Multiple origins, one evolutionary trajectory: gradual evolution characterizes distinct lineages of allotetraploid Brachypodium.

The "genomic shock" hypothesis posits that unusual challenges to genome integrity such as whole genome duplication may induce chaotic genome restructuring. Decades of research on polyploid genomes have revealed that this is often, but not always the case. While some polyploids show major chromosomal rearrangements and derepression of transposable elements in the immediate aftermath of whole genome duplication, others do not. Nonetheless, all polyploids show gradual diploidization over evolutionary time. To evaluate these hypotheses, we produced a chromosome-scale reference genome for the natural allotetraploid grass Brachypodium hybridum, accession "Bhyb26." We compared 2 independently derived accessions of B. hybridum and their deeply diverged diploid progenitor species Brachypodium stacei and Brachypodium distachyon. The 2 B. hybridum lineages provide a natural timecourse in genome evolution because one formed 1.4 million years ago, and the other formed 140 thousand years ago. The genome of the older lineage reveals signs of gradual post-whole genome duplication genome evolution including minor gene loss and genome rearrangement that are missing from the younger lineage. In neither B. hybridum lineage do we find signs of homeologous recombination or pronounced transposable element activation, though we find evidence supporting steady post-whole genome duplication transposable element activity in the older lineage. Gene loss in the older lineage was slightly biased toward 1 subgenome, but genome dominance was not observed at the transcriptomic level. We propose that relaxed selection, rather than an abrupt genomic shock, drives evolutionary novelty in B. hybridum, and that the progenitor species' similarity in transposable element load may account for the subtlety of the observed genome dominance.

Modelling of gene loss propensity in the pangenomes of three Brassica species suggests different mechanisms between polyploids and diploids.

Plant genomes demonstrate significant presence/absence variation (PAV) within a species; however, the factors that lead to this variation have not been studied systematically in Brassica across diploids and polyploids. Here, we developed pangenomes of polyploid Brassica napus and its two diploid progenitor genomes B. rapa and B. oleracea to infer how PAV may differ between diploids and polyploids. Modelling of gene loss suggests that loss propensity is primarily associated with transposable elements in the diploids while in B. napus, gene loss propensity is associated with homoeologous recombination. We use these results to gain insights into the different causes of gene loss, both in diploids and following polyploidization, and pave the way for the application of machine learning methods to understanding the underlying biological and physical causes of gene presence/absence.

Gradual polyploid genome evolution revealed by pan-genomic analysis of Brachypodium hybridum and its diploid progenitors.

Our understanding of polyploid genome evolution is constrained because we cannot know the exact founders of a particular polyploid. To differentiate between founder effects and post polyploidization evolution, we use a pan-genomic approach to study the allotetraploid Brachypodium hybridum and its diploid progenitors. Comparative analysis suggests that most B. hybridum whole gene presence/absence variation is part of the standing variation in its diploid progenitors. Analysis of nuclear single nucleotide variants, plastomes and k-mers associated with retrotransposons reveals two independent origins for B. hybridum, ~1.4 and ~0.14 million years ago. Examination of gene expression in the younger B. hybridum lineage reveals no bias in overall subgenome expression. Our results are consistent with a gradual accumulation of genomic changes after polyploidization and a lack of subgenome expression dominance. Significantly, if we did not use a pan-genomic approach, we would grossly overestimate the number of genomic changes attributable to post polyploidization evolution.

Homoeologous exchange is a major cause of gene presence/absence variation in the amphidiploid Brassica napus.

Homoeologous exchanges (HEs) have been shown to generate novel gene combinations and phenotypes in a range of polyploid species. Gene presence/absence variation (PAV) is also a major contributor to genetic diversity. In this study, we show that there is an association between these two events, particularly in recent Brassica napus synthetic accessions, and that these represent a novel source of genetic diversity, which can be captured for the improvement of this important crop species. By assembling the pangenome of B. napus, we show that 38% of the genes display PAV behaviour, with some of these variable genes predicted to be involved in important agronomic traits including flowering time, disease resistance, acyl lipid metabolism and glucosinolate metabolism. This study is a first and provides a detailed characterization of the association between HEs and PAVs in B. napus at the pangenome level.

The high-quality genome of Brassica napus cultivar 'ZS11' reveals the introgression history in semi-winter morphotype.

