Integration of the FISH pachytene and genetic maps of Medicago truncatula

A molecular cytogenetic map of Medicago truncatula (2n = 2x = 16) was constructed on the basis of a pachytene DAPI karyogram. Chromosomes at this meiotic prophase stage are 20 times longer than at mitotic metaphase, and display a well differentiated pattern of brightly fluorescing heterochromatin segments. We describe here a pachytene karyogram in which all chromosomes can be identified based on chromosome length, centromere position, heterochromatin patterns, and the positions of three repetitive sequences (5S rDNA, 45S rDNA and the MtR1 tandem repeat), visualized by fluorescence in situ hybridization (FISH). We determined the correlation between genetic linkage groups and chromosomes by FISH mapping of bacterial artificial chromosome (BAC) clones, with two to five BACs per linkage group. In the cytogenetic map, chromosomes were numbered according to their corresponding linkage groups. We determined the relative positions of the 20 BACs and three repetitive sequences on the pachytene chromosomes, and compared the genetic and cytological distances between markers. The mapping resolution was determined in a euchromatic part of chromosome 5 by comparing the cytological distances between FISH signals of clones of a BAC contig with their corresponding physical distance, and showed that resolution in this region is about 60 kb. The establishment of this FISH pachytene karyotype, with a far better mapping resolution and detection sensitivity compared to those in the highly condensed mitotic metaphase complements, has created the basis for the integration of molecular, genetic and cytogenetic maps in M. truncatula.     

An integrated molecular cytogenetic map of Cucumis sativus L. chromosome 2

Background

Integration of molecular, genetic and cytological maps is still a challenge for most plant species. Recent progress in molecular and cytogenetic studies created a basis for developing integrated maps in cucumber (Cucumis sativus L.).

Results

In this study, eleven fosmid clones and three plasmids containing 45S rDNA, the centromeric satellite repeat Type III and the pericentriomeric repeat CsRP1 sequences respectively were hybridized to cucumber metaphase chromosomes to assign their cytological location on chromosome 2. Moreover, an integrated molecular cytogenetic map of cucumber chromosomes 2 was constructed by fluorescence in situ hybridization (FISH) mapping of 11 fosmid clones together with the cucumber centromere-specific Type III sequence on meiotic pachytene chromosomes. The cytogenetic map was fully integrated with genetic linkage map since each fosmid clone was anchored by a genetically mapped simple sequence repeat marker (SSR). The relationship between the genetic and physical distances along chromosome was analyzed.

Conclusions

Recombination was not evenly distributed along the physical length of chromosome 2. Suppression of recombination was found in centromeric and pericentromeric regions. Our results also indicated that the molecular markers composing the linkage map for chromosome 2 provided excellent coverage of the chromosome.     

A molecular cytogenetic map of sorghum chromosome 1. Fluorescence in situ hybridization analysis with mapped bacterial artificial chromosomes

We used structural genomic resources for Sorghum bicolor (L.) Moench to target and develop multiple molecular cytogenetic probes that would provide extensive coverage for a specific chromosome of sorghum. Bacterial artificial chromosome (BAC) clones containing molecular markers mapped across sorghum linkage group A were labeled as probes for fluorescence in situ hybridization (FISH). Signals from single-, dual-, and multiprobe BAC-FISH to spreads of mitotic chromosomes and pachytene bivalents were associated with the largest sorghum chromosome, which bears the nucleolus organizing region (NOR). The order of individual BAC-FISH loci along the chromosome was fully concordant to that of marker loci along the linkage map. In addition, the order of several tightly linked molecular markers was clarified by FISH analysis. The FISH results indicate that markers from the linkage map positions 0.0-81.8 cM reside in the short arm of chromosome 1 whereas markers from 81.8-242.9 cM are located in the long arm of chromosome 1. The centromere and NOR were located in a large heterochromatic region that spans approximately 60% of chromosome 1. In contrast, this region represents only 0.7% of the total genetic map distance of this chromosome. Variation in recombination frequency among euchromatic chromosomal regions also was apparent. The integrated data underscore the value of cytological data, because minor errors and uncertainties in linkage maps can involve huge physical regions. The successful development of multiprobe FISH cocktails suggests that it is feasible to develop chromosome-specific "paints" from genomic resources rather than flow sorting or microdissection and that when applied to pachytene chromatin, such cocktails provide an especially powerful framework for mapping. Such a molecular cytogenetic infrastructure would be inherently cross-linked with other genomic tools and thereby establish a cytogenomics system with extensive utility in development and application of genomic resources, cloning, transgene localization, development of plant "chromonomics," germplasm introgression, and marker-assisted breeding. In combination with previously reported work, the results indicate that a sorghum cytogenomics system would be partially applicable to other gramineous genera.     

