Mapping of high temperature growth genes derived from industrial yeast strains

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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. - PowerPoint PPT Presentation

Text of Mapping of high temperature growth genes derived from industrial yeast strains

  • Mapping of high temperature growth genes derived from industrial yeast strainsJustin Goh, Richard GardnerSchool of Biological Sciences, University of Auckland

  • 15C40CWide temperature tolerance of Saccharomyces cerevisiae has industrial applications

  • Two strains of S. cerevisiae can ferment well at high temperatureCO2CO2CO2CO2

  • Aim: To map some of the major genes involved in high temperature growth (htg)

  • AL3 and KK:YS1 are heterozygous

    Microsatellite markerC3C5C8C4091cAT4AT2Scaat3009C267CMATMATaAL3 (heterozygous)108, 120172130, 143259290292, 296357, 366356, 370382, 449439, 445468492KK (heterozygous)111, 114119127, 130none239266, 288350395396, 405398, 442468492

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

  • Homozygous derivatives can ferment nearly as well at heterozygous parent

    Microsatellite markerC3C5C8C4091cAT4AT2Scaat3009C267CMATMATaAL3 (heterozygous)108, 120172130, 143259290292, 296357, 366356, 370382, 449439, 445468492AL3h (homozygous)120172143none290296357370449445468492KK (heterozygous)111, 114119127, 130none239266, 288350395396, 405398, 442468492KKh (homozygous)114119130none239288350395396398468492

  • Cross AL3h and KKh to S288c standard laboratory strain to map htg genes

  • Phenotyping: High temperature fermentation vs growthColony growth at 40C vs.315 tubes1 week9 L sugar mediumMany weighings1 plate48 h0.02 L sugar mediumSingle scoringTo phenotype 100 progenyFermentation at 40C

  • Phenotyping high temperature growthMeasure progeny for colony growth at optimal and stressful high temperatures40C 48h37C 24h41C 48h28C 24h

  • Qualitative assessment of htg

  • Quantify growth by pixel intensity of colony spots of scanned plate

  • Calculate high temperature growth ability as ratio of growth compared to 28C

    AL3h

    S288C

    F1 hybrid0.84

    0

    0.980.91

    0

    1.310.01

    0

    0.91vs.Htg = Sum of ratios of pixel intensities40C 48h37C 24h41C 48h28C 24hHtg

  • Scheme for crossing & backcrossing homozygous strains to S288cF1 hybridMAT/a URA/ura HO/hoMAT ura hoMAT/a ura HOMATa ura hoScreen 100 F1 haploid progeny for colony growth at 40CBest F1 segregantHomozygous spores

  • Crossing & backcrossing of Htg strains to S288cBC segregantsMAT lys hoMATa ura hoMAT lys hoMATa lys ura hoBackcross (BC)MAT/a LYS/lys URA/ura ho

  • Verify crossing & backcrossing by microsattelite genotyping

    Microsattelite markerC3C5C8C4091cAT4AT2Scaat3009C267CMATMATaS288C120174130240302296357407443415468492AL3 (heterozygous)108, 120172130, 143259290292, 296357, 366356, 370382, 449439, 445468492AL3h (homozygous)120172143none290296357370449445468492F1 hybrid120172, 174130, 143240290, 302296357370, 407443, 449415, 445468492F1 segregant120174143240290296357370449445none492BC120174130, 143240290, 302296357370, 407443, 449415, 445468492BC segregant120174143240302296357370443415none492KK (heterozygous)111, 114119127, 130none239266, 288350395396, 405398, 442468492KKh (homozygous)114119130none239288350395396398468492F1 hybrid114, 120119, 174130240302288, 296350, 357395, 407396, 443398, 415468492F1 segregant120119130240302296357395443398none492BC120119, 174130240302296357395, 406443398, 415468492BC segregant120119130240302296357395443415none492

  • Phenotypic distribution of htg of backcrossed segregantsBC segregantsBackcross (BC)

  • Phenotypic distribution of htg of backcrossed segregantsBC segregantsBackcross (BC)KKh

  • Positive heterosis in F1 hybrids suggests htg is co-dominant & both parents contribute107

    106

    105

    104Dilution series 41C 48h107

    106

    105

    104

  • Only a few genes may be required for high temperature growth37/184 segregants() 2.340/184 segregants9/184 segregants() 4.3615/184 segregants41C40C() 2.2() 3.6

  • Two major genes for high temperature growth were recently mappedF1 hybridStandard laboratory strainHomozygous derivative of a clinical isolateSinha et al (2008)

  • Major genes affecting htg have no obvious link to function post-transcriptional regulationStandard laboratory strainHomozygous derivative of a clinical isolatePost-transcriptional regulation of HO mRNAPost-transcriptional regulation of tRNA & rRNAMKT1 and NCS2 alleles from YJM parent important for htg in F1 hybrid

  • AL3

    KK:YS1YJM 421Alcohol distillery - BrazilKodo ko jaanr fermented finger millet beverage Hypothesis: the major htg genes in AL3h and KKh are different from YJM 421

  • Genotyping of MKT1 and NCS2 in BC segregants that are Htg+ and Htg-20 High pool20 Low poolIf 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 pool20 Low pool

  • Inheritance of parental alleles of MKT1 and NCS2 determined using RFLPE.g. Amplify 900 bp region of NCS2 and cut with Tsp5091

  • Clear association with MKT1 and NCS2 in KK

    High pool #NCS2MKT11234567891011121314151617181920

    Low pool #NCS2MKT11234567891011121314151617181920

  • and in AL3 BC segregants

    High pool #NCS2MKT11234567891011121314151617181920

    Low pool #NCS2MKT11234567891011121314151617181920

  • MKT1 and NCS2 are linked on chrom 14AL3h BC segregantsKKh BC segregants

    High pool #NCS2MKT11234567891011121314151617181920

    Low pool #NCS2MKT11234567891011121314151617181920

    High pool #NCS2MKT11234567891011121314151617181920

    Low pool #NCS2MKT11234567891011121314151617181920

  • BC segregantsBackcross (BC)non-htg BC segregantFix htg+ derived MKT1 & NCS2 alleles in next cross find other htg genesBC F1 hybrid

  • Identify htg genes from S288C(Both parents have same MKT1, NCS2 loci)KKh

  • non-htg BC segregantGenotype high & low pool segregants using high-density microarraysHigh poolLow pool

  • High-density tiling microarrays map ALL SNPs in a segregating crossOverlapping 25 bp oligomers, 5 bp apart 5x coverage of entire genomeHigh density Affmetrix tiling miroarray based on S288c

  • ConclusionsHtg phenotype is quick and reproducible to measure 100s 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

  • Current workUse 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

    Say here that although you will be talking mostly about growth, your initial screen was for fermentation.**Yeast haploid cells that are isolated and are wild type for HO spontaneously switch mating type and undergo mother-daughter mating to produce a homozygous progeny.I dont know why some of the boxes are not coloured blue and green here. Maybe colour them all, since you have not yet introduced the lab strain?Assuming unlinked, additive, not epistatic*Cite paper.To achieve the second goal, you would need to cross the best F1 segregant back to KK and try to map genes in there that keep it above KK. See last slide added.For AL3 the difference does not look to be big enough.Alternatively could cross KK/AL3 with an S288C that has got the good alleles of MKT1 and NCS2, and map it in the F1 segregants. Then you would get all Htg loci from either parent, without any confounding effects from the two major loci.