A new DNA extraction method for high-throughput marker analysis in a large-genome species such as Triticum aestivum

Gene mapping and marker‐assisted selection in complex, polyploid genomes still relies strongly on restriction fragment length polymorphism (RFLP) analysis, as conversion of RFLP to polymerase chain reaction (PCR) markers can be very difficult. DNA extraction in amounts suitable for RFLP analysis represents the most time‐consuming and labour‐intensive step in molecular marker analysis of plant populations. In this paper, a new flexible method for plant DNA extraction is presented. It allows a high‐throughput of samples in a short time without the need for freezing or lyophilizing the plant material. The method allows the isolation of genomic DNA with a yield of 100 μg for a minimal amount of 200 mg of leaf material. This is sufficient for work with large‐genome plant species such as hexaploid wheat, where 20 μg of genomic DNA are required for a single RFLP analysis.     

高通量的PCR模板植物基因组DNA制备方法

制备大量生物样品的模板DNA用于PCR检测是费时费人工的工作。本文介绍一种高通量的植物基因组DNA (gDNA)快速制备及其用于PCR基因型检测的操作方法。将一小段单子叶植物苗叶片(长度约30 mm或40 mm,与96方孔板的孔深大致相同) 或一小块(约2~4 mg)双子叶植物叶片放入96方孔板的各孔中、放入一粒钨合金珠和150 µL制备缓冲液,盖好硅橡胶盖,在涡旋器振动3~5 min破碎组织。用96针复制器(或多通道移液器)转移每样品约0.5~1.0 µL此粗制gDNA溶液(含有2~3 ng gDNA µL^-1)到96孔PCR板的反应液中,适合用各种类型的PCR标记(如简单序列重复SSR,插入缺失InDel等)进行基因型检测,或较大的DNA片段(>1 kb)的扩增,可以得到良好的效果。本方法的关键是控制合适的破碎叶片量与制备溶液量比例(约2~5 mg, 但不超过10 mg 150 µL^-1溶液),以及不要加入过多量的gDNA (不超过PCR反应液量的1/10),以免带入过量的杂质抑制PCR。因此,这种从种植材料,制备gDNA, 转移样品gDNA,到PCR都是96格式化操作的高通量、低成本方法适合于大量植物样品的规模化的基因型检测。     

Application of SSR markers and real-time PCR for variety identification in azuki-bean [Vigna angularis (Willd.) Ohwi and Ohashi]

The azuki bean in Korea consists of seven domestic varieties which have been developed and registered for the public during last 25 years. Here, we present a simple but reliable method to screen and identify Korean azuki bean varieties. A method based on simple sequence repeat (SSR) markers is widely used for prominent gene identification and variety discrimination. In molecular biology, real-time polymerase chain reaction (PCR) is a laboratory technique based on the polymerase chain reaction that is used to amplify and simultaneously quantify a targeted DNA molecule. It enables easy detection of a specific sequence in a DNA sample without performing electrophoresis and further processes. For separation of seven Korean azuki bean varieties, 110 unique azuki bean SSR markers from an (AG)n-enriched library were selected, synthesized and used for polymerase chain reaction (PCR). Data were taken through acrylamide gel electrophoresis and automated multi-capillary electrophoresis system for selection of specific markers and then changed into proper formats for data mining analysis. Ten primer pairs that showed high polymorphism were chosen for the indepth study. These ten primers were re-amplified with real-time PCR and checked the cycle threshold (Ct) and temperature (Tm) for comparison of amplification sequence in seven varieties. Consequently, a total of 20 alleles and 6 SSR primers were detected from the standard PCR amplification. Within these 6 primers, 7 alleles of 3 SSR primers were isolated for variety identification. From real-time PCR results, 3 SSR primers were selected as efficient markers for discrimination of seven Korean azuki bean varieties. The approach described here could be applied in monitoring our varieties and can be adapted in the azuki bean breeding program.     

Efficient low-cost DNA extraction and multiplex fluorescent PCR method for marker-assisted selection in breeding

Marker‐assisted selection (MAS) is an increasingly important tool in current breeding efforts for improved crop plants and animal breeds. It enables detection of favourable alleles in early developmental stages and thus may result in substantial cost savings. Until now, however, the high costs of the required chemicals and materials, together with the still very labour‐intensive methods, have been an obstacle to widespread application of MAS. A new multiplex‐polymerase chain reaction (PCR)‐based method has been developed for reliable low‐cost, high‐throughput screening. By its use 3366 apple seedlings were screened with an average hands‐on time from DNA extraction to data ready for analysis of < 4 h per 96 plants, and at a cost below US$ 0.5 per marker per plant. Factors that have a strong effect on segregation ratios such as elevated levels of outcrossing are easily detected, as a significant correlation was observed between deviation from expected segregation ratios in some affected markers and the level of outcrossing in a cross. The new method is suitable for many crop species and, provided that suitable buffers are used for DNA extraction, for animals too.     

