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Polyploidy is a major evolutionary force that has shaped plant diversity. However, the various pathways toward polyploid formation and interploidy gene flow remain poorly understood. Here, we demonstrated that the immediate progeny of allotriploid AAC Brassica (obtained by crossing allotetraploid Brassica napus and diploid Brassica rapa ) was predominantly aneuploids with ploidal levels ranging from near-triploidy to near-hexaploidy, and their chromosome numbers deviated from the theoretical distribution toward increasing chromosome numbers, suggesting that they underwent selection. Karyotype and phenotype analyses showed that aneuploid individuals containing fewer imbalanced chromosomes had higher viability and fertility. Within three generations of self-fertilization, allotriploids mainly developed into near or complete allotetraploids similar to B. napus via gradually increasing chromosome numbers and fertility, suggesting that allotriploids could act as a bridge in polyploid formation, with aneuploids as intermediates. Self-fertilized interploidy hybrids ultimately generated new allopolyploids carrying different chromosome combinations, which may create a reproductive barrier preventing allotetraploidy back to diploidy and promote gene flow from diploids to allotetraploids. These results suggest that the maintenance of a proper genome balance and dosage drove the recurrent conversion of allotriploids to allotetraploids, which may contribute to the formation and evolution of polyploids.

Ethiopian mustard (Brassica carinata) is an ancient crop with remarkable stress resilience and a desirable seed fatty acid profile for biofuel uses. Brassica carinata is one of six Brassica species that share three major genomes from three diploid species (AA, BB, and CC) that spontaneously hybridized in a pairwise manner to form three allotetraploid species (AABB, AACC, and BBCC). Of the genomes of these species, that of B. carinata is the least understood. Here, we report a chromosome scale 1.31-Gbp genome assembly with 156.9-fold sequencing coverage for B. carinata, completing the reference genomes comprising the classic Triangle of U, a classical theory of the evolutionary relationships among these six species. Our assembly provides insights into the hybridization event that led to the current B. carinata genome and the genomic features that gave rise to the superior agronomic traits of B. carinata. Notably, we identified an expansion of transcription factor networks and agronomically important gene families. Completion of the Triangle of U comparative genomics platform has allowed us to examine the dynamics of polyploid evolution and the role of subgenome dominance in the domestication and continuing agronomic improvement of B. carinata and other Brassica species., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Plant genomes demonstrate significant presence/absence variation (PAV) within a species; however, the factors that lead to this variation have not been studied systematically in Brassica across diploids and polyploids. Here, we developed pangenomes of polyploid Brassica napus and its two diploid progenitor genomes B. rapa and B. oleracea to infer how PAV may differ between diploids and polyploids. Modelling of gene loss suggests that loss propensity is primarily associated with transposable elements in the diploids while in B. napus, gene loss propensity is associated with homoeologous recombination. We use these results to gain insights into the different causes of gene loss, both in diploids and following polyploidization, and pave the way for the application of machine learning methods to understanding the underlying biological and physical causes of gene presence/absence., (© 2021 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Understanding the evolutionary history of crops, including identifying wild relatives, helps to provide insight for conservation and crop breeding efforts. Cultivated Brassica oleracea has intrigued researchers for centuries due to its wide diversity in forms, which include cabbage, broccoli, cauliflower, kale, kohlrabi, and Brussels sprouts. Yet, the evolutionary history of this species remains understudied. With such different vegetables produced from a single species, B. oleracea is a model organism for understanding the power of artificial selection. Persistent challenges in the study of B. oleracea include conflicting hypotheses regarding domestication and the identity of the closest living wild relative. Using newly generated RNA-seq data for a diversity panel of 224 accessions, which represents 14 different B. oleracea crop types and nine potential wild progenitor species, we integrate phylogenetic and population genetic techniques with ecological niche modeling, archaeological, and literary evidence to examine relationships among cultivars and wild relatives to clarify the origin of this horticulturally important species. Our analyses point to the Aegean endemic B. cretica as the closest living relative of cultivated B. oleracea, supporting an origin of cultivation in the Eastern Mediterranean region. Additionally, we identify several feral lineages, suggesting that cultivated plants of this species can revert to a wild-like state with relative ease. By expanding our understanding of the evolutionary history in B. oleracea, these results contribute to a growing body of knowledge on crop domestication that will facilitate continued breeding efforts including adaptation to changing environmental conditions., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

There is an increasing awareness that as a result of structural variation, a reference sequence representing a genome of a single individual is unable to capture all of the gene repertoire found in the species. A large number of genes affected by presence/absence and copy number variation suggest that it may contribute to phenotypic and agronomic trait diversity. Here we show by analysis of the Brassica oleracea pangenome that nearly 20% of genes are affected by presence/absence variation. Several genes displaying presence/absence variation are annotated with functions related to major agronomic traits, including disease resistance, flowering time, glucosinolate metabolism and vitamin biosynthesis., Competing Interests: W.R.M. has participated in Illumina-sponsored meetings over the past 4 years and has received travel reimbursement and an honorarium for presenting at these events. Illumina had no role in decisions relating to the study/work to be published, data collection, analysis of data and the decision to publish. W.R.M. has participated in Pacific Biosciences-sponsored meetings over the past 3 years and received travel reimbursement for presenting at these events. W.R.M. is a founder and shared holder of Orion Genomics, which focuses on plant genomics and cancer genetics. W.R.M. is an SAB member for RainDance Technologies, Inc. The remaining authors declare no competing financial interests.

