Evolution and taxonomic split of the model grass Brachypodium distachyon

Background and Aims

Brachypodium distachyon is being widely investigated across the world as a model plant for temperate cereals. This annual plant has three cytotypes (2n =  10, 20, 30) that are still regarded as part of a single species. Here, a multidisciplinary study has been conducted on a representative sampling of the three cytotypes to investigate their evolutionary relationships and origins, and to elucidate if they represent separate species.

Methods

Statistical analyses of 15 selected phenotypic traits were conducted in individuals from 36 lines or populations. Cytogenetic analyses were performed through flow cytometry, fluorescence in situ hybridization (FISH) with genomic (GISH) and multiple DNA sequences as probes, and comparative chromosome painting (CCP). Phylogenetic analyses were based on two plastid (ndhF, trnLF) and five nuclear (ITS, ETS, CAL, DGAT, GI) genes from different Brachypodium lineages, whose divergence times and evolutionary rates were estimated.

Key Results

The phenotypic analyses detected significant differences between the three cytotypes and demonstrated stability of characters in natural populations. Genome size estimations, GISH, FISH and CCP confirmed that the 2n = 10 and 2n = 20 cytotypes represent two different diploid taxa, whereas the 2n = 30 cytotype represents the allotetraploid derived from them. Phylogenetic analysis demonstrated that the 2n = 20 and 2n = 10 cytotypes emerged from two independent lineages that were, respectively, the maternal and paternal genome donors of the 2n = 30 cytotype. The 2n = 20 lineage was older and mutated significantly faster than the 2n = 10 lineage and all the core perennial Brachypodium species.

Conclusions

The substantial phenotypic, cytogenetic and molecular differences detected among the three B. distachyon sensu lato cytotypes are indicative of major speciation processes within this complex that allow their taxonomic separation into three distinct species. We have kept the name B. distachyon for the 2n = 10 cytotype and have described two novel species as B. stacei and B. hybridum for, respectively, the 2n = 20 and 2n = 30 cytotypes.     

Comparative analysis of rDNA distribution in chromosomes of various species of Brassicaceae

BACKGROUND AND AIMS: The Brassicaceae family encompasses numerous species of great agronomic importance, belonging to such genera, as Brassica, Raphanus, Sinapis and Armoracia. Many of them are characterized by extensive intraspecific diversity of phenotypes. The present study focuses on the polymorphism of number, appearance and chromosomal localization of ribosomal DNA (rDNA) sites and, when possible, in relation to polyploidy, in 42 accessions of Brassica species and ten accessions of Diplotaxis, Eruca, Raphanus and Sinapis species. METHODS: Chromosomal localization of ribosomal DNA was carried out using dual colour fluorescence in situ hybridization (FISH) with 5S rDNA and 25S rDNA sequences as probes on enzymatically digested root-tip meristematic cells. KEY RESULTS: Loci for 5S and 18S-5.8S-25S rDNA were determined for the first time in six taxa, and previously unreported rDNA constellations were described in an additional 12 accessions. FISH revealed frequent polymorphism in number, appearance and chromosomal localization of both 5S and 25S rDNA sites. This phenomenon was most commonly observed in the A genome of Brassica, where it involves exclusively pericentromeric sites of 5S and 25S rRNA genes. The intraspecific polymorphism was between subspecies/varieties or within a variety or cultivar (i.e. interindividual). CONCLUSIONS: The number of rDNA sites can differ up to 5-fold in species with the same chromosome number. In addition to the eight previously reported chromosomal types with ribosomal genes, three new variant types are described. The extent of polymorphism is genome dependent. Comparing the A, B and C genomes revealed the highest rDNA polymorphism in the A genome. The loci carrying presumably inactive ribosomal RNA genes are particularly prone to polymorphism. It can also be concluded that there is no obvious polyploidization-related tendency to reduce the number of ribosomal DNA loci in the allotetraploid species, when compared with their putative diploid progenitors. The observed differences are rather caused by the prevailing polymorphism within the diploids and allotetraploids. This would make it difficult to predict expected numbers of rDNA loci in natural polyploids.     

