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.     

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.     

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.     

Genetic changes in a novel breeding population of Brassica napus synthesized from hundreds of crosses between B. rapa and B. carinata

Introgression of genomic variation between and within related crop species is a significant evolutionary approach for population differentiation, genome reorganization and trait improvement. Using the Illumina Infinium Brassica 60K SNP array, we investigated genomic changes in a panel of advanced generation new‐type Brassica napus breeding lines developed from hundreds of interspecific crosses between 122 Brassica rapa and 74 Brassica carinata accessions, and compared them with representative accessions of their three parental species. The new‐type B. napus population presented rich genetic diversity and abundant novel genomic alterations, consisting of introgressions from B. rapa and B. carinata, novel allelic combinations, reconstructed linkage disequilibrium patterns and haplotype blocks, and frequent deletions and duplications (nonrandomly distributed), particularly in the C subgenome. After a much shorter, but very intensive, selection history compared to traditional B. napus, a total of 15 genomic regions with strong selective sweeps and 112 genomic regions with putative signals of selective sweeps were identified. Some of these regions were associated with important agronomic traits that were selected for during the breeding process, while others were potentially associated with restoration of genome stability and fertility after interspecific hybridization. Our results demonstrate how a novel method for population‐based crop genetic improvement can lead to rapid adaptation, restoration of genome stability and positive responses to artificial selection.     

Homoeolog Expression Is Modulated Differently by Different Subgenomes in <Emphasis Type="Italic">Brassica napus</Emphasis> Hybrids and Allotetraploids

Synthetic and natural allotetraploid Brassica napus (2n?=?38, AACC) have been widely used as a model to study the genetic changes associated with allopolyploidization; however, there has been little research on the homoeolog expression patterns and the roles of cis and trans regulation. Herein, homoeolog expression patterns were assessed by using RNA-seq for two interspecific hybrids (A_nC_o with the extracted A subgenome from natural B. napus, and A_rC_o with the A subgenome from extant B. rapa), synthetic and natural allopolyploids (C_oC_oA_rA_r and A_nA_nC_nC_n), and the diploid parents. The ranges of homoeolog expression bias decreased after hybridization, whereas the extents of homoeolog expression bias and non-conserved expression, especially transgressive expression, increased over evolutionary time. Despite sharing the same C subgenome parent, these two hybrids showed different homolog expression patterns in many respects. In A_nC_o, the trans-regulatory factors from C_o subgenome tended to cause downregulation of A_n subgenome homoeologs, but trans-regulatory factors from the A_n subgenome acted as both activators and repressors, and such asymmetric effects of trans-regulatory factors might explain why the homoeolog expression was biased toward the C subgenome after genome merger. No significant asymmetric effects of trans-regulatory factors were found in A_rC_o, which was consistent with the overall balanced expression of homoeologs. These results suggested that A subgenomes with different regulatory systems might act differently in modulating homoeolog expression after merger with the C subgenome, resulting in either balanced or unbalanced homoeolog expression biases.     

Trigenomic Bridges for Brassica Improvement

We introduce and review Brassica crop improvement via trigenomic bridges. Six economically important Brassica species share three major genomes (A, B, and C), which are arranged in diploid (AA, BB, and CC) and allotetraploid (AABB, AACC, and BBCC) species in the classical triangle of U. Trigenomic bridges are Brassica interspecific hybrid plants that contain the three genomes in various combinations, either triploid (ABC), unbalanced tetraploid (e.g., AABC), pentaploid (e.g., AABCC) or hexaploid (AABBCC). Through trigenomic bridges, Brassica breeders can access all the genetic resources in the triangle of U for genetic improvement of existing species and development of new agricultural species. Each of the three Brassica genomes occurs in several species, where they are distinguished as subgenomes with a tag to identify the species of origin. For example, the A subgenome in B. juncea (2n = AABB) is denoted as A^j and the A subgenome in B. napus (2n = AACC) as Aⁿ. Trigenomic bridges have been used to increase genetic diversity in allopolyploid Brassica crop species, such as a new-type B. napus with subgenomes from B. rapa (A^r) and B. carinata (C^c). Recently, trigenomic bridges from several sources have been crossed together as the ‘founders’ of a potentially new allohexaploid Brassica species (AABBCC). During meiosis in a trigenomic bridge, crossovers are expected to form between homologous chromosomes of related subgenomes (for example A^r and Aⁿ), but cross-overs may also occur between non-homologous chromosomes (for example between A and C genome chromosomes). Irregular meiosis is a common feature of new polyploids, and any new allotetraploid or allohexaploid Brassica genotypes derived from a trigenomic bridge must achieve meiotic stability through a process of diploidisation. New sequencing technologies, at the genomic and epigenomic level, may reveal the genetic and molecular basis of diploidization, and accelerate selection of stable allotetraploids or allohexaploids. Armed with new genetic resources from trigenomic bridges, Brassica breeders will be able to improve yield and broaden adaptation of Brassica crops to meet human demands for food and biofuel, particularly in the face of abiotic constraints caused by climate change.     

