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
Arabidopsis wild-type
Fusion mutants
FIDDLEHEAD defined a class of mutations that resulted in ectopic organ fusion
Two heads are better than one...
hothead - phenotype
• Organ fusion • Pollen hydration on vegetative surfaces • Increased rate of chlorophyll extraction • Limited self-fertility
hth-10
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 verses Cross-fertilizing
Progeny should all be homozygous hothead
Stable Inheritance
100%
parent
progeny
But, some progeny are wild-type!
90%
10%
Mechanism?
Arabidopsis wild-type Fusion mutant
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
Allele „A‟
PCR amplify
(using specific primers)
Allele „a‟
Restriction Enzyme Digestion
Gel electrophoresis
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.
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?)
Embryonic revertants
hth-4/hth-4 HTH/HTH parent # of embryos % 2 <1
HTH/hth 57 16
hth/hth 301 84
•Revertants detected as embryos cannot be
due to contaminating seed
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
Gene conversion with other family members
HTH hth-10 At HTH-like At HTH-like At HTH-like At HTH-like At HTH-like At HTH-like At HTH-like 1 2 3 4 5 6 7 ACT ACT GTT CCA CCA GTC GCC GCT GTC GTT GTT GTT CCT CCT GTG GTA GTT GTT GGA GAA GGG CAA CAA GGT GGA GGG GGA ATT ATT ATT GTT GTT GTT ATC ATC ATC ACA ACA ACC GTA GCA ACT ACC ACA ACA
•None of these sequences could provide correction
of the mutant nucleotide without introducing other changes
Instability of molecular markers in hth background
Marker
AG GAPC GL1
hth/hth
4% (7/186) 4% (9/242) 1% (1/90)
HTH/HTH
0% (0/190) 0% (0/190) 0% (0/196)
Location in gene intron intron 3’-UTR
HTH
RGA UFO
2% (10/484)
3% (14/402) 4% (16/438)
0% (0/590)
0% (0/386) 0% (0/196)
exon
exon 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?
What might this mean?
“@$#?!$”
•Organisms could carry
sequence information for more than two alleles •Mechanism might help selffertilizing species avoid the negative consequences of inbreeding
Should you worry about your ancestry?
How long does the allele cache persist? Is it quality cache?
Acknowledgements
National Science Foundation Collaborator: Bob Pruitt
People: Katie Krolikowski Ryan Lee Tina Nussbaum Wagler Liese Pruitt Trulie Thorley Mirayda Torres Jen Victor Jessica Young