Highly Universal DNA Barcoding Primers of ITS2 for Gymnosperms*

doi:10.7677/ynzwyj201313190

Abstract

Abstract:

DNA barcoding is an approach for rapid and accurate species identification using a standard DNA region. Ideal barcodes should have high level of universality in PCR and sequencing. Although the nuclear ribosomal DNA (nrDNA) internal transcribed spacers 2 (ITS2) was proposed as potential DNA barcode for seed plants, including gymnosperms, no suitable ITS2 primer pair for gymnosperms available shows high universality in either aspect. To obtain a highly universal ITS2 primer pair, we newly designed three forward primers for ITS2 based on sequences in the 5.8S gene greatly conserved among 55 genera of gymnosperms. Combined with previously published reverse primers of ITS, seven candidate primer combinations for ITS2 were assessed for their universality. A total of 56 species, representing 40 genera from all the 12 families of the eight orders of gymnosperms, were sampled in this study. Three ITS2 primer combinations, 5.8SR/ITS4, 5.8SRa/ITS4, and 5.8SF2/S3R, were excluded due to their low universality or because of yielding double bands in the PCR in the familylevel assessment. The remaining four primer combinations, GYM_5.8SF1/ITS4, GYM_5.8SF1/S3R, GYM_5.8SF2/ITS4, and S2F/S3R, showed high PCR universality (100%) on all 56 sampled species. Based on our overall assessment of PCR and sequencing universality, brightness of PCR bands, and sequence quality and coverage, we propose the primer combination GYM_5.8SF2/ITS4 as ITS2 barcode for gymnosperms because it was shown to be the most “universal”.

Key words: DNA barcoding, Gymnosperms, ITS2, Universal primer, Species identification

摘要：

DNA条形码技术是利用标准DNA片段进行准确快速鉴定物种的一种方法，理想的DNA条形码片段应具有高通用性。虽然核糖体DNA内部转录间隔区II (ITS2) 被建议作为种子植物有效的DNA条形码，但目前裸子植物还没有通用性高的引物可用。为获得高通用性的ITS2引物，本研究基于裸子植物55个属的5.8S基因的保守序列区设计了3个正向引物，与已有的ITS反向引物组合，组成了7对ITS2引物进行通用性的评价。选取了裸子植物8目、12科和40属的56个种用于本文的研究。引物组合5.8SR/ITS4、5.8SRa/ITS4和5.8SF2/S3R因为在科水平评价中通用性低或者产生的PCR产物有双带，因而排除在全部物种水平上进一步评价。其余4对引物（GYM_5.8SF1/ITS4、GYM_5.8SF1/S3R、GYM_5.8SF2/ITS4和S2F/S3R）在56个物种的PCR检测中，均有100%的扩增率。基于PCR产物的亮度、序列质量和正反向引物覆盖率的综合评价，建议引物GYM_5.8SF2/ITS4作为裸子植物条形码片段ITS2最好的通用引物。

关键词: DNA条形码, 裸子植物, ITS2, 引物通用性, 物种鉴定

CLC Number:

Q 781

LI Yan-, Wu Ding-, GAO Lian-Ming. Highly Universal DNA Barcoding Primers of ITS2 for Gymnosperms*[J]. Plant Diversity, 2013, 35(6): 751-760.

李燕1、2, 吴丁3, 高连明1. 裸子植物DNA条形码ITS2高通用性引物的筛选[J]. Plant Diversity, 2013, 35(6): 751-760.

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