苹果不同HRM反应体系分析效果评价

高分辨率熔解曲线(high resolution melting,HRM)分析是一种新型的DNA多态性快速筛查技术。本研究以苹果品种"富士"和"舞姿"为试材,用来自苹果基因组的SSR标记CH03d11对不同反应体积、不同DNA浓度以及不同PCR退火程序下的HRM分型效果进行了测试与评估。结果表明:采用5μL反应体积可以获得与10μL或20μL反应体积相同的检测效果。在5μL反应体积中,DNA浓度小到0.25ng/μL时,PCR扩增及随后的HRM分析效果依然良好。另外,研究表明,采用降落PCR扩增模式也能取得良好的HRM多态性检测结果。     

LAMP检测无乳链球菌方法的建立及应用

以无乳链球菌纤连蛋白fbs基因为主要研究对象,采取环介导等温扩增技术(Loop-Mediated Isothermal Amplification,LAMP),针对fbs基因的6个区域设计4条特异性引物,利用一种链置换DNA聚合酶(Bst DNA polymerase)在63℃保温1 h,通过荧光显色即可完成对无乳链球菌的检测工作.结果显示,LAMP方法能够特异性检测fbs基因,其检测灵敏度是常规PCR方法的100倍,并与实时荧光定量PCR方法相当.所建立的针对无乳链球菌fbs基因的LAMP检测方法具有高度的特异性及稳定性,结果可靠,非常适合无乳链球菌的快速检测.     

Stability of RAPD markers for determining cultivar specific DNA profiles in rye (Secale cereale L.)

Summary Cultivar specific DNA profiles in rye were revealed by polymerase chain reaction (PCR) using randomly amplified polymorphic DNA (RAPD) sequences. Ten base primers were used for the amplification of genomic DNA of rye cultivars by PCR. RAPD analysis was found to be reproducible among samples between PCR runs. When amplification profiles of different rye cultivars were compared using various primers, the overall profiles were cultivar specific. However, not all primers revealed polymorphisms. These primers appear to amplify conserved sequences in all rye cultivars. Intracultivar studies were conducted on two of the cultivars. In the cultivar Imperial, no polymorphisms were observed among ten plants analyzed with five primers. In the cultivar Balboa, polymorphisms were observed among fifty plants with four of the ten primers analyzed. Despite the small amount of intracultivar variability, RAPD analysis has the potential to be a rapid and reliable method of cultivar identification in this outcrossing species.     

Random amplified polymorphic DNA analysis of Australian rice (Oryza sativa L.) varieties

Summary The genetic relationships between rice varieties were analysed by using the polymerase chain reaction (PCR), with arbitrary oligonucleotide primers in the random amplified polymorphic DNA (RAPD) method. PCR with 22 arbitrary primers applied to 37 varieties produced 144 useful markers, of which 67% were polymorphic. Thus, with selected primers sufficient polymorphism could be detected to allow identification of individual varieties. Visual examination of electrophoresis gels and analysis of banding patterns confirmed that commercial Australian and USA lines and their relatives were very closely related, with similarity indices of 88–97%. Three varieties originating from more distant geographical centres were easily distinguished, producing variety-specific amplification profiles and expressing a lower similarity index of 80% to all other varieties tested. PCR offers a potentially simple, rapid and reliable method for rice genotype identification and recognition of lines that could contribute genetic diversity to new commercial varieties.Abbreviations PCR Polymerase Chain Reaction - RAPD Random Amplified Polymorphic DNA     

Linkage groups of isozymes,RFLP and RAPD markers in carrot (Daucus carota L. sativus)

