Will that be allele cache or Mendelian credit? Evidence for

the inheritance of ancestral sequences in Arabidopsis.

Susan J. LollePurdue UniversityNational Science Foundation

Arabidopsis wild-type Fusion mutants

FIDDLEHEAD defined a class of mutations that resulted in ectopic organ fusion

Two heads are better than one...

hth-10

hothead - phenotype

• Organ fusion

• Pollen hydration on vegetative surfaces

• Increased rate of chlorophyll extraction

• Limited self-fertility

All hothead mutations are recessive

Both copies of the gene must have a mutation to reveal the mutant phenotype

Fundamentals of Mendelian Genetics

• Two alleles per locus• Alleles can be dominant

or recessive• Alleles are stable from one

generation to the next, whether or not they contribute to the observed phenotype

Self-fertilizing versesCross-fertilizing

Progeny should all be homozygous hothead

parent

100%

progeny

Stable Inheritance

But, some progeny are wild-type!

90%

10%

Arabidopsis wild-type Fusion mutant

Mechanism?

Sequencing of HOTHEAD

•All mutant alleles contain a single point mutation in the HOTHEAD gene•Columbia and Landsberg wild-type alleles very similar; Ws quite different

Using Molecular Markers to Genotype

PCR amplify(using specific primers)

Restriction EnzymeDigestion

Gel electrophoresis

Allele ‘A’ Allele ‘a’

Does phenotype relate to genotype?

Does phenotype relate to genotype?

What does the sequence tell us?

I don’t know if this is such a wise thing to

do, Bob.

•Seed contamination due to wild-type plants grown in close proximity to mutants•Elevated levels of outcrossing in hothead mutants (who is the daddy?)

Is reversion really contamination?Is this a mistake?

•Revertants detected as embryos cannot be due to contaminating seed

Embryonic revertants

hth-4/hth-4 parent

HTH/HTH HTH/hth hth/hth

# of embryos

2 57 301

% <1 16 84

Explanations for Genetic Instability

•Transposon-induced alleles

•Inverted or direct repeats at the locus

•Epigenetic alleles

•High rate of random mutation

•Gene conversion

What does the sequence tell us?

•None of these sequences could provide correction of the mutant nucleotide without introducing other changes

Gene conversion with other family members

HTH ACT GTT GGA ATT ACAhth-10 ACT GTT GAA ATT ACAAt HTH-like 1 GTT GTT GGG ATT ACCAt HTH-like 2 CCA CCT CAA GTT GTAAt HTH-like 3 CCA CCT CAA GTT GCAAt HTH-like 4 GTC GTG GGT GTT ACTAt HTH-like 5 GCC GTA GGA ATC ACCAt HTH-like 6 GCT GTT GGG ATC ACAAt HTH-like 7 GTC GTT GGA ATC ACA

Instability of molecular markers in hth background

Marker hth/hth HTH/HTHLocation in

gene

AG 4% (7/186) 0% (0/190) intron

GAPC 4% (9/242) 0% (0/190) intron

GL1 1% (1/90) 0% (0/196) 3’-UTR

HTH 2% (10/484) 0% (0/590) exon

RGA 3% (14/402) 0% (0/386) exon

UFO 4% (16/438) 0% (0/196) exon

Where did these sequences come from?

• They were not present in the parental genome molecular and genetic evidence

• They were present in the ancestors molecular and genetic evidence

A DNA template cannot be detected in the parental genome

Evidence strongly supports a ‘template-directed’ process.

Is the template RNA?

Where is it?

How long does it persist?

•Organisms could carry sequence information for more than two alleles•Mechanism might help self-fertilizing species avoid the negative consequences of inbreeding

What might this mean?“@$#?!$”

Should you worry about your ancestry?

How long does the allele cache persist?

Is it quality cache?

National Science Foundation

Acknowledgements

Collaborator:Bob Pruitt

People:Katie Krolikowski

Ryan LeeTina Nussbaum

WaglerLiese Pruitt

Trulie ThorleyMirayda Torres

Jen VictorJessica Young