In situ analysis of epigenetic modifications in the chromatin of Brachypodium distachyon embryos

Epigenetic modifications of the chromatin structure are crucial for many biological processes and act on genes during the development and germination of seeds. The spatial distribution of 3 epigenetic markers, i.e. H4K5ac, H3K4me2 and H3K4me1 was investigated in ‘matured,’ ‘dry,’ ‘imbibed” and ‘germinating’ embryos of a model grass, Brachypodium. Our results indicate that the patterns of epigenetic modification differ in the various types of tissues of embryos that were analyzed. Such a tissue-specific manner of these modifications may be linked to the switch of the gene expression profiles in various organs of the developing embryo.     

Brachypodium distachyon as a new model system for understanding iron homeostasis in grasses: phylogenetic and expression analysis of Yellow Stripe-Like (YSL) transporters

Background and Aims

Brachypodium distachyon is a temperate grass with a small stature, rapid life cycle and completely sequenced genome that has great promise as a model system to study grass-specific traits for crop improvement. Under iron (Fe)-deficient conditions, grasses synthesize and secrete Fe(III)-chelating agents called phytosiderophores (PS). In Zea mays, Yellow Stripe1 (ZmYS1) is the transporter responsible for the uptake of Fe(III)–PS complexes from the soil. Some members of the family of related proteins called Yellow Stripe-Like (YSL) have roles in internal Fe translocation of plants, while the function of other members remains uninvestigated. The aim of this study is to establish brachypodium as a model system to study Fe homeostasis in grasses, identify YSL proteins in brachypodium and maize, and analyse their expression profiles in brachypodium in response to Fe deficiency.

Methods

The YSL family of proteins in brachypodium and maize were identified based on sequence similarity to ZmYS1. Expression patterns of the brachypodium YSL genes (BdYSL genes) were determined by quantitative RT–PCR under Fe-deficient and Fe-sufficient conditions. The types of PS secreted, and secretion pattern of PS in brachypodium were analysed by high-performance liquid chromatography.

Key Results

Eighteen YSL family members in maize and 19 members in brachypodium were identified. Phylogenetic analysis revealed that some YSLs group into a grass-specific clade. The Fe status of the plant can regulate expression of brachypodium YSL genes in both shoots and roots. 3-Hydroxy-2′-deoxymugineic acid (HDMA) is the dominant type of PS secreted by brachypodium, and its secretion is diurnally regulated.

Conclusions

PS secretion by brachypodium parallels that of related crop species such as barley and wheat. A single grass species-specific YSL clade is present, and expression of the BdYSL members of this clade could not be detected in shoots or roots, suggesting grass-specific functions in reproductive tissues. Finally, the Fe-responsive expression profiles of several YSLs suggest roles in Fe homeostasis.     

Comparative genome analysis of Solanum lycopersicum and Solanum tuberosum

Solanum lycopersicum and Solanum tuberosum are agriculturally important crop species as they are rich sources of starch, protein, antioxidants, lycopene, beta-carotene, vitamin C, and fiber. The genomes of S. lycopersicum and S. tuberosum are currently available. However the linear strings of nucleotides that together comprise a genome sequence are of limited significance by themselves. Computational and bioinformatics approaches can be used to exploit the genomes for fundamental research for improving their varieties. The comparative genome analysis, Pfam analysis of predicted reviewed paralogous proteins was performed. It was found that S. lycopersicum proteins belong to more families, domains and clans in comparison with S. tuberosum. It was also found that mostly intergenic regions are conserved in two genomes followed by exons, intron and UTR. This can be exploited to predict regions between genomes that are similar to each other and to study the evolutionary relationship between two genomes, leading towards the development of disease resistance, stress tolerance and improved varieties of tomato.     