Allotetraploid oilseed rape (Brassica napus L.) is an agriculturally important crop. Cultivation and breeding of B. napus by humans has resulted in numerous genetically diverse morphotypes with optimized agronomic traits and ecophysiological adaptation. To further understand the genetic basis of diversification and adaptation, we report a draft genome of an Asian semi-winter oilseed rape cultivar 'ZS11' and its comprehensive genomic comparison with the genomes of the winter-type cultivar 'Darmor-bzh' as well as two progenitors. The integrated BAC-to-BAC and whole-genome shotgun sequencing strategies were effective in the assembly of repetitive regions (especially young long terminal repeats) and resulted in a high-quality genome assembly of B. napus 'ZS11'. Within a short evolutionary period (~6700 years ago), semi-winter-type 'ZS11' and the winter-type 'Darmor-bzh' maintained highly genomic collinearity. Even so, certain genetic differences were also detected in two morphotypes. Relative to 'Darmor-bzh', both two subgenomes of 'ZS11' are closely related to its progenitors, and the 'ZS11' genome harbored several specific segmental homoeologous exchanges (HEs). Furthermore, the semi-winter-type 'ZS11' underwent potential genomic introgressions with B. rapa (Ar ). Some of these genetic differences were associated with key agronomic traits. A key gene of A03.FLC3 regulating vernalization-responsive flowering time in 'ZS11' was first experienced HE, and then underwent genomic introgression event with Ar , which potentially has led to genetic differences in controlling vernalization in the semi-winter types. Our observations improved our understanding of the genetic diversity of different B. napus morphotypes and the cultivation history of semi-winter oilseed rape in Asia.

Surviving a Genome Collision: Genomic Signatures of Allopolyploidization in the Recent Crop Species Brassica napus.

Polyploidization has played a major role in crop plant evolution, leading to advantageous traits that have been selected by humans. Here, we describe restructuring patterns in the genome of L., a recent allopolyploid species. Widespread segmental deletions, duplications, and homeologous chromosome exchanges were identified in diverse genome sequences from 32 natural and 20 synthetic accessions, indicating that homeologous exchanges are a major driver of postpolyploidization genome diversification. Breakpoints of genomic rearrangements are rich in microsatellite sequences that are known to interact with the meiotic recombination machinery. In both synthetic and natural , a subgenome bias was observed toward exchanges replacing larger chromosome segments from the C-subgenome by their smaller, homeologous A-subgenome segments, driving postpolyploidization genome size reduction. Selection in natural favored segmental deletions involving genes associated with immunity, reproduction, and adaptation. Deletions affecting mismatch repair system genes, which are assumed to control homeologous recombination, were also found to be under selection. Structural exchanges between homeologous subgenomes appear to be a major source of novel genetic diversity in de novo allopolyploids. Documenting the consequences of genomic collision by genomic resequencing gives insights into the adaptive processes accompanying allopolyploidization.

Plant genetics. Early allopolyploid evolution in the post-Neolithic Brassica napus oilseed genome.

Oilseed rape (Brassica napus L.) was formed ~7500 years ago by hybridization between B. rapa and B. oleracea, followed by chromosome doubling, a process known as allopolyploidy. Together with more ancient polyploidizations, this conferred an aggregate 72× genome multiplication since the origin of angiosperms and high gene content. We examined the B. napus genome and the consequences of its recent duplication. The constituent An and Cn subgenomes are engaged in subtle structural, functional, and epigenetic cross-talk, with abundant homeologous exchanges. Incipient gene loss and expression divergence have begun. Selection in B. napus oilseed types has accelerated the loss of glucosinolate genes, while preserving expansion of oil biosynthesis genes. These processes provide insights into allopolyploid evolution and its relationship with crop domestication and improvement.

Transcriptome and methylome profiling reveals relics of genome dominance in the mesopolyploid Brassica oleracea.

