Morphological traits of five Toxicodendron accessions
The leaf length, leaflet number, leaflet length and leaflet width were compared among the five accessions (Table 1), the results showed that the leaf length of triploid T. vernicifluum was significantly higher than other four accessions. The leaflet number, leaflet length and leaflet width were significantly lower than T. vernicifluum cv. Dahongpao and T. vernicifluum cv. Hongpigaobachi, and higher than T. vernicifluum and T. succedaneum. The leaf shape index of TZT, DHP and GBC (2.03, 2.11 and 2.18, respectively) were lower than TZG (3.21) and TRB (3.31). In addition, all the morphological traits both showed that there were no significant difference between T. vernicifluum cv. Dahongpao and T. vernicifluum cv. Hongpigaobachi.
Table 1
Leaf morphological characteristics of five Toxicodendron accessions
Accessions | Leaf length | Leaflet number | Leaflet length | Leaflet width | Leaf shape index |
TZT | 33.67 ± 4.19A | 8.56 ± 0.92C | 11.29 ± 1.52B | 5.60 ± 0.62B | 2.03 ± 0.17B |
DHP | 29.41 ± 5.97B | 10.97 ± 1.73AB | 12.97 ± 1.83A | 6.21 ± 1.00A | 2.11 ± 0.20B |
GBC | 31.02 ± 6.93AB | 10.04 ± 1.78B | 14.38 ± 2.31A | 6.62 ± 0.54A | 2.18 ± 0.35B |
TZG | 25.17 ± 4.17C | 10.93 ± 2.52AB | 9.77 ± 1.81D | 3.05 ± 0.42C | 3.21 ± 0.37A |
TRB | 21.08 ± 3.70D | 12.22 ± 2.76A | 9.28 ± 1.42D | 2.86 ± 0.56C | 3.31 ± 0.48A |
TZT: triploid T. vernicifluum, DHP: T. vernicifluumcv. Dahongpao, GBC: T. vernicifluum cv. Hongpigaobachi, TZG: T. vernicifluum, TRB: T. succedaneum |
Features of Toxicodendron accessions chloroplast genome
In this study, all the cpDNAs showed a typical circular tetramerous structure, consisting of a pair of inverted repeats (IRs), a large single copy region (LSC), and a small single copy region (SSC) (Fig. 1). The size of cpDNA and its regions were all similar across different Toxicodendron accessions (Table 2). The cpDNA length of Toxicodendron ranges from 158,221 bp (triploid T. vernicifluum) to 159,710 bp (T. vernicifluum). The size of the IR region ranges from 26,462 bp (triploid T. vernicifluum) to 26,534 bp (T. succedaneum), while the SSC and LSC size varies from 18,346 bp (triploid T. vernicifluum) to 19,074 bp (T. vernicifluum cv. Dahongpao and T. vernicifluum cv. Hongpigaobachi) and from 86,951 bp (triploid T. vernicifluum) to 87,636 bp (T. vernicifluum) (Table 2).
The base composition of cp genome sequence was analyzed and found to be 30.7% A, 19.3% C, 18.6% G and 31.4% Tin triploid T. vernicifluum (Table 3). The overall GC content was 38.0%, which was very close to those of other Toxicodendron, e.g., T. vernicifluum cv. Hongpigaobachi (38.0%), T. vernicifluum (37.9%), T. succedaneum (37.9%), T. vernicifluum cv. Dahongpao (38.0%) [24] and T. vernicifluum (37.9%) [25]. Furthermore, the GC contents are unevenly distributed across regions of the cp genome, which were found 36.1%, 42.9% and 32.7% for the LSC, IR and SSC regions, respectively in triploid T. vernicifluum (Table 2). The triploid T. vernicifluum cpDNA consists of 116 unique genes (Fig. 1 and Table S1), including 82 protein-coding genes, 30 tRNA genes and 4 rRNA genes. In the chloroplast genomes of triploid T. vernicifluum, 14 genes (atpF, ndhA, ndhB, petB, petD, rpl2, rpl16, rpoC1, rps16, trnA-UGC, trnG-UCC, trnI-GAU, trnL-UAA and trnV-GAC) contain one intron and while three genes (clpP, rps12 and ycf3) contain two introns.
