Table 3. Nucleotide differences and clustering results for HRM discrimination of known haplotype.

Sample ID of the accessions that were PCR amplified in replicates of 16, the number of replicates that successfully amplified during PCR and subject to HRM analysis is given (N), followed by HRM haplotype discrimination (sensitivity, specificity and accuracy), the grouping of each replicate into a HRM cluster is provided for each haplotype amplified per primer combination (clusters 1–11), a summary of the nucleotide differences between haplotypes is also provided.

Primer pair	N	Sen	Spe	Acc	HRM grouping of replicates into cluster 1–cluster 11											Nucleotide difference summary
					1	2	3	4	5	6	7	8	9	10	11
MLT C1-MLT C4 (150 bp) (TrnG intron)																19	20	72	205
																T	A	T	A
Haplotype A	14	71	94	88	2	10		2								G	T	G	.
Haplotype B	16	69	44	52	11		4		1							.	.	.	.
Haplotype C	11	91	49	58	10	1										.	.	.	C
Haplotype D	11	73	44	50	8	1	3									.	.	G	.
MLT C3-MLT C4 (236 bp) (TrnG intron)																41	48-55	62
																T	#	A
Haplotype A	14	100	43	57	14											.	#	T
Haplotype B	16	56	27	36	9	6	1									.	–	.
Haplotype C	12	58	79	75	4	7		1								G	#	.
Haplotype D	14	79	36	46	11	3										.	#	.
																# = AAAAATTG
MLT M1-MLT M2 (170 bp) (pctL-psbE intergenic spacer)																84	88	110	118
																G	G	G	A
Haplotype A	15	93	98	97		14	1									A	.	.	.
Haplotype B	16	94	67	74	15			1								.	.	.	.
Haplotype C	14	93	98	97		1	13									.	T	.	.
Haplotype D	16	94	67	74	15			1								.	.	T	G
MLT S1-MLT S2 (217 bp) (atpI-atpH intergenic spacer)																53	54	62-80	95
																T	A	–	G
Haplotype A	12	100	100	100			12									.	.	–	.
Haplotype B	15	100	100	100	15											.	.	–	A
Haplotype C	11	100	100	100				11								A	C	–	.
Haplotype D	14	100	100	100		14										A	C	#	.
																# = TTCATAGATAACTAGTTAG
MLT S1-MLT S4 (527 bp) (atpI-atpH intergenic spacer)																75	76	86-104	117	267	281	287	382	477-481
																T	A	–	G	C	C	T	C	*
Haplotype A	14	100	79	83	14											.	.	–	.	T	.	.	.	–
Haplotype B	10	80	100	98					8	2						.	.	–	A	.	.	.	.	–
Haplotype C	14	100	79	83	14											.	.	–	.	T	.	.	.	*
Haplotype D	14	86	100	98			12				2					A	C	#	.	.	T	G	.	*
Haplotype E	16	100	100	100		16										.	.	–	A	.	.	.	A	–
Haplotype F	14	71	100	95				10				2	2			A	C	–	.	.	T	G	.	*
																# = CATAGATAACTAGTTAGTT, * = TTTTC
MLT S3-MLT S4 (310 bp) (atpI-atpH intergenic spacer)																52	66	72	167	262-266
																C	C	T	C	–
Haplotype A	15	100	100	100		15										T	.	.	.	–
Haplotype B	12	92	100	95				11			1					.	.	.	.	–
Haplotype C	11	91	100	95					10	1						T	.	.	.	#
Haplotype D	16	100	100	100	16											.	T	G	.	#
Haplotype E	14	93	100	95			13					1				.	.	.	A	–
																# = TTTTC
MLT U1-MLT U2 (345 bp) (ndhA intron)																15	22	47	79	135-141	149	172	183	220	253	289
																T	T	G	C	#	C	A	A	G	T	A
Haplotype A	16	100	100	100	16											.	.	.	.	#	.	.	.	.	.	.
Haplotype B	11	73	100	96				8			3					.	C	.	A	#	A	C	G	.	G	T
Haplotype C	15	93	100	99		14									1	C	.	.	.	#	.	.	.	.	.	.
Haplotype D	16	63	100	91					10	3		2	1			.	.	A	.	#	.	.	.	.	.	.
Haplotype E	12	92	100	99			11							1		.	.	A	.	–	.	.	.	A	.	.
																# = TATCCCC
MLT V1-MLT V2 (340 bp) (rpl32-trnL intergenic spacer)																34-38	56	104
																#	T	T
Haplotype A	14	86	65	71	12	2										–	.	.
Haplotype B	10	91	99	98			10									#	.	.
Haplotype C	12	92	66	73	12											#	A	A
Haplotype D	10	100	89	92		10										#	A	.
																# = ATTATT