DNA Barcoding in Plants: Choosing rbcL, matK, and ITS2

Most teams searching DNA barcoding in plants face the same choice: rely on the rbcL + matK plastid barcode, or add ITS2 to push species-level resolution—especially for herbs and processed botanicals. This practical guide explains when each marker wins, how to avoid typical lab pitfalls, and how to report results that reviewers trust. If you need an end-to-end option, our Plant DNA Barcoding Service and DNA Barcoding Service provide study design, validated primers, and audit-ready reports.

Quick Answer: What Works for Plants

One-line takeaway: For land plants, the community standard is rbcL + matK; ITS2 often boosts species discrimination and is widely used for herbal authentication and mixed botanical inputs.

Conceptual overview of plant DNA barcoding and marker choices (rbcL, matK, ITS/ITS2) across taxa, from amplification to reference matching (Applied Sciences, 2024, MDPI).

Why this matters:

• The right loci reduce PCR retries and re-sequencing.
• Match rates improve in BOLD and GenBank, making calls easier to defend.
• Reports align better with QA needs and procurement audits.

What each locus contributes—at a glance

• rbcL (plastid, coding): Very high universality and clean alignments; ideal "backbone" marker. Resolution can be limited among very close relatives.
• matK (plastid, coding): Faster evolving than rbcL; boosts species separation, but some clades need optimized primers or primer cocktails.
• ITS2 (nuclear ribosomal spacer): Strong species-level power in medicinal plants and diverse angiosperms; widely used for herbal materials and processed botanicals. Best paired with rbcL/matK for defensible calls.

Scatterplots of maximum intraspecific vs. minimum interspecific K2P distances showing barcode gaps across single and multi-locus plant barcodes; ITS2 alone resolves 93.2% of species (Wu X. et al. (2021) BMC Plant Biology).

rbcL vs matK vs ITS2: What Each Marker Brings

rbcL: The Universal Workhorse

Why teams start here. rbcL amplifies consistently across major plant groups and aligns cleanly. It's excellent for vouchers, large reference builds, and compliance settings where recoverability matters.

When rbcL is enough

• Leaves or tissues from well-sampled genera
• Routine curation where genus-level resolution is acceptable
• Regional reference projects prioritizing consistent coverage

matK: The Plastid "Sharpening Lens"

Why add matK. matK evolves faster and often separates congeners that rbcL cannot. Historically, amplification was uneven; multiplexable matK primer cocktails improved success across angiosperms, cutting troubleshooting time.

Where matK shines

• Floras rich in look-alike species
• Herbarium projects seeking stronger species resolution
• Seeding robust regional or product-line libraries

ITS2: Resolution for Herbs and Complex Mixes

Why plant labs lean on ITS2. Large datasets in medicinal plants show high species-level identification rates and good amplification from processed material. ITS2 helps detect substitutions and adulterants that plastid barcodes may blur.

Caveats to manage

• The nuclear ribosomal cistron can include paralogues or pseudogenes.
• Use conservative primer design and QC filters; flag ambiguous profiles for confirmatory testing.

Lab Reality: Primer Success, Reference Depth, and Reporting

Primer Strategy That Avoids Rework

• Voucher workflow: start with rbcL + matK; keep a matK primer cocktail in reserve; add ITS2 if close relatives remain unresolved.
• Processed botanicals: lead with ITS2 for species separation; confirm with rbcL/matK when chemistry allows, adding a second line of evidence for supply-chain defensibility.
• Backup plan: carry alternative primers per locus; pre-define when to pivot to shorter amplicons or mini-barcodes for degraded inputs.

Reference Libraries: Use Both BOLD and GenBank

• Cross-check sequences in BOLD Systems and GenBank to reduce misidentifications.
• Report accession numbers and, where applicable, BIN clusters; if BINs and Latin names diverge, present both and explain the limitation.
• Maintain a brief methods note: locus, primers, platform, length windows, orientation checks, and match thresholds.

