DATA REPORT article

Front. Plant Sci., 13 July 2026

Sec. Functional and Applied Plant Genomics

Volume 17 - 2026 | https://doi.org/10.3389/fpls.2026.1837075

The complete chloroplast genome of Trachyspermum ammi reveals a species-specific inverted repeat expansion

  • 1. ICAR-National Institute for Plant Biotechnology, New Delhi, India

  • 2. Division of Biochemistry, ICAR-Indian Agricultural Research Institute, New Delhi, India

  • 3. ICAR-National Research Centre on Seed Spices, Ajmer, India

1 Introduction

Trachyspermum ammi (L.) Sprague, commonly known as ajwain or carom, is an aromatic annual herb in the family Apiaceae, subfamily Apioideae, and one of the most commercially significant seed spice crops of South Asia and the Middle East (). The plant is native to Egypt and the eastern Mediterranean, and is extensively cultivated across the arid and semi-arid regions of India (particularly Gujarat and Rajasthan), Pakistan, Iran, Afghanistan, and Iraq (; ). T. ammi produces characteristic ovoid, greyish-brown mericarps (commonly called seeds) that are highly valued in Indian culinary traditions and across Ayurvedic, Siddha, and Unani systems of medicine (; ). The fruits possess a pungent, thyme-like aroma attributable to essential oils dominated by thymol, a monoterpene phenol, alongside γ-terpinene, p-cymene, and other volatile compounds (). Ethnobotanically, ajwain is used for the treatment of digestive disorders (flatulence, indigestion, dyspepsia), respiratory ailments (asthma, bronchial conditions), abdominal pain, and gynaecological complaints, among other conditions (; ; ).

Despite the considerable economic, medicinal, and cultural importance of T. ammi, its genomic resources remain limited. Previous molecular studies have been largely restricted to transcriptome-based investigations of thymol biosynthesis (), inflorescence development (), and the identification of SSR molecular markers (). The complete chloroplast genome sequence of T. ammi has not been fully characterized to date. A ~154 kb T. ammi chloroplast genome (NC_047246.1) is available in NCBI and has been reported as complete. This may represent a partial or differently assembled genome, as it is notably smaller (~154 kb) than our assembly and the independently sequenced T. ammi accession PV794607.

Chloroplast genomes (plastomes) in angiosperms are typically circular, quadripartite molecules of 120–170 kb, encoding 120–130 genes encompassing components of the photosynthetic apparatus, ribosomal proteins, RNA polymerase subunits, ribosomal RNAs, and transfer RNAs (; ). The canonical quadripartite architecture comprises a large single-copy (LSC) region and a small single-copy (SSC) region separated by a pair of inverted repeats (IRA and IRB). Their slow evolutionary rate, maternal inheritance, low recombination frequency, and largely conserved gene order have established plastomes as primary markers for phylogenetic reconstruction and species-level barcoding across angiosperms (; ; ).

Within Apiaceae, subfamily Apioideae is exceptional among angiosperms for the frequency and magnitude of structural variation in the plastome, particularly dynamic shifts in the inverted repeat (IR) boundaries (). documented at least ten independent expansions and contractions of the IR across Apioideae, with boundary shifts ranging from ~1 to 16 kb. Genera such as Angelica, Ferula, Peucedanum, Melanosciadium, Ligusticum, Sanicula, Glehnia, and Cnidium have all been reported to exhibit significant IR boundary polymorphism (; ). These IR boundary shifts are consequential because they alter gene copy number; genes transferred from the single-copy LSC into the duplicated IR are subject to IR-mediated gene conversion, which tends to homogenise the two IR copies and may reduce substitution rates in the captured genes ().

In the present study, we report the complete chloroplast genome of T. ammi cv. AA-1 and compare with those of T. scaberulum (NC_070346) and T. triradiatum (PV794612) to characterise the structural basis of the observed genome size difference and assess its evolutionary significance within the genus. This resource provides a foundational genomic reference for future research on T. ammi genetics, molecular breeding, pharmacological enhancement, and phylogenomics of Apioideae.

