Analysis of phaseolin gene copy number by quantitative PCR

The 7S globulin phaseolin is the most abundant seed protein in common bean (Phaseolus vulgaris) accounting for close to 50% of total seed protein in cultivated varieties. Two different types of phaseolin genes encode two phaseolin subtypes, α- and β-. Their coding sequence is distinguished by the presence of a 27 bp repeat in α-phaseolin (Slightom et al. 1985; Kami and Gepts 1994). The α- and β-phaseolin genes are more dissimilar in their promoters and 5'-untranslated regions (Slightom et al. 1983; Anthony et al. 1990). Phaseolins are encoded by a single complex locus in the genome, predicted to contain multiple genes in tandem. Southern hybridization studies showed that the phaseolin multigene family consists of approximately seven members (Talbot et al. 1984). It is notably difficult to assemble next generation sequencing read data for loci with highly repetitive gene sequences such as those of storage protein genes (Dong et al. 2016). The first assembly of the reference genome G19833 contained unique copies of α- and β-phaseolin on chromosome 7, on opposite strands approximately 120 kb apart (Schmutz et al. 2014). A newly released version (v2.1) contains three α- and two β-phaseolin genes (Fig. 1). We used the quantitative polymerase chain reaction (qPCR) according to Ingham et al. (2001), in the genetic stock SARC1 which contains both α- and β-phaseolin (Osborn et al. 2003), to determine the number of copies of these genes.