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Bradeen Lab

Goal:  Describe RB structure and sequence from

          
Solanaceous spp. (SOLAR)

Objectives:

Phase One:  LR-PCR for allelic mining

technical optimization, strategies for use

application in S. bulbocastanum

empirical testing of evolutionary hypotheses

Phase Two:  BAC-based RB recovery in Solanum

primitive potato: S. bulbocastanum

advanced potato: S. demissum

tomato

eggplant

Phase Three:  RB recovery in other Solanaceous spp.

Phase One: LR-PCR for allelic mining

Where we were (~2 years ago):

LR-PCR used to clone RB (complementation)

Song et al. PNAS 100:9128-9133

RB

LR-PCR

Phase One: LR-PCR for allelic mining

Where we were (~2 years ago):

LR-PCR used to clone RB (complementation)

Evolutionary comparisons of RB and rb paralogs:

   RB is a Type II resistance gene

~present at high frequency

~order within cluster conserved

~sequence conserved

~function retained

Song et al. PNAS 100:9128-9133

Phase One: LR-PCR for allelic mining

technical optimization, strategies for use

1) DNA extraction: 7 methods

2) 4 long range Taqs

3) Primer design

4) 2 cloning vectors

5) Sequence fidelity:  3 independent clones =
100%

6) Sequencing strategy: primer walking

Manuscript currently being prepared (to be submitted
this fall)

application in S. bulbocastanum

Phase One: LR-PCR for allelic mining

44 genotype reference population:

~ Within vs. between population

~ Entire geographic range

~ 3 subspecies

~ (tied to total genome diversity study)

Results: LR-PCR gene fragment amplified in 41% (18/44)

     ~ replicated

     ~ 2 primer pairs tested

Pha