Overcoming polyploidy pitfalls: a user guide for effective SNP conversion into KASP markers in wheat.

KEY MESSAGE: Conversion of SNP chip assays into locus-specific KASP markers requires adapted strategies in polyploid species with high genome homeology. Procedures are exemplified by QTL-associated SNPs in hexaploid wheat. Kompetitive allele-specific PCR (KASP) markers are commonly used in marker-assisted commercial plant breeding due to their cost-effectiveness and throughput for high sample volumes. However, conversion of trait-linked SNP markers from array-based SNP detection technologies into KASP markers is particularly challenging in polyploid crop species, due to the presence of highly similar homeologous and paralogous genome sequences. We evaluated strategies and identified key requirements for successful conversion of Illumina Infinium assays from the wheat 90 K SNP array into robust locus-specific KASP markers. Numerous examples showed that commonly used software for semiautomated KASP primer design frequently fails to achieve locus-specificity of KASP assays in wheat. Instead, alignment of SNP probes with multiple reference genomes and Sanger sequencing of relevant genotypes, followed by visual KASP primer placement, was critical for locus-specificity. To identify KASP assays resulting in false calling of heterozygous individuals, validation of KASP assays using extended reference genotype sets including heterozygous genotypes is strongly advised for polyploid crop species. Applying this strategy, we developed highly reproducible, stable KASP assays that are predictive for root biomass QTL haplotypes from highly homoeologous wheat chromosome regions. Due to their locus-specificity, these assays predicted root biomass considerably better than the original trait-associated markers from the Illumina array.

Not a load of rubbish: simulated field trials in large-scale containers.

Assessment of yield performance under fluctuating environmental conditions is a major aim of crop breeders. Unfortunately, results from controlled-environment evaluations of complex agronomic traits rarely translate to field performance. A major cause is that crops grown over their complete lifecycle in a greenhouse or growth chamber are generally constricted in their root growth, which influences their response to important abiotic constraints like water or nutrient availability. To overcome this poor transferability, we established a plant growth system comprising large refuse containers (120 L 'wheelie bins') that allow detailed phenotyping of small field-crop populations under semi-controlled growth conditions. Diverse winter oilseed rape cultivars were grown at field densities throughout the crop lifecycle, in different experiments over 2 years, to compare seed yields from individual containers to plot yields from multi-environment field trials. We found that we were able to predict yields in the field with high accuracy from container-grown plants. The container system proved suitable for detailed studies of stress response physiology and performance in pre-breeding populations. Investment in automated large-container systems may help breeders improve field transferability of greenhouse experiments, enabling screening of pre-breeding materials for abiotic stress response traits with a positive influence on yield.

Oilseed rape: learning about ancient and recent polyploid evolution from a recent crop species.

Oilseed rape (Brassica napus) is one of our youngest crop species, arising several times under cultivation in the last few thousand years and completely unknown in the wild. Oilseed rape originated from hybridisation events between progenitor diploid species B. rapa and B. oleracea, both important vegetable species. The diploid progenitors are also ancient polyploids, with remnants of two previous polyploidisation events evident in the triplicated genome structure. This history of polyploid evolution and human agricultural selection makes B. napus an excellent model with which to investigate processes of genomic evolution and selection in polyploid crops. The ease of de novo interspecific hybridisation, responsiveness to tissue culture, and the close relationship of oilseed rape to the model plant Arabidopsis thaliana, coupled with the recent availability of reference genome sequences and suites of molecular cytogenetic and high-throughput genotyping tools, allow detailed dissection of genetic, genomic and phenotypic interactions in this crop. In this review we discuss the past and present uses of B. napus as a model for polyploid speciation and evolution in crop species, along with current and developing analysis tools and resources. We further outline unanswered questions that may now be tractable to investigation.

Regional association analysis delineates a sequenced chromosome region influencing antinutritive seed meal compounds in oilseed rape.

This study describes the use of regional association analyses to delineate a sequenced region of a Brassica napus chromosome with a significant effect on antinutritive seed meal compounds in oilseed rape. A major quantitative trait locus (QTL) influencing seed colour, fibre content, and phenolic compounds was mapped to the same position on B. napus chromosome A9 in biparental mapping populations from two different yellow-seeded × black-seeded B. napus crosses. Sequences of markers spanning the QTL region identified synteny to a sequence contig from the corresponding chromosome A9 in Brassica rapa. Remapping of sequence-derived markers originating from the B. rapa sequence contig confirmed their position within the QTL. One of these markers also mapped to a seed colour and fibre QTL on the same chromosome in a black-seeded × black-seeded B. napus cross. Consequently, regional association analysis was performed in a genetically diverse panel of dark-seeded, winter-type oilseed rape accessions. For this we used closely spaced simple sequence repeat (SSR) markers spanning the sequence contig covering the QTL region. Correction for population structure was performed using a set of genome-wide SSR markers. The identification of QTL-derived markers with significant associations to seed colour, fibre content, and phenolic compounds in the association panel enabled the identification of positional and functional candidate genes for B. napus seed meal quality within a small segment of the B. rapa genome sequence.