BACKGROUND: Brassica oleracea is a valuable vegetable species that has contributed to human health and nutrition for hundreds of years and comprises multiple distinct cultivar groups with diverse morphological and phytochemical attributes. In addition to this phenotypic wealth, B. oleracea offers unique insights into polyploid evolution, as it results from multiple ancestral polyploidy events and a final Brassiceae-specific triplication event. Further, B. oleracea represents one of the diploid genomes that formed the economically important allopolyploid oilseed, Brassica napus. A deeper understanding of B. oleracea genome architecture provides a foundation for crop improvement strategies throughout the Brassica genus. RESULTS: We generate an assembly representing 75% of the predicted B. oleracea genome using a hybrid Illumina/Roche 454 approach. Two dense genetic maps are generated to anchor almost 92% of the assembled scaffolds to nine pseudo-chromosomes. Over 50,000 genes are annotated and 40% of the genome predicted to be repetitive, thus contributing to the increased genome size of B. oleracea compared to its close relative B. rapa. A snapshot of both the leaf transcriptome and methylome allows comparisons to be made across the triplicated sub-genomes, which resulted from the most recent Brassiceae-specific polyploidy event. CONCLUSIONS: Differential expression of the triplicated syntelogs and cytosine methylation levels across the sub-genomes suggest residual marks of the genome dominance that led to the current genome architecture. Although cytosine methylation does not correlate with individual gene dominance, the independent methylation patterns of triplicated copies suggest epigenetic mechanisms play a role in the functional diversification of duplicate genes.

Meiotic gene evolution: can you teach a new dog new tricks?

Meiosis, the basis of sex, evolved through iterative gene duplications. To understand whether subsequent duplications have further enriched the core meiotic "tool-kit," we investigated the fate of meiotic gene duplicates following whole genome duplication (WGD), a common occurrence in eukaryotes. We show that meiotic genes return to a single copy more rapidly than genome-wide average in angiosperms, one of the lineages in which WGD is most vividly exemplified. The rate at which duplicates are lost decreases through time, a tendency that is also observed genome-wide and may thus prove to be a general trend post-WGD. The sharpest decline is observed for the subset of genes mediating meiotic recombination; however, we found no evidence that the presence of these duplicates is counterselected in two recent polyploid crops selected for fertility. We therefore propose that their loss is passive, highlighting how quickly WGDs are resolved in the absence of selective duplicate retention.

Prevalence of gene expression additivity in genetically stable wheat allohexaploids.

The reprogramming of gene expression appears as the major trend in synthetic and natural allopolyploids where expression of an important proportion of genes was shown to deviate from that of the parents or the average of the parents. In this study, we analyzed gene expression changes in previously reported, highly stable synthetic wheat allohexaploids that combine the D genome of Aegilops tauschii and the AB genome extracted from the natural hexaploid wheat Triticum aestivum. A comprehensive genome-wide analysis of transcriptional changes using the Affymetrix GeneChip Wheat Genome Array was conducted. Prevalence of gene expression additivity was observed where expression does not deviate from the average of the parents for 99.3% of 34,820 expressed transcripts. Moreover, nearly similar expression was observed (for 99.5% of genes) when comparing these synthetic and natural wheat allohexaploids. Such near-complete additivity has never been reported for other allopolyploids and, more interestingly, for other synthetic wheat allohexaploids that differ from the ones studied here by having the natural tetraploid Triticum turgidum as the AB genome progenitor. Our study gave insights into the dynamics of additive gene expression in the highly stable wheat allohexaploids.

Duplication and partitioning in evolution and function of homoeologous Q loci governing domestication characters in polyploid wheat.

The Q gene encodes an AP2-like transcription factor that played an important role in domestication of polyploid wheat. The chromosome 5A Q alleles (5AQ and 5Aq) have been well studied, but much less is known about the q alleles on wheat homoeologous chromosomes 5B (5Bq) and 5D (5Dq). We investigated the organization, evolution, and function of the Q/q homoeoalleles in hexaploid wheat (Triticum aestivum L.). Q/q gene sequences are highly conserved within and among the A, B, and D genomes of hexaploid wheat, the A and B genomes of tetraploid wheat, and the A, S, and D genomes of the diploid progenitors, but the intergenic regions of the Q/q locus are highly divergent among homoeologous genomes. Duplication of the q gene 5.8 Mya was likely followed by selective loss of one of the copies from the A genome progenitor and the other copy from the B, D, and S genomes. A recent V(329)-to-I mutation in the A lineage is correlated with the Q phenotype. The 5Bq homoeoalleles became a pseudogene after allotetraploidization. Expression analysis indicated that the homoeoalleles are coregulated in a complex manner. Combined phenotypic and expression analysis indicated that, whereas 5AQ plays a major role in conferring domestication-related traits, 5Dq contributes directly and 5Bq indirectly to suppression of the speltoid phenotype. The evolution of the Q/q loci in polyploid wheat resulted in the hyperfunctionalization of 5AQ, pseudogenization of 5Bq, and subfunctionalization of 5Dq, all contributing to the domestication traits.

