Comparing and phylogenetic analysis chloroplast genome of three Achyranthes species

In this study, the chloroplast genome sequencing of the Achyranthes longifolia, Achyranthes bidentata and Achyranthes aspera were performed by Next-generation sequencing technology. The results revealed that there were a length of 151,520 bp (A. longifolia), 151,284 bp (A. bidentata), 151,486 bp (A. aspera), respectively. These chloroplast genome have a highly conserved structure with a pair of inverted repeat (IR) regions (25,150 bp; 25,145 bp; 25,150 bp), a large single copy (LSC) regions (83,732 bp; 83,933 bp; 83,966 bp) and a small single copy (SSC) regions (17,252 bp; 17,263 bp; 17,254 bp) in A. bidentate, A. aspera and A. longifolia. There were 127 genes were annotated, which including 8 rRNA genes, 37 tRNA genes and 82 functional genes. The phylogenetic analysis strongly revealed that Achyranthes is monophyletic, and A. bidentata was the closest relationship with A. aspera and A. longifolia. A. bidentata and A. longifolia were clustered together, the three Achyranthes species had the same origin, then the gunes of Achyranthes is the closest relative to Alternanthera, and that forms a group with Alternanthera philoxeroides. The research laid a foundation and provided relevant basis for the identification of germplasm resources in the future.

Scientific RepoRtS | (2020) 10:10818 | https://doi.org/10.1038/s41598-020-67679-y www.nature.com/scientificreports/ analysis chloroplast genome of Achyranthes species. By comparing the chloroplast genome sequences of plants, we can clearly observe the differences among the genomes of different species at the molecular level, and use them as the basis for species division and identification. The chloroplast genome of A.bidentata was reported in research of Park 30 and Li 31 Park et al. found that the chloroplast genome of Korean A.bidentata has the same structural characteristics of angiosperms, while there were the same results of Hubei A.bidentata in the research of Li 31 . With the emergence of chloroplast genome sequence, chloroplast genome is also expected to help solve the deeper system development branch. The phylogenetic analysis of chloroplast genome sequence was used to evaluate the evolutionary relationship among species now. In this study, the complete chloroplast genome sequences information of A. bidentate, A. longifolia and A. aspera were obtained by sequencing the whole chloroplast genome, and comparative analyses of their structure and function. All of these will provide valuable reference information for species evolution and phylogeny of Achyranthes, and provide new reference for the identification and development of plant resources in the future.

Results
Achyranthes Chloroplast (CP) Genome structure and content. In this study, the whole chloroplast genome sequence of 3 Achyranthes species were obtained by sequencing and submitted to the NCBI database with the GenBank accession number MN953049 (A. longifolia), MN953050 (A. bidentata), MN953051 (A. aspera). Then these sequences were analyzed by various means. In this study, in order to validate the assembly sequences of Chloroplast genome of the three Achyranthes species, the junction sequences of LSC-IRb, IRb-SSC, SSC-IRa and IRa-LSC regions from three species were amplified and compared with assembly sequences. The results showed that the junction sequences of PCR amplification and assembly sequences were consistent up 99% or more. The results also indicated that the assembly sequences are accurate and reliable. The partial Blast results and DNA peak map of junction sequences was listed in Table S1. The results show that the chloroplast genome of them had a typical quadripartite structure (Fig. 1).
The results also indicated that the complete chloroplast genome had a length of 151,520 bp (A. longifolia), 151,284 bp (A. bidentata), 151,486 bp (A. aspera), respectively (Fig. 1).The structure of chloroplast genome included a small single-copy (SSC) region, a large single-copy (LSC) region and two inverted repeat (IR) regions. The GC content was 34.1-34.2% in LSC region. The GC content was 30% in SSC region, and the GC content was 42.5% in the IR regions. Thus, the GC content was the lowest in the SSC region. In addition, the total GC content was 36.4% (A. longifolia), 36.5% (A. bidentata), 36.5% (A. aspera), respectively ( Table 1).