Comparative mapping of quantitative trait loci involved in heterosis for seedling and yield traits in oilseed rape (Brassica napus L.).

Little is known about the genetic control of heterosis in the complex polyploid crop species oilseed rape (Brassica napus L.). In this study, two large doubled-haploid (DH) mapping populations and two corresponding sets of backcrossed test hybrids (THs) were analysed in controlled greenhouse experiments and extensive field trials for seedling biomass and yield performance traits, respectively. Genetic maps from the two populations, aligned with the help of common simple sequence repeat markers, were used to localise and compare quantitative trait loci (QTL) related to the expression of heterosis for seedling developmental traits, plant height at flowering, thousand seed mass, seeds per silique, siliques per unit area and seed yield. QTL were mapped using data from the respective DH populations, their corresponding TH populations and from mid-parent heterosis (MPH) data, allowing additive and dominance effects along with digenic epistatic interactions to be estimated. A number of genome regions containing numerous heterosis-related QTL involved in different traits and at different developmental stages were identified at corresponding map positions in the two populations. The co-localisation of per se QTL from the DH population datasets with heterosis-related QTL from the MPH data could indicate regulatory loci that may also contribute to fixed heterosis in the highly duplicated B. napus genome. Given the key role of epistatic interactions in the expression of heterosis in oilseed rape, these QTL hotspots might harbour genes involved in regulation of heterosis (including fixed heterosis) for different traits throughout the plant life cycle, including a significant overall influence on heterosis for seed yield.

Chromosomal distribution of 18S-25S rDNA in four Lupinus species visualized by fluorescence in situ hybridization.

The number and position of 18S-25S rDNA sites in 4 selected Lupinus species are reported for the first time. L. atlanticus, L. subcarnosus and L. paniculatus had two rDNA loci, while L. albus exhibited only one loci. Among these 4 species, all of them exhibited one large pair of strong signals that extends from the short arm to a NOR on a chromosome satellite. L. atlanticus, L. subcarnosus, L. paniculatus had one more locus of 18-25S rDNA, but a pair of weak hybridization signals were observed in L. paniculatus when 18S-25S rDNA was used as probe. The results are discussed in terms of the evolutionary relationships among these species.

Identification of quantitative trait loci for resistance against Verticillium longisporum in oilseed rape (Brassica napus).

Verticillium longisporum is one of the major pathogens of oilseed rape (Brassica napus; genome AACC, 2n = 38) in Europe. Current European cultivars possess only a low level of resistance against V. longisporum, meaning that heavy infection can cause major yield losses. The aim of this study was to identify quantitative trait loci (QTL) for resistance against V. longisporum as a starting point for marker-assisted breeding of resistant cultivars. Resistance QTL were localized in a segregating oilseed rape population of 163 doubled haploid (DH) lines derived by microspore culture from the F1 of a cross between two B. napus breeding lines, one of which exhibited V. longisporum resistance derived by pedigree selection from a resynthesized B. napus genotype. A genetic map was constructed comprising 165 restriction fragment length polymorphism, 94 amplified fragment length polymorphism and 45 simple sequence repeats (SSR) markers covering a total of 1,739 cM on 19 linkage groups. Seedlings of the DH lines and parents were inoculated with V. longisporum isolates in four greenhouse experiments performed in Sweden during autumn 1999. In three of the experiments the DH lines were inoculated with a mixture of five isolates, while in the fourth experiment only one of the isolates was used. The intention was to simulate four different environments with variable disease pressure, while still maintaining uniform conditions in each environment to enable reliable disease scoring. The disease index (DI) was calculated by scoring symptoms on a total of 21 inoculated plants per line in comparison to 21 noninoculated plants per line. Using the composite interval mapping procedure a total of four different chromosome regions could be identified that showed significant QTL for resistance in more than one environment. Two major QTL regions were identified on the C-genome linkage groups N14 and N15, respectively; each of these QTL consistently exhibited significant effects on resistance in multiple environments. The presence of flanking markers for the respective QTL was associated with a significant reduction in DI in the inoculated DH lines.

Association of gene-linked SSR markers to seed glucosinolate content in oilseed rape (Brassica napus ssp. napus).