The genome of the mesopolyploid crop species Brassica rapa.

We report the annotation and analysis of the draft genome sequence of Brassica rapa accession Chiifu-401-42, a Chinese cabbage. We modeled 41,174 protein coding genes in the B. rapa genome, which has undergone genome triplication. We used Arabidopsis thaliana as an outgroup for investigating the consequences of genome triplication, such as structural and functional evolution. The extent of gene loss (fractionation) among triplicated genome segments varies, with one of the three copies consistently retaining a disproportionately large fraction of the genes expected to have been present in its ancestor. Variation in the number of members of gene families present in the genome may contribute to the remarkable morphological plasticity of Brassica species. The B. rapa genome sequence provides an important resource for studying the evolution of polyploid genomes and underpins the genetic improvement of Brassica oil and vegetable crops.

The impact of Ty3-gypsy group LTR retrotransposons Fatima on B-genome specificity of polyploid wheats.

BACKGROUND: Transposable elements (TEs) are a rapidly evolving fraction of the eukaryotic genomes and the main contributors to genome plasticity and divergence. Recently, occupation of the A- and D-genomes of allopolyploid wheat by specific TE families was demonstrated. Here, we investigated the impact of the well-represented family of gypsy LTR-retrotransposons, Fatima, on B-genome divergence of allopolyploid wheat using the fluorescent in situ hybridisation (FISH) method and phylogenetic analysis. RESULTS: FISH analysis of a BAC clone (BAC_2383A24) initially screened with Spelt1 repeats demonstrated its predominant localisation to chromosomes of the B-genome and its putative diploid progenitor Aegilops speltoides in hexaploid (genomic formula, BBAADD) and tetraploid (genomic formula, BBAA) wheats as well as their diploid progenitors. Analysis of the complete BAC_2383A24 nucleotide sequence (113,605 bp) demonstrated that it contains 55.6% TEs, 0.9% subtelomeric tandem repeats (Spelt1), and five genes. LTR retrotransposons are predominant, representing 50.7% of the total nucleotide sequence. Three elements of the gypsy LTR retrotransposon family Fatima make up 47.2% of all the LTR retrotransposons in this BAC. In situ hybridisation of the Fatima_2383A24-3 subclone suggests that individual representatives of the Fatima family contribute to the majority of the B-genome specific FISH pattern for BAC_2383A24. Phylogenetic analysis of various Fatima elements available from databases in combination with the data on their insertion dates demonstrated that the Fatima elements fall into several groups. One of these groups, containing Fatima_2383A24-3, is more specific to the B-genome and proliferated around 0.5-2.5 MYA, prior to allopolyploid wheat formation. CONCLUSION: The B-genome specificity of the gypsy-like Fatima, as determined by FISH, is explained to a great degree by the appearance of a genome-specific element within this family for Ae. speltoides. Moreover, its proliferation mainly occurred in this diploid species before it entered into allopolyploidy.Most likely, this scenario of emergence and proliferation of the genome-specific variants of retroelements, mainly in the diploid species, is characteristic of the evolution of all three genomes of hexaploid wheat.

The Ma gene for complete-spectrum resistance to Meloidogyne species in Prunus is a TNL with a huge repeated C-terminal post-LRR region.

