Heterotic patterns in rapeseed (<Emphasis Type="Italic">Brassica napus</Emphasis> L.): I. Crosses between spring and Chinese semi-winter lines

Chinese semi-winter rapeseed is genetically diverse from Canadian and European spring rapeseed. This study was conducted to evaluate the potential of semi-winter rapeseed for spring rapeseed hybrid breeding, to assess the genetic effects involved, and to estimate the correlation of parental genetic distance (GD) with hybrid performance, heterosis, general combining ability (GCA) and specific combining ability (SCA) in crosses between spring and semi-winter rapeseed lines. Four spring male sterile lines from Germany and Canada as testers were crossed with 13 Chinese semi-winter rapeseed lines to develop 52 hybrids, which were evaluated together with their parents and commercial hybrids for seed yield and oil content in three sets of field trials with 8 environments in Canada and Europe. The Chinese parental lines were not adapted to local environmental conditions as demonstrated by poor seed yields per se. However, the hybrids between the Chinese parents and the adapted spring rapeseed lines exhibited high heterosis for seed yield. The average mid-parent heterosis was 15% and ca. 50% of the hybrids were superior to the respective hybrid control across three sets of field trials. Additive gene effects mainly contributed to hybrid performance since the mean squares of GCA were higher as compared to SCA. The correlation between parental GD and hybrid performance and heterosis was found to be low whereas the correlation between GCA_{(f + m)} and hybrid performance was high and significant in each set of field trials, with an average of r = 0.87 for seed yield and r = 0.89 for oil content, indicating that hybrid performance can be predicted by GCA_{(f + m)}. These results demonstrate that Chinese semi-winter rapeseed germplasm has a great potential to increase seed yield in spring rapeseed hybrid breeding programs in Canada and Europe.     

Genome‐wide selection footprints and deleterious variations in young Asian allotetraploid rapeseed

Brassica napus (AACC, 2n = 38) is an important oilseed crop grown worldwide. However, little is known about the population evolution of this species, the genomic difference between its major genetic groups, such as European and Asian rapeseed, and the impacts of historical large‐scale introgression events on this young tetraploid. In this study, we reported the de novo assembly of the genome sequences of an Asian rapeseed (B. napus), Ningyou 7, and its four progenitors and compared these genomes with other available genomic data from diverse European and Asian cultivars. Our results showed that Asian rapeseed originally derived from European rapeseed but subsequently significantly diverged, with rapid genome differentiation after hybridization and intensive local selective breeding. The first historical introgression of B. rapa dramatically broadened the allelic pool but decreased the deleterious variations of Asian rapeseed. The second historical introgression of the double‐low traits of European rapeseed (canola) has reshaped Asian rapeseed into two groups (double‐low and double‐high), accompanied by an increase in genetic load in the double‐low group. This study demonstrates distinctive genomic footprints and deleterious SNP (single nucleotide polymorphism) variants for local adaptation by recent intra‐ and interspecies introgression events and provides novel insights for understanding the rapid genome evolution of a young allopolyploid crop.     

Comparison of rapeseed cultivars and resynthesized lines based on allozyme and RFLP markers

It has frequently been suggested to use the resynthesis of rapeseed (Brassica napus) from B. campestris and B. oleracea to broaden its genetic base. The objective of the present study is twofold: (1) to compare the genetic variation within resynthesized rapeseed with a world-wide collection of oilseed rape cultivars, and (2) to compare genetic distances estimated from RFLP markers with distances estimated from a relatively small number of allozyme markers. We investigated 17 resynthesized lines and 24 rapeseed cultivars. Genetic distances were estimated either based on the electrophoresis of seven allozymes, with a total of 38 different bands, or based on RFLP data of 51 probe/enzyme combinations, with a total of 355 different bands. The results of allozyme and RFLP analyses agreed reasonably well. Genetic distances, estimated from two independent sets of RFLP data with 25 and 26 probe/enzyme combinations respectively, were highly correlated; hence about 50 RFLP markers are sufficient to characterize rapeseed material with a large genetic diversity. The cultivars were clustered into three groups: (1) spring rapeseed of European and Northern American origin, (2) winter rapeseed of European and Northern American origin, and (3) rapeseed of Asian origin. Several of the resynthesized rapeseed lines were similar to European winter rapeseed cultivars, whereas others had quite unique patterns. It is concluded, that resynthesized rapeseed is a valuable source for broadening the genetic variation in present breeding material of Brassica napus. However, different lines differ widely in their suitability for this purpose.     

