Construction of an EcoRI restriction map of Mycoplasma pneumoniae and localization of selected genes.

A restriction map of the genome of Mycoplasma pneumoniae, a small human pathogenic bacterium, was constructed by means of an ordered cosmid library which spans the complete bacterial chromosome. The positions of 143 endonuclease EcoRI restriction fragments were determined and aligned with the physical map. In addition, restriction sites for the rare-cutting enzymes XhoI (25 sites), ApaI (13 sites), NotI (2 sites), and SfiI (2 sites) were included. The resulting map consists of 185 restriction sites, has a mean resolution of 4.4 kbp, and predicts a genome size of 809 kbp. In addition, several genes were identified and mapped to their respective genomic EcoRI restriction fragments.     

The human mitochondrial ribosomal protein genes: mapping of 54 genes to the chromosomes and implications for human disorders.

Mitochondria possess their own translational machinery, which is composed of components distinct from their cytoplasmic counterparts. To investigate the possible involvement of mitochondrial ribosomal defects in human disease, we mapped nuclear genes that encode mitochondrial ribosomal proteins (MRPs). We generated sequence-tagged sites (STSs) of individual MRP genes that were able to be detected by PCR. They were placed on an STS content map of the human genome by typing of radiation hybrid panels. We located 54 MRP genes on the STS-content map and assigned these genes to cytogenetic bands of the human chromosomes. Although mitochondria are thought to have originated from bacteria, in which the genes encoding ribosomal proteins are clustered into operons, the mapped MRP genes are widely dispersed throughout the genome, suggesting that transfer of each MRP gene to the nuclear genome occurred individually. We compared the assigned positions with candidate regions for mendelian disorders and found certain genes that might be involved in particular diseases. This map provides a basis for studying possible roles of MRP defects in mitochondrial disorders.     

Conservation of Arabidopsis flowering genes in model legumes

The model plants Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) have provided a wealth of information about genes and genetic pathways controlling the flowering process, but little is known about the corresponding pathways in legumes. The garden pea (Pisum sativum) has been used for several decades as a model system for physiological genetics of flowering, but the lack of molecular information about pea flowering genes has prevented direct comparison with other systems. To address this problem, we have searched expressed sequence tag and genome sequence databases to identify flowering-gene-related sequences from Medicago truncatula, soybean (Glycine max), and Lotus japonicus, and isolated corresponding sequences from pea by degenerate-primer polymerase chain reaction and library screening. We found that the majority of Arabidopsis flowering genes are represented in pea and in legume sequence databases, although several gene families, including the MADS-box, CONSTANS, and FLOWERING LOCUS T/TERMINAL FLOWER1 families, appear to have undergone differential expansion, and several important Arabidopsis genes, including FRIGIDA and members of the FLOWERING LOCUS C clade, are conspicuously absent. In several cases, pea and Medicago orthologs are shown to map to conserved map positions, emphasizing the closely syntenic relationship between these two species. These results demonstrate the potential benefit of parallel model systems for an understanding of flowering phenology in crop and model legume species.     

Genes and genomes: Towards construction of an overlapping YAC library of the Arabidopsis thaliana genome

Arabidopsis thaliana (Thale cress, Arabidopsis) is an ideal model organism for the molecular genetic analysis of many plant processes. The availability of a complete physical map would greatly facilitate the gene cloning steps in these studies. The small genome size of Arabidopsis makes the construction of such a map a feasible goal. One of the approaches to construct an overlapping library of the Arabidopsis genome takes advantage of the many mapped markers and the availability of Arabidopsis yeast artificial chromosome (YAC) libraries. Mapped molecular markers are used to identify corresponding YAC clones and thereby place them on the genetic map. Subsequently, these YAC clones provide the framework for directed walking experiments aimed at closing the gaps between the YAC contigs. Adopting this strategy, YAC clones comprising about 10% of the genome have been assigned to the top halves of Arabidopsis chromosomes 4 and 5. Extensive walking experiments in a 10 cM interval of chromosome 4 have resulted in two contiguous regions in the megabase size range.     

Mapping of 19032 mouse cDNAs on mouse chromosomes

Finding genes by the positional candidate approach requires abundant cDNAs mapped to chromosomes. To provide such important information, we computationally mapped 19032 of our mouse cDNAs to mouse chromosomes by using data from public databases. We used 2 approaches. In the first, we integrated the mapping data of cDNAs on the human genome, known gene-related data, and comparative mapping data. From this, we calculated map positions on the mouse chromosomes. For this first approach, we developed a simple and powerful criterion to choose the correct map position from candidate positions in sequence homology searches. In the second approach, we related cDNAs to expressed sequence tags (EST) previously mapped in radiation hybrid experiments. We discuss improving the mapping by combining the 2 methods.     

