DNA Barcode Reference Library Construction and Genetic Diversity and Structure Analysis of Amomum Villosum Lour. Populations in Guangdong Province

Lu Gong Guangdong Provincial Hospital of Traditional Chinese Medicine https://orcid.org/0000-0002-12450506 Danchun Zhang Guangdong Provincial Hospital of Traditional Chinese Medicine Xiaoxia Ding Guangdong Provincial Hospital of Traditional Chinese Medicine Wan Guan Guangdong Provincial Hospital of Traditional Chinese Medicine Xiaohui Qiu Guangdong Provincial Hospital of Traditional Chinese Medicine Zhihai Huang (  zhhuang7308@163.com ) Guangdong Provincial Hospital of Traditional Chinese Medicine


Introduction
Amomum villosum Lour. is a medicinal plant of Zingiberaceae family mainly grown in southern China. Its ripe and dried fruit Amomi Fructus is the famous traditional Chinese medicine (TCM) with the effects of dampening appetite, warming the spleen to stop diarrhea, regulating qi and relieving the fetus. Modern pharmacological studies show that, Amomi Fructus has great activities of anti-ulceration, anti-diarrhea, anti-in ammatory and antimicrobial [1]. In addition, Amomi Fructus is also widely used in food, liquors, and tea as the health product and condiment. Yangchun City located in Guangdong Province is considered the Daodi (genuine) producing area of Amomi Fructus for its high quality. With the rapid development of the city as well as traditional Chinese medicine business, the habitat of A. villosum has been frequently destroyed that seriously threatened its germplasm resources [2]. In 2016, Amomi Fructus from Yangchun was selected as one of the eight legally protected TCM varieties in Guangdong Province [3].
The genetic diversity of a species is the basis for its survival and evolution, which is of great signi cance to the analysis of evolutionary polymorphism, genetic relationship, and optimization of germplasm resources and protection of populations. Polymerase Chain Reaction (PCR)-based molecular markers have been widely used in the analysis of plant genetic diversity. Among them, Inter-Simple Sequence Repeat (ISSR) is fast and e cient, with the characteristics of high polymorphism, high reliability, low cost, and does not require pre-determination of target sequence information [4,5]. Another marker, DNA barcodes, proposed in 2003 can not only be used in biological identi cation, but also in genetic diversity analysis [6,7].
COI was an e cient species identi cation tool for animals and frequently used in genetic diversity analysis of animals [8]. In plants, however, low substitution rates of mitochondrial DNA have made it unsuitable, and some other barcoding regions were researched as alternatives [9]. In the current study, ISSR and ve DNA barcodes ITS2, psbA-trnH, ITS, matK and rbcL were used to investigate the genetic diversity of seven populations of A. villosum in Guangdong Province, especially in the Daodi producing area of Amomi Fructus, Yangchun City. It will provide insights into the identi cation, conservation, breeding and cultivation of A. villosum.

Plant Material Sampling
A total of 141 samples of 7 A. villosum populations were collected in Guangdong Province from August to November 2018. Six of them were in Yangchun City and the other one was in Maoming City. Population ZY was collected from a deserted germplasm garden containing A. villosum plants coming from Guangdong (Jinhuakeng), Yunnan and Guangxi provinces and a foreign country Myanmar. The sampled plants were identi ed by Huang Zhihai, the chief Chinese pharmacist of the Second Clinical College of Guangzhou University of Chinese Medicine. Fresh and healthy leaves were removed from the plants, dried and preserved in silica gel right now in the eld, then stored in an ultra-low temperature refrigerator (eppendorf, Hamburg, Germany) when came back to the laboratory. Detailed information and geographic location of the samples see Table 1 and Fig. 1.

