Robert T. Gaeta

Genomic changes in resynthesized Brassica napus and their effect on gene expression and phenotype.

Many previous studies have provided evidence for genome changes in polyploids, but there are little data on the overall population dynamics of genome change and whether it causes phenotypic variability. We analyzed genetic, epigenetic, gene expression, and phenotypic changes in ∼50 resynthesized Brassica napus lines independently derived by hybridizing double haploids of Brassica oleracea and Brassica rapa. A previous analysis of the first generation (S0) found that genetic changes were rare, and cytosine methylation changes were frequent. Our analysis of a later generation found that most S0 methylation changes remained fixed in their S5 progeny, although there were some reversions and new methylation changes. Genetic changes were much more frequent in the S5 generation, occurring in every line with lines normally distributed for number of changes. Genetic changes were detected on 36 of the 38 chromosomes of the S5 allopolyploids and were not random across the genome. DNA fragment losses within lines often occurred at linked marker loci, and most fragment losses co-occurred with intensification of signal from homoeologous markers, indicating that the changes were due to homoeologous nonreciprocal transpositions (HNRTs). HNRTs between chromosomes A1 and C1 initiated in early generations, occurred in successive generations, and segregated, consistent with a recombination mechanism. HNRTs and deletions were correlated with qualitative changes in the expression of specific homoeologous genes and anonymous cDNA amplified fragment length polymorphisms and with phenotypic variation among S5 polyploids. Our data indicate that exchanges among homoeologous chromosomes are a major mechanism creating novel allele combinations and phenotypic variation in newly formed B. napus polyploids.

Homoeologous shuffling and chromosome compensation maintain genome balance in resynthesized allopolyploid Brassica napus

Polyploidy has contributed to the evolution of eukaryotes, particularly flowering plants. The genomic consequences of polyploidy have been extensively studied, but the mechanisms for chromosome stability and diploidization in polyploids remain largely unknown. By using new cytogenetic tools to identify all of the homoeologous chromosomes, we conducted a cytological investigation of 50 resynthesized Brassica napus allopolyploids across generations S0:1 to S5:6 and in the S10:11 generation. Changes in copy number of individual chromosomes were detected in the S0:1 generation and increased in subsequent generations, despite the fact that the mean chromosome number among lines was approximately 38. The chromosome complement of individual plants (segregants) ranged from 36 to 42, with a bias toward the accumulation of extra chromosomes. Karyotype analysis of the S10:11 generation detected aneuploidy and inter- and intragenomic rearrangements, chromosome breakage and fusion, rDNA changes, and loss of repeat sequences. Chromosome sets with extensive homoeology showed the greatest instability. Dosage balance requirements maintained chromosome numbers at or near the tetraploid level, and the loss and gain of chromosomes frequently involved homoeologous chromosome replacement and compensation. These data indicate that early generations of resynthesized B. napus involved aneuploidy and gross chromosomal rearrangements, and that dosage balance mechanisms enforced chromosome number stability. Seed yield and pollen viability were inversely correlated with increasing aneuploidy, and the greatest fertility was observed in two lines that were additive for parental chromosomes. These data on resynthesized B. napus and the correlation of fertility with additive karyotypes cast light on the origins and establishment of natural B. napus.

Homoeologous recombination in allopolyploids: the polyploid ratchet

Polyploidization and recombination are two important processes driving evolution through the building and reshaping of genomes. Allopolyploids arise from hybridization and chromosome doubling among distinct, yet related species. Polyploids may display novel variation relative to their progenitors, and the sources of this variation lie not only in the acquisition of extra gene dosages, but also in the genomic changes that occur after divergent genomes unite. Genomic changes (deletions, duplications, and translocations) have been detected in both recently formed natural polyploids and resynthesized polyploids. In resynthesized Brassica napus allopolyploids, there is evidence that many genetic changes are the consequence of homoeologous recombination. Homoeologous recombination can generate novel gene combinations and phenotypes, but may also destabilize the karyotype and lead to aberrant meiotic behavior and reduced fertility. Thus, natural selection plays a role in the establishment and maintenance of fertile natural allopolyploids that have stabilized chromosome inheritance and a few advantageous chromosomal rearrangements. We discuss the evidence for genome rearrangements that result from homoeologous recombination in resynthesized B. napus and how these observations may inform phenomena such as chromosome replacement, aneuploidy, non-reciprocal translocations and gene conversion seen in other polyploids.