Quality Controls That Keep IDs Defensible

• Extraction blanks and no-template controls to catch contamination early
• Replicate PCRs for borderline DNA or rare targets
• Length windows and orientation checks before database queries
• A pre-agreed ambiguity policy (e.g., genus-level call with rationale)

For teams that prefer a turnkey approach, our BOLD/GenBank Best Practices guidance and service workflows standardize these steps and shorten time-to-result.

Plant-Specific Pitfalls & Mixed-Product Caveats

Plastid–Nuclear Discordance Happens

Hybridization, introgression, or p Plastid barcodes trace maternal lineages olyploidy can blur plastid signals. When rbcL/matK disagree with morphology or geography, add ITS2 or an additional locus and report the conflict transparently.

Plastid phylogeny of Berberis illustrating polyphyly and lineage complexity that can lead to plastid–nuclear discordance in plant barcoding (Sun Y. et al. (2019) Frontiers in Plant Science).

ITS2 Paralogues and Pseudogenes

Watch for unusual base composition, frameshifts, or out-of-clade hits. Conservative pipelines screen problematic copies and avoid overconfident calls. When in doubt, require a second locus.

Degraded Teas, Tinctures, and Mixed Botanicals

Shorter amplicons improve recovery in harsh matrices. Besides ITS2 shorts, the trnL P6 loop is a well-known mini-barcode for mixed plant materials and environmental samples. It increases detection but offers lower species-level power—disclose that limit.

Supply-Chain Defensibility

• Keep voucher-linked references when possible.
• Use two-locus confirmation for high-risk SKUs.
• Document chain-of-custody and acceptance thresholds in the report.

Decision Guide & Comparison

Fast Picks

• Audit/voucher curation: run rbcL + matK; add ITS2 for stubborn clades or look-alikes.
• Herbal authentication/processed botanicals: use ITS2 first, then confirm with rbcL/matK.
• Reference library builds or regional flora projects: combine rbcL + matK + ITS2 to seed a resilient, multi-locus repository.

Marker Comparison for Plant Projects

Call to action: Start a scoped project through our Plant DNA Barcoding Service—we'll design the primer plan, define QC, and deliver voucher-backed, BOLD/GenBank-cross-checked reports.

FAQs: Plant DNA Barcoding Markers

Putting It All Together: A Plant-Focused Workflow You Can Defend

1. Define the decision. Are you curating vouchers, authenticating herbs, or building a regional reference? Your objective sets markers and acceptance rules.
2. Select your marker set. Start with rbcL + matK for vouchers or flora builds; lead with ITS2 for herbal authentication and processed goods; combine when in doubt.
3. Lock in primers. Adopt a matK primer cocktail; carry backups for each locus; pre-approve a pivot to shorter amplicons for degraded inputs.
4. Control your QC. Use extraction blanks, NTCs, replicate PCRs on borderline samples, enforce length windows, and check orientation before search.
5. Search both databases. Query BOLD and GenBank; record accessions/BINs and explain disagreements or low coverage.
6. Report clearly. Provide locus, primers, platform, top hits with % identity and coverage, and a concise interpretation with caveats. Add a "next step" when confidence is limited.
7. Close the loop. For contentious cases, add ITS2 or a secondary locus; for harsh matrices, try mini-barcodes like trnL P6 and disclose resolution limits.

Ready to move from plan to data? Our Plant DNA Barcoding Service integrates marker selection, primer design, QC gates, and BOLD/GenBank cross-checking into an auditable workflow with clear timelines. For cross-kingdom portfolios, our broader DNA Barcoding Service aligns animal, plant, and fungal markers under one reporting standard.

Related Resources

• DNA Barcoding 101: Definition, Steps, and How It Works
• Marker Selection Cheat Sheet: COI vs rbcL/matK vs ITS
• Building and Using Reference Libraries: BOLD & GenBank Best Practices