Table 2.

PCR conditions and primers used for amplification and sequencing of Phytophthora isolates from Clade 10.

Locus	Primer names	Primer sequences (5’-3’)	Orientation	Annealing temperature (°C)	Extension time (s)	Reference for primer sequences
βtub a,b	TUBUF2	CGGTAACAACTGGGCCAAGG	Forward	68	12	Kroon et al. (2004)
	TUBUR1	CCTGGTACTGCTGGTACTCAG	Reverse
	Btub_F1A	GCCAAGTTCTGGGARGTSAT	Forward	66	15	Blair et al. (2008)
	Btub_R1A	CCTGGTACTGCTGGTAYTCMGA	Reverse
cox1 d	OomCoxI-Levup a	TCAWCWMGATGGCTTTTTTCAAC	Forward	60	10	Robideau et al. (2011)
	OomCoxI-Levlo a	CYTCHGGRTGWCCRAAAAACCAAA	Reverse
	COXF4N c	GTATTTCTTCTTTATTAGGTGC	Forward	50	65	Kroon et al. (2004)
	COXR4N c	CGTGAACTAATGTTACATATAC	Reverse
	OomCoxI-Levup c	TCAWCWMGATGGCTTTTTTCAAC	Forward	50	80	Robideau et al. (2011), this study.
	FM83 Oom c	CHCCNATAAARAATAACCARAARTG	Reverse
	FM84 c	TTTAATTTTTAGTGCTTTTGC	Forward	50	95	Martin & Tooley (2003), this study.
	FM83 Oom c	CHCCNATAAARAATAACCARAARTG	Reverse
tef-1α a	EF1A_FL	GGTCACCTGATCTACAAGTGC	Forward	60	15	Blair et al. (2008)
	EF1A_RL	CCTTCTTGTTCACCGACTTG	Reverse
enl e	Enl_for d	CTTTGACTCGCGTGGCAAC	Forward	55–58	90	Blair et al. (2008)
	Enl_FY d	CAACCCSACCGTYGAGGT	Forward
	Enl_rev	CCTCCTCAATACGMAGAAGC	Reverse
hsp90 a	HSP90_F1int	CAAGGTGATCCCGGACAAGGC	Forward	63–66	15	Blair et al. (2008)
	HSP90R1	ACACCCTTGACRAACGACAG	Reverse
ITS a	ITS1	TCCGTAGGTGAACCTGCGG	Forward	63–65	12	White et al. (1990), Cooke et al. (2000)
	ITS4 f	TCCTCCGCTTATTGATATGC	Reverse
	ITS6 f	GAAGGTGAAGTCGTAACAAGG	Forward
LSU a,g	CTB6	GCATATCAATAAGCGGAGG	Forward	53	20	Garbelotto et al. (1997), Hopple & Vilgalys (1994)
	LR3 h	CCGTGTTTCAAGACGGG	Reverse
	LR3R h	GTCTTGAAACACGGACC	Forward
	LR7	TACTACCACCAAGATCT	Reverse
nadh1 c	NADHF1	CTGTGGCTTATTTTACTTTAG	Forward	50	65	Kroon et al. (2004)
	NADHR1	CAGCAGTATACAAAAACCAAC	Reverse
ras-ypt1 a	Ypt1F	CGACCATYGGYGTKGACTTT	Forward	60–62	7	Chen & Roxby (1996), this study
	Ypt_820	CCATCATCATGAADGCYTTYTCR	Reverse
rpl10 a	60SL10_for	GCTAAGTGTTACCGTTTCCAG	Forward	62–64	7	Martin & Tooley (2003)
	60SL10_rev	ACTTCTTGGAGCCCAGCAC	Reverse
rps10 i	rps10_DB_FOR	GTTGGTTAGAGYARAAGACT	Forward	48	30	Foster et al. (2022)
	rps10_DB_REV	RTAYACTCTAACCAACTGAGT	Reverse
tigA c	Tig_FY_Oom	TYGTGGGCGGHAAYTGGAA	Forward	60–63	120	This study
	G3P_for_Oom h	TBGCBATYAAYGGHTTYGG	Forward
	Tig_rev_Oom h	CCRAADCCRTTRATVGCVA	Reverse
	G3PDH_rev_Oom	DCCCCACTCRTTGTCRTACCAM	Reverse

^a PCR protool 1: 20 µl volume containing 10.4 pl H₂O, 4 µl Q5 Reaction Buffer (5X), 1 µl of each primer (10 µM), 0.4 µl deoxynucleotide (dNTP) mixture (Meridian Bioscience, Memphis, USA) (2.5 mM each), 0.2 µl of Q5 High-Fidelity DNA Polymerase (2 U/µl) (New England Biolabs, Ipswich, USA), and 3 µl of gDNA. Initial denaturation for 30 s at 98 °C ; 35 cycles consisting of 5 s at 98 °C, 20 s at optimised annealing temperafore for each primer set optimised length of extension at 72 °C ; 2 min at 72 °C for final extension.

^b Two primer pairs Were used separately: TUBUF2/TUBUR1 or Btub_F1A/Btub_R1A.

^c PCR protocol 2: 20 µl volume containing 10 µl H₂O, 4 µl PrimeSTAR GXL Buffer (5X), 0.8 µl of each primer, 1.6 µl dNTP mixture, 0.4 µl PrimeSTAR GXL DNA Polymerase (1.25 U/µl) (TaKaRa Bio, Kusatsu, Shiga, Japan), and 3 µl of gDNA. Initial denaturation for 5s at 98 °C; 35 cycles consisting of 10 s at 98 °C, 15 s at optimised annealing temperature, optimised length of extension at 68 °C; 5 min at 68 °C for final extension.

^d COX4FN/COX4RN primers were used to obtain the amplicons for sequencing. For samples that did not amplify with COX4N primers, two sets of alternative primers (OomCoxI-Levup/FM83_Oom; FM84/FM83_Oom) were used.

^e Two primer combinations were used separately: Enl_for/Enl_rev or Enl_FY/Enl_rev.

^f Two primer combinations were used separately: ITS1/ITS4 or ITS6/ITS4.

^g Double concentration of Q5 polymerase.

^h Primers used exclusively for sequencing.

ⁱ PCR protocol 3: 20 µl volume containing 6.2 µl H₂O, 10 µl OneTaq Hot Start Quick-Load 2X Master Mix with Standard Buffer (New England Biolabs, Ipswich, USA) 0.4 µl of each primer, and 3 µl of gDNA. Initial denaturation for 5 s at 98 °C; 35 cycles consistin g of 30 s at 98 °C, 30 s at optimised ann ealing temperature, optimised length of extension at 72 °C; 7 min at 72 °C for final extension.