Development of four phylogenetically-arrayed BAC libraries and sequence of the APA locus in Phaseolus vulgaris

The APA family of seed proteins consists of three subfamilies, in evolutionary order of hypothesized appearance: phytohaemagglutinins (PHA), α-amylase inhibitors (αAI), and arcelins (ARL). The APA family plays a defensive role against mammalian and insect seed predation in common bean (Phaseolus vulgaris L.). The main locus (APA) for this gene family is situated on linkage group B4. In order to elucidate the pattern of duplication and diversification at this locus, we developed a BAC library in each of four different Phaseolus genotypes that represent presumptive steps in the evolutionary diversification of the APA family. Specifically, BAC libraries were established in one P. lunatus (cv. ‘Henderson: PHA+ αAI− ARL−) and three P. vulgaris accessions (presumed ancestral wild G21245 from northern Peru: PHA+ αAI + ARL−; Mesoamerican wild G02771: PHA+ αAI + ARL+; and Mesoamerican breeding line BAT93: PHA+ αAI + ARL−). The libraries were constructed after HindIII digestion of high molecular weight DNA, obtained with a novel nuclei isolation procedure. The frequency of empty or cpDNA-sequence-containing clones in all libraries is low (generally <1%). The Henderson, G21245, and G02771 libraries have a 10× genome coverage, whereas the BAT93 library has a 20× coverage to allow further, more detailed genomic analysis of the bean genome. The complete sequence of a 155 kbp-insert clone of the G02771 library revealed six sequences belonging to the APA gene family, including members of the three subfamilies, as hypothesized. The different subfamilies were interspersed with retrotransposon sequences. In addition, other sequences were identified with similarity to chloroplast DNA, a dehydrin gene, and the Arabidopsis flowering D locus. Linkage between the dehydrin gene and the D1711 RFLP marker identifies a potential syntenic region between parts of common bean linkage group B4 and cowpea linkage group 2