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.

Plant comparative genetics after 10 years.

The past 10 years have seen the discovery of unexpected levels of conservation of gene content and gene orders over millions of years of evolution within grasses, crucifers, legumes, some trees, and Solanaceae crops. Within the grasses, which include the three 500-million-ton-plus-per-year crops (wheat, maize, and rice), and the crucifers, which include all the Brassica crops, colinearity looks good enough to do most map-based cloning only in the small genome model species, rice and Arabidopsis. Elsewhere, knowledge gained in a few major crops is being pooled and applied across the board. The extrapolation of information from the well-studied species to orphan crops, which include many tropical species, is providing a solid base for their improvement. Genome rearrangements are giving new insights into evolution. In fact, comparative genetics is the key that will unlock the secrets of crop plants with genomes larger than that of humans.

Cereal genome evolution. Grasses, line up and form a circle.

The genomes of six major grass species can be aligned by dissecting the individual chromosomes into segments and rearranging these linkage blocks into highly similar structures.

L-RCA (ligation-rolling circle amplification): a general method for genotyping of single nucleotide polymorphisms (SNPs).

A flexible, non-gel-based single nucleotide polymorphism (SNP) detection method is described. The method adopts thermostable ligation for allele discrimination and rolling circle amplification (RCA) for signal enhancement. Clear allelic discrimination was achieved after staining of the final reaction mixtures with Cybr-Gold and visualisation by UV illumination. The use of a compatible buffer system for all enzymes allows the reaction to be initiated and detected in the same tube or microplate well, so that the experiment can be scaled up easily for high-throughput detection. Only a small amount of DNA (i.e. 50 ng) is required per assay, and use of carefully designed short padlock probes coupled with generic primers and probes make the SNP detection cost effective. Biallelic assay by hybridisation of the RCA products with fluorescence dye-labelled probes is demonstrated, indicating that ligation-RCA (L-RCA) has potential for multiplexed assays.

Relationship between chromosome 9 of maize and wheat homeologous group 7 chromosomes.

Comparison of the genetic map of maize chromosome 9 with maps of wheat chromosomes has revealed a high degree of colinearity between maize chromosome 9 and the group 4 and 7 chromosomes of wheat. The order of DNA markers on the short arm and a proximal region of the long arm of the genetic map of maize chromosome 9 is highly conserved with the marker order on the short arm and proximal region of the long arm of the genetic map of the wheat homeologous group 7 chromosomes. A major part of the long arm of the genetic map of maize chromosome 9 is homeologous with a short segment in the proximal region of the long arm of the genetic map of the wheat group 4 chromosomes. Evidence is also presented that maize chromosome 9 has diverged from the wheat group 7 chromosomes by both a pericentric and a paracentric inversion. The paracentric inversion is probably unique to maize among the major cereal genomes.

Development of simple sequence repeat markers from bacterial artificial chromosomes without subcloning.

Simple sequence repeats (SSRs) were isolated from pearl millet bacterial artificial clones (BACs) without any subcloning steps. SSR sequences were targeted using 3' end-anchored SSR primers. Flanking sequences were isolated by suppression PCR. In this pilot study, 25 SSR markers have been developed from 40 BAC pools, comprising a total of 384 clones. This novel way to develop new markers has the added advantage that mapping the SSR markers will anchor individual BACs to the genetic maps and, thus, facilitate the construction of BAC contigs.

Allelic variation at the linked AP1 and PhyC loci in hexaploid wheat is associated but not perfectly correlated with vernalization response.

Vernalization requirement is an important trait in temperate crop plants such as wheat and must be considered when selecting varieties for cultivation under different climatic conditions. To determine the growth habit of wheat varieties, plants need to be grown under different vernalization regimes, a lengthy but necessary process for breeders involved in crossing winter with spring germplasm. If haplotypes can be associated with growth habit, then molecular marker assays that are reliable, cheap, and quick can be developed to assist in the selection of plants with the desired phenotype. We have analyzed 81 accessions that have different vernalization requirements and putative different origins of spring habit for sequence variation at the Apetala1 (AP1) locus, which underlies Vrn-1, and at the linked Phytochrome C (PhyC) locus. Good correspondence was found between the AP1 genotype and the PhyC haplotype for 77 of the 81 accessions. Two varieties displayed a recombination event between the AP1 and PhyC loci, and one variety carried a recombinant PhyC gene. In addition, one variety carried an apparent AP1 winter allele, but displayed the Vrn-A1 spring habit. The PhyC haplotype for this variety also indicated the presence of a Vrn-A1 spring allele. Our data suggest that both the AP1 promoter region and PhyC SNPs can be used as diagnostic markers for vernalization response at the vrn-A1 locus, but that neither are perfect tags.

