APPENDIX.
Polymorphic markers used to distinguish FL and AB alleles in the F2 generation
| Genea | Primers (5′ → 3′) | Amplicon length | Restrictionb enzyme | Allele cut | Linkage group | Marker no. | Position (cM) |
|---|---|---|---|---|---|---|---|
| A. Gene-based codominant markers | |||||||
| Ws5096 | GATGATCGATCTGGAGCACATGGCTAAAACAGGCTACTTCA | 331 | HinfI | AB | 2 | 8 | 50.0 |
| Ws13043 | CGGCCGTGACTTATGCTAAAGCAGGAACTCCGCCATAATA | 182 | NA | NA | 2 | 17 | 123.8 |
| 278c | |||||||
| UbcD4 | CCAGCAGTGGATATGGGTGTGCGGCTCAAATCCCTAAAA | 398 | EcoRV | FL | 2 | 18 | 124.6 |
| timeless | GGTGGTGATCTGGAGCAAATCCACGCTTGGGTTGAGCAGAT | 624 | BsmBI | AB | 3 | 1 | 0.0 |
| Ws10014 | CCCCAATGGTGATGTTTCTCAGCCCGATTTTTGGAATGAT | 503 | EcoRI | AB | 3 | 2 | 4.1 |
| WsRpL17 | TCCGCAAAGCAATATTACCGGCTTGTGTCGAGCACACATT | 499 | SfaNI | FL | 3 | 4 | 18.0 |
| Ws31075 | AAGGTTGGCTGTCCATTCACGAAACGGTTTTCGTTTCGAG | 488 | BccI | AB | 3 | 5 | 22.6 |
| l(1)G0156 | GAGGCGGTTGATGTAACTCCTGGATCCTGCACCATATTCA | 540 | AciI | AB | 3 | 12 | 53.1 |
| Ws13994 | CGGAAAAAGTCCAAATGTTGTTTTTGGTTCACCTGGATCGT | 258 | HindIII | FL | 3 | 13 | 54.3 |
| Marker name/selective primersd | Dominant population | Linkage group | Marker no. | Position (cM) |
|---|---|---|---|---|
| B. Dominant AFLP markers | ||||
| EAGCMCTA.o | AB | 1 | 1 | 0.0 |
| EAGCMCTA.c | AB | 1 | 2 | 13.4 |
| EAGCMCTT.s | AB | 1 | 3 | 17.1 |
| EACCMCTT.i | AB | 1 | 4 | 25.9 |
| EAACMCTA.l | FL | 1 | 5 | 33.7 |
| EAGCMCTA.h | FL | 2 | 1 | 0.0 |
| EAGCMCTA.i | AB | 2 | 2 | 5.1 |
| EAACMCTA.f | AB | 2 | 3 | 13.9 |
| EAGCMCTA.g | FL | 2 | 4 | 16.2 |
| EAGCMCTT.k | AB | 2 | 5 | 21.8 |
| EACCMCTT.c | FL | 2 | 6 | 27.1 |
| EAGCMCTA.m | FL | 2 | 7 | 47.3 |
| EAACMCTA.e | FL | 2 | 9 | 66.0 |
| EACCMCTT.o | FL | 2 | 10 | 84.6 |
| EACCMCTT.q | FL | 2 | 11 | 88.7 |
| EAGCMCTT.t | FL | 2 | 12 | 92.8 |
| EAGCMCTT.o | AB | 2 | 13 | 95.8 |
| EAGCMCTA.d | FL | 2 | 14 | 101.9 |
| EACCMCTT.a | FL | 2 | 15 | 108.0 |
| EAGCMCTT.n | AB | 2 | 16 | 117.0 |
| EAGCMCTT.h | AB | 2 | 19 | 134.1 |
| EAACMCTA.d | AB | 2 | 20 | 151.2 |
| EAGCMCTA.b | FL | 3 | 3 | 14.7 |
| EAGCMCTT.q | AB | 3 | 6 | 31.1 |
| EAGCMCTT.i | FL | 3 | 7 | 31.7 |
| EACCMCTT.l | FL | 3 | 8 | 38.9 |
| EAACMCTA.h | FL | 3 | 9 | 42.5 |
| EAGCMCTT.r | AB | 3 | 10 | 42.9 |
| EACCMCTT.m | AB | 3 | 11 | 48.6 |
| EAACMCTA.m | AB | 3 | 14 | 56.7 |
| EAGCMCTA.a | AB | 3 | 15 | 58.7 |
| EAGCMCTT.v | FL | 3 | 16 | 61.0 |
| EAGCMCTT.y | AB | 3 | 17 | 65.0 |
| EAGCMCTA.n | FL | 3 | 18 | 67.4 |
| EAGCMCTT.j | AB | 3 | 19 | 85 |
| EAACMCTA.c | AB | 3 | 20 | 102.0 |
The genes are named according to the putative ortholog from TBLASTX results of the W. smithii sequence against the genome of D. melanogaster. The numbered genes are unannotated on FlyBase and the prefix Cg- has been replaced by Ws- for W. smithii. The GenBank accession nos. for the nine genes, in descending order, are EF094841–EF094856 and AY943312.
The restriction endonuclease used in genotyping (see materials and methods for details).
The polymorphism for Ws13043 is a 96-bp insertion/deletion. Genotyping in the F2 was performed by scoring length differences of PCR amplicons instead of by restriction endonuclease digestion.
The EcoRI and MseI core primer sequences for both the pre- and selective amplification steps of the AFLP protocol are 5′-GACTGCGTACCAATTC-3′ and 5′-GATGAGTCCTGAGTAA-3′, respectively. The three bases after the letters E and M in each marker name denote the three arbitrary nucleotides added to the 3′-end of the core sequence for selective amplification. The lowercase letter at the end of the name distinguishes between multiple markers generated by the same set of selective primers.