Unlocking the barley genome by chromosomal and comparative genomics

We used a novel approach that incorporated chromosome sorting, next-generation sequencing, array hybridization, and systematic exploitation of conserved synteny with model grasses to assign ~86% of the estimated ~32,000 barley (Hordeum vulgare) genes to individual chromosome arms. Using a series of bioinformatically constructed genome zippers that integrate gene indices of rice (Oryza sativa), sorghum (Sorghum bicolor), and Brachypodium distachyon in a conserved synteny model, we were able to assemble 21,766 barley genes in a putative linear order. We show that the barley (H) genome displays a mosaic of structural similarity to hexaploid bread wheat (Triticum aestivum) A, B, and D subgenomes and that orthologous genes in different grasses exhibit signatures of positive selection in different lineages. We present an ordered, information-rich scaffold of the barley genome that provides a valuable and robust framework for the development of novel strategies in cereal breeding.     

Exploring the genomes: From Arabidopsis to crops

Model systems have played a crucial role for understanding biological processes at genetic, molecular and systems levels. Arabidopsis thaliana is one of the best studied model species for higher plants. Large genomic resources and mutant collections made Arabidopsis an excellent source for functional and comparative genomics. Rice and Brachypodium have a great potential to become model systems for grasses. Given the agronomic importance of grass crops, it is an attractive strategy to apply knowledge from Arabidopsis to grasses. Despite many efforts successful reports are sparse. Knowledge transfer should generally work best between orthologous genes that share functionality and a common ancestor. In higher plants, however, recent genome projects revealed an active and rapid evolution of genome structure, which challenges the concept of one-to-one orthologous mates between two species. In this study, we estimated on the example of protein families that are involved in redox related processes, the impact of gene expansions on the success rate for a knowledge transfer from Arabidopsis to the grass species rice, sorghum and Brachypodium. The sparse synteny between dicot and monocot plants due to frequent rearrangements, translocations and gene losses strongly impairs and reduces the number of orthologs detectable by positional conservation. To address the limitations of sparse synteny and expanded gene families, we applied for the detection of orthologs in this study orthoMCL, a sequence-based approach that allows to group closely related paralogs into one orthologous gene cluster. For a total of 49 out of 170 Arabidopsis genes we could identify conserved copy numbers between the dicot model and the grass annotations whereas approximately one third (34.7%, 59 genes) of the selected Arabidopsis genes lack an assignment to any of the grass genome annotations. The remaining 62 Arabidopsis genes represent groups that are considerably biased in their copy numbers between Arabidopsis and all or most of the three grass genomes.     

Comparative Sequence Analysis of the Ghd7 Orthologous Regions Revealed Movement of Ghd7 in the Grass Genomes

Ghd7 is an important rice gene that has a major effect on several agronomic traits, including yield. To reveal the origin of Ghd7 and sequence evolution of this locus, we performed a comparative sequence analysis of the Ghd7 orthologous regions from ten diploid Oryza species, Brachypodium distachyon, sorghum and maize. Sequence analysis demonstrated high gene collinearity across the genus Oryza and a disruption of collinearity among non-Oryza species. In particular, Ghd7 was not present in orthologous positions except in Oryza species. The Ghd7 regions were found to have low gene densities and high contents of repetitive elements, and that the sizes of orthologous regions varied tremendously. The large transposable element contents resulted in a high frequency of pseudogenization and gene movement events surrounding the Ghd7 loci. Annotation information and cytological experiments have indicated that Ghd7 is a heterochromatic gene. Ghd7 orthologs were identified in B. distachyon, sorghum and maize by phylogenetic analysis; however, the positions of orthologous genes differed dramatically as a consequence of gene movements in grasses. Rather, we identified sequence remnants of gene movement of Ghd7 mediated by illegitimate recombination in the B. distachyon genome.     