Background: Brassica oleracea is a valuable vegetable species that has contributed to human health and nutrition for hundreds of years and comprises multiple distinct cultivar groups with diverse morphological and phytochemical attributes. In addition to this phenotypic wealth, B. oleracea offers unique insights into polyploid evolution, as it results from multiple ancestral polyploidy events and a final Brassiceae-specific triplication event. Further, B. oleracea represents one of the diploid genomes that formed the economically important allopolyploid oilseed, Brassica napus. A deeper understanding of B. oleracea genome architecture provides a foundation for crop improvement strategies throughout the Brassica genus., Results: We generate an assembly representing 75% of the predicted B. oleracea genome using a hybrid Illumina/Roche 454 approach. Two dense genetic maps are generated to anchor almost 92% of the assembled scaffolds to nine pseudo-chromosomes. Over 50,000 genes are annotated and 40% of the genome predicted to be repetitive, thus contributing to the increased genome size of B. oleracea compared to its close relative B. rapa. A snapshot of both the leaf transcriptome and methylome allows comparisons to be made across the triplicated sub-genomes, which resulted from the most recent Brassiceae-specific polyploidy event., Conclusions: Differential expression of the triplicated syntelogs and cytosine methylation levels across the sub-genomes suggest residual marks of the genome dominance that led to the current genome architecture. Although cytosine methylation does not correlate with individual gene dominance, the independent methylation patterns of triplicated copies suggest epigenetic mechanisms play a role in the functional diversification of duplicate genes.

Polyploidization has provided much genetic variation for plant adaptive evolution, but the mechanisms by which the molecular evolution of polyploid genomes establishes genetic architecture underlying species differentiation are unclear. Brassica is an ideal model to increase knowledge of polyploid evolution. Here we describe a draft genome sequence of Brassica oleracea, comparing it with that of its sister species B. rapa to reveal numerous chromosome rearrangements and asymmetrical gene loss in duplicated genomic blocks, asymmetrical amplification of transposable elements, differential gene co-retention for specific pathways and variation in gene expression, including alternative splicing, among a large number of paralogous and orthologous genes. Genes related to the production of anticancer phytochemicals and morphological variations illustrate consequences of genome duplication and gene divergence, imparting biochemical and morphological variation to B. oleracea. This study provides insights into Brassica genome evolution and will underpin research into the many important crops in this genus.

Premise of the Study: Cruciferous vegetables, many of which are in the genus Brassica (Brassicaceae), are prized for their nutritive value and have been cultivated for thousands of years. There are numerous wild northwestern Mediterranean species in the tribe Brassiceae, and it is therefore assumed this center of diversity is also the region of origin. Within the tribe, the Nigra and Oleracea clades contain the three diploid Brassica crops, B. oleracea, B. rapa, and B. nigra. These three species hybridized in the past to form the tetraploid crop species B. juncea, B. carinata, and B. napus. Collectively, these crop Brassicas have been thought to be closely related because they can still hybridize., Methods: Using a combination of molecular phylogenetics, diversification analysis, and historical biogeography, we evaluated the relationships and origins of four nested clades: the tribe Brassiceae, the Nigra-Oleracea clade, the core Oleracea (includes B. oleracea + B. rapa and their respective wild relatives), and Brassica oleracea and relatives., Key Results: We found evidence that the tribe originated around the intersection forming between the Arabian Peninsula and Saharan Africa approximately 24 million years ago (Mya). Our data also suggest that the maternal genomes of the three diploid crop Brassicas are not closely related and that the Nigra-Oleracea clade diverged 20 Mya. Finally, our analyses indicate that the core Oleracea lineage giving rise to B. oleracea + B. rapa originated ≈3 Mya in the northeastern Mediterranean, from where ancestors of B. oleracea spread through Europe and B. rapa to Asia., Conclusions: These results challenge previous hypotheses about the biogeographic origins of the tribe Brassiceae and the crop Brassica species and appear to be correlated with major geological and climatic events in the Mediterranean basin.

