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The complete plastid genome of Cotinus coggygria and phylogenetic analysis of the Anacardiaceae

Abstract Cotinus coggygria Scop. (Anacardiaceae) is an important ornamental tree with beautiful characteristics that is grown in China. In this study, the complete plastid genome of C. coggygria was sequenced and assembled. This genome was 158,843 bp in size and presented a typical tetrad structure, consisting of a large single-copy region (87,121 bp), a pair of inverted repeat regions (26,829 bp), and a small single-copy region (18,064 bp). A total of 134 genes were annotated, including 88 protein-coding genes, 38 tRNA genes, and 8 rRNA genes. We observed a deletion that caused the loss of the rpl32 gene, and a small expansion of IR regions resulted in the trnH gene accessing IR regions; two copies were obtained. Phylogenetic analysis showed that C. coggygria was most closely related to Pistacia, with 100% bootstrap support within Anacardiaceae. In this study, we report the plastid genome of Cotinus species for the first time, which provides insight into the evolution of the plastid genome in Anacardiaceae and promotes the understanding of Cotinus plants.

Fresh leaves of C. coggygria were collected from Mount Jinyun (geospatial coordinates: N29.842889, E106.394527), Chongqing, China. The samples were deposited in the Herbarium of Southwest University, Chongqing, with the accession number SWU-CQ2. Total genomic DNA was extracted using the CTAB method (Arseneau et al., 2017). The total DNA was ultrasonically fragmented. A DNA library with an insert size of 350 bp was constructed using the NEBNext® library building kit (Emerman et al., 2017) and was sequenced using the HiSeq Xten PE150 sequencing platform. Sequencing produced a total of 5.9 Gb raw data with 21,318,311 raw reads. Clean data were obtained using Trimmomatic (Bolger et al., 2014): we removed the low-quality sequences with more than 5% bases being "N", and more than 50% of the total bases had a quality value of Q < 19. Ultimately, 21,200,559 clean reads were obtained after trimming.
De novo genome assembly from the clean data was accomplished utilizing NOVOPlasty v2.7.2 (Dierckxsens et al., 2017), with a k-mer length of 39 bp and a sequence fragment of the rbcL gene from maize as the seed sequence. A total of 286,982 reads were used in the final plastid assembly, and a circular genome was obtained. The average sequence coverage was 646. Bowtie2 (v2. 0.1) (Langmead et al., 2009) was used to ensure the correctness of the assembly by mapping all clean reads to the assembled genome sequence. The CPGAVAS2 (Shi et al., 2019) program was used to annotate the genome with the reference genome (Pistacia chinensis, GenBank: NC_046786.1). Apollo (Misra and Harris, 2006) was used to manually edit any annotations with problems. The genome maps were drawn in OGDRAW (Greiner et al., 2019). The genome sequence and raw sequencing data have been deposited in GenBank with the accession numbers MT876478 and SRR13076877.

