Marker Selection Cheat Sheet: COI vs rbcL/matK vs ITS

Short answer: If you need a fast, defensible way to choose DNA barcoding markers, start with COI for animals, rbcL + matK for land plants, and ITS/ITS2 for fungi and select plant groups. Use mini-barcodes (100–300 bp) when DNA is degraded. This page links to our step-by-step workflow and the DNA Barcoding Service for intake, locus recommendations, and reporting templates.

Marker decision map for DNA barcoding: COI for animals, rbcL + matK for plants, ITS/ITS2 for fungi, and mini-barcodes (100–300 bp) for degraded samples.

How to Use This Cheat Sheet

This selector maps taxon → marker(s) → amplicon size → primer options → reference depth → notes so you avoid trial-and-error. For samples that contain DNA from multiple organisms (water filters, soil, feces), switch to metabarcoding rather than single-specimen barcoding; see DNA Barcoding vs Metabarcoding. For process and QC details, use How Does DNA Barcoding Work?

Quick Selector Matrix

Use case	Primary marker(s)	Typical amplicon (bp)	Primer examples	Reference depth	Notes
Animals (general)	COI	~658	LCO1490/HCO2198 ("Folmer")	Very high for many phyla	Widely validated for animal identification.
Fish & seafood	COI (mini-COI for processed)	~650; mini 100–300	FishF2_t1/VF2_t1 cocktails; jgLCO/jgHCO variants	High and growing	Cocktails improve recovery; mini-COI helps degraded tissue.
Land plants (routine)	rbcL + matK	rbcL ~600; matK ~700–900	rbcLa-F / rbcLa-R; matK 390F/1326R (or KIM sets)	rbcL broad; matK variable	rbcL amplifies reliably; matK adds discrimination.
Botanicals / medicinal herbs	rbcL + matK ± ITS/ITS2	ITS2 ~200–400	ITS1/ITS4 or ITS2 universal sets	Good in many clades	ITS/ITS2 often improves separation among close relatives.
Fungi (incl. lichens)	ITS (ITS1/ITS2)	~300–700	ITS1F/ITS4 (and variants)	Extensive	ITS is the primary fungal barcode.
Degraded DNA / archival	Mini-barcodes	100–300	Short COI or plastid/ITS fragments	Varies	Proven approach for archival or processed samples.

Tip: Keep the table handy in your lab wiki. When in doubt, send your target taxa and sample matrix to our team via the DNA Barcoding Service for a tailored marker set.

Animals: Why COI Still Leads—and When to Tweak It

Why COI: COI balances universality and variation, supporting broad cross-taxon amplification and species-level resolution. The classic LCO1490/HCO2198 ("Folmer") pair targets a ~658 bp region that works across many metazoans. For clades where Folmer underperforms, use degenerate or short-amplicon alternatives such as mlCOIintF + jgHCO2198 (~313 bp) to lift success in diverse animal groups.

When to adjust:

Fish and seafood barcoding: Start with FishF2_t1/VF2_t1 primer cocktails, which improve recovery across families. In cooked or heavily processed fillets, switch to mini-COI (100–300 bp) to overcome fragmentation.
Groups with mitochondrial quirks: If you suspect introgression, NUMTs, or recent radiations, add a second locus or confirm with morphology. Document evidence in your report rather than relying on a single percent identity.

Good practice:

Match against both curated and broad databases (e.g., BOLD Systems and GenBank).
Favor voucher-linked references and verify geographic plausibility.
Keep chromatogram QC logs and aligned length metrics with every reported ID.

Land Plants: rbcL + matK First, with ITS/ITS2 as a Targeted Booster

Resolution (%) of genera, species, and infraspecific taxa for rbcL, matK, and rbcL + matK across the Arctic flora dataset, showing higher identification performance when combining loci. (Saarela J.M. et al. (2013) PLOS ONE)

Core barcode: The community standard for land plants is rbcL + matK. In practice, rbcL amplifies widely and anchors comparisons; matK often improves species-level discrimination but can be more variable to amplify.

Primer picks that travel well:

rbcL: rbcLa-F / rbcLa-R perform well across many families.
matK: 390F/1326R is common; KIM primer sets can further extend coverage across angiosperms.