RFLP and AFLP Analysis of Inter- and Intraspecific Variation of Brassica rapa and B. napus Shows that B. rapa Is an Important Genetic Resource for B. napus Improvement

Fingerprinting of 29 accessions of oilseed rape, including seven accessions of Brassica napus, and 22 accessions of B. rapa (B. campestris) from Europe, North America, and China was analyzed using RFLP and AFLP markers. In total, 1 477 polymorphic RFLP bands and 183 polymorphic AFLP bands from 166 enzyme-probe combinations and two pairs of AFLP primers, respectively, were scored for the 29 accessions. On average, RFLP analysis showed that the Arabidopsis EST probe detected more polymorphic bands in Brassica than the random genomic probe performed. More polymorphic RFLP markers were detected with the digestion of EcoR I or BamH I than HindIII. According to the number of bands amplified from each accession, the copy numbers of each gene in the genomes of B. rapa and B. napus were estimated. The average copy numbers in B. rapa of China, B. rapa of Europe, and B. napus, were 3.2, 3.1, and 2.9, respectively. Genetic distance based on the AFLP data was well correlated with that based on the RFLP data (r = 0.72, P<0.001), but 0.39 smaller on average. Genetic diversity analysis showed that Chinese B. rapa was more polymorphic than Chinese B. napus and European materials. Some European B. napus accessions were clustered into European B. rapa, which were distinctly different from Chinese B. napus. The larger variations of Chinese accessions of B. rapa suggest that they are valuable in oilseed rape breeding. Novel strategies to use intersubgenomic heterosis between genome of B. rapa (A^rA^r) and genome of B. napus (AⁿAⁿCⁿCⁿ) were elucidated.     

Identification and characterization of improved nitrogen efficiency in interspecific hybridized new-type Brassica napus

Background and Aims

Oilseed rape (Brassica napus) is an important oil crop worldwide. The aim of this study was to identify the variation in nitrogen (N) efficiency of new-type B. napus (genome A^rA^rC^cC^c) genotypes, and to characterize some critical physiological and molecular mechanisms in response to N limitation.

Methods

Two genotypes with contrasting N efficiency (D4-15 and D1-1) were identified from 150 new-type B. napus lines, and hydroponic and pot experiments were conducted. Root morphology, plant biomass, N uptake parameters and seed yield of D4-15 and D1-1 were investigated. Two traditional B. napus (genome AⁿAⁿCⁿCⁿ) genotypes, QY10 and NY7, were also cultivated. Introgression of exotic genomic components in D4-15 and D1-1 was evaluated with molecular markers.

Key Results

Large genetic variation existed among traits contributing to the N efficiency of new-type B. napus. Under low N levels at the seedling stage, the N-efficient new-type D4-15 showed higher values than the N-inefficient D1-1 line and the traditional B. napus QY10 and NY7 genotypes with respect to several traits, including root and shoot biomass, root morphology, N accumulation, N utilization efficiency (NutE), N uptake efficiency (NupE), activities of nitrate reductase (NR) and glutamine synthetase (GS), and expression levels of N transporter genes and genes that are involved in N assimilation. Higher yield was produced by the N-efficient D4-15 line compared with the N-inefficient D1-1 at maturity. More exotic genome components were introgressed into the genome of D4-15 (64·97 %) compared with D1-1 (32·23 %).

Conclusions

The N-efficient new-type B. napus identified in this research had higher N efficiency (and tolerance to low-N stress) than traditional B. napus cultivars, and thus could have important potential for use in breeding N-efficient B. napus cultivars in the field.     