Low rates of expression profile divergence in highly expressed genes and tissue-specific genes during mammalian evolution

Evolutionary rates provide important information about the pattern and mechanism of evolution. Although the rate of gene sequence evolution has been well studied, the rate of gene expression evolution is poorly understood. In particular, it is unclear whether the gene expression level and tissue specificity influence the divergence of expression profiles between orthologous genes. Here we address this question using a microarray data set comprising the expression signals of 10,607 pairs of orthologous human and mouse genes from over 60 tissues per species. We show that the level of gene expression and the degree of tissue specificity are generally conserved between the human and mouse orthologs. The rate of gene expression profile change during evolution is negatively correlated with the level of gene expression, measured by either the average or the highest level among all tissues examined. This is analogous to the observation that the rate of gene (or protein) sequence evolution is negatively correlated with the gene expression level. The impacts of the degree of tissue specificity on the evolutionary rate of gene sequence and that of expression profile, however, are opposite. Highly tissue-specific genes tend to evolve rapidly at the gene sequence level but slowly at the expression profile level. Thus, different forces and selective constraints must underlie the evolution of gene sequence and that of gene expression.     

Intraspecific sequence comparisons reveal similar rates of non-collinear gene insertion in the B and D genomes of bread wheat

ABSTRACT: BACKGROUND: Polyploidization is considered one of the main mechanisms of plant genome evolution. The presence of multiple copies of the same gene reduces selection pressure and permits sub-functionalization and neo-functionalization leading to plant diversification, adaptation and speciation. In bread wheat, polyploidization and the prevalence of transposable elements resulted in massive gene duplication and movement. As a result, the number of genes which are non-collinear to genomes of related species seems markedly increased in wheat. RESULTS: We used new-generation sequencing (NGS) to generate sequence of a Mb-sized region from wheat chromosome arm 3DS. Sequence assembly of 24 BAC clones resulted in two scaffolds of 1,264,820 and 333,768 bases. The sequence was annotated and compared to the homoeologous region on wheat chromosome 3B and orthologous loci of Brachypodium distachyon and rice. Among 39 coding sequences in the 3DS scaffolds, 32 have a homoeolog on chromosome 3B. In contrast, only fifteen and fourteen orthologs were identified in the corresponding regions in rice and Brachypodium, respectively. Interestingly, five pseudogenes were identified among the non-collinear coding sequences at the 3B locus, while none was found at the 3DS locus. CONCLUSION: Direct comparison of two Mb-sized regions of the B and D genomes of bread wheat revealed similar rates of non-collinear gene insertion in both genomes with a majority of gene duplications occurring before their divergence. Relatively low proportion of pseudogenes was identified among non-collinear coding sequences. Our data suggest that the pseudogenes did not originate from insertion of non-functional copies, but were formed later during the evolution of hexaploid wheat. Some evidence was found for gene erosion along the B genome locus.     

Conserved noncoding sequences among cultivated cereal genomes identify candidate regulatory sequence elements and patterns of promoter evolution

Surveys for conserved noncoding sequences (CNS) among genes from monocot cereal species were conducted to assess the general properties of CNS in grass genomes and their correlation with known promoter regulatory elements. Initial comparisons of 11 orthologous maize-rice gene pairs found that previously defined regulatory motifs could be identified within short CNS but could not be distinguished reliably from random sequence matches. Among the different phylogenetic footprinting algorithms tested, the VISTA tool yielded the most informative alignments of noncoding sequence. VISTA was used to survey for CNS among all publicly available genomic sequences from maize, rice, wheat, barley, and sorghum, representing >300 gene comparisons. Comparisons of orthologous maize-rice and maize-sorghum gene pairs identified 20 bp as a minimal length criterion for a significant CNS among grass genes, with few such CNS found to be conserved across rice, maize, sorghum, and barley. The frequency and length of cereal CNS as well as nucleotide substitution rates within CNS were consistent with the known phylogenetic distances among the species compared. The implications of these findings for the evolution of cereal gene promoter sequences and the utility of using the nearly completed rice genome sequence to predict candidate regulatory elements in other cereal genes by phylogenetic footprinting are discussed.     