A new recombined double low restorer line for the Ogu-INRA cms in rapeseed (Brassica napus L.)

A major objective of breeders using the Ogu-INRA cytoplasmic male sterility (cms) system in rapeseed (Brassica napus L.) is to obtain double low restorer lines with a shorter introgression and a good agronomic value. The development of low glucosinolate content (low GC) restorer lines often occurs through the deletion of a part of the introgression. One of these lines has lost the radish Pgi-2 allele expression, without recovering that of the rapeseed Pgi-2 allele. This line shows a defect in the meiotic transmission of the restorer gene Rfo and a very poor agronomic value. We initiated a programme to force non-spontaneous recombination between this Rfo-carrying introgression and the rapeseed homologous chromosome from a low GC B. napus line. Gamma ray irradiation was used to induce chromosome breakage just prior meiosis aiming at just such a recombination. Low GC cms plants were crossed with the pollen of irradiated plants that were heterozygous for this introgression. The F₂ families were scored for their vigour, transmission rate of Rfo and female fertility. One family of plants, R2000, showed an improved behaviour for these three traits. This family presented a unique combination of molecular markers when compared to other rapeseed restorers analysed, which suggests that the recombination event allowed the recovery of B. oleracea genetic information that was originally replaced by the radish introgression in the original restorers. This resulted in a duplicated region (originating from radish and B. oleracea) on the chromosome carrying the introgression in the R2000 family.Electronic Supplementary Material Supplementary material is available for this article at     

Origin of new <Emphasis Type="Italic">Brassica</Emphasis> types from a single intergeneric hybrid between <Emphasis Type="Italic">B. rapa</Emphasis> and <Emphasis Type="Italic">Orychophragmus violaceus</Emphasis> by rapid chromosome evolution and introgression

Many novel lines were established from an intergeneric mixoploid between Brassica rapa (2n = 20) and Orychophragmus violaceus (2n = 24) through successive selections for fertility and viability. Pedigrees of individual F₂ plants were advanced to the 10th generation by selfing. Their breeding habit was self-compatible and different from the self-incompatibility of their female parent B. rapa, and these lines were reproductively isolated to different degrees from B. rapa and B. napus. The lines with high productivity showed not only a wide spectrum of phenotypes but also obvious variations in fatty acid profiles of seed oil and glucosinolate contents in seed meal. These lines had 2n = 36, 37, 38, 39 and 40, with 2n = 38 being most frequent (64.56%), and no intact O. violaceus chromosomes were detected by genomic in situ hybridization (GISH) analysis. Amplified fragment length polymorphism (AFLP) analyses revealed a high extent of variation in genomic compositions across all the lines. O. violaceus-specific bands, deleted bands in B. rapa and novel bands for two parents were detected in these lines, with novel bands being the most frequent. The morphological and genetic divergence of these novel types derived from a single hybrid is probably due to rapid chromosomal evolution and introgression, and provides new genetic resources for rapeseed breeding.     

