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Finding close flanking markers

Thomas Mitchell-Olds tmo at POPGEN.BIOLOGY.UMT.EDU
Wed Dec 23 13:19:24 EST 1992

Recently zrsung at insect.berkeley.edu asked:

>Dear members of Arabidopsis community,
>I'm in a desperate need of new (not listed on H. Goodman &
>E.Meyerowitz maps) RFLP markers on chromosome 5.They should be close to
>the RFLP  marker m247. 
>Also, I'm  looking for YAC clones for the marker m247.
>Any information regarding this matter will be appreciated.
> Chang-Hsien Yang, Dept.of Plant Biology, UC Berkeley /

In the absence of mapped markers that are very close to an area of interest, 
a strategy based on bulk segregant analysis may be helpful.   Michelmore 
(PNAS 88:9828) and Skolnik (PNAS 89:1477) have had good success with a similar methods.

1) Obtain the Dupont recombinant inbred lines from the Ohio stock center.

2) Get the RFLP genotypes for these lines from the Arabidopsis database.

3) For markers near your region of interest, identify recombinant inbred
lines fixed for each allele at nearby markers.  Make pooled DNA from individuals
in each group, and compare RAPD markers between the two groups.  Polymorphic markers
in the region of interest will show differences between the two DNA pools,
while markers elsewhere in the genome will give identical banding patterns. 
For example, assume the Arabidopsis genome is 500 cM, and your bulk segregant
analysis compared individuals differing over 10 cM.  On average, 2% of your
RAPD markers will be in the 10 cM region near your gene.  Assuming two
polymorphic bands per RAPD primer, you could screen 500 primers (1,000 loci),
and find about 20 markers near your gene.  On average, several markers should be less
than 0.5 cM from your gene.  You have a 95% chance that your gene of interest is
flanked by two RAPD markers defining an interval SMALLER than 0.75 cM.  These
nearby RAPDs can then be used to identify cosmids or YACs.  This strategy would
be much easier if DuPont would release information on their mapped primers.

Good luck!

Tom Mitchell-Olds
tmo at selway.umt.edu

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