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Rapeseed is a staple oilseed crop of enormous economic importance worldwide. The oil derived from crushing harvested seed is a major provider of calorific value to the human food chain, with variations in fatty acid profile including combinations of erucic acid, oleic and linolenic acid that are of industrial value as oils, lubricants, surfactants and high-value plastics (Metzger & Bornscheuer, 2006). There is an increasing need to focus rapeseed crop improvement on optimizing performance characteristics such as fatty acid composition, seed oil content and yield. More generally, crop genetic improvement needs to expand the available variation present within the existing gene pool in order to make significant advances in key traits. For many years, mutant populations generated with chemical mutagens such as ethylmethanesulfonate (EMS) and N-ethyl-N-nitrosourea (ENU) have been used to induce valuable variations in crop genomes (Konzak et al., 1976). However, whilst precursory construction of an EMS population for B. napus has recently been reported (Wu et al., 2007a), the resultant mutant populations have not been large enough to generate sufficient variation, nor has an efficient method for screening such variation been developed. There remains a requirement for development of large mutant populations, along with suitable high-efficiency screening methods suitable for rapeseed improvement.
Functional characterization of genes is an important component of plant genomic research and is increasingly informing crop improvement. Loss of function mutants are of particular interest since they provide valuable evidence for the role of specific genes in regulatory, developmental, biochemical and metabolic networks. Several general methods such as insertional mutagenesis and RNA interference have been used to obtain reduction-of-function or knockout mutations, and used successfully in model plants such as Arabidopsis (Arabidopsis thaliana) (Long & Coupland, 1998) and crops such as rice (Waterhouse et al., 1998). However, there are particular challenges in applying these approaches to rapeseed research and crop improvement, primarily because of either reliance on Agrobacterium T-DNA vectors for transmission or lack of availability of endogenous tagging systems. Transgenesis is inefficient in B. napus, and endogenous tagging is not accepted by regulatory bodies and consumers in most countries. TILLING (targeting-induced local lesions in genomes) is a more recent reverse genetic approach that has successfully been applied to both model plants such as Arabidopsis (McCallum et al., 2000) and crops such as rice (Till et al., 2007), maize (Till et al., 2004b) and wheat (Slade et al., 2005). TILLING is based on screening populations of plants that have been treated with traditional chemical mutagens that cause point mutations, followed by discovery in genes of interest using a very sensitive detection method. The main advantage is the ability to accumulate an allelic series of mutants with a range of modified functions, from wild-type to almost loss of function (Slade et al., 2005). The original TILLING method involves seeds that are mutagenized by treatment with EMS, which primarily introduces G/C to A/T transitions. The resulting M1 plants are self-fertilized, and M2 individuals are used to prepare DNA samples for mutational screening, whilst an inventory of their seeds is established for future and downstream research (Henikoff et al., 2004). The mutational screening relies on an enzyme which cleaves singleton DNA mismatches, as described by Oleykowski et al. (1998). Since TILLING relies on use of chemical mutagens that induce genome lesions randomly, it is relatively easy to construct a sufficiently large mutant population that has a high probability of containing mutations in all gene loci. Moreover, TILLING is applicable to targeting induction of mutations in genes of interest within the genome of almost any plant species (Till et al., 2006). Species with duplicated genomes are more tolerant of induced mutation as a result of the functional redundancy conferred by paralogous sets of genes (Slade et al., 2005). Since the rapeseed genome is particularly complex, it is likely to be quite tolerant of mutations induced by EMS. These considerations suggest that TILLING is better suited to rapeseed genomic research and trait improvement than other methods reported to date.