Reconstituting the genome of a young allopolyploid crop,Brassica napus,with its related species

Brassica napus (AⁿAⁿCⁿCⁿ) is an important worldwide oilseed crop, but it is a young allotetraploid with a short evolutionary history and limited genetic diversity. To significantly broaden its genetic diversity and create a novel heterotic population for sustainable rapeseed breeding, this study reconstituted the genome of B. napus by replacing it with the subgenomes from 122 accessions of Brassica rapa (A^rA^r) and 74 accessions of Brassica carinata (B^cB^cC^cC^c) and developing a novel gene pool of B. napus through five rounds of extensive recurrent selection. When compared with traditional B. napus using SSR markers and high‐throughput SNP/Indel markers through genotyping by sequencing, the newly developed gene pool and its homozygous progenies exhibited a large genetic distance, rich allelic diversity, new alleles and exotic allelic introgression across all 19 AC chromosomes. In addition to the abundant genomic variation detected in the AC genome, we also detected considerable introgression from the eight chromosomes of the B genome. Extensive trait variation and some genetic improvements were present from the early recurrent selection to later generations. This novel gene pool produced equally rich phenotypic variation and should be valuable for rapeseed genetic improvement. By reconstituting the genome of B. napus by introducing subgenomic variation within and between the related species using intense selection and recombination, the whole genome could be substantially reorganized. These results serve as an example of the manipulation of the genome of a young allopolyploid and provide insights into its rapid genome evolution affected by interspecific and intraspecific crosses.     

Development of a population for substantial new type Brassica napus diversified at both A/C genomes

Intersubgenomic heterosis in rapeseed has been revealed in previous studies by using traditional Brassica napus (AⁿAⁿCⁿCⁿ) to cross partial new type B. napus with A^r/C^c introgression from the genomes of B. rapa and B. carinata, respectively. To further enlarge the genetic basis of B. napus and to facilitate a sustained heterosis breeding in rapeseed, it is crucial to create a population for substantial new type B. napus diversified at both A/C genomes. In this experiment, hundreds of artificial hexaploid plants (A^rA^rB^cB^cC^cC^c) involving hundreds of B. carinata/B. rapa combinations were first crossed with elite lines of partial new type B. napus. The pentaploid plants (AABCC) were open-pollinated in isolated conditions, and their offspring were successively self-pollinated and intensively selected for two generations. Thereafter, a population of substantial new type B. napus mainly with a genomic composition of A^rA^rC^cC^c harbouring genetic diversity from 25 original cultivars of B. rapa and 72 accessions of B. carinata was constructed. The population was cytologically verified to have the correct chromosome constitution of AACC and differed genetically from traditional B. napus, in terms of the genome components of A^r/C^c and B^c as well as the novel genetic variations induced by the interspecific hybridisation process. Synchronously, rich phenotypic variation with plenty of novel valuable traits was observed in the population. The origin of the novel variations and the value of the population are discussed.     

Intersubgenomic heterosis in seed yield potential observed in a new type of Brassica napus introgressed with partial Brassica rapa genome

This paper reports the observation on the intersubgenomic heterosis for seed yield among hybrids between natural Brassica napus (AⁿAⁿCⁿCⁿ) and a new type of B. napus with introgressions of genomic components of Brassica rapa (A^rA^r). This B. napus was selected from the progeny of B. napus × B. rapa and (B. napus × B. rapa) × B. rapa based on extensive phenotypic and cytological observation. Among the 129 studied partial intersubgenomic hybrids, which were obtained by randomly crossing 13 lines of the new type of B. napus in F₃ or BC₁F₃ to 27 cultivars of B. napus from different regions as tester lines, about 90% of combinations exceeded the yield of their respective tester lines, whereas about 75% and 25% of combinations surpassed two elite Chinese cultivars, respectively. This strong heterosis was further confirmed by reevaluating 2 out of the 129 combinations in a successive year and by surveying hybrids between 20 lines of the new type of B. napus in BC₁F₅ and its parental B. napus in two locations. Some DNA segments from B. rapa were identified with significant effects on seed yield and yield components of the new type of B. napus in BC₁F₅ and intersubgenomic hybrids in positive or negative direction. It seems that the genomic components introgressed from B. rapa contributed to improvement of seed yield of rapeseed.     

