Abstract:

Objective In order to establish a method of obtaining high quality DNA templates from tiny thick-chitin arthropods without grinding or damaging morphology of the voucher specimens. Methods Tiny soil oribatid mites with thick-chitin were used as research materials. We used chitinase to digest the external chitin skeleton first,after the digestion,the individuals were treated with lysate. Amplification efficiency of the DNA barcodes were used as the indicator for DNA template quality. Working concentration and condition of chitinase were optimized. Morphology of the samples treated with or without the chitinase were compared. Results High quality DNA template could be obtained with the method we established,working concentration of chitinase was optimized to 1 mg/ml, and working time was optimized to 24 h. The morphology of oribatid mites treated with the chitinase has no obvious changes compared to the untreated ones, and could be used in the morphology species identification. Conclusion Chitinase is first applied to the digestion of chitin in the extraction of template DNA, high quality DNA could be obtained without damaging the morphological characteristics, after the extraction, the tiny arthropods could be mounted on the slides and kept as voucher specimen.

Key words: Thick chitin, Tiny arthropods, No damage, Voucher specimens

摘要：

目的 针对具厚几丁质外壳的微小节肢动物，研究一种无需研磨、不损坏凭证标本形态的高质量DNA模板的获取方法。方法 以具有厚几丁质外壳的土壤甲螨为材料，用几丁质酶消化其外壳，然后利用裂解液对整个个体进行裂解，以DNA条形码片段的扩增结果作为判断模板质量的依据，探讨几丁质酶的最佳工作浓度和工作时间；比较几丁质酶处理前后标本的外部形态。结果 该方法能获得高质量的DNA模板，在25℃，pH值6.0时，几丁质酶最佳浓度为1 mg/ml，最佳作用时间为24 h。经几丁质酶处理后，螨的外部形态保存完整，与处理前无明显差别，可以用于形态鉴定。结论 该方法首次将几丁质酶消化动物几丁质外壳与提取模板DNA相结合，不仅能保证微小节肢动物的形态特征不被破坏，而且获取的DNA模板质量高，作为凭证标本的微小节肢动物可以制成玻片标本保存，保证了凭证标本的形态完整性。

关键词: 厚几丁质外壳, 微小节肢动物, 无损伤, 凭证标本