Summary Genetic and linkage analysis of marker loci were performed with 4 selfed progenies, derived from single plant (I_0/1 lines) of carrot (Daucus carota L. sativus). The analysis of 58 markers included 1 morphological marker, 10 isozyme loci, 14 RFLPs, 28 RAPD markers, and 6 isolated PCR fragments used as RFLP probes. Linkage analysis was carried out with the MAPMAKER program and resulted in the construction of 8 linkage groups containing 55 markers with an average distance of 13.1 cM, 3 marker loci remained unlinked. 24% of the markers deviated significantly from the expected Mendelian ratios (1:2:1 or 3:1) due to gametic or zygotic selection. It was shown that isolated PCR amplification products can be used as RFLP probes to detect polymorphisms for a certain locus in progenies where the corresponding RAPD pattern is monomorphic or no amplification product is observed. Since carrot has a relative small genome the probability of amplifying repetitive DNA sequences is comparatively low. Thus PCR amplification products represent an additional useful source of RFLP probes.     

余甘子SRAP反应体系的优化

本文报道通过正交设计和单因素优化实验建立余甘子SRAP-PCR优化体系。实验表明PCR反应各因素（模板DNA、Mg2+、dNTPs、引物）对扩增结果均有不同的影响。采用50 ng模板DNA,2.0 mmol?L-1 Mg2+, 0.4 mmol?L-1 dNTPs, 0.3 μmol?L-1 引物的20 μL反应体系可扩增出最清晰丰富的多样性条带。使用该优化体系检测12份余甘子种质资源样品,结果稳定可信,适用于分子遗传研究。     

农杆菌介导轮状病毒抗原蛋白VP4基因遗传转化花生的研究

选用鲁花14与花育23花生品种, 通过农杆菌介导开展了轮状病毒抗原蛋白VP4基因遗传转化研究, 从转化植株中随机选取26株表现Kan抗性植株进行npt II基因的PCR检测, 结果有22株能扩增出620 bp左右的npt II基因条带, 阳性率约为84.62%。提取npt II基因显示为阳性的植株叶片DNA作模板, 用VP4基因特异引物进行PCR扩增, 经琼脂糖凝胶电泳分析, 所有npt II基因阳性的植株均扩增出了约2 350 bp的特异性条带, 而野生型没有。对部分转基因植株进一步进行PCR-Southern杂交和Southern杂交分析, 发现转基因植株中出现了阳性杂交信号, 表明VP4基因的确已经整合到花生的基因组中, 并且是1~2个拷贝。用RT-PCR分析了11株转G1VP4基因的植株, 证明插入鲁花14中的VP4基因已经正常转录, 利用Western blot方法检测筛选到的4株转基因花生, 分别提取其蛋白, 在30 kD处出现特异性蛋白条带, 这为获得转基因创新种质材料奠定了基础。     

农杆菌介导轮状病毒抗原蛋白VP4基因遗传转化花生的研究

选用鲁花14与花育23花生品种, 通过农杆菌介导开展了轮状病毒抗原蛋白VP4基因遗传转化研究, 从转化植株中随机选取26株表现Kan抗性植株进行npt II基因的PCR检测, 结果有22株能扩增出620 bp左右的npt II基因条带, 阳性率约为84.62%。提取npt II基因显示为阳性的植株叶片DNA作模板, 用VP4基因特异引物进行PCR扩增, 经琼脂糖凝胶电泳分析, 所有npt II基因阳性的植株均扩增出了约2 350 bp的特异性条带, 而野生型没有。对部分转基因植株进一步进行PCR-Southern杂交和Southern杂交分析, 发现转基因植株中出现了阳性杂交信号, 表明VP4基因的确已经整合到花生的基因组中, 并且是1~2个拷贝。用RT-PCR分析了11株转G1VP4基因的植株, 证明插入鲁花14中的VP4基因已经正常转录, 利用Western blot方法检测筛选到的4株转基因花生, 分别提取其蛋白, 在30 kD处出现特异性蛋白条带, 这为获得转基因创新种质材料奠定了基础。     

Extraction of Cotton Fiber DNA and Its Application in Traceability

[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.     