An insertional mutagenesis system for analyzing the Chinese cabbage genome using Agrobacterium T-DNA

In this study, we applied insertional mutagenesis using Agrobacterium transfer DNA to functionally characterize the gene of Brassica rapa L. ssp. pekinensis. The specific objectives were to: (i) develop and apply a gene tagging system using plasmid rescue and inverse PCR, (ii) select and analyze mutant lines, and (iii) analyze the phenotypic characteristics of mutants. A total of 3,400 insertional mutant lines were obtained from the Chinese cabbage cultivar, ’seoul’, using optimized condition. Plasmid rescue was performed successfully for transgenic plants with multiple T-DNA insertions, and inverse PCR was performed for plants with a single copy. The isolated flanking DNA sequences were blasted against the NCBI database and mapped to a linkage map. We determined the genetic loci in B. rapa with two methods: RFLP using the rescue clones themselves and sequence homology analysis to the B. rapa sequence database by queries of rescued clones sequences. Compared to wild type, the T₁ progenies of mutant lines showed variable phenotypes, including hairless and wrinkled leaves, rosette-type leaves, and chlorosis symptoms. T-DNA inserted mutant lines were the first population that we developed and will be very useful for functional genomics studies of Chinese cabbage.     

A large-scale introgression of genomic components of Brassica rapa into B. napus by the bridge of hexaploid derived from hybridization between B. napus and B. oleracea

Brassica rapa (AA) has been used to widen the genetic basis of B. napus (AACC), which is a new but important oilseed crop worldwide. In the present study, we have proposed a strategy to develop new type B. napus carrying genomic components of B. rapa by crossing B. rapa with hexaploid (AACCCC) derived from B. napus and B. oleracea (CC). The hexaploid exhibited large flowers and high frequency of normal chromosome segregation, resulting in good seed set (average of 4.48 and 12.53 seeds per pod by self and open pollination, respectively) and high pollen fertility (average of 87.05 %). It was easy to develop new type B. napus by crossing the hexaploid with 142 lines of B. rapa from three ecotype groups, with the average crossability of 9.24 seeds per pod. The genetic variation of new type B. napus was diverse from that of current B. napus, especially in the A subgenome, revealed by genome-specific simple sequence repeat markers. Our data suggest that the strategy proposed here is a large-scale and highly efficient method to introgress genomic components of B. rapa into B. napus.     

Quantitative trait analysis of seed yield and other complex traits in hybrid spring rapeseed (Brassica napus L.): 1. Identification of genomic regions from winter germplasm

The introgression of winter germplasm into spring canola (Brassica napus L.) represents a novel approach to improve seed yield of hybrid spring canola. In this study, quantitative trait loci (QTL) for seed yield and other traits were genetically mapped to determine the effects of genomic regions introgressed from winter germplasm into spring canola. Plant materials used comprised of two populations of doubled haploid (DH) lines having winter germplasm introgression from two related French winter cultivars and their testcrosses with a spring line used in commercial hybrids. These populations were evaluated for 2 years at two locations (Wisconsin, USA and Saskatchewan, Canada). Genetic linkage maps based on RFLP loci were constructed for each DH population. Six QTL were detected in the testcross populations for which the winter alleles increased seed yield. One of these QTL explained 11 and 19% of the phenotypic variation in the two Canadian environments. The winter allele for another QTL that increased seed yield was linked in coupling to a QTL allele for high glucosinolate content, suggesting that the transition of rapeseed into canola could have resulted in the loss of favorable seed yield alleles. Most QTL for which the introgressed allele decreased seed yield of hybrids mapped to genomic regions having homoeologous non-reciprocal transpositions. This suggests that allelic configurations created by these rearrangements might make an important contribution to genetic variation for complex traits in oilseed B. napus and could account for a portion of the heterotic effects in hybrids. Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Biomass yield and heterosis of crosses within and between European winter cultivars of turnip rape (<Emphasis Type="Italic">Brassica rapa</Emphasis> L.)

