Direct access to millions of mutations by whole genome sequencing of an oilseed rape mutant population.

Induced mutations are an essential source of genetic variation in plant breeding. Ethyl methanesulfonate (EMS) mutagenesis has been frequently applied, and mutants have been detected by phenotypic or genotypic screening of large populations. In the present study, a rapeseed M2 population was derived from M1 parent cultivar 'Express' treated with EMS. Whole genomes were sequenced from fourfold (4×) pools of 1988 M2 plants representing 497 M2 families. Detected mutations were not evenly distributed and displayed distinct patterns across the 19 chromosomes with lower mutation rates towards the ends. Mutation frequencies ranged from 32/Mb to 48/Mb. On average, 284 442 single nucleotide polymorphisms (SNPs) per M2 DNA pool were found resulting from EMS mutagenesis. 55% of the SNPs were C → T and G → A transitions, characteristic for EMS induced ('canonical') mutations, whereas the remaining SNPs were 'non-canonical' transitions (15%) or transversions (30%). Additionally, we detected 88 725 high confidence insertions and deletions per pool. On average, each M2 plant carried 39 120 canonical mutations, corresponding to a frequency of one mutation per 23.6 kb. Approximately 82% of such mutations were located either 5 kb upstream or downstream (56%) of gene coding regions or within intergenic regions (26%). The remaining 18% were located within regions coding for genes. All mutations detected by whole genome sequencing could be verified by comparison with known mutations. Furthermore, all sequences are accessible via the online tool 'EMSBrassica' (http://www.emsbrassica.plantbreeding.uni-kiel.de), which enables direct identification of mutations in any target sequence. The sequence resource described here will further add value for functional gene studies in rapeseed breeding.

A rhomboid-like protease gene from an interspecies translocation confers resistance to cyst nematodes.

Plant-parasitic nematodes are severe pests in crop production worldwide. Chemical control of nematodes has been continuously reduced in recent decades owing to environmental and health concerns. Therefore, breeding nematode-resistant crops is an important aim if we are to secure harvests. The beet cyst nematode impairs root development and causes severe losses in sugar beet production. The only sources for resistance are distantly related wild species of the genus Patellifolia. Nematode resistance had been introduced into the beet genome via translocations from P. procumbens. We sequenced three translocations and identified the translocation breakpoints. By comparative sequence analysis of three translocations, we localized the resistance gene Hs4 within a region c. 230 kb in size. A candidate gene was characterized by CRISPR-Cas-mediated knockout and overexpression in susceptible roots. The gene encodes a rhomboid-like protease, which is predicted to be bound to the endoplasmic reticulum. Gene knockout resulted in complete loss of resistance, while overexpression caused resistance. The data confirm that the Hs4 gene alone protects against the pest. Thus, it constitutes a previously unknown mechanism of plants to combat parasitic nematodes. Its function in a nonrelated species suggests that the gene can confer resistance in crop species from different plant families.

Elevating seed oil content in a polyploid crop by induced mutations in SEED FATTY ACID REDUCER genes.

Plant-based oils are valuable agricultural products, and seed oil content (SOC) is the major yield component in oil crops. Increasing SOC has been successfully targeted through the selection and genetic modification of oil biosynthesis. The SOC in rapeseed declined during the seed maturation and eventually caused the final accumulated seed oil quantity. However, genes involved in oil degradation during seed maturity are not deeply studied so far. We performed a candidate gene association study using a worldwide collection of rapeseed germplasm. We identified SEED FATTY ACID REDUCER (SFAR) genes, which had a significant effect on SOC and fatty acid (FA) composition. SFAR genes belong to the GDSL lipases, and GDSL lipases have a broad range of functions in plants. After quantification of gene expression using RNA-seq and quantitative PCR, we used targeted (CRISPR-Cas mediated) and random (chemical) mutagenesis to modify turnover rates of seed oil in winter rapeseed. For the first time, we demonstrate significant increase of SOC in a crop after knocking out members of the BnSFAR4 and BnSFAR5 gene families without pleiotropic effects on seed germination, vigour and oil mobilization. Our results offer new perspectives for improving oil yield by targeted mutagenesis.

Identification of phytic acid mutants in oilseed rape (Brassica napus) by large-scale screening of mutant populations through amplicon sequencing.

Brassica napus (oilseed rape) is an important oil crop in temperate regions, which originated from hybridization of Brassica oleracea and Brassica rapa. Owing to its polyploidy, the functional study of single genes is cumbersome. Phytic acid is considered as an antinutritive compound, and we aimed to knock out the underlying synthesis and transporter genes to identify low phytic acid mutants. We implemented a high-throughput next-generation sequencing screening protocol for an ethylmethane sulfonate population of 7680 plants in six gene families (BnMIPS, BnMIK, Bn2-PGK, BnIPK1, BnIPK2, and BnMRP5) with two paralogues for each gene. A total of 1487 mutations were revealed, and the vast majority (96%) were confirmed by Sanger sequencing. Furthermore, the characterization of double mutants of Bn.2-PGK2 showed a significant reduction of phytic acid contents. We propose to use three-dimensional pooling combined with amplicon stacking and next-generation sequencing to identify mutations in polyploid oilseed rape in a fast and cost-effective manner for complex metabolic pathways. Furthermore, the mutants identified in Bn2-PGK2 might be a very valuable resource for industrial production of oilseed rape protein for human consumption.

