Mapping of high temperature Mapping of high temperature growth genes derived from growth genes derived from
industrial yeast strainsindustrial yeast strains
Justin Goh, Richard Gardner
School of Biological Sciences, University of Auckland
15ºC 40ºC
Wide temperature tolerance ofWide temperature tolerance of Saccharomyces cerevisiaeSaccharomyces cerevisiae has industrial has industrial
applicationsapplications
Two strains of Two strains of S. cerevisiae S. cerevisiae can ferment can ferment well at high temperaturewell at high temperature
CO2
CO2CO2
CO2
AL3
Alcohol distillery - Brazil
KK:YS1
Kodo ko jaanr – fermented finger millet beverage
Aim: To map some of the major genes Aim: To map some of the major genes involved in high temperature growth (htg)involved in high temperature growth (htg)
AL3 and KK:YS1 are heterozygousAL3 and KK:YS1 are heterozygous
Microsatellite
marker C3 C5 C8 C4 091c AT4 AT2 Scaat3 009C 267C MATα MATa
AL3 (heterozygous)
108, 120 172
130, 143 259 290
292, 296
357, 366
356, 370
382, 449
439, 445 468 492
KK (heterozygous)
111, 114 119
127, 130 none 239
266, 288 350 395
396, 405
398, 442 468 492
Obtained homozygous derivatives of AL3 Obtained homozygous derivatives of AL3 and KK:YS1 by tetrad dissection and KK:YS1 by tetrad dissection
Heterozygous parent
Screen among homozygous progeny
for a fermentation phenotype as good as
the parent strain
Homozygous derivatives can ferment Homozygous derivatives can ferment nearly as well at heterozygous parentnearly as well at heterozygous parent
Microsatellite marker C3 C5 C8 C4 091c AT4 AT2 Scaat3 009C 267C MATα MATa
AL3 (heterozygous)
108, 120 172
130, 143 259 290 292, 296 357, 366 356, 370 382, 449 439, 445 468 492
AL3h (homozygous) 120 172 143 none 290 296 357 370 449 445 468 492
KK (heterozygous)
111, 114 119
127, 130 none 239 266, 288 350 395 396, 405 398, 442 468 492
KKh (homozygous) 114 119 130 none 239 288 350 395 396 398 468 492
Cross AL3h and KKh to S288c – standard Cross AL3h and KKh to S288c – standard laboratory strain – to map htg geneslaboratory strain – to map htg genes
Phenotyping: High temperature Phenotyping: High temperature fermentation vs growthfermentation vs growth
Colony growth at 40°C
vs.
315 tubes1 week
9 L sugar mediumMany weighings
1 plate48 h
0.02 L sugar mediumSingle scoring
To phenotype 100 progeny
Fermentation at 40°C
Phenotyping high temperature growthPhenotyping 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
Qualitative assessment of htgQualitative assessment of htg
Quantify growth by pixel intensity of Quantify growth by pixel intensity of colony spots of scanned platecolony spots of scanned plate
Calculate high temperature growth ability Calculate high temperature growth ability as ratio of growth compared to 28°Cas 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 48h37°C 24h 41°C 48h28°C 24h Htg
Scheme for crossing & backcrossing Scheme for crossing & backcrossing homozygous strains to S288chomozygous strains to S288c
F1 hybridMATα/a URA/ura HO/ho
MATα ura hoMATα/a ura HO MATa ura ho
S288c (Sequenced lab strain)
MATα ura ho
KKhMATα/a HO
AL3hMATα/a HO
Screen 100 F1 haploid progeny for colony
growth at 40°C
Best F1 segregant
Homozygous spores
Crossing & backcrossing of Htg strains to Crossing & backcrossing of Htg strains to S288cS288c
S288cMATα 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
Verify crossing & backcrossing by Verify crossing & backcrossing by microsattelite genotypingmicrosattelite genotyping
Microsattelite
marker C3 C5 C8 C4 091c AT4 AT2 Scaat3 009C 267C MATα MATa
S288C 120 174 130 240 302 296 357 407 443 415 468 492
AL3 (heterozygous)
108, 120 172
130, 143 259 290
292, 296
357, 366
356, 370
382, 449
439, 445 468 492
AL3h (homozygous) 120 172 143 none 290 296 357 370 449 445 468 492
F1 hybrid 120172, 174
130, 143 240
290, 302 296 357
370, 407
443, 449
415, 445 468 492
F1 segregant 120 174 143 240 290 296 357 370 449 445 none 492
BC 120 174130, 143 240
290, 302 296 357
370, 407
443, 449
415, 445 468 492
BC segregant 120 174 143 240 302 296 357 370 443 415 none 492
KK (heterozygous)
111, 114 119
127, 130 none 239
266, 288 350 395
396, 405
398, 442 468 492
KKh (homozygous) 114 119 130 none 239 288 350 395 396 398 468 492
F1 hybrid
114, 120
119, 174 130 240 302
288, 296
350, 357
395, 407
396, 443
398, 415 468 492
F1 segregant 120 119 130 240 302 296 357 395 443 398 none 492
BC 120119, 174 130 240 302 296 357
395, 406 443
398, 415 468 492
BC segregant 120 119 130 240 302 296 357 395 443 415 none 492
