体系缺乏的问题,探索了适用于棉纤维DNA的提取方法,旨在建立以棉纤维DNA为介质鉴定棉花品种真实性及产地溯源的体系。【方法】通过改进和优化提取方法,提取了开花后不同时间棉纤维及不同年份皮棉的DNA,以其作为模板,进行了常规聚合酶链式反应(Polymerase chain reaction,PCR)扩增;并选取13对简单重复序列(Simple sequence repeat,SSR)引物对鲁1127、石抗126和瑞杂816棉纤维DNA进行SSR-PCR扩增。【结果】此DNA提取法可提取出不同发育时期和不同年份的棉纤维DNA,随着纤维发育成熟及年代增加,所提取的DNA含量尽管有所降低,但仍能满足下游试验需求;所提取的棉纤维DNA可进行常规PCR扩增,且PCR扩增条带清晰;以13对棉花SSR引物对鲁1127、石抗126和瑞杂816棉纤维DNA进行SSR-PCR扩增,均可扩增出清晰的多态性谱带,且易于区分。【结论】该提取法适合提取棉花纤维的基因组DNA,且满足于常规PCR扩增和SSR分子标记。本研究证实基于棉纤维DNA分子标记用于棉花品种溯源切实可行,并有望为保障我国棉花产业安全提供技术支持。 相似文献
10.
《分子植物育种》2020,(17)
SSR-PCR反应体系的建立与优化是凤仙花属植物(Impatiens L.) SSR标记研究的基础,本研究以8种凤仙花属植物为试材,对Mg~(2+)、Taq酶、dNTPs浓度、DNA模板、引物浓度等5因素进行L_(16)(4~5)正交试验,探讨适合凤仙花属植物的SSR-PCR反应体系,并对主要因素进行优化。结果表明:Mg~(2+)、Taq酶和dNTPs浓度3个因素对凤仙花属SSR-PCR扩增结果有显著影响,影响程度为Mg2+Taq酶dNTPs浓度DNA模板引物浓度;20μL为最佳反应体系,其中,2.6 mmol/L (含10×PCR Buffer)的Mg~(2+),1.6 mmol/L的dNTPs,2μmol/L的引物,60 ng的DNA,0.1 U的Taq酶,ddH_2O补齐剩余部分。利用优化后的反应体系对同属54种材料进行PCR扩增,扩增产物在130～200 bp,具4个等位基因,目标条带清晰,多态性良好。该体系的建立可为今后利用SSR标记对凤仙花种质鉴定、遗传多样性分析、系统发育研究、遗传图谱构建、基因定位和分子标记辅助育种等研究提供了依据。 相似文献
11.
[Objective] To address the questions of lacking monitoring system for origins of varieties, this study explored a DNA extraction method suitable for cotton fiber, aiming to establish an identification system that uses cotton fiber DNA as a medium to trace the authenticity and cultivar of cotton varieties. [Method] By improving and optimizing the extraction method, the DNAs of cotton fibers at different days post anthesis and lint from different years were extracted and used as templates for routine polymerase chain reaction (PCR) amplification. 13 pairs of simple sequence repeat(SSR) primers were selected for SSR-PCR amplification using the DNA of Lu 1127, Shikang 126 and Ruiza 816. [Result] The DNA extraction method can extract cotton fiber DNAs of different developmental stages and different years. With the development of fiber, although the extracted DNA content is reduced, it can still meet the needs of downstream experiments. The extracted cotton fiber DNAs can be subjected to conventional PCR amplification, the PCR amplified bands were clear; 13 pairs of cotton SSR primers were used for SSR-PCR amplification of cotton fiber DNAs of Lu 1127, Shikang 126 and Ruiza 816, and a clear polymorphic band could be amplified and easily distinguished. [Conclusion] This extraction method is suitable for extracting genomic DNA of cotton fiber, and is satisfied with conventional PCR amplification with SSR markers. It is confirmed that the cotton fiber DNA molecular markers are feasible for tracing the source of cotton varieties, and it is expected to provide technical support for ensuring the safety of cotton industry. 相似文献
12.
以PCR为目的的大豆叶片DNA提取方法的比较研究 总被引：16，自引：0，他引：16
模板DNA的质量直接影响PCR扩增的结果,而不同提取方法及其缓冲液的成份与浓度对提取DNA的质量有重要影响。本文以5个栽培大豆品种的叶片为材料,比较分析了SDS与CTAB两种提取方法以及不同浓度CTAB提取缓冲液对所提取的DNA质量的影响,并通过PCR进行检验。实验结果表明：用1％(W/V)、2％(W/V)浓度的CTAB提取缓冲液和1．25％(WV)SDS提取缓冲液所提取的大豆叶片DNA的质量较好,均能满足PCR扩增模板的需求,其中以1．25％(W／V)SDS提取得到的大豆叶片DNA质量最好,以其为模板扩增的效果最佳,而4％浓度的CTAB不适宜提取大豆叶片DNA。 相似文献
13.
