SUPPLEMENTAL MATERIALS AND METHODS
Identification of SNPs physically linked to previously identified genetic
markers: Previous mapping efforts of the allorecognition trait relied on two Amplified
Fragment Length Polymorphisms (AFLPs), designated 194 and 174, one co-dominant
PCR marker designated 18, and one dominant PCR marker, R28, that together defined a
3.4 cM region. A fifth dominant AFLP marker (F29) was also identified but was
determined to segregate only with the f haplotype and therefore could not be used for
mapping in the backcross since all individuals carried the f haplotype (CADAVID et al.
2004).
Poudyal et al. (2007) described the cloning of the AFLP 194 marker and
extension of this genomic sequence in the r haplotype as well as the identification of the
sequence of a corresponding f allele. Analysis of that sequence revealed only one SNP.
Therefore additional primers were designed to extend this sequence by the Universal
GenomeWalker™ method (Clontech Laboratories). A 2.2 kb extended sequence of this
region identified an additional 12 SNPs in the 194 genomic region. In the marker 18
sequence, three SNPs were identified that were suitable for the hME assay. No SNPs
were known for the short (80bp) r-segregating sequence of marker 28. The
GenomeWalker™ method was used to extend the sequence in this region and genomic
products were sequenced and PCR primers were designed to amplify a 0.8 kb region in
ff/ff and rr/rr DNA. Sequencing of these products identified a total of eight SNPs.
Marker 174 was a 247 bp AFLP present only in the r haplotype. In order to clone
and sequence 174, the fragment was generated as described by Cadavid et al. (2004) with
the exception that the primers used in the selective amplification contained no fluorescent
label. The AFLP fragments were separated by gel electrophoresis using 3% Agarose,
Resolute GPG (American Bioanalytical, Natick, MA). Those fragments corresponding to
the predicted size of the 174 marker (247 bp) were excised from a gel and purified using
the Qiaquick® Gel Extraction Kit (Qiagen, Valencia, CA) and cloned into the pCRIITOPO TA vector (Invitrogen) and transformed into Subcloning Efficiency™ DH5™
Competent Cells (Invitrogen). Because these clones still represented independent AFLP
products of similar size, the clones containing 174 were identified by performing a PCR
using the fluorescence-labeled primers originally used for selective amplification in the
AFLP procedure (CADAVID et al. 2004). The products were run on a 6% acrylamide gel
and analyzed to identify clones containing a 247 bp fragment, which were then
sequenced. The insert of a positive clone was also used as a probe in a Southern blot
containing selective amplification products (from the AFLP procedure) of 3 homozygous
ff/ff individuals and 4 heterozygous rr/ff individuals to verify the correct band had been
identified.
Primers designed to amplify the 174 r sequence also amplified a fragment in ff/ff
DNA. This product was subsequently used to physically map and clone the alr2
chromosomal region using fosmids and BAC libraries described in NICOTRA (2007).
End-sequences generated in the course of this study were used to design primers that
amplified corresponding fragments from the f and r haplotypes. These PCR products
were sequenced and a total of 25 SNPs were identified.
Marker 29 was a dominant AFLP marker linked to the f haplotype that was cloned
and sequenced by Cadavid et al. (2004). This DNA was used as a probe to screen the
fosmid library described by Nicotra (2007). A fosmid (pYU1527) was recovered and
end-sequenced to design probes that were hybridized to the ff/ff genomic BAC library.
Thirteen hybridizing BACs were identified and end-sequenced. PCR primers were
designed to the end sequence and tested on rr/rr genomic DNA. These products were
sequenced and compared to the ff/ff sequence, which led to the identification of 36 SNPs.
LITERATURE CITED
CADAVID, L. F., A. E. POWELL, M. L. NICOTRA, M. MORENO and L. W. BUSS, 2004 An
invertebrate histocompatibility complex. Genetics 167: 357-365.
NICOTRA, M. L., 2007 Positional cloning of a cnidarian allorecognition locus. Ph.D.
Thesis, Yale University, New Haven, CT.
POUDYAL, M., S. ROSA, A. E. POWELL, M. MORENO, S. L. DELLAPORTA et al., 2007
Embryonic chimerism does not induce tolerance in an invertebrate model organism. Proc.
Natl. Acad. Sci. USA 104: 4559-4564.