Segmental structure of the Brassica napus genome based on comparative analysis with Arabidopsis thaliana

Over 1000 genetically linked RFLP loci in Brassica napus were mapped to homologous positions in the Arabidopsis genome on the basis of sequence similarity. Blocks of genetically linked loci in B. napus frequently corresponded to physically linked markers in Arabidopsis. This comparative analysis allowed the identification of a minimum of 21 conserved genomic units within the Arabidopsis genome, which can be duplicated and rearranged to generate the present-day B. napus genome. The conserved regions extended over lengths as great as 50 cM in the B. napus genetic map, equivalent to approximately 9 Mb of contiguous sequence in the Arabidopsis genome. There was also evidence for conservation of chromosome landmarks, particularly centromeric regions, between the two species. The observed segmental structure of the Brassica genome strongly suggests that the extant Brassica diploid species evolved from a hexaploid ancestor. The comparative map assists in exploiting the Arabidopsis genomic sequence for marker and candidate gene identification within the larger, intractable genomes of the Brassica polyploids.     

BAC contig development by fingerprint analysis in soybean.

We constructed a soybean bacterial artificial chromosome (BAC) library suitable for map-based cloning and physical mapping in soybean. This library consists of approximately 40 000 clones (4-5 genome equivalents) stored individually in 384-well microtiter dishes. A random sampling of 224 clones yielded an average insert size of 150 kb, giving a 98% probability of recovering any specific sequence. We screened the library for seven single or very low copy genie or genomic sequences using the polymerase chain reaction (PCR) and found between one and seven BACs for each of the seven sequences. When testing the library with a portion of the soybean psbA chloroplast gene, we found less than 1% chloroplast DNA representation. We also screened the library for eight different classes of disease resistance gene analogs (RGAs) and identified BACs containing all RGAs except class 8. We arranged nine of the class 1 RGA BACs and six of the class 3 RGA BACs into individual contigs based on fingerprint patterns observed after Southern probing of restriction digests of the member BACs with a class-specific sequence. This resulted in the partial localization of the different multigene family sequences without precise definition of their exact positions. Using PCR-based end rescue techniques and RFLP mapping of BAC ends, we mapped individual BACs of each contig onto linkage group J of the soybean public map. The class 1 contig mapped to the region on linkage group J that contains several disease resistance genes. The class 1 contig extended approximately 400 kb. The arrangement of the BACs within this contig has been confirmed using PCR. One end of the class 1 contig core BAC mapped to two positions on linkage group J and cosegregated with two class 1 RGA loci, suggesting that this segment is within an area of regional duplication.     

Genetic architecture of flowering time in maize as inferred from quantitative trait loci meta-analysis and synteny conservation with the rice genome

Genetic architecture of flowering time in maize was addressed by synthesizing a total of 313 quantitative trait loci (QTL) available for this trait. These were analyzed first with an overview statistic that highlighted regions of key importance and then with a meta-analysis method that yielded a synthetic genetic model with 62 consensus QTL. Six of these displayed a major effect. Meta-analysis led in this case to a twofold increase in the precision in QTL position estimation, when compared to the most precise initial QTL position within the corresponding region. The 62 consensus QTL were compared first to the positions of the few flowering-time candidate genes that have been mapped in maize. We then projected rice candidate genes onto the maize genome using a synteny conservation approach based on comparative mapping between the maize genetic map and japonica rice physical map. This yielded 19 associations between maize QTL and genes involved in flowering time in rice and in Arabidopsis. Results suggest that the combination of meta-analysis within a species of interest and synteny-based projections from a related model plant can be an efficient strategy for identifying new candidate genes for trait variation.     

The Arabidopsis TAC Position Viewer: a high‐resolution map of transformation‐competent artificial chromosome (TAC) clones aligned with the Arabidopsis thaliana Columbia‐0 genome

We present a high‐resolution map of genomic transformation‐competent artificial chromosome (TAC) clones extending over all Arabidopsis thaliana (Arabidopsis) chromosomes. The Arabidopsis genomic TAC clones have been valuable genetic tools. Previously, we constructed an Arabidopsis genomic TAC library consisting of more than 10 000 TAC clones harboring large genomic DNA fragments extending over the whole Arabidopsis genome. Here, we determined 13 577 end sequences from 6987 Arabidopsis TAC clones and mapped 5937 TAC clones to precise locations, covering approximately 90% of the Arabidopsis chromosomes. We present the large‐scale data set of TAC clones with high‐resolution mapping information as a Java application tool, the Arabidopsis TAC Position Viewer, which provides ready‐to‐go transformable genomic DNA clones corresponding to certain loci on Arabidopsis chromosomes. The TAC clone resources will accelerate genomic DNA cloning, positional walking, complementation of mutants and DNA transformation for heterologous gene expression.     

