SNP typing for germplasm identification of Amomum villosum Lour. Based on DNA barcoding markers.

Qionglin Huang,Xinye Ma,Ruoting Zhan,Hui Xu,Weiwen Chen,Zhonggang Duan,Jinfen Yang,Frederick C C Leung

doi:10.1371/journal.pone.0114940

Abstract

Amomum villosum Lour., produced from Yangchun, Guangdong Province, China, is a Daodi medicinal material of Amomi Fructus in traditional Chinese medicine. This herb germplasm should be accurately identified and collected to ensure its quality and safety in medication. In the present study, single nucleotide polymorphism typing method was evaluated on the basis of DNA barcoding markers to identify the germplasm of Amomi Fructus. Genomic DNA was extracted from the leaves of 29 landraces representing three Amomum species (A. villosum Lour., A. xanthioides Wall. ex Baker and A. longiligulare T. L. Wu) by using the CTAB method. Six barcoding markers (ITS, ITS2, LSU D1–D3, matK, rbcL and trnH-psbA) were PCR amplified and sequenced; SNP typing and phylogenetic analysis were performed to differentiate the landraces. Results showed that high-quality bidirectional sequences were acquired for five candidate regions (ITS, ITS2, LSU D1–D3, matK, and rbcL) except trnH-psbA. Three ribosomal regions, namely, ITS, ITS2, and LSU D1–D3, contained more SNP genotypes (STs) than the plastid genes rbcL and matK. In the 29 specimens, 19 STs were detected from the combination of four regions (ITS, LSU D1–D3, rbcL, and matK). Phylogenetic analysis results further revealed two clades. Minimum-spanning tree demonstrated the existence of two main groups: group I was consisting of 9 STs (ST1–8 and ST11) of A. villosum Lour., and group II was composed of 3 STs (ST16–18) of A. longiligulare T.L. Wu. Our results suggested that ITS and LSU D1–D3 should be incorporated with the core barcodes rbcL and matK. The four combined regions could be used as a multiregional DNA barcode to precisely differentiate the Amomi Fructus landraces in different producing areas.

Highlights

Amomum villosum Lour. belonging to the monophyletic Zingiberaceae family is a valuable herbaceous plant in southern China [1]
The lengths of the acquired sequences ranged from 444 bp for ITS2 to 818 bp for matK
All of the heterozygous sites were manually edited and confirmed by bidirectional sequence chromatograms in repeated amplifications and multiple samples

Summary

Introduction

Amomum villosum Lour. belonging to the monophyletic Zingiberaceae family is a valuable herbaceous plant in southern China [1]. The ripe fruits of A. villosum Lour., A. xanthioides Wall. Ex Baker and A. longiligulare T.L. Wu are used as Amomi Fructus, one of the traditional Chinese medicines. Amomi Fructus produced from A. villosum Lour. In Yangchun City, located in Guangdong Province, is considered a Daodi (‘‘genuine’’) medicinal material [1, 3] and is sold at a higher price. Produced in Guangxi Province and Yunnan Province, as well as those of A. xanthioides Wall. Ex Baker or A. longiligulare T.L. Wu, are traded as Daodi medicinal materials of Amomi Fructus because of highly similar morphological traits. Amomi Fructus landraces should be identified using an accurate and reliable method to ensure quality and safety in medication

Methods

Results

Conclusion