PCR-compatible genomic DNA isolation from different tissues of rice (Oryza sativa) for SSR fingerprinting

Q3 Agricultural and Biological Sciences Eurasian Journal of Biosciences Pub Date : 2012-07-01 DOI:10.5053/EJOBIOS.2012.6.0.10
R. Roychowdhury, J. Karmakar, N. Dey
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引用次数: 14

Abstract

Background: In the genomic era, polymerase chain reaction (PCR) based DNA marker analysis is widely used for several crop plants, especially in rice (Oryza sativa), for several improvemental aspects. Such study requires a fast, inexpensive, and suitable DNA isolation protocol having several overall advantages. The aim of this work was to standardize a DNA isolation protocol for rice which should be simple, cost-effective, high throughput, PCR compatible, and needs a small amount of plant tissues without using liquid nitrogen. Materials and Methods: To fulfill such a desired goal, genomic DNA was isolated from different tissues (seedling, leaf, root, grain, kernel, straw, and embryogenic callus) of the rice plant following a modified protocol. The isolated DNA was subjected to PCR amplification with a reported trait linked rice-microsatellite (RM) marker. Results: The quality and quantity of the isolated genomic DNA from this modified protocol proved to be comparable with the other standardized protocols. The microsatellite based DNA fingerprint shows reproducible bands from different isolated DNA tissue sources. Conclusions: This mini prep cost-effective protocol was standardized with few milligrams of fresh and dried tissues, it does not require liquid nitrogen, can handle large number of samples in a working day per worker, and be efficiently applied to rice. The protocol has now also been applied in other plants like wheat and mungbean yielding about 0.55 μg of high molecular weight DNA from 100 mg of plant material with negligible RNA contamination.
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水稻(Oryza sativa)不同组织基因组DNA的pcr兼容分离及SSR指纹图谱分析
背景:在基因组时代,基于聚合酶链反应(PCR)的DNA标记分析被广泛应用于几种作物,特别是水稻(Oryza sativa),用于几个改良方面。这样的研究需要一种快速、廉价和合适的DNA分离方案,该方案具有几个总体优势。本工作的目的是建立一种简单、经济、高通量、PCR兼容、不使用液氮且需要少量植物组织的水稻DNA分离方案。材料和方法:为了实现这一期望的目标,按照修改的方案从水稻植株的不同组织(幼苗、叶片、根、籽粒、籽粒、秸秆和胚性愈伤组织)中分离基因组DNA。将分离的DNA用报道的性状连锁水稻微卫星(RM)标记进行PCR扩增。结果:该改进方案分离的基因组DNA的质量和数量证明与其他标准化方案相当。基于微卫星的DNA指纹图谱显示了不同分离DNA组织来源的可重复性条带。结论:该迷你准备方案具有标准化、低成本、低成本的特点,鲜干纸巾用量少,不需要液氮,每个工人在一个工作日内可处理大量样品,可有效应用于水稻。该方案现已应用于小麦和绿豆等其他植物,从100毫克植物材料中产生约0.55 μg的高分子量DNA, RNA污染可以忽略不计。
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来源期刊
Eurasian Journal of Biosciences
Eurasian Journal of Biosciences Agricultural and Biological Sciences-Agricultural and Biological Sciences (all)
CiteScore
1.20
自引率
0.00%
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0
期刊介绍: EurAsian Journal of BioSciences (Abbrev. Eurasia J Biosci or EJOBIOS) is an international, refereed electronic journal. It publishes the results of original research in the field of biological sciences restricted tomorphology, physiology, genetics, taxonomy, ecology and biogeography of both prokaryotic and eucaryotic organisms. The journal encourages submission of manuscripts dealing with plant biology, animal biology, plant physiology, microbiology, hydrobiology, ecology and environmental science, ethnobiology, biodiversity and conservation biology. EurAsian Journal of BioSciences publishes original articles in the following areas: -Agriculture, Fisheries & Food -Anatomy & Morphology -Behavioural Sciences -Biology, Biochemistry and Biotechnology -Biophysics -Biology Education -Cellular Biology and Anatomical Sciences -Ecology, Evolution & Environment -Entomology -Forestry -General Biology -Genetics & Heredity -Life Sciences - Other topics -Microbiology and Immunology -Molecular Biology -Mycology -Palaeontology -Parasitology -Pharmacology & Pharmacy -Physiology and Related Sciences -Plant Sciences -Toxicology -Veterinary Sciences -Virology -Zoology
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