Root-knot nematode (RKN) Meloidogyne species are major polyphagous pests of most crops worldwide, and cultivars with durable resistance are urgently needed because of nematicide bans. The Ma gene from the Myrobalan plum (Prunus cerasifera) confers complete-spectrum, heat-stable, and high-level resistance to RKN, which is remarkable in comparison with the Mi-1 gene from tomato (Solanum lycopersicum), the sole RKN resistance gene cloned. We report here the positional cloning and the functional validation of the Ma locus present at the heterozygous state in the P.2175 accession. High-resolution mapping totaling over 3,000 segregants reduced the Ma locus interval to a 32-kb cluster of three Toll/Interleukin1 Receptor-Nucleotide Binding Site-Leucine-Rich Repeat (LRR) genes (TNL1-TNL3), including a pseudogene (TNL2) and a truncated gene (TNL3). The sole complete gene in this interval (TNL1) was validated as Ma, as it conferred the same complete-spectrum and high-level resistance (as in P.2175) using its genomic sequence and native promoter region in Agrobacterium rhizogenes-transformed hairy roots and composite plants. The full-length cDNA (2,048 amino acids) of Ma is the longest of all Resistance genes cloned to date. Its TNL structure is completed by a huge post-LRR (PL) sequence (1,088 amino acids) comprising five repeated carboxyl-terminal PL exons with two conserved motifs. The amino-terminal region (213 amino acids) of the LRR exon is conserved between alleles and contrasts with the high interallelic polymorphisms of its distal region (111 amino acids) and of PL domains. The Ma gene highlights the importance of these uncharacterized PL domains, which may be involved in pathogen recognition through the decoy hypothesis or in nuclear signaling.

Genome-wide gene expression changes in genetically stable synthetic and natural wheat allohexaploids.

*The present study aims to understand regulation of gene expression in synthetic and natural wheat (Triticum aestivum) allohexaploids, that combines the AB genome of Triticum turgidum and the D genome of Aegilops tauschii; and which we have recently characterized as genetically stable. *We conducted a comprehensive genome-wide analysis of gene expression that allowed characterization of the effect of variability of the D genome progenitor, the intergenerational stability as well as the comparison with natural wheat allohexaploid. We used the Affymetrix GeneChip Wheat Genome Array, on which 55 049 transcripts are represented. *Additive expression was shown to represent the majority of expression regulation in the synthetic allohexaploids, where expression for more than c. 93% of transcripts was equal to the mid-parent value measured from a mixture of parental RNA. This leaves c. 2000 (c. 7%) transcripts, in which expression was nonadditive. No global gene expression bias or dominance towards any of the progenitor genomes was observed whereas high intergenerational stability and low effect of the D genome progenitor variability were revealed. *Our study suggests that gene expression regulation in wheat allohexaploids is established early upon allohexaploidization and highly conserved over generations, as demonstrated by the high similarity of expression with natural wheat allohexaploids.

Newly synthesized wheat allohexaploids display progenitor-dependent meiotic stability and aneuploidy but structural genomic additivity.

To understand key mechanisms leading to stabilized allopolyploid species, we characterized the meiotic behaviour of wheat allohexaploids in relation to structural and genetic changes. For that purpose, we analysed first generations of synthetic allohexaploids obtained through interspecific hybridization, followed by spontaneous chromosome doubling, between several genotypes of Triticum turgidum and Aegilops tauschii wheat species, donors of AB and D genomes, respectively. As expected for these Ph1 (Pairing homoeologous 1) gene-carrying allopolyploids, chromosome pairing at metaphase I of meiosis essentially occurs between homologous chromosomes. However, the different synthetic allohexaploids exhibited progenitor-dependent meiotic irregularities, such as incomplete homologous pairing, resulting in univalent formation and leading to aneuploidy in the subsequent generation. Stability of the synthetic allohexaploids was shown to depend on the considered genotypes of both AB and D genome progenitors, where few combinations compare to the natural wheat allohexaploid in terms of regularity of meiosis and euploidy. Aneuploidy represents the only structural change observed in these synthetic allohexaploids, as no apparent DNA sequence elimination or rearrangement was observed when analysing euploid plants with molecular markers, developed from expressed sequence tags (ESTs) as well as simple sequence repeat (SSR) and transposable element sequences.

Impact of transposable elements on the organization and function of allopolyploid genomes.

Transposable elements (TEs) represent an important fraction of plant genomes and are likely to play a pivotal role in fuelling genome reorganization and functional changes following allopolyploidization. Various processes associated with allopolyploidy (i.e. genetic redundancy, bottlenecks during the formation of allopolyploids or genome shock following genome merging) may allow accumulation of TE insertions. Our objective in carrying out a survey of the literature and a comparative analysis across different allopolyploid systems is to shed light on the structural, epigenetic and functional modifications driven by TEs during allopolyploidization and subsequent diploidization. The available evidence indicates that TE proliferation in the short or the long term after allopolyploidization may be restricted to a few TEs, in specific polyploid systems. By contrast, data indicate major structural changes in the TE genome fraction immediately after allopolyploidization, mainly through losses of TE sequences as a result of recombination. Emerging evidence also suggests that TEs are targeted by substantial epigenetic changes, which may impact gene expression and genome stability. Furthermore, TEs may directly or indirectly support the evolution of new functionalities in allopolyploids during diploidization. All data stress allopolyploidization as a shock associated with drastic genome reorganization. Mechanisms controlling TEs during allopolyploidization as well as their impact on diploidization are discussed.