The use of random amplified polymorphic DNA markers in wheat.

An evaluation was made of the use of random amplified polymorphic DNA (RAPD) as a genetic marker system in wheat. Reproducible amplification products were obtained from varietal, homozygous single chromosome recombinant line and wheat/alien addition line genomic DNA with selected primers and rigorously optimized reaction conditions. Factors influencing the RAPD patterns are DNA concentration, Mg(2+) concentration, polymerase concentration and denaturing temperature. In wheat, the non-homoeologous, non-dose responsive and dominant behaviour of RAPD products devalues their use as genetic markers for the construction of linkage maps, and the high probability that the amplified fragments derive from repetitive DNA limits their use as a source of conventional RFLP probes. However, RAPD markers will most certainly find many applications in the analysis of genotypes where single chromosomes or chromosome segments are to be manipulated.

Preparation of plant DNA for separation by pulsed field gel electrophoresis.

A method was developed for the preparation of completely intact plant DNA embedded in agarose, and suitable for restriction enzyme digestion. Digestion with restriction enzyme was carried out according to modified protocols of Anand and Kenwrick et al. The new method of DNA isolation allows the separation of high molecular weight plant DNA by pulsed field gel electrophoresis.

Comparative genetics in the grasses.

Comparative genetic studies have demonstrated that gene content and orders are highly conserved, both at the map and megabase level, between different species within the grass family. Integration of the genetic maps of rice, foxtail millet, sugar cane, sorghum, maize, the Triticeae cereals and oats into a single synthesis reveals that some chromosome arrangements characterise taxonomic groups, while others have arisen during or after speciation. A detailed analysis of the comparative maps of seven species, belonging to three subfamilies, and their applications are described below.

Comparative genetics in the grasses.

Genetic mapping of wheat, maize, and rice and other grass species with common DNA probes has revealed remarkable conservation of gene content and gene order over the 60 million years of radiation of Poaceae. The linear organization of genes in some nine different genomes differing in basic chromosome number from 5 to 12 and nuclear DNA amount from 400 to 6,000 Mb, can be described in terms of only 25 "rice linkage blocks." The extent to which this intergenomic colinearity is confounded at the micro level by gene duplication and micro-rearrangements is still an open question. Nevertheless, it is clear that the elucidation of the organization of the economically important grasses with larger genomes, such as maize (2n = 10, 4,500 Mb DNA), will, to a greater or lesser extent, be predicted from sequence analysis of smaller genomes such as rice, with only 400 Mb, which in turn may be greatly aided by knowledge of the entire sequence of Arabidopsis, which may be available as soon as the turn of the century. Comparative genetics will provide the key to unlock the genomic secrets of crop plants with bigger genomes than Homo sapiens.

Characterization of loci containing microsatellite sequences among Canadian wheat cultivars.

Two microsatellite sequences, one within a γ-gliadin locus and another within a low molecular weight glutenin locus, were characterized on a set of 16 wheat lines. The wheat lines analyzed were primarily Canadian cultivars or breeding lines. A high level of variation was detected, especially between the Canadian Prairie Spring and the Canadian Western Red Spring Wheat classes. Markers based on microsatellite sequence sites appear to be more informative on closely related germplasm than either RFLP- or RAPD-based markers. The applicability of these markers across a wide spectrum of classes and cultivars provides a starting point for developing a point of delivery wheat class identification system.

Comparative RFLP maps of the homoeologous group-2 chromosomes of wheat, rye and barley.

