Chloroplast genome sequence of triploid Toxicodendron vernicifluum and comparative analyses with other lacquer chloroplast genomes
BMC Genomics volume 24, Article number: 56 (2023)
Toxicodendron vernicifluum, belonging to the family Anacardiaceae, is an important commercial arbor species, which can provide us with the raw lacquer, an excellent adhesive and painting material used to make lacquer ware. Compared with diploid, triploid lacquer tree has a higher yield of raw lacquer and stronger resistance to stress. Triploid T. vernicifluum was a newly discovered natural triploid lacquer tree. However, the taxonomy of triploid T. vernicifluum has remained uncertain. Here, we sequenced and analyzed the complete chloroplast (cp) genome of triploid T. vernicifluum and compared it with related species of Toxicodendron genus based on chloroplast genome and SSR markers.
The plastome of triploid T. vernicifluum is 158,221 bp in length, including a pair of inverted repeats (IRs) of 26,462 bp, separated by a large single-copy region of 86,951 bp and a small single-copy region of 18,346 bp. In total, 132 genes including 87 protein-coding genes, 37 tRNA genes and 8 rRNA genes were identified in the triploid T. vernicifluum. Among these, 16 genes were duplicated in the IR regions, 14 genes contain one intron, while three genes contain two introns. After nucleotide substitutions, seven small inversions were analyzed in the chloroplast genomes, eight hotspot regions were found, which could be useful molecular genetic markers for future population genetics. Phylogenetic analyses showed that triploid T. vernicifluum was a sister to T. vernicifluum cv. Dahongpao and T. vernicifluum cv. Hongpigaobachi. Moreover, phylogenetic clustering based on the SSR markers showed that all the samples of triploid T. vernicifluum, T. vernicifluum cv. Dahongpao and T. vernicifluum cv. Hongpigaobachi in one group, while the samples of T. vernicifluum and T. succedaneum in another group, which is consistent with the cp genome and morphological analysis.
The current genomic datasets provide pivotal genetic resources to determine the phylogenetic relationships, variety identification, breeding and resource exploitation, and future genetic diversity-related studies of T. vernicifluum.
Polyploidy, that is having multiple sets of chromosomes as a consequence of whole-genome duplication, is common in nature and provides a major mechanism for adaptation and speciation [1, 2]. The polyploidy genotypes may lead to differences in morphology, physiology and molecular characteristics, etc . Toxicodendron vernicifluum, belonging to the family Anacardiaceae, is a deciduous tree species with a toxic sap . The resin and sap which was extracted from lacquer trees were used as paint for culture assets and lacquer wares, making it a natural material of cultural and social significance [5,6,7]. In addition, lacquer tree is also used as a food additive, natural dye, or in herbal medicine to improve blood circulation and to prevent blood stasis, while the metabolic extract of the leaves has neuroprotective and anti-inflammatory activity . The economic value of different varieties of lacquer can be judged by lacquer yield, the growth rate of lacquer and genetic diversity .
Compared with the diploid lacquer tree, the triploid lacquer tree has a higher yield of raw lacquer and stronger resistance to stress . T. vernicifluum cv. Dahongpao (2n=3x=45) was the first nature triploid lacquer tree which was discovered at Bashan Mountain in Shaanxi Province, China and now is widely introduced into the planting area of lacquer tree [11, 12]. Han et al  determined the pseudo-polyploidy of T. vernicifluum was a natural triploid lacquer (2n=3x=45) by observation of the stomatal characteristic of leaf lower epidermis, analysis of lower epidermis, flow cytometry and measurement of genome size, and it was named as triploid T. vernicifluum. However, as an economic tree species, T. vernicifluum has been widely introduced and cultivated , which lead to the taxonomy classification was difficult to resolve because of considerable phenotypic variability with overlapping morphologies.