The gene content and sequence of three Achyranthes species chloroplast genome are relatively conservative. Each of the three Achyranthes species chloroplast genome were predicted to encode 127 genes, including 82 protein-coding genes, 37 tRNA genes and 8 rRNA genes (Table 2). These genes classified to 17 groups according to their function. Additionally, the IRs regions contain 6 protein-coding and 7 tRNA genes, and the LSC and SSC region contain 67 and 11 protein-coding genes, respectively, meanwhile, the LSC and SSC region included 29 and one tRNA genes, respectively (Fig. 1).
Totally, there were 15 intron-containing genes, containing five tRNA genes and ten protein-coding genes ( Table 3). Then thirteen genes included one intron, and the remaining two genes (ycf3 and clpP) included two introns of these 15 genes. The length of intron in trnK-UUU gene is largest, which was approximately 2,483 bp, and it is same to A. longifolia of Achyranthes, but there is a length of 2,480 bp in intron of trnK-UUU gene in A. aspera.
Long repeat structure analysis. In the 3 Achyranthes species chloroplast genome, there were 50 repeats were detected, which contained forward repeats, reverse repeats, complement repeats and palindromic repeats (Fig. 2). Then, there were 19 forward repeats, 2 reverse repeats, one complement repeats and 28 palindromic repeats in A. longifolia chloroplast genome. And there were 18 forward repeats, 6 reverse repeats, one complement repeats and 25 palindromic repeats in A. bidentata chloroplast genome. Then there were 20 forward repeats, 3 reverse repeats and 27 palindromic repeats in A. aspera chloroplast genome. However, there was no complement repeats in A. aspera chloroplast genome. These results also presented that it had a length about 20-29 bp in most forward repeats of three Achyranthes chloroplast genome. Then the length of most reverse repeats is below 19 bp, and the length of complement repeats is only below 19 bp, the length of most palindromic repeats was 20-29 bp in A. bidentata chloroplast genome. However, there was a different phenomenon in the A. longifolia and A. aspera chloroplast genome. In the A. longifolia chloroplast genome, the length of most reverse repeats and complement repeats were about 20-29 bp. Then the length of most reverse repeats was about 20-29 bp and there was no complement repeats in A. aspera chloroplast genome.
Simple sequence repeat (SSR) analysis. Simple sequence repeats (SSR), it was known as a microsatellite, including 1-6 nucleotides, and it was widely distributed the genome. In our study, the SSRs were analyzed in the Achyranthes chloroplast genome (Fig. 3), and the numbers and distributions of the SSRs were very similar in the three chloroplast genomes, but there were some differences. comparative chloroplast genomic analysis in three Achyranthes species. In this study, the comparison of structure among three Achyranthes chloroplast genomes were performed. The result indicated that there was a length of 151,520 bp (A. longifolia), 151,284 bp (A. bidentata), 151,486 bp (A. aspera) in these Achyranthes chloroplast genome, and the length of IRs regions of A. bidentata is 25,150 bp, which has the same length with A. longifoli. And it had the smallest SSC region among these sequenced chloroplast genomes of Achyranthes.
In addition, to analysis the DNA sequences divergence of related species, other chloroplast DNAs was premeditated using mVISTA, and with the chloroplast genome of A. bidentata as a reference (Fig. 4). The results showed that the LSC and SSC regions were no more difference than a pair of IRs regions in length. Besides, the coding regions were less flexible than the noncoding regions, and the highly divergent regions was found in the intergenic spaces amongst these Achyranthes chloroplast genomes. Phylogenetic analysis. Now there are more and more studies using complete chloroplast genome sequences to evaluate phylogenetic relationships between medicinal plants. Understanding the phylogenetic relationships between Achyranthes species and other Amaranthaceae could provide favorable guidance into the related angiosperm species. In this study, in order to analyze the phylogenetic relationships of Achyranthes species, the chloroplast genome sequences among 16 angiosperm species form NCBI (Fig. 6). On the basis of       www.nature.com/scientificreports/ the same origin, then the gunes of Achyranthes is the closest relative to Alternanthera, and that forms a group with Alternanthera philoxeroides. In addition, these results also provided effective evidence that the evolution of A. bidentata and A. longifolia occurred in the same direction.