Comparative Structural and Functional Characterization of Sorghum and Maize Duplications Containing Orthologous Myb Transcription Regulators of 3-Deoxyflavonoid Biosynthesis

Sequence characterization of the genomic region of sorghum yellow seed 1 shows the presence of two genes that are arranged in a head to tail orientation. The two duplicated gene copies, y1 and y2 are separated by a 9.084 kbp intergenic region, which is largely composed of highly repetitive sequences. The y1 is the functional copy, while the y2 may represent a pseudogene; there are several sequence indels and rearrangements within the putative coding region of y2. The y1 gene encodes a R2R3 type of Myb domain protein that regulates the expression of chalcone synthase, chalcone isomerase and dihydroflavonol reductase genes required for the biosynthesis of 3-deoxyflavonoids. Expression of y1 can be observed throughout the plant and it represents a combination of expression patterns produced by different alleles of the maize p1. Comparative sequence analysis within the coding regions and flanking sequences of y1, y2 and their maize and teosinte orthologs show local rearrangements and insertions that may have created modified regulatory regions. These micro-colinearity modifications possibly are responsible for differential patterns of expression in maize and sorghum floral and vegetative tissues. Phylogenetic analysis indicates that sorghum y1 and y2 sequences may have arisen by gene duplication mechanisms and represent an evolutionarily parallel event to the duplication of maize p2 and p1 genes.     

Dynamic gene copy number variation in collinear regions of grass genomes

A salient feature of genomes of higher organisms is the birth and death of gene copies. An example is the alpha prolamin genes, which encode seed storage proteins in grasses (Poaceae) and represent a medium-size gene family. To better understand the mechanism, extent, and pace of gene amplification, we compared prolamin gene copies in the genomes of two different tribes in the Panicoideae, the Paniceae and the Andropogoneae. We identified alpha prolamin (setarin) gene copies in the diploid foxtail millet (Paniceae) genome (490 Mb) and compared them with orthologous regions in diploid sorghum (730 Mb) and ancient allotetraploid maize (2,300 Mb) (Andropogoneae). Because sequenced genomes of other subfamilies of Poaceae like rice (389 Mb) (Ehrhartoideae) and Brachypodium (272 Mb) (Pooideae) do not have alpha prolamin genes, their collinear regions can serve as "empty" reference sites. A pattern emerged, where genes were copied and inserted into other chromosomal locations followed by additional tandem duplications (clusters). We observed both recent (species-specific) insertion events and older ones that are shared by these tribes. Many older copies were deleted by unequal crossing over of flanking sequences or damaged by truncations. However, some remain intact with active and inactive alleles. These results indicate that genomes reflect only a snapshot of the gene content of a species and are far less static than conventional genetics has suggested. Nucleotide substitution rates for active alpha prolamins genes were twice as high as for low copy number beta, gamma, and delta prolamin genes, suggesting that gene amplification accelerates the pace of divergence.     

Lr34 multi-pathogen resistance ABC transporter: molecular analysis of homoeologous and orthologous genes in hexaploid wheat and other grass species

The Triticum aestivum (bread wheat) disease resistance gene Lr34 confers durable, race non-specific protection against three fungal pathogens, and has been a highly relevant gene for wheat breeding since the green revolution. Lr34, located on chromosome 7D, encodes an ATP-binding cassette (ABC) transporter. Both wheat cultivars with and without Lr34-based resistance encode a putatively functional protein that differ by only two amino acid polymorphisms. In this study, we focused on the identification and characterization of homoeologous and orthologous Lr34 genes in hexaploid wheat and other grasses. In hexaploid wheat we found an expressed and putatively functional Lr34 homoeolog located on chromosome 4A, designated Lr34-B. Another homoeologous Lr34 copy, located on chromosome 7A, was disrupted by the insertion of repetitive elements. Protein sequences of LR34-B and LR34 were 97% identical. Orthologous Lr34 genes were detected in the genomes of Oryza sativa (rice) and Sorghum bicolor (sorghum). Zea mays (maize), Brachypodium distachyon and Hordeum vulgare (barley) lacked Lr34 orthologs, indicating independent deletion of this particular ABC transporter. Lr34 was part of a gene-rich island on the wheat D genome. We found gene colinearity on the homoeologous A and B genomes of hexaploid wheat, but little microcolinearity in other grasses. The homoeologous LR34-B protein and the orthologs from rice and sorghum have the susceptible haplotype for the two critical polymorphisms distinguishing the LR34 proteins from susceptible and resistant wheat cultivars. We conclude that the particular Lr34-haplotype found in resistant wheat cultivars is unique. It probably resulted from functional gene diversification that occurred after the polyploidization event that was at the origin of cultivated bread wheat.     

