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SNP Typing for Germplasm Identification of Amomum villosum Lour. Based on DNA Barcoding Markers

机译：SNP分型用于砂仁种质鉴定。基于DNA条形码标记

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摘要

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.
机译：阳春砂仁是中国广东省阳春市产的道地药材。该草药种质应准确鉴定和收集，以确保其在药物治疗中的质量和安全性。在本研究中，基于DNA条形码标记对单核苷酸多态性分型方法进行了评估，以鉴定Amomi Fructus的种质。使用CTAB方法从代表三种砂仁种的29种地方品种的叶子中提取基因组DNA，该种砂仁种包括A. villosum Lour。，A。xanthioides Wall。ex Baker和A. longiligulare T. L. Wu）。 PCR扩增并测序了六个条形码标记（ITS，ITS2，LSU D1-D3，matK，rbcL和trnH-psbA）。进行SNP分型和系统发育分析以区分地方品种。结果表明，除trnH-psbA外，五个候选区域（ITS，ITS2，LSU D1-D3，matK和rbcL）均获得了高质量的双向序列。 ITS，ITS2和LSU D1-D3这三个核糖体区比质体基因rbcL和 matK 含有更多的SNP基因型（STs）。在这29个标本中，从四个区域（ITS， LSU D1-D3， rbcL 和 matK ）的组合中检测到19个ST。系统发育分析结果进一步揭示了两个进化枝。最小生成树表明存在两个主要组：第一组由 A的9个ST（ST1-8和ST11）组成。绒毛，第二组由三个 A的ST（ST16-18）组成。长廊吴。我们的结果表明，ITS和 LSU D1-D3应该与核心条形码 rbcL 和 matK 结合使用。这四个组合区域可以用作多区域DNA条形码，以精确地区分不同产地的Amomi Fructus地方品种。

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