Genetic maps of the homoeologous group-2 chromosomes were constructed, comprising 114 loci in wheat and 34 loci in rye. These include the genes coding for sucrose synthase, sedoheptulose-1,7-bisphosphatase, a bZIP protein (EmBP-1), a peroxidase and an abscisic acid-induced protein (#7). Overall, gene orders are highly conserved in the genomes of wheat, barley and rye, except for the distal ends of chromosome arms 2BS and 2RS, which are involved in interchromosomal, probably evolutionary, translocations. Clustering of loci in the centromeric regions of the maps, resulting from the concentration of recombination events in the distal chromosomal regions, is observed in wheat and rye, but not in barley. Furthermore, loci for which homoeoloci can be detected in rye and barley tend to lie in the centromeric regions of the maps, while non-homoeologous and wheat-specific loci tend to be more evenly distributed over the genetic maps. Mapping of the group-2 chromosomes in the intervarietal 'Timgalen' x 'RL4137' cross revealed that the T. timopheevi chromosome segment introgressed into chromosome 2B in 'Timgalen' is preferentially transmitted. Recombination is also greatly reduced in that segment.

Arabidopsis-rice: will colinearity allow gene prediction across the eudicot-monocot divide?

With the genomic sequencing of Arabidopsis nearing completion and rice sequencing very much in its infancy, a key question is whether we can exploit the Arabidopsis sequence to identify candidate genes for traits in cereal crops using a map-based approach. This requires the existence of colinearity between the Arabidopsis and cereal genomes, represented by rice, which is readily detectable using currently available resources, that is, Arabidopsis genomic sequence, rice ESTs, and genetic and physical maps. A detailed study of the colinearity remaining between two small regions of Arabidopsis chromosome 1 and rice suggests that at least in these regions of the Arabidopsis genome, conservation of gene orders with rice has been eroded to the point that it is no longer identifiable using comparative mapping. Although our analysis does not preclude that tracts of colinear gene orders may be identified using sequence comparisons or may exist in other regions of the rice and Arabidopsis genomes, it is unlikely that the extent of colinearity will be sufficient to allow map-based cross-species gene prediction and isolation. Our research also highlights the difficulties encountered in identifying orthologs using BLAST searches in incomplete sequence databases. This complicates the interpretation of comparative data among highly divergent species and limits the exploitation of Arabidopsis sequence in monocot studies.

Use of RFLPs to determine the chromosome composition of tetraploid triticale (A/B)(A/B)RR.

Tetraploid triticale, (A/B)(A/B)RR (2n = 28), is a botanical novelty, an amphiploid composed of a diploid rye and a 14 chromosome wheat genome made up of chromosomes of the A and B genomes of tetraploid wheat. Restriction fragment length polymorphism (RFLP) markers were used to elucidate the chromosome composition of the mixed wheat genome of 35 different tetraploid triticale lines. Of 128 possible A/B chromosome pair combinations, only 6 were found among these lines, with a prevalence of the 1A, 2A, 3B, 4B, 5B, 6B, and 7B karyotype. In most triticale lines stable wheat genomes made up of only homologous A or B genome chromosome pairs were identified, however, in some lines homoeologous chromosome pairs were found. In this paper we demonstrate that RFLPs can be used successfully as an alternative to C-banding for the identification of the chromosome composition of tetraploid triticale and discuss the possible selective advantage of specific chromosome composition.

Differentiation between wheat chromosomes 4B and 4D.

A linkage map based on homoeologous recombination, induced by the absence of the Ph1 locus, between chromosome 4D of Triticum aestivum L. (genomes AABBDD) and chromosome 4B of T. turgidum L. (genomes AABB) was compared with a linkage map of chromosome 4Am of T. monococcum L. and a consensus map of chromosomes 4B and 4D of T. aestivum based on homologous recombination. The 4D/4B homoeologous map was only one-third the length of the homologous maps and all intervals were reduced relative to the 4B-4D consensus map. After the homoeologous map was corrected for this overall reduction in recombination, the distribution of recombination in the short arm was similar in both types of maps. In the long arm, homoeologous recombination declined disproportionally in the distal to proximal direction. This gradient was shown to be largely caused by severe segregation distortion reflecting selection against 4D genetic material. The segregation distortion had a maximum that coincided with the centromere and likely had a polygenic cause. Chromosomes 4D and 4B were colinear and recombination between them occurred in almost all intervals where homologous recombination occurred. These findings suggest that these chromosomes are not differentiated structurally and that the differentiation is not segmental. In the presence of Ph1, metaphase I chromosome pairing between chromosomes composed of homologous and differentiated regions correlated with the lengths of the homologous regions. No compensatory allocation of crossovers into the homologous regions was detected. In this respect, the present results are in dramatic contrast with the crossover allocation into the pseudoautosomal region in the mammalian male meiosis.