Breeding of oilseed rape (Brassica napus ssp. napus) has evoked a strong bottleneck selection towards double-low (00) seed quality with zero erucic acid and low seed glucosinolate content. The resulting reduction of genetic variability in elite 00-quality oilseed rape is particularly relevant with regard to the development of genetically diverse heterotic pools for hybrid breeding. In contrast, B. napus genotypes containing high levels of erucic acid and seed glucosinolates (++ quality) represent a comparatively genetically divergent source of germplasm. Seed glucosinolate content is a complex quantitative trait, however, meaning that the introgression of novel germplasm from this gene pool requires recurrent backcrossing to avoid linkage drag for high glucosinolate content. Molecular markers for key low-glucosinolate alleles could potentially improve the selection process. The aim of this study was to identify potentially gene-linked markers for important seed glucosinolate loci via structure-based allele-trait association studies in genetically diverse B. napus genotypes. The analyses included a set of new simple-sequence repeat (SSR) markers whose orthologs in Arabidopsis thaliana are physically closely linked to promising candidate genes for glucosinolate biosynthesis. We found evidence that four genes involved in the biosynthesis of indole, aliphatic and aromatic glucosinolates might be associated with known quantitative trait loci for total seed glucosinolate content in B. napus. Markers linked to homoeologous loci of these genes in the paleopolyploid B. napus genome were found to be associated with a significant effect on the seed glucosinolate content. This example shows the potential of Arabidopsis-Brassica comparative genome analysis for synteny-based identification of gene-linked SSR markers that can potentially be used in marker-assisted selection for an important trait in oilseed rape.

Broadening the Genetic Basis of Verticillium longisporum Resistance in Brassica napus by Interspecific Hybridization.

ABSTRACT Verticillium wilt caused by the vascular fungal pathogen Verticillium longisporum is one of the most important pathogens of oilseed rape (Brassica napus sp. oleifera) in northern Europe. Because production of this major oilseed crop is expanding rapidly and no approved fungicides are available for V. longisporum, long-term control of the disease can only be achieved with cultivars carrying effective quantitative resistance. However, very little resistance to V. longisporum is available within the gene pool of oilseed rape, meaning that interspecific gene transfer from related species is the only possibility for broadening levels of resistance in current varieties. The amphidiploid species B. napus can be resynthesized by crossing the two progenitor species Brassica oleracea and Brassica rapa, hence resistant accessions of these two diploid species can be used as resistance donors. In this study a total of 43 potential B. rapa and B. oleracea resistance donors were tested with regard to their reaction to a mixture of two aggressive V. longisporum isolates, and resistances from diverse lines were combined by embryo rescue-assisted interspecific hybridization in resynthesized rapeseed lines. Progenies from crosses of the two B. rapa gene bank accessions 13444 and 56515 to the B. oleracea gene bank accessions BRA1008, CGN14044, 8207, BRA1398, and 7518 showed a broad spectrum of resistance in pathogenicity tests. Of 45 tested resynthesized lines, 41 lines exhibited a significantly higher level of resistance than the moderately V. longisporum-tolerant oilseed rape cultivar Express. These lines represent a promising basis for the combination of different resistance resources in new varieties.

Genetic mapping of agronomic traits in false flax (Camelina sativa subsp. sativa).

The crucifer oilseed plant false flax (Camelina sativa subsp. sativa) possesses numerous valuable agronomic attributes that make it attractive as an alternative spring-sown crop for tight crop rotations. The oil of false flax is particularly rich in polyunsaturated C18-fatty acids, making it a valuable renewable feedstock for the oleochemical industry. Because of the minimal interest in the crop throughout the 20th century, breeding efforts have been limited. In this study, a genetic map for C. sativa was constructed, using amplified fragment length polymorphism (AFLP) markers, in a population of recombinant inbred lines that were developed, through single-seed descent, from a cross between 'Lindo' and 'Licalla', 2 phenotypically distinct parental varieties. Three Brassica simple sequence repeat (SSR) markers were also integrated into the map, and 1 of these shows linkage to oil-content loci in both C. sativa and Brassica napus. Fifty-five other SSR primer combinations showed monomorphic amplification products, indicating partial genome homoeology with the Brassica species. Using data from field trials with different fertilization treatments (0 and 80 kg N/ha) at multiple locations over 3 years, the map was used to localize quantitative trait loci (QTLs) for seed yield, oil content, 1000-seed mass, and plant height. Some yield QTLs were found only with the N0 treatment, and might represent loci contributing to the competitiveness of false flax in low-nutrient soils. The results represent a starting point for future marker-assisted breeding.

Behaviour of Sinapis alba chromosomes in a Brassica napus background revealed by genomic in-situ hybridization.