Although TILLING is potentially very powerful, there are some difficulties in applying the method to rapeseed. The most challenging issues arise from the need to develop gene locus-specific primer pairs within the complex rapeseed genome. B. napus (AACC, 2n = 4x = 38; c. 1150 Mbp) is an amphidiploid species originating from a spontaneous hybridization of Brassica rapa (AA, 2n = 2x = 20) and Brassica oleracea (CC, 2n = 2x = 18) (U, 1935), where the oginating diploids have been retained essentially intact (Li et al., 2005). Moreover, the Arabidopsis, B. rapa and B. oleracea genomes appear to have arisen from a common hexaploid ancestor c. 14.5–20.4 million yr ago (Lysak et al., 2005). Sequential duplication events within ancestral genomes have resulted in regions that are represented as single copies within Arabidopsis, being present as three segmental copies within the B. rapa and B. oleracea genomes. This compounds the complexity of the B. napus A and C genomes (Mayer et al., 2001; Park et al., 2005; Yang et al., 2006). From recent comparative genomic studies (Park et al., 2005; Town et al., 2006), it is possible to infer the presence of six paralogous regions in B. napus, with random gene loss resulting in, on average, four intact gene copies, with additional genomic rearrangements contributing further pseudogene fragments. Among the intact paralogous genes, some are able to be distinguished based on differences such as sequence lengths and intron composition. In such cases, it is relatively trivial to obtain gene-specific primer pairs based on sequence polymorphisms. Slade et al. (2005) have reported successful screening using a TILLING approach for this class of multi-paralogous gene, in both hexaploid and tetraploid wheat. For multi-paralogous genes that differ by only a few nucleotides among different paralogues, it is very difficult to design gene-specific primer pairs to screen for mutations, and also difficult to identify mutations reliably in distinct paralogues.
We were motivated to overcome the difficulties associated with application of TILLING in polyploid species, owing to the potential benefits for functional gene analysis in the closest crop relatives to the model Arabidopsis, and specifically for potential application in genetic crop improvement of B. napus. We therefore tested our system by focusing on screening using TILLING for mutations in the gene FAE1 (fatty acid elongase1), which had previously been shown by Southern blotting analysis to be present with at least two very similar paralogous copies within the B. napus genome (Barret et al., 1998). FAE1 is the key gene in seed erucic acid biosynthesis in rapeseed (Roscoe et al., 2001), and was originally cloned in Arabidopsis by directed transposon tagging with the maize element Activator (Ac) (James et al., 1995). The product of the gene is a condensing enzyme that extends the chain length of fatty acids from C18 to C20 and C22 (Lassner et al., 1996). More recently, the restriction enzyme AvrII has been used to discriminate between the two genes present in the A and C genomes of the two B. napus cultivars Zhongyou 821 and Zhongshuang No. 9, which possess high (HEA) and low seed erucic acid (LEA) content respectively. The B. napus FAE1 gene copies are intronless, with 98% sequence similarity between the paralogues (Wu et al., 2007b). A reference doubled haploid population (TNDH) developed by our laboratory from Ningyou7 (a Chinese semi-winter cultivar) and Tapidor (a European winter cultivar) was used to detect functional QTL for seed erucic acid content, with evidence from four independent field environments (Qiu et al., 2006). Two major seed erucic acid content QTL were detected, one located on linkage group/chromosome A8 (A genome) and the other on C3 (C genome), accounting for c. 71% of the genetic variation. Taken together, such evidence indicates there are likely to be two intact highly identical functional FAE1 gene paralogues in B. napus, one in each of the A and C genomes.
Here we report development of a TILLING platform for B. napus, involving generation of two structured EMS mutagenized populations derived from homozygous cv. Ningyou7 (one parent of the TNDH mapping population). In order to demonstrate the utility of these populations for gene discovery, a forward genetic screen was carried out, resulting in a large number of novel phenotypes. Reverse genetics was tested within a potentially multi-paralogous gene system. Comparative genomic sequence analysis involved screening of a B. napus cv. Tapidor (the other parent of the TNDH population) BAC library to obtain a more precise estimate of paralogous FAE1 gene copy. BAC-derived locus-specific sequences were then mapped to the A8 and C3 chromosomes within the TNDH mapping population (Qiu et al., 2006). Reverse genetic screening for FAE1 mutants by TILLING resulted in identification of 19 mutations from 1344 M2 plants, of which three had altered function as revealed by marked changes in the accumulation of erucic acid.
