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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