Structural characterization of Brachypodium genome and its syntenic relationship with rice and wheat

Brachypodium distachyon (Brachypodium) has been recently recognized as an emerging model system for both comparative and functional genomics in grass species. In this study, 55,221 repeat masked Brachypodium BAC end sequences (BES) were used for comparative analysis against the 12 rice pseudomolecules. The analysis revealed that ~26.4% of BES have significant matches with the rice genome and 82.4% of the matches were homologous to known genes. Further analysis of paired-end BES and ~1.0 Mb sequences from nine selected BACs proved to be useful in revealing conserved regions and regions that have undergone considerable genomic changes. Differential gene amplification, insertions/deletions and inversions appeared to be the common evolutionary events that caused variations of microcolinearity at different orthologous genomic regions. It was found that ~17% of genes in the two genomes are not colinear in the orthologous regions. Analysis of BAC sequences also revealed higher gene density (~9 kb/gene) and lower repeat DNA content (~13.1%) in Brachypodium when compared to the orthologous rice regions, consistent with the smaller size of the Brachypodium genome. The 119 annotated Brachypodium genes were BLASTN compared against the wheat EST database and deletion bin mapped wheat ESTs. About 77% of the genes retrieved significant matches in the EST database, while 9.2% matched to the bin mapped ESTs. In some cases, genes in single Brachypodium BACs matched to multiple ESTs that were mapped to the same deletion bins, suggesting that the Brachypodium genome will be useful for ordering wheat ESTs within the deletion bins and developing specific markers at targeted regions in the wheat genome.     

The compact Brachypodium genome conserves centromeric regions of a common ancestor with wheat and rice

The evolution of five chromosomes of Brachypodium distachyon from a 12-chromosome ancestor of all grasses by dysploidy raises an interesting question about the fate of redundant centromeres. Three independent but complementary approaches were pursued to study centromeric region homologies among the chromosomes of Brachypodium, wheat, and rice. The genes present in pericentromeres of the basic set of seven chromosomes of wheat and the Triticeae, and the 80 rice centromeric genes spanning the CENH3 binding domain of centromeres 3, 4, 5, 7, and 8 were used as “anchor” markers to identify centromere locations in the B. distachyon chromosomes. A total of 53 B. distachyon bacterial artificial chromosome (BAC) clones anchored by wheat pericentromeric expressed sequence tags (ESTs) were used as probes for BAC-fluorescence in situ hybridization (FISH) analysis of B. distachyon mitotic chromosomes. Integrated sequence alignment and BAC-FISH data were used to determine the approximate positions of active and inactive centromeres in the five B. distachyon chromosomes. The following syntenic relationships of the centromeres for Brachypodium (Bd), rice (R), and wheat (W) were evident: Bd1-R6, Bd2-R5-W1, Bd3-R10, Bd4-R11-W4, and Bd5-R4. Six rice centromeres syntenic to five wheat centromeres were inactive in Brachypodium chromosomes. The conservation of centromere gene synteny among several sets of homologous centromeres of three species indicates that active genes can persist in ancient centromeres with more than 40 million years of shared evolutionary history. Annotation of a BAC contig spanning an inactive centromere in chromosome Bd3 which is syntenic to rice Cen8 and W7 pericentromeres, along with BAC FISH data from inactive centromeres revealed that the centromere inactivation was accompanied by the loss of centromeric retrotransposons and turnover of centromere-specific satellites during Bd chromosome evolution.     

Genetic interactions between wheat and rye genomes in triticale

Summary Six primary triticale lines were produced from two advanced breeding lines of Triticum durum and three inbred genotypes of Secale cereale. The wheat and rye parents and the triticale derivatives were crossed in all possible combinations within each species group. Chiasma and univalent frequency of parents and hybrids were determined. The primary triticale lines had more univalents and less chiasmata per pollen mother cell than the corresponding wheat and rye parents together. The parental wheat F₁ exhibited negative heterosis for chiasma frequency whereas all rye hybrids had much higher chiasma frequencies than their inbred parents. Triticale F₁s generally showed lower chiasma frequencies and more univalents than their parents, but the degree of pairing failure was dependent upon which of the parental species within the triticale, wheat or rye, was in the heterozygous state. F₁s with heterozygous wheat genome only showed the least reduction in chiasma number (presumably caused by gene actions within the wheat genome), while F₁s with heterozygous rye genome showed high reduction in chiasma frequency and an increase in pairing failure (induced by negative interactions between the heterozygous rye and the wheat genome in triticale). A high correlation was found between the frequency of undisturbed pollen mother cells and the frequency of aneuploids in the subsequent generation. A higher number of aneuploids occurred in those populations which were heterozygous for the rye genome.     