Chloroplast (cp) genomes have assembled notable contributions in diverse plant families, setting evolutionary within phylogenetic clades [15,16,17], because the lack of recombination and maternal transmission means the chloroplast genome is also useful for tracking source populations [18,19,20] and chloroplast genomes variation could provide valuable genetic markers for the analysis of polyploids . In addition, the cp genome plays an important role in reconstruction of the plant phylogeny and understanding the origins of economically important cultivated species and changes that have taken place during domestication . Microsatellites are powerful markers to detect genetic variation, because of their high mutation rate and high polymorphism, allowing us to distinguish between closely related individuals, and thus to assess parentage and kinship relationships .
In this study, we reported the characteristics of the complete chloroplast genome sequences of triploid T. vernicifluum for the first time and compared with other Toxicodendron cp genomes to investigate the relationship among chloroplast genome. To understand the relationships of the triploid T. vernicifluum, we constructed the phylogenetic tree using their fully sequenced chloroplast genome sequenced and SSR molecular markers. The results will provide a theoretical basis for variety identification, breeding and resource exploitation.
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 (TZT) was significantly higher than other four accessions. The leaflet number, leaflet length and leaflet width were significantly lower than T. vernicifluum cv. Dahongpao (DHP) and T. vernicifluum cv. Hongpigaobachi (GBC), and higher than T. vernicifluum (TZG) and T. succedaneum (TRB). 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 (DHP) and T. vernicifluum cv. Hongpigaobachi (GBC).
General features of Toxicodendron chloroplast genome
The results showed that the cp genomes of triploid T. vernicifluum (TZT), T. vernicifluum cv. Hongpigaobachi (GBC), T. succedaneum (TRB) and T. vernicifluum (TZG) have a typical circular tetramerous structure like other related plants [24, 25], with a genome size of 158,221 bp, 159,571 bp, 159,636 bp and 159,710 bp, respectively. Both the cp genomes comprised of four distinctive parts in which the LSC (86,951 bp, 87,475 bp, 87,523 bp and 87,636 bp) and SSC (18, 346 bp, 19,074 bp, 19,045 bp and 19, 041 bp) are separated by two IRs (26,462 bp, 26,511 bp, 26,534 bp and 26,525 bp) (Fig. 1, Table 2).
The four cp genomes were analyzed and compared with T. succedaneum (Ts), T. griffithii (Tg), T. sylvestre (Tsy), T. diversilobum (Tdi), and T. vernicifluum cv. Yanggangdamu (Tyg) which belong to the same genus. The nine Toxicodendron cp genomes have minor differences in length, except TZT. The size of these cp genomes ranges from 158,221 bp (TZT) to 159,710 bp (TZG and Ts). The size of the IR region ranges from 26,462 bp (TZT) to 26,534 bp (TRB), while the SSC and LSC size varies from 18,346 bp (TZT) to 19,074 bp (DHP, GBC and Tyg) and from 86,951 bp (TZT) to 87,693 bp (Tdi) (Table 2). The overall GC content in the whole genome sequences was practically identical among these plastomes (37.9-38.0%). Furthermore, the GC contents are unevenly distributed across regions of the cp genome, which were found 36.0-36.1%, 42.9-43.0% and 32.4-32.7% for the LSC, IR and SSC regions, respectively (Table 2).