Genome-wide exploration of the molecular evolution and regulatory network of mitogen-activated protein kinase cascades upon multiple stresses in Brachypodium distachyon

Background

Brachypodium distachyon is emerging as a widely recognized model plant that has very close relations with several economically important Poaceae species. MAPK cascade is known to be an evolutionarily conserved signaling module involved in multiple stresses. Although the gene sequences of MAPK and MAPKK family have been fully identified in B. distachyon, the information related to the upstream MAPKKK gene family especially the regulatory network among MAPKs, MAPKKs and MAPKKKs upon multiple stresses remains to be understood.

Results

In this study, we have identified MAPKKKs which belong to the biggest gene family of MAPK cascade kinases. We have systematically investigated the evolution of whole MAPK cascade kinase gene family in terms of gene structures, protein structural organization, chromosomal localization, orthologs construction and gene duplication analysis. Our results showed that most BdMAPK cascade kinases were located at the low-CpG-density region, and the clustered members in each group shared similar structures of the genes and proteins. Synteny analysis showed that 62 or 21 pairs of duplicated orthologs were present between B. distachyon and Oryza sativa, or between B. distachyon and Arabidopsis thaliana respectively. Gene expression data revealed that BdMAPK cascade kinases were rapidly regulated by stresses and phytohormones. Importantly, we have constructed a regulation network based on co-expression patterns of the expression profiles upon multiple stresses performed in this study.

Conclusions

BdMAPK cascade kinases were involved in the signaling pathways of multiple stresses in B. distachyon. The network of co-expression regulation showed the most of duplicated BdMAPK cascade kinase gene orthologs demonstrated their convergent function, whereas few of them developed divergent function in the evolutionary process. The molecular evolution analysis of identified MAPK family genes and the constructed MAPK cascade regulation network under multiple stresses provide valuable information for further investigation of the functions of BdMAPK cascade kinase genes.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1452-1) contains supplementary material, which is available to authorized users.     

Chromosome identification and karyotype analysis of Podophyllum hexandrum Roxb. ex Kunth using FISH

Podophyllum hexandrum is an important high altitude medicinal plant from Himalaya. Somatic chromosomes of this species were studied to delineate and physical mapping of repetitive rDNA sites to provide landmarks in chromosome identification. The karyotype formula of this species was found to be 6m + 2sm + 2st + 2t with secondary constriction in the chromosome 1 and 7. The FISH analysis of rDNA sites showed 4 sites for 18S rDNA and 2 sites for 5S rDNA. The chromosome number 1, 2, 5 and 6 can be identified based on 18S rDNA sites in their short arm and chromosome 1 and 2 can be identified by 5S rDNA site in the centromere region. The estimated genome size of this plant is 16.07 pg (1C).     

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.     

Tandem 5S ribosomal RNA genes and the spacer region sequences of three Japanese Laminaria species

The organization of 5S ribosomal RNA genes (rDNA) was examined for threeJapanese Laminaria species, L. japonica, L.religiosa and L. ochotensis. The linkage of 5SrDNA with the 18S-5.8S-25S rDNAs unit known in the brown algaScytosiphon lomentaria could not be detected inLaminaria. Instead, the tandem repeats of 5S rDNA were notassociated with the 18S-5.8S-25S rDNAs unit. The nucleotide sequence of 5S rDNAwas completely identical among these three species and its length was 118bp. However, a difference of nucleotide arrangement was detectedinthe spacer region of the tandemly repeated 5S rDNAs. Several nucleotideinsertion / deletions and substitutions were confirmed between differentindividuals of L. japonica, which were collected from notonly disjunct localities, but also the same locality. The lengths of the spacerregion of L. japonica, L. religiosaand L. ochotensis were 247–252 bp, 232bp and 252 bp, respectively.     