新疆野生欧洲李ISSR-PCR反应体系的建立与优化

为建立适合新疆野生欧洲李的ISSR-PCR反应体系,本研究以野生欧洲李为供试材料,采用L₁₆(4⁵)正交试验设计和单因素试验设计相结合的方法,对影响野生欧洲李ISSR-PCR反应体系的5个因素(DNA模板,Taq酶,dNTPs,引物,Mg²⁺)进行筛选与优化,对16条引物的退火温度进行筛选。结果表明,Taq酶对PCR扩增反应的影响最大,野生欧洲李ISSR-PCR最佳反应体系为:总体积20μL,5 U Taq酶0.10μL,10 mmol/L引物1.00μL,2.5 mmol/L dNTPs 1.50μL,10×Buffer(含Mg²⁺)2.00μL,50 ng/μL DNA模板1.00μL,双蒸水14.40μL。建立的ISSR-PCR反应体系经过22份野生欧洲李样品验证,表明反应体系稳定可靠,可用于后续遗传多样性研究,为野生欧洲李种质资源的保护和利用提供理论参考。     

木薯SRAP扩增体系的建立与优化

建立适宜木薯DNA的SRAP扩增体系,为木薯分子标记和基因图谱的构建打下基础。以木薯基因组DNA为模板,采用序列相关扩增多态性(sequence related amplified polymorphism,SRAP)技术对木薯DNA进行PCR扩增,逐级优化反应参数。最佳SRAP-PCR反应体系(10Ll)为：DNA (50ng/μl) 0.5μl、10×PCR buffer (Mg2+) 1.0μl、dNTPs (20mM) 0.2μl、primer (50ng) 0.3μl、Taq polymerase (5U/μl) 0.2μl。该程序和体系能很好地满足木薯基因组SRAP扩增的要求,SRAP标记能够很好应用于木薯遗传研究。     

一种适于PCR扩增的蓖麻基因组DNA提取方法

报道了一种从蓖麻叶片中提取DNA的改良SDS方法,该方法提取的DNA经琼脂糖凝胶电泳检测,获得的DNA条带较亮且无明显拖尾现象。利用ISSR引物对提取的蓖麻基因组DNA进行PCR检测,能够获得清晰稳定的条带。说明该方法提取的蓖麻基因组DNA能够满足PCR反应的需要。     

Evaluation of 'quick and dirty' DNA extraction methods for marker-assisted selection in rice (Oryza sativa L.)

Six simple methods for extracting DNA from rice seedlings were evaluated for marker‐assisted selection (MAS). The assessment of each method was based on PCR amplification of SSR markers, DNA yield and purity, time and cost. Based on these criteria, two methods were selected as being superior to other methods. The best two methods included the standard method developed at the International Rice Research Institute (IRRI), which utilizes a sodium dodecyl sulfate extraction buffer followed by chloroform/isoamyl alcohol extraction and a previously published method using sodium hydroxide and Tris. These two methods produced nearly identical PCR amplification results. The sodium hydroxide method is considerably simpler, quicker and cheaper than the standard IRRI method, and may be particularly useful for many applications of MAS or high‐resolution mapping. This method was also adapted into an effective high throughput method utilizing 96‐well plates emphasizing its versatility.     

香菇反转录转座子间扩增多态性（IRAP）PCR反应体系的研究

摘 要：为了开发香菇反转录转座子间扩增多态性标记(IRAP),建立稳定的IRAP-PCR反应体系,本文对影响IRAP-PCR的主要因素进行了优化筛选。确定了最佳反应体系为：20μl反应体系中,包含30 ng模板DNA,0.30μmol/L引物, 0.3mmol/L dNTPs,2.0 mmol/L Mg2+及0.75UTaq酶。梯度PCR试验筛选得到的最佳退火温度为56.1℃。采用上述最佳反应体系和引物LTR1L-MarY1L对香菇18个菌株进行了IRAP-PCR扩增,验证了该体系的可靠性。     

道地中药材川贝母特异性PCR鉴别方法研究

为建立快速检测中药材川贝母成分的特异性PCR法,从NCBI数据库下载植物HMGR基因序列,根据同源性区域设计兼并引物,对川贝母及其易混品鳞茎基因组进行扩增。通过比对川贝母特异性DNA片段区域,设计引物扩增获得川贝母基原物种特异核酸片段。从川贝母基原鳞茎中提取DNA,设计特异引物对BMH-TF/BMH-TR,优化PCR扩增条件,通过特异性、灵敏度实验验证,建立特异性PCR检测体系。采用该方法从川贝母基原中扩增出一条120 bp左右条带,而非川贝母样品、空白对照在相同条件下无扩增条带,检测灵敏度为2 ng/μL,检测下限为4 ng。对10个市售药材的检测结果证明,该方法简便可行、重现性好。该PCR法可快速、准确鉴别川贝母基原,具有操作简便、快速、特异性高的优点。     

Enhancement of RAPD Analysis by Restriction-endonuclease Digestion of Template DNA in Wheat

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.