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mapping

scolnipa at esvax.dnet.dupont.com scolnipa at esvax.dnet.dupont.com
Mon Jan 18 12:00:39 EST 1993


A few comments on the December 23 message posted by Tom Mitchell-Olds
concerning the identification of new RAPD markers near a locus of interest:
For the purpose of chromosome walking, we feel it is more efficient to use 
a population that is segregating for a marker, mutation, or allele of 
interest in a bulked or pooling approach, rather than using regional fixed-
allele information from the recombinant inbred (RI) collection.  See Patton 
et. al. 1991 MGG 227:337-347 for an example of the bulked F2 approach used in 
Arabidopsis.  The markers mapped in the RI collection are a good starting 
point only if you have the same pair of alleles in your cross.  You will, 
however, have to screen more primers if you want markers tightly linked to 
your gene of interest.

Not all primers identify polymorphisms.  Only 245 RAPD primers out of 1200
tested showed mappable polymorphisms and each polymorphic primer, on 
average showed 1.6 polymorphisms (Reiter et. al. PNAS 89:1477-1481, 1992).  
Therefore, screening 500 new primers will reveal only 163 new polymorphisms.  
If evenly spaced throughout a 500 cM map, there would be 1 new marker per 3cM.
If you started with 500 new primers, AND ALL SHOWED POLYMORPHISMS, the 
theoretical degree of saturation is on the order of 0.63 cM.  However, based 
on Reiter et al. results, you would need to screen 2450 primers to find 500 
primers that show polymorphisms.  That would entail setting up 4900 reactions, 
using over $3,000 of Taq polymerase, and running over 75 gels 
(64 well capacity).  Bulking or pooling at a particular locus does not change 
the distribution of these new markers, it only allows you to easily identify 
the markers linked to that locus.

Tom suggested that the sequences of the RAPD primers should be distributed 
and we fully agree.  However, we felt that they would not be useful in the 
absence of information identifying the RAPD band mapped.  We are beginning the 
laborious process of digitizing the images of the RAPD patterns for deposit in 
the AATDB.  However, in the meantime, we have compiled and sent to the AATDB 
all primer sequence information and RI scoring data. The RI collection has 
already been deposited at the seed stock center.  In addition, we have allowed 
over 50 different labs to come here and screen the DuPont T-DNA insertional 
collection, and have mapped many clones.  We hope that our openness will 
stimulate others to provide equal access to their sequences, strains and 
collections.

The DuPont Arabidopsis Group
 Pablo Scolnik
 Tim Caspar
 David Patton
 Sandra Russell 




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