1. Mapping of high temperature growth genes derived from industrial yeast strains Justin Goh, Richard Gardner School of Biological Sciences, University of Auckland

2. 15ºC 40ºC Wide temperature tolerance of Saccharomyces cerevisiae has industrial applications

3. Two strains of S. cerevisiae can ferment well at high temperature CO 2

4. AL3 Alcohol distillery - Brazil KK:YS1 Kodo ko jaanr – fermented finger millet beverage Aim: To map some of the major genes involved in high temperature growth (htg)

5. AL3 and KK:YS1 are heterozygous Microsatellite marker C3C5C8C4091cAT4AT2Scaat3009C267CMATαMATa AL3 (heterozygous) 108, 120172 130, 143259290 292, 296 357, 366 356, 370 382, 449 439, 445468492 KK (heterozygous) 111, 114119 127, 130none239 266, 288350395 396, 405 398, 442468492

6. Obtained homozygous derivatives of AL3 and KK:YS1 by tetrad dissection Heterozygous parent Screen among homozygous progeny for a fermentation phenotype as good as the parent strain

7. Homozygous derivatives can ferment nearly as well at heterozygous parent Microsatellite marker C3C5C8C4091cAT4AT2Scaat3009C267CMATαMATa AL3 (heterozygous) 108, 120172 130, 143259290292, 296357, 366356, 370382, 449439, 445468492 AL3h (homozygous)120172143none290296357370449445468492 KK (heterozygous) 111, 114119 127, 130none239266, 288350395396, 405398, 442468492 KKh (homozygous)114119130none239288350395396398468492

8. Cross AL3h and KKh to S288c – standard laboratory strain – to map htg genes

9. Phenotyping: High temperature fermentation vs growth Colony growth at 40°C vs. 315 tubes 1 week 9 L sugar medium Many weighings 1 plate 48 h 0.02 L sugar medium Single scoring To phenotype 100 progeny Fermentation at 40°C

10. Phenotyping high temperature growth Measure progeny for colony growth at optimal and stressful high temperatures 40°C 48h 37°C 24h 41°C 48h 28°C 24h

11. Qualitative assessment of htg

12. Quantify growth by pixel intensity of colony spots of scanned plate

13. Calculate high temperature growth ability as ratio of growth compared to 28°C AL3h S288C F1 hybrid 0.84 0 0.98 0.91 0 1.31 0.01 0 0.91 vs. 1.75 0 3.2 Htg = Sum of ratios of pixel intensities 40°C 48h 37°C 24h 41°C 48h 28°C 24h Htg

14. Scheme for crossing & backcrossing homozygous strains to S288c F1 hybrid MATα/a URA/ura HO/ho MATα ura ho MATα/a ura HO MATa ura ho S288c (Sequenced lab strain) MATα ura ho KKh MATα/a HO AL3h MATα/a HO Screen 100 F1 haploid progeny for colony growth at 40°C Best F1 segregant Homozygous spores

15. Crossing & backcrossing of Htg strains to S288c S288c MATα lys ho BC segregants MATα lys ho MATa ura ho MATα lys ho MATa lys ura ho MATa ura ho Best F1 segregant Backcross (BC) MATα/a LYS/lys URA/ura ho

16. Verify crossing & backcrossing by microsattelite genotyping Microsattelite marker C3C5C8C4091cAT4AT2Scaat3009C267CMATαMATa S288C 120174130240302296357407443415468492 AL3 (heterozygous) 108, 120172 130, 143259290 292, 296 357, 366 356, 370 382, 449 439, 445468492 AL3h (homozygous) 120172143none290296357370449445468492 F1 hybrid 120 172, 174 130, 143240 290, 302296357 370, 407 443, 449 415, 445468492 F1 segregant 120174143240290296357370449445none 492 BC 120174 130, 143240 290, 302296357 370, 407 443, 449 415, 445468492 BC segregant 120174143240302296357370443415 none492 KK (heterozygous) 111, 114119 127, 130none239 266, 288350395 396, 405 398, 442468492 KKh (homozygous) 114119130none239288350395396398468492 F1 hybrid 114, 120 119, 174130240302 288, 296 350, 357 395, 407 396, 443 398, 415468492 F1 segregant 120119130240302296357395443398none 492 BC 120 119, 174130240302296357 395, 406443 398, 415468492 BC segregant 120119130240302296357395443415 none492

17. Phenotypic distribution of htg of backcrossed segregants S288c F1 hybrid BC segregants S288c Best F1 segregant Backcross (BC) 40°C 48h 37°C 24h 41°C 48h S288c AL3h F1 hybrid Best F1 segregant BC AL3h