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.     

A resource of mapped dissociation launch pads for targeted insertional mutagenesis in the Arabidopsis genome

We describe a new resource for targeted insertional mutagenesis in Arabidopsis using a maize (Zea mays) Activator/Dissociation (Ds) two-element system. The two components of the system, T-DNA vectors carrying a Ds launch pad and a stable Activator transposase source, were designed to simplify selection of transposition events and maximize their usefulness. Because Ds elements preferentially transpose to nearby genomic sites, they can be used in targeted mutagenesis of linked genes. To efficiently target all genes throughout the genome, we generated a large population of transgenic Arabidopsis plants containing the Ds launch pad construct, identified lines containing single Ds launch pad inserts, and mapped the positions of Ds launch pads in 89 lines. The integration sites of the Ds launch pads were relatively evenly distributed on all five chromosomes, except for a region of chromosomes 2 and 4 and the centromeric regions. This resource therefore provides access to the majority of the Arabidopsis genome for targeted tagging.     

Linkage mapping of genes controlling resistance to white rust (Albugo candida) in Brassica rapa (syn. campestris) and comparative mapping to Brassica napus and Arabidopsis thaliana.

Genes for resistance to white rust (Albugo candida) in oilseed Brassica rapa were mapped using a recombinant inbred (RI) population and a genetic linkage map consisting of 144 restriction fragment length polymorphism (RFLP) markers and 3 phenotypic markers. Young seedlings were evaluated by inoculating cotyledons with A. candida race 2 (AC2) and race 7 (AC7) and scoring the interaction phenotype (IP) on a 0-9 scale. The IP of each line was nearly identical for the two races and the population showed bimodal distributions, suggesting that a single major gene (or tightly linked genes) controlled resistance to the two races. The IP scores were converted to categorical resistant and susceptible scores, and these data were used to map a single Mendelian gene controlling resistance to both races on linkage group 4 where resistance to race 2 had been mapped previously. A quantitative trait loci (QTL) mapping approach using the IP scores detected the same major resistance locus for both races, plus a second minor QTL effect for AC2 on linkage group 2. These results indicate that either a dominant allele at a single locus (Acal) or two tightly linked loci control seedling resistance to both races of white rust in the biennial turnip rape cultivar Per. The map positions of white rust resistance genes in B. rapa and Brassica napus were compared and the results indicate where additional loci that have not been mapped may be located. Alignment of these maps to the physical map of the Arabidopsis genome identified regions to target for comparative fine mapping using this model organism.     

Physical mapping of genes to specific chromosomes in Dictyostelium discoideum.

Cloned genes were used to probe a highly redundant library of large cloned fragments of the Dictyostelium discoideum genome carried in yeast artificial chromosomes (YACs). Each gene recognized several independent YAC clones, thereby grouping them into a contig. Individual YACs were arranged within the contig by positioning genes relative to rare restriction sites and the YAC ends. Genes that had been previously assigned to one of the six linkage groups by parasexual genetics were used to establish physically mapped regions on specific chromosomes. Previously unmapped genes were assigned to specific chromosomes when they recognized members of a mapped contig. Linkage was confirmed by congruence of large-scale restriction maps centered on either the previously mapped or the newly mapped genes. At present, the chromosome-assigned map segments comprise approximately 50% of the genome. About half of each map segment is covered by overlapping YACs.     

Map positions of 47 Arabidopsis sequences with sequence similarity to disease resistance genes

Map positions have been determined for 42 non-redundant Arabidopsis expressed sequence tags (ESTs) showing similarity to disease resistance genes (R-ESTs), and for three Pto-like sequences that were amplified with degenerate primers. Employing a PCR-based strategy, yeast artificial chromosome (YAC) clones containing the EST sequences were identified. Since many YACs have been mapped, the locations of the R-ESTs could be inferred from the map positions of the YACs. R-EST clones that exhibited ambiguous map positions were mapped as either cleavable amplifiable polymorphic sequence (CAPS) or restriction fragment length polymorphism (RFLP) markers using F₈ (Ler x Col-0) recombinant inbred (RI) lines. In all cases but two, the R-ESTs and Pto-like sequences mapped to single, unique locations. One R-EST and one Pto-like sequence each mapped to two locations. Thus, a total of 47 loci were identified in this study. Several R-ESTs occur in clusters suggesting that they may have arisen via gene duplication events. Interestingly, several R-ESTs map to regions containing genetically defined disease resistance genes. Thus, this collection of mapped R-ESTs may expedite the isolation of disease resistance genes. As the cDNA sequencing projects have identified an estimated 63% of Arabidopsis genes, a very large number of R-ESTs (～95), and by inference disease resistance genes of the leucine-rich repeat-class probably occur in the Arabidopsis genome.     