2 Materials and methods

2.1 Plant material and DNA extraction

Seeds of Trachyspermum ammi cultivar AA-1 were surface-sterilized and germinated on moistened filter paper at 25 ± 2 °C under a 16 h light/8 h dark photoperiod. Young leaves were harvested from two-week-old seedlings, immediately snap-frozen in liquid nitrogen, and stored at −80 °C until use. Total genomic DNA was extracted following a modified cetyltrimethylammonium bromide (CTAB) protocol (). DNA quality was assessed by measuring the A260/A280 absorbance ratio on a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA) and by 1% agarose gel electrophoresis. Only samples with an A260/A280 ratio of 1.8–2.0 and a minimum yield of 1.5 μg were used for library preparation.

2.2 Library preparation and sequencing

Sequencing libraries were constructed from high-quality genomic DNA using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, UK) according to the manufacturer’s instructions, incorporating end-repair, dA-tailing, adapter ligation, and PCR enrichment steps. Paired-end sequencing (2 × 150 bp) was performed on the Illumina NovaSeq 6000 platform (Illumina, USA) at a commercial sequencing facility.

2.3 Chloroplast genome assembly and annotation

Raw FASTQ reads were quality-assessed using FastQC v0.11.9 (; https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Adapter sequences and low-quality bases (Phred score < 20) were removed using Trim Galore v0.6.7 (; https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) with default parameters. Chloroplast reads were assembled de novo using GetOrganelle v1.7.5 (). Assembly graph circularity and completeness were verified using Bandage v0.8.1 (). Genome annotation was performed using three complementary tools to maximise accuracy: GeSeq v2.03 () with default eudicot reference sets, CPGAVAS2 (), and PGA (). Annotation conflicts between the three tools were resolved by manual inspection, with particular attention to start and stop codon positions, CDS reading frames, intron–exon boundaries, and gene strand orientations. The final annotated sequence was visualized as a circular map using CPGView (). The complete annotated chloroplast genome sequence of T. ammi cv. AA-1 has been deposited in the NCBI GenBank database under accession number PZ091433; raw whole-genome sequencing reads are available under BioProject ID PRJNA1437723.

2.4 Comparative analysis of inverted repeat boundaries

To investigate inverted repeat contraction and expansion of the chloroplast genome of T. ammi, the boundaries of the large single-copy (LSC) region, small single-copy (SSC) region, and the two IR regions (IRA and IRB) were compared across four Trachyspermum plastomes: T. ammi cv. AA-1 (PZ091433, this study), T. ammi isolate YL11021 (PV794607), T. scaberulum (NC_070346), and T. triradiatum isolate YL09151 (PV794612). To ensure consistency, all the genomes were reoriented relative to the rbcL gene using the Rotate v1.0 tool () and subsequently reannotated using GeSeq v2.06 (). IR boundary positions were determined from the annotated repeat region features and confirmed by examining the positions of known boundary-flanking genes (rpoA at the LSC/IRB junction; ycf1 at the IRB/SSC junction; ndhF in the SSC; rpl2 and rpl23 at the IRA/LSC junction). The IR boundary positions and junction gene configurations of all four genomes were visualized using CPJSdraw v1.0.0 ().

3 Preliminary data analysis

The complete chloroplast genome of T. ammi cv. AA-1 (GenBank accession PZ091433) is a circular molecule of 160,370 bp (Figure 1A). It exhibits the typical quadripartite structure consisting of a large single-copy (LSC) region of 78,260 bp, a small single-copy (SSC) region of 17,180 bp, and a pair of inverted repeats (IRA and IRB) of 32,465 bp each. The plastome of T. ammi is larger than those typically reported for most Apiaceae (; ). This increased genome size is largely attributable to the species-specific expansion of the IR, a type of structural variation extensively documented in Apiaceae (). The overall GC content is 37.73%, which falls within the range commonly reported for eudicot plastomes (; ). As expected, GC content varies across regions, being highest in the IR (41.41%), moderate in the LSC (36.04%), and lowest in the SSC (31.50%). These patterns are consistent with those observed in other members of the Apiaceae family (). Genome annotation revealed 113 unique genes, including 79 protein-coding genes, 30 tRNA genes, and 4 rRNA genes (Table 1). This gene content is typical for the Apiaceae subfamily Apioideae (; ). Fifteen protein-coding genes and six tRNA genes contain introns. Among them, clpP and ycf3 each harbour two introns, while atpF, rpoC1, rpl2, rpl16, petB, petD, rps16, ndhA, and ndhB contain one intron each. The maturase gene matK is nested within a large group IIA intron of trnK-UUU, a conserved structural arrangement found across angiosperm plastomes. The rps12 gene is trans-spliced, with its 5′ exon located in the LSC and the 3′ exons duplicated in the IR regions ().

