DNA barcode reference libraryconstruction and population genetic diversity and structure analysis of Amomum villosum Lour. in Daodi production area
Authors: Danchun Zhang , Ding Xiaoxia , Guan Wan , Huang Juan , Su He , Qiu Xiaohui , Huang Zhi hai , Gong Lu 
Published: December 2020

Abstract
Background: The Amomum villosum has the situation that it is inferior and other other varieties are used as A. villosum in the market. In order to develop and utilize the genuine medicinal materials A. villosum, this experiment aims to carry out the identification and research of variety of the A. villosum and analyze its genetic diversity, constructing the DNA barcode database of the genuine medicinal materials A. villosum in Guangdong Province and providing recommendations for populations planting, which will be critical to the further research of A. villosum. (2) Methods: A total of 141 samples of A. villosum were analyzed by DNA barcoding to construct DNA barcode database. The genetic diversity of A. villosum sampled from 7 populations in Guangdong Province was detected based on ISSR molecular marker technology. (3) Results: The success rates of PCR amplification and sequencing of five barcodes of A. villosumwas rbcL> ITS > ITS2 >psbA-trnH>matK. 141 samples of A. villosum from 7 populations in Guangdong Province were used to construct a reference DNA barcode database containing 531 sequences. The results of genetic diversity were as follow, the number of alleles Na ranged from 1.2879 to 1.7121, the effective number of alleles Ne ranged from 1.1848 to 1.4240, the gene diversity index (H) ranged from 0.2536 to 0.1117, and the Shannon index (I) ranged from 0.3816 to 0.1658, whichindicatedthegenetic diversity of A.Villosum was rich. The total genetic diversity among the 7 populations (Ht) was 0.3299, the genetic diversity within the populations (Hs) was 0.1819, and the gene differentiation coefficient (Gst) was 0.4487. AMOVA showed that the genetic variation within the populations and the genetic variation between the populations accounted for 68.74% (P<0.05) and 31.26% (P<0.05) respectively, indicating that the genetic variation of A. villosum was mainly within the populations. The gene flow Nm was 0.6143.The genetic distance of the 7 populations was 0.0844 - 0.3347, and the genetic identity was 0.7156 - 0.9191, confirming that the genetic relationship of each population was relatively close. The 7 populations were significantly grouped in the cluster analysis and the genetic level of each population from high to low was as follow: ZY (National Highway Roadside) > ZJD (Zhongjiaodong) > GY (Geopark) >MM (Dianbai) > YC (Dadong Village) > XFC (Xingfu Village) > TK (Tankui Village). There was no correlation between the geographic distance and the degree of genetic differentiation among populations. Conclusion: By amplifying and sequencing five barcodes of ITS2, psbA-trnH, ITS, matK and rbcL, a reference DNA barcode database of A. villosum with 531 sequences was constructed. The results of genetic diversity showed that it is necessary to take appropriate in situ protection measures for the populations of A. villosum in Yangchun City and increase the genetic exchange between populations to improve the genetic diversity of A. villosum.