Following polyploidy, duplicate genes are often deleted, and if they are not, then duplicate regulatory regions are sometimes lost. By what mechanism is this loss and what is the chance that such a loss removes function? To explore these questions, we followed individual Arabidopsis thaliana-A. thaliana conserved noncoding sequences (CNSs) into the Brassica ancestor, through a paleohexaploidy and into Brassica rapa. Thus, a single Brassicaceae CNS has six potential orthologous positions in B. rapa; a single Arabidopsis CNS has three potential homeologous positions. We reasoned that a CNS, if present on a singlet Brassica gene, would be unlikely to lose function compared with a more redundant CNS, and this is the case. Redundant CNSs go nondetectable often. Using this logic, each mechanism of CNS loss was assigned a metric of functionality. By definition, proved deletions do not function as sequence. Our results indicated that CNSs that go nondetectable by base substitution or large insertion are almost certainly still functional (redundancy does not matter much to their detectability frequency), whereas those lost by inferred deletion or indels are approximately 75% likely to be nonfunctional. Overall, an average nondetectable, once-redundant CNS more than 30 bp in length has a 72% chance of being nonfunctional, and that makes sense because 97% of them sort to a molecular mechanism with "deletion" in its description, but base substitutions do cause loss. Similarly, proved-functional G-boxes go undetectable by deletion 82% of the time. Fractionation mutagenesis is a procedure that uses polyploidy as a mutagenic agent to genetically alter RNA expression profiles, and then to construct testable hypotheses as to the function of the lost regulatory site. We show fractionation mutagenesis to be a "deletion machine" in the Brassica lineage.

Background: Evolution of the Brassica species has been recursively affected by polyploidy events, and comparison to their relative, Arabidopsis thaliana, provides means to explore their genomic complexity., Results: A genome-wide physical map of a rapid-cycling strain of B. oleracea was constructed by integrating high-information-content fingerprinting (HICF) of Bacterial Artificial Chromosome (BAC) clones with hybridization to sequence-tagged probes. Using 2907 contigs of two or more BACs, we performed several lines of comparative genomic analysis. Interspecific DNA synteny is much better preserved in euchromatin than heterochromatin, showing the qualitative difference in evolution of these respective genomic domains. About 67% of contigs can be aligned to the Arabidopsis genome, with 96.5% corresponding to euchromatic regions, and 3.5% (shown to contain repetitive sequences) to pericentromeric regions. Overgo probe hybridization data showed that contigs aligned to Arabidopsis euchromatin contain ~80% of low-copy-number genes, while genes with high copy number are much more frequently associated with pericentromeric regions. We identified 39 interchromosomal breakpoints during the diversification of B. oleracea and Arabidopsis thaliana, a relatively high level of genomic change since their divergence. Comparison of the B. oleracea physical map with Arabidopsis and other available eudicot genomes showed appreciable 'shadowing' produced by more ancient polyploidies, resulting in a web of relatedness among contigs which increased genomic complexity., Conclusions: A high-resolution genetically-anchored physical map sheds light on Brassica genome organization and advances positional cloning of specific genes, and may help to validate genome sequence assembly and alignment to chromosomes.All the physical mapping data is freely shared at a WebFPC site (http://lulu.pgml.uga.edu/fpc/WebAGCoL/brassica/WebFPC/; Temporarily password-protected: account: pgml; password: 123qwe123.

Brassica carinata, an allotetraploid with B and C genomes, has a number of traits that would be valuable to introgress into B. napus. Interspecific hybrids were created between B. carinata (BBCC) and B. napus (AACC), using an advanced backcross approach to identify and introgress traits of agronomic interest from the B. carinata genome and to study the genetic changes that occur during the introgression process. We mapped the B and C genomes of B. carinata with SSR markers and observed their introgression into B. napus through a number of backcross generations, focusing on a BC(3) and BC(3)S(1) sibling family. There was close colinearity between the C genomes of B. carinata and B. napus and we provide evidence that B. carinata C chromosomes pair and recombine normally with those of B. napus, suggesting that similar to other Brassica allotetraploids no major chromosomal rearrangements have taken place since the formation of B. carinata. There was no evidence of introgression of the B chromosomes into the A or C chromosomes of B. napus; instead they were inherited as whole linkage groups with the occasional loss of terminal segments and several of the B-genome chromosomes were retained across generations. Several BC(3)S(1) families were analyzed using SSR markers, genomic in situ hybridization (GISH) assays, and chromosome counts to study the inheritance of the B-genome chromosome(s) and their association with morphological traits. Our work provides an analysis of the behavior of chromosomes in an interspecific cross and reinforces the challenges of introgressing novel traits into crop plants., (© 2011 by the Genetics Society of America)

The Brassica B-genome species possess many valuable agronomic and disease resistance traits. To transfer traits from the B genome of B. carinata into B. napus, an interspecific cross between B. napus and B. carinata was performed and a doubled haploid (DH) population was generated from the BC2S3 generation. Successful production of interspecific DH lines as identified using B-genome microsatellite markers is reported. Five percent of DH lines carry either intact B-genome chromosomes or chromosomes that have deletions. All of the DH lines have linkage group J13/B7 in common. This was further confirmed using B. nigra genomic DNA in a fluorescent in situ hybridization assay where the B-genome chromosomes were visualized and distinguished from the A- and C-genome chromosomes. The 60 DH lines were also evaluated for morphological traits in the field for two seasons and were tested for resistance to blackleg, caused by Leptosphaeria maculans, under greenhouse conditions. Variation in the DH population followed a normal distribution for several agronomic traits and response to blackleg. The lines with B-genome chromosomes were significantly different (p < 0.01) from the lines without B-genome chromosomes for both morphological and seed quality traits such as days to flowering, days to maturity, and erucic acid content.

