In this study, five DNA barcodes ITS2, psbA-trnH, ITS, matK and rbcL were amplified and sequenced from 141 individuals of 7 A. villosum populations and finally 531 sequences were obtained. Thus a local DNA barcode reference library of A. villosum in its Daodi production area was constructed. Many DNA barcodes reference libraries have been constructed for the purpose of a more rapid and accurate species identification. And DNA barcoding has been used in A. villosum identification [10–12].COI was an efficient species identification tool and frequently used in genetic diversity analysis of animals [13–17]. In plants, however, low substitution rates of mitochondrial DNA have made it unsuitable, and some other barcoding regions were searched as alternatives just like we used in the current study[18–20]. An ideal DNA barcode should be easily retrievable and bidirectionally sequenced, and provide maximal discrimination among species. Among the five barcodes, the highest PCR amplification and sequencing success rate for A. villosum was rbcL. For a more comprehensive assessment of discrimination power of DNA barcodes, there needs more investigations of the DNA barcodes with adulterants of A. villosum included.
We aligned the sequences within the DNA barcodes, and no variation sites of the five barcodes were found. Therefore, genetic diversity could not be analyzed by these DNA barcodes. The main reason for Intra-specificdivergence could occur at a very high rate within geographically isolated populations. But genetic diversity at the sub-specific level is best explored with a multi-locus approach such as fingerprinting techniques . Consequently, we may say that ITS method ismore suitable for genetic diversity analyses of population with wide geographic areas.
For species identification, geographic structure and plant diversity of the populations might be the problems for the barcoding approach and these problems have to be dealt at the library construction stage. Here, no polymorphisms were examined in any DNA barcode sequence indicating that these DNA barcodes were suitable for A. villosum identification. How much variation is actually needed to separate species is not known with certainty because intra-specific sampling has generally been limited to narrow geographic locales.
Molecular markers have been used extensively to determine genetic diversity and genetic relationships in plant science[27, 28]. Moradkhani etal. expressed that the ISSR marker was used among marker systems as a desirable marker in a wide range of genetic variations in various plants. In the ISSR marker, most amplified fragments were between 200 and 1500 bp.
The richer the genetic diversity of species, the stronger the ability of species to adapt to the natural environment. The level of genetic diversity of plants can be influenced by a variety of factors, including the breeding system, the mechanism of seed transmission, geographical distribution, and natural selection. The species with high genetic variation can resist the survival pressure caused by various environmental changes. The loss of genetic diversity will reduce the ability of species to adapt to environmental changes and affect the survival ability of species. Therefore, it is helpful to evaluate the genetic diversity level of A. villosum populations to analyze its evolutionary potential and provide reference for the identification, preservation and utilization of the germplasm resources of A. villosum. This provides a reference for increasing the genetic diversity and planting of A. villosum in different populations.
According to the ISSR markers, there was a high level of inter-population genetic variability and a relatively low level of genetic diversity within populations. Then we analyzed the genetic diversity and genetic structure of A. villosum populations by ISSR marker through the whole genome. Genetic diversity parameters indicated that the genetic diversity of germplasm materials of A.villosum in Yangchun was relatively rich (PPB = 47.19%, H = 0.1820, I = 0.2689).The results of Gst (55.13%) and AMOVA (68.74%) showed that more genetic variations existed in within populations.
Our results show that ISSR markers can effectively reveal the polymorphism among materials. Genetic diversity also differed somewhat within the 7 populations in this study. ISSR markers indicated that population ZY had the highest genetic diversity and TK had the lowest. This variation may be due to human activity, random genetic drift and/or inbreeding variation.
The higher the genetic differentiation index between populations, the more obvious the differentiation between populations and the more genetic difference between populations.Wright et al.believed that the Gst value of genetic differentiation coefficient is between 0 and 0.05, and the genetic differentiation of the populations is weak; between 0.05 and 0.15, the genetic differentiation of the populations is moderate; between 0.15 and 0.25, meaninga large genetic differentiation of the populations; when the Gst value is higher than 0.25, the differentiation is extremely large. According to Nei's analysis of genetic diversity, the Gst value among A. villosum populations was 0.448, which was greater than 0.25, finding that the genetic differentiation between the populations was extremely large. AMOVA analysis showed that genetic variation within populations of A. villosum accounted for 68.74% (P < 0.05) of the total genetic variation in the populations, and genetic variation among populations accounted for 31.26% (P < 0.05), indicating that most of the genetic variation of A. villosum occurred within the populations. Gene flow is the movement of genes within and between populations, and its intensity has an important effect on populations differentiation. In this study, the gene flow Nm between different populations was 0.6143. According to Slatkin, the fraction of Nm > 1 between any population reflects that it is resistant to the influence of genetic drift, with sufficient communication and no obvious differentiation, so it can prevent populations segmentation. Therefore, the Nm value of 0.6143 indicated that genetic drift was the main factor affecting the genetic variation between populations and the genetic communication between populations is difficult, and high genetic variation was maintained within the population, which can be considered as an independent population. It was also confirmed by the genetic differentiation index between populations. The similarity and genetic relationship between plant populations can be expressed through genetic distance. Some scholars believe that genetic distance and geographic distance have a positive correlation. While some scholars also believe that geographic distance and genetic distance are not significantly correlated.
The genetic distance of 7 populations was between 0.0844 and 0.3347, and the genetic similarity coefficient was between 0.7156 and 0.9191, indicating that the kinship of the populations was relatively close. If gene flow and seed transmission through the mating system are the main causes of populations variation, the closer the geographic distance between populations, the smaller the genetic differentiation. However, the result of the Mantel test indicated that the distribution of genetic diversity among populations may not be explained by obvious geographic distance and this results can be explained by enhancing the geographical distribution of gene flow, therefore, we can analyze the results through the grouping situation generated by the PCoA diagram and UPGMA. The populations in this study were clustered according to the similarity of habitats, regardless of geographic location, and populations with similar habitats are clustered together first, and 7 populations were clearly divided into three major clusters: ZJD, TK and ZY were clustered together, GY, MM and YC were clustered together, while XFC was a separate cluster. It could be an introduction problem. Isolates in different groups had a similarity range of 78–92%, this high level of genetic diversity can be obtained through a series of evolutionary processes, including mutation, recombination and migration. The genetic level among populations was consistent with the results of PCoA and UPGMA cluster analysis revealed by ISSR markers.
In this study, ISSR marker technology is used to preliminarily analyze the genetic relationship of 7 populations by analysing band polymorphism, populations polymorphism, populations clustering based on genetic distance, populations genetic distance and genetic consistency, clustering result and PCoA, which can all verify that 7 populations have certain genetic diversity and ability to resist external invasion, which is undoubtedly a good news for the protection and cultivation of germplasm. It provides a theoretical basis for further research on the classification of A. villosum populations and lays a theoretical foundation for the protection and sustainable utilization of germplasm resources of Southern Medicine A. villosum.