Control of flowering time and spike development in cereals: the earliness per se Eps-1 region in wheat, rice, and Brachypodium

The earliness per se gene Eps-A ^m 1 from diploid wheat Triticum monococcum affects heading time, spike development, and spikelet number. In this study, the Eps1 orthologous regions from rice, Aegilops tauschii, and Brachypodium distachyon were compared as part of current efforts to clone this gene. A single Brachypodium BAC clone spanned the Eps-A ^m 1 region, but a gap was detected in the A. tauschii physical map. Sequencing of the Brachypodium and A. tauschii BAC clones revealed three genes shared by the three species, which showed higher identity between wheat and Brachypodium than between them and rice. However, most of the structural changes were detected in the wheat lineage. These included an inversion encompassing the wg241-VatpC region and the presence of six unique genes. In contrast, only one unique gene (and one pseudogene) was found in Brachypodium and none in rice. Three genes were present in both Brachypodium and wheat but were absent in rice. Two of these genes, Mot1 and FtsH4, were completely linked to the earliness per se phenotype in the T. monococcum high-density genetic map and are candidates for Eps-A ^m 1. Both genes were expressed in apices and developing spikes, as expected for Eps-A ^m 1 candidates. The predicted MOT1 protein showed amino acid differences between the parental T. monococcum lines, but its effect is difficult to predict. Future steps to clone the Eps-A ^m 1 gene include the generation of mot1 and ftsh4 mutants and the completion of the T. monococcum physical map to test for the presence of additional candidate genes.     

Ultraconserved elements between the genomes of the plants Arabidopsis thaliana and rice

Ultraconserved elements, sequences with 100% identity with no insertions or deletions between genomes, have been found in both vertebrate and invertebrate genomes; whether plant genomes contain ultraconserved elements, however, is unknown. We consequently compared the genomes of Arabidopsis thaliana and rice, which diverged about 200 million years ago, and identified 25 ultraconserved elements that are longer than 100 bp. Similar to those previously found, ultraconserved elements in plants tend to occur in clusters and locate at noncoding regions; nevertheless, they have many distinct features. For instance, the longest ultraconserved element between the 2 plant genomes is 1491 bp, much longer than the longest one (779 bp) between the human and rodent genomes. Some biological implications are discussed, but the functions of these plant ultraconserved elements and the reasons why they are practically frozen during the evolution of millions of years remain a mystery.     

PCR-based identification of wheat genomes

We describe a PCR system that distinguishes the A, B and D genomes in wheat DNA extracts. PCRs were directed at the ‘non-transcribed spacer’ regions of the rDNA loci. The spacers within the D genome locus have a 71-bp insertion that is absent from the corresponding A and B loci PCR product sizes therefore enable D- and D+ genomes to be distinguished. The A and B genomes can be differentiated by PCR with an internal primer which does not anneal to A genome sequences. This work is relevant to the ancient ecology of wheat, as it is often difficult to determine ploidy level from morphological examination of archaeobotanical remains.     

Orthology relations, symbolic ultrametrics, and cographs

Orthology detection is an important problem in comparative and evolutionary genomics and, consequently, a variety of orthology detection methods have been devised in recent years. Although many of these methods are dependent on generating gene and/or species trees, it has been shown that orthology can be estimated at acceptable levels of accuracy without having to infer gene trees and/or reconciling gene trees with species trees. Thus, it is of interest to understand how much information about the gene tree, the species tree, and their reconciliation is already contained in the orthology relation on the underlying set of genes. Here we shall show that a result by Böcker and Dress concerning symbolic ultrametrics, and subsequent algorithmic results by Semple and Steel for processing these structures can throw a considerable amount of light on this problem. More specifically, building upon these authors’ results, we present some new characterizations for symbolic ultrametrics and new algorithms for recovering the associated trees, with an emphasis on how these algorithms could be potentially extended to deal with arbitrary orthology relations. In so doing we shall also show that, somewhat surprisingly, symbolic ultrametrics are very closely related to cographs, graphs that do not contain an induced path on any subset of four vertices. We conclude with a discussion on how our results might be applied in practice to orthology detection.     

