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Transcriptional variation in cis and trans,genetics of quantitative traits.
• Anne Genisell, Rita Graze & Lauren McIntyre
UG Florida
• Rita Graze, Maria Persianinova & Olga Barmina
• Artyom Kopp & Ryan Bickel
• Transcriptional variation in cis and trans
• QG of divergence between species
• QG of variation within species
• cis or trans? • allele specific?
Agilent 60 bp Splicing Arrays, 8 R I Lines:
1. Genetic variation
2. Infinitesimal model 1 cis; zillion trans.
3. ‘Large trans-effect’ model (g: P0, P1, O0, O1)
3685 cis trans cis-by-trans Infinitesimal 206 79 63 Large-effect 1275 44 79 Overlap 189 0 5
ijiij lY
ijkijjiijk cttcY
• cis or trans? • allele specific?
ijiiijk glgY )(
Morley et al. 2004: 14 human families;
3,554 genes with significant genetic variation;
984 QTLs 19% cis, 78% trans, 3% both;
(co-localized)
An average QTL accounts for ~50% of variation.
Power? LOW (5%) overestimation
VARIABLE explains co-localization?
• cis or trans?
hybrids
Wittkopp, Haerun, Clark, 2004 mel
sim
• cis or trans? • allele specific?
Genes with mismatches:
O S O*S
mau sim hybMichalak & Noor 2003
• cis or trans? • allele specific?
• cis or trans? • allele specific?
• cis or trans? • allele specific?
False positive
False negative
GCRMA 12.5% 61.8%
MAS 5.0
12.5% 65.6%
ln(PM) 18.7% 59.9%
ln(PM/MM)
18.4% 57.2%
Norm. cis trans cis & trans
GCRMA 3 8 1
MAS 5.0 10 16 1
ln(PM) 2 8 1
ln(PM/MM)
7 69 5
• Transcriptional variation in cis and trans
• QG of divergence between species
• QG of variation within species
Divergence in sex comb tooth number.
D. simulans
D. mauritiana
Chromosome III:
LOD
cM
Sex Comb Tooth Number
tra Antp
Reproduced from: True et al. (1997)
Introgression line mapping
e+
P{lacW}76C
P{lacW} [76C]
e [93C]
e+
+
D. simulans
D. mauritiana
Obtained recombinants between two flanking visible markers:
62 nearly isogenic lines :
Introgression line mapping results
Marker Type III SS F value P Effectc SEc t valuec
93C (ebony)a 79.67 157.54 4.23X10-35 .54 .043 12.55
84A (Scr)b 39.29 77.70 2.18X10-18 .58 .065 8.81
P{lacW}76Ca 25.52 50.47 1.57X10-12 .41 .058 7.10
82C (Dip2)b 12.25 24.22 9.15X10-7 -.43 .087 -4.92
77B (rdgC)b 7.25 14.34 .0002 -.24 .064 -3.79
79D (Ten-m)b 2.2 4.36 .04 .14 .065 2.09
{Sig. Interactions
76C 77B 79D 80A 82C 84A 93C
P{lacW}76C ** rdgC * Ten-m * CG11367
Dip2 ** Scr ** ebony **
a)
+ + ++- -76C 77B 79D 80A 82C 84A 93C
P{lacW}76C ** rdgC * Ten-m * CG11367
Dip2 ** Scr ** ebony **
a)
+ + ++- -
a)
+ + ++- -
a)a)
+ + ++- - .54 .58.43.14.24.41
Comparing expression T1 & T2 pupal legs
• Pupal legs were collected at 16h post pupariation
• For each leg and each species, legs from multiple males were pooled into 3 biological replicates each.