Analysis of sequence, map position, and gene expression reveals conserved essential genes for iron uptake in Arabidopsis and tomato

Arabidopsis (Arabidopsis thaliana) and tomato (Lycopersicon esculentum) show similar physiological responses to iron deficiency, suggesting that homologous genes are involved. Essential gene functions are generally considered to be carried out by orthologs that have remained conserved in sequence and map position in evolutionarily related species. This assumption has not yet been proven for plant genomes that underwent large genome rearrangements. We addressed this question in an attempt to deduce functional gene pairs for iron reduction, iron transport, and iron regulation between Arabidopsis and tomato. Iron uptake processes are essential for plant growth. We investigated iron uptake gene pairs from tomato and Arabidopsis, namely sequence, conserved gene content of the regions containing iron uptake homologs based on conserved orthologous set marker analysis, gene expression patterns, and, in two cases, genetic data. Compared to tomato, the Arabidopsis genome revealed more and larger gene families coding for the iron uptake functions. The number of possible homologous pairs was reduced if functional expression data were taken into account in addition to sequence and map position. We predict novel homologous as well as partially redundant functions of ferric reductase-like and iron-regulated transporter-like genes in Arabidopsis and tomato. Arabidopsis nicotianamine synthase genes encode a partially redundant family. In this study, Arabidopsis gene redundancy generally reflected the presumed genome duplication structure. In some cases, statistical analysis of conserved gene regions between tomato and Arabidopsis suggested a common evolutionary origin. Although involvement of conserved genes in iron uptake was found, these essential genes seem to be of paralogous rather than orthologous origin in tomato and Arabidopsis.     

Comparative genomics of grasses tolerant to aluminum

The family Poaceae includes over 10,000 species, among which are the most economically important cereals: maize, sorghum, rice, wheat, rye, barley, and oat. These cereals are very important components of human and animal food. Although divergence of the members of this family occurred about 40 million years ago, comparative genome analyses demonstrated that gene orders among species of this family remain largely conserved, which can be very useful for understanding their roles and evolution. Even with an intricate evolutionary history in which chromosome fragments, losses and duplications have to be considered at the ploidy level, grasses present a genetic model system for comparative genomics. The availability of mapped molecular markers, rice genome sequences and BAC and EST libraries from several grass species, such as rice, wheat, sorghum, and maize, facilitates biology and phylogeny studies of this group. The value of using information from different species in modern plant genetics is unquestionable, especially in the study of traits such as tolerance to aluminum in soils, which affects plant growth and development. Comparative genomic approaches to aluminum tolerance can identify genomic regions and genes responsible for aluminum tolerance in grasses.     