Gene space dynamics during the evolution of Aegilops tauschii, Brachypodium distachyon, Oryza sativa, and Sorghum bicolor genomes

Nine different regions totaling 9.7 Mb of the 4.02 Gb Aegilops tauschii genome were sequenced using the Sanger sequencing technology and compared with orthologous Brachypodium distachyon, Oryza sativa (rice), and Sorghum bicolor (sorghum) genomic sequences. The ancestral gene content in these regions was inferred and used to estimate gene deletion and gene duplication rates along each branch of the phylogenetic tree relating the four species. The total gene number in the extant Ae. tauschii genome was estimated to be 36,371. The gene deletion and gene duplication rates and total gene numbers in the four genomes were used to estimate the total gene number in each node of the phylogenetic tree. The common ancestor of the Brachypodieae and Triticeae lineages was estimated to have had 28,558 genes, and the common ancestor of the Panicoideae, Ehrhartoideae, and Pooideae subfamilies was estimated to have had 27,152 or 28,350 genes, depending on the ancestral gene scenario. Relative to the Brachypodieae and Triticeae common ancestor, the gene number was reduced in B. distachyon by 3,026 genes and increased in Ae. tauschii by 7,813 genes. The sum of gene deletion and gene duplication rates, which reflects the rate of gene synteny loss, was correlated with the rate of structural chromosome rearrangements and was highest in the Ae. tauschii lineage and lowest in the rice lineage. The high rate of gene space evolution in the Ae. tauschii lineage accounts for the fact that, contrary to the expectations, the level of synteny between the phylogenetically more related Ae. tauschii and B. distachyon genomes is similar to the level of synteny between the Ae. tauschii genome and the genomes of the less related rice and sorghum. The ratio of gene duplication to gene deletion rates in these four grass species closely parallels both the total number of genes in a species and the overall genome size. Because the overall genome size is to a large extent a function of the repeated sequence content in a genome, we suggest that the amount and activity of repeated sequences are important factors determining the number of genes in a genome.     

Attempts to induce homoeologous pairing between wheat and Agropyron cristatum genomes.

Agropyron cristatum (2n = 4x = 28, PPPP) possesses potentially valuable traits that could be used in wheat (Triticum aestivum) improvement through interspecific hybridization. Homoeologous pairing between wheat chromosomes and P chromosomes added to wheat in a set of wheat - A. cristatum addition lines was assessed. First, the Ph-suppressing effect of P chromosomes (except 7P) was analyzed. It was concluded that this system is polygenic with no major gene, and consequently, has no prospect in the transfer of alien genes from wild relatives. In a second step, the potential of the deletion ph1b of the Ph1 gene for inducing P-ABD pairing was evaluated. Allosyndetic associations between P and ABD genomes are very rare. This very low level of pairing is likely due to divergence in the repeated sequences between Agropyron and wheat genomes. Development of translocation lines using ionizing radiation seems to be a more suitable technique than homoeologous recombination to exploit the A. cristatum genome in wheat improvement.     

Further evidence of microcolinearity between barley and rice genomes at two orthologous regions

Two genetic markers, BCD135 and RZ567 were used to select clones from genomic BAC libraries of barley and rice for sequencing and subsequent sequence comparisons. A set of two orthologous BACs each from barley and rice was selected by hybridization with BCD135 and RZ567 cDNA probes. A total of 556-kb stretch including two barley BACs (773K135 and 745C13) and two orthologous rice BACs (24K23 and 49D11) was completely sequenced. Comparative sequence analysis between orthologous BACs from the two species revealed presence of two conserved genes at BCD135 region and only one gene at the RZ567 regions. The two conserved genes were in the same order and orientation in both the species however, separated by significantly larger distance in barley. The larger distance between two barley genes was mainly due to presence of different retrotransposable elements and their derivatives (78.9% of the intergenic region) that expanded the barley BCD135 region at the rate of 9.1X. An additional gene of unknown function was also inserted along with several retrotransposable elements between two conserved genes at barley BCD135 region. More genome expansion rate (10X) around barley RZ567 locus was estimated by extremely high proportion (> 70%) of retrotransposons. Among different retrotransposons, the Sabrina elements rather than BARE were more prevalent in both the regions. Contrary to it, the BCD135 region of rice was composed of only 17.1% retrotransposable elements and no significant retrotransposons except 14 miniature inverted transposable elements (MITEs) were observed in its RZ567 region. The sequence comparison between orthologous regions of rice and barley genomes was useful for gene identification and determination of individual gene structure indicating the possibility of effective utilization of rice genome sequences in understanding the large genome of barley. (The sequence data described in this paper have been submitted to the GenBank data library under the accession no. AF474072 (773K14), AF474071 (745C13), AF480497 (24K23) and AF480496 (49D11)).     