Genomic in-situ hybridization (GISH) was applied to study the behaviour of addition chromosomes in first and second backcross (BC) progenies of hybrids between Brassica napus ssp. napus L. (AACC, 2n = 38) and Sinapis alba L. (SS, 2n = 24) produced by electrofusion. With GISH using genomic DNA of S. alba was used as probe it was possible to clearly distinguish both of the parental genomes and effectively monitor the fate of S. alba chromosomes in the BC(1) and BC(2) progenies. GISH analysis confirmed the sesquidiploid genome composition (AACCS) of the BC(1) progenies, which contained 38 chromosomes from B. napus and 12 chromosomes from S. alba. Genome painting in the pollen mother cells (PMCs) of the BC(1) plants revealed intergenomic association between B. napus and S. alba chromosomes, whereby a maximum of 4 trivalents between AC and S chromosomes were identified at metaphase I. In the BC(2) progenies, aneuploids with different numbers of additional chromosomes from S. alba, ranging from 1 to 7, were confirmed. Three putative monosomic alien addition lines were characterized, and the results are discussed with respect to the potential for intergenomic chromosome recombination.

Cytogenetic characterization and fae1 gene variation in progenies from asymmetric somatic hybrids between Brassica napus and Crambe abyssinica.

Sexual progenies of asymmetric somatic hybrids between Brassica napus and Crambe abyssinica were analyzed with respect to chromosomal behavior, fae1 gene introgression, fertility, and fatty-acid composition of the seed. Among 24 progeny plants investigated, 11 plants had 38 chromosomes and were characterized by the occurrence of normal meiosis with 19 bivalents. The other 13 plants had more than 38 chromosomes, constituting a complete chromosomal set from B. napus plus different numbers of additional chromosomes from C. abyssinica. The chromosomes of B. napus and C. abyssinica origin could be clearly discriminated by genomic in situ hybridization (GISH) in mitotic and meiotic cells. Furthermore, meiotic GISH enabled identification of intergenomic chromatin bridges and of asynchrony between the B. napus and C. abyssinca meiotic cycles. Lagging, bridging and late disjunction of univalents derived from C. abyssinica were observed. Analysis of cleaved amplified polymorphic sequence (CAPS) markers derived from the fae1 gene showed novel patterns different from the B. napus recipient in some hybrid offspring. Most of the progeny plants had a high pollen fertility and seed set, and some contained significantly greater amounts of seed erucic acid than the B. napus parent. This study demonstrates that a part of the C. abyssinica genome can be transferred into B. napus via asymmetric hybridization and maintained in sexual progenies of the hybrids. Furthermore, it confirms that UV irradiation improves the fertility of the hybrid and of its sexual progeny via chromosomal elimination and facilitates the introgression of exotic genetic material into crop species.

Identifying the chromosomes of the A- and C-genome diploid Brassica species B. rapa (syn. campestris) and B. oleracea in their amphidiploid B. napus.

Oilseed rape ( Brassica napus L.) is an amphidiploid species that originated from a spontaneous hybridisation of Brassica rapa L. (syn. campestris) and Brassica oleracea L., and contains the complete diploid chromosome sets of both parental genomes. The metaphase chromosomes of the highly homoeologous A genome of B. rapa and the C genome of B. oleracea cannot be reliably distinguished in B. napus because of their morphological similarity. Fluorescence in situ hybridisation (FISH) with 5S and 25S ribosomal DNA probes to prometaphase chromosomes, in combination with DAPI staining, allows more dependable identification of Brassica chromosomes. By comparing rDNA hybridisation and DAPI staining patterns from B. rapa and B. oleracea prometaphase chromosomes with those from B. napus, we were able to identify the putative homologues of B. napus chromosomes in the diploid chromosome sets of B. rapa and B. oleracea, respectively. In some cases, differences were observed between the rDNA hybridisation patterns of chromosomes in the diploid species and their putative homologue in B. napus, indicating locus losses or alterations in rDNA copy number. The ability to reliably identify A and C genome chromosomes in B. napus is discussed with respect to evolutionary and breeding aspects.

Chromosomal localization and characterization of rDNA loci in the Brassica A and C genomes.

Using fluorescence in situ hybridization, we located ribosomal DNA loci on prometaphase chromosomes of the diploid species Brassica rapa and Brassica oleracea and their amphidiploid Brassica napus. Based on comparisons of chromosome morphology and hybridization patterns, we characterized the individual B. napus rDNA loci according to their presumed origins in the Brassica A and C genomes. As reported in other studies, the sum of rDNA loci observed on B. rapa (AA genome) and B. oleracea (CC genome) chromosomes was one greater than the total number of loci seen in their amphidiploid B. napus (AACC). Evidence is presented that this reduction in B. napus rDNA locus number results from the loss of the smallest A genome rDNA site in the amphidiploid.