Functional innovations of three chronological mesohexaploid Brassica rapa genomes

Background

The Brassicaceae family is an exemplary model for studying plant polyploidy. The Brassicaceae knowledge-base includes the well-annotated Arabidopsis thaliana reference sequence; well-established evidence for three rounds of whole genome duplication (WGD); and the conservation of genomic structure, with 24 conserved genomic blocks (GBs). The recently released Brassica rapa draft genome provides an ideal opportunity to update our knowledge of the conserved genomic structures in Brassica, and to study evolutionary innovations of the mesohexaploid plant, B. rapa.

Results

Three chronological B. rapa genomes (recent, young, and old) were reconstructed with sequence divergences, revealing a trace of recursive WGD events. A total of 636 fast evolving genes were unevenly distributed throughout the recent and young genomes. The representative Gene Ontology (GO) terms for these genes were ‘stress response’ and ‘development’ both through a change in protein modification or signaling, rather than by enhancing signal recognition. In retention patterns analysis, 98% of B. rapa genes were retained as collinear gene pairs; 77% of those were singly-retained in recent or young genomes resulting from death of the ancestral copies, while others were multi-retained as long retention genes. GO enrichments indicated that single retention genes mainly function in the interpretation of genetic information, whereas, multi-retention genes were biased toward signal response, especially regarding development and defense. In the recent genome, 13,302, 5,790, and 20 gene pairs were multi-retained following Brassica whole genome triplication (WGT) events with 2, 3, and 4 homoeologous copies, respectively. Enriched GO-slim terms from B. rapa homomoelogues imply that a major effect of the B. rapa WGT may have been to acquire environmental adaptability or to change the course of development. These homoeologues seem to more frequently undergo subfunctionalization with spatial expression patterns compared with other possible events including nonfunctionalization and neofunctionalization.

Conclusion

We refined Brassicaceae GB information using the latest genomic resources, and distinguished three chronologically ordered B. rapa genomes. B. rapa genes were categorized into fast evolving, single- and multi-retention genes, and long retention genes by their substitution rates and retention patterns. Representative functions of the categorized genes were elucidated, providing better understanding of B. rapa evolution and the Brassica genus.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-606) contains supplementary material, which is available to authorized users.     

Karyotype Analysis of Gossypium arboreum × G. bickii by Genome in situ Hybridization

By using genome in situ hybridization (GISH) on root somatic chromosomes of allotetraploid derived from the cross Gossypium arboreum × G. bickii with genomic DNA (gDNA) of G. bickii as a probe, two sets of chromosomes, consisting of 26 chromosomes each, were easily distinguished from each other by their distinctive hybridization signals. GISH analysis directly proved that the hybrid GarboreumxG. bickii is an allotetraploid amphiploid. The karyotype formula of the species was 2n = 4x = 52 = 46m (4sat) + 6sm (4sat). We identified four pairs of satellites with two pairs in each sub-genome. FISH analysis using 45S rDNA as a probe showed that the cross G. arboreumxG. bickii contained 14 NORs. At least five pairs of chromosomes in the G sub-genome showed double hybridization (red and blue) in their long arms, which indicates that chromatin introgression from the A sub-genome had occurred.     

Identification of all homoeologous chromosomes of newly synthetic allotetraploid <Emphasis Type="Italic">Cucumis</Emphasis> × <Emphasis Type="Italic">hytivus</Emphasis> and its wild parent reveals stable subgenome structure

Allopolyploidy and homoeologous recombination are two important processes in reshaping genomes and generating evolutionary novelties. Newly formed allopolyploids usually display chromosomal perturbations as a result of pairing errors at meiosis. To understand mechanisms of stabilization of allopolyploid species derived from distant chromosome bases, we investigated mitotic stability of a synthetic Cucumis allotetraploid species in relation to meiosis chromosome behavior. The Cucumis × hytivus is an allotetraploid synthesized from interspecific hybridization between cucumber (Cucumis sativus, 2n = 14) and its wild relative Cucumis hystrix (2n = 24) followed by spontaneous chromosome doubling. In the present study, we analyzed the wild parent C. hystrix and the latest generation of C. hytivus using GISH (genomic in situ hybridization) and cross-species FISH (fluorescence in situ hybridization). The karyotype of C. hystrix was constructed with two methods using cucumber fosmid clones and repetitive sequences. Using repeat-element probe mix in two successive hybridizations allowed for routine identification of all 19 homoeologous chromosomes of allotetraploid C. hytivus. No aneuploids were identified in any C. hytivus individuals that were characterized, and no large-scale chromosomal rearrangements were identified in this synthetic allotetraploid. Meiotic irregularities, such as homoeologous pairing, were frequently observed, resulting in univalent and intergenomic multivalent formation. The relatively stable chromosome structure of the synthetic Cucumis allotetraploid may be explained by more deleterious chromosomal viable gametes compared with other allopolyploids. The knowledge of genetic and genomic information of Cucumis allotetraploid species could provide novel insights into the establishment of allopolyploids with different chromosome bases.     