CRISPR-Cas9 Targeted Mutagenesis Leads to Simultaneous Modification of Different Homoeologous Gene Copies in Polyploid Oilseed Rape (Brassica napus).

In polyploid species, altering a trait by random mutagenesis is highly inefficient due to gene redundancy. We have stably transformed tetraploid oilseed rape (Brassica napus) with a CRISPR-Cas9 construct targeting two ALCATRAZ (ALC) homoeologs. ALC is involved in valve margin development and, thus, contributes to seed shattering from mature fruits. Knocking out ALC would increase shatter resistance to avoid seed loss during mechanical harvest. We obtained a transgenic T1 plant with four alc mutant alleles by the use of a single target sequence. All mutations were stably inherited to the T2 progeny. The T2 generation was devoid of any wild-type alleles, proving that the underlying T1 was a nonchimeric double heterozygote. T-DNA and ALC loci were not linked, as indicated by random segregation in the T2 generation. Hence, we could select double mutants lacking the T-DNA already in the first offspring generation. However, whole-genome sequencing data revealed at least five independent insertions of vector backbone sequences. We did not detect any off-target effects in two genome regions homologous to the target sequence. The simultaneous alteration of multiple homoeologs by CRISPR-Cas9 mutagenesis without any background mutations will offer new opportunities for using mutant genotypes in rapeseed breeding.

The effect of INDEHISCENT point mutations on silique shatter resistance in oilseed rape (Brassica napus).

KEY MESSAGE: This study elucidates the influence of indehiscent mutations on rapeseed silique shatter resistance. A phenotype with enlarged replum-valve joint area and altered cell dimensions in the dehiscence zone is described. Silique shattering is a major factor reducing the yield stability of oilseed rape (Brassica napus). Attempts to improve shatter resistance often include the use of mutations in target genes identified from Arabidopsis (Arabidopsis thaliana). A variety of phenotyping methods assessing the level of shatter resistance were previously described. However, a comparative and comprehensive evaluation of the methods has not yet been undertaken. We verified the increase of shatter resistance in indehiscent double knock-down mutants obtained by TILLING with a systematic approach comparing three independent phenotyping methods. A positive correlation of silique length and shatter resistance was observed and accounted for in the analyses. Microscopic studies ruled out the influence of different lignification patterns. Instead, we propose a model to explain increased shattering resistance of indehiscent rapeseed mutants by altered cell shapes and sizes within the contact surfaces of replum and valves.

Reduced glucosinolate content in oilseed rape (Brassica napus L.) by random mutagenesis of BnMYB28 and BnCYP79F1 genes.

The presence of anti-nutritive compounds like glucosinolates (GSLs) in the rapeseed meal severely restricts its utilization as animal feed. Therefore, reducing the GSL content to < 18 µmol/g dry weight in the seeds is a major breeding target. While candidate genes involved in the biosynthesis of GSLs have been described in rapeseed, comprehensive functional analyses are missing. By knocking out the aliphatic GSL biosynthesis genes BnMYB28 and BnCYP79F1 encoding an R2R3 MYB transcription factor and a cytochrome P450 enzyme, respectively, we aimed to reduce the seed GSL content in rapeseed. After expression analyses on single paralogs, we used an ethyl methanesulfonate (EMS) treated population of the inbred winter rapeseed 'Express617' to detect functional mutations in the two gene families. Our results provide the first functional analysis by knock-out for the two GSL biosynthesis genes in winter rapeseed. We demonstrate that independent knock-out mutants of the two genes possessed significantly reduced seed aliphatic GSLs, primarily progoitrin. Compared to the wildtype Express617 control plants (36.3 µmol/g DW), progoitrin levels were decreased by 55.3% and 32.4% in functional mutants of BnMYB28 (16.20 µmol/g DW) and BnCYP79F1 (24.5 µmol/g DW), respectively. Our study provides a strong basis for breeding rapeseed with improved meal quality in the future.

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

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.

Genomic background selection to reduce the mutation load after random mutagenesis.

Random mutagenesis is a standard procedure to increase allelic variation in a crop species, especially in countries where the use of genetically modified crops is limited due to legal constraints. The chemical mutagen EMS is used in many species to induce random mutations throughout the genome with high mutation density. The major drawback for functional analysis is a high background mutation load in a single plant that must be eliminated by subsequent backcrossing, a time and resource-intensive activity. Here, we demonstrate that genomic background selection combined with marker-assisted selection is an efficient way to select individuals with reduced background mutations within a short period. We identified BC1 plants with a significantly higher share of the recurrent parent genome, thus saving one backcross generation. Furthermore, spring rapeseed as the recurrent parent in a backcrossing program could accelerate breeding by reducing the generation cycle. Our study depicts the potential for reducing the background mutation load while accelerating the generation cycle in EMS-induced winter oilseed rape populations by integrating genomic background selection.