Application of Brachypodium to the genetic improvement of wheat roots

To meet the demands of a larger and more affluent global population, wheat yields must increase faster this century than last, with less irrigation, fertilizer, and land. Modelling and experiments consistently demonstrate a large potential for increasing wheat productivity by improving root systems; however, application of research to new varieties is slow because of the inherent difficulties associated with working underground. This review makes the case for the use of the model grass Brachypodium distachyon to simplify root research and accelerate the identification of genes underlying wheat root improvement. Brachypodium is a small temperate grass with many genomic, genetic, and experimental resources that make it a tractable model plant. Brachypodium and wheat have very similar root anatomies which are distinct from rice root anatomy that is specialized to help it overcome anaerobic conditions associated with submerged roots. As a dicotyledonous plant, Arabidopsis has an even more divergent root system that features a tap root system and cambia with secondary growth, both of which are lacking in the grasses. The major advantage of Brachypodium is its small stature that allows the adult grass root system to be readily phenotyped, unlike rice and maize. This will facilitate the identification of genes in adult roots that greatly influence yield by modulating water uptake during flowering and grain development. A summary of the advantages of Brachypodium for root studies is presented, including the adult root system architecture and root growth during grain development. Routes to translate discoveries from Brachypodium to wheat are also discussed.     

A comparison of rice chloroplast genomes

Using high quality sequence reads extracted from our whole genome shotgun repository, we assembled two chloroplast genome sequences from two rice (Oryza sativa) varieties, one from 93-11 (a typical indica variety) and the other from PA64S (an indica-like variety with maternal origin of japonica), which are both parental varieties of the super-hybrid rice, LYP9. Based on the patterns of high sequence coverage, we partitioned chloroplast sequence variations into two classes, intravarietal and intersubspecific polymorphisms. Intravarietal polymorphisms refer to variations within 93-11 or PA64S. Intersubspecific polymorphisms were identified by comparing the major genotypes of the two subspecies represented by 93-11 and PA64S, respectively. Some of the minor genotypes occurring as intravarietal polymorphisms in one variety existed as major genotypes in the other subspecific variety, thus giving rise to intersubspecific polymorphisms. In our study, we found that the intersubspecific variations of 93-11 (indica) and PA64S (japonica) chloroplast genomes consisted of 72 single nucleotide polymorphisms and 27 insertions or deletions. The intersubspecific polymorphism rates between 93-11 and PA64S were 0.05% for single nucleotide polymorphisms and 0.02% for insertions or deletions, nearly 8 and 10 times lower than their respective nuclear genomes. Based on the total number of nucleotide substitutions between the two chloroplast genomes, we dated the divergence of indica and japonica chloroplast genomes as occurring approximately 86,000 to 200,000 years ago.     

The rice mitochondrial genomes and their variations

Based on highly redundant and high-quality sequences, we assembled rice (Oryza sativa) mitochondrial genomes for two cultivars, 93-11 (an indica variety) and PA64S (an indica-like variety with maternal origin of japonica), which are paternal and maternal strains of an elite superhybrid rice Liang-You-Pei-Jiu (LYP-9), respectively. Following up with a previous analysis on rice chloroplast genomes, we divided mitochondrial sequence variations into two basic categories, intravarietal and intersubspecific. Intravarietal polymorphisms are variations within mitochondrial genomes of an individual variety. Intersubspecific polymorphisms are variations between subspecies among their major genotypes. In this study, we identified 96 single nucleotide polymorphisms (SNPs), 25 indels, and three segmental sequence variations as intersubspecific polymorphisms. A signature sequence fragment unique to indica varieties was confirmed experimentally and found in two wild rice samples, but absent in japonica varieties. The intersubspecific polymorphism rate for mitochondrial genomes is 0.02% for SNPs and 0.006% for indels, nearly 2.5 and 3 times lower than that of their chloroplast counterparts and 21 and 38 times lower than corresponding rates of the rice nuclear genome, respectively. The intravarietal polymorphism rates among analyzed mitochondrial genomes, such as 93-11 and PA64S, are 1.26% and 1.38% for SNPs and 1.13% and 1.09% for indels, respectively. Based on the total number of SNPs between the two mitochondrial genomes, we estimate that the divergence of indica and japonica mitochondrial genomes occurred approximately 45,000 to 250,000 years ago.     

