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Non-destructive DNA extraction from herbarium specimens: a method particularly suitable for plants with small and fragile leaves.
Journal of Plant Research ( IF 2.8 ) Pub Date : 2019-11-23 , DOI: 10.1007/s10265-019-01152-4
Norimasa Sugita 1 , Atsushi Ebihara 1 , Tsuyoshi Hosoya 1 , Utsugi Jinbo 2 , Shingo Kaneko 3 , Takahide Kurosawa 3 , Masanori Nakae 2 , Tomohisa Yukawa 1
Affiliation  

Protocols for DNA extraction from plants generally involve physical and chemical destruction of tissues. Use of these conventional methods precludes preservation of morphological information from herbarium specimens, especially for small plants with few leaves, and reduces the voucher value of specimens. Here, we developed a new, non-destructive DNA extraction protocol (Protocol 1) that only needs a small piece of leaf (< 25 mm2) to obtain DNA suitable for DNA sequencing from fragile herbarium specimens. The protocol was very simple and rapid; an extraction buffer was placed on the leaf surface of an intact specimen for 30 min at room temperature (20 °C). The quality of extracted DNA was checked by PCR amplification of two standard plant DNA barcode regions, the maturase K gene (matK, ca. 850 bp) and the ribulose-1,5-bisphosphatecarboxylase/oxygenase gene (rbcL, ca. 550 bp), for 14 vascular plant species encompassing various taxonomic groups. The protocol retrieved sequences from 80.0% of specimens for matK and 46.2% of specimens for rbcL. Placing of the extraction buffer onto specimens did not cause any tears or deformation, but caused discoloration in some plants. To improve DNA yield for specimens incompatible with Protocol 1, we developed an alternative protocol for DNA extraction with minimally invasive destruction of specimens (Protocol 2). In this protocol, a cut leaf was immersed in the extraction buffer for 30 min and stored subsequently in a fragment pocket on the specimen sheet. This alternative method retrieved matK sequences from 80.0% of specimens and rbcL sequences from 92.8% of specimens. The combination of Protocols 1 and 2 enabled us to obtain matK sequences from 90.0% of specimens and rbcL sequences form 92.8% of specimens. The new protocols facilitate the use of museum specimens for use of DNA of museum specimens while still preserving morphological information.

中文翻译:

从植物标本室标本中进行无损DNA提取:一种特别适用于叶片细小而脆弱的植物的方法。

从植物中提取DNA的方案通常涉及组织的物理和化学破坏。这些传统方法的使用会阻止保存标本室标本的形态信息,尤其是对于叶片很少的小型植物,并且会降低标本的凭证价值。在这里,我们开发了一种新的,无损的DNA提取方案(协议1),该方案仅需一小片叶子(<25 mm2)即可从脆弱的植物标本室标本中获得适合进行DNA测序的DNA。该协议非常简单快捷。将提取缓冲液在室温(20°C)下放置在完整样本的叶片表面上30分钟。通过PCR扩增两个标准植物DNA条码区域(成熟酶K基因(matK,约850 bp)和核糖1,5-双磷酸羧化酶/加氧酶基因(rbcL,约550 bp),适用于14个维管植物物种,包括各种生物分类群。该方案从用于matK的80.0%样本和用于rbcL的46.2%样本中检索序列。将提取缓冲液放置在样品上不会引起任何撕裂或变形,但会导致某些植物变色。为了提高不符合方案1的标本的DNA产量,我们开发了另一种方案以最小程度地破坏标本而对DNA进行提取(方案2)。在该方案中,将切下的叶子在提取缓冲液中浸泡30分钟,然后将其存储在标本薄片上的碎片袋中。这种替代方法从80.0%的标本中检索了matK序列,从92.8%的标本中检索了rbcL序列。协议1和协议2的组合使我们能够从90.0%的样本中获得matK序列,而rbcL序列从92.8%的样本中获得。新的协议促进了博物馆标本的使用和博物馆标本DNA的使用,同时仍保留了形态信息。
更新日期:2019-11-01
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