The ten Toxicodendron cp genomes encoded the same 132 functional genes, consisting of 87 protein-coding genes, 8 rRNA genes and 37 tRNA genes and a total of 16 genes were duplicated in the IR regions (Table 2 and Table S1). For both accessions, 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, while three genes (clpP, rps12 and ycf3) contain two introns (Table S1)
SSR polymorphisms and long repeat analyses
The number of cp genome SSRs (cpSSRs) ranged from 52 to 70 among the ten Toxicodendron plastomes (Table 3). The number of cpSSRs in the four accessions TZT, DHP, GBC and Tyg were similar (52), and the numbers of cpSSRs in TZG (70) was the same as that in Tsy (70). The mononucleotide repeat (P1) number with the highest variability ranged from 45 (TZT) to 60 (TZG and Tsy), and most of the P1s were composed of poly A and T repeats. Dinucleotide (P2) repeat sequences in the ten accessions were AT or TA repeats. Only one pentanucleotide (P5) repeat was detected in Tdi. Within the ten plastomes, SSR loci were primarily in the LSC region (Table 3 and Table S2). In addition, 6 (TRB, TZG, Ts and Tsy) and 7 (TZT, DHP, GBC, Tg, Tdi and Tsy) SSR loci were detected in the protein-coding genes rpoC2, atpB, ycf1, ndhI (only in DHP, GBC and Tyg) and ndhF (only in Tg), other situated in intergenic spacers and introns (Table S2)
In the ten Toxicodendron plastomes, four repeat types were detected using REputer software (Fig. 2A). We found 50 repeats in DHP (22 F, 1R, 1 C and 26 P), GBC (22 F, 1R, 1 C and 26 P) and Tyg (21 F, 1R, 1 C and 27 P), which was lower than those found in the other seven accessions. 52 repeat (22 F, 1 C and 29 P) was found in the plastomes of TZT. In addition, the majority of repeats varied from 30 to 39 bp in length (Fig. 2B).
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]. The border regions of the ten Toxicodendron cp genomes were compared. The IR regions’ lengths correlate across all the compared cp genomes with only slight expansion and contraction (Fig. 3), ranged from 26,462 bp (TZT) to 26,534 bp (TRB) in size, of which rps19, rpl2, ycf1, ndhF and trnH genes were present at the junctions of the LSC/IR and SSC/IR borders. At the LSC/IRb junction (JLB), the gene rps19 in the LSC of TZT, DHP, GBC, TRB, TZG, Ts, Tg, Tsy and Tdi contracted 71 to125 bp, whereas Tyg extended 4 bp into the IR region. The gene rpl2 in the IRa region also contracted by a different number of bases (65-103 bp). The distance between trnH and JLA is 11-64 bp. In contrast, the SSC/IR boundary regions were relatively stable. The ycf1 gene is located at the IRa/SSC (JSA) border in the ten cp genomes, and the junctions of IRa/SSC (JSA) located in ycf1 within the SSC and IRa regions almost had the same length. The gene ycf1 in the IRb region and gene ndhF in the SSC region interlaced at the IRb/SSC (JSB) border and ycf1 in the SSC region was astride the border of SSC/IRa (JSA).
Comparative analysis of genome structure
To investigate the intergeneric divergence of cp genome sequences, the percentage of identity was plotted for ten Toxicodendron accessions using mVISTA program with DHP as a reference. The alignment revealed high sequence similarity across the ten cp genomes and no rearrangement occurred (Fig. 4), which suggests that they are highly conserved.
The cp genome sequences of the ten Toxicodendron accessions were aligned by MAUVE, and DHP was used as a reference to compare the gene orders among these cp genomes (Fig. 5). The results showed that all sequences show perfect synteny conservation with no inversion or rearrangements.
The nucleotide variability (Pi) values of the ten cp genomes were calculated with the DnaSP software. A total of 1794 polymorphic sites were detected and the Pi values ranged from 0.0000 to 0.0240, with the mean of 0.0037. Eight hotspots (Pi>0.0120) were uncovered among the ten cp genomes. They are four intergenic spacers (trnH-psbA, trnK-rps16, ycf4-cemA, petL-psbJ) from the LSC region, three intergenic spacers (ndhF-rpl32, trnL-ndhD and ndhD-ndhE) and one gene regions (ycf1) from the SSC regions (Fig. 6). The results indicated that the non-coding regions exhibited more variations than the coding regions. The sequences of these highly variable regions could be developed as barcodes for species identification, phylogenetic analysis, and population genetics research in Toxicodendron.