New genes in the left arm of the bacteriophage phi 80 chromosome.

We describe the isolation and partial genetic characterization of 247 amber (suppressor-sensitive) mutants of temperate bacteriophage phi 80 of Escherichia coli. Of these 247 mutants, the mutations of 201 mapped to the left arm of the phi 809 chromosome and the mutations of 39 mapped to the right arm of the genome. Complementation tests among these and previously described left arm mutants defined five additional genes in the left arm of the chromosome. The positions of these genes are consistent with the hypothesis that four of them represent functions essential for phi 80 tail assembly and one represents a capsid assembly function, probably the major coat protein. The identification of these genes brings the phi 80 genome into closer correspondence with the organization of the phage lambda genome. Two- and three-factor crosses performed between mutants with defects in each of the previously identified genes and mutants with defects in the five new genes allowed us to construct a consistent, roughly additive recombination map of the left arm of the bacteriophage phi 80 genome.     

Comparative physical mapping of segments of the genome of Brassica oleracea var. alboglabra that are homoeologous to sequenced regions of chromosomes 4 and 5 of Arabidopsis thaliana

Due to their relatedness to Arabidopsis thaliana (Arabidopsis), the cultivated Brassica species represent the first group of crops with which to evaluate comparative genomics approaches to understanding biological processes and manipulating traits. We have constructed a high-quality binary BAC library (JBo) from genomic DNA of Brassica oleracea var. alboglabra, in order to underpin such investigations. Using the Arabidopsis genome sequence and clones from the JBo library, we have analysed aspects of gene conservation and microsynteny between a 222 kb region of the genome of Arabidopsis and homoeologous segments of the genome of B. oleracea. All 19 predicted genes tested were found to hybridize to clones in the JBo library, indicating a high level of gene conservation. Further analyses and physical mapping with the BAC clones identified allowed us to construct clone contig maps and analyse in detail the gene content and organization in the set of paralogous segments identified in the genome of B. oleracea. Extensive divergence of gene content was observed, both between the B. oleracea paralogous segments and between them and their homoeologous segment within the genome of Arabidopsis. However, the genes present show highly conserved collinearity with their orthologues in the genome of Arabidopsis. We have identified one example of a Brassica gene in a non-collinear position and one rearrangement. Some of the genes not present in the discernible homoeologous regions appear to be located elsewhere in the B. oleracea genome. The implications of our findings for comparative map-based cloning of genes from crop species are discussed.     

Map positions of SFR genes in relation to other freezing-related genes of Arabidopsis thaliana

We determined the map positions of seven SFR genes and compared them to the positions of 51 genes suspected of involvement in freezing tolerance in Arabidopsis thaliana. The SFR genes were recognized by the freezing sensitivity of mutants; the others (including 14 whose map positions we have determined) were genes whose expression is induced by low temperature, genes involved in abscisic acid (ABA) biosynthesis and perception, and genes involved in tolerance of oxidative stress. The comparison of map positions indicated a limited set of potential identities, some of which were eliminated by further mapping or by an allelism test.     

A complete map of the human ribosomal protein genes: assignment of 80 genes to the cytogenetic map and implications for human disorders

Mapping of the human ribosomal protein (RP) genes has been completed, and all 80 different genes were placed on a cytogenetic map of the human genome. Because of the existence of processed pseudogenes, the localization of the RP genes was complicated, and five genes had remained to be mapped. Here we developed a novel strategy to identify sequence-tagged sites (STSs) at introns of the RP genes, and we localized RPL14, RPL22, RPL35, RPL36, and RPL39 within the chromosomes by radiation hybrid mapping. Unlike the case of eubacteria or archaebacteria, human RP genes are widely scattered about the genome. Together with the previous results, both sex chromosomes and 20 autosomes (all but chromosomes 7 and 21) were found to carry one or more RP genes. To explore the possible involvement of RP genes in human disorders, all 80 genes were assigned to cytogenetic bands according to a published cytogenetic BAC-STS map of the human genome. We compared the assigned positions with candidate regions for Mendelian disorders and found certain genes that might be involved in particular human disorders.     

Integration of CAPS markers into the RFLP map generated using recombinant inbred lines of Arabidopsis thaliana

Konieczny and Ausubel have described a technique whereby Arabidopsis thaliana loci can be rapidly mapped to one of the ten chromosome arms using a small number of F2 progeny from crosses between the ecotypes Landsberg erecta and Columbia. The technique involves the use of 18 co-dominant, cleaved amplified polymorphic sequence (CAPS) markers which are evenly distributed throughout the Arabidopsis genome. We have mapped these 18 markers using recombinant inbred (RI) lines generated in our laboratory. These data enable a better integration of loci mapped relative to the CAPS markers into the restriction fragment length polymorphism (RFLP) map generated using Arabidopsis RI lines.