Genetic mapping, cloning, and functional characterization of the BnaX.VTE4 gene encoding a γ-tocopherol methyltransferase from oilseed rape

Rapeseed (Brassica napus L.) is one of the major oilseed crops and an important source for tocopherols known as vitamin E in human nutrition. Increasing the tocopherol content and altering the tocopherol composition is a major goal in rapeseed breeding. The genes encoding enzymes from the tocopherol pathway have been cloned from model species. However, only scant data about tocopherol genes from crop species have been available. We have cloned four sequences of a gene family from B. napus with homology to the Arabidopsis thaliana VTE4 gene. The sequences were amplified by PCR with primers derived from the A. thaliana gene. BAC-clones were isolated to analyze the genomic structure of the BnaX.VTE4-loci. In contrast to the A. thaliana gene all B. napus sequences have two additional introns. For functional analysis, the BnaA.VTE4.a1 sequence was transformed into A. thaliana. Seeds from transgenic offspring showed a 50-fold increase of the alpha-tocopherol fraction which is in accordance with the predicted function of the gene. A marker assay was established and the BnaA.VTE4.a1 sequence was mapped to the end of chromosome A02 of the Tapidor x Ningyou7 genetic map, where also two QTL for alpha-tocopherol content had been mapped. Thus, the BnaA.VTE4.a1 gene is a promising candidate for these QTL and can be used for marker assisted selection for alpha-tocopherol content in rapeseed.