[Zbrafish] Mapcrosses and Positional Cloning

[Thomas Bartman]

Hi all.

We're having trouble with our positional cloning project, and we 
hoping for some input on the following two questions:

1) Are others noticing a great deal of misassembly to the genome 
still?  It seems that every new release of the assembly, our markers 
jump all over.  For example, on Zv5 we had two markers 500 kb apart, 
which went to 12 megabases apart on Zv6.  We kept walking, and got 
down to 4 MB, but then Zv7 pushed those 7 MB apart.  Furthermore, Zv7 
has three of our favorite markers in order A-B-C on the genome, but 
our data has them A-C-B.

(as an aside, we are looking at another gene, and have found it (all 
800 bp) with the identical DNA sequence on two different chromosomes 
on Zv7.  Even if these are redundant copies, how likely is it that 
they will match at 800 of 800 bp, or is this a sign of further misassembly?)

2) This is the crossing strategy we used.  Are there any glaring 
errors in this that could be causing us problems or are we o.k.?

         a) A female AB carrying the mutation was crossed to a male 
WIK.  Embryos were collected for positional cloning from this 
mapcross.  However, we ran out of embryo DNA and the mapcross line 
got old and stopped laying well.  So,

         b) A male from the above mapcross was backcrossed to a 
female WIK.  We continue to collect embryos from this mapcross backcross.

I wasn't sure if I read somewhere if it makes a great difference if 
the crosses to the mapping line are against females or males.

Thanks for any advice, comments.

Tom



Thomas Bartman, M.D., Ph.D.
Divisions of Neonatology, Pulmonary Biology, and Developmental Biology
Cincinnati Children's Hospital Medical Center
3333 Burnet Ave, MLC 7009
Cincinnati, OH  45229-3039

Office: 513-636-9902 
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