Towards comprehensive integration and curation of chloroplast genomes

Dear Editor,

Chloroplasts are semi‐autonomous genetic organelles that contain their own DNA. Since the first chloroplast genome was sequenced in 1986, chloroplast genomes have been extensively utilized as fundamental tools in plant phylogenetics and genetically modified to produce protein drugs, especially in the fight against COVID‐19 (Daniell et al., 2021, 2022). More chloroplast genome sequences could not only help us learn more about plant diversity and evolution (Daniell et al., 2016), but they could also help chloroplast biotechnological applications by codon optimization and identifying non‐conserved intergenic spacer regions and regulatory sequences that are needed for genetic engineering (Daniell et al., 2021). Consequently, the chloroplast genomes of numerous plant species, particularly economically significant crops, have been continuously sequenced. Powered by high‐throughput sequencing, over 7000 plant chloroplast genomes have been deposited in the National Center for Biotechnology Information (NCBI) organelle genome database, of which over 50% have been sequenced in the last 3 years (1082, 1175, and 1539 were sequenced in 2019, 2020, and 2021, respectively). With the accumulation of data, inaccurate taxonomic information (Locatelli et al., 2020), disunity of genomic terms (Abeysooriya et al., 2021), and other attendant problems have emerged, provoking significant challenges in employing chloroplast genomes. Many efforts have been made, but current databases still suffer from a lack of comprehensiveness and data curation, as well as incomplete data collection. Most existing curated databases are taxon‐specific (e.g., cpGDB for spermatophytes (Singh et al., 2020) and OGDA for algae (Liu et al., 2020)) or limited to certain data types (e.g., ChloroMitoSSRDB for simple sequence repeats [SSRs; Sablok et al., 2015]), which could be further improved by incorporating more comprehensive data. Additionally, each published organelle genome database covers only a fraction of chloroplasts, and thousands of chloroplast genomes are still dispersed in different nucleotide databases. Therefore, there is an urgent need to establish an integrated portal with a comprehensive collection and curation of chloroplast genomes.

Here, we developed the Chloroplast Genome Information Resource (CGIR), an integrated platform (https://ngdc.cncb.ac.cn/cgir) comprising 19 388 chloroplast genome assemblies and their corresponding meta‐information (Figure 1a). The CGIR comprises five modules: (1) genomes, (2) genes, (3) SSRs, (4) barcodes, and (5) DNA signature sequences (DSSs; Figure 1b). The ‘Genomes’ module displayed 19 388 chloroplast assemblies from 11 946 different species (Figure 1c). Noticeably, among all assemblies, we sequenced 1170 assemblies from 718 species, of which the chloroplast genomes from 307 species were reported for the first time, including one family (Juncaginaceae) and 53 genera. In addition to boosting the number of sequenced species, newly added assemblies allow a group of species to have chloroplasts from many individuals. Compared to the NCBI Organelle Genome Database and CpGDB, with only one component for each species, multiple assemblies with explicit taxonomic information can provide more information for plant phylogeny. The taxonomic information of assemblies was curated in accordance with The Catalogue of Life Checklist 2021 to eliminate disunity across different databases and contributors (Figure 1d). Functional information on plant species was integrated into the CGIR according to the World Checklist of Useful Plant Species. The ‘Genes’ module contains information on genes as well as their associated coding DNA sequence (CDS) and protein sequence (Figure 1e). To ensure a high‐quality dataset, we first unified gene names by curating incorrect capitalization, spelling mistakes, extra characters in gene names, and synonymous gene names (Figure 1f). More importantly, not only was uniformity achieved, but corrections were also made. For example, the gene NADH–ubiquinone oxidoreductase chain 6 (nad6) should be encoded in the mitochondrial genome. However, this gene was observed in some chloroplast genome annotations, such as Bulbophyllum reptans (GenBank accession: NC_058531.1). By manual curation, we confirmed that nad6 in NC_058531.1 was ndhG (Figure 1f).

Figure 1.

Architecture of the CGIR. (a) Design and construction of the CGIR, (b) the CGIR homepage, (c) the Genome module, (d) the curation model of taxonomic information, (e) the Gene module, (f) the curation model of gene annotation, (g) the Barcode module, (h) the DSS module, (i) the SSR module, (j) the Taxonomy tree view, (k) the Download module, (l) BarcodeBlast, (m) BarcodeFinder, and (n) the statistics of CGIR.

To better utilize these chloroplast genomes, the remaining three modules contained three commonly used DNA markers developed based on chloroplast genomes. The ‘Barcodes’ module contains DNA barcodes extracted from 29 different loci using the electronic PCR approach (Figure 1g), making the CGIR an excellent complement to traditional DNA barcode databases (e.g., Barcode of Life Data System [BOLD; Ratnasingham and Hebert, 2007]), which are mainly from rbcL and matK loci. The ‘DSS’ module contains the candidate DSSs from all species with more than one chloroplast assembly deposited in the CGIR (Figure 1h). DSS is a species‐level marker that can be used as a complement to conventional DNA markers (Hua et al., 2022). The ‘SSR’ module comprises 7 284 705 SSRs and their associated primers (Figure 1i), far exceeding that of any other plastid SSR database.

In addition, the CGIR provides various methods for viewing, searching, and downloading data. To help users find the genome of a certain taxon, the ‘Genomes’ module allows users to search by species name, as well as class, order, genus, and family names. The synonyms are also listed in the search results, enabling researchers to determine whether to use these assemblies (Figure 1c). Because chloroplast data are always used in interspecies comparisons, the CGIR also provides a taxonomy tree view for users who are concerned with specific aspects of chloroplast data (e.g., rbcL gene, CDS sequences) in higher taxa (Figure 1j). Using this view, users can browse, search, and retrieve gene, barcode, and DSS data at any taxonomic level. A separate download module is also provided for easy data downloads (Figure 1k). Additionally, the ‘BarcodeBLAST’ tool allows users to search their barcode sequences against those deposited in the CGIR using BLAST (Figure 1l), and the ‘BarcodeFinder’ tool can help users to identify barcode regions in their uploaded chloroplast sequences (Figure 1m).

In general, the integration of high‐throughput sequencing, public genomic resources, and careful manual curation guaranteed both the quantity and quality of chloroplast data in the CGIR, making it the largest comprehensive chloroplast repository available (Figure 1n). The CGIR will be a valuable resource for researchers working on phylogenetics and chloroplast genetic engineering. The curated taxonomy information and molecular markers are of tremendous value to plant phylogenetics; the labelled featured plants and corrected gene information will assist researchers in identifying suitable research objects and locating intergenic spacer regions, both of which are necessary for designing chloroplast engineering vectors (Daniell et al., 2021). In future, the CGIR will be continuously updated to incorporate more types of data.

Conflict of interests

The authors declare no conflict of interest.

Author contributions

Y.Y., S.S., Z.Z., and L.H. designed the project. Z.H. and C.J. wrote the manuscript. Z.C., Y.Z., C.J., and Y.J. collected the samples. Z.H., Z.C., and D.T. curated the data. D.T., S.S., and Z.Z. constructed the database. All authors read and approved the manuscript.