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Mapping of high temperature growth genes derived from industrial yeast strains

Mapping of high temperature growth genes derived from industrial yeast strains. Justin Goh , Richard Gardner School of Biological Sciences, University of Auckland. Wide temperature tolerance of Saccharomyces cerevisiae has industrial applications. 15ºC. 40ºC.

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Mapping of high temperature growth genes derived from industrial yeast strains

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

  2. Wide temperature tolerance ofSaccharomycescerevisiae has industrial applications 15ºC 40ºC

  3. Two strains of S. cerevisiaecan ferment well at high temperature CO2 CO2 CO2 CO2

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

  5. AL3 and KK:YS1 are heterozygous

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

  7. Homozygous derivatives can ferment nearly as well at heterozygous parent

  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 Fermentation at 40°C vs. To phenotype 100 progeny 1 plate 48 h 0.02 L sugar medium Single scoring 315 tubes 1 week 9 L sugar medium Many weighings

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

  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 Htg 41°C 48h 37°C 24h 40°C 48h 28°C 24h AL3h S288C F1 hybrid 0.91 0 1.31 0.01 0 0.91 0.84 0 0.98 vs. Htg = Sum of ratios of pixel intensities 1.75 0 3.2

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

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

  16. Verify crossing & backcrossing by microsattelite genotyping

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

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

  19. Positive heterosis in F1 hybrids suggests htg is co-dominant & both parents contribute 37°C 24h 40°C 48h 41°C 48h Dilution series 41°C 48h 107 106 105 104 107 106 105 104 S288cKKhF1 S288cAL3h F1 S288c BC AL3h BC Best F1 segregant Best F1 segregant F1 hybrid KKh F1 hybrid S288c 37°C 24h 40°C 48h 41°C 48h

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

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

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

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

  24. Genotyping of MKT1 and NCS2 in BC segregants that are Htg+ and Htg- 20 Low pool 20 Low pool 20 High pool 20 High 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 S288c BC AL3h BC Best F1 segregant Best F1 segregant F1 hybrid KKh F1 hybrid S288c

  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 KKhS288cF1F1 s BCBCsegregants→

  26. Clear association with MKT1 and NCS2 in KK 41°C 48h 40°C 48h 37°C 24h S288c orKKh

  27. …and in AL3 BC segregants 41°C 48h 40°C 48h 37°C 24h S288c orAL3h

  28. MKT1 and NCS2 are linked on chrom 14 KKhBC segregants AL3h BC segregants

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

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

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

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

  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

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