A mutation screening platform for rapeseed (Brassica napus L.) and the detection of sinapine biosynthesis mutants

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.

Production of a High-Efficiency TILLING Population through Polyploidization

“…The vertical bar connects instances of the same species. Ai, Peanut (Knoll et al, 2011); AtC, Arabidopsis Col-0 (Greene et al, 2003); AtL, Arabidopsis Ler (Martín et al, 2009), Bn, canola (Harloff et al, 2012); Br, B. rapa (Stephenson et al, 2010); Gm, soybean (Cooper et al, 2008) (Till et al, 2004b). [See online article for color version of this figure. ] with EMS) was germinated and grown.…”

“…The highest mutation densities were obtained in polyploids ( Fig. 1), hexaploid wheat (Triticum aestivum; Slade et al, 2005;Uauy et al, 2009), and tetraploid canola (Brassica napus; Wang et al, 2008;Harloff et al, 2012), displaying the highest number at approximately 40 mutations Mb -1 of diploid DNA (i.e. for each of the ancestral genomes of the polyploid).…”

Recent Biotechnological Advances in the Improvement of Cassava

“…TILLING involves induction of mutations in the plant genome using classical mutagenesis approaches followed by traditional or high throughput deep sequencing to identify the mutations in the gene of interest [77][78][79]. This technique has been used in allele discovery in different plant species [80][81][82][83]. EcoTILLING, which is an adaptation of the TILLING, is used in detecting rare single nucleotide polymorphism (SNPs) or small INDELs in target genes in natural populations [84].…”

Self-Incompatibility in the Brassicaceae

“…TILLING extends genomic resources, particularly in organisms lacking reverse-genetic tools, where mutants with a range of phenotypic severity are highly desirable. Since the inception of TILLING, this method has been applied to various organisms including Cucumis melo L. (González et al 2011 ), Solanum lycopersium (Minioa et al 2010 ), B. napus (Wang et al 2008 ;Harloff et al 2012 ), B. oleracea (Himelblau et al 2009 ), B. rapa (Stephenson et al 2010 ), Lotus japonicus (Perry et al 2009 ), Zea mays , Oryza sativa (Till et al 2007 ), Drosophila (Winkler et al 2005 ), and zebrafi sh (Wienholds et al 2003 ).…”