Figure 1

Table 1

FunctionGene groupGenes
Photosynthesis
PhotosynthesisPhotosystem I (5)psaA, psaB, psaC, psaI, psaJ
Photosystem II (15)psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ
F-type ATP synthase (6)atpA, atpB, atpE, atpFb, atpH, atpI
NADH dehydrogenase (11)ndhAb, ndhBba, ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK
Cytochrome b6/f complex (6)petA, petBb, petDb, petG, petL, petN
Large subunit of RuBisCO (1)rbcL
Genetic apparatus
TranscriptionRNA polymerase (4)rpoAa, rpoB, rpoC1b, rpoC2
Ribosomal proteinsLarge subunit (9)rpl2ba, rpl14a, rpl16b, rpl20, rpl22a, rpl23a, rpl32, rpl33, rpl36a
Small subunit (13)rps2, rps3a, rps4, rps7a, rps8a, rps11a, rps12da, rps14, rps15, rps16b, rps18, rps19a
Translation initiation factor (1)infAa
Structural RNAs
Structural RNAsTransfer RNAs (30)trnA-UGCba, trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnG-GCC, trnG-UCCb, trnH-GUG, trnI-CAUa, trnI-GAUba, trnK-UUUb, trnL-CAAa, trnL-UAAb, trnL-UAG, trnM-CAU, trnN-GUUa, trnP-UGG, trnQ-UUG, trnR-ACGa, trnR-UCU, trnS-GCU, trnS-GGA, trnS-UGA, trnT-GGU, trnT-UGU, trnV-GACa, trnV-UACb, trnW-CCA, trnY-GUA, trnfM-CAU
Ribosomal RNAs (4)rrn4.5a, rrn5a, rrn16a, rrn23a
Other functions
Post-transcriptional modificationMaturase (1)matK
OtherATP-dependent protease (1)clpPc
Acetyl-CoA carboxylase (1)accD
Envelope membrane protein (1)cemA
Cytochrome c haem attachment (1)ccsA
Genes of unknown or hypothetical function
Unknown/hypotheticalHypothetical chloroplast reading frames (3)ycf1, ycf2a, ycf3c, ycf15a

Gene composition of the chloroplast genome of Trachyspermum ammi cv. AA-1.

a

Genes duplicated in both inverted repeat regions (IRA and IRB).

b

Gene contains one intron.

c

Gene contains two introns.

d

Gene is trans-spliced; the 5′ exon of rps12 is in the LSC and the duplicated 3′ exons are in the IR regions.

The most distinctive feature of the T. ammi plastome is a pronounced species-specific expansion of the inverted repeat. This IR expansion is consistent across two independent T. ammi accessions (PZ091433 and PV794607) that share 100% sequence identity, confirming that it is a stable species level characteristic rather than an assembly artefact. Each IR measures 32,465 bp, which is ~7,598 bp larger than those of its congeners, T. scaberulum (24,867 bp) and T. triradiatum (24,985 bp). Consequently, the IR regions constitute 40.5% of the total T. ammi genome, compared with 32.6% in T. scaberulum and 32.7% in T. triradiatum.

At 32,465 bp, the IR of T. ammi is among the largest reported in the subfamily Apioideae. Similar IR sizes have been reported in Ferula sinkiangensis, Melanosciadium pimpinelloideum, and other Ferula species, whereas even larger IRs occur in the long-type plastome of Peucedanum japonicum (; ; ; ; ). However, unlike Ferula and Angelica, where large IRs are shared across multiple species and likely represent ancestral expansions, the enlarged IR of T. ammi appears to be species-specific. Both sequenced congeners retain the typical Apioideae IR size of approximately 25 kb, suggesting that the expansion occurred after the divergence of T. ammi from the common ancestor of the genus.