18. Phenotypic distribution of htg of backcrossed segregants S288c F1 hybrid BC segregants S288c Best F1 segregant Backcross (BC) S288c KKh F1 hybrid Best F1 segregant BC KKh 40°C 48h 37°C 24h 41°C 48h

19. Positive heterosis in F1 hybrids suggests htg is co-dominant & both parents contribute 40°C 48h 37°C 24h 41°C 48h S288c AL3h F1 hybrid Best F1 segregant BC S288c KKh F1 hybrid Best F1 segregant BC 40°C 48h 37°C 24h 41°C 48h S288c AL3h F1 S288c KKh F1 10 7 10 6 10 5 10 4 Dilution series 41°C 48h 10 7 10 6 10 5 10 4

20. Only a few genes may be required for high temperature growth 40°C 48h 37°C 24h 41°C 48h S288c AL3h F1 hybrid Best F1 segregant BC S288c KKh F1 hybrid Best F1 segregant BC 40°C 48h 37°C 24h 41°C 48h 37/184 segregants (½) 2.3 40/184 segregants 9/184 segregants (½) 4.36 15/184 segregants 41°C 40°C (½) 2.2 (½) 3.6

21. Two major genes for high temperature growth were recently mapped S288c YJM 421 F1 hybrid Standard laboratory strain Homozygous derivative of a clinical isolate Sinha et al (2008)

22. Major genes affecting htg have no obvious link to function – “post-transcriptional regulation” S288c YJM 421 Standard laboratory strain Homozygous derivative of a clinical isolate MKT1 NCS2 MKT1 NCS2 Post-transcriptional regulation of HO mRNA Post-transcriptional regulation of tRNA & rRNA MKT1 and NCS2 alleles from YJM parent important for htg in F1 hybrid

23. AL3 KK:YS1 YJM 421 Alcohol distillery - Brazil Kodo ko jaanr – fermented finger millet beverage Hypothesis: the major htg genes in AL3h and KKh are different from YJM 421

24. Genotyping of MKT1 and NCS2 in BC segregants that are Htg+ and Htg- S288c AL3h F1 hybrid Best F1 segregant BC S288c KKh F1 hybrid Best F1 segregant BC 20 High pool 20 Low pool If AL3h and KKh have different major genes for htg than YJM 421, then the MKT1 and NCS2 alleles from the htg parent and S288c should not be linked in BC segregants from high and low pool 20 High pool 20 Low pool

25. Inheritance of parental alleles of MKT1 and NCS2 determined using RFLP E.g. Amplify 900 bp region of NCS2 and cut with Tsp5091 KK KKh S288c F1 F1 s BC BC segregants →

26. Clear association with MKT1 and NCS2 in KK High pool #NCS2MKT1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Low pool # NCS2MKT1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 S288c or KKh 40°C 48h 37°C 24h 41°C 48h

27. …and in AL3 BC segregants 40°C 48h 37°C 24h 41°C 48h High pool #NCS2MKT1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 S288c or AL3h Low pool # NCS2MKT1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

28. MKT1 and NCS2 are linked on chrom 14 High pool #NCS2MKT1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Low pool # NCS2MKT1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 High pool #NCS2MKT1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Low pool # NCS2MKT1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 AL3h BC segregants KKh BC segregants

29. S288c F1 hybrid BC segregants S288c Best F1 segregant Backcross (BC) AL3h Best F1 segregant non-htg BC segregant Fix htg+ derived MKT1 & NCS2 alleles in next cross → find other htg genes BC F1 hybrid 40°C 48h 37°C 24h 41°C 48h S288c AL3h F1 hybrid Best F1 segregant BC non-htg BC segregant ?

30. Identify htg genes from S288C (Both parents have same MKT1, NCS2 loci) S288c KKh F1 hybrid Best F1 segregant 40°C 48h 37°C 24h 41°C 48h S288c F1 hybrid Best F1 segregant KKh BC segregants Backcross (BC) KKh ?

31. S288c AL3h F1 hybrid Best F1 segregant BC non-htg BC segregant Genotype high & low pool segregants using high-density microarrays High pool Low pool ?

32. High-density tiling microarrays map ALL SNPs in a segregating cross Overlapping 25 bp oligomers, 5 bp apart → 5x coverage of entire genome High density Affmetrix tiling miroarray based on S288c

33. Conclusions Htg phenotype is quick and reproducible to measure → 100’s of progeny can be tested to map major genes Both S288c and industrial parent contribute genes for htg as shown by positive heterosis in F1 hybrid Crossing with S288c has identified the NCS2-MKT1 region as important for Htg in two industrial yeasts from geographically & environmentally diverse habitats

34. Current work Use selected individuals from the backcrossed strains to map additional genes for Htg - in industrial parents - from S288c Test selected backcrossed individuals to see if the MKT1 and NCS2 alleles also contribute to high temperature fermentation