Homoeologous regions of Brassica genomes were analyzed at the sequence level. These represent segments of the Brassica A genome as found in Brassica rapa and Brassica napus and the corresponding segments of the Brassica C genome as found in Brassica oleracea and B. napus. Analysis of synonymous base substitution rates within modeled genes revealed a relatively broad range of times (0.12 to 1.37 million years ago) since the divergence of orthologous genome segments as represented in B. napus and the diploid species. Similar, and consistent, ranges were also identified for single nucleotide polymorphism and insertion-deletion variation. Genes conserved across the Brassica genomes and the homoeologous segments of the genome of Arabidopsis thaliana showed almost perfect collinearity. Numerous examples of apparent transduplication of gene fragments, as previously reported in B. oleracea, were observed in B. rapa and B. napus, indicating that this phenomenon is widespread in Brassica species. In the majority of the regions studied, the C genome segments were expanded in size relative to their A genome counterparts. The considerable variation that we observed, even between the different versions of the same Brassica genome, for gene fragments and annotated putative genes suggest that the concept of the pan-genome might be particularly appropriate when considering Brassica genomes.

"Synthetic" allopolyploids recreated by interspecific hybridization play an important role in providing novel genomic variation for crop improvement. Such synthetic allopolyploids often undergo rapid genomic structural variation (SV). However, how such SV arises, is inherited and fixed, and how it affects important traits, has rarely been comprehensively and quantitively studied in advanced generation synthetic lines. A better understanding of these processes will aid breeders in knowing how to best utilize synthetic allopolyploids in breeding programs. Here, we analyzed three genetic mapping populations (735 DH lines) derived from crosses between advanced synthetic and conventional Brassica napus (rapeseed) lines, using whole-genome sequencing to determine genome composition. We observed high tolerance of large structural variants, particularly toward the telomeres, and preferential selection for balanced homoeologous exchanges (duplication/deletion events between the A and C genomes resulting in retention of gene/chromosome dosage between homoeologous chromosome pairs), including stable events involving whole chromosomes ("pseudoeuploidy"). Given the experimental design (all three populations shared a common parent), we were able to observe that parental SV was regularly inherited, showed genetic hitchhiking effects on segregation, and was one of the major factors inducing adjacent novel and larger SV. Surprisingly, novel SV occurred at low frequencies with no significant impacts on observed fertility and yield-related traits in the advanced generation synthetic lines. However, incorporating genome-wide SV in linkage mapping explained significantly more genetic variance for traits. Our results provide a framework for detecting and understanding the occurrence and inheritance of genomic SV in breeding programs, and support the use of synthetic parents as an important source of novel trait variation. [ABSTRACT FROM AUTHOR]

The study of domestication contributes to our knowledge of evolution and crop genetic resources. Human selection has shaped wild Brassica rapa into diverse turnip, leafy, and oilseed crops. Despite its worldwide economic importance and potential as a model for understanding diversification under domestication, insights into the number of domestication events and initial crop(s) domesticated in B. rapa have been limited due to a lack of clarity about the wild or feral status of conspecific noncrop relatives. To address this gap and reconstruct the domestication history of B. rapa , we analyzed 68,468 genotyping-by-sequencing-derived single nucleotide polymorphisms for 416 samples in the largest diversity panel of domesticated and weedy B. rapa to date. To further understand the center of origin, we modeled the potential range of wild B. rapa during the mid-Holocene. Our analyses of genetic diversity across B. rapa morphotypes suggest that noncrop samples from the Caucasus, Siberia, and Italy may be truly wild, whereas those occurring in the Americas and much of Europe are feral. Clustering, tree-based analyses, and parameterized demographic inference further indicate that turnips were likely the first crop type domesticated, from which leafy types in East Asia and Europe were selected from distinct lineages. These findings clarify the domestication history and nature of wild crop genetic resources for B. rapa , which provides the first step toward investigating cases of possible parallel selection, the domestication and feralization syndrome, and novel germplasm for Brassica crop improvement. [ABSTRACT FROM AUTHOR]

Summary: Many crops are polyploid or have a polyploid ancestry. Recent phylogenetic analyses have found that polyploidy often preceded the domestication of crop plants. One explanation for this observation is that increased genetic diversity following polyploidy may have been important during the strong artificial selection that occurs during domestication.In order to test the connection between domestication and polyploidy, we identified and examined candidate genes associated with the domestication of the diverse crop varieties of Brassica rapa. Like all 'diploid' flowering plants, B. rapa has a diploidized paleopolyploid genome and experienced many rounds of whole genome duplication (WGD). We analyzed transcriptome data of more than 100 cultivated B. rapa accessions.Using a combination of approaches, we identified > 3000 candidate genes associated with the domestication of four major B. rapa crop varieties. Consistent with our expectation, we found that the candidate genes were significantly enriched with genes derived from the Brassiceae mesohexaploidy. We also observed that paleologs were significantly more diverse than non‐paleologs.Our analyses find evidence for that genetic diversity derived from ancient polyploidy played a key role in the domestication of B. rapa and provide support for its importance in the success of modern agriculture. [ABSTRACT FROM AUTHOR]

