Table 3. pmr1 mutations identified in this study.

Evolved clone		Sequencing method	pmr1 mutation	Amino acid change
C10	A	WGS	C2027T	A676V
C10	B	WGS	None	n/a
C10	C	WGS	None	n/a
C10	D	Sanger, PMR1 PCR	None	n/a
C10	E	Sanger, PMR1 PCR	None	n/a
C10	F	Sanger, PMR1 PCR	None	n/a
C10	K	Sanger, PMR1 PCR	None	n/a
C10	L	Sanger, PMR1 PCR	None	n/a
C10	M	Sanger, PMR1 PCR	None	n/a
C10	N	Sanger, PMR1 PCR	A778C	T260P
C16	A	WGS	C2027T	A676V
C16	G	WGS	T-220A*
C16	H	Sanger, PMR1 PCR	G1348T	D450Y
C16	I	Sanger, PMR1 PCR	G1121T	G374V
C16	J	Sanger, PMR1 PCR	C1532T	S511F
C16	N	Sanger, PMR1 PCR	A778C	T260P
I10	A	WGS	T459A	C153→stop codon
I10	B	WGS	T412C	S138P
I10	C	WGS	T2G	start codon disruption
I10	D	WGS	T2213C	F738S
I10	E	WGS	A1349T	D450V
I10	F	Sanger, PMR1 PCR	A557G	D186G
I10	G	Sanger, PMR1 PCR	G533C	R178T
I10	I	Sanger, PMR1 PCR	+ 2(AA) at bp 658	frameshift, amino acid 220
I10	J	Sanger, PMR1 PCR	A631G	K211E
I10	K	Sanger, PMR1 PCR	A1981C	K661Q
I10	L	Sanger, PMR1 PCR	C1508T	A503V
I10	M	Sanger, PMR1 PCR	G1051C	A351P
I10	Q	Sanger, PMR1 PCR	none	n/a
I10	O	Sanger, PMR1 PCR	none	n/a
I16	F	Sanger, PMR1 PCR	A557G	D186G
I16	H	WGS	C554T	A185V
I16	D	WGS	G3T	start codon disruption
I16	B	Sanger, PMR1 PCR	T412C	S138P
I16	I	Sanger, PMR1 PCR	+ 2(AA) at bp 658	frameshift, amino acid 220
I16	N	Sanger, PMR1 PCR	T2062G	L688V
I16	O	Sanger, PMR1 PCR	none	n/a
I16	P	Sanger, PMR1 PCR	G2031T	M677I

Clones from the indicated compatible (C) and incompatible (I) lines at Transfer 10 or 16 were analyzed by DNA sequencing. WGS indicates whole genome sequencing that was confirmed by Sanger sequencing the PCR-amplified PMR1 locus. “Sanger, PMR1 PCR” indicates that the PMR1 locus was amplified by PCR and sequenced by the Sanger method. pmr1 mutations are indicated by the wild-type sequence, followed by base pair or amino acid position, and then the mutant sequence. n/a, not applicable. *T to A substitution 220 bp upstream of the PMR1 start codon.