We investigated SNPs, the most abundant type of mutation, in the ten 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, one Ts were detected in Tyg-DHP, as well as 121 (67 Ts and 54 Tv), 227 (114 Ts and 113 Tv), 102 (46 Ts and 56 Tv), 239 (119 Ts and 120 Tv), 209 (111 Ts and 98 Tv), 244 (124 Ts and 120 Tv) and 199 (95 Ts and 104 Tv) SNPs were detected in the combinations of TZT-DHP, TZG-DHP, TRB-DHP, Ts-DHP, Tg-DHP, Tsy-DHP and Tdi-DHP (Table 4). Furthermore, 5 (4 Ts and 1 Tv), 260 (96 Ts and 164 Tv), 462 (181 Ts and 281 Tv), 126 (10 Ts and 116 Tv), 462 (177 Ts and 285Tv), 430 (145 Ts and 285 Tv), 463 (181 Ts and 282 Tv), 324 (118 Ts and 206Tv) and 1 (1 Tv) SNPs were detected in noncoding regions among the nine comparative combinations, respectively (Table S3).
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 . Seven small inversions were identified in the Toxicodendron cp genomes and their inverted repeating flanking sequences formed stem-loop structures (Fig. 7). All the inversions were located in noncoding regions including 6 in intergenic spacers (ccsA-ndhD, trnS-psbZ, atpF-atpH, trnW-trnP, trnG-trnR and trnQ-psbK) and one in intron regions (rpl16 intron). Among the seven small inversions, six were located in LSC region, one in SSC region (rpl16 intron). The small inversions from trnW-trnP occurred only in DHP, GBC, TZT and Tyg. The small inversions from atpF-atpH, trnW-trnP, trnG-trnR and trnQ-psbK and rpl16 intron have been occurred in DHP, GBC and TZT.
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 12 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 outgroup (Fig. 8). 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 ten Toxicodendron species were divided into two main clades. Clade I contained six accessions (T. vernicifluum cv. Dahongpao, T. vernicifluum cv. Hongpigaobachi, T. vernicifluum cv. Yanggangdamu, triploid T. vernicifluum, T. diversilobum and T. griffithii) and the results showed that T. vernicifluum cv. Dahongpao is closely related to T. vernicifluum cv. Hongpigaobachi and sister to triploid T. vernicifluum and T. vernicifluum cv. Yanggangdamu. Clade II consists of T. succedaneum (MT211614), T. vernicifluum, T. sylvestre and T. succedaneum (OP235437). The phylogenetic tree was very helpful for us to understand the phylogenetic relationship among more Toxicodendron species.
Genetic distance among the 15 samples was calculated according to the software GenoDive (Table S4) . 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 samples of T. succedaneum (T. succedaneum 1#, T. succedaneum 2# and T. succedaneum 3#) were included in subgroup 1, and the three samples 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 (Fig. 9).
There are obvious differences in leaf morphology between diploid and polyploidy, which is one of the simpler methods to identify plant ploidy by morphological differences . Leaf shape index (length/width) is an important parameter of leaf shape. The larger the leaf shape index of plant is, the longer and narrower the leaves are, while the smaller the leaf shape index is, the rounder the leaves are . Liu et al  used the observational method and paraffin section technique to analyze the intraspecific differences in four kinds of Toxicodendron species and the results showed that the sharp differences presented in height, shape, bark and leaves of the four varieties. In this study, morphological traits of five Toxicodendron accessions were determined and the leaf length of triploid T. vernicifluum, T. vernicifluum cv. Dahongpao and T. vernicifluum cv. Hongpigaobachi were significantly higher than T. vernicifluum and T. succedaneum. Therefore, the morphological characteristics of diploid and triploid can be distinguished by the leaf shape characteristics . In addition, all four morphological characteristics (leaf length, leaflet number, leaflet length and width) were shown that T. vernicifluum cv. Dahongpao and T. vernicifluum cv. Hongpigaobachi had no significant difference, and there were no significant difference between T. vernicifluum and T. succedaneum in the morphological traits of leaflet number, leaflet length and width. In conclusion, certain accessions are similar in terms of morphological characteristics.