DNA extraction, PCR ampli cation and sequencing
The total DNA was extracted using a DP305 plant DNA kit (TIANGEN Biotech Co., Ltd., Beijing, China). The NanoDrop2000 ultra-micro ultriolet spectrophotometer (Thermo Scienti c, MIT, USA) was used to determine the DNA concentration and purity. The PCR ampli cation reaction system of the experiment contained 2 Taq PCR Mix 12.5 µL, forward primer (2.5 µM) 1.0 µL, reverse primer (2.5 µM) 1.0 µL, genomic DNA 2.0 µL and added up to 25 µL with ddH 2 O. The primer sequences and ampli cation conditions of different DNA barcodes were shown in Table S1. All ampli cation reactions were completed on the ProFlex PCR instrument (Life Technologies, New York, USA). PCR products were sent to Shanghai Majorbio Pharmaceutical Biotechnology Co., Ltd Guangzhou Branch to be sequenced.
ISSR-PCR ampli cation system 100 ISSR universal primer sequences published by Columbia University were screened [10]. And 6 primers that produced clear and reproducible banding patterns were selected (Table 3). ISSR-PCR ampli cations were performed on 38 individuals randomly chosen from seven A. villosum populations ( Table 4). The 20μL ISSR reaction volume including 10μL2X PCR Mix (containing dye, MgCl 2 , dNTPs), 2μL template DNA, 1μLISSR selected primer and 7μL ddH 2 O. The ISSR PCR ampli cation was programmed in the ProFlex thermocycler as follows: predenaturation at 94 ℃ for 5 min, 35 cycles of denaturation at 94 ℃ for 45s, annealing at the 46.83 -56.60 ℃ of the primer for 45s, extension at 72 ℃ for 2min, with a nal extension at 72 ℃ for 5 min and preservation at 4 ℃. ISSR-PCR products were separated on a 2% agarose gel stained with Goldview by electrophoresis in 1×TAE buffer at 80 V. The gels were visualized under UV light and photographed with a ChemiDoc imaging system (Bio-Rad, California, USA). The molecular weights of ISSR-PCR products were estimated using a 100bp Plus DNA Ladder (TIANGEN Biotech Co., Ltd., Beijing, China).

Data analysis
The two-way sequenced peaks of DNA barcodes were evaluated and assembled by CondonCode Aligner v8.0.1 software [11]. Low-quality areas at both ends of the assembled sequences were removed. ITS2 barcodes were annotated by cutting off the conserved 5.8S and 28S motifs based on HMM [12] at the ITS2 database [13]. Mega6.0 software was used to align DNA barcode sequences and calculate sequence statistics including the base composition ratio, GC content, heterotopic site information, conservative site and parsimony informative sites [14]. Haplotype sequences for each barcodes were exhibited in the two-dimensional code picture. In the picture, each vertical line represented a nucleotide base , and the two-dimensional code on the right could be scanned directly with the DNA sequence.
Reproducible ISSR-PCR bands were determined with the help of the GelPro32 software and manual correction. These clear bands were scored as either present (1) or absent (0), thus generating an ISSR phenotype data matrix. And the data matrix was imported in Popgene32 software to analyze genetic diversity and genetic structure [15]. Genetic diversity parameters included percentage of polymorphic sites (PPB), number of alleles (Na), effective number of alleles (Ne), Nei's gene diversity index (H) and Shannon's polymorphism information index (I) were calculated. Genetic structure parameters including Nei's gene differentiation coe cient (Gst), total population genetic diversity (Ht), intra-group genetic diversity (Hs) and gene ow (Nm) were calculated. GenAlEx 6.502 software was used to estimate the components of genetic variance within and among populations by analysis of molecular variance (AMOVA) and to assess the correlation between population genetic distance and geographic distance by We also analyzed the sequences of each barcode we obtained. All the sequences of the ve barcodes had no variation sites, showing a strong conservation. Among the ve DNA barcodes, ITS2 had the shortest sequence length and the highest GC content. The sequences of each barcode were shown in Fig. 2.

ISSR polymorphism and genetic diversity
We established the ISSR-PCR system for A. villosum. The ISSR-PCR fragments of A. villosum populations ranged from 300bp to 2000 bp. An example of ampli ed pro les was shown in Fig. S1. A total of 66 ISSR bands were generated from A. villosum populations by the selected 6 primers. Among them, 56 bands were polymorphic, thus the percentage of polymorphic bands was 84.85% for all the seven A. villosum populations. For each primer, it ampli ed 6-14 bands with the polymorphic ratio was 64.29%-100.0%. The results were shown in Table 3.
Genetic diversity information of A. villosum populations analyzed by Popgene 32 was shown in Table 4 (Table 6). Additionally, the gene ow (Nm) among different populations was 0.6143.