Resources and transgenesis techniques for functional genomics in Xenopus

Recent developments in genomic resources and high‐throughput transgenesis techniques have allowed Xenopus to ‘metamorphose’ from a classic model for embryology to a leading‐edge experimental system for functional genomics. This process has incorporated the fast‐breeding diploid frog, Xenopus tropicalis, as a new model‐system for vertebrate genomics and genetics. Sequencing of the X. tropicalis genome is nearly complete, and its comparison with mammalian sequences offers a reliable guide for the genome‐wide prediction of cis‐regulatory elements. Unique cDNA sets have been generated for both X. tropicalis and X. laevis, which have facilitated non‐redundant, systematic gene expression screening and comprehensive gene expression analysis. A variety of transgenesis techniques are available for both X. laevis and X. tropicalis, and the appropriate procedure may be chosen depending on the purpose for which it is required. Effective use of these resources and techniques will help to reveal the overall picture of the complex wiring of gene regulatory networks that control vertebrate development.     

Chromosomal-level genome assembly of the orchid tree Bauhinia variegata (Leguminosae; Cercidoideae) supports the allotetraploid origin hypothesis of Bauhinia

Cercidoideae, one of the six subfamilies of Leguminosae, contains one genus Cercis with its chromosome number 2n = 14 and all other genera with 2n = 28. An allotetraploid origin hypothesis for the common ancestor of non-Cercis genera in this subfamily has been proposed; however, no chromosome-level genomes from Cercidoideae have been available to test this hypothesis. Here, we conducted a chromosome-level genome assembly of Bauhinia variegata to test this hypothesis. The assembled genome is 326.4 Mb with the scaffold N50 of 22.1 Mb and contains 37,996 protein-coding genes. The Ks distribution between gene pairs in the syntenic regions indicates two whole-genome duplications (WGDs): one is B. variegata-specific, and the other is shared among core eudicots. Although Ks between gene pairs generated by the recent WGD in Bauhinia is greater than that between Bauhinia and Cercis, the WGD was not detected in Cercis, which can be explained by an accelerated evolutionary rate in Bauhinia after divergence from Cercis. Ks distribution and phylogenetic analysis for gene pairs generated by the recent WGD in Bauhinia and their corresponding orthologs in Cercis support the allopolyploidy origin hypothesis of Bauhinia. The genome of B. variegata also provides a genomic resource for dissecting genetic basis of its ornamental traits.     

Lineage-Specific Tandem Repeats Riding on a Transposable Element of MITE in <Emphasis Type="Italic">Xenopus</Emphasis> Evolution: A New Mechanism for Creating Simple Sequence Repeats

Xstir is a repetitive DNA sequence element that is extremely amplified as a common component of two different structures: a tandem repeat (Xstir array) and a MITE (miniature inverted-repeat transposable element) in the genome of Xenopus laevis. To elucidate the origin and evolutionary history of Xstir-related sequences, we investigated their species specificity among three Xenopus species (X. laevis, X. borealis, and X. tropicalis). Analyses by sequence alignment and digestion with restriction enzymes of genomic Xstir-related sequences revealed that the MITE (Xmix MITE) was well conserved among the three Xenopus species, with small lineage-specific differences. On the other hand, the tandem repeat element (tropXstir) in X. tropicalis was different from the Xstir that X. laevis and X. borealis have in common. Both sequences of Xstir and tropXstir were, however, different segments of the Xmix MITE. The results suggest that these tandem repeats were formed by partial tandem duplication of the MITE internal sequence in each lineage of X. tropicalis and of X. borealis/X. laevis after their branching. A molecular mechanism for creating and elongating the tandem repeats from the MITE is proposed.Reviewing Editor: Dr. Jerzy Jurka     