The chloroplast genomes of plants are a valuable resource for developing molecular markers to study intra-species and interspecies evolution [34, 35]. In this study, we sequenced and annotated the cp genomes of four Toxicodendron accessions and compared their characteristics with those of six other related species from Toxicodendron. Like other angiosperms, Toxicodendron cp genome is a circular double-standed DNA molecular, exhibiting a conserved quadripartite structure [24, 25, 36], and the ten cp genomes were relatively conserved, which is consistent with the slow rates of sequence and structure evolution of plant plastomes [37, 38]. The cp genomes ranged from 158,221 bp (triploid T. vernicifluum) to 159,710 bp [T. vernicifluum and T. succedaneum (Ts)], with a difference in size of 1489 bp. Large variation in LSC length (742 bp) accounted for most of this genome-wide variation, which indicated that variation in genome length appears to be mainly caused by variation in LSC length . The GC content of the ten Toxicodendron accessions ranged from 37.9-38.0%, which is closely related to species affinity . High GC content is conducive to the stability of the genome and maintaining the complexity of the sequence. The four rRNAs genes have high GC content, which results are a high GC content in the IR regions . Usually, a higher GC content indicated a more stable genome sequence .
The expansion and contraction of IR and SC boundaries are thought to be the main cause of CP genome size changes, although CP genomes in plants are highly conserved . The change of the IR and SC junction is a common phenomenon and plays an important role in evolution [43, 44]. After comparing CP genomes among the ten Toxicodendron accessions in this study, changes in the IR (26,462-26,534 bp) and boundary between the IR and LSC or SSC of the cp genome of ten accessions were found to be small. Some variations were observed in these cp genomes, mainly due to variation in the LSC regions rather than contraction and expansion of IR region [45, 46]. Comparison between IR regions and the chloroplast genomes of T. vernicifluum cv. Dahongpao and T. vernicifluum cv. Hongpigaobachi showed similarity, which was the same as for the boundary gene of LSC/IRa, SSC/IRa and LSC/IRb regions and the size of genes and fragments in two adjacent regions. Therefore, we speculate that the genetic relationship between T. vernicifluum cv. Dahongpao and T. vernicifluum cv. Hongpigaobachi is closer than other accessions, and our subsequent phylogenetic analysis validates our inference.
Previous studies have shown that tRNA activity may be a key factor triggering the inversions events . Small inversions in the cp genome of angiosperms are ubiquitous and commonly associated with a hairpin secondary structure and are generally detected by performing pairwise comparisons between sequences of closely related taxa . Many small inversions are generated by parallel or back mutation events during chloroplast genome evolution [26, 48]. In the present study, seven small inversions were discovered based on the sequence alignment of the ten cp genomes and the inversion in ccsA-ndhD have been reported in other studies [16, 49,50,51]. trnW-trnP were specific in T. vernicifluum cv. Dahongpao and T. vernicifluum cv. Hongpigaobachi, triploid T. vernicifluum and T. vernicifluum cv. Yanggangdamu. The small inversions from atpF-atpH, trnW-trnP, trnG-trnR and trnQ-psbK and rpl16 intron have been occurred in T. vernicifluum cv. Dahongpao and T. vernicifluum cv. Hongpigaobachi and triploid T. vernicifluum. These small inversion regions will provide abundant information for marker development in phylogenetic analyses of related Toxicodendron species [48, 52, 53]. However, small inversions of noncoding sequences may influence sequence alignment and character interpretation in phylogeny reconstructions, so caution is necessary when using cp noncoding sequences for phylogenetic analysis [51, 54].
To solve phylogenetic problems at the species level, or to identify species using DNA barcodes, the high evolutionary rates regions were needed to identify . Eight intergenic spacer regions including trnH-psbA, trnK-rps16, ycf4-cemA, petL-psbJ, ndhF-rpl32, trnL-ndhD, ndhD-ndhE and one gene regions ycf1 are highly variable regions in the Toxicodendron cp genome. These variable regions may also be useful for assessing phylogenetic relationships and interspecific differences of Toxicodendron species . Among these, trnH-psbA, ycf4-cemA and ycf1 have been regarded as a candidate DNA barcodes for Toxicodendron and other plants [25, 55, 57].