Because of its high growth rate at low temperatures in early spring, there is renewed interest in Brassica rapa as a winter crop for biomass production in Europe. The available cultivars are not developed for this purpose however. An approach for breeding bioenergy cultivars of B. rapa could be to establish populations from two or more different cultivars with high combining ability. The objective of this study was to evaluate the heterosis for biomass yield in the European winter B. rapa genepool. The genetic variation and heterosis of the biomass parameters: dry matter content, fresh and dry biomass yields were investigated in three cultivars representing different eras of breeding by comparing full-sibs-within and full-sibs-between the cultivars. Field trials were performed at two locations in Germany in 2005–2006. Mean mid-parent heterosis was low with 2.5% in fresh and 3.0% in dry biomass yield in full-sibs-between cultivars. Mean values of individual crosses revealed a higher variation in mid-parent heterosis ranging from 14.6% to −7.5% in fresh biomass yield and from 19.7% to −12.7% in dry biomass yield. The low heterosis observed in hybrids between European winter cultivars can be explained by the low genetic variation between these cultivars as shown earlier with molecular markers. In conclusion, a B. rapa breeding program for biomass production in Europe should not only use European genetic resources, but should also utilize the much wider worldwide variation in this species.     

Detection,introgression and localization of genes conferring specific resistance to <Emphasis Type="Italic">Leptosphaeria maculans</Emphasis> from <Emphasis Type="Italic">Brassica rapa</Emphasis> into <Emphasis Type="Italic">B. napus</Emphasis>

Blackleg (stem canker) caused by the fungus Leptosphaeria maculans is one of the most damaging diseases of oilseed rape (Brassica napus). Crop relatives represent a valuable source of “new” resistance genes that could be used to diversify cultivar resistance. B. rapa, one of the progenitors of B. napus, is a potential source of new resistance genes. However, most of the accessions are heterozygous so it is impossible to directly detect the plant genes conferring specific resistance due to the complex patterns of avirulence genes in L. maculans isolates. We developed a strategy to simultaneously characterize and introgress resistance genes from B. rapa, by homologous recombination, into B. napus. One B. rapa plant resistant to one L. maculans isolate was used to produce B. rapa backcross progeny and a resynthesized B. napus plant from which a population of doubled haploid lines was derived after crossing with natural B. napus. We then used molecular analyses and resistance tests on these populations to identify and map the resistance genes and to characterize their introgression from B. rapa into B. napus. Three specific genes conferring resistance to L. maculans (Rlm1, Rlm2 and Rlm7) were identified in B. rapa. Comparisons of genetic maps showed that two of these genes were located on the R7 linkage group, in a region homologous to the region on linkage group N7 in B. napus, where these genes have been reported previously. The results of our study offer new perspectives for gene introgression and cloning in Brassicas.     

Genetic relationships within Brassica rapa as inferred from AFLP fingerprints

Amplified fragment length polymorphism (AFLP) markers were employed to assess the genetic diversity amongst two large collections of Brassica rapa accessions. Collection A consisted of 161 B. rapa accessions representing different morphotypes among the cultivated B. rapa, including traditional and modern cultivars and breeding materials from geographical locations from all over the world and two Brassica napus accessions. Collection B consisted of 96 accessions, representing mainly leafy vegetable types cultivated in China. On the basis of the AFLP data obtained, we constructed phenetic trees using mega 2.1 software. The level of polymorphism was very high, and it was evident that the amount of genetic variation present within the groups was often comparable to the variation between the different cultivar groups. Cluster analysis revealed groups, often with low bootstrap values, which coincided with cultivar groups. The most interesting information revealed by the phenetic trees was that different morphotypes are often more related to other morphotypes from the same region (East Asia vs. Europe) than to similar morphotypes from different regions, suggesting either an independent origin and or a long and separate domestication and breeding history in both regions.     

Cytoplasmic male sterility in rapeseed (Brassica napus L.)