Table 2
Features of cp genomes of five Toxicodendron accessions
Genome Feature | TZT | DHP | GBC | TRB | TZG |
Length/bp | 158,221 | 159,571 | 159,571 | 159,636 | 159,710 |
LSC/bp | 86,951 | 87,475 | 87,475 | 87,523 | 87,636 |
IR/bp | 26,462 | 26,511 | 26,511 | 26,534 | 26,525 |
SSC/bp | 18,346 | 19,074 | 19,074 | 19,045 | 19,041 |
GC/% | 38.0 | 38.0 | 38.0 | 37.9 | 37.9 |
GC in LSC/% | 36.1 | 36.1 | 36.1 | 36.0 | 36.0 |
GC in IR/% | 42.9 | 43.0 | 43.0 | 43.0 | 42.9 |
GC in SSC/% | 32.7 | 32.6 | 32.6 | 32.4 | 32.5 |
TZT: triploid T. vernicifluum, DHP: T. vernicifluumcv. Dahongpao, GBC: T. vernicifluum cv. Hongpigaobachi, TRB: T. succedaneum, TZG: T. vernicifluum |
Table 3
Base compositions of cp genomes of five Toxicodendron accessions
Accessions | Region | A | C | G | T |
TZT | LSC | 31.3 | 18.5 | 17.5 | 32.6 |
IR | 28.4 | 22.3 | 20.7 | 28.6 |
SSC | 33.6 | 17.0 | 15.7 | 33.7 |
Total | 30.7 | 19.3 | 18.6 | 31.4 |
DHP | LSC | 31.3 | 18.6 | 17.6 | 32.6 |
IR | 28.6 | 20.7 | 22.3 | 28.4 |
SSC | 33.8 | 17.0 | 15.7 | 33.5 |
Total | 30.7 | 19.3 | 18.6 | 31.3 |
GBC | LSC | 31.3 | 18.5 | 17.6 | 32.6 |
IR | 28.6 | 20.7 | 22.3 | 28.4 |
SSC | 33.8 | 17.0 | 15.7 | 33.5 |
Total | 30.7 | 19.3 | 18.6 | 31.3 |
TRB | LSC | 31.4 | 18.5 | 17.5 | 32.6 |
IR | 28.6 | 20.7 | 22.3 | 28.4 |
SSC | 34.0 | 16.9 | 15.5 | 33.6 |
Total | 30.7 | 19.3 | 18.6 | 31.4 |
TZG | LSC | 31.4 | 18.5 | 17.5 | 32.6 |
IR | 28.7 | 20.7 | 22.3 | 28.4 |
SSC | 34.0 | 16.9 | 15.6 | 33.5 |
Total | 30.8 | 19.3 | 18.6 | 31.4 |
Ir Expansion And Contraction
Although cp genomes are highly conserved in terms of genomic structure and size, the IR/SC junction position change caused by expansion and contraction of the IR/SC boundary regions was usually considered as a primary mechanism in creating the length variation of the higher plant cp genomes [26–27]. We investigated the position of genes at the junction regions of five chloroplast genomes: T. vernicifluum cv. Dahongpao, T. vernicifluum cv. Hongpigaobachi, triploid T. vernicifluum, T. vernicifluum and T. succedaneum (Fig. 2). At the LSC/IR junction of five accessions, the rpl2 gene was duplicated at the IR/SSC junction completely and included in the IR region and the rpl2 gene in the T. vernicifluum cv. Dahongpao and T. vernicifluum cv. Hongpigaobachi was shifted by 67 bp from IR to LSC at the LSC/IR border and 66 bp, 88 bp and 103 bp from IR to LSC in the triploid T. vernicifluum, T. vernicifluum and T. succedaneum, respectively. The ycf1 gene is located at the IRa/SSC border in the five cp genomes, and the junctions of IRa/SSC located in ycf1 within the SSC and IRa regions almost had the same length (4560 bp and 1107 bp) except T. succedaneum (4560 bp and 1101 bp). The gene ycf1 in the IRb region and gene ndhF in the SSC region interlaced at the IRb/SSC border and ycf1 in the SSC region was astride the border of SSC/IRa. Gene ndhF and ycf1 in the SSC region extended the same number of bases among the five accessions (42 bp and 1107 bp), except T. succedaneum (36 bp and 1101 bp). Therefore, we may infer that the expansion and contraction of IR region in triploid T. vernicifluum chloroplast genome is fairly stable compared to that of the other chloroplast genomes in Toxicodendron.
Comparative Analysis Of Genome Structure
To investigate the intergeneric divergence of cp genome sequences, the percentage of identity was plotted for five Toxicodendron accessions using mVISTA program with DHP as a reference. The alignment revealed high sequence similarity across the five cp genomes and no rearrangement occurred (Fig. 3), which suggests that they are highly conserved. Non-coding and SC regions exhibit higher divergence levels than coding and IR regions, respectively.
The cp genome sequences of the five Toxicodendron accessions were aligned by MAUVE, and DHP was used as a reference to compare the gene orders among these cp genomes (Fig. 4). The results showed that all sequences show perfect synteny conservation with no inversion or rearrangements.