Testing for the Footprint of Sexually Antagonistic Polymorphisms in the Pseudoautosomal Region of a Plant Sex Chromosome Pair

The existence of sexually antagonistic (SA) polymorphism is widely considered the most likely explanation for the evolution of suppressed recombination of sex chromosome pairs. This explanation is largely untested empirically, and no such polymorphisms have been identified, other than in fish, where no evidence directly implicates these genes in events causing loss of recombination. We tested for the presence of loci with SA polymorphism in the plant Silene latifolia, which is dioecious (with separate male and female individuals) and has a pair of highly heteromorphic sex chromosomes, with XY males. Suppressed recombination between much of the Y and X sex chromosomes evolved in several steps, and the results in Bergero et al. (2013) show that it is still ongoing in the recombining or pseudoautosomal, regions (PARs) of these chromosomes. We used molecular evolutionary approaches to test for the footprints of SA polymorphisms, based on sequence diversity levels in S. latifolia PAR genes identified by genetic mapping. Nucleotide diversity is high for at least four of six PAR genes identified, and our data suggest the existence of polymorphisms maintained by balancing selection in this genome region, since molecular evolutionary (HKA) tests exclude an elevated mutation rate, and other tests also suggest balancing selection. The presence of sexually antagonistic alleles at a locus or loci in the PAR is suggested by the very different X and Y chromosome allele frequencies for at least one PAR gene.     

Globin evolution in the genusXenopus: Comparative analysis of cDNAs coding for adult globin polypeptides ofXenopus borealis andXenopus tropicalis

Summary Globin mRNAs ofXenopus borealis andXenopus tropicalis have been cloned and sequenced. The nucleotide and derived amino acid sequences were compared with each other and with already available data fromXenopus laevis. This analysis rendered clear evidence that the common ancestor ofX. laevis andX. borealis, but not ofX. tropicalis, had lost one amino acid of the -globins prior to a genome duplication event that preceded the segregation of the former two species. Replacement-site substitutions were used to calculate a rough time scale of genome duplication and species segregation. The results suggest an ancient separation between theX. laevis and theX. tropicalis groups occurring approximately 110–120 million years ago. Analysis of the amino acid chains demonstrated various alterations. However, some functional domains, like heme-binding sites and₁₂ contact sites, were subject to a high degree of conservation, indicating the existence of functional constraints on them also in the genusXenopus.     

Association between simple sequence repeat-rich chromosome regions and intergenomic translocation breakpoints in natural populations of allopolyploid wild wheats

Background and Aims

Repetitive DNA sequences are thought to be involved in the formation of chromosomal rearrangements. The aim of this study was to analyse the distribution of microsatellite clusters in Aegilops biuncialis and Aegilops geniculata, and its relationship with the intergenomic translocations in these allotetraploid species, wild genetic resources for wheat improvement.

Methods

The chromosomal localization of (ACG)_n and (GAA)_n microsatellite sequences in Ae. biuncialis and Ae. geniculata and in their diploid progenitors Aegilops comosa and Aegilops umbellulata was investigated by sequential in situ hybridization with simple sequence repeat (SSR) probes and repeated DNA probes (pSc119·2, Afa family and pTa71) and by dual-colour genomic in situ hybridization (GISH). Thirty-two Ae. biuncialis and 19 Ae. geniculata accessions were screened by GISH for intergenomic translocations, which were further characterized by fluorescence in situ hybridization and GISH.

Key Results

Single pericentromeric (ACG)_n signals were localized on most U and on some M genome chromosomes, whereas strong pericentromeric and several intercalary and telomeric (GAA)_n sites were observed on the Aegilops chromosomes. Three Ae. biuncialis accessions carried 7U^b–7M^b reciprocal translocations and one had a 7U^b–1M^b rearrangement, while two Ae. geniculata accessions carried 7U^g–1M^g or 5U^g–5M^g translocations. Conspicuous (ACG)_n and/or (GAA)_n clusters were located near the translocation breakpoints in eight of the ten translocated chromosomes analysed, SSR bands and breakpoints being statistically located at the same chromosomal site in six of them.

