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
The DuPont Arabidopsis Group