Fingerprint of Biomphalaria arabica, the intermediate host of Schistosoma mansoni in Saudi Arabia, using RAPD-PCR

In the time schistosomisis control programs are implemented in many countries, schistosomiasis continues to spread throughout the world. Among these control strategies is the vector control. Within this context, analysis of the genetic variability of the intermediate host snails is important because it allows identification of specific sequences of the genome of this mollusk related to determine their fingerprint. We investigated Biomphalaria arabica, which is found in Saudi Arabia, the intermediate host of Schistosoma mansoni infection. Genetic fingerprint was studied by RAPD-PCR using our own different random primers as well as published primers. The electrophoretic patterns resulting from amplification showed specific polymorphic markers of B. arabica. This information will be helpful in the identification of the snails and demonstrating that RAPD-PCR is an appropriate and efficient methodological approach for establishment of genetic barcode development.     

Characterization of the complete mitochondrial genomes of Cnaphalocrocis medinalis and Chilo suppressalis (Lepidoptera: Pyralidae)

The complete mitochondrial genomes (mitogenomes) of Cnaphalocrocis medinalis and Chilo suppressalis (Lepidoptera: Pyralidae) were determined and analyzed. The circular genomes were 15,388 bp long for C. medinalis and 15,395 bp long for C. suppressalis. Both mitogenomes contained 37 genes, with gene order similar to that of other lepidopterans. Notably, 12 protein-coding genes (PCGs) utilized the standard ATN, but the cox1 gene used CGA as the initiation codon; the cox1, cox2, and nad4 genes in the two mitogenomes had the truncated termination codons T, T, and TA, respectively, but the nad5 gene was found to use T as the termination codon only in the C. medinalis mitogenome. Additionally, the codon distribution and Relative Synonymous Codon Usage of the 13 PCGs in the C. medinalis mitogenome were very different from those in other pyralid moth mitogenomes. Most of the tRNA genes had typical cloverleaf secondary structures. However, the dihydrouridine (DHU) arm of the trnS1(AGN) gene did not form a stable stem-loop structure. Forty-nine helices in six domains, and 33 helices in three domains were present in the secondary structures of the rrnL and rrnS genes of the two mitogenomes, respectively. There were four major intergenic spacers, except for the A+T-rich region, spanning at least 12 bp in the two mitogenomes. The A+T-rich region contained an 'ATAGT(A)'-like motif followed by a poly-T stretch in the two mitogenomes. In addition, there were a potential stem-loop structure, a duplicated 25-bp repeat element, and a microsatellite '(TA)(13)' observed in the A+T-rich region of the C. medinalis mitogenome. A poly-T motif, a duplicated 31-bp repeat element, and a 19-bp triplication were found in the C. suppressalis mitogenome. However, there are many differences in the A+T-rich regions between the C. suppressalis mitogenome sequence in the present study and previous reports. Finally, the phylogenetic relationships of these insects were reconstructed based on amino acid sequences of mitochondrial 13 PCGs using Bayesian inference and maximum likelihood methods. These molecular-based phylogenies support the traditional morphologically based view of relationships within the Pyralidae.     

Comparative analysis of mitochondrial genomes between the hau cytoplasmic male sterility (CMS) line and its iso-nuclear maintainer line in Brassica juncea to reveal the origin of the CMS-associated gene orf288

Background

Cytoplasmic male sterility (CMS) is not only important for exploiting heterosis in crop plants, but also as a model for investigating nuclear-cytoplasmic interaction. CMS may be caused by mutations, rearrangement or recombination in the mitochondrial genome. Understanding the mitochondrial genome is often the first and key step in unraveling the molecular and genetic basis of CMS in plants. Comparative analysis of the mitochondrial genome of the hau CMS line and its maintainer line in B. juneca (Brassica juncea) may help show the origin of the CMS-associated gene orf288.

