Hi, here is my question. I need to map a recessive mutation and have narrow it between -16 and -14 in LgII by SNP. Someone told me I can cross my mutant with hawaii strain, single F2 m/m recombinants, check F3 SNP at -16 and -14 again and mark the plates which have Hawaii SNP at -16 or -14. Then, cross these F3 animals (with Hawaii SNP at -16 or -14) with Hawaii strain again to further narrow down the mutation. I do not quite understand why crossing back to hawaii again could narrow the region. what kind of detection should I do?
Also, are there other ways to narrow the region?
So that i understand better what has been proposed…you’re contemplating crossing your N2 background recessive mutant with CB4856 to generate (amongst others) homozygous mutants? Then you would look for left/right flanking CB4856 SNPs to mark the endpoints? Of course, you can also use heterozygotes to mark left and right boundaries for the mutation.
If you do the F3 backcross as suggested, would this not generate a proportion of m/m animals with the ROI flanked by CB4856 DNA (and SNPs) and further refine the endpoints?
I don’t see good arguments for crossing in the Hawaiian strain to your recombinant animals; it’s not a sensible way to get more recombination events and narrow down your interval, or at least it doesn’t offer any advantages over what you’re already doing. As Steve says, you could use this approach to get your mutation marked on both sides with Hawaiian markers, although the only reason I can think of that you’d want this is to cross them off again in an unusually thorough backcrossing strategy.
I’ve only come up with two possible arguments for doing this cross, neither of them any good:
Doing the cross will give you animals with which to more easily narrow down the recombination breakpoint, by looking for N2-derived markers in recombinant/Hawaiian hets, as an alternative to scoring for the presence of Hawaiian-derived markers in parental/recombinant hets or to homozygosing the recombinant chromosome. But this isn’t really efficient: you’d have to score individuals (because of the parental/Hawaiian cross-progeny), which would be about a much work as homozygosing the recombinant chromosome, and if you wanted to save a recombinant chromosome (while ordering a new set of primers, for example) you’d still have to homozygose it.
It could be a way to quickly homozygose your recombinant chromosome: once you have a recombinant/Hawaiian het, pick a phenotypically m/m homozygote and you’ve almost certainly homozygosed the recombinant chromosome, without doing more PCRs. But identifying that recombinant/Hawaiian het will require PCRing individuals - so you might as well homozygose the recombinant chromosome in the first place.
Note you can combine (1) and (2) and it becomes a little more attractive: in theory you could slightly (and I’m talking mere days) speed up your mapping by more easily scoring N2-derived markers while you were in the process of homozygosing your recombinant chromosome for possible later use. But I’m still not convinced it’s a good approach. I’d love to hear of some clever reason for doing this cross that I’ve missed, of course.
RE other ways to narrow down the mutation: if there are conveniently placed visible markers flanking your mutation - a dpy and an unc, for example - you could build a triply marked chromosome dpy m unc and put it over the Hawaiian chromosome (or make dpy (Hawaiian) unc and put it over your mutation), and pick Unc-non-Dpy or Dpy-non-Unc, thereby greatly enriching for chromosomes with recombination events close to your mutation. This can be more efficient than looking blindly for nearby recombination events, if you want really fine mapping. Since you’ve got a mutation you can score reliably in F2s, you might also consider a bulked-segregant approach such as that described by Doutsidou et al