Conserve primers for sequencing complete ungulate mitochondrial cytochrome c oxidase I (COI) gene from problematic and decomposed biological samples

Figures & data

Table 1. Six primers designed for amplification of either complete or partial fragment of mtDNA COI gene.

S. No.	Primer name	Sequence (5′→3′)
1	COI (5298-323) F1	TACAGTCTAATGCTTCACTCAGCCA
2	COI (5577-599) F2	GTACCGCTAATAATTGGTGCTCC
3	COI (5599-571) R1	GGGGCAATTATTAGGGGAACTAGTCA
4	COI (6024-046) F3	CAACACTTGTTCTGATTCTTCGG
5	COI (6052-027) R2	GGGTGGCCAAAGAATCAGAACAAGTG
6	COI (6897-876) R3	GGGGGTTCGATTCCTTCCTTTC

Primer positions were derived from the complete mtDNA sequence of Rusa unicolor swinhoei (EF035448).

Figure 1. Image of the agarose gel showing amplification result of multiplex PCR reaction with the DNA extracted from partially decomposed tissue of a verity of ungulate species as labelled at each lane. (A) PCR amplification with F1, R1, R2 and R3 primers; (B) amplification with F1, F2, R2 and R3 primers; (C) amplification with F1, F2, R2 and R3 and (D) amplification with F1, F2, F3 and R3. Numeric are the base pair size of respective amplicons.

Table 2. All possible combinations of the primers during PCR amplification with tested samples and expected amplicon lengths.

S. No.	Primer combination	Expected amplicon length(s)	Sample type
1	F1 and R1	301 bp	T/B/H/A/Bo/F
2	F1 and R2	754 bp	T/B/H/A/Bo
3	F1 and R3	1599 bp	T/B
4	F2 and R2	475 bp	T/B/H/A/Bo/F
5	F2 and R3	1320 bp	T/B
6	F3 and R3	873 bp	T/B/H/A
In multiplex reactions
7	F1, R1, R2 and R3	301, 754 and 1599 bp
8	F2, R2 and R3	475 and 1320 bp
9	F1, F2, R2 and R3	475, 754, 1320 and 1599 bp
10	F1, F2, F3 and R3	873, 1320 and 1599 bp

Where T, B, H, A, Bo and F stand for fresh and partially decomposed tissue, blood, hairs, antlers, bone and faecal samples respectively.

Figure 2. Phylogenetic tree based on neighbour-joining method using MEGA 7. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches.

Table 3. List of ungulate species used for validation of primers.

S. No.	Amplification and sequencing with ungulate species	NCBI accession No.
1	Sambar (Rusa unicolor)	KT372090
2	Indian wild pig (Sus scrofa)	KT372091
3	Domestic pig (Sus scrofa)	KT372092
4	Barking deer (Muntiacus muntjak)	KT372093
5	Nilgai (Boselaphus tragocamelus)	KT372094
6	Hog deer (Axis porcinus)	KT372095
7	Swamp deer (Rucervus duvaucelii)	KT372096
8	Four-horned antelope (Tetracerus quadricornis)	KT372097
9	Chital (Axis axis)	KT372098
10	Chinkara (Gazella bennettii)	KT372099
11	Blackbuck (Antelope cervicapra)	KT372100

Alphanumeric are the NCBI accession numbers of our sequences.