Table 2.

PCR Primers and PCR/RFLP Typing Parameters for Y-Chromosome SNP Analysis

	Parameter
	PCR			PCR-RFLP
	Primer(5′→3′)				Fragment(s)(bp)
Marker	Forward	Reverse	Tanna(°C)	Enzyme	Ancestral	Mutant
M4 A→G	TCCTAGGTTATGATTACAGAGCG	GGCACAAGCTGTTCCAGTACA	60	NdeI	164	80 and 84
M5 C→T	GGTTTATACTGACCTGCCAATGT	CTATTACCAAAGGTTTGTGTTAGG	59b	…c	…c	…c
M9 C→G	GCAGCATATAAAACTTTCAGG	GAAATGCATAATGAAGTAAGCG	54	HinfI	100 and 64	164
M16 C→A	ATATTGTTATGTCATTTGAACCCAGG	TGTTCTATTAAAAGCTGACAAATCCAA	59	…c	…c	…c
M21 A→T	AAGCCCTTGATTTTTATTTATC	AACAGCAGATTTGAGCAGG	51d	…c	…c	…c
M119 A→C	CGCAGTGCTATGTGTTTATTTG	GTTATGGGTTATTCCAATTCAGC	56e	CviTI	31 and 62	93
M122 T→C	GTTGCCTTTTGGAAATGAATAAATC	CACTTGCTCTGTGTTAGAAAAGATAGC	58	Hsp92II	58 and 51	109
RPS4Y711 C→T	CTGTACTTACTTTTATCTCCTC	CAGCAACAGTAAGTCGAATG	54	BslI	34 and 57	91

^aAnnealing temperature.

^bTouchdown PCR: decrease of T_ann, in increments of 0.5°C, starting from 64°C, for the first 10 cycles, followed by 20 cycles at 59°C.

^cTyped only via SSO hybridization (see table 3).

^dTouchdown PCR: decrease of T_ann, in increments of 0.4°C, starting from 58°C, for the first 18 cycles, followed by 30–40 cycles at 51°C.

^eTouchdown PCR: decrease of T_ann, in increments of 0.4°C, starting from 63°C, for the first 18 cycles, followed by 30–40 cycles at 56°C.