Will that be allele cache or Mendelian credit? Evidence for the inheritance of ancestral sequences in Arabidopsis.

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Will that be allele cache or Mendelian credit?
Evidence for the inheritance of ancestral
sequences in Arabidopsis.
Susan J. Lolle Purdue University National Science
Foundation
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Arabidopsis wild-type
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FIDDLEHEAD defined a class of mutations that
resulted in ectopic organ fusion
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Two heads are better than one...
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hothead - phenotype

• Organ fusion
• Pollen hydration on vegetative surfaces
• Increased rate of chlorophyll extraction
• Limited self-fertility

hth-10
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All hothead mutations are recessive
Both copies of the gene must have a mutation to
reveal the mutant phenotype
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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

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Self-fertilizing versesCross-fertilizing
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Progeny should all be homozygous hothead
Stable Inheritance
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But, some progeny are wild-type!
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Mechanism?
Arabidopsis wild-type
Fusion mutant
?
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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

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Using Molecular Markers to Genotype
Allele A
Allele a
PCR amplify (using specific primers)
Restriction Enzyme Digestion
Gel electrophoresis
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Does phenotype relate to genotype?
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Does phenotype relate to genotype?
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What does the sequence tell us?
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I dont know if this is such a wise thing to do,
Bob.
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Is reversion really contamination?Is this a
mistake?

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

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Embryonic revertants
hth-4/hth-4 parent HTH/HTH HTH/hth hth/hth
of embryos 2 57 301
lt1 16 84

• Revertants detected as embryos cannot be due to
  contaminating seed

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Explanations for Genetic Instability
What does the sequence tell us?

• Transposon-induced alleles
• Inverted or direct repeats at the locus
• Epigenetic alleles
• High rate of random mutation
• Gene conversion

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Gene conversion with other family members
HTH ACT GTT GGA ATT ACA hth-10
ACT GTT GAA ATT ACA At HTH-like 1 GTT GTT GGG ATT
ACC At HTH-like 2 CCA CCT CAA GTT GTA At HTH-like
3 CCA CCT CAA GTT GCA At HTH-like 4 GTC GTG GGT
GTT ACT At HTH-like 5 GCC GTA GGA ATC ACC At
HTH-like 6 GCT GTT GGG ATC ACA At HTH-like 7 GTC
GTT GGA ATC ACA

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

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Instability of molecular markers in hth background
Marker hth/hth HTH/HTH Location 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
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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

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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?

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What might this mean?

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

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Should you worry about your ancestry?
How long does the allele cache persist?
Is it quality cache?
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Acknowledgements

• National Science Foundation

Collaborator Bob Pruitt