Genes that are inherently subject to strong selective constraints tend to be overretained in duplicate after polyploidy. They also continue to experience similar, but somewhat relaxed, constraints after that polyploidy event. We sought to assess for how long the influence of polyploidy is felt on these genes' selective pressures. We analyzed two nested polyploidy events in Brassicaceae: theAt-a genome duplication that is the most recent polyploidy in themodel plant Arabidopsis thaliana and a more recent hexaploidy shared by thegenus Brassica andits relatives. By comparing the strength and direction of thenatural selection acting at the population andat the species level, we find evidence for continued intensified purifying selection acting on retained duplicates from both polyploidies even down to the present. The constraint observed in preferentially retained genes is not a result of the polyploidy event: the orthologs of such genes experience even stronger constraint in nonpolyploid outgroup genomes. In both the Arabidopsis and Brassica lineages, we further find evidence for segregating mildly deleterious variants, confirming that the population-level data uncover patterns not visiblewith between-species comparisons. Using the A. thalianametabolic network, we also explored whether network position was correlated with the measured selective constraint. At both the population and species level, nodes/genes tended to show similar constraints to their neighbors. Our results paint a picture of the long-lived effects of polyploidy on plant genomes, suggesting that even yesterday's polyploids still have distinct evolutionary trajectories. [ABSTRACT FROM AUTHOR]

Demographic modelling is often used with population genomic data to infer the relationships and ages among populations. However, relatively few analyses are able to validate these inferences with independent data. Here, we leverage written records that describe distinct Brassica rapa crops to corroborate demographic models of domestication. Brassica rapa crops are renowned for their outstanding morphological diversity, but the relationships and order of domestication remain unclear. We generated genomewide SNPs from 126 accessions collected globally using high-throughput transcriptome data. Analyses of more than 31,000 SNPs across the B. rapa genome revealed evidence for five distinct genetic groups and supported a European-Central Asian origin of B. rapa crops. Our results supported the traditionally recognized South Asian and East Asian B. rapa groups with evidence that pak choi, Chinese cabbage and yellow sarson are likely monophyletic groups. In contrast, the oil-type B. rapa subsp. oleifera and brown sarson were polyphyletic. We also found no evidence to support the contention that rapini is the wild type or the earliest domesticated subspecies of B. rapa. Demographic analyses suggested that B. rapa was introduced to Asia 2,400-4,100 years ago, and that Chinese cabbage originated 1,200-2,100 years ago via admixture of pak choi and European-Central Asian B. rapa. We also inferred significantly different levels of founder effect among the B. rapa subspecies. Written records from antiquity that document these crops are consistent with these inferences. The concordance between our age estimates of domestication events with historical records provides unique support for our demographic inferences. [ABSTRACT FROM AUTHOR]

The crop species Brassica rapa L. has significant economic importance around the world. However, the global distribution and complex evolutionary history of the species has made investigating its genetic population structure difficult. Crop domestication and improvement has resulted in extreme phenotypic diversity and subspecies that are used for oilseed, food for human consumption, and fodder for livestock. These subspecies include the oilseed morphotypes. oleifera (turnip rape), ssp. dichotoma (brown sarson/toria), ssp. trilocularis (yellow sarson); ssp. rapa (turnip); and Asian leafy vegetables ssp. pekinensis (Chinese cabbage), ssp. chinensis (bok choy), ssp. nipposinica (mizuna/mibuna), ssp. rapifera (rapini/broccoli rabe), ssp. narinosa (tatsoi), ssp parachinensis (choy sum), and ssp. perviridis (komatsuna). To date, studies have had insufficient sampling to determine the relationship of all morphotypes, especially oilseed morphotypes, and questions remain over the contribution of morphotype and geographic origin to population structure. We used genotyping-by-sequencing to score 18,272 single nucleotide polymorphism markers in a globally diverse panel of 333 B. rapa National Plant Germplasm System accessions that included 10 recognized subspecies. Our population genetic and phylogenetic analyses were broadly congruent and revealed five subpopulations that were largely reflective of morphotype and geography. These subpopulations were 1. European turnips/oilseed, 2. Asian turnips/oilseed, 3. yellow/brown sarson (ssp. trilocularis and ssp. dichotoma), 4. Chinese cabbage (ssp. pekinensis), and 5. bok choy, choy sum, and tatsoi (ssp. chinensis, ssp. parachinensis, ssp. narinosa). Additionally, we found evidence of polyphyly and/or paraphyly, particularly for oilseed morphotypes (ssp. oleifera and ssp. dichotoma) and turnips. The results of this study have provided improved resolution to the genetic and phylogenetic relationships of subspecies within the species B. rapa. Understanding of these relationships is key to the future genetic study and improvement of this globally important crop species. [ABSTRACT FROM AUTHOR]