• The mRNA was amplified and hybridized to Drosophila 2.0 Affymetrix GeneChips®
T2
D. mauritiana
T1
3331
32
3331
32
D. simulans
3432
33
T1
3432
33
T2
---.0008m---Transcription FactorMorphogenesis60E2Dll
---.0008m---Ubiquitin conjugationUbiquitin cycle32A5UbcD2
---.0008m---UnknownUnknown2B1CG14795
---.0004m---Transporter activityTransport67D8CG6640
---.0002m---UnknownUnknown38A8CG16772
.0002g------UnknownUnknown83E8CG15186
.0018g.0003m---Takeout familyUnknown82E4CG2016
.0008g.0044m---Fibronectin type IIICell adhesion95E1kal-1
.0002g.0065m---Transcription FactorMorphogenesis69C8ara
<.0001g---.2-,gNucleic acid bindingDNA repair43E18phr
.005g.0006m---MAB-21 relatedUnknown5D1CG4766
.0002g.0002m---UnknownUnknown94A11CG138571,2,3
.0003g<.0001m.05m,-UnknownUnknown18A3CG14191
<.0001g.0001m.05-,gUnknownUnknown84E6CG27911,3
<.0001g<.0001m.2m, .05gFibronectin type IIIOlfactory learning60C3Nord1,3
<.0001g<.0001m.05m,gTranscription factorA/P axis specification84A5Scr1,3
<.0001g<.0001m.05m,gUnknownUnknown72E1CG4962
PgPmFDRm,gMolecular FunctionBiological ProcessBandGene
---.0008m---Transcription FactorMorphogenesis60E2Dll
---.0008m---Ubiquitin conjugationUbiquitin cycle32A5UbcD2
---.0008m---UnknownUnknown2B1CG14795
---.0004m---Transporter activityTransport67D8CG6640
---.0002m---UnknownUnknown38A8CG16772
.0002g------UnknownUnknown83E8CG15186
.0018g.0003m---Takeout familyUnknown82E4CG2016
.0008g.0044m---Fibronectin type IIICell adhesion95E1kal-1
.0002g.0065m---Transcription FactorMorphogenesis69C8ara
<.0001g---.2-,gNucleic acid bindingDNA repair43E18phr
.005g.0006m---MAB-21 relatedUnknown5D1CG4766
.0002g.0002m---UnknownUnknown94A11CG138571,2,3
.0003g<.0001m.05m,-UnknownUnknown18A3CG14191
<.0001g.0001m.05-,gUnknownUnknown84E6CG27911,3
<.0001g<.0001m.2m, .05gFibronectin type IIIOlfactory learning60C3Nord1,3
<.0001g<.0001m.05m,gTranscription factorA/P axis specification84A5Scr1,3
<.0001g<.0001m.05m,gUnknownUnknown72E1CG4962
PgPmFDRm,gMolecular FunctionBiological ProcessBandGene
Combining mapping & array results:
76C 77B 79D 80A 82C 84A 93C
P{lacW}76C ** rdgC * Ten-m * CG11367
Dip2 ** Scr ** ebony **
a)
+ + ++- -76C 77B 79D 80A 82C 84A 93C
P{lacW}76C ** rdgC * Ten-m * CG11367
Dip2 ** Scr ** ebony **
a)
+ + ++- -
a)
+ + ++- -
a)a)
+ + ++- -
Gene Band Biological Process Molecular Function FDRm,g Pm Pg
CG4962 72E1 Unknown Unknown .05m,g <.0001m <.0001g
CG6640 67D8 Transport Transporter activity .05m, .1g <.0001m .0143g
ara 69C8 Morphogenesis Transcription Factor .05m, .1g .0026m .0068g
kal-1 95E1 Cell adhesion Fibronectin type III .05m,g .0014m .0014g
CG15186 83E8 Unknown Unknown .05-,g --- <.0001g
CG2791 84E6 Unknown Unknown .1-,g --- .006g
CG2016 82E4 Unknown Takeout family .2m,- .0282m ---
CG13857 94A11 Unknown Unknown .2m,- .03m ---
&
• We examined five insertions in or near CG2791,
two affect sex comb number and morphology.
Is CG2791 a sex comb gene?
dsx CG2791
Os-C
1. P{SUPor-P}KG01932 reduces tooth number and
has weakly penetrant effects on the sex comb.
1
2
2. PBac{5HPw [+])A146 reduces tooth number slightly
and causes curving in the top portion of the sex comb.
Tibia
Metatarsus
T2
CG2791 is expressed in T1 and not in T2
Tarsus
24 hours post-pupariation
T1
CG2791 is expressed on the ventral anterior surface of T1,but not T2.
sex comb
PBac{GAL4D, EYFP} CG2791PL00285/UAS-LacZ
PBac{GAL4D, EYFP} CG2791PL00285/UAS-LacZ
What’s the proof?
- have gone all the way from between species morphological differences
to QTLs
to fine-mapping
to the gene.
Transformation? (position effect)
Gene replacement (not D. melanogaster)
Modification of allele-specific expression?
• Transcriptional variation in cis and trans
• QG of divergence between species
• QG of variation within species
Building a genotype to phenotype map
bab1 bab2
Low LD (200 b) enables precise association
inferences
AbdBDsxF
bab
Yellow
Gene functioning might be assayed at the right time in the right tissues…
AbdBDsxF
Yellow
bab
Snp1Snp2 Snp3 Snp4Snp...indel1indel1bab
variation
Phenotype
… to build the genotype to phenotype map.