Results

Through next-generation sequencing, the B. juncea hau CMS mitochondrial genome was assembled into a single, circular-mapping molecule that is 247,903 bp in size and 45.08% in GC content. In addition to the CMS associated gene orf288, the genome contains 35 protein-encoding genes, 3 rRNAs, 25 tRNA genes and 29 ORFs of unknown function. The mitochondrial genome sizes of the maintainer line and another normal type line “J163-4” are both 219,863 bp and with GC content at 45.23%. The maintainer line has 36 genes with protein products, 3 rRNAs, 22 tRNA genes and 31 unidentified ORFs. Comparative analysis the mitochondrial genomes of the hau CMS line and its maintainer line allowed us to develop specific markers to separate the two lines at the seedling stage. We also confirmed that different mitotypes coexist substoichiometrically in hau CMS lines and its maintainer lines in B. juncea. The number of repeats larger than 100 bp in the hau CMS line (16 repeats) are nearly twice of those found in the maintainer line (9 repeats). Phylogenetic analysis of the CMS-associated gene orf288 and four other homologous sequences in Brassicaceae show that orf288 was clearly different from orf263 in Brassica tournefortii despite of strong similarity.

Conclusion

The hau CMS mitochondrial genome was highly rearranged when compared with its iso-nuclear maintainer line mitochondrial genome. This study may be useful for studying the mechanism of natural CMS in B. juncea, performing comparative analysis on sequenced mitochondrial genomes in Brassicas, and uncovering the origin of the hau CMS mitotype and structural and evolutionary differences between different mitotypes.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-322) contains supplementary material, which is available to authorized users.     

Comparative molecular cytogenetic analyses of a major tandemly repeated DNA family and retrotransposon sequences in cultivated jute Corchorus species (Malvaceae)

Background and Aims

The cultivated jute species Corchorus olitorius and Corchorus capsularis are important fibre crops. The analysis of repetitive DNA sequences, comprising a major part of plant genomes, has not been carried out in jute but is useful to investigate the long-range organization of chromosomes. The aim of this study was the identification of repetitive DNA sequences to facilitate comparative molecular and cytogenetic studies of two jute cultivars and to develop a fluorescent in situ hybridization (FISH) karyotype for chromosome identification.

Methods

A plasmid library was generated from C. olitorius and C. capsularis with genomic restriction fragments of 100–500 bp, which was complemented by targeted cloning of satellite DNA by PCR. The diversity of the repetitive DNA families was analysed comparatively. The genomic abundance and chromosomal localization of different repeat classes were investigated by Southern analysis and FISH, respectively. The cytosine methylation of satellite arrays was studied by immunolabelling.

Key Results

Major satellite repeats and retrotransposons have been identified from C. olitorius and C. capsularis. The satellite family CoSat I forms two undermethylated species-specific subfamilies, while the long terminal repeat (LTR) retrotransposons CoRetro I and CoRetro II show similarity to the Metaviridea of plant retroelements. FISH karyotypes were developed by multicolour FISH using these repetitive DNA sequences in combination with 5S and 18S–5·8S–25S rRNA genes which enable the unequivocal chromosome discrimination in both jute species.

Conclusions

The analysis of the structure and diversity of the repeated DNA is crucial for genome sequence annotation. The reference karyotypes will be useful for breeding of jute and provide the basis for karyotyping homeologous chromosomes of wild jute species to reveal the genetic and evolutionary relationship between cultivated and wild Corchorus species.     