Comparative cytogenetic analysis of the genomes of the model grass Brachypodium distachyon and its close relatives

Background and Aims

Brachypodium is a small genus of temperate grasses that comprises 12–15 species. Brachypodium distachyon is now well established as a model species for temperate cereals and forage grasses. In contrast to B. distachyon, other members of the genus have been poorly investigated at the chromosome level or not at all.

Methods

Twenty accessions comprising six species and two subspecies of Brachypodium were analysed cytogenetically. Measurements of nuclear genome size were made by flow cytometry. Chromosomal localization of 18–5·8–25S rDNA and 5S rDNA loci was performed by dual-colour fluorescence in situ hybridization (FISH) on enzymatically digested root-tip meristematic cells. For comparative phylogenetic analyses genomic in situ hybridization (GISH) applied to somatic chromosome preparations was used.

Key Results

All Brachypodium species examined have rather small genomes and chromosomes. Their chromosome numbers and genome sizes vary from 2n = 10 and 0·631 pg/2C in B. distachyon to 2n = 38 and 2·57 pg/2C in B. retusum, respectively. Genotypes with 18 and 28 chromosomes were found among B. pinnatum accessions. GISH analysis revealed that B. pinnatum with 28 chromosomes is most likely an interspecific hybrid between B. distachyon (2n = 10) and B. pinnatum (2n = 18). Two other species, B. phoenicoides and B. retusum, are also allopolyploids and B. distachyon or a close relative seems to be one of their putative ancestral species. In chromosomes of all species examined the 45S rDNA loci are distally distributed whereas loci for 5S rDNA are pericentromeric.

Conclusions

The increasing significance of B. distachyon as a model grass emphasizes the need to understand the evolutionary relationships in the genus Brachypodium and to ensure consistency in the biological nomenclature of its species. Modern molecular cytogenetic techniques such as FISH and GISH are suitable for comparative phylogenetic analyses and may provide informative chromosome- and/or genome-specific landmarks.     

Orthology,paralogy and proposed classification for paralog subtypes

The paper clears up confusions about the concepts of orthology and paralogy, particularly in cases involving gene family expansions. The terms ‘inparalog’ and ‘outparalog’ are defined to distinguish ancient paralogs from lineage-specific ones.     

Detection of loci controlling seed dormancy on group 4 chromosomes of wheat and comparative mapping with rice and barley genomes

Three quantitative trait loci (QTLs) controlling seed dormancy were detected on group 4 chromosomes of wheat (Triticum aestivum L.) using 119 doubled haploid lines (DHLs) derived from a cross between AC Domain and Haruyutaka. A major QTL, designated QPhs.ocs-4A.1, was identified within the marker interval between Xcdo795 and Xpsr115 in the proximal region of the long arm of chromosome 4A. Two minor QTLs, QPhs.ocs-4B.2 on 4B and QPhs.ocs-4D.2 on 4D, were flanked by common markers, Xbcd1431.1 and Xbcd1431.2 in the terminal region of the long arms, suggesting a homoeologous relationship. These three QTLs explained more than 80% of the total phenotypic variance in seed dormancy of DHLs grown in the field and under glasshouse conditions. The AC Domain alleles at the three QTLs contributed to increasing seed dormancy. Comparative maps across wheat, barley and rice demonstrated the possibility of a homoeologous relationship between QPhs.ocs-4A.1 and the barley gene SD4, while no significant effects of the chromosome regions of wheat and barley orthologous to rice chromosome 3 region carrying a major seed dormancy QTL were detected. Received: 5 June 2000 / Accepted: 31 August 2000     

Homoeologous relationships of rice, wheat and maize chromosomes

A set of cDNA clones, which had previously been mapped onto wheat chromosomes, was genetically mapped onto the chromosomes of rice. The resulting comparative maps make it possible to estimate the degree of linkage conservation between these two species. A number of chromosomal rearrangements, some of which must have involved interchromosomal translocations, differentiate the rice and wheat genomes. However, synteny of a large proportion of the loci appears to be conserved between the two species. The results of this study, combined with those from a recently published comparative map of the rice and maize genomes, suggest that rice, wheat and maize share extensive homoeologies in a number of regions in their genomes. Some chromosomes (e.g. chromosome 4 in rice, chromosomes 2 and 2S in wheat and maize, respectively) may have escaped major rearrangement since the divergence of these species from their last common ancestor. Comparative maps for rice, wheat and maize should make it possible to begin uniting the genetics of these species and allow for transfer of mapping information (including centromere positions) and molecular marker resources (e.g. RFLP probes) between species. In addition, such maps should shed light on the nature of chromosome evolution that accompanied the radiation of grasses in the early stages of plant diversification.