The nucleotide variability (Pi) values of the eight cp genomes were calculated with the DnaSP software. A total of 1252 polymorphic sites were detected and the Pi values ranged from 0.00001 to 0.0060. Five Pi value peaks (trnK-rps16, ycf4-cemA, psbL-petL, ndhF-ccsA and ccsA-ndhA) were recognized as divergence hotspots (Fig. 5). All hotspots were identified as intergenic spacers, which echoed the finding that the non-coding regions exhibited more variations than the coding regions. Overall, the LSC showed more divergence than the SSC and the IRs. The sequences of these highly variable regions could be developed as barcodes for species identification, phylogenetic analysis, and population genetics research.
We investigated SNPs, the most abundant type of mutation, in the five cp genomes, with T. vernicifluum cv. Dahongpao (DHP) as the reference. In the gene-coding regions, we detected two SNPs in the comparative combination of GBC-DHP, including one transition (Ts) and one transversion (Tv) SNPs, as well as 121 (67 Ts and 54 Tv), 227 (114 Ts and 113 Tv) and 102 (46 Ts and 56 Tv) SNPs were detected in the combinations of TZT-DHP, TZG-DHP and TRB-DHP (Table 4). Furthermore, 5 (4 Ts and 1 Tv), 260 (96 Ts and 164 Tv), 462 (181 Ts and 281 Tv) and 126 (10 Ts and 116 Tv) SNPs were detected in noncoding regions among the four comparative combinations, respectively (Table S2).
Table 4
Transitions (Ts) and transversions (Tv) in the protein-coding regions of the four plastomes, compared with DHP
Treat | Ts | | Tv | Total |
A-G | C-T | | A-T | A-C | T-G | G-C |
GBC-DHP | 0 | 1 | | 0 | 1 | 0 | 0 | 2 |
TZT-DHP | 31 | 36 | | 5 | 22 | 19 | 8 | 121 |
TZG-DHP | 54 | 60 | | 7 | 34 | 63 | 9 | 227 |
TRB-DHP | 22 | 24 | | 6 | 12 | 28 | 10 | 102 |
It has been reported that each small inversion is commonly associated with a hairpin secondary structure in the chloroplast genomes [26, 28]. Small inversions are generally detected by performing pairwise comparisons between sequences of closely related taxa [26]. Seven small inversions were identified in the Toxicodendron cp genomes and their inverted repeating flanking sequences formed stem-loop structures (Fig. 6). All the inversions were located in noncoding regions including 6 in space (ccsA-ndhD, trnS-psbZ, atpF-atpH, trnW-trnP, trnG-trnR and trnQ-psbK) and one in intron regions (rpl16 intron). Four inversions (ccsA-ndhD, trnS-psbZ, trnG-trnR and trnQ-psbK) were specific to one species. For example, the inversion in ccsA-ndhD and trnS-psbZ were specific to TZT, whereas inversions in trnG-trnR and trnQ-psbK occurred in T. vernicifluum. This suggests that these inversions are polymorphic in one species.
Phylogenetic Analysis Based On The Chloroplast Complete Genome And Ssr Molecular Markers
To identified the phylogenetic position of triploid T. vernicifluum in Toxicodendron, we used the 10 cp genomes to phylogenetic analyse. Maximum likelihood (ML) and Bayesian inference (BI) were used to construct phylogenetic tree with Pistacia weinmaniifolia and Mangifera indica as out-groups (Fig. 7). The topologies of the ML and BI trees were nearly identical, which both showed that Toxicodendron species formed a monophyletic clade (BS = 100, PP = 1). The eight Toxicodendron species were divided into two main clades. Clade I contained four accessions (T. vernicifluum cv. Dahongpao, T. vernicifluum cv. Hongpigaobachi, T. vernicifluum cv. Yanggangdamu, and triploid T. vernicifluum) and the results showed that T. vernicifluum cv. Dahongpao is closely related to T. vernicifluum cv. Hongpigaobachi and sister to triploid T. vernicifluum. Clade II consist of T. succedaneum 1#, T. vernicifluum, T. sylvestre and T. succedaneum and T. vernicifluum can be distinguished with T. succedaneum. The phylogenetic tree was very helpful for us to understand the phylogenetic relationship among more Toxicodendron species.
Genetic distance among the 15 accessions was calculated according to the software GenoDive (Table S3) [29]. Based on Nei’s genetic distance coefficient, a dendrogram was obtained using UPGMA cluster analysis. With this result, two groups could be distinguished. The first group was further divided into two subgroups, three accessions (T. succedaneum 1#, T. succedaneum 2# and T. succedaneum 3#) were included in subgroup 1, and the three accessions of T. vernicifluum (T. vernicifluum 1#, 2# and 3#) were included in the subgroup II. Group II included three accessions of T. vernicifluum cv. Dahongpao, T. vernicifluum cv. Hongpigaobachi and triploid T. vernicifluum, and T. vernicifluum cv. Dahongpao was sister to triploid T. vernicifluum, while the three accessions can be distinguished from triploid T. vernicifluum, and T. vernicifluum cv. Dahongpao (Fig. 8).