This study performs the first detailed study of the genome resources of C. chinense, although conservation strategies should have implemented due to its economically important value [4, 5]. Both C. chinense-ZJWZ and JPBB that possess chloroplast genome lengths of 151,024 bp and 151,097 bp have sharing identical gene contents information , which then causes the divergence hotspots could not be detected in C. chinense. Nevertheless, both sequences have harbored 19 highly variable loci (Table 2) that might serve the potential mutational hotspots. Comprehensive mutational hotspot markers screening from the whole chloroplast genome is useful in polymorphism sites identification to elucidate the evolutionary and resolving controversial in phylogenetic relationships, hybridization issues, and biogeography [26, 27]. For instance, 20 mutational hotspots were respectively recognized for Artemisia scoparia , A. maritima and A. absinthium . Meanwhile, Kim et al.  compared 21 Artemisia (32 accessions) and suggested the markers accD and ycf1 may represent the potential markers to be tested for the whole Asteraceae. Recognition of these two markers seem to be line with several other studies on the genera in Asteraceae, where either one of both markers were observed in the suggestion list [31, 32]. The marker ycf1 is included among the 19 divergent hotspots although it possesses a lower nucleotide diversity (Pi = 0.0004), imply the potential application of these markers over all species of Asteraceae. Overall, narrow nucleotide diversities for C. chinense are observed (Table 2), likely because of comparative analyses was made within species that sampling differently from two localities. The highly divergent hotspots usually are identified between closer species [30, 31, 32]. Furthermore, the chloroplast genomes of same species were relatively conserved, exhibited in less remarkable polymorphism. Thus, the listed mutational hotspots regions in this study, though with low nucleotide diversity, could still apply for inter-population genetic study and phylogeographic study to test the biogeography origin.
Nine polymorphism cpSSRs observed from both genome of C. chinense-ZJWZ and-JPBB are mononucleotide tandem repeats with intraspecific variation of polyA (polyadenine) represents the most repeated motif in six primer sets (Table 1). Overall, the repeat motif is varied between 10 and 12 nucleotides, with either polyA or polyT shown as the content. Among reported Asteraceae, the identified loci with abundance A/T content were also present for A. scoparia . Moreover, mononucleotide SSRs were also the most frequent identified sequence in Artemisia species [28, 29], though multiple-nucleotide type SSRs may sometime present in least frequency. A distribution pattern of continual repeat sequences of polyT (polythymine) following by polyA are occurred in atpA-trnR (Table 1). Among the cpSSRs markers, three primer sets of which rpoC2-rps2 (cpSSR2), atpA-trnR (cpSSR4), and ycf1 (cpSSR9) were also suggested for mutational hotspots (Table 1). Repeat sequences have been proven crucial in chloroplast genome arrangement and sequence variation . Further, the variable repeat sequences between lineages allow it significances used as microsatellites markers for genetic diversity, and population genetics studies of plant species [28, 33].
In this study, the employment of genome skimming data using CandiSSR represents the first-ever study in Asteraceae to identify the appropriate polymorphic nSSRs for C. chinense. Estimation on the expected heterozygosity that shown significant deviation on four loci (CC19, CC32, CC55 and CC66) may not only due to the presence of an excess homozygotes. Other factors including Wahlund effect, inbreeding, null alleles, and sampling effect are also the potential causes to the deviation [34, 35]. Attempt of 10 selected nSSRs tested for the transferability of loci among the populations of C. chinense is perfectly successful, whereas only four nSSRs is applicable for Artemisia stolonifera and A. argyi (Table 4). Verification of transferability loci onto other species would allow further understanding of phylogenetic relationship at both inter and intra level . Thus, it is believed that more transferability markers could be select from the remaining 123 markers in this study. The approaches of applying nSSRs from various employment have been developed in Asteraceae for Chresta , Solidago , as well as to study the hybridization of two Tithonia species . Application of nSSRs were also used for other plant species such as transferability test in Sanguinaria , and genetic structures studies in Salix , Euptelea , and Engelhardia . The 133 successfully developed polymorphic nucleotide microsatellite markers can be further applied to reveal the genetic diversity, population structure, and to develop effective conservation as well as management strategies for C. chinense. This approach is applicable to other plants species.