Conclusions

Intergenomic translocation breakpoints are frequently mapped to SSR-rich chromosomal regions in the allopolyploid species examined, suggesting that microsatellite repeated DNA sequences might facilitate the formation of those chromosomal rearrangements. The (ACG)_n and (GAA)_n SSR motifs serve as additional chromosome markers for the karyotypic analysis of UM genome Aegilops species.     

Cytogenetic evidence of mixed disomic and polysomic inheritance in an allotetraploid (AABB) Musa genotype

Background and Aims

Edible bananas originated mainly from two wild species, Musa acuminata Colla (AA) and Musa balbisiana Colla (BB), and triploid cultivars with an AAA, AAB or ABB genome are the most widely used. In the present study, chromosome pairing affinities are investigated in a sterile AB Indian variety and in its fertile colchicine-induced allotetraploid (AABB) derivative to determine the inheritance pattern of the tetraploid genotype. The potential implications of interspecific recombination and chromosomal composition of diploid gametes for Musa improvement are presented.

Methods

The pairing of different chromosome sets at diploid and tetraploid levels was investigated through a combination of conventional cytogenetic and genomic in-situ hybridization (GISH) analyses of meiotic chromosomes, leading to a likelihood model of the pairing behaviour. GISH analysis of mitotic chromosomes was also conducted to reveal the chromosome constitution of hybrids derived from crosses involving the allotetraploid genotype.

Key Results

Analysis of chromosome associations at both ploidy levels suggested that the newly formed allotetraploid behaves as a ‘segmental allotetraploid’ with three chromosome sets in a tetrasomic pattern, three sets in a likely disomic pattern and the five remaining sets in an intermediate pattern. Balanced and unbalanced diploid gametes were detected in progenies, with the chromosome constitution appearing to be more homogenous in pollen than in ovules.

Conclusions

Colchicine-induced allotetraploids in Musa provide access to the genetic background of natural AB varieties. The segmental inheritance pattern exhibited by the AABB allotetraploid genotype implies chromosome exchanges between M. acuminata and M. balbisiana species and opens new horizons for reciprocal transfer of valuable alleles.     

Additions, Losses, and Rearrangements on the Evolutionary Route from a Reconstructed Ancestor to the Modern Saccharomyces cerevisiae Genome

Comparative genomics can be used to infer the history of genomic rearrangements that occurred during the evolution of a species. We used the principle of parsimony, applied to aligned synteny blocks from 11 yeast species, to infer the gene content and gene order that existed in the genome of an extinct ancestral yeast about 100 Mya, immediately before it underwent whole-genome duplication (WGD). The reconstructed ancestral genome contains 4,703 ordered loci on eight chromosomes. The reconstruction is complete except for the subtelomeric regions. We then inferred the series of rearrangement steps that led from this ancestor to the current Saccharomyces cerevisiae genome; relative to the ancestral genome we observe 73 inversions, 66 reciprocal translocations, and five translocations involving telomeres. Some fragile chromosomal sites were reused as evolutionary breakpoints multiple times. We identified 124 genes that have been gained by S. cerevisiae in the time since the WGD, including one that is derived from a hAT family transposon, and 88 ancestral loci at which S. cerevisiae did not retain either of the gene copies that were formed by WGD. Sites of gene gain and evolutionary breakpoints both tend to be associated with tRNA genes and, to a lesser extent, with origins of replication. Many of the gained genes in S. cerevisiae have functions associated with ethanol production, growth in hypoxic environments, or the uptake of alternative nutrient sources.     

Evolution of satellite DNA sequences in two tribes of Bovidae: A cautionary tale

Two clones, Bt1 from Bos taurus and Om1 from Ovis orientalis musimon, were used as probes for hybridization on genomic DNA and on metaphase chromosomes in members of Bovini and Caprini tribes. Bt1 and Om1 are sequences respectively belonging to the 1.715 and 1.714 DNA satellite I families. Southern blots and fluorescence in situ hybridization experiments showed completely coherent results: the Bovini probe Bt1 hybridized only to members of the Bovini tribe and not to members of Caprini. Likewise, the Caprini probe Om1 hybridized only to members of the Caprini tribe and not to members of Bovini. Hybridization signals were detected in the heterochromatic regions of every acrocentric autosome, except for two pairs of autosomes from Capra hircus that did not show hybridization to probe Om1. No signal was detected on X and Y chromosomes or on bi-armed autosomes. Remarkably, probe Om1 showed almost 100% homology with a bacterial sequence reported in Helicobacter pylori.     