Analysis of Gene Expression in Resynthesized Brassica napus Allopolyploids Using Arabidopsis 70mer Oligo Microarrays

Background Studies in resynthesized Brassica napus allopolyploids indicate that homoeologous chromosome exchanges in advanced generations (S5∶6) alter gene expression through the loss and doubling of homoeologous genes within the rearrangements. Rearrangements may also indirectly affect global gene expression if homoeologous copies of gene regulators within rearrangements have differential affects on the transcription of genes in networks. Methodology/Principal Findings We utilized Arabidopsis 70mer oligonucleotide microarrays for exploring gene expression in three resynthesized B. napus lineages at the S0∶1 and S5∶6 generations as well as their diploid progenitors B. rapa and B. oleracea. Differential gene expression between the progenitors and additive (midparent) expression in the allopolyploids were tested. The S5∶6 lines differed in the number of genetic rearrangements, allowing us to test if the number of genes displaying nonadditive expression was related to the number of rearrangements. Estimates using per-gene and common variance ANOVA models indicated that 6–15% of 26,107 genes were differentially expressed between the progenitors. Individual allopolyploids showed nonadditive expression for 1.6–32% of all genes. Less than 0.3% of genes displayed nonadditive expression in all S0∶1 lines and 0.1–0.2% were nonadditive among all S5∶6 lines. Differentially expressed genes in the polyploids were over-represented by genes differential between the progenitors. The total number of differentially expressed genes was correlated with the number of genetic changes in S5∶6 lines under the common variance model; however, there was no relationship using a per-gene variance model, and many genes showed nonadditive expression in S0∶1 lines. Conclusions/Significance Few genes reproducibly demonstrated nonadditive expression among lineages, suggesting few changes resulted from a general response to polyploidization. Furthermore, our microarray analysis did not provide strong evidence that homoeologous rearrangements were a determinant of genome-wide nonadditive gene expression. In light of the inherent limitations of the Arabidopsis microarray to measure gene expression in polyploid Brassicas, further studies are warranted.

Synthetic Chromosome Platforms in Plants

Synthetic chromosomes provide the means to stack transgenes independently of the remainder of the genome. Combining them with haploid breeding could provide the means to transfer many transgenes more easily among varieties of the same species. The epigenetic nature of centromere formation complicates the production of synthetic chromosomes. However, telomere-mediated truncation coupled with the introduction of site-specific recombination cassettes has been used to produce minichromosomes consisting of little more than a centromere. Methods that have been developed to modify genes in vivo could be applied to minichromosomes to improve their utility and to continue to increase their length and genic content. Synthetic chromosomes establish the means to add or subtract multiple transgenes, multigene complexes, or whole biochemical pathways to plants to change their properties for agricultural applications or to use plants as factories for the production of foreign proteins or metabolites.

Engineered Minichromosomes in Plants

Engineered minichromosomes provide the ability to target transgenes to a defined insertion position for predictable expression on an independent chromosome. This technology promises to provide a means to add many genes to a synthetic chromosome in sequential manner. An additional advantage is that the multiple transgenes will not be inserted into the normal chromosomes and thus will not exhibit linkage drag when converging the transgenes to different germplasm nor will they be mutagenic. Telomere truncation coupled with the introduction of site-specific recombination cassettes has proven to be an easy method to produce minichromosomes. Telomere truncation results from the transformation of plasmids carrying a block of telomere repeats at one end. Minichromosomes consisting of little more than a centromere have been produced for B chromosomes of maize. Such small chromosomes have been studied for their meiotic behavior, which differs from normal sized chromosomes in that homologue pairing is rare or nonexistent and sister chromatid cohesion fails at meiosis I. Potential modifications of the minichromosomes that can address these issues are discussed. Minichromosomes can be recovered from transformed plants that are polyploid or that carry an additional chromosome as the preferred target for truncation. Site-specific recombination has been demonstrated to operate on these terminally located sites. By introducing normal B chromosomes into lines with engineered mini-B chromosomes, the latter can be increased in copy number, which provides the potential to augment the expression of the introduced genes. Because the vast majority of plant species have the same telomere sequence, the truncating transgenes should be effective in most plants to generate engineered minichromosomes. Such chromosomes establish the means to add or subtract multiple transgenes, multigene complexes, or whole biochemical pathways to plants to change their properties for agronomic applications or to use plants as factories for the production of foreign proteins or metabolites.

