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.