Karyotype differentiation of four Cestrum species (Solanaceae) revealed by fluorescent chromosome banding and FISH

The karyotypes of four South American species of Cestrum (C. capsulare,C. corymbosum,C. laevigatum and C. megalophylum) were studied using conventional staining, C-CMA/DAPI chromosome banding and FISH with 45S and 5S rDNA probes. The karyotypes showed a chromosome number of 2n = 2x = 16, with metacentric chromosomes, except for the eighth submeta- to acrocentric pair. Several types of heterochromatin were detected, which varied in size, number, distribution and base composition. The C-CMA(+) bands and 45S rDNA were located predominantly in terminal regions. The C-CMA (+) /DAPI (+) bands appeared in interstitial and terminal regions, and the C-DAPI (+) bands were found in all chromosome regions. The 5S rDNA sites were observed on the long arm of pair 8 in all species except C. capsulare, where they were found in the paracentromeric region of the long arm of pair 4. The differences in band patterns among the species studied here, along with data from other nine species reported in the literature, suggest that the bands are dispersed in an equilocal and non-equilocal manner and that structural rearrangements can be responsible for internal karyotype diversification. However, it is important to point out that the structural changes involving repetitive segments did not culminate in substantial changes in the general karyotype structure concerning chromosome size and morphology.     

Mapping of the 18S and 5S ribosomal RNA genes in the fish Prochilodus argenteus Agassiz, 1829 (Characiformes,Prochilodontidae)

A single NOR-bearing chromosome pair was identified by silver nitrate staining in a previous study of the fish Prochilodus argenteus from the S ã o Francisco River (MG, Brazil), with a third metacentric chromosome sporadically bearing active NOR. The present study focused on an analysis of the chromosomal localization of both the major (45S) and the minor (5S) rRNA genes using FISH. The use of the 18S rDNA probe confirmed the previous Ag-NOR sites interstitially located in a large metacentric pair and also identified up to three other sites located in the telomeric regions of distinct chromosomes, characterizing an interindividual variation of these sites. In addition, the 5S rDNA site was revealed adjacent to the major NOR site, identified at the end of the large Ag-NOR bearing metacentric chromosome. In a few metaphases, an additional weak hybridization signal was observed in a third chromosome, possibly indicating the presence of another 5S rDNA cluster. Despite a lower karyotype diversification (2n=54 and FN=108) often observed among species of Prochilodontidae, variations involving both 45S and 5S rRNA genes could play an important role in their chromosome diversification.     

Evolution of genome size and chromosome number in the carnivorous plant genus Genlisea (Lentibulariaceae), with a new estimate of the minimum genome size in angiosperms

MethodsNuclear genome sizes were measured from cultivated plant material for a comprehensive sampling of taxa, including nearly half of all species of Genlisea and representing all major lineages. Flow cytometric measurements were conducted in parallel in two laboratories in order to compare the consistency of different methods and controls. Chromosome counts were performed for the majority of taxa, comparing different staining techniques for the ultrasmall chromosomes.ConclusionsGenlisea is an ideal candidate model organism for the understanding of genome reduction as the genus includes species with both relatively large (∼1700 Mbp) and ultrasmall (∼61 Mbp) genomes. This comparative, phylogeny-based analysis of genome sizes and karyotypes in Genlisea provides essential data for selection of suitable species for comparative whole-genome analyses, as well as for further studies on both the molecular and cytogenetic basis of genome reduction in plants.     

Nuclear DNA content in allopolyploid species and synthetic hybrids in the grass genus Paspalum

DNA content was estimated by flow cytometry in seventeen taxa from the Dilatata, Quadrifaria and Paniculata groups of Paspalum and five synthetic hybrids. Results were compared to known genome constitutions and phylogenetic relationships. DNA 2C-values ranged from 1.24 pg in diploid P. juergensii to 3.79 pg in a hexaploid biotype of P. dilatatum. The I genome of three Quadrifaria diploids is 1.2 to 1.5-fold larger than the J genome of P. juergensii (Paniculata). The 2C-values of the IIJJ tetraploids of the Dilatata group are lower than expected based on putative genome donors. Reduction of genome sizes could have occurred after the formation of the allopolyploids of the Dilatata group. The DNA content of all synthetic hybrids is in accordance with the sum of parental C-values. The interactions driving genome downsizing may operate differently during the transition from diploidy to polyploidy than on subsequent increases in ploidy level.