Single-nucleotide polymorphism frequency in a set of selected lines of bread wheat (Triticum aestivum L.).

Information on single-nucleotide polymorphisms (SNPs) in hexaploid bread wheat is still scarce. The goal of this study was to detect SNPs in wheat and examine their frequency. Twenty-six bread wheat lines from different origins worldwide were used. Specific PCR-products were obtained from 21 genes and directly sequenced. SNPs were discovered from the alignment of these sequences. The overall sequence polymorphism observed in this sample appears to be low; 64 single-base polymorphisms were detected in approximately 21.5 kb (i.e., 1 SNP every 335 bp). The level of polymorphism is highly variable among the different genes studied. Fifty percent of the genes studied contained no sequence polymorphism, whereas most SNPs detected were located in only 2 genes. As expected, taking into account a synthetic line created with a wild Triticum tauschii parent increases the level of polymorphism (101 SNPs; 1 SNP every 212 bp). The detected SNPs are available at http://urgi.versailles.inra.fr/GnpSNP">http://urgi.versailles.inra.fr/GnpSNP. Data on linkage disequilibrium (LD) are still preliminary. They showed a significant level of LD in the 2 most polymorphic genes. To conclude, the genome size of hexaploid wheat and its low level of polymorphism complicate SNP discovery in this species.

Types and rates of sequence evolution at the high-molecular-weight glutenin locus in hexaploid wheat and its ancestral genomes.

The Glu-1 locus, encoding the high-molecular-weight glutenin protein subunits, controls bread-making quality in hexaploid wheat (Triticum aestivum) and represents a recently evolved region unique to Triticeae genomes. To understand the molecular evolution of this locus region, three orthologous Glu-1 regions from the three subgenomes of a single hexaploid wheat species were sequenced, totaling 729 kb of sequence. Comparing each Glu-1 region with its corresponding homologous region from the D genome of diploid wheat, Aegilops tauschii, and the A and B genomes of tetraploid wheat, Triticum turgidum, revealed that, in addition to the conservation of microsynteny in the genic regions, sequences in the intergenic regions, composed of blocks of nested retroelements, are also generally conserved, although a few nonshared retroelements that differentiate the homologous Glu-1 regions were detected in each pair of the A and D genomes. Analysis of the indel frequency and the rate of nucleotide substitution, which represent the most frequent types of sequence changes in the Glu-1 regions, demonstrated that the two A genomes are significantly more divergent than the two B genomes, further supporting the hypothesis that hexaploid wheat may have more than one tetraploid ancestor.

Advanced resources for plant genomics: a BAC library specific for the short arm of wheat chromosome 1B.

Common wheat (Triticum aestivum L., 2n = 6x = 42) is a polyploid species possessing one of the largest genomes among the cultivated crops (1C is approximately 17 000 Mb). The presence of three homoeologous genomes (A, B and D), and the prevalence of repetitive DNA make sequencing the wheat genome a daunting task. We have developed a novel 'chromosome arm-based' strategy for wheat genome sequencing to simplify this task; this relies on sub-genomic libraries of large DNA inserts. In this paper, we used a di-telosomic line of wheat to isolate six million copies of the short arm of chromosome 1B (1BS) by flow sorting. Chromosomal DNA was partially digested with HindIII and used to construct an arm-specific BAC library. The library consists of 65 280 clones with an average insert size of 82 kb. Almost half of the library (45%) has inserts larger than 100 kb, while 18% of the inserts range in size between 75 and 100 kb, and 37% are shorter than 75 kb. We estimated the chromosome arm coverage to be 14.5-fold, giving a 99.9% probability of identifying a clone corresponding to any sequence on the short arm of 1B. Each chromosome arm in wheat can be flow sorted from an appropriate cytogenetic stock, and we envisage that the availability of chromosome arm-specific BAC resources in wheat will greatly facilitate the development of ready-to-sequence physical maps and map-based gene cloning.