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We have successfully constructed two rapeseed mutant populations suitable for both forward and reverse genetic detection methods. In the forward genetic screening of mutants, a large number of novel mutated phenotypes were identified, and many of these represent a valuable genetic resource affecting crop traits such as male sterility, reduced stature and plants with increased oil content. In the reverse genetic TILLING of mutants, we screened 1344 M2 plants representing 1344 distinct lines (i.e. siblings were not screened), and detected a total of 19 FAE1 mutations, three of which had a change of function. In addition, in silico Ecotilling analysis demonstrated a clear distinction between sites of natural and induced SNPs. This is very encouraging for the application of TILLING within rapeseed crop genetic improvement as a highly efficient method for generating new genetic resources for breeding. We are also able to calculate that if a 1kb gene fragment were to be screened within the 0.6% EMS population, with a single plant selected from each line, then c. 100 mutations would be detected following a complete screening of the 0.6% EMS population (a total of 3926 lines). This suggests that the population carries a high mutation load, and is a valuable resource for breeding and research. However, for practical application of induced mutation within crop improvement, it is necessary to adopt a process that reduces the background caused by the mutations at unlinked loci. Such a schema should involve backcrossing to the WT or target elite cultivar, using molecular marker and phenotypic tracking for recurrent selection of the mutant locus. It is also important to eliminate epistatic effects and carry out controlled self-pollination of the candidate mutant plant before segregation analysis of the mutation identified and modified phenotype. Such information can be substantiated by analysis of transcript profile and in vitro protein expression for the target gene (Heckmann et al., 2006). We conclude that the populations generated are efficient for exploring novel sources of variation using both forward and reverse genetics approaches to screening mutants.
Although we were able to demonstrate development of an efficient new research platform through discovery of three novel induced functional low erucic acid mutant alleles, it is important to determine the optimal mutagen dosage for B. napus. The EMS concentration is a key factor in developing such populations, with higher concentrations leading to higher mutation frequencies. However, any higher mutation load achieved is offset by the effect of EMS on reducing seed set, viability and germination (Greene et al., 2003; Kim et al., 2006). For B. napus there have been no prior reports of an optimal mutagen dosage to guide development of a forward genetics or reverse genetics TILLING population. Here, we found that at EMS concentration of 0.6% yielded a calculated mutation density of one per 41.5 kb in each plant, compared with almost a third of that in the population treated at 0.3% EMS. These estimates may not accurately reflect the true mutation density of the two EMS populations, as it was not possible to screen the first 50 bp in a TILLING gel. However, the mutation frequencies within our two B. napus mutants were within the range of those reported elsewhere for monocot and dicot species, with one mutation per 24 kb reported in hexaploid wheat, one per 40 kb in tetraploid wheat (Slade et al., 2005), 300 kb in rice (Till et al., 2007), 100 kb in barley (Hordeum vulgare L.) (Caldwell et al., 2004), and 170 kb in Arabidopsis (Greene et al., 2003). In the forward genetic investigation, 23.25% M2 plants displayed observable mutant phenotypes in the 0.6% EMS population, compared with 12.98% in the 0.3% EMS population. These estimates may be low and not reflect the true mutant load, as some plants may carry more than one mutant phenotype. Moreover, when M2 plants were grown in field conditions, we found that the performance within the 0.3% EMS population was stronger than the 0.6% population. At concentrations of EMS above 0.6% we found that M1-treated Ningyou7 plants set almost no seed. Therefore, taking into account the mutation frequency and the performance of mutagenized plants in the field, we consider an EMS concentration between 0.3 and 0.6% to be optimal for construction of mutant populations in B. napus and probably also in other amphidploids of the Brassica triangle of U, for example B. juncea (2n = 36; AB genomes) and B. carinata (2n = 34; BC genomes).
Since the original report of TILLING in 2000, the technique has successfully been applied to many crops, notably in rice and wheat (Slade et al., 2005; Till et al., 2007). However, we are not aware of other reports to date of its application within any Brassica species. The main challenges arise from the complex genome of B. napus in which most of genes are multi-paralogous. This reduces the ease with which gene locus-specific primers can be designed, and also limits the ability to locate the mutations unequivocally to specific paralogous copies. Here we have successfully demonstrated that TILLING technology can also be applied in polyploid crops such as B. napus that comprise multiple closely related complex segmentally duplicated genomes. We have been able to allocate target genes within the B. napus genome into two classes. The first class comprises those genes which occur as multi-paralogous copies distinguished by clear sequence differences; and the second class includes multi-paralogous genes that possess only minor sequence differences between different copies. It is a relatively trivial exercise to design gene-specific primers for the first class. However, for the second class we have developed a novel strategy that is based on using a primer pair that is designed to amplify all paralogous copies of the gene simultaneously, albeit from individual rather than pooled DNA samples. The procedure is based on single plant TILLING, to identify mutations in situations where two or more very similar paralogous gene copies exist in a genome. We made use of existing SNPs as a positive control, and demonstrated the ability to distinguish novel mutations, based on use of primer pairs designed to amplify both FAE1 paralogues simultaneously. Slade et al. (2005) reported that using a primer pair that could amplify at least two gene copies would be less efficient. However, in our experiments we found that these types of primers provide a powerful approach to mutant detection. The precondition for successful screening of mutations using this approach is that the target fragment should be highly conserved amongst the different paralogues. In the case of FAE1, we found just five SNPs between the two paralogues in the 426 bp target region. These five SNPs were also detected as background signals in the Li-Cor gel images, but did not detract from our ability to detect novel induced mutations (Fig. 5). Within the TILLING gel, the cleaved bands that were present in all lanes with positions differing between 700 and 800 nm are predicted to arise from the SNPs that exist between the two FAE1 paralogues. However, the cleaved bands that were detected in only one lane within the 700 nm image, together with the corresponding cleaved band in the 800 nm image, indicated discovery of a novel mutation. Therefore, we can conclude that with a modified TILLING primer design we are able to screen for mutations in any gene in the EMS population of B. napus, even though this consists of very complex duplicated genomes. This modified method may improve and widen the application of TILLING technology for crop genomic research and improvement.
