Mapping Mutations in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 17

given site. If you are working with a recessive mutation, then linkage to a SNP is
indicated by an increase in the ratio of N2:CB in the lysate made from New
animals. If you are working with a dominant mutation, then linkage is indicated
by an increase in the ratio of CB:N2 in the lysate made from non-New worms. If
two unlinked mutations are responsible for the New phenotype, then you are
likely to see evidence of linkage to two different SNPs. Note that Seidel et al.
(2008) have shown that the distal left arm of chromosome I is refractory to
becoming homozygous for CB4856 sequences after intercrossing between N2
and CB4856.
B. After Bulked Lysates
Thousands of sites that are polymorphic between N2 and CB4856 have been
identified and these can be accessed through WormBase. For extensive lists of
SNPs and methods for their detection, see Davis et al. (2005) , Fuhrman et al.
(2008) , Jakubowski and Kornfeld (1999) , Shelton (2006) , Swan et al. (2002) , and
Wicks et al. (2001) . These SNPs can be used for bulked lysate analysis, or they can
be used to analyze lysates made from individual animals if one wishes to obtain
multipoint mapping data. However, fairly large sample sizes are needed in order to
achieve high resolution when multipoint mapping is done this way.
In order to achieve high-resolution SNP mapping data, it is necessary to place the
new-1(*) mutation on the same chromosome as a marker mutation. This is typically
done using a standard three-point cross ( Fig. 3 ). For example, if SNP mapping data
indicated that new-1 is situated within/near the chromosome II gene cluster, then one
would construct a strain of genotype new-1(*)/dpy-10(e124) unc-4(e120), then pick
Dpy non-Unc and Unc non-Dpy recombinants (e124 and e120 are both recessive). If
new-1 is to the left of dpy-10, then approximately all of the Unc non-Dpy recombi-
nants will carry new-1(*) on a recombinant chromosome of genotype new-1(*)
unc-4(e120).Ifnew-1 is to the right of unc-4, then approximately all of the Dpy
non-Unc recombinants will carry new-1(*) on the recombinant chromosome. If new-
1 is between dpy-10 and unc-4, some recombinants of each class will carry new-1(*)
and some will not. Thus, this procedure not only generates a chromosome where
new-1(*) is adjacent to a recessive visible marker, but also provides information
about map position.
In order to perform SNP mapping, CB4856 males are crossed with a strain that
carries a marked new-1(*) chromosome, for example, dpy-10(e124) new-1(*),to
generate F1s of genotype dpy-1(e124) new-1(*)/dpy-10(+) new-1(+). This strain is
not balanced, but it can be propagated by cloning wild-type animals and verifying
that they segregate Dpy New progeny. Mapping is then done by cloning Dpy non-
New and New non-Dpy recombinants, making lysates and scoring SNPs by PCR.
Given the high density of SNPs, this is a very powerful method for gene mapping.
However, depending on the nature of the New and marker phenotypes, picking
recombinants can be tedious and/or difficult, so this method is being replaced by
WGS technology whenever possible.