Mapping of homoeologous chromosome exchanges influencing quantitative trait variation in Brassica napus

Genomic rearrangements arising during polyploidization are an important source of genetic and phenotypic variation in the recent allopolyploid crop Brassica napus. Exchanges among homoeologous chromosomes, due to interhomoeologue pairing, and deletions without compensating homoeologous duplications are observed in both natural B. napus and synthetic B. napus. Rearrangements of large or small chromosome segments induce gene copy number variation (CNV) and can potentially cause phenotypic changes. Unfortunately, complex genome restructuring is difficult to deal with in linkage mapping studies. Here, we demonstrate how high‐density genetic mapping with codominant, physically anchored SNP markers can detect segmental homoeologous exchanges (HE) as well as deletions and accurately link these to QTL. We validated rearrangements detected in genetic mapping data by whole‐genome resequencing of parental lines along with cytogenetic analysis using fluorescence in situ hybridization with bacterial artificial chromosome probes (BAC‐FISH) coupled with PCR using primers specific to the rearranged region. Using a well‐known QTL region influencing seed quality traits as an example, we confirmed that HE underlies the trait variation in a DH population involving a synthetic B. napus trait donor, and succeeded in narrowing the QTL to a small defined interval that enables delineation of key candidate genes.     

Proteomic Changes in Newly Synthesized <Emphasis Type="Italic">Brassica napus</Emphasis> Allotetraploids and Their Early Generations

Polyploidy is a prominent process in higher plants and is often described as a genomic shock that may induce stress and defense responses. The Brassica napus allotetraploid model was chosen to investigate the proteomic modifications that occur during allopolyploid formation. Large-scale analysis of the proteome from the leaves of B. napus was performed and compared with the homozygous diploid progenitors, Brassica rapa and Brassica oleracea, and among the proteomic changes in B. napus in the early generations (F₁–F₄). The abundance of all these differentially expressed proteins in the F₁ generation differed from that of the corresponding proteins expressed in its progenitors, some of which relatively deviated from mid-parent predictions, exhibiting somewhat non-additive expression repatterning. Proteomic changes in the resynthesized B. napus from the first to the fourth generations were detected, which indicated that gene silencing was a permanent phenomenon and it could be reactivated at any moment. Although leaf proteins were extensively modified in synthetic B. napus, the distribution of the “housekeeping” proteins was not disturbed. Moreover, no evidence of chaos or large disorder was observed after the merging of the two genomes. Instead, a novel order quickly developed, which might evolve in further generations of synthetic B. napus.     

Genomic changes at early stages of formation of allopolyploid <Emphasis Type="Italic">Aegilops longissima × Triticum urartu</Emphasis>

Using the model of synthetic allopolyploid Aegilops longissima TL05 × Triticum urartu TMU06 of the first generation, the degree and character of changes in subtelomeric, microsatellite and random amplified DNA sequences (RAPD) on early stage of polyploidization was estimated. Study of genome changes was performed by comparing PCR fragments of the allopolyploid and its parental forms. For analysis of subtelomeric DNA, we used 66 pairs of primers composed of 11 singular primers designed for these chromosomal regions sequences of cereals. RAPD analysis was performed with usage of 38 primers, in microsatellite (SSR) analysis 23 primer pairs were used. RAPD analysis appeared to be a more effective PCR-based method to identify genome changes. Absence of some RAPD fragments typical for parental genome in allopolyploid TL05 × TMU06 was shown using 13 primers of 38 (34%), and with usage of subtelomeric primers the changes in PCR fragments were shown only for one of 66 pairs of primers (1.5%). SSR loci were stable during the polyploidization process. Subsequent analysis of PCR fragments absent in the synthetic allopolyploid showed that high level of genome changes in RAPD analysis is probably connected with more effective ability of this method to reveal point mutations. Some data was found suggesting that not all genome changes observed in experimentally synthesized allopolyploids of the first generation are consequences of coadaptation of few genomes in one nucleus.     