For rapeseed, the ability to achieve zero or low seed erucic acid (LEA) content has long been a major breeding objective, since erucic acid has been regarded as an antinutritional component in seed oil (Vles et al., 1978). In the 1960s, the first variant with LEA content was found in the animal feed rape cultivar ‘Liho’, with the first LEA rapeseed ‘Oro’ being derived following introduction of the LEA variation from ‘Liho’ (Downey & Craig, 1964). Subsequently, almost all LEA rapeseed cultivars have carried the LEA gene source from ‘Liho’ or ‘Oro’, and so this single genetic resource has probably contributed considerably to an inbreeding effect and associated genetic erosion through linkage drag in rapeseed breeding programmes (Sharpe & Lydiate, 2003). Although breeders have successfully developed many LEA rapeseed cultivars, new sources of LEA are still required to modulate the relevant pathways within the seed oil fatty acid synthesis and modification pathways (Barker et al., 2007). Wu et al. (2008) reported discovery of a four base deletion in the FAE1 gene that resulted in LEA content within six Chinese rapeseed cultivars. However, this new source of LEA may still contain ‘Liho’ or ‘Oro’ genetic background, as so many LEA rapeseed cultivars have derived from material including the parent ‘Liho’ or ‘Oro’.
In the work reported here, the WT genotype of our EMS mutant population was Ningyou7, which is a high erucic acid (HEA) content cultivar in China, distinct from many other modern rapeseed cultivars (Meng et al., 1996). This cultivar has little or no allelic genetic background in common with ‘Liho’ or ‘Oro’, and so the three novel LEA content rapeseed alleles induced by TILLING in this research represent a unique genetic resource. The gene locus-specific primer analysis confirmed that the mutant a8fae1-599 and c3fae1-400 were separately associated with bna8.fae1and bnC3.fae1, respectively. Seed erucic acid content in rapeseed has been shown to be primarily controlled by these two genes (Barret et al., 1998; Qiu et al., 2006; Wu, Y et al., 2007; Wu et al., 2008). Thus a cross between these two mutants would be expected to generate a new LEA cultivar within what is otherwise a HEA background. In conclusion, the three new LEA mutants, L080-1, N004-1 and L297-1, that were discovered by TILLING could be used as a new genetic resource for LEA rapeseed breeding without collateral inbreeding effect and genetic erosion.
In conclusion, we have reported the application of TILLING to the generation and identification of a novel LEA genetic resource for rapeseed improvement. Although TILLING was reported initially as a reverse genetic approach for Arabidopsis genomic research (McCallum et al., 2000), it will increasingly be very powerful to combine these two approaches to crop functional genomics and genetic improvement. Alongside this work focused on TILLING, QTL for rapeseed seed oil and erucic acid content have been detected in the TNDH genetic mapping population, followed by development of near-isogenic lines (NILs) to resolve these two regions (F. Tian & J. Meng, unpublished). Fine mapping of the two QTLs has resulted in FAE1 and several other genes being proposed as major candidates contributing to seed oil content (unpublished data). To validate gene function for these candidates, TILLING will now be the method of choice in B. napus, rather than conventional transgene knock-down approaches. Knockout of candidate gene function via TILLING overcomes complications associated with RNAi or anti-sense in a multi-paralogous system, and has the advantage of greatly reducing the time required to carry out such research. Taken together, we propose that the combination of QTL resolution making use of comparative genomic information and TILLING of candidates provides a powerful new approach to functional crop genomics, with the advantages of both forward and reverse genetics, and that this will accelerate genetic crop improvement.