High-resolution pachytene chromosome mapping of bacterial artificial chromosomes anchored by genetic markers reveals the centromere location and the distribution of genetic recombination along chromosome 10 of rice

Large-scale physical mapping has been a major challenge for plant geneticists due to the lack of techniques that are widely affordable and can be applied to different species. Here we present a physical map of rice chromosome 10 developed by fluorescence in situ hybridization (FISH) mapping of bacterial artificial chromosome (BAC) clones on meiotic pachytene chromosomes. This physical map is fully integrated with a genetic linkage map of rice chromosome 10 because each BAC clone is anchored by a genetically mapped restriction fragment length polymorphism marker. The pachytene chromosome-based FISH mapping shows a superior resolving power compared to the somatic metaphase chromosome-based methods. The telomere-centromere orientation of DNA clones separated by 40 kb can be resolved on early pachytene chromosomes. Genetic recombination is generally evenly distributed along rice chromosome 10. However, the highly heterochromatic short arm shows a lower recombination frequency than the largely euchromatic long arm. Suppression of recombination was found in the centromeric region, but the affected region is far smaller than those reported in wheat and barley. Our FISH mapping effort also revealed the precise genetic position of the centromere on chromosome 10.     

Development of one set of chromosome-specific microsatellite-containing BACs and their physical mapping in Gossypium hirsutum L.

Fluorescence in situ hybridization (FISH), using bacterial artificial chromosome (BAC) clone as probe, is a reliable cytological technique for chromosome identification. It has been used in many plants, especially in those containing numerous small chromosomes. We previously developed eight chromosome-specific BAC clones from tetraploid cotton, which were used as excellent cytological markers for chromosomes identification. Here, we isolated the other chromosome-specific BAC clones to make a complete set for the identification of all 26 chromosome-pairs by this technology in tetraploid cotton (Gossypium hirsutum L.). This set of BAC markers was demonstrated to be useful to assign each chromosome to a genetic linkage group unambiguously. In addition, these BAC clones also served as convenient and reliable landmarks for establishing physical linkage with unknown targeted sequences. Moreover, one BAC containing an EST, with high sequence similarity to a G. hirsutum ethylene-responsive element-binding factor was located physically on the long arm of chromosome A7 with the help of a chromosome-A7-specific BAC FISH marker. Comparative analysis of physical marker positions in the chromosomes by BAC-FISH and genetic linkage maps demonstrated that most of the 26 BAC clones were localized close to or at the ends of their respective chromosomes, and indicated that the recombination active regions of cotton chromosomes are primarily located in the distal regions. This technology also enables us to make associations between chromosomes and their genetic linkage groups and re-assign each chromosome according to the corresponding genetic linkage group. This BAC clones and BAC-FISH technology will be useful for us to evaluate grossly the degree to which a linkage map provides adequate coverage for developing a saturated genetic map, and provides a powerful resource for cotton genomic researches.     

Integrated cytogenetic map of mitotic metaphase chromosome 9 of maize: resolution,sensitivity, and banding paint development

To study the correlation of the sequence positions on the physical DNA finger print contig (FPC) map and cytogenetic maps of pachytene and somatic maize chromosomes, sequences located along the chromosome 9 FPC map approximately every 10 Mb were selected to place on maize chromosomes using fluorescent in situ hybridization (FISH). The probes were produced as pooled polymerase chain reaction products based on sequences of genetic markers or repeat-free portions of mapped bacterial artificial chromosome (BAC) clones. Fifteen probes were visualized on chromosome 9. The cytological positions of most sequences correspond on the pachytene, somatic, and FPC maps except some probes at the pericentromeric regions. Because of unequal condensation of mitotic metaphase chromosomes, being lower at pericentromeric regions and higher in the arms, probe positions are displaced to the distal ends of both arms. The axial resolution of FISH on somatic chromosome 9 varied from 3.3 to 8.2 Mb, which is 12-30 times lower than on pachytene chromosomes. The probe collection can be used as chromosomal landmarks or as a "banding paint" for the physical mapping of sequences including transgenes and BAC clones and for studying chromosomal rearrangements.     

High-resolution single-copy gene fluorescence in situ hybridization and its use in the construction of a cytogenetic map of maize chromosome 9

High-resolution cytogenetic maps provide important biological information on genome organization and function, as they correlate genetic distance with cytological structures, and are an invaluable complement to physical sequence data. The most direct way to generate a cytogenetic map is to localize genetically mapped genes onto chromosomes by fluorescence in situ hybridization (FISH). Detection of single-copy genes on plant chromosomes has been difficult. In this study, we developed a squash FISH procedure allowing successful detection of single-copy genes on maize (Zea mays) pachytene chromosomes. Using this method, the shortest probe that can be detected is 3.1 kb, and two sequences separated by approximately 100 kb can be resolved. To show the robust nature of this protocol, we localized nine genetically mapped single-copy genes on chromosome 9 in one FISH experiment. Integration of existing information from genetic maps and the BAC contig-based physical map with the cytological structure of chromosome 9 provides a comprehensive cross-referenced cytogenetic map and shows the dramatic reduction of recombination in the pericentromeric heterochromatic region. To establish a feasible mapping system for maize, we also developed a probe cocktail for unambiguous identification of the 10 maize pachytene chromosomes. These results provide a starting point toward constructing a high-resolution integrated cytogenetic map of maize.     