The internal transcribed spacers of the nuclear ribosomal RNA gene cluster, termed ITS1 and ITS2, are the most frequently used nuclear markers for phylogenetic analyses across many eukaryotic groups including most plant families. The reasons for the popularity of these markers include: 1.) Ease of amplification due to high copy number of the gene clusters, 2.) Available cost-effective methods and highly conserved primers, 3.) Rapidly evolving markers (i.e. variable between closely related species), and 4.) The assumption (and/or treatment) that these sequences are non-functional, neutrally evolving phylogenetic markers. Here, our analyses of ITS1 and ITS2 for 50 species suggest that both sequences are instead under selective constraints to preserve proper secondary structure, likely to maintain complete self-splicing functions, and thus are not neutrally-evolving phylogenetic markers. Our results indicate the majority of sequence sites are co-evolving with other positions to form proper secondary structure, which has implications for phylogenetic inference. We also found that the lowest energy state and total number of possible alternate secondary structures are highly significantly different between ITS regions and random sequences with an identical overall length and Guanine-Cytosine (GC) content. Lastly, we review recent evidence highlighting some additional problematic issues with using these regions as the sole markers for phylogenetic studies, and thus strongly recommend additional markers and cost-effective approaches for future studies to estimate phylogenetic relationships. [ABSTRACT FROM AUTHOR]

Whole-genome duplications happen repeatedly in a typical flowering plant lineage. Following most ancient tetraploidies, the two subgenomes are distinguishable because one subgenome, the dominant subgenome, tends to have more genes than the other subgenome. Additionally, among retained pairs, the gene on the dominant subgenome tends to be expressed more than its recessive homeolog. Using comparative genomics, we show that genome dominance is heritable. The dominant subgenome of one postpolyploidy event remains dominant through a subsequent polyploidy event. We show that transposon-derived 24-nt RNAs target and cover the upstream region of retained genes preferentially when located on the recessive subgenome, and with little regard for a gene's level of expression. We hypothesize that small RNA (smRNA)-mediated silencing of transposons near genes causes position-effect down-regulation. Unlike 24-nt smRNA coverage, transposon coverage tracks gene expression, so not all transposons behave identically. We propose that successful ancient tetraploids begin as wide crosses between two lines, each evolved for different tradeoffs between transposon silencing and negative position effects on gene expression. We hypothesize that following a chaotic wide-cross/new tetraploid period, genes acquire their new expression balances based on differences in transposon coverage in the parents. We envision patches of silenceable transposon as quantitative c/s-regulators of baseline transcription rate. Attractive solutions to heterosis and the C-value paradox are mentioned. [ABSTRACT FROM AUTHOR]

Brassicas boast an enormous diversity of economically important products. Strikingly, relationships among main lineages are still unknown. The phylogeny of the tribe Brassiceae (Brassicaceae) was reconstructed for 89 species using four plastid regions (rpl32-trnL, atpI-atpH,psbD-trnT,ycf6-psbM). Representatives of almost all genera were sampled, covering the entire natural range of the tribe from Central Asia to the western Mediterranean, including four previously unsampled genera (sAmmosperma, Eremophyton, Fezia, Pseudofortuynia). Phylogenetic analysis recovered eight well-supported clades in the tribe including a new African clade (Henophyton) comprised of genera that have not been previously sampled. Relationships within and between the eight major clades are strongly supported for the first time. The earliest-divergent lineages in the tribe are the clades Vella and Zilla. Henophyton is sister to the clade that includes the lineages Nigra, Crambe, Cakile, Savignya, and Oleracea. Core Brassiceae--a new clade defined here--is comprised of two subclades: (1) Nigra, Crambe, and Cakile and (2) Savignya and Oleracea. Within the Oleracea lineage, a new Core Oleracea clade is defined. Several genera were confirmed to be polyphyletic or paraphyletic, including Brassica, Eracastrum, Sinapis, Diplotaxis and Cakile. Economically important species belonging to the genera Brassica were primarily distributed across the two most species-rich lineages, Nigra or Oleracea. Collectively, the finding of these novel clades has numerous taxonomic implications. [ABSTRACT FROM AUTHOR]