Flow sorting of C-genome chromosomes from wild relatives of wheat Aegilops markgrafii,Ae. triuncialis and Ae. cylindrica,and their molecular organization

Background and Aims Aegilops markgrafii (CC) and its natural hybrids Ae. triuncialis (U^tU^tC^tC^t) and Ae. cylindrica (D^cD^cC^cC^c) represent a rich reservoir of useful genes for improvement of bread wheat (Triticum aestivum), but the limited information available on their genome structure and the shortage of molecular (cyto-) genetic tools hamper the utilization of the extant genetic diversity. This study provides the complete karyotypes in the three species obtained after fluorescent in situ hybridization (FISH) with repetitive DNA probes, and evaluates the potential of flow cytometric chromosome sorting.Methods The flow karyotypes obtained after the analysis of 4'',6-diamidino-2-phenylindole (DAPI)-stained chromosomes were characterized and the chromosome content of the peaks on the flow karyotypes was determined by FISH. Twenty-nine conserved orthologous set (COS) markers covering all seven wheat homoeologous chromosome groups were used for PCR with DNA amplified from flow-sorted chromosomes and genomic DNA.Key Results FISH with repetitive DNA probes revealed that chromosomes 4C, 5C, 7C^t, T6U^tS.6U^tL-5C^tL, 1C^c and 5D^c could be sorted with purities ranging from 66 to 91 %, while the remaining chromosomes could be sorted in groups of 2–5. This identified a partial wheat–C-genome homology for group 4 and 5 chromosomes. In addition, 1C chromosomes were homologous with group 1 of wheat; a small segment from group 2 indicated 1C–2C rearrangement. An extensively rearranged structure of chromosome 7C relative to wheat was also detected.Conclusions The possibility of purifying Aegilops chromosomes provides an attractive opportunity to investigate the structure and evolution of the Aegilops C genome and to develop molecular tools to facilitate the identification of alien chromatin and support alien introgression breeding in bread wheat.     

Seed colour loci, homoeology and linkage groups of the C genome chromosomes revealed in Brassica rapa-B. oleracea monosomic alien addition lines

Background and Aims

Brassica rapa and B. oleracea are the progenitors of oilseed rape B. napus. The addition of each chromosome of B. oleracea to the chromosome complement of B. rapa results in a series of monosomic alien addition lines (MAALs). Analysis of MAALs determines which B. oleracea chromosomes carry genes controlling specific phenotypic traits, such as seed colour. Yellow-seeded oilseed rape is a desirable breeding goal both for food and livestock feed end-uses that relate to oil, protein and fibre contents. The aims of this study included developing a missing MAAL to complement an available series, for studies on seed colour control, chromosome homoeology and assignment of linkage groups to B. oleracea chromosomes.

Methods

A new batch of B. rapa–B. oleracea aneuploids was produced to generate the missing MAAL. Seed colour and other plant morphological features relevant to differentiation of MAALs were recorded. For chromosome characterization, Snow''s carmine, fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH) were used.

Key Results

The final MAAL was developed. Morphological traits that differentiated the MAALs comprised cotyledon number, leaf morphology, flower colour and seed colour. Seed colour was controlled by major genes on two B. oleracea chromosomes and minor genes on five other chromosomes of this species. Homoeologous pairing was largely between chromosomes with similar centromeric positions. FISH, GISH and a parallel microsatellite marker analysis defined the chromosomes in terms of their linkage groups.

Conclusions

A complete set of MAALs is now available for genetic, genomic, evolutionary and breeding perspectives. Defining chromosomes that carry specific genes, physical localization of DNA markers and access to established genetic linkage maps contribute to the integration of these approaches, manifested in the confirmed correspondence of linkage groups with specific chromosomes. Applications include marker-assisted selection and breeding for yellow seeds.