Phenotypic distribution of htg of Phenotypic distribution of htg of backcrossed segregantsbackcrossed segregants
S288c
F1 hybrid
BC segregants
S288c Best F1 segregant
Backcross (BC)
40°C 48h37°C 24h 41°C 48h
S288c
AL3h
F1 hybridBest F1 segregant
BC
AL3h
Phenotypic distribution of htg of Phenotypic distribution of htg of backcrossed segregantsbackcrossed segregants
S288c
F1 hybrid
BC segregants
S288cBest F1
segregant
Backcross (BC)
S288c
KKh F1 hybridBest F1 segregant
BC
KKh
40°C 48h37°C 24h 41°C 48h
Positive heterosis in F1 hybrids suggests Positive heterosis in F1 hybrids suggests htg is co-dominant & both parents htg is co-dominant & both parents
contributecontribute
40°C 48h37°C 24h 41°C 48h
S288c
AL3h
F1 hybridBest F1 segregant
BCS288c
KKh F1 hybridBest F1 segregant
BC
40°C 48h37°C 24h 41°C 48h
S288c AL3h F1S288c KKh F1
107
106
105
104
Dilution series 41°C 48h
107
106
105
104
Only a few genes may be required for high Only a few genes may be required for high temperature growthtemperature growth
40°C 48h37°C 24h 41°C 48h
S288c
AL3h
F1 hybridBest F1 segregant
BCS288c
KKh F1 hybridBest F1segregant
BC
40°C 48h37°C 24h 41°C 48h
37/184 segregants
(½) 2.3
40/184 segregants
9/184 segregants
(½) 4.36
15/184 segregants41°C
40°C
(½) 2.2
(½) 3.6
Two major genes for high temperature growth Two major genes for high temperature growth were recently mappedwere recently mapped
S288c YJM 421
F1 hybrid
Standard laboratory
strain
Homozygous derivative of a clinical isolate
Sinha et al (2008)
Major genes affecting htg have no obvious link Major genes affecting htg have no obvious link to function – “post-transcriptional regulation”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
AL3
KK:YS1
YJM 421Alcohol distillery - Brazil
Kodo ko jaanr – fermented finger millet beverage
Hypothesis: the major htg genes in AL3h Hypothesis: the major htg genes in AL3h and KKh are different from YJM 421and KKh are different from YJM 421
Genotyping of MKT1 and NCS2 in BC Genotyping of MKT1 and NCS2 in BC segregants that are Htg+ and Htg-segregants that are Htg+ and Htg-
S288c
AL3h
F1 hybridBest F1 segregant
BCS288c
KKh F1 hybridBest 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
Inheritance of parental alleles of MKT1 and Inheritance of parental alleles of MKT1 and NCS2 determined using RFLPNCS2 determined using RFLP
E.g. Amplify 900 bp region of NCS2 and cut with Tsp5091
KK KKh S288c F1 F1 s BC BC segregants →
Clear association with MKT1 and NCS2 in Clear association with MKT1 and NCS2 in KKKK
High pool # NCS2 MKT11 2 3 4 5 6 7 8 9
10 11 12 13 14 15 16 17 18 19 20
Low pool # NCS2 MKT11 2 3 4 5 6 7 8 9
10 11 12 13 14 15 16 17 18 19 20
S288c or KKh
40°C 48h37°C 24h 41°C 48h
……and in AL3 BC segregantsand in AL3 BC segregants
40°C 48h37°C 24h 41°C 48h
High pool # NCS2 MKT11 2 3 4 5 6 7 8 9
10 11 12 13 14 15 16 17 18 19 20
S288c or AL3h
Low pool # NCS2 MKT11 2 3 4 5 6 7 8 9
10 11 12 13 14 15 16 17 18 19 20
MKT1 and NCS2 are linked on chrom 14MKT1 and NCS2 are linked on chrom 14
High pool # NCS2 MKT11 2 3 4 5 6 7 8 9
10 11 12 13 14 15 16 17 18 19 20
Low pool # NCS2 MKT11 2 3 4 5 6 7 8 9
10 11 12 13 14 15 16 17 18 19 20
High pool # NCS2 MKT11 2 3 4 5 6 7 8 9
10 11 12 13 14 15 16 17 18 19 20
Low pool # NCS2 MKT11 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
S288c
F1 hybrid
BC segregants
S288cBest F1
segregant
Backcross (BC)
AL3h
Best F1 segregantnon-htg BC segregant
Fix htg+ derived MKT1 & NCS2 alleles in Fix htg+ derived MKT1 & NCS2 alleles in next cross → find other htg genesnext cross → find other htg genes
BC F1 hybrid
40°C 48h37°C 24h 41°C 48h
S288cAL3h
F1 hybridBest F1 segregant
BCnon-htg BC segregant
?
Identify htg genes from S288CIdentify htg genes from S288C(Both parents have same MKT1, NCS2 loci)(Both parents have same MKT1, NCS2 loci)
S288c KKh F1 hybridBest F1 segregant
40°C 48h37°C 24h 41°C 48h
S288c
F1 hybrid
Best F1 segregant
KKh
BC segregants
Backcross (BC)
KKh
?
S288c
AL3h
F1 hybridBest F1 segregant
BC
non-htg BC segregant
Genotype high & low pool segregants Genotype high & low pool segregants using high-density microarraysusing high-density microarrays
High poolLow pool
?
High-density tiling microarrays map ALL High-density tiling microarrays map ALL SNPs in a segregating crossSNPs in a segregating cross
Overlapping 25 bp oligomers, 5 bp apart → 5x coverage of entire genome
High density Affmetrix tiling miroarray based on S288c
ConclusionsConclusions
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
Current workCurrent 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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