When to add ITS/ITS2:

Use ITS/ITS2 when working with closely related species, such as medicinal herbs with known substitution risks.
Validate performance in your focal clade. ITS can outperform plastid barcodes in some groups but is not universal across all plants.

Reporting notes for botanicals:

If multiple congeners cluster tightly, state the best-supported species and note plausible alternatives.
Provide aligned length, identity, and references; avoid over-confident calls when reference coverage is thin.

Fungi: Use ITS as the Primary Barcode

Why ITS: A large benchmarking effort identified nuclear ribosomal ITS as the universal fungal barcode. COI is not recommended for fungi due to introns an amplification issues. Most labs start with ITS1F/ITS4 or close variants and tune amplicon length by group.

Practical tips:

For environmental or mixed fungal material, consider amplicon length that matches your sequencing read length to minimize trimming loss.
Maintain a curated internal list of successful primer sets by lineage, and retire those that underperform in your matrices.

Examples of barcode-gap classifications (good, intermediate, poor) across Basidiomycota genera, illustrating when full ITS or sub-regions (ITS1/ITS2) perform best. (Badotti F. et al. (2017) BMC Microbiology)

Degraded DNA: Plan Mini-Barcodes from the Start

Formalin-exposed tissues, museum specimens, and processed foods often yield fragmented DNA. Mini-barcodes in the 100–300 bp range preserve discriminatory signal while increasing amplification and sequencing success.

Schematic of multiple mini-barcode primer targets within the standard fish COI region (127–314 bp), facilitating DNA recovery from degraded or processed material (Shokralla S. et al. (2015) Scientific Reports). Schematic map of multiple mini-barcode primer targets positioned within the standard fish COI region (127–314 bp), enabling recovery from degraded or processed material. (Shokralla S. et al. (2015) Scientific Reports)

Where mini-barcodes shine:

Seafood authenticity and other processed food audits.
Museum and archival material where preservation chemistry complicaes extraction.
Field-collected swabs and trace materials with low template concentration.

Design notes:

Keep amplicons short and primers robust to common inhibitors.
Expect to report with slightly lower aligned length; compensate with strong curation and multiple evidence lines.

Primer Crib Sheet (Fast Choices That Work Often)

Animals (COI, general): LCO1490/HCO2198; for broad metazoans or degraded DNA, try mlCOIintF + jgHCO2198 (~313 bp).
Fish: FishF2_t1/VF2_t1 primer cocktails to improve family-level coverage.
Plants (rbcL): rbcLa-F / rbcLa-R as a reliable universal set.
Plants (matK): 390F/1326R or KIM primer sets; be prepared for variable success.
Fungi (ITS): ITS1F/ITS4; adjust amplicon length for your lineage and platform.

For a deeper walkthrough of acceptance criteria, QC thresholds, and data hand-off, use How Does DNA Barcoding Work?.

Reference Depth and Curation: When to Trust the Match

Curated libraries matter. Use BOLD Systems for barcode-centric matching and GenBank for broad coverage. Where animal COI species names are unsettled, the BIN (Barcode Index Number) acts as a stable cluster label that often aligns with species boundaries.

Avoid common pitfalls:

Over-interpreting a single number: Identity values without aligned length and gap pattern can mislead.
Trusting unvouchered records: Prefer sequences tied to vouchers and georeferenced metadata.
Thin references: Where a clade is undersampled, consider a second locus or expert taxonomic review.

What to include in your report:

Which database(s) you searched and on what date.
Alignment length and identity for each locus used.
Voucher accessions or catalog numbers, where available.

Reporting That Stakeholders Can Audit (and Accept)

Stakeholders need clarity and traceability. Keep your language specific and testable:

Describe what was tested (marker, amplicon size, primers), how it was matched (databases, algorithms), and why the reported ID is the best-supported conclusion.
If taxonomy is unsettled, cite the BIN (for animal COI) and summarize competing hypotheses rather than forcing a single name.
Include a one-page summary table listing sample IDs, loci, aligned length, top match identity, database source, and interpretation notes.

If you need a starting point, our DNA Barcoding Service can supply an editable report template aligned to COI/rbcL/matK/ITS workflows and your sector's documentation needs.