Targeted analysis of orthologous phytochrome A regions of the sorghum,maize, and rice genomes using comparative gene-island sequencing

A "gene-island" sequencing strategy has been developed that expedites the targeted acquisition of orthologous gene sequences from related species for comparative genome analysis. A 152-kb bacterial artificial chromosome (BAC) clone from sorghum (Sorghum bicolor) encoding phytochrome A (PHYA) was fully sequenced, revealing 16 open reading frames with a gene density similar to many regions of the rice (Oryza sativa) genome. The sequences of genes in the orthologous region of the maize (Zea mays) and rice genomes were obtained using the gene-island sequencing method. BAC clones containing the orthologous maize and rice PHYA genes were identified, sheared, subcloned, and probed with the sorghum PHYA-containing BAC DNA. Sequence analysis revealed that approximately 75% of the cross-hybridizing subclones contained sequences orthologous to those within the sorghum PHYA BAC and less than 25% contained repetitive and/or BAC vector DNA sequences. The complete sequence of four genes, including up to 1 kb of their promoter regions, was identified in the maize PHYA BAC. Nine orthologous gene sequences were identified in the rice PHYA BAC. Sequence comparison of the orthologous sorghum and maize genes aided in the identification of exons and conserved regulatory sequences flanking each open reading frame. Within genomic regions where micro-colinearity of genes is absolutely conserved, gene-island sequencing is a particularly useful tool for comparative analysis of genomes between related species.     

Evolutionary significance of gene expression divergence

Recent large-scale studies of evolutionary changes in gene expression among mammalian species have led to the proposal that gene expression divergence may be neutral with respect to organismic fitness. Here, we employ a comparative analysis of mammalian gene sequence divergence and gene expression divergence to test the hypothesis that the evolution of gene expression is predominantly neutral. Two models of neutral gene expression evolution are considered: 1-purely neutral evolution (i.e., no selective constraint) of gene expression levels and patterns and 2-neutral evolution accompanied by selective constraint. With respect to purely neutral evolution, levels of change in gene expression between human-mouse orthologs are correlated with levels of gene sequence divergence that are determined largely by purifying selection. In contrast, evolutionary changes of tissue-specific gene expression profiles do not show such a correlation with sequence divergence. However, divergence of both gene expression levels and profiles are significantly lower for orthologous human-mouse gene pairs than for pairs of randomly chosen human and mouse genes. These data clearly point to the action of selective constraint on gene expression divergence and are inconsistent with the purely neutral model; however, there is likely to be a neutral component in evolution of gene expression, particularly, in tissues where the expression of a given gene is low and functionally irrelevant. The model of neutral evolution with selective constraint predicts a regular, clock-like accumulation of gene expression divergence. However, relative rate tests of the divergence among human-mouse-rat orthologous gene sets reveal clock-like evolution for gene sequence divergence, and to a lesser extent for gene expression level divergence, but not for the divergence of tissue-specific gene expression profiles. Taken together, these results indicate that gene expression divergence is subject to the effects of purifying selective constraint and suggest that it might also be substantially influenced by positive Darwinian selection.     

The colinearity of the Sh2/A1 orthologous region in rice, sorghum and maize is interrupted and accompanied by genome expansion in the triticeae

The Sh2/A1 orthologous region of maize, rice, and sorghum contains five genes in the order Sh2, X1, X2, and two A1 homologs in tandem duplication. The Sh2 and A1 homologs are separated by approximately 20 kb in rice and sorghum and by approximately 140 kb in maize. We analyzed the fate of the Sh2/A1 region in large-genome species of the Triticeae (wheat, barley, and rye). In the Triticeae, synteny in the Sh2/A1 region was interrupted by a break between the X1 and X2 genes. The A1 and X2 genes remained colinear in homeologous chromosomes as in other grasses. The Sh2 and X1 orthologs also remained colinear but were translocated to a nonhomeologous chromosome. Gene X1 was duplicated on two nonhomeologous chromosomes, and surprisingly, a paralog shared homology much higher than that of the orthologous copy to the X1 gene of other grasses. No tandem duplication of A1 homologs was detected but duplication of A1 on a nonhomeologous barley chromosome 6H was observed. Intergenic distances expanded greatly in wheat compared to rice. Wheat and barley diverged from each other 12 million years ago and both show similar changes in the Sh2/A1 region, suggesting that the break in colinearity as well as X1 duplications and genome expansion occurred in a common ancestor of the Triticeae species.