Development of SCAR markers linked to zt-2, one of the genes controlling absence of tannins in faba bean

Abstract

Faba beans (Vicia faba L.) have a great potential as a protein-rich fodder crop, but anti-nutritional factors such as condensed tannins reduce the biological value of their protein. Tannins can be removed from seeds by any of the two complementary genes, zt-1 and zt-2, which also determine white-flowered plants. The less common gene, zt-2, is also associated with increased protein levels and energy values and reduced fibre content of the seeds. To identify a cost-effective marker linked to zt-2, we analysed a segregating F2 population derived from the cross between the coloured flower and high tannin content genotype Vf6 and a zt-2 line. By using Bulked Segregant Analysis (BSA), five RAPD markers linked in coupling and repulsion phase to zt-2 were identified and their conversion into Sequence Characterised Amplified Regions (SCARs) was attempted. Amplification of the SCARS was more consistent, although the initial polymorphism was lost. Restriction digestion of SCAR SCAD16589 with AluI (SCAD16-A), Bsp120I (SCAD16-B) and HinfI (SCAD16-H) revealed clear differences due to the amplification of different loci. The consensus sequence of these CAPs (Cleavage Amplification Polymorphisms) markers allowed discrimination of three bands from which two new forward SCAR primers were developed based on specific sequences from zero tannin and high tannin content genotypes. To improve the efficiency of the marker screening, a multiplex PCR was developed that allowed the simultaneous amplification of the SCAR with the same advantages as a codominant marker. Marker validation was carried out with a new F2 population segregating for flower colour and tannin content, underscoring the potential of these markers in breeding selection to introgress the zt-2 gene for the development of new tannin free faba bean cultivars.