A mutation screening platform for rapeseed (Brassica napus L.) and the detection of sinapine biosynthesis mutants.
Theor Appl Genet
; 124(5): 957-69, 2012 Mar.
Article
in En
| MEDLINE
| ID: mdl-22198204
ABSTRACT
We developed two mutant populations of oilseed rape (Brassica napus L.) using EMS (ethylmethanesulfonate) as a mutagen. The populations were derived from the spring type line YN01-429 and the winter type cultivar Express 617 encompassing 5,361 and 3,488 M(2) plants, respectively. A high-throughput screening protocol was established based on a two-dimensional 8× pooling strategy. Genes of the sinapine biosynthesis pathway were chosen for determining the mutation frequencies and for creating novel genetic variation for rapeseed breeding. The extraction meal of oilseed rape is a rich protein source containing about 40% protein. Its use as an animal feed or human food, however, is limited by antinutritive compounds like sinapine. The targeting-induced local lesions in genomes (TILLING) strategy was applied to identify mutations of major genes of the sinapine biosynthesis pathway. We constructed locus-specific primers for several TILLING amplicons of two sinapine synthesis genes, BnaX.SGT and BnaX.REF1, covering 80-90% of the coding sequences. Screening of both populations revealed 229 and 341 mutations within the BnaX.SGT sequences (135 missense and 13 nonsense mutations) and the BnaX.REF1 sequences (162 missense, 3 nonsense, 8 splice site mutations), respectively. These mutants provide a new resource for breeding low-sinapine oilseed rape. The frequencies of missense and nonsense mutations corresponded to the frequencies of the target codons. Mutation frequencies ranged from 1/12 to 1/22 kb for the Express 617 population and from 1/27 to 1/60 kb for the YN01-429 population. Our TILLING resource is publicly available. Due to the high mutation frequencies in combination with an 8× pooling strategy, mutants can be routinely identified in a cost-efficient manner. However, primers have to be carefully designed to amplify single sequences from the polyploid rapeseed genome.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Genetic Variation
/
Breeding
/
Choline
/
Brassica napus
/
Mutation
Type of study:
Diagnostic_studies
/
Screening_studies
Language:
En
Journal:
Theor Appl Genet
Year:
2012
Type:
Article
Affiliation country:
Germany