Summary Restriction patterns of chloroplast (cp) and mitochondrial (mt) DNA in Brassica napus rapeseed reveal the alloplasmic nature of cytoplasmic male sterility in this crop. Both the Shiga and Bronowski systems probably exploit cytoplasmic diversity in B. napus cultivars arising from introgression of cytoplasm from the other rapeseed species, B. campestris. Nuclear genes specific to these systems do not cause sterility in maintainers (Bronowski and Isuzu-natane) because they have a campestris cytoplasm, but give rise to sterility in napus cytoplasms. In the course of hybridization to napus cultivars a line with the triazine resistant cytoplasm (a campestris cytoplasm) has undergone an alteration in the mt genome rendering its restriction pattern more similar than previously to that of napus. The alteration may be an inversion between 7.2 and 3.4 kb in length.     

<Emphasis Type="Bold">A user guide to the</Emphasis><Emphasis Type="BoldItalic">Brassica</Emphasis><Emphasis Type="Bold">60K Illumina Infinium</Emphasis>™ <Emphasis Type="Bold">SNP genotyping array</Emphasis>

The Brassica napus 60K Illumina Infinium? SNP array has had huge international uptake in the rapeseed community due to the revolutionary speed of acquisition and ease of analysis of this high-throughput genotyping data, particularly when coupled with the newly available reference genome sequence. However, further utilization of this valuable resource can be optimized by better understanding the promises and pitfalls of SNP arrays. We outline how best to analyze Brassica SNP marker array data for diverse applications, including linkage and association mapping, genetic diversity and genomic introgression studies. We present data on which SNPs are locus-specific in winter, semi-winter and spring B. napus germplasm pools, rather than amplifying both an A-genome and a C-genome locus or multiple loci. Common issues that arise when analyzing array data will be discussed, particularly those unique to SNP markers and how to deal with these for practical applications in Brassica breeding applications.     

Characterisation of the radish introgression carrying the Rfo restorer gene for the Ogu-INRA cytoplasmic male sterility in rapeseed (Brassica napus L.)

 Bulked segregant analysis and comparative mapping were applied to identify molecular markers linked to the Rfo restorer gene used for the Ogu-INRA cytoplasmic male-sterility system in rapeseed. These markers were then used to localise the radish introgression on the B. napus genetic map constructed from the cross ‘Darmor.bzh’ x ’Yudal’. The introgression mapped on the DY15 linkage group. From the comparison of this latter group to the linkage group constructed on a F₂ progeny segregating for the radish introgression, it was concluded that the introgression had occurred through homoeologous recombination, that it was not distal and that it had replaced a B. napus region of around 50 cM. A QTL involved in aliphatic seed glucosinolate content was located on the DY15 linkage group at a position corresponding to one end of the introgression. The DNA markers identified in this study are being used in map-based cloning of the Rfo gene and in marker-assisted selection. Received: 3 December 1997 / Accepted: 17 December 1997     

Extending the rapeseed gene pool with resynthesized <Emphasis Type="Italic">Brassica napus</Emphasis> II: Heterosis

Hybrid breeding relies on the combination of parents from two differing heterotic groups. However, the genetic diversity in adapted oilseed rape breeding material is rather limited. Therefore, the use of resynthesized Brassica napus as a distant gene pool was investigated. Hybrids were derived from crosses between 44 resynthesized lines with a diverse genetic background and two male sterile winter oilseed rape tester lines. The hybrids were evaluated together with their parents and check cultivars in 2 years and five locations in Germany. Yield, plant height, seed oil, and protein content were monitored, and genetic distances were estimated with molecular markers (127 polymorphic RFLP fragments). Resynthesized lines varied in yield between 40.9 dt/ha and 21.5 dt/ha, or between 85.1 and 44.6% of check cultivar yields. Relative to check cultivars, hybrids varied from 91.6 to 116.6% in yield and from 94.5 to 103.3% in seed oil content. Mid-parent heterosis varied from −3.5 to 47.2% for yield. The genetic distance of parental lines was not significantly correlated with heterosis or hybrid yield. Although resynthesized lines do not meet the elite rapeseed standards, they are a valuable source for hybrid breeding due to their large distance from present breeding material and their high heterosis when combined with European winter oilseed rape.