Faithful inheritance of cytosine methylation patterns in repeated sequences of the allotetraploid tobacco correlates with the expression of DNA methyltransferase gene families from both parental genomes

The widespread occurrence of epigenetic alterations in allopolyploid species deserves scrutiny that DNA methylation systems may be perturbed by interspecies hybridization and polyploidization. Here we studied the genes involved in DNA methylation in Nicotiana tabacum (tobacco) allotetraploid containing S and T genomes inherited from Nicotiana sylvestris and Nicotiana tomentosiformis progenitors. To determine the inheritance of DNA methyltransferase genes and their expression patterns we examined three major DNA methyltransferase families (MET1, CMT3 and DRM) from tobacco and the progenitor species. Using Southern blot hybridization and PCR-based methods (genomic CAPS), we found that the parental loci of these gene families are retained in tobacco. Homoeologous expression was found in all tissues examined (leaf, root, flower) suggesting that DNA methyltransferase genes were probably not themselves targets of uniparental epigenetic silencing for over thousands of generations of allotetraploid evolution. The level of CG and CHG methylation of selected high-copy repeated sequences was similar and high in tobacco and its diploid progenitors. We speculate that natural selection might favor additive expression of parental DNA methyltransferase genes maintaining high levels of DNA methylation in tobacco, which has a repeat-rich heterochromatic genome. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AM946602–AM946620 and FM872474–FM872476.     

Molecular analysis of genomic changes in synthetic autotetraploid Phlox drummondii Hook

Polyploidization is a major force that has shaped the evolution of flowering plants. Newly‐generated polyploids have been used to investigate the changes that occur in genomes immediately after polyploidization. The present research programme created colchitetraploids in Phlox drummondii and followed them through different generations (C₀, C₁, C₂, and C₃) to determine genomic changes. Genomic changes that followed autopolyploidization were investigated using amplified fragment length polymorphism (AFLP) and methylation‐sensitive amplified polymorphism (MSAP) technologies. The markers for which variation was evident were characterized by cloning and sequencing. Dot‐blotting was used to confirm increased or decreased abundance of the fragments for which variation was noted. We used bisulfite sequencing to determine changes in the levels of DNA methylation in the vicinities of the fragments that show variation. Our data indicate that these areas were more methylated in C₂ and C₃ colchitetraploids than in C₀ and C₁ colchitetraploids. The overall levels of methylation polymorphism found in C₂ and C₃ colchitetraploids were 1.0% and 1.65%, respectively. Changes were observed in both coding and noncoding regions. Some MSAP fragments that showed variation were similar to retrotransposons. Bisulfite sequencing indicated that CHH sites accounted for all the observed variation, whereas CG and CHG did not show any variation. The present study suggests that the genomic changes that accompany polyploidization in synthetic autopolyploids resemble those in allopolyploids. However, the extent of variation is lower in synthetic autopolyploids than in allopolyploids. These changes may help in the stabilization of the genomes of neo‐autopolyploids. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110 , 591–605.     

Analysis of the Brassica oleracea genome by the generation of B. campestris-oleracea chromosome addition lines: characterization by isozymes and rDNA genes

Summary This study aimed at generating chromosome addition lines and disclosing genome specific markers in Brassica. These stocks will be used to study genome evolution in Brassica oleracea L., B. campestris L. and the derived amphidiploid species B. napus L. B. campestris-oleracea monosomic and disomic chromosome addition plants were generated by crossing and backcrossing the natural amphidiploid B. napus to the diploid parental species B. campestris. The pollen viability of the derived sesquidiploid and hyperploid ranged from 63% to 88%, while the monosomic and disomic addition plants had an average pollen fertility of 94% and 91%, respectively. The addition lines were genetically characterized by genome specific markers. The isozymes for 6PGD, LAP, PGI and PGM, and rDNA Eco RI restriction fragments were found to possess the desired genome specificity. Duplicated loci for several of these markers were observed in B. campestris and B. oleracea, supporting the hypothesis that these diploid species are actually secondary polyploids. A total of eight monosomic and eight disomic addition plants were identified and characterized on the basis of these markers. Another 51 plants remained uncharacterized due to the lack of additional markers. rDNA genes were found to be distributed in more than one chromosome, differing in its restriction sites. Intergenomic recombination for some of the markers was detected at frequencies between 6% and 20%, revealing the feasibility of intergenomic gene transfer.     

Genomic changes at early stages of formation of allopolyploid Aegilops longissima x Triticum urartu