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Bacterial artificial chromosome (BAC) contigs have been genetically mapped to the 10 linkage groups of Brassica rapa by BAC end sequences (BES). To integrate the genetic, physical, and cytogenetic maps, fluorescence in situ hybridization (FISH) was used to anchor the assembly of BAC contigs onto Brassica chromosomes using representative BACs. This BAC-FISH approach can be used to identify chromosome arms on separate mitotic metaphase chromosomes or to map multiple BACs to single long pachytene chromosomes. As part of an international consortium that is sequencing the B. rapa genome, we integrated the linkage and physical maps with the B. rapa cytogenetic map for chromosome A7 by hybridizing BACs to mitotic chromosomes and along the length of pachytene chromosome spreads. A total of 31 BACs that were putatively located on A7 were used as probes for FISH analyses; however, only 19 BACs mapped unambiguously to A7 while the remaining BACs either mapped to other chromosomes or hybridized to multiple locations. We then created a multicolor FISH cocktail of 16 BAC probes to simultaneously hybridize the entire length of the A7 chromosome. We successfully applied the 16 A7 BAC probe mix to B. rapa, B. oleracea, and domesticated and resynthesized genotypes of B. napus to demonstrate that this approach can facilitate studies of genome evolution by integrating the genetic, physical, and cytogenetic maps among closely related species of Brassica. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Homoeologous regions of Brassica genomes were analyzed at the sequence level. These represent segments of the Brassica A genome as found in Brassica rapa and Brassica napus and the corresponding segments of the Brassica C genome as found in Brassica oleracea and B. napus. Analysis of synonymous base substitution rates within modeled genes revealed a relatively broad range of times (0.12 to 1.37 million years ago) since the divergence of orthologous genome segments as represented in B. napus and the diploid species. Similar, and consistent, ranges were also identified for single nucleotide polymorphism and insertion-deletion variation. Genes conserved across the Brassica genomes and the homoeologous segments of the genome of Arabidopsis thaliana showed almost perfect collinearity. Numerous examples of apparent transduplication of gene fragments, as previously reported in B. oleracea, were observed in B. rapa and B napus, indicating that this phenomenon is widespread in Brassica species. In the majority of the regions studied, the C genome segments were expanded in size relative to their A genome counterparts. The considerable variation that we observed, even between the different versions of the same Brassica genome, for gene fragments and annotated putative genes suggest that the concept of the pan-genome might be particularly appropriate when considering Brassica genomes [ABSTRACT FROM AUTHOR]

Background: The Brassica species, related to Arabidopsis thaliana, include an important group of crops and represent an excellent system for studying the evolutionary consequences of polyploidy. Previous studies have led to a proposed structure for an ancestral karyotype and models for the evolution of the B. rapa genome by triplication and segmental rearrangement, but these have not been validated at the sequence level. Results: We developed computational tools to analyse the public collection of B. rapa BAC end sequence, in order to identify candidates for representing collinearity discontinuities between the genomes of B. rapa and A. thaliana. For each putative discontinuity, one of the BACs was sequenced and analysed for collinearity with the genome of A. thaliana. Additional BAC clones were identified and sequenced as part of ongoing efforts to sequence four chromosomes of B. rapa. Strikingly few of the 19 inter-chromosomal rearrangements corresponded to the set of collinearity discontinuities anticipated on the basis of previous studies. Our analyses revealed numerous instances of newly detected collinearity blocks. For B. rapa linkage group A8, we were able to develop a model for the derivation of the chromosome from the ancestral karyotype. We were also able to identify a rearrangement event in the ancestor of B. rapa that was not shared with the ancestor of A. thaliana, and is represented in triplicate in the B. rapa genome. In addition to inter-chromosomal rearrangements, we identified and analysed 32 BACs containing the end points of segmental inversion events. Conclusion: Our results show that previous studies of segmental collinearity between the A. thaliana, Brassica and ancestral karyotype genomes, although very useful, represent over-simplifications of their true relationships. The presence of numerous cryptic collinear genome segments and the frequent occurrence of segmental inversions mean that inference of the positions of genes in B. rapa based on the locations of orthologues in A. thaliana can be misleading. Our results will be of relevance to a wide range of plants that have polyploid genomes, many of which are being considered according to a paradigm of comprising conserved synteny blocks with respect to sequenced, related genomes. [ABSTRACT FROM AUTHOR]

Allopolyploid formation requires the adaptation of two nuclear genomes within a single cytoplasm, which may involve programmed genetic and epigenetic changes during the initial generations following genome fusion. To study the dynamics of genome change, we synthesized 49 isogenic Brassica napus allopolyploids and surveyed them with 76 restriction fragment length polymorphism (RFLP) probes and 30 simple sequence repeat (SSR) primer pairs. Here, we report on the types and distribution of genetic and epigenetic changes within the S1 genotypes. We found that insertion/deletion (indel) events were rare, but not random. Of the 57,710 (54,383 RFLP and 3,327 SSR) parental fragments expected among the amphidiploids, we observed 56,676 or 99.9%. Three loci derived from Brassica rapa had indels, and one indel occurred repeatedly across 29% (14/49) of the lines. Loss of one parental fragment was due to the 400-bp reduction of a guanine-adenine dinucleotide repeat-rich sequence. In contrast to the 4% (3/76) RFLP probes that detected indels, 48% (35/73) detected changes in the CpG methylation status between parental genomes and the S1 lines. Some loci were far more likely than others to undergo epigenetic change, but the number of methylation changes within each synthetic polyploid was remarkably similar to others. Clear de novo methylation occurred at a much higher frequency than de novo demethylation within allopolyploid sequences derived from B. rapa. Our results suggest that there is little genetic change in the So generation of resynthesized B. napus polyploids. In contrast, DNA methylation was altered extensively in a pattern that indicates tight regulation of epigenetic changes. [ABSTRACT FROM AUTHOR]