Discussion
In this study, the chloroplast genome of three Achyranthes species was analyzed, the results showed that the three Achyranthes species in this study were content with the characteristics of angiosperms both in structure and content. The typical quadripartite structure of the Achyranthes chloroplast genome are consistent with the characteristics of the chloroplast genome in medicinal angiosperms 32 . The GC content was lower than the AT content in the chloroplast genome of three Achyranthes species, and all these proved that there was no significant difference in chloroplast genomes among three Achyranthes species. The phenomenon was universal in other angiosperms chloroplast genomes [33][34][35] . And the results also showed that the GC content the highest in the IR regions, which may be caused by the presence of large amounts of rRNA in the IR regions. The specific reasons will require further research. And the results of coding regions and the highly divergent regions amongst these Achyranthes chloroplast genomes, were also found in other plants chloroplast genomes [36][37][38][39] . The length of exons and introns in genes were important information in plant chloroplast genome. In this study, the results showed that there were one gene (rps12) included three exons, and two genes (ycf3 and clpP) included two introns in three Achyranthes chloroplast genome. The rps12 gene is a trans-spliced gene with the 5′ end located in the LSC region and duplicated 3′ ends in the IRs regions 40 . Moreover, it has been reported that ycf3 is a gene closely related to photosynthesis 41,42 . Consequently, the attainment of ycf3 gene will contribute to the further investigation of chloroplast in Achyranthes. The ycf1 gene also played a vital role in the chloroplast genome, there were the related reports on gene function of ycf1, these reports revealed ycf1 is an important pseudogene for the chloroplast genome variation and encoding of Tic214 in plants 43,44 .
According to the previous reports, these introns played a vital role in the regulation of the gene expression 45 , which could adjust the level of the gene expression in a special spatiotemporal 46,47 . Moreover, we found that some phenomenon in the chloroplast genomes, such as the intron or gene losses [48][49][50] , and the regulating function of intron have been found in many plants chloroplast genome 51,52 . However, now there were no related research on the introns regulation mechanism of Achyranthes. Therefore, we could attain more useful information through the further studies of introns in the chloroplast genomes. The information of chloroplast genome could provide important theoretical basis for plant resource identification, especially medicinal plants.
Long repeats and the SSRs of the chloroplast genome were the vital information for identification of plant germplasm resources and molecular markers. Studies have shown that there are more than 30 bases of 14 repeats in S.miltiorrhiza chloroplast genome, similar, the repeats of ≥ 30 bases were 16, 15 and 16 in the chloroplast of A. longifolia, A. bidentate and A. aspera, respectively. The results of this study show that genes with long repeat sequences may be very suitable for genetic marker identification of related species, and the specific role needs to be proved by subsequent studies.
SSRs play a vital role in the chloroplast genomes. Due to its extreme variability, it was used to genetic research [53][54][55] . Previous report showed that the SSR was commonly distributed the genome, and the SSR was www.nature.com/scientificreports/ widely used to the genetic population structure and maternity analysis because of its unique uniparental in inheritance. Previous studies have shown that the mononucleotides were the most abundant repeats in A.formosae, and there was the same phenomenon in the three Achyranthes chloroplast genome. Therefore, the study of the chloroplast genome SSRs will greatly promote the investigation of species identification, genetic diversity and evolutionary process in Achyranthes 56,57 . Previous research had shown that IRs regions were the most conserved regions in the chloroplast genome 19 . Its contraction and expansion at the borders is a general evolutionary event, and which represent the dominant reason for the size variation and rearrangement of the chloroplast genome [58][59][60] . There were many reports that the chloroplast gene had a conservation order in most land plants, but there were also reports that many sequences were rearranged in the chloroplast genomes of most plant species, then the IR contraction and expansions with inversions, the inversions in the LSC region and the re-inversion in the SSC region were included [61][62][63] , and some reports showed that the extensive rearrangements in the chloroplast genome of Trachelium caeruleum are associated with repeats and tRNA genes 64 . Because of the sequence rearrangements that modification of chloroplast genome structure in associated species may be related to the plant genetic diversity information, so it can be used for molecular identification and evolutionary research 65 .