Comparative analysis of the IR boundaries (Figure 1B) shows that the LSC/IRB junction in T. ammi has shifted inward by ~7,321 bp into the LSC region compared to other species of the Trachyspermum genus. As a result, the RNA polymerase gene rpoA now straddles the new junction, with only its 5′ end (218 bp) remaining in the LSC. This boundary shift has led to the duplication of 10 ribosomal protein genes (rps11, rpl36, infA, rps8, rpl14, rpl16, rps3, rpl22, rps19, and rpl2), which remain single copy in the LSC of T. scaberulum and T. triradiatum.

The molecular mechanism most widely accepted to explain IR boundary shifts in angiosperm plastomes is illegitimate recombination between short dispersed repeats located near the IR/single-copy junctions, followed by IR-mediated gene conversion that progressively extends one IR copy at the expense of the adjacent single-copy region (; ). The IR expansion observed in T. ammi is consistent with this model and appears to be restricted to the LSC/IRB boundary. In contrast, the SSC region remains highly conserved among the three examined Trachyspermum species, measuring 17,180 bp in T. ammi, 17,120 bp in T. scaberulum, and 17,112 bp in T. triradiatum. Likewise, the IRB/SSC boundary gene ycf1 exhibits an identical arrangement in all three species. Such asymmetric expansion affecting only the LSC/IR boundary is characteristic of IR expansion events documented across Apioideae (; ).

A major consequence of this expansion is the duplication of ten ribosomal protein genes (rps11, rpl36, infA, rps8, rpl14, rpl16, rps3, rpl22, rps19, and rpl2) that were ancestrally located in the LSC. These genes are now incorporated into the duplicated IR and therefore exist as two identical copies. Genes located within IR regions are subject to continuous copy correction through IR-mediated gene conversion, a process that suppresses mutation accumulation and maintains sequence identity between the two IR copies (; ; ). Across land plants, synonymous substitution rates in IR-located genes are approximately 3.7-fold lower than those of genes residing in single-copy regions (). Accordingly, the duplicated ribosomal protein genes in T. ammi are expected to experience stronger purifying selection and maintain higher sequence fidelity than their homologues in T. scaberulum and T. triradiatum.

The enlarged IR, together with the unique boundary configuration involving rpoA and the duplicated ribosomal protein genes, represents a useful molecular character for species identification and phylogenetic studies within Trachyspermum genus. Further sampling of additional species will be necessary to determine whether this structural rearrangement is unique to T. ammi or shared with closely related taxa within the tribe Ammineae.

Statements

Data availability statement

The datasets presented in this study can be found in the NCBI (https://www.ncbi.nlm.nih.gov). The annotated chloroplast genome sequence is available in the GenBank under accession number PZ091433, and the raw sequencing data are available under BioProject ID PRJNA1437723.

Author contributions

RN: Conceptualization, Data curation, Formal analysis, Methodology, Supervision, Writing – original draft, Writing – review & editing. VP: Methodology, Writing – original draft, Writing – review & editing. NM: Writing – original draft, Writing – review & editing. SC: Writing – original draft, Writing – review & editing.

Funding

The author(s) declared that financial support was not received for this work and/or its publication.

Acknowledgments

Research was supported by the Indian Council of Agricultural Research, Department of Agricultural Research and Education, Government of India.

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

The author(s) declared that generative AI was not used in the creation of this manuscript.

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All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

References

Summary

Keywords

ajwain, Trachyspermum ammi, Apiaceae, Apioideae, chloroplast genome, inverted repeat expansion, seed spice crop, plastome

Citation

Nagar R, Patial V, M. S. N and Choudhary S (2026) The complete chloroplast genome of Trachyspermum ammi reveals a species-specific inverted repeat expansion. Front. Plant Sci. 17:1837075. doi: 10.3389/fpls.2026.1837075

Received

23 March 2026

Revised

16 June 2026

Accepted

17 June 2026

Published

13 July 2026

Volume

17 - 2026

Edited by

Wei Tong, Anhui Agricultural University, China

Reviewed by

Linhe Sun, Jiangsu Province and Chinese Academy of Sciences, China

Dahe Qiao, Guizhou Province Academy of Agricultural Science, China

Updates

Copyright

*Correspondence: Ramawatar Nagar,

Disclaimer

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.

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