Plastome (plastid genome) sequences provide valuable information for understanding the phylogenetic relationships and evolutionary history of plants. Although the rapid development of high-throughput sequencing technology has led to an explosion of plastome sequences, annotation remains a significant bottleneck for plastomes. User-friendly batch annotation of multiple plastomes is an urgent need. We introduce Plastid Genome Annotator (PGA), a standalone command line tool that can perform rapid, accurate, and flexible batch annotation of newly generated target plastomes based on well-annotated reference plastomes. In contrast to current existing tools, PGA uses reference plastomes as the query and unannotated target plastomes as the subject to locate genes, which we refer to as the reverse query-subject BLAST search approach. PGA accurately identifies gene and intron boundaries as well as intron loss. The program outputs GenBank-formatted files as well as a log file to assist users in verifying annotations. Comparisons against other available plastome annotation tools demonstrated the high annotation accuracy of PGA, with little or no post-annotation verification necessary. Likewise, we demonstrated the flexibility of reference plastomes within PGA by annotating the plastome of Rosa roxburghii using that of Amborella trichopoda as a reference. The program, user manual and example data sets are freely available at https://github.com/quxiaojian/PGA . PGA facilitates rapid, accurate, and flexible batch annotation of plastomes across plants. For projects in which multiple plastomes are generated, the time savings for high-quality plastome annotation are especially significant.

The plastid genome (plastome) represents an indispensable molecular resource for studying plant phylogeny and evolution. Although plastome size is much smaller than that of nuclear genomes, accurately and efficiently annotating and utilizing plastome sequences remain challenging. Therefore, a streamlined phylogenomic pipeline spanning plastome annotation, phylogenetic reconstruction and comparative genomics would greatly facilitate research utilizing this important organellar genome. Here, we develop PlastidHub, a novel web application employing innovative tools to analyze plastome sequences. In comparison with existing tools, key novel functionalities in PlastidHub include: (1) standardization of quadripartite structure; (2) improvement of annotation flexibility and consistency; (3) quantitative assessment of annotation completeness; (4) diverse extraction modes for canonical and specialized sequences; (5) intelligent screening of molecular markers for biodiversity studies; (6) gene-level visual comparison of structural variations and annotation completeness. PlastidHub features cloud-based web applications that do not require users to install, update, or maintain tools; detailed help documents including user guides, test examples, a static pop-up prompt box, and dynamic pop-up warning prompts when entering unreasonable parameter values; batch processing capabilities for all tools; intermediate results for secondary use; and easy-to-operate task flows between file upload and download. A key feature of PlastidHub is its interrelated task-based user interface design. Give that PlastidHub is easy to use without specialized computational skills or resources, this new platform should be widely used among botanists and evolutionary biologists, improving and expediting research employing the plastome. PlastidHub is available at https://www.plastidhub.cn.

Background: Viola philippica Cav. is the only source plant of “Zi Hua Di Ding”, which is a Traditional Chinese Medicine (TCM) that is utilized as an antifebrile and detoxicant agent for the treatment of acute pyogenic infections. Historically, many Viola species with violet flowers have been misused in “Zi Hua Di Ding”. Viola have been recognized as a taxonomically difficult genera due to their highly similar morphological characteristics. Here, all common V. philippica adulterants were sampled. A total of 24 complete chloroplast (cp) genomes were analyzed, among these 5 cp genome sequences were downloaded from GenBank and 19 cp genomes, including 2 “Zi Hua Di Ding” purchased from a local TCM pharmacy, were newly sequenced. Results: The Viola cp genomes ranged from 156,483 bp to 158,940 bp in length. A total of 110 unique genes were annotated, including 76 protein-coding genes, 30 tRNAs, and four rRNAs. Sequence divergence analysis screening identified 16 highly diverged sequences; these could be used as markers for the identification of Viola species. The morphological, maximum likelihood and Bayesian inference trees of whole cp genome sequences and highly diverged sequences were divided into five monophyletic clades. The species in each of the five clades were identical in their positions within the morphological and cp genome tree. The shared morphological characters belonging to each clade was summarized. Interestingly, unique variable sites were found in ndhF, rpl22, and ycf1 of V. philippica, and these sites can be selected to distinguish V. philippica from samples all other Viola species, including its most closely related species. In addition, important morphological characteristics were proposed to assist the identification of V. philippica. We applied these methods to examine 2 “Zi Hua Di Ding” randomly purchased from the local TCM pharmacy, and this analysis revealed that the morphological and molecular characteristics were valid for the identification of V. philippica. Conclusions: This study provides invaluable data for the improvement of species identification and germplasm of V. philippica that may facilitate the application of a super-barcode in TCM identification and enable future studies on phylogenetic evolution and safe medical applications.

Eragrostideae Stapf, the second-largest tribe in Chloridoideae (Poaceae), is a taxonomically complex tribe. In this study, chloroplast genomes of 13 Eragrostideae species were newly sequenced and used to resolve the phylogenetic relationships within Eragrostideae. Including seven reported chloroplast genomes from Eragrostideae, the genome structure, number and type of genes, codon usage, and repeat sequences of 20 Eragrostideae species were analyzed. The length of these chloroplast genomes varied from 130,773 bp to 135,322 bp. These chloroplast genomes showed a typical quadripartite structure, including a large single-copy region (77,993–80,643 bp), a small single-copy region (12,410–12,668 bp), and a pair of inverted repeats region (19,394–21,074 bp). There were, in total, 129–133 genes annotated in the genome, including 83–87 protein-coding genes, eight rRNA genes, and 38 tRNA genes. Forward and palindromic repeats were the most common repeat types. In total, 10 hypervariable regions (rpl22, rpoA, ndhF, matK, trnG–UCC-trnT–GGU, ndhF–rpl32, ycf4–cemA, rpl32–trnL–UAG, trnG–GCC–trnfM–CAU, and ccsA–ndhD) were found, which can be used as candidate molecular markers for Eragrostideae. Phylogenomic studies concluded that Enneapogon diverged first, and Eragrostis including Harpachne is the sister to Uniola. Furthermore, Harpachne harpachnoides is considered as a species of Eragrostis based on morphological and molecular evidence. In addition, the interspecies relationships within Eragrostis are resolved based on complete chloroplast genomes. This study provides useful chloroplast genomic information for further phylogenetic analysis of Eragrostideae.