With the continuous development of next generation sequencing technology, especially the application of second-generation sequencing technology, chloroplast genome sequencing has become simpler and easier than first generation sequencing. Moreover, at present, more and more researches have used the complete CP genome sequence to evaluate the phylogenetic relationship between angiosperms. In this study, The ML phylogenetic tree showed that there were divided into 13 clades among these analyzed species, and the results showed that there was a strong sister relationship between A. bidentate and A. longifolia. The chloroplast genomes were vital genomic resources for the reconstruction of precise high-resolution phylogenies 66 . As a member of the Amaranthaceae family, Achyranthes species contained vital genetic resources for the evolution and development of other species 67,68 . The Achyranthes species and Alternanthera philoxeroides come from a monophyletic group, which is consistent with the results of Park 30 . However, the A.bidentate formed a group with Cyathula capitata and with 100% bootstrap in the research of Li 31 . Combined with our phylogenetic analysis and Li's research results, it is speculated that there may be a far-reaching relationship between A.bidentata of Hubei and A.bidentata from other regions, indicating that geographic isolation may have a greater impact on the interspecific relationship of Achyranthes. And in this study, we found that in the Amaranthaceae, each genus is basically clustered independently, indicating that there was a good monophyletic separation in this family.
At present, there are three species of Achyranthes species in China, and most of the studies are concentrated on A. bidentate and A. aspera in the world. Some studies have shown that the combined extract of Lycii Radicis Cortex and A. japonica had the effect of anti-osteoporosis 69 , in addition, it was also found that tannins isolated from leaf callus cultures of A.aspera and O.basilicum had the ability of anti-inflammatory and promoting wound healing 70 . Then some studies have also shown that the quality of chicken can be affected by adding the extract of A. japonica to chicken feed 71 . Therefore, it is speculated whether the addition of A. japonica extract to human diet will also affect the body muscle quality, which needs further research to prove. All these studies provided theoretical support for the research and development of Achyranthes in the future. Now it has been shown that chloroplast genome can be used as super barcode to identify plant species 72 . According to our phylogenetic analysis of the chloroplast genome of three Achyranthes species, we speculated that the chloroplast genome of Achyranthes might be an important marker for species identification. Further research is needed to study this conjecture. The study results are of great value to the evaluation of genetic diversity and phylogenetic research of Achyranthes in the future. However, unfortunately, our study did not fully understand the relationship between genera. In addition, our phylogenetic study only is based on the chloroplast genome. If we want to fully understand the phylogeny of species in Amaranthaceae and even Centrospermae, we may need to analyze the nuclear genes of plants, and more genera should be included in the future. Nevertheless, our phylogeny research provided valuable resources for the classification, phylogeny and evolutionary history of Achyranthes.
conclusions Achyranthes L. is the extremely important medicinal plant. The chloroplast genome contains a large amount of available genetic information. At present, there is almost no research on the chloroplast genome of Achyranthes genus around the world. Consequently, it is extremely important to explore the genetic evolution and phylogeny by studying the genetic information of chloroplast genome of Achyranthes. In this study, the chloroplast genome sequencing of the three Achyranthes species was performed by next generation sequencing technology, the complete chloroplast genome sequence was obtained of the Achyranthes. This is an important finding about complete chloroplast genome of Achyranthes in China. The result revealed that the chloroplast genome of A. bidentata has a highly conserved structure, it was similar to angiosperms. Then we also determined the SSR, protein-coding gene sequence and repeated sequences, the phylogenetic analysis shows that there was a closer relationship between A. bidentata and A. longifolia. These results will offer the correlative supportable evidences and lay a solid foundation for the development of chloroplast genome of Amaranthaceae plants.

Materials and methods
Materials and DNA extraction. Fresh materials leaves of the A. bidentate were collected from Wuzhi County, Jiaozuo City, Henan Province of China (N35° 04′ 43.03″, E113° 24′ 7.69″), and A. aspera and A. longifolia were obtained from the field in Tongbai County, Nanyang City, Henan province in China (N32° 38′ 56.23″, E113° 43′ 50.46″). The fresh leaves of plant materials were quickly frozen with liquid nitrogen immediately after picking and cleaning, and kept in low temperature and dark. Total genomic DNA of them were extracted with Plant