Genome Insight Plant Genetics 2
The complete plastome sequence of C. coggygria is 158,843 bp in size and presents a typical tetrad structure, consisting of a large single-copy (LSC) region, a pair of inverted repeat (IR) regions, and a small single-copy (SSC) region, with lengths of 87,121 bp, 26,829 bp, and 18,064 bp, respectively. This structure is similar to those of other plants of Anacardiaceae. A total of 134 genes were annotated in the plastome of C. coggygria (Table S1, Figure 1), including 88 protein-coding genes, 38 tRNA genes, and 8 rRNA genes. We noted that the rpl32 gene was not annotated in this plastome. The rpl32 gene encodes a ribosomal protein and is the structural constituent of the 50S ribosome, which is considered to be involved in the biological process of translation (Weglöhner and Subramanian, 1993;Gaudet et al., 2011). We retrieved the sequence of rpl32 from a closely related species (Pistacia chinensis) and performed a Bowtie 2 search on the complete raw data. Only 5 reads were partially mapped, and the sequence source of the unmapped part could not be identified after BLAST (Altschul et al., 1990) retrieval. We found no evidence to support transfer to the nucleus; therefore, we hypothesized that the deletion caused the species to completely lose the gene.
The IRscope program (Amiryousefi et al., 2018) was used to visualize IR boundaries. The IR boundaries of C. coggygria and seven other taxa from Anacardiaceae were analyzed, and the results are shown in Figure 2. In the SSC/ IRa boundary, the ycf1 gene spans this boundary and is mostly located in the SSC region, and it overlaps with IRa by 1097-1104 bp. Spondias bahiensis is an exception, and the overlap is 1401 bp. At the IRb/SSC boundary, ycf1 pseudogenes are produced by inverted repeats. Additionally, the ndhF gene crosses the IRb/SSC boundary (except for Sclerocarya birrea) and forms a partial overlapping region with the ycf1 pseudogenes. In general, there are no significant differences in SSC/IR boundaries.
At the LSC/IR boundary, we observed changes in the position of the rps19 gene in different taxa. We found that rps19 genes are completely located in the IR region in Spondias and Sclerocarya species; as a result, there are two complete copies in their genomes. In addition, the rps19 gene spans the LSC/IRb boundary and is partially duplicated in the IRa regions in Anacardium and Mangifera species. However, in Toxicodendron, Rhus and Pistacia species, we observed that the rps19 genes are located in the LSC region and are annotated as pseudogenes. Moreover, we observed a small expansion of IR regions in C. coggygria that resulted in the capture of the trnH and rps19 genes. The latter gene, rps19 in C. coggygria, is also a fragment and is annotated as a pseudogene. The rps19 gene encodes a ribosomal protein that is the structural constituent of the 40S ribosome, which is considered to be involved in the biological processes of translation and ribosomal small subunit assembly (Sánchez et al., 1996;Gaudet et al., 2011). Previously, rps19 was also observed to be a pseudogene in Gentiana species (Sun et al., 2018).
To evaluate the divergence of plastomes among Anacardiaceae species, six plastomes (C. coggygria, P. chinensis, R. potaninii, T. sylvestre, S. birrea and S. bahiensis) were compared using shuffle-LAGAN mode in mVISTA (Brudno et al., 2003;Frazer et al., 2004) to identify interspecific variations. The reference annotation is P. chinensis. In addition, M. indica and A. occidentale were excluded from the analysis because it has been previously reported that their plastomes contain specific inversions in the LSC region or that there is a migration of DNA sequences from mitochondria in IR regions (Rabah et al., 2017). Therefore, they will not be discussed here. The IR regions are highly conserved compared to the SSC and LSC regions (Figure 3). Most of the regions with large divergence are observed in the intergenic regions, including trnH-psbA, psbM-trnD, trnT-psbD, trnT-trnL, ycf4-cemA, psbF-petL and ndhF-rpl32-trnL. In particular, a deletion is present between the ndhF and trnL genes and results in the complete loss of the rpl32 gene in C. coggygria. Among the protein-coding genes, rpoC2, rps19 and ycf1 show high sequence differences. To determine the phylogenetic position of C. coggygria in Anacardiaceae, we reconstructed maximum likelihood (ML) trees based on the plastome sequences. The plastid genome sequences of 16 species belonging to the family Anacardiaceae were downloaded from GenBank. Two Burseraceae species, Boswellia sacra and Canarium album, were used as outgroups. A detailed list of all plastid genomes analyzed in this paper is provided in Table S3. The complete plastid genome sequences were aligned using MAFFT online version 7.471 (Rozewicki et al., 2019). To avoid negative effects of sequence inversion and migration sequences from mitochondria on phylogenetic reconstruction, a total of 1,477 highly conservative blocks were identified using Gblock (Castresana, 2000;Talavera and Castresana, 2007) with the default setting. The Gblock alignment (138,527 bp) accounted for 71% of the original alignment (194,522 bp). These selected blocks were used to construct the phylogenetic trees separately using the maximum likelihood (ML) method implemented in RaxML (v8.2.4) (Stamatakis, 2014). The parameters were "raxmlHPC-PTHREADS-SSE3 -f a -N 1000 -m PROTGAMMALGX/GTRGAMMA -x 551314260 -p 551314260". Bootstrap analysis was performed with 1,000 replicates. The results showed that all nodes had bootstrap support of 100%, indicating the reliability of phylogenetic recovery (Figure 4). In our phylogenetic trees, Cotinus and Pistacia were closely related. They belong to the tribe Rhoeae, together with Rhus and Toxicodendron. Unfortunately, few plastomes have been sequenced in Anacardiaceae, and we cannot describe the phylogenetic relationships of other Anacardiaceae plants in more detail based on plastome sequences. The application of NGS technology in Anacardiaceae plants is not sufficient; more plastome sequencing needs to be carried out in the future to improve the plastome resources of Anacardiaceae plants. Figure 3 -Comparison of the cp genomes in six Anacardiaceae species by using mVISTA. The genes were represented as gray arrows on the top of the alignment. Different regions are labeled with different colors. The pink regions are "Conserved Non-Coding Sequences" (CNS), the dark blue regions are exons, and the light-blue regions are tRNA or rRNA. 50% and 100% refer to the similarity among sequences. Gray arrows above the aligned sequences represent genes and their orientation. In particular, two genes (rps19 and rpl32) are circled in red, as they are quite different in these taxa. The deletion of a large fragments in C. coggygria led to the loss of rpl32 gene.

Availability of data and materials
The annotated plastome sequences and raw sequencing data of C. coggygria were deposited in GenBank (https://www. ncbi.nlm.nih.gov/) with the accession numbers MT876478 and SRR13076877. All the samples were deposited in the Herbarium of Southwest University, Chongqing, China. All other data and materials generated in this manuscript are available from the corresponding author upon reasonable request. Figure 4 -Phylogenetic relationships of species from Anacardiaceae family inferred using Maximum likelihood (ML) method. The phylogenetic tree was constructed by using the selected conserved blocks based on complete plastome sequences among the 18 plastomes. A total of 1,477 highly conservative blocks were identified by using Gblock with the default setting. Gblocks alignment (138,527 bp) accounts for 71% of the original alignment (194,522 bp). The number at the bottom of the scale, 0.005, means that the length of the branch represents the replacement frequency of bases at each site of the genome at 0.005. Bootstrap values were calculated from 1000 replicates. Two taxa from Burseraceae, namely, B. sacra and C. album were used as outgroups.

Supplementary material
The following online material is available for this article: Table S1 -Gene composition in the chloroplast genome of C. coggygria. Table S2 -The Simple Sequence Repeats (SSRs) identified in the plastome of C. coggygria. Table S3 -List of plastid genomes used for phylogenetic analysis.

Associate Editor: Rogério Margis
License information: This is an open-access article distributed under the terms of the Creative Commons Attribution License (type CC-BY), which permits unrestricted use, distribution and reproduction in any medium, provided the original article is properly cited.