3种PCR程序扩增甜菜SSR及InDel标记的比较 总被引：1，自引：1，他引：0
本研究旨在扩增出条带清晰、非特异性条带少的SSR/InDel标记产物。选择来自6个国家的12份不同基因型甜菜品种,利用SSR及InDel两种标记方法,Touch-down PCR、梯度PCR及常规固定退火温度PCR三种反应程序扩增产物。通过比较扩增产物的多态性、稳定性、清晰度选择适合两种分子标记方法的PCR反应程序。结果表明:Touch-down PCR法可以通过一次反应即顺利扩增出带型清晰,无无效带,扩增效果稳定的样本产物。梯度PCR法扩增结果不稳定,有非特异性条带出现并且弥散现象严重。固定退火温度PCR扩增结果因引物不同而差异较大,同时伴有弥散现象。Touch-down PCR扩增产物特异性,稳定性均高于梯度PCR及固定退火温度PCR,且较梯度PCR法省略了摸索最适退火温度的过程。因此,推荐使用Touch-down PCR程序进行甜菜SSR与InDel分子标记法研究。 相似文献
14.
Giuseppe Mandolino Andrea Carboni Manuela Bagatta V.M. Cristiana Moliterni Paolo Ranalli 《Euphytica》2002,126(2):211-218
DNA from female and male hemp (Cannabis sativa L.) plants belonging to nine different varieties were screened with180 RAPD primers in a search for sex-associated DNA markers.
About 1500bands were produced in total, nine primers were found yielding one or two DNA bands amplified in all nine male DNA
bulks and absent in all female DNA bulks. These putatively male-associated markers were then scored in three different F1progenies,
deriving from a cross between a common male parent and three different female plants. The sex of the progeny was accurately
scored on the basis of the floral phenotype, and the presence of the nine male-associated markers was verified by RAPD analysis.
In all three progenies examined, all the male plants showed the DNA markers previously identified by bulk segregant analysis
(BSA) on the hemp varieties, while all the female plants lacked them. The fact that the association between these markers
and the staminate phenotype is found when examining male plants of distantly related varieties, and that such linkage is never
broken when different progenies are examined, strongly supports the hypothesis that the markers found are physically located
on the Y chromosome, in a region excluded from recombination during meiosis. Another marker was shown to be present in the
male parent, in all the male plants of each progeny, and in 50% of the female progenies, while it was absent in the female
parent; the possible occurrence of markers deriving from multiple amplification sites of the genome is discussed.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
15.
芝麻SSR检测体系的优化与应用 总被引：1，自引：1，他引：0
为了建立一套适用于芝麻SSR标记检测的技术体系,以5个芝麻新品种为试验材料,选用35对SSR引物,从反应体系、反应程序、电泳、银染4个环节进行优化。结果表明：反应总体积为10 μL,优化后的基本成分及用量分别为25 mmol/L Mg2+ 1.2 μL,10 mmol/L dNTPs 0.3 μL,5 U/μL Taq酶0.3 μL,50 ng/μL Primer 0.9 μL,10×Buffer 0.7 μL,25 ng/μL DNA模板2.5 μL,ddH2O 4.1 μL。反应程序为：94℃ 3 min;94℃ 1 min,60℃ 30 s,72℃ 45 s,10个循环;94℃ 30 s,57℃ 30 s,72℃ 45 s,30个循环;72℃ 5 min。PCR产物用6%变性聚丙烯酰胺凝胶电泳,硝酸银快速染色检测效果良好。筛选出多态性较强的SSR引物24对,用这些引物对20个品种进行扩增,初步分析了SSR标记应用于芝麻DNA指纹分析的潜力。 相似文献
16.