A- or C-chromosomes, does it matter for the transfer of transgenes from Brassica napus

Introgression of genes from allotetraploid Brassica napus into its diploid wild relative B. rapa is generally considered to be inevitable. As a means to minimize a potential ecological risk in environments where B. rapa is growing, the insertion of transgenes into chromosome regions of B. napus with a very low probability of transfer to backcross generations with B. rapa has been proposed. Recently, the progeny of four backcross generations between transgenic herbicide-tolerant B. napus and B. rapa was studied in selection experiments (Metz et al. 1997). The rapid decrease in the frequency of herbicide-tolerant plants was explained by selection against the C-chromosomes of B. napus in favor of the homeologous A-chromosomes. Obviously, such C-chromosomes could be potential candidates as safe integration sites for transgenes. We considered these safety aspects using a simple population genetic model. Theory and experiments, however, do not favor the chromosomes of B. napus as safe candidates with respect to the introgression of transgenes into wild populations of B. rapa. Received: 5 July 1999 / Accepted: 29 July 1999     

Broadening the avenue of intersubgenomic heterosis in oilseed Brassica

Accumulated evidence has shown that each of the three basic Brassica genomes (A, B and C) has undergone profound changes in different species, and has led to the concept of the “subgenome”. Significant intersubgenomic heterosis was observed in hybrids between traditional Brassica napus and first generation lines of new type B. napus. The latter were produced by the partial introgression of subgenomic components from different species into B. napus. To increase the proportion of exotic subgenomic components and thus achieve stronger heterosis, lines of first generation new type B. napus were intercrossed with each other, and subjected to intensive marker-assisted selection to develop the second generation of new type B. napus. The second generation showed better agronomic traits and a higher proportion of introgression of subgenomic components than did the first generation. Compared with the commercial hybrid and the hybrids produced with the first generation new type B. napus, the novel hybrids showed stronger heterosis for seed yield during the 2 years of field trials. The extent of heterosis showed a significant positive correlation with the introgressed subgenomic components in the parental new type B. napus. To increase the content of the exotic subgenomic components further and to allow sustainable breeding of novel lines of new type B. napus, we initiated the development of a gene pool for new type B. napus that contained a substantial amount of genetic variation in the A^r and C^c genome. We discuss new approaches to broaden the avenue of intersubgenomic heterosis in oilseed Brassica.     

Characterization of directly transformed weedy <Emphasis Type="Italic">Brassica rapa</Emphasis> and introgressed <Emphasis Type="Italic">B. rapa</Emphasis> with Bt <Emphasis Type="Italic">cry1Ac</Emphasis> and <Emphasis Type="Italic">gfp</Emphasis> genes

Crop to weed transgene flow, which could result in more competitive weed populations, is an agricultural biosafety concern. Crop Brassica napus to weedy Brassica rapa hybridization has been extensively characterized to better understand the transgene flow and its consequences. In this study, weedy accessions of B. rapa were transformed with Bacillus thuringiensis (Bt) cry1Ac- and green fluorescence protein (gfp)-coding transgenes using Agrobacterium to assess ecological performance of the wild biotype relative to introgressed hybrids in which the transgenic parent was the crop. Regenerated transgenic B. rapa events were characterized by progeny analysis, Bt protein enzyme-linked immunosorbent assay (ELISA), Southern blot analysis, and GFP expression assay. GFP expression level and Bt protein concentration were significantly different between independent transgenic B. rapa events. Similar reproductive productivity was observed in comparison between transgenic B. rapa events and B. rapa × B. napus introgressed hybrids in greenhouse and field experiments. In the greenhouse, Bt transgenic plants experienced significantly less herbivory damage from the diamondback moth (Plutella xylostella). No differences were found in the field experiment under ambient, low, herbivore pressure. Directly transformed transgenic B. rapa plants should be a helpful experimental control to better understand crop genetic load in introgressed transgenic weeds.     

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.     

A review of sources of resistance to turnip yellows virus (TuYV) in Brassica species

Turnip yellows virus (TuYV; previously known as beet western yellows virus) causes major diseases of Brassica species worldwide resulting in severe yield-losses in arable and vegetable crops. It has also been shown to reduce the quality of vegetables, particularly cabbage where it causes tip burn. Incidences of 100% have been recorded in commercial crops of winter oilseed rape (Brassica napus) and vegetable crops (particularly Brassica oleracea) in Europe. This review summarises the known sources of resistance to TuYV in B. napus (AACC genome), Brassica rapa (AA genome) and B. oleracea (CC genome). It also proposes names for the quantitative trait loci (QTLs) responsible for the resistances, Tu rnip Y ellows virus R esistance (TuYR), that have been mapped to at least the chromosome level in the different Brassica species. There is currently only one known source of resistance deployed commercially (TuYR1). This resistance is said to have originated in B. rapa and was introgressed into the A genome of oilseed rape via hybridisation with B. oleracea to produce allotetraploid (AACC) plants that were then backcrossed into oilseed rape. It has been utilised in the majority of known TuYV-resistant oilseed rape varieties. This has placed significant selection pressure for resistance-breaking mutations arising in TuYV. Further QTLs for resistance to TuYV (TuYR2-TuYR9) have been mapped in the genomes of B. napus, B. rapa and B. oleracea and are described here. QTLs from the latter two species have been introgressed into allotetraploid plants, providing for the first time, combined resistance from both the A and the C genomes for deployment in oilseed rape. Introgression of these new resistances into commercial oilseed rape and vegetable brassicas can be accelerated using the molecular markers that have been developed. The deployment of these resistances should lessen selection pressure for resistance-breaking isolates of TuYV and thereby prolong the effectiveness of each other and extant resistance.     