Novel phenotypes often arise in generations immediately following polyploidization. Previous studies have shown that separate lineages derived from a resynthesized Brassica napus allopolyploid rapidly evolved heritable differences in flowering time. These early-flowering and late-flowering polyploid lines were expected to be genetically identical because they were derived from a single, chromosome-doubled amphihaploid plant. In this study, we investigated the molecular genetic basis for these flowering time differences. We assessed the diploid B. rapa and B. oleracea parents and the early- and late-flowering B. napus lineages for changes in genome structure, and for changes in transcript levels of four sets of FLOWERING LOCUS C ( FLC) genes. No such changes were observed for BnFLC1, but we detected chromosomal rearrangements (e.g. de novo non-reciprocal transpositions) and changes in transcript level for BnFLC2 and BnFLC3 between the early- and late-flowering B. napus. A chromosomal rearrangement of a genomic segment containing BnFLC3 was responsible for 29% of the phenotypic variation among the B. napus lines. Expression of BnFLC5 was silenced in all polyploids, although no changes in genome structure were detected. An ongoing investigation of 50 identical B. napus allopolyploids may further reveal the dynamics of changes in phenotype, genome and transcriptome at the early stages in polyploid evolution. © 2004 The Linnean Society of London, Biological Journal of the Linnean Society, 2004, 82, 675–688. [ABSTRACT FROM AUTHOR]

The crop species within the genus Brassica have highly replicated genomes. Three base ‘diploid’ species, Brassica oleracea, B. nigra and B. rapa, are likely ancient polyploids, and three derived allopolyploid species, B. carinata, B. juncea and B. napus, are created from the interspecific hybridization of these base genomes. The base Brassica genome is thought to have hexaploid ancestry, and both recent and ancient polyploidization events have been proposed to generate a large number of genome rearrangements and novel genetic variation for important traits. Here, we revisit and refine these hypotheses. We have examined the B. oleracea linkage map using the Arabidopsis thaliana genome sequence as a template and suggest that there is strong evidence for genome replication and rearrangement within the base Brassicas, but less evidence for genome triplication. We show that novel phenotypic variation within the base Brassicas can be achieved by replication of a single gene, BrFLC, that acts additively to influence flowering time. Within the derived allopolyploids, intergenomic heterozygosity is associated with higher seed yields. Some studies have reported that de novo genomic variation occurs within derived polyploid genomes, whereas other studies have not detected these changes. We discuss reasons for these different findings. Large translocations and tetrasomic inheritance can explain some but not all genomic changes within the polyploids. Transpositions and other small-scale sequence changes probably also have contributed to genomic novelty. Our results have shown that the Brassica genomes are remarkably plastic, and that polyploidy generates novel genetic variation through gene duplication, intergenomic heterozygosity and perhaps epigenetic change. © 2004 The Linnean Society of London, Biological Journal of the Linnean Society, 2004, 82, 665–674. [ABSTRACT FROM AUTHOR]

Synthetic oligonucleotides (oligos) represent an attractive alternative to cDNA amplicons for spotted microarray analysis in a number of model organisms, including Arabidopsis, C. elegans, Drosophila, human, mouse and yeast. However, little is known about the relative effectiveness of 60–70-mer oligos and cDNAs for detecting gene expression changes. Using 192 pairs of Arabidopsis thaliana cDNAs and corresponding 70-mer oligos, we performed three sets of dye-swap experiments and used analysis of variance ( anova) to compare sources of variation and sensitivities for detecting gene expression changes in A. thaliana, A. arenosa and Brassica oleracea. Our major findings were: (1) variation among different RNA preparations from the same tissue was small, but large variation among dye-labellings and slides indicates the need to replicate these factors; (2) sources of variation were similar for experiments with all three species, suggesting these feature types are effective for analysing gene expression in related species; (3) oligo and cDNA features had similar sensitivities for detecting expression changes and they identified a common subset of significant genes, but results from quantitative RT-PCR did not support the use of one over the other. These findings indicate that spotted oligos are at least as effective as cDNAs for microarray analyses of gene expression. We are using oligos designed from ∼26 000 annotated genes of A. thaliana to study gene expression changes in Arabidopsis and Brassica polyploids. [ABSTRACT FROM AUTHOR]

The genome sequence of the paleohexaploid Brassica rapa shows that fractionation is biased among the three subgenomes and that the least fractionated subgenome has approximately twice as many orthologs as its close (and relatively unduplicated) relative Arabidopsis than had either of the other two subgenomes. One evolutionary scenario is that the two subgenomes with heavy gene losses (I and II) were in the same nucleus for a longer period of time than the third subgenome (III) with the fewest gene losses. This "two-step" hypothesis is essentially the same as that proposed previously for the eudicot paleohexaploidy; however, the more recent nature of the B. rapa paleohexaploidy makes this model more testable. We found that subgenome II suffered recent small deletions within exons more frequently than subgenome I, as would be expected if the genes in subgenome I had already been near maximally fractionated before subgenome III was introduced. We observed that some sequences, before these deletions, were flanked by short direct repeats, a unique signature of intrachromosomal illegitimate recombination. We also found, through simulations, that short--single or two-gene--deletions appear to dominate the fractionation patterns in B. rapa. We conclude that the observed patterns of the triplicated regions in the Brassica genome are best explained by a two-step fractionation model. The triplication and subsequent mode of fractionation could influence the potential to generate morphological diversity--a hallmark of the Brassica genus. [ABSTRACT FROM AUTHOR]