Complete chloroplast genome provides sufficient information sites for resolving phylogenetic relationships of plant, and have been examined to be effective in the ability of differentiation in lower taxonomic levels and provide valuable data for resolving complex evolutionary relationships [58, 59]. Within the Anacardiaceae, Wang et al  indicated that T. vernicifluum cv. Yanggangdamu was more closely to R. chinensis and two Pistacia species, due to lack of more Toxicodendron cp genomes. In the current study, ten Toxicodendron accessions were used to reconstruct the phylogenetic, two distinct clades were recognized: one consisting of T. vernicifluum cv. Dahongpao, T. vernicifluum cv. Hongpigaobachi, T. vernicifluum cv. Yanggangdamu, triploid T. vernicifluum T. diversilobum and T. griffithii and the other comprised of the remaining accessions. The result is consistent with previous studies, which shown that T. sylvestre was more closely related to T. succedaneum based on five nuclear and chloroplast DNA regions .
Compared to morphological trait classification systems, molecular markers can reveal genetic differences at the DNA reveal genetic differences and are effective for evaluating the genetic diversity of germplasm in breeding programs . SSR markers were testified to be a more advanced tool than all of these markers, which are very suitable for the identification and classification of Toxicodendron species . In our study, five accessions can be distinguished clearly by these SSR markers. All the samples of T. vernicifluum cv. Dahongpao, T. vernicifluum cv. Hongpigaobachi, and triploid T. vernicifluum grouped together in one group, and all the samples of T. vernicifluum and T. succedaneum grouped together in another group, which is consistent with the cp genome and morphological analysis.
The current study primarily explored the chloroplast genome of triploid T. vernicifluum and compared it with related species of Toxicodendron genus. The size of genome, structure and organization of gene were shown to be conservative, which is similar to those reported Toxicodendron cp genomes. Eight hotspot regions were identified and may be utilized as potential molecular markers for population genetic and phylogenetic studies in Toxicodendron. Phylogenetic analysis based on cp genomes and SSR markers both showed that triploid T. vernicifluum can be distinguished with T. vernicifluum cv. Dahongpao, while T. vernicifluum cv. Dahongpao had a close relationship with T. vernicifluum cv. Hongpigaobachi. Therefore, complete cp genomes is useful for species identification, taxonomic clarification, and genomic evolutionary analysis. Further research on the relationships within Toxicodendron genus and the identification of ploidy should incorporate morphology and genome wide analyses to enhance the results.
Materials and methods
In this study, triploid sumac of T. vernicifluum cv. Dahongpao (DHP) and triploid T. vernicifluum (TZT) were cultivated in the germplasm resource nursery in Yangling Shaanxi Province (108.08E, 34.27N), and Zhaotong Yunnan Province (103.72E, 27.34 N), respectively [11, 13]. Three diploid sumac of T. vernicifluum cv. Hongpigaobachi (GBC) were cultivated in the germplasm resource nursery in Yangling Shaanxi Province, T. succedaneum (TRB) and T. vernicifluum (TZG) were cultivated in the germplasm resource nursery in Southwest Forestry University, in Kunming Yunnan Province (102.76 E, 25.06 N) which were introduced from Changsha Hunan Province and Wenshan Yunnan Province, respectively. These five accessions (DHP, GBC, TZT, TRB and TZG) were used for morphological characterization (Table 1) and SSR molecular markers analysis. GBC, TZT, TRB and TZG were sequenced and combined with the six previously sequenced accessions DHP, T. succedaneum (Ts), T. griffithii (Tg), T. sylvestre (Tsy), T. diversilobum (Tdi) and T. vernicifluum cv. Yanggangdamu (Tyg) for comparative analysis of chloroplast genome (Table 2).
In order to compare the differences of leaf shapes among the five accessions (DHP, GBC, TZT, TRB and TZG), three trees per accession were selected for data collection on leaves, and five foliar traits (leaf length, leaflet number, leaflet length, leaflet width; and leaf shape index) were measured. All variables were measured using a ruler (0.1 mm resolution) and are recorded in mm and each sample was tested for three biological replicates.