Using the model of synthetic allopolyploid Aegilops longissima TL05 x Triticum urartu TMU06 of the first generation, the degree and character of changes in subtelomeric, microsatellite and random amplified DNA sequences (RAPD) on early stage of polyploidization was estimated. Study of genome changes was performed by comparing of PCR spectra obtained while amplifying genome DNA of allopolyploid and its parental forms. For analysis of subtelomeric DNA, we used 66 pairs of primers composed of 11 singular primers designed for subtelomere DNA sequences of cereals. RAPD analysis was performed with usage of 38 primers, in microsatellite (SSR) analysis 23 primer pairs were used. RAPD analysis appeared to be a more effective PCR-based method to identify genome changes. Absence of some PCR fragments typical for parental genome in RAPD specters of allopolyploid TL05 x TMU06 was shown using 13 primers of 38 (34%), and with usage of subtelomere primers such changes in PSR specters were shown only for one of 66 pays of primers (1.5%). SSR loci were stable during the polyploidization process. Subsequent analysis of PCR fragments absent in specter of synthetic allopolyploid showed that high level of genome changes in RAPD analysis is probably connected with more effective ability of this method to reveal point mutations. Some data was found suggesting that not all genome changes observed in experimentally synthesized allopolyploids of the first generation are consequences of coadaptation of few genomes in one nucleus.     

Development of synthetic Brassica amphidiploids by reciprocal hybridization and comparison to natural amphidiploids

In a previous study we proposed that cytoplasmic genomes have played an important role in the evolution of Brassica amphidiploid species. Based on this and other studies, we hypothesized that interactions between the maternal cytoplasmic genomes and the paternal nuclear genome may cause alterations in genome structure and/or gene expression of a newly synthesized amphidiploid, which may play an important role in the evolution of natural amphidiploid species. To test this hypothesis, a series of synthetic amphidiploids, including all three analogs of the natural amphidiploids B. napus, B. juncea, and B. Carinata and their reciprocal forms, were developed. These synthetic amphidiploids were characterized for morphological traits, chromosome number, and RFLPs revealed by chloroplast, mitochondrial, and nuclear DNA clones. The maternal transmission of chloroplast and mitochondrial genomes was observed in all of the F₁ hybrids examined except one hybrid plant derived from the B. rapa x B. oleracea combination, which showed a biparental transmission of organelles. However, the paternal chloroplast and mitochondrial genomes were not observed in the F₂ progeny. Nuclear genomes of synthetic amphidiploids had combined RFLP patterns of their parental species for all of the nuclear DNA clones examined. A variation in fertility was observed among self-pollinated progenies of single amphidiploids that had completely homozygous genome constitutions. Comparisons between natural and synthetic amphidiploids based on restriction fragment length polymorphism (RFLP) patterns indicated that natural amphidiploids are considerably more distant from the progenitor diploid species than the synthetic amphidiploids. The utility of these synthetic amphidiploids for investigating the evolution of amphidiploidy is discussed.     

Analysis of DNA methylation patterns of PLBs derived from <Emphasis Type="Italic">Cymbidium hybridium</Emphasis> based on MSAP

The best known and most thoroughly studied epigenetic phenomenon is DNA methylation, which plays an important role in regulating gene expression during plant regeneration and development. In this study, the methylation-sensitive amplified polymorphism (MSAP) technique was carried out to determine differences in methylation profiles between two forms of protocorm-like bodies (PLBs), continuously proliferating PLBs (cPLBs) and spontaneously-differenting PLBs (sdPLBs), derived from cultures of Cymbidium hybridium. A total of 72 selective primer combinations were used to assess the status of cytosine methylation of DNA in these tissues. Of 4,440 fragments obtained 911 fragments, each representing a recognition site cleaved by one or both of the isoschizomers (Hpa II and Msp I), were amplified and were significantly different between the two forms of PLBs. Frequency of total and full-methylation of cPLBs and sdPLBs were 26.7/12.2%, 24.1/11.1%, respectively. In addition, 14 types of MSAP patterns detected in the two forms of PLBs belonged to two classes, type I and II. Sequencing of 14 differentially methylated fragments and their subsequent blast search revealed that cytosine methylated 5′-CCGG-3′ sequences were equally distributed in the coding and non-coding regions. Southern blotting was conducted to verify the methylation polymorphism.     

Cytological study of interspecific hybrid between Brassica campestris x B. hirta (Sinapis alba)

Interspecific hybrids from the crossing Brassica campestris x B. hirta are reported in our study for the first time. F₁ plants were obtained by using ovary culture. The phenotype of hybrids was similar to B. napus; the plants were self-fertile. Investigation of meiotic division and nuclear DNA content measurements showed the amphidiploid origin of these hybrids. The relationship between genome A and D, as well as the spontaneous amphidiploidization of the hybrids, are discussed.     

