# Presentation: 132453

## 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

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

## Two heads are better than one...

## hothead - phenotype

*hth-10*

*hothead* - phenotype

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

## All hothead mutations are recessive

- 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

- Self-fertilizing versesCross-fertilizing

## Progeny should all be homozygous hothead 

- Stable Inheritance

## But, some progeny are wild-type! 

- But, some progeny are wild-type!

## 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

- PCR amplify
- (using specific primers)

- Restriction Enzyme
- Digestion

- 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?)

- 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 G**_**G**_**A ATT ACA**
***hth-10*****        ACT GTT G**_**A**_**A ATT ACA**
**At HTH-like 1**** ****GT****T GTT G****G****G ****ATT AC****C**
**At HTH-like 2**** ****C****C****A CC****T**** ****CA****A**** ****G****TT**** ****GT****A**
**At HTH-like 3**** ****C****C****A CC****T**** ****CA****A**** ****G****TT**** ****G****CA**
**At HTH-like 4**** ****GTC ****GT****G ****G**_**G**_**T G****TT AC****T**
**At HTH-like 5**** ****G****C****C ****GT****A ****G**_**G**_**A AT****C ****AC****C**
**At HTH-like 6**** ****G****CT GTT G**_**G**_**G ****AT****C ****ACA**
**At HTH-like 7**** ****GTC ****GTT G**_**G**_**A AT****C ****ACA**

## 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 |

## 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

- 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

- National Science Foundation

- Acknowledgements

- Collaborator:
- Bob Pruitt