Fresh leaves of T. vernicifluum cv. Hongpigaobachi (GBC), triploid T. vernicifluum (TZT), T. succedaneum (TRB) and T. vernicifluum (TZG) were collected from different regions and quickly frozen in liquid nitrogen, and stored at ultra-low-temperature refrigerator at -80℃ until use. The voucher specimen deposited in the Herbarium of Southwest Forestry University. The total genomic DNA was extracted with the TGuide plant genomic DNA prep kit (Tiangen Biotech, Beijing, China) and DNA quality was inspected in 0.8% agarose gels, DNA quantification was performed using a NanoDrop spectrophotometer, DNA samples were stored at -80℃ at the Key Laboratory of State Forestry Administration on Biodiversity Conservation in Southwest China, Southwest Forestry University, Kunming, China.
Plastid genome sequencing, assembly and annotation
Total DNA was used to generate libraries with an average insert size of 350 bp with the Illumina Novaseq 6000 platform. Approximately 8 Gb raw data were produced with 150 bp pair-end read lengths. GetOrganelle software  was used to assemble the complete cp genome of the four accessions, with Pistacia weinmaniifolia as the reference. Geneious R8 (Biomatters Ltd, Auckland, New Zealand) software was used for initial cp genome annotation. Start and stop condos were checked and adjusted manually when necessary by comparing them to the reference genome P. weinmaniifolia. The tRNA genes were further confirmed through online tRNA scane-SE web servers . The gene map of annotated Toxicodendron chloroplast genome was drawn by OGdraw online . The annotated sequences have been deposited to the NCBI GenBank database under the accession numbers OP235457, OP271782, OP279729 and OP279730 (Corresponding to T. succedaneum (TRB), triploid T. vernicifluum (TZT), T. vernicifluum cv. Hongpigaobachi (GBC) and T. vernicifluum (TZG)).
Identification of simple sequence repeats (SSRs) and long sequence repeats
Chloroplast SSR loci in ten Toxicodendron cp genomes were detected using MISA  with the minimal repeat number set to 10, 6, 5, 5, 5 and 5 for mononucleotide (momo-), dinucleotide (di-), trinucleotide (tri-), tetranucleotide (tetra-), pentanucleotide (penta-), and hexanucleotide (hexa-) nucleotide sequences, respectively.
Morever, the online REPuter  software was used to estimate and locate forward (F), reverse (R), complemented (C) and palindromic (P) repeats. The following settings for repeat identification were used: (1) Hamming distance equal to 3; (2) minimal repeat size was set to 30 bp; (3) 90% or greater sequence identity.
Comparative analysis of the chloroplast genomes
To investigate divergence in cp genomes, identity across the whole cp genomes was visualized using the online genome comparison tool mVISTA viewer with Shuffle-LAGAN mode among the ten accessions with T. vernicifluum cv. Dahongpao (DHP)  as a reference to show inter-and intraspecific variations . The software MAUVE alignment  was employed to analyze and compare the plastome structure of triploid T. vernicifluum with the other accessions of Toxicodendron. Furthermore, events of IR expansion and contraction were compared between these accessions; the junction regions between the IR, SSC, and LSC of ten accessions were compared using the online program IR scope .
To identify the mutational hotspot regions for Toxicodendron, we calculated nucleotide diversity (Pi) across the whole plastome. The plastome sequences were aligned using MAFFT version 7 software  and the nucleotide diversity was detected using DnaSP version 5 software  with sliding window strategy. The step size was set to 200 bp, with a 600 bp window length. Single nucleotide polymorphisms (SNPs) were detected using the “find variation” function in Geneious.