MSAP markers and global cytosine methylation in plants: a literature survey and comparative analysis for a wild‐growing species

Methylation of DNA cytosines affects whether transposons are silenced and genes are expressed, and is a major epigenetic mechanism whereby plants respond to environmental change. Analyses of methylation‐sensitive amplification polymorphism (MS‐AFLP or MSAP) have been often used to assess methyl‐cytosine changes in response to stress treatments and, more recently, in ecological studies of wild plant populations. MSAP technique does not require a sequenced reference genome and provides many anonymous loci randomly distributed over the genome for which the methylation status can be ascertained. Scoring of MSAP data, however, is not straightforward, and efforts are still required to standardize this step to make use of the potential to distinguish between methylation at different nucleotide contexts. Furthermore, it is not known how accurately MSAP infers genome‐wide cytosine methylation levels in plants. Here, we analyse the relationship between MSAP results and the percentage of global cytosine methylation in genomic DNA obtained by HPLC analysis. A screening of literature revealed that methylation of cytosines at cleavage sites assayed by MSAP was greater than genome‐wide estimates obtained by HPLC, and percentages of methylation at different nucleotide contexts varied within and across species. Concurrent HPLC and MSAP analyses of DNA from 200 individuals of the perennial herb Helleborus foetidus confirmed that methyl‐cytosine was more frequent in CCGG contexts than in the genome as a whole. In this species, global methylation was unrelated to methylation at the inner CG site. We suggest that global HPLC and context‐specific MSAP methylation estimates provide complementary information whose combination can improve our current understanding of methylation‐based epigenetic processes in nonmodel plants.     

The allotetraploid <Emphasis Type="Italic">Arabidopsis thaliana–Arabidopsis lyrata</Emphasis> subsp. <Emphasis Type="Italic">petraea</Emphasis> as an alternative model system for the study of polyploidy in plants

Polyploidy is known to be common in plants and recent work has focused on the rapid changes in genome structure and expression that occur upon polyploidization. In Arabidopsis, much of this work has been done on a synthetic allotetraploid obtained by crossing a tetraploid Arabidopsis thaliana (2n = 4x = 20) with A. arenosa (2n = 4x = 32). To explore an alternative route to polyploidy in this model species, we have developed a synthetic allopolyploid by crossing two diploid species: A. thaliana (2n = 2x = 10) and Arabidopsis lyrata subsp. petraea (2n = 2x = 16). F₁ hybrids were easy to obtain and phenotypically more similar to A. lyrata. Spontaneous chromosome doubling events occurred in about 25% of the F₁s, thus restoring fertility. The resulting allotetraploids (2n = 26) exhibited many genomic changes typically reported upon polyploidization. Nucleolar dominance was observed as only the A. lyrata rDNA loci were expressed in the F₁ and allotetraploids. Changes in the degree of methylation were observed at almost 25% of the loci examined by MSAP analysis. Finally, structural genomic alterations did occur as a large deletion covering a significant portion of the upper arm of chromosome II was detected but no evidence of increased mobility of transposons was obtained. Such allotetraploids derived from two parents with sequenced (or soon to be sequenced) genomes offer much promise in elucidating the various changes that occur in newly synthesized polyploids.     

The allopolyploid origin of kiwifruit,Actinidia deliciosa (Actinidiaceae)

The genetic origin of kiwifruit (Actinidia deliciosa var.deliciosa) was studied using phylogenetic analysis of DNA sequences derived from the polygalacturonase gene. Results indicate that hexaploid kiwifruit had an allopolyploid origin with the diploidA. chinensis contributing one genome (genome A) and another (as yet unidentified) diploid species contributing a second genome (genome B). The results leave open the question of whether a third, distinct species contributed to the hexaploid kiwifruit genome. A tetraploid race ofA. chinensis is also suggested to be allopolyploid containing genomes A and B.     

Genetic Changes Following Hybridization and Genome Doubling in Synthetic Brassica napus

Genetic changes were investigated in two sets of independently synthesized Brasscia napus allopolyploids by the AFLP approach in the present study. We found that 1.17 % of the loci showed genetic changes following both hybridization and genome doubling in the synthesized B. napus F04J2 relative to its diploid progenitors, B. rapa (AA genome) and B. oleracea (CC genome). No significant difference between the proportion of A-genome-specific genetic changes and that of C-genome-specific genetic changes was detected in B. napus F04J2. Approximately 0.6 % of the loci displayed genetic changes following somatic genome doubling in the amphidiploid B. napus DCE11 relative to the amphihaploid in the dimorphic plants. This study showed that rapid genetic changes occurred after hybridization and/or genome doubling in synthesized B. napus allopolyploids and indicated that both hybridization and genome doubling could affect the genomic architecture in newly formed allopolyploids.