Supplementary Data 1.
Conversion of AFLP marker into single locus PCR-based marker: The PCR products
were run on 6% polyacrylamide gel (19:1) and stained according to the Silver Sequence
DNA sequencing system (Promega, Carlsbad, CA, USA). Seven bands of interest were
excised from gels and squashed in micro-centrifuge tubes containing 20μl of distilled
water. The solution was used as the template DNA for PCR to re-amplify the fragment.
The PCR products were cloned into a pGEM-T Easy vector according to the protocol of
the manufacture (Promega, WI, USA). The clones containing the correct PCR fragment
size were sequenced using Big-Dye Cycle Sequencing Kits and an ABI 310 DNA
sequencer (Applied Biosystems). The primers (outer gene-specific primer and nested
gene-specific primer) were designated to the internal sequences.
DNA adjacent to the AFLP fragment was obtained by anchor PCR using the
Genome Walker Kit (Clontech, Palo Alto, CA, USA). Using genomic DNA of cultivated
azuki bean and its wild ancestor, libraries of uncloned, adaptor-ligated genomic DNA
libraries were prepared for the parents using four restriction enzymes (SwaI, EcoRV,
ScaI, SspI). PCR with an outer gene-specific primer in combination with the Genome
Walker outer adapter primer was performed on the eight libraries (two parents x four
restriction enzymes). The nested PCR using nested gene-specific and nested adaptor
primers were performed on the first PCR product. Strong bands amplified from both
parents with the same restriction enzymes were purified from gels through a column
(Sigma, USA) and used as template DNA for the direct sequencing with nested genespecific primer and nested adapter primers. When an insertion or deletion was found
between two parents in comparison with the obtained sequence information, a primer pair
was designed so that the polymorphic region could be amplified, and the F2 population
subsequently analyzed. When polymorphism associated with differences in the
recognition site of restriction enzymes were found between two parents, PCR using a
nested gene-specific primer and a nested adaptor primer was performed on the DNA of F2
plants, and PCR products were treated with the restriction enzyme to detect CAPS
polymorphisms.