Pairing and recombination at meiosis of Brassica rapa (AA) × Brassica napus (AACC) hybrids

Interspecific crosses contribute significantly to plant evolution enabling gene exchanges between species. The efficiency of interspecific crosses depends on the similarity between the implicated genomes as high levels of genome similarity are required to ensure appropriate chromosome pairing and genetic recombination. Brassica napus (AACC) is an allopolyploid, resulting from natural hybridization between Brassica rapa (AA) and Brassica oleracea (CC), both being diploid species derived from a common ancestor. To study the relationships between genomes of these Brassica species, we have determined simultaneously the pairing and recombination pattern of A and C chromosomes during meiosis of AAC triploid hybrids, which result from the interspecific cross between natural B. napus and B. rapa. Different AAC triploid hybrids and their progenies have been analysed using cytogenetic, BAC-FISH, and molecular techniques. In 71% of the pollen mother cells, homologous A chromosomes paired regularly, and usually one chromosome of each pair was transmitted to the progeny. C chromosomes remained mainly univalent, but were involved in homoeologous pairing in 21.5% of the cells, and 13% of the transmitted C chromosomes were either recombined or broken. The rate of transmission of C chromosomes depended on the identity of the particular chromosome and on the way the hybrid was crossed, as the male or as the female parent, to B. napus or to B. rapa. Gene transfers in triploid hybrids are favoured between A genomes of B. rapa and B. napus, but also occur between A and C genomes though at lower rates.     

Reproduction and cytogenetic characterization of interspecific hybrids derived from crosses between Brassica carinata and B. rapa

The tri-genomic hybrid (ABC, 2n=27) between Brassica carinata (BBCC, 2n=34) and B. rapa (AA, 2n=20) is a unique material for studying genome relationships among Brassica species and a valuable bridge for transferring desirable characteristics from one species to the other within the genus Brassica. The crossability between B. carinata and B. rapa was varied with the cultivar of B. rapa. Hybrid pollen mother cells (PMCs), confirmed by morphological observation and molecular marker assay, could be grouped into 20 classes on the basis of chromosome pairing configurations. More than 30% of the PMCs had nine or more bivalents. Genomic in situ hybridization confirmed that two of the bivalents most likely belonged to the B genome. Nearly one-half of the PMCs had trivalents (0–2) and quadrivalents (0–2), which revealed partial homology among the A, B, and C genomes and suggested that there is a good possibility to transfer genes by means of recombination among the three genomes. The advantages of using the tri-genomic hybrids as bridge material for breeding new types of B. napus are discussed.     

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. Received: 13 July 2001 / Accepted: 23 August 2001     

Sequence‐level comparative analysis of the Brassica napus genome around two stearoyl‐ACP desaturase loci

We conducted a sequence‐level comparative analyses, at the scale of complete bacterial artificial chromosome (BAC) clones, between the genome of the most economically important Brassica species, Brassica napus (oilseed rape), and those of Brassica rapa, the genome of which is currently being sequenced, and Arabidopsis thaliana. We constructed a new B. napus BAC library and identified and sequenced clones that contain homoeologous regions of the genome including stearoyl‐ACP desaturase‐encoding genes. We sequenced the orthologous region of the genome of B. rapa and conducted comparative analyses between the Brassica sequences and those of the orthologous region of the genome of A. thaliana. The proportion of genes conserved (～56%) is lower than has been reported previously between A. thaliana and Brassica (～66%). The gene models for sets of conserved genes were used to determine the extent of nucleotide conservation of coding regions. This was found to be 84.2 ± 3.9% and 85.8 ± 3.7% between the B. napus A and C genomes, respectively, and that of A. thaliana, which is consistent with previous results for other Brassica species, and 97.5 ± 3.1% between the B. napus A genome and B. rapa, and 93.1 ± 4.9% between the B. napus C genome and B. rapa. The divergence of the B. napus genes from the A genome and the B. rapa genes was greater than anticipated and indicates that the A genome ancestor of the B. napus cultivar studied was relatively distantly related to the cultivar of B. rapa selected for genome sequencing.