Development and validation of a gene-targeted dCAPS marker for marker-assisted selection of low-alkaloid content in seeds of narrow-leafed lupin (Lupinus angustifolius l.)

Abstract

Low-alkaloid content is an important breeding target to improve the quality of lupin seeds. An APETALA2/ethylene response transcription factor, RAP2-7, is likely a candidate gene for the major alkaloid locus iucundus, and plays a crucial role in regulation of seed alkaloid content in narrow-leafed lupin (NLL; Lupinus angustifolius L.). Here, we exploited a single-nucleotide polymorphism within RAP2-7 credibly associated with seed alkaloid content, to develop the co-dominant derived cleaved amplified polymorphic sequence (dCAPS) marker iuc_RAP2-7. Marker validation in 202 NLL accessions demonstrated that seed alkaloid content ≥0.9% of the seed dry weight was associated with the high-alkaloid marker band (Iucundus genotypes), whereas alkaloid content up to 0.5% of the seed dry weight was associated with the low-alkaloid marker band (iucundus genotypes). Within a given detection limit, iuc_RAP2-7 unambiguously identified all but three low-alkaloid accessions. The latter accessions apparently have a different regulatory mechanism for seed alkaloid content because the RAP2-7 gene/putative promoter sequence and expression of alkaloid-associated genes in the leaves of the three ambiguous accessions were similar to those of bitter Iucundus lines. We consider the iuc_RAP2-7 marker is a powerful tool that will facilitate NLL marker-assisted selection by rapid rejection of bitter Iucundus genotypes and thus accelerate development of new low-alkaloid cultivars.