G. Barcaccia L. Pallottini M. Soattin R. Lazzarin P. Parrini M. Lucchin 《Plant Breeding》2003,122(2):178-183
Red or variegated chicory (Cichorium intybus L., 2n = 2 ×= 18) native to, and very extensively cultivated in north‐eastern Italy as a leafy vegetable, locally called ‘radicchio’, includes different types which represent valuable high‐quality crops. The five major types of radicchio cultivated in the Veneto region were investigated by polymerase chain reaction (PCR)‐derived markers. The experimental material was represented by two outbred populations (one of ‘Variegato di Castel‐franco’ (CF) and one of ‘Rosso di Verona’ (VR)) and by eight inbred lines (three of early ‘Rosso di Treviso’ (TVP), three of late ‘Rosso di Treviso’ (TVT) and two of ‘Rosso di Chioggia’ (CH)). A total of 96 individual plant DNAs and 16 bulked DNAs of six plants each were assayed. The different types were well distinguished from one another if analysed by means of bulks using amplified fragment length polymorphism markers at the population level, while they were not if analysed at the individual level using random amplified polymorphic DNA, inter‐simple sequence repeat and Arbitrarily Primed (AP)‐PCR markers. The genetic variation was shown to be much higher within types than between types. This result suggests that, in each radicchio type, populations produced by breeders through controlled intercrossing (VR and CF) or repeated selfing (TVP, TVT and CH) conserved their well‐separated gene pools over the years. The setting up of a molecular reference system seems to be feasible and suitable both for the precise identification of the single types of radicchio and for the evaluation of the extent of natural hybridization that can occur between different types. 相似文献
17.
为了建立Lm型雌性系蓖麻不同发育时期的标雌/单雌/两性系花序MSAP反应体系,提取不同类型不同发育时期的花序基因组DNA,混合成基因池;用EcoRⅠ和HpaⅡ/MspⅠ的组合,12 h可将DNA酶切完全;16℃条件下,将酶切产物与EcoRⅠ、HpaⅡ/MspⅠ接头过夜连接;连接产物用于预扩增。优化后的预扩增反应体系为10×PCR Buffer 2.5μL、d NTPs(10 mmol/L)2.5μL、Mg2+(25 mmol/L)2.0μL、模板2.0μL、E0扩增引物(10pmol/μL)1.5μL、H0扩增引物(10 pmol/μL)1.5μL、LA Taq(5 U/μL)0.25μL、dd H2O 12.75μL。预扩增产物稀释10倍后用于选择性扩增。优化后的选择性扩增体系为10×PCR Buffer 2.5μL、d NTPs(10 mmol/L)2.0μL、Mg2+(25mmol/L)4.0μL、模板3.0μL、EX扩增引物(10 pmol/μL)1.0μL、H/MX扩增引物(10 pmol/μL)1.0μL、LA Taq(5U/μL)0.30μL、dd H2O 11.2μL。 相似文献
18.
Enhancement of RAPD Analysis by Restriction-endonuclease Digestion of Template DNA in Wheat 总被引：1，自引：0，他引：1
C.R. Riede D.J. Fairbanks W.R. Andersen R.L. Kehrer L.R. Robison 《Plant Breeding》1994,113(3):254-257
Random amplified polymorphic DNA (RAPD) analysis has proven to be an effective procedure for molecular marker applications in plant breeding, although non-specific amplification may limit its utility in some species. The objective of this study was to determine the effectiveness of restriction-endonuclease digestion of template DNA for elimination of non-specific amplification and generation of heritable RAPD markers. Restriction endonucleases digested wheat DNA to completion in amplification buffer, suggesting that the restriction endonuclease can be added directly to the reaction mixture prior to amplification. A 1-h 37°C step was programmed into the thermal cycler for restriction-endonuclease digestion which was followed immediately by amplification. Non-specific amplification was reduced and DNA marker patterns were altered in digested samples when compared to undigested samples. Genetic segregation of two polymorphic markers tested in F5 inbred progeny fit expected 1:1 ratios. These results suggest that heritable DNA markers may be generated with reduction in non-specific amplification when restriction-endonuclease digestion of template DNA is conducted as part of the RAPD procedure. 相似文献
19.
太行花DNA提取的优化和适用分子标记检测 总被引：1，自引：1，他引：0
比较了常规CTAB法、改良CTAB法和SDS法对太行花叶片总DNA的提取效果,并对改良CTAB法提取的DNA在多种分子标记中的适用性进行了测试。结果表明：常规CTAB法提取的DNA难以完全溶解,且有褐化现象;SDS法提取的DNA产率及纯度都很低;改良CTAB法提取的DNA产率高且稳定,无明显降解,杂质少,OD260/OD280比值在1.8左右。以改良CTAB法提取的DNA为模板,应用叶绿体和线粒体通用引物扩增出了特异性的高效产物,ISSR和RAPD引物对总DNA的扩增也获得理想结果。因此,改良CTAB法适用于太行花总DNA提取,其产物能满足核、叶绿体和线粒体基因组分子实验的要求。 相似文献