Amplification of SSR marker
The materials used were T. vernicifluum cv. Dahongpao (DHP), triploid T. vernicifluum (TZT), T. vernicifluum cv. Hongpigaobachi (GBC), T. succedaneum (TRB) and T. vernicifluum (TZG). Three samples per accession were selected for SSR analysis. The fresh leaves from each sample were immediately dried with silica gel for further DNA extraction. Total genomic DNAs were extracted from each sample using the TGuide plant genomic DNA prep kit (Tiangen Biotech, Beijing, China), DNA quality was inspected in 0.8% agarose gels, and DNA quantification was performed using a NanoDrop spectrophotometer. A total of 116 SSR primers [73,74,75,76,77] were used to identify the polymorphic markers among 15 samples, 7 primer pairs amplified well-distributed fragments with good distinctness. Sequence information of the SSRs was listed in Table 5, and the forward of which (F) was labeled 5(6)-carboxyfluorescein (FAM) by the 5’-terminal. PCR reactions were carried out in 25 µL, containing 12.5 µL 2×PCR Mix, 1 µL of each primer, respectively, 1 µL template DNA. The PCR cycling conditions were as follows: pre-denaturation at 94 ℃ for 4 min, followed by 35 cycles of denaturation at 94 ℃ for 30 s, annealing at the corresponding temperature (52 ℃-61 ℃) for 30 s, extension at 72 ℃ for 1 min and the final extension for 10 min at 72 ℃. Amplified SSR alleles were rechecked in 1.5 % binary gels by electrophoresis at 80 volts for 50 min and gel documentation was performed with Gel Logic 200 imaging system. Capillary electrophoresis-based fragment analyses of single pollen amplified SSR alleles were conducted on an ABI3730XL Genetic Analyzer following the manufacture’s instruction to generate GeneScan files. Scoring of alleles was done using ‘GeneMaker’ version 2.2. Genetic distance among the 15 samples was calculated according to the software GenoDive  and the cluster analysis was carried out based on the matrix using MEGA 5.0.
Phylogenetic analysis was conducted using the complete chloroplast genome sequences of the ten Toxicodendron accessions mentioned above, with two Anacardiaceae species [Pistacia weinmaniifolia (MF630953) and Mangifera indica (KY635882)] that were used as outgroup. The nucleotide sequences were aligned using MAFFT version 7 software . The phylogenetic analyses were performed using maximum likelihood (ML) and Bayesian inference (BI). ModelFinder  was used to select the best-fit model with default setting and the maximum likelihood (ML) analysis was performed using IQ-TREE 1.5.5  with 1000 bootstrap replications. The BI analysis was performed by Mybayes 3.2.6 . The jModelTest 2.0 program  was used to determine the best-fitting model for each dataset based on the Akaike information criterion and the optimal model of “GTR+F+R3”. The Markov chain Monte Carlo (MCMC) algorithm was run for 1,000,000 generations, and a burn-in of 25% was used for the analysis.
Availability of data and materials
The datasets generated during the current study are available in the NCBI GenBank (Accession number OP235457, OP271782, OP279729, OP279730)
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This work was supported by Yunnan Provincial Expert Workstation (202005AF150020) and Yunnan Provincial Ten-Thousand Program -Industry Leading Talents (YNWR-CYJS-81210420) and Applied Basic Research Foundation of Yunnan Province (202101AU070144).
Ethics approval and consent to participate
We have obtained the permission of Southwest Forestry University to collect the species of Toxicodendron. The collection and usage of plant specimens in current study complied with the IUCN Policy Statement on Research Involving Species at Risk of Extinction and is allowed by the Convention on the Trade in Endangered Species of Wild Fauna and Flora. Ethical approval was not applicable for this study.
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List of genes in the Toxicodendron chloroplast genomes. Table S2. SSR repeats in the ten cp genomes. Table S3. Ts and Tv in non-coding regions of the nine plastomes, compared with DHP. Table S4. Genetic distance among the 15 samples.
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Zong, D., Qiao, Z., Zhou, J. et al. Chloroplast genome sequence of triploid Toxicodendron vernicifluum and comparative analyses with other lacquer chloroplast genomes. BMC Genomics 24, 56 (2023). https://doi.org/10.1186/s12864-023-09154-2
- Triploid T. vernicifluum
- Chloroplast genome
- Phylogeny analyses
- SSR markers