In vivo modification of a maize engineered minichromosome

Engineered minichromosomes provide efficient platforms for stacking transgenes in crop plants. Methods for modifying these chromosomes in vivo are essential for the development of customizable systems for the removal of selection genes or other sequences and for the addition of new genes. Previous studies have demonstrated that Cre, a site-specific recombinase, could be used to modify lox sites on transgenes on maize minichromosomes; however, these studies demonstrated somatic recombination only, and modified minichromosomes could not be recovered. We describe the recovery of an engineered chromosome composed of little more than a centromere plus transgene that was derived by telomere-mediated truncation. We used the fiber fluorescence in situ hybridization technique and detected a transgene on the minichromosome inserted among stretches of CentC centromere repeats, and this insertion was large enough to suggest a tandem insertion. By crossing the minichromosome to a plant expressing Cre-recombinase, the Bar selection gene was removed, leaving behind a single loxP site. This study demonstrates that engineered chromosomes can be modified in vivo using site-specific recombinases, a demonstration essential to the development of amendable chromosome platforms in plants.

Multiple maize minichromosomes in meiosis

In this study, four distinct minichromosomes derived from the maize B chromosome, were increased in copy number using the B chromosomes accumulation mechanism, namely nondisjunction at the second pollen mitosis and preferential fertilization of the egg. These minichromosomes provide the unique opportunity to examine the behavior of many copies of a single chromosome in an otherwise diploid background. While multiple copies were associated in multivalent configurations, they often dissociated into univalents or bivalents prior to metaphase I. The largest minis behavior closely resembled the progenitor B chromosome, but all smaller chromosomes showed failure of sister chromatid cohesion. In addition to the meiotic behavior, we observed many anomalies of univalent behavior and possible heterochromatic fusions of B repeat associated heterochromatin.

Structural and Functional Evolution of Resynthesized Polyploids

Polyploidy is widespread among the flowering plants. While many extant plant species show evidence of polyploidy in their genomes, there is still much to be learned regarding the role it has played in phenotypic evolution and speciation. The Brassica genus has polyploidy at multiple levels: the genomes of diploid species show evidence of repeated rounds of ancient polyploidization, and the agronomically important diploid species (Brassica rapa, Brassica oleracea, and Brassica nigra) may hybridize to form allopolyploids (Brassica napus, Brassica juncea, and Brassica carinata). The phenotypic diversity among these six domesticated species is spectacular. Research has provided evidence that gene and genome redundancy contribute significantly to the variation observed among Brassica species. The relative ease by which Brassica allopolyploids can be resynthesized has allowed them to emerge as an efficient model for studying the consequences of polyploidization. Studies on resynthesized B. napus polyploids have reported on homoeologous genome rearrangements and epigenetic changes, as well as changes in gene expression, protein expression, and phenotypic variation in the early generations following hybridization and polyploidization. In contrast, studies in resynthesized B. juncea polyploids show little evidence for rapid changes, although a recent report indicated changes within the organelle genomes. The differences between these two resynthesized allopolyploids may be attributed to the differing degrees of similarity between their diploid progenitor genomes (those of B. napus being more similar to each other than those of B. juncea). It may also be due to differences in genetic variation for homoeologous pairing control in these two species. These studies demonstrate that polyploidization may have immediate genetic and phenotypic consequences, particularly in B. napus. The variation that results from a polyploidization event may be critical in the early establishment and evolution of new polyploids; however, selection must have played a critical role in the establishment of natural Brassica polyploids, as their genomes appear relatively stable. Future studies of resynthesized Brassica allopolyploids should include tests for the effects of progenitor genotypes, selection for fertility under field-style conditions, and use genomic approaches that have up to now been limited to studies of polyploid Arabidopsis suecica.

Recovery of a telomere-truncated chromosome via a compensating translocation in maize

Maize-engineered minichromosomes are easily recovered from telomere-truncated B chromosomes but are rarely recovered from A chromosomes. B chromosomes lack known genes, and their truncation products are tolerated and transmitted during meiosis. In contrast, deficiency gametes resulting from truncated A chromosomes prevent their transmission. We report here a de novo compensating translocation that permitted recovery of a large truncation of chromosome 1 in maize. The truncation (trunc-1) and translocation with chromosome 6 (super-6) occurred during telomere-mediated truncation experiments and were characterized using single-gene fluorescent in situ hybridization (FISH) probes. The truncation contained a transgene signal near the end of the broken chromosome and transmitted together with the compensating translocation as a heterozygote to approximately 41%-55% of progeny. Transmission as an addition chromosome occurred in ~15% of progeny. Neither chromosome transmitted through pollen. Transgene expression (Bar) cosegregated with trunc-1 transcriptionally and phenotypically. Meiosis in T1 plants revealed eight bivalents and one tetravalent chain composed of chromosome 1, trunc-1, chromosome 6, and super-6 in diplotene and diakinesis. Our data suggest that de novo compensating translocations allow recovery of truncated A chromosomes by compensating deficiency in female gametes and by affecting chromosome pairing and segregation. The truncated chromosome can be maintained as an extra chromosome or together with the super-6 as a heterozygote.