Genetic distance and cluster analysis
Genetic distance is the main indicator to examine the degree of genetic differentiation and the relationship between groups. The genetic distances of 7 populations were between 0.0844 and 0.3347 (Table 7). Among them, the smallest genetic distance was between ZJD and TK population (0.0844), and the largest one was between XFC and YC population (0.3347). Mantel test carried out with NTSYS-pc 2.0 indicated that the genetic distance and geographical distance were not signi cantly correlated (r = 0.02698, P = 0.5504) (Fig. 3).
The UPGMA clustering map of populations based on genetic similarity coe cient was constructed using the data of Nei's genetic distance of seven populations (Fig. 4). A. villosum populations were divided into three groups at the similarity coe cient of 0.84. Three populations ZJD, TK and ZY formed one group. Three populations GY, MM and YC formed another group. One population XFC formed a single group. The results of PCoA based on the unbiased pair Фst matrix of Nei were consistent with UPGMA cluster analysis. And the rst two principal components accounted for 48.04% of the total variation of ISSR markers indicating from PCoA (Fig. 5). . An ideal DNA barcode should be easily retrievable and bi-directionally sequenced, and provide maximal discrimination among species [9]. From our results, the highest PCR ampli cation and sequencing success rate of the ve DNA barcodes for A. villosum was rbcL. For a more comprehensive assessment of discrimination power of DNA barcodes, it needs more investigations for discriminating power with adulterants of A. villosum included.

Discussion
DNA markers especially ITS sequences are used in plant population genetic analysis [22]. In this study, we aligned the sequences within the DNA barcodes, but no variation sites of the ve barcodes were found. Therefore, genetic diversity could not be analyzed by these DNA barcode markers. gene ow of A. villosum populations is probably due to its pollination style and the habitat fragmentation. A. villosum is extremely di cult to self-pollinate caused by its morphological structure of owers. Due to lacking of pollinating insects, A. villosum in Guangdong mainly relies on arti cial pollination. The gene ow is consequently restricted to a small population. Another reason is that, habitat fragmentation of A. villosum in Yangchun is likely to reduce the exchange of spores between populations.
Amomi Fructus has been used in China for more than a thousand years and was mainly imported from abroad for a long history period. A. villosum has been cultivated in Yangchun for some 200 years and then was introduced into Guangxi, Yunnan and Fujian provinces in southern China [29]. The species with high genetic variation can resist the survival pressure caused by various environmental changes. A. villosum populations in Guangdong Province especially in the Daodi producing area of Amomi Fructus, Yangchun City, have high genetic diversity inferred from our study. And Yangchun has expanded the planting area of A. villosum in recent years. These are good news for A. villosum protection and cultivation. Though, considering the small populations and the increasingly fragmented habitat as well as the lower gene ow of A. villosum, we need to take continuous measures to protect the genetic resources of the species.

Conclusion
In the present study, a local DNA barcode reference library containing 531 sequences were constructed from 141 samples of seven A. villosum populations in Guangdong Province. On the other hand, the A. villosum populations have high genetic diversity but the gene ow was weak revealed by ISSR markers. The genetic relationship of each population was relatively close. More measures are needed to protect the genetic resources of A. villosum in Guangdong province, especially in Amomi Fructus's Daodi producing area, Yangchun city.

Declarations
Ethics approval and consent to participate Not applicable.

Consent for publication
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Availability of data and materials
All data generated or analyzed during this study are included in this published article and its supplementary information les except for some ISSR-PCR electrophoretic maps. They are available from the corresponding author on reasonable request.   Figure 1 Geographical distribution of collected A. villosum populations Note: The designations employed and the presentation of the material on this map do not imply the expression of any opinion whatsoever on the part of Research Square concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors.