Table 2. Primer details for each DNA barcoding locus.

Table includes: the Genome Acquisition Lab (GAL) or Barcoding hub responsible for a given taxon group; taxon group with defined sets of DNA barcoding loci; DNA barcoding database used for search queries (multiple databases searched for some groups); forwards and reverse primers separated by a slash; ENA marker sequence checklist classification for depositing the data on ENA; primer sequences; locus description and notes providing detailed information and typical amplicon size.

GAL or Barcoding hub	Taxon group	DNA barcoding loci	DNA barcoding database	Primers	ENA marker sequence checklist	Forward Primer		Reverse Primer		Locus description and notes
GAL or Barcoding hub	Taxon group	DNA barcoding loci	DNA barcoding database	Primers	ENA marker sequence checklist	Name	Sequence	Name	Sequence	Locus description and notes
RBG Edinburgh	Seed plants	ITS2	BOLD/NCBI	ITS_S2F/ITS_S3R	ITS rDNA	ITS_S2F	ATGCGATACTTGGTGTGAAT	ITS_S3R	GACGCTTCTCCAGACTACAAT	Nuclear internal transcribed spacer 2, flanked by partial 5.8S and 26S
	Seed plants	rbcL	BOLD/NCBI	rbcLa-F/ rbcLajf634R;	Single CDS genomic DNA	rbcLa-F	ATGTCACCACAAACAGAGACTAAAG	rbcLajf634R	GAAACGGTCTCTCCAACGCAT	Partial sequence (607bp) of plastid- encoded rubisco gene
	Ferns, Lycophytes	rbcL	BOLD/NCBI	rbcLa-F/ rbcLajf634R	Single CDS genomic DNA	rbcLa-F	ATGTCACCACAAACAGAGACTAAAG	rbcLajf634R	GAAACGGTCTCTCCAACGCAT	Partial sequence (607bp) of plastid- encoded rubisco gene
	Ferns, Lycophytes	trnL-trnF	BOLD/NCBI	trnL.C_bryo/ trnFGAAf	Multi- Locus Marker	trnL.C_bryo	CGAAATTGGTAGACGCTGCG	trnFGAAf	ATTTGAACTGGTGACACGAG	Partial sequence of the first exon of the plastid trnL gene, the intragenic spacer, the second exon of the trnL gene, the trnL-trnF intergenic spacer, and partial sequence of the trnF gene
	Liverworts, Mosses, Hornworts	ITS2	BOLD/NCBI	ITS2.seqF/ ITS.4bryo	ITS rDNA	ITS2.seqF	AACAACTCTCAGCAACGG	ITS.4bryo	TCCTCCGCTTAGTGATATGC	Nuclear internal transcribed spacer 2, flanked by partial 5.8S and 26S
		rbcL	BOLD/NCBI	rbcLa-F/ rbcLajf634R	Single CDS genomic DNA	rbcLa-F	ATGTCACCACAAACAGAGACTAAAG	rbcLajf634R	GAAACGGTCTCTCCAACGCAT	Partial sequence (607bp) of plastid- encoded rubisco gene
		psbA-trnH	BOLD/NCBI	psbA501F/trnHR	Multi- Locus Marker	psbA501F	TTTCTCAGACGGTATGCC	trnHR	GAACGACGGGAATTGAAC	c. 500 bases of plastid-encoded photosystem gene, short intergenic spacer and partial trnH gene
	Lichen- forming fungi	ITS	BOLD/ NCBI/ UNITE	ITS1-F/ITS4	ITS rDNA	ITS1-F	CTTGGTCATTTAGAGGAAGTAA	ITS4	TCCTCCGCTTATTGATATGC	Internal transcribed spacer 1, 5.8S and internal transcribed spacer 2, flanked by partial 18S and 26S
		nuLSU	BOLD/ NCBI/ UNITE	LROR/LR5	rRNA Gene	LROR	ACCCGCTGAACTTAAGC	LR5	ATCCTGAGGGAAACTTC	Nuclear large subunit ribosomal RNA, partial sequence
		mtSSU	BOLD/ NCBI/ UNITE	mrSSU1/ mrSSU2R	rRNA Gene	mrSSU1	AGCAGTGAGGAATATTGGTC	mrSSU2R	CCTTCGTCCTTCAACGTCAG	Mitochondrial small subunit rDNA, partial sequence; trialling a different reverse primer (mtSSU3R) for a slightly longer fragment
NHM	Arthropoda, Mollusca	COI	BOLD/NCBI	Primer cocktail: LCO1490 + LepF/HCO2198 + LepR	COI gene	LCO1490	GGTCAACAAATCATAAAGATATTGG	HCO2198	TAAACTTCAGGGTGACCAAAAA ATCA	Partial sequence (c. 710bp) of COI gene
	Arthropoda, Mollusca	COI	BOLD/NCBI	Primer cocktail: LCO1490 + LepF/HCO2198 + LepR	COI gene	Lep_F1	ATTCAACCAATCATAAAGATATTGG	Lep_R1	TAAACTTCTGGATGTCCAAAAA ATCA	Partial sequence (c.648bp) of COI gene
	Gastropods	16S	BOLD/NCBI	16SAR_gpd_ S/16SBR_gpd_S	rRNA Gene	16SAR_gpd_S	CGCCTGTTTAWCAAAAACAT	16SBR_gpd_S	CCGGTYTGAACTCAGATCAGAT CAYGT	Partial sequence (c. 570 bp) of 16S rRNA gene
	Vertebrates	CO1	BOLD/NCBI	C_VF1LFt1 – C_VR1LRt1 (Mammal cocktail); LepF1_t1, VF1_t1, VF1d_t1, VF1i_t1, LepR1_t1, VR1d_t1, VR1_t1, VR1i_t1	COI gene	LepF1_t1	TGTAAAACGACGGCCAGTAT TCAAC CAATCATAAAGATATTGG	LepR1_t1	CAGGAAACAGCTATGACTAAAC TTC TGGATGTCCAAAAAATCA	Partial sequence (c. 700bp) of COI gene
						VF1_t1	TGTAAAACGACGGCCAGTTCT CAAC CAACCACAAAGACATTGG	VR1d_t1	CAGGAAACAGCTATGACTAGAC TTC TGGGTGGCCRAARAAYCA	Partial sequence (c. 700bp) of COI gene
						VF1d_t1	TGTAAAACGACGGCCAGTTCT CAAC CAACCACAARGAYATYGG	VR1_t1	CAGGAAACAGCTATGACTAGAC TTC TGGGTGGCCAAAGAATCA	Partial sequence (c. 700bp) of COI gene
						VF1i_t1	TGTAAAACGACGGCCAGTTCT CAAC CAACCAIAAIGAIATIGG	VR1i_t1	CAGGAAACAGCTATGACTAGAC TTC TGGGTGICCIAAIAAICA	Partial sequence (c. 700bp) of COI gene
Oxford	Amoebozoa, Alveolata, Cercozoa, Chlorophyta, Cryptophyta, Euglenozoa	18S (V4,V9)	SILVA/PR2/ NCBI	TAReuk454FWD1/ TAReukREV3, E572F/E1009R, 1389F/1510R, 1391F/EUKBr	rRNA Gene	TAReuk454FWD1	CCAGCASCYGCGGTAATTCC	TAReukREV3	ACTTTCGTTCTTGATYRA	Partial sequence (c.417 bp) of 18s sRNAgene (V4)
						E572F	CYGCGGTAATTCCAGCTC	E1009R	CRAAGAYGATYAGATACCRT	Partial sequence (c.440bp) of 18s sRNAgene (V4)
						1389F	TTGTACACACCGCCC	1510R	CCTTCYGCAGGTTCACCTAC	Partial sequence (c.170 bp) of 18s sRNAgene (V9)
						1391F	GTACACACCGCCCGTC	EUKBr	TGATCCTTCTGCAGG TTCACCTAC	Partial sequence (c.170 bp) of 18s sRNAgene (V9)
	Ochrophyta	18S (V4,V9)	SILVA/PR2/ NCBI	TAReuk454FWD1/ TAReukREV3, E572F/E1009R, D512/D978rev, 1389F/1510R, 1391F/EUKBr	rRNA Gene	TAReuk454FWD1	CCAGCASCYGCGGTAATTCC	TAReukREV3	ACTTTCGTTCTTGATYRA	Partial sequence (c.417 bp) of 18s sRNAgene (V4)
						E572F	CYGCGGTAATTCCAGCTC	E1009R	CRAAGAYGATYAGATACCRT	Partial sequence (c.440bp) of 18s sRNAgene (V4)
						D512	TTGTACACACCGCCC	D978rev	GACTACGATGGTATCTAATC	Partial sequence (c. 400bp) of 18s sRNAgene (V4)
						1389F	TTGTACACACCGCCC	1510R	CCTTCYGCAGGTTCACCTAC	Partial sequence (c.170 bp) of 18s sRNAgene (V9)
						1391F	GTACACACCGCCCGTC	EUKBr	TGATCCTTCTGCAGGTTCACCT AC	Partial sequence (c.170 bp) of 18s sRNAgene (V9)
RBG Kew	Fungi	ITS	NCBI/ UNITE	ITS1F/ITS4	ITS rDNA	ITS1F	CTTGGTCATTTAGAGGAAGTAA	ITS4	TCCTCCGCTTATTGATATGC	Internal transcribed spacer 1, 5.8S and internal transcribed spacer 2 (c. 500bp)
MBA	Phaeophytes	COI	BOLD/NCBI	GazF2/GazR2	COI gene	GazF2	CCAACCAYAAAGATATWGGTAC	GazR2	GGATGACCAAARAACCAAAA	Partial sequence (c. 700 bp) michondrial cytochrome oxidase I gene 5P end
	Phaeophytes, Rhodophytes	COI	BOLD/NCBI	GWSFn/GWSRx	COI gene	GWSFn	TCAACAAAYCAYAAAGATATYGG	GWSRx	ACTTCTGGRTGICCRAARAAYCA	Partial sequence (c. 700 bp) michondrial cytochrome oxidase I gene 5P end
	Phaeophytes, Rhodophytes	LSU D2/D3	BOLD/NCBI	T16N/T24U	rRNA Gene	T16N	AMAAGTACCRYGAGGGAAAG	T24U	SCWCTAATCATTCGCTTTACC	D2/D3 region partial sequence (c. 200 bp) of large subunit ribosomal DNA gene
	Rhodophytes	rbcL	BOLD/NCBI	rbcLa-F/rbcLa-R	Single CDS genomic DNA	rbcLa-F	ATGTCACCACAAACAGAGACTAAAGC	rbcLa-R	GTAAAATCAAGTCCACCRCG	Partial sequence (c. 500bp) of plastid-encoded rubisco gene
		rbcL	BOLD/NCBI	F57/rbcLrevNEW	Single CDS genomic DNA	F57	GTAATTCCATATGCTAAAATGGG	rbcLrevNEW	ACATTTGCTGTTGGAGTYTC	Plastid enclosed rubisco large subunit gene (c. 1,300 bp)
		rbcL	BOLD/NCBI	F57/TLR1	Single CDS genomic DNA	F57	GTAATTCCATATGCTAAAATGGG	TLR1	AAYTCWGCTCTTTCRTAYAT	Plastid enclosed rubisco large subunit gene (c. 650 bp) partial 5P end
		rbcL	BOLD/NCBI	F57/TLR4	Single CDS genomic DNA	F57	GTAATTCCATATGCTAAAATGGG	TLR4	AAYTCWGCCCTTTCRTACAT	Plastid enclosed rubisco large subunit gene (c. 650 bp) partial 5P end
		rbcL	BOLD/NCBI	AcroF1/ rbcLrevNEW	Single CDS genomic DNA	AcroF1	AGCTCAAGCCGCAGCAGGAG	rbcLrevNEW	ACATTTGCTGTTGGAGTYTC	Plastid enclosed rubisco large subunit gene (c. 650 bp) partial 3P end
		rbcL	BOLD/NCBI	TLF1/rbcLrevNEW	Single CDS genomic DNA	TLF1	TCYCARCCWTTYATGCGCTGG	rbcLrevNEW	ACATTTGCTGTTGGAGTYTC	Plastid enclosed rubisco large subunit gene (c. 650 bp) partial 3P end
		rbcL	BOLD/NCBI	M13LF3/M13RX	Single CDS genomic DNA	M13LF3	TGTAAAACGACGGCCAGTACH AAYCAYAARGATATHGG	M13RX	CAGGAAACAGCTATGACACTTC TGGRTGICCRAARAAYCA	Plastid enclosed rubisco large subunit gene (c. 1,300 bp)
	Chlorophyte macroalgae	tufA	BOLD/NCBI	tufGF4/tufAR	Single CDS genomic DNA	tufGF4	GGNGCNGCNCAAATGGAYGG	tufAR	CCTTCNCGAATMGCRAAWCGC	Partial sequence elongation factor Tu (c. 600 bp)
	Chlorophyte macroalgae	rbcL	BOLD/NCBI	GrbcLFi/1385R	Single CDS genomic DNA	GrbcLFi	TCTCARCCWTTYATGCGTTGG	1385R	AATTCAAATTTAATTTCTTTCC	Plastid enclosed rubisco large subunit gene (c. 650 bp) partial 3P end
	Alismatales (seagrasses)	ITS	BOLD/NCBI	P674/P675	ITS rDNA	P674	CCTTATCATTTAGAGGAAGGAG	P675	TCCTCCGCTTATTGATATGC	Internal transcribed spacer 1, 5.8S and internal transcribed spacer 2 (c. 700bp)
	Cryptophytes	psaA	SILVA/PR2/ NCBI	psaA.F529/ psaA.1748R	Single CDS genomic DNA	psaA.F529	GGWTGGTTYCAYTAYCAYAARKCWGC	psaA.1748R	CCCAWGCHGAWMYTTGRCAW GTWCC	Partial sequence photosystem I P700 chlor-ophyll a apoprotein A1 (c. 1050 bp)
	Protists	18S V4	SILVA/PR2/ NCBI	E572F/E1009R	rRNA Gene	E572F	CYGCGGTAATTCCAGCTC	E1009R	AYGGTATCTRATCRTCTTYG	Partial sequence 18S small subunit V4 region (c.450 bp)
	Protists	18S V9	SILVA/PR2/ NCBI	1389F/1510R	rRNA Gene	1389F	TTGTACACACCGCCC	1510R	CCTTCYGCAGGTTCACCTAC	Partial sequence 18S small subunit V4 region (c.150 bp)
	Marine fungi and lichens	ITS	NCBI/ Species Fungorum/ UNITE	ITS1F/ITS4	ITS rDNA	ITS1F	CTTGGCATTTAGAGGAAGTAA	ITS4	TCCTCCGCTTATTGATATGC	Internal transcribed spacer 1, 5.8S and internal transcribed spacer 2 (c. 500bp)
	Marine fungi and lichens	18S	NCBI/ Species Fungorum/ UNITE	NS1/FR1	rRNA Gene	NS1	GTAGTCATATGCTTGTCTC	FR1	AICCATTCAATCGGTAIT	Partial sequence 18S small subunit V2/V9 region (c.1650 bp)