PCR thermo-cycling conditions N of cycles. Isolate Locus Amplification primers. Method. Final step

Transcription

1 Table S4 PCR-based methods and thermo-cycling conditions for determining the HD1, HD2, STE3_Mr4, Mr_Ph4 and STE3_Mr1 alleles, transcription of A and B mating genes, TAIL-PCR and SSR analysis in the M. roreri isolates 2 MCA 04 MCA 04 HD1 Mr_HD1_1F/ Mr_HD1_1R HD2 Mr_HD2_F/ Mr_HD2_R Cycling s 2 C/ 0 C/ Direct sequencing (a) with primers: Mr_HD1_1F Mr_HD1_1R Mr_HD1_1F_Internal1 Mr_HD1_1R_Internal2 Mr_HD2_F Mr_HD2_R Mr_HD2_IntF Mr_HD2_IntR CBS MCA 263 MCA 270 HD1 and HD2 Mr_HD1_4F/ Mr_HD2_R Mr_HD1_4F Mr_HD2_R Mr_HD1_2R Mr_HD2_2F Mr_HD2_2R Mr_HD2_Int_F Mr_HD2_IntR JD3 JD4 2 CBS MCA 293 CBS MCA 02 HD1 and HD2 Mr_HD1_4F/ Mr_HD2_R Mr_HD1_4F Mr_HD2_R Mr_HD1_Int_B_F Mr_HD1_Int_B2_F Mr_HDs_R Mr_HDs_F Mr_HD2_2R Mr_HD2_IntR Mr_HD2_Int_B_F

2 JD7 CBS 1386 CBS N6 Pa V3 H1 P N1 CBS 1386 C CBS MCA 294 MCA 2978 MCA 2974 MCA 00 MCA 01 MCA 03 1 DIS 122 DIS 116e E24 C10 HD1 and HD2 HD1 and HD2 Mr_HD2_Int_B_F/ Mr_HD2_3R Mr_HDs_F/ Mr_HD2_IntR Mr_HD1_Int_B2_F/ Mr_HD2_2R Mr_HD2_Int_B_F/ Mr_HDs_R Mr_HD1_6F/ Mr_HD1_6F/ Mr_HDs_F/ Mr_HD2_IntR Cycling s Mr_HD2_Int_B_F Mr_HD2_3R Mr_HDs_F Mr_HD2_IntR Mr_HD1_Int_B2_F Mr_HD2_2R Mr_HD2_Int_B_F Mr_HDs_R Mr_HD1_6F Digestion with restriction enzymes: BsiHKAI StuI

3 MCA 270 MCA 04 CBS CBS CBS CBS MCA 263 MCA 293 MCA 2974 CBS MCA 2978 MCA 00 MCA 01 MCA 02 C MCA 03 MCA 294 CBS 1386 CBS 1386 JD7 1 2 JD4 JD3 All isolates STE3_Mr4 Mr_Ph4 STE3_Mr4 Mr_Ph4 STE3_Mr1 Mr_Rec4_F2/ Mr_Rec4_R2 Mr_Ph4_A1_F/ Mr_Ph4_A1_R Mr_R4_A2_F/ Mr_R4_A2_R Mr_Ph4_A2_F/ Mr_Ph4_A2_R Mr_Receptor_F/ Mr_Receptor_R Cycling s 2 C/ Mr_Rec4_F2 Mr_Rec4_R2 Mr_Ph4_A1_F Mr_Ph4_A1_R Mr_R4_A2_F Mr_R4_A2_R Mr_Ph4_A2_F Mr_Ph4_A2_R Mr_Receptor_F Mr_Receptor_R Mr_Recep_IntF

4 CBS 1386 CBS MCA 01 CBS Isolates listed in Supplementary Table S1 (b) HD1.1and HD1.2 HD2.1 and HD2.2 STE3_Mr4.1 STE3_Mr4.2 Mr_HD1_Int_B_F/ Mr_HD2_Int_B_F / Mr_HD2_Int_R Mr_Rec4_F2/ Mr_Rec4_R2 Mr_R4_A2_F/ Mr_R4_A2_R Mr_SSR1-Mr_SSR Mr_SSR#_F/ Mr_SSR#_R Selected loci after initial screening (see Supplementary Table S3) FL-M13/ M13-Mr_SSR#_F/ Mr_SSR#_R Cycling s 40s C/ 2 C/ 8 10min 10min RT-PCR Initial SSR screening visualization method: 4% SFR agarose gel Full SSR screening method: M13 method (c) (Schuelke, 2000) ABI 37XL Genetic Analyzer

5 3min 3min 3min C/ 3min 62 C/ 62 C/ 3min First round of TAIL-PCR (see Singer and Burke, 2003). One single reaction consisted of: Apex TaqRED Master Mix 2X AD Mix primer Template (0.8ng DNA/µl) Total µl Second round of TAIL-PCR using a 1/100 dilution of product of first round as template. One single reaction consisted of: Apex TaqRED Master Mix 2X AD Mix primer Template Total µL Products were run a 1% agarose gel and bands were extracted from gel with Promega Wizard SV Gel and PCR Clean-Up System. Then bands were sequenced with specific primer. Third round of TAIL-PCR using a 1/0 dilution of product of second round as template. One single reaction consisted of: Apex TaqRED Master Mix 2X AD Mix primer Template Total µl Sequencing occurred as for products of second run of TAIL-PCR (a) In all instances where alleles where determined by direct sequencing PCR cocktails were as follows: 12. µl of master mix (long-range PCRs were conducted with DreamTaq PCR Master Mix 2X (Thermo Fisher Scientific, Waltham, MA; short products (< 1Kb), with Apex TaqRED Master Mix 2X (Genesee Scientific, San Diego, CA), 1.2 µl each of the forward and reverse primers (10 µm) and 10 µl of DNA template (0. ng/ µl). Products were sequenced with amplification primers at Beckman Coulter Co. (Brea, CA); sequences were edited in Sequencher.2.3 (Gene Codes Co., Ann Arbor, MI). (b) In the full SSR screening, isolates E24, DIS 116e, P, N6, Pa, H1 and N1 were not genotyped due to lack of DNA. # on primer names represents the number of the SSR marker within the developed (for example M13-Mr_SSR1_F/ Mr_SSR1_R and so on). In the full screening three primers were used: M13 fluorescently labeled (FL-M13), M13-tailed forward primer (M13-Mr_SSR#_F) and reverse primer (Mr_SSR#_R). PCR cocktails in the SSR screening consisted of 12. µl of Apex TaqRED Master Mix (2X), ~ µl of the M13-tailed forward primer, ~ µl of one M13 fluorescently labeled forward primer, 1.2 µl of the reverse primer and 10 µl DNA template (0. ng/ µl). (c) In the M13 method the universal M13 (-21) primer ('-TGTAAAACGACGGCCAGT- 3') was '- labeled with a specific fluorescent dye from the DS-33 dye set from Applied Biosystems, Life Technologies, Carlsbad, CA, (6-FAM TM, blue; VIC, green; NED TM, yellow; and PET, red). Then, the 11 selected forward primers were tailed at their ' ends with the M13 (-21) sequence. Because four dye colors were used, four PCR products each labeled with a different color were pooled and diluted to 1/100. Then, 2 µl of this mix was added to 10 µl of a solution composed of 14.2 µl of GeneScan -00 LIZ Size Standard in 1 ml of Hi-Di TM Formamide, and run on an ABI 37XL Genetic Analyzer. Alleles were scored with Peak Scanner v1.0.