Troubleshooting Signals (Choose, Don't Chase)

rbcL amplifies; matK fails: Keep the rbcL result. Try alternative matK primers (e.g., KIM sets). Consider adding ITS/ITS2 for extra resolution rather than forcing matK in stubborn taxa.
COI fails in certain animal clades: Use degenerate or mini-COI primers. Check for inhibitors or poor DNA integrity, especially in preserved or processed tissues.
Ambiguous top hits: Do not rely only on the first record. Review the top set, check geography and vouchers, and add a second locus if the signal is weak.
Mixed DNA in the extract: Classic barcoding expects a dominant template. For composites, switch to metabarcoding with a validated pipeline.

Example Decisions

Museum beetle leg, 20+ years old

Start with mini-COI to maximize recovery from old tissue. If discrimination is borderline, layer a second short mitochondrial fragment or confirm with expert morphology.

Dried herbal powder (authentication)

Run rbcL + matK and add ITS2 to improve separation among near relatives often involved in substitution. Phrase results with best-supported species and plausible alternatives.

Frozen fish fillet (retail audit)

Use COI with fish primer cocktails. If traces suggest degradation, switch to mini-COI. Prioritize voucher-linked references and document commercial name vs. scientific name mapping in your notes.

FAQs

1) What is the best single marker for land plants—rbcL or matK?

Neither wins alone. rbcL amplifies more consistently across clades, while matK often improves species-level discrimination. The community standard is to use both as the core plant barcode.

2) When should I add ITS/ITS2 to plant barcoding?

Add ITS/ITS2 when you need more separation among closely related species, such as medicinal herbs or genera with recent radiations. Validate within your focal clade before scaling.

3) My DNA is degraded—how short can I go?

Use mini-barcodes in the 100–300 bp range. They extend barcoding to archival and processed materials while retaining useful signal.

4) Which COI primers should I start with for animals or fish?

Begin with LCO1490/HCO2198 for general COI. For fish, FishF2_t1/VF2_t1 primer cocktails often yield higher success across families. For broad metazoans or degraded DNA, mlCOIintF + jgHCO2198 (~313 bp) is a reliable short-amplicon option.

5) How do I report results when names are disputed or in flux?

Provide aligned length and identity, cite your database sources, and include the BIN(Barcode Index Number) for animal COI as a stable cluster label until taxonomy settles.

What to Do Next

Read How Does DNA Barcoding Work? to set acceptance criteria and QC steps before your first PCR.
Share your target taxa, matrix, and decision context via the DNA Barcoding Service. We will recommend markers, primers, and a reporting format aligned to your goals.

RUO reminder: Our all services and deliverables are provided for research use only and are not intended for clinical applications.

Related Resources

References

Saarela, J.M., Sokoloff, P.C., Gillespie, L.J. et al. DNA Barcoding the Canadian Arctic Flora: Core Plastid Barcodes (rbcL + matK) for 490 Vascular Plant Species. PLOS ONE 8(10), e77982 (2013).
Chen, S., Yao, H., Han, J. et al. Validation of the ITS2 Region as a Novel DNA Barcode for Identifying Medicinal Plant Species. PLOS ONE 5(1), e8613 (2010).
Badotti, F., de Oliveira, F.S., Garcia, C.F. et al. Effectiveness of ITS and Sub-regions as DNA Barcode Markers for the Identification of Basidiomycota (Fungi). BMC Microbiology 17, 42 (2017).
Shokralla, S., Hellberg, R.S., Handy, S.M. et al. A DNA Mini-Barcoding System for Authentication of Processed Fish Products. Scientific Reports 5, 15894 (2015).
Hebert, P.D.N., Cywinska, A., Ball, S.L. et al. Biological Identifications Through DNA Barcodes. Proceedings of the Royal Society B 270, 313–321 (2003).
CBOL Plant Working Group. A DNA Barcode for Land Plants. Proceedings of the National Academy of Sciences 106(31), 12794–12797 (2009).
Leray, M., Yang, J.Y., Meyer, C.P. et al. A New Versatile Primer Set Targeting a Short Fragment of the Mitochondrial COI Region for Metabarcoding Metazoan Diversity. Frontiers in Zoology 10, 34 (2013).

* Designed for biological research and industrial applications, not intended for individual clinical or medical purposes.