Structural evolution of wheat chromosomes 4A, 5A, and 7B and its impact on recombination.

The construction of comparative genetic maps of chromosomes 4A(m) and 5A(m) of Triticum monococcum and chromosomes of homoeologous groups 4, 5 and 7 of T. aestivum has provided insight into the evolution of these chromosomes. The structures of chromosomes 4A, 5A and 7B of modern-day hexaploid bread wheat can be explained by a 4AL/5AL translocation that occurred at the diploid level and is present both in T. monococcum and T. aestivum. Three further rearrangements, a 4AL/7BS translocation, a pericentric inversion and a paracentric inversion, have taken place in the tetraploid progenitor of hexaploid wheat. These structural rearrangements and the evolution of chromosomes 4A, 5A and 7B of bread wheat are discussed. The presence of the 4AL/5AL translocation in several Triticeae genomes raises two questions - which state is the more primitive, and is the translocation of mono- or poly-phylogenetic origin?The rearrangements that have occurred in chromosome 4A resulted in segments of both arms having different positions relative to the telomere, compared to 4A(m) and to 4B and 4D. Comparisons of map length in these regions indicate that genetic length is a function of distance from the telomere, with the distal regions showing the highest recombination.

Application of two microsatellite sequences in wheat storage proteins as molecular markers.

In eukaryotes, tandem arrays of simple-sequence repeat sequences can find applications as highly variable and multi-allelic PCR-based genetic markers. In hexaploid bread wheat, a large-genome inbreeding species with low levels of RFLP, di- and trinucleotide tandem repeats were found in 22 published gene sequences, two of which were converted to PCR-based markers. These were shown to be genome-specific and displayed high levels of variation. These characteristics make them especially suitable for intervarietal breeding applications.

Multiple cDNAs of wheat voltage-dependent anion channels (VDAC): isolation, differential expression, mapping and evolution.

The mitochondrial outer membrane of eukaryotic cells contains voltage-dependent anion channels (VDAC) also termed porins. Three cDNAs from wheat (Triticum aestivum) were isolated and sequenced (Tavdac 1-3). They share 65% similarity of their amino acid sequences, and therefore they probably represent isoforms. The deduced amino acid sequence of one of the cDNAs was found to be identical to the purified VDAC protein from wheat mitochondria [8]. Secondary structure analysis of the deduced amino acid sequences of the three vdac cDNAs revealed a characteristic alpha helix at their N-terminal and beta-barrel cylinders characteristic of VDAC channels. The Tavdac cDNAs are differentially expressed in meristematic tissues. The transcript levels of Tavdac 1 in all wheat tissues is at least 2.5-fold higher than Tavdac 2 and Tavdac 3. Tavdac 2 has a low level of expression in all floral tissues whereas Tavdac 3 is highly expressed in anthers. This is the first report on differential expression of vdac genes in plants. The Tavdac genes have been mapped on the wheat genome. Tavdac 1 is located on the long arm of chromosome 5, Tavdac 2 on the long arm of chromosome 1 and Tavdac 3 on the long arm of chromosome 3. A phylogenetic reconstruction indicates that vdac genes underwent numerous duplication events throughout their evolution. All duplications occurred after the separation of plants from animals and fungi, and no orthologous genes are shared among phyla. Within plants, some of the vdac gene duplications probably occurred before the monocotydelon-dicotydelon split.

Cloning and genetic mapping of wheat telomere-associated sequences.

Wheat telomere-associated sequences (TASs) were cloned using a Vectorette approach and sequenced. Reverse primers specific to the TASs were combined with labelled degenerate telomere primers in PCR reactions containing total genomic DNA as template. Amplification products were separated on sequencing gels. In total, seventeen primer combinations provided 47 polymorphic fragments. Nine of these mapped beyond the most distal RFLP markers and defined the ends of seven chromosome arms. Seven of the nine terminal fragments were derived from a 118-bp tandem repeat, indicating that subtelomeric tandem repeat sequences provide an efficient means to target chromosome ends. A telomere cloning strategy and the terminal and interstitial location of TASs are discussed.