An Integrated Genetic and Cytogenetic Map for Zhikong Scallop,Chlamys farreri,Based on Microsatellite Markers

The reliability of genome analysis and proficiency of genetic manipulation requires knowledge of the correspondence between the genetic and cytogenetic maps. In the present study, we integrated cytogenetic and microsatellite-based linkage maps for Zhikong scallop, Chlamys farreri. Thirty-eight marker-anchored BAC clones standing for the 19 linkage groups were used to be FISH probes. Of 38 BAC clones, 30 were successfully located on single chromosome by FISH and used to integrate the genetic and cytogenetic map. Among the 19 linkage groups, 12 linkage groups were physically anchored by 2 markers, 6 linkage groups were anchored by 1 marker, and one linkage group was not anchored any makers by FISH. In addition, using two-color FISH, six linkage groups were distinguished by different chromosomal location; linkage groups LG6 and LG16 were placed on chromosome 10, LG8 and LG18 on chromosome 14. As a result, 18 of 19 linkage groups were localized to 17 pairs of chromosomes of C. farreri. We first integrated genetic and cytogenetic map for C. farreri. These 30 chromosome specific BAC clones in the cytogenetic map could be used to identify chromosomes of C. farreri. The integrated map will greatly facilitate molecular genetic studies that will be helpful for breeding applications in C. farreri and the upcoming genome projects of this species.     

Chromatin structure and physical mapping of chromosome 6 of potato and comparative analyses with tomato

Potato (Solanum tuberosum) has the densest genetic linkage map and one of the earliest established cytogenetic maps among all plant species. However, there has been limited effort to integrate these maps. Here, we report fluorescence in situ hybridization (FISH) mapping of 30 genetic marker-anchored bacterial artificial chromosome (BAC) clones on the pachytene chromosome 6 of potato. The FISH mapping results allowed us to define the genetic positions of the centromere and the pericentromeric heterochromatin and to relate chromatin structure to the distribution of recombination along the chromosome. A drastic reduction of recombination was associated with the pericentromeric heterochromatin that accounts for ~28% of the physical length of the pachytene chromosome. The pachytene chromosomes 6 of potato and tomato (S. lycopersicum) share a similar morphology. However, distinct differences of heterochromatin distribution were observed between the two chromosomes. FISH mapping of several potato BACs on tomato pachytene chromosome 6 revealed an overall colinearity between the two chromosomes. A chromosome inversion was observed in the euchromatic region of the short arms. These results show that the potato and tomato genomes contain more chromosomal rearrangements than those reported previously on the basis of comparative genetic linkage mapping.     

A plant-transformation-competent BIBAC/BAC-based map of rice for functional analysis and genetic engineering of its genomic sequence.

Sequencing of the rice genome has provided a platform for functional genomics research of rice and other cereal species. However, multiple approaches are needed to determine the functions of its genes and sequences and to use the genome sequencing results for genetic improvement of cereal crops. Here, we report a plant-transformation-competent, binary bacterial artificial chromosome (BIBAC) and bacterial artificial chromosome (BAC) based map of rice to facilitate these studies. The map was constructed from 20 835 BIBAC and BAC clones, and consisted of 579 overlapping BIBAC/BAC contigs. To facilitate functional analysis of chromosome 8 genomic sequence and cloning of the genes and QTLs mapped to the chromosome, we anchored the chromosomal contigs to the existing rice genetic maps. The chromosomal map consists of 11 contigs, 59 genetic markers, and 36 sequence tagged sites, spanning a total of ca. 38 Mb in physical length. Comparative analysis between the genetic and physical maps of chromosome 8 showed that there are 3 "hot" and 2 "cold" spots of genetic recombination along the chromosomal arms in addition to the "cold spot" in the centromeric region, suggesting that the sequence component contents of a chromosome may affect its local genetic recombination frequencies. Because of its plant transformability, the BIBAC/BAC map could provide a platform for functional analysis of the rice genome sequence and effective use of the sequencing results for gene and QTL cloning and molecular breeding.     

A fine physical map of the rice chromosome 5

A fine physical map of the rice (Oryza sativa spp. Japonica var. Nipponbare) chromosome 5 with bacterial artificial chromosome (BAC) and PI-derived artificial chromosome (PAC) clones was constructed through integration of 280 sequenced BAC/PAC clones and 232 sequence tagged site/expressed sequence tag markers with the use of fingerprinted contig data of the Nipponbare genome. This map consists of five contigs covering 99% of the estimated chromosome size (30.08 Mb). The four physical gaps were estimated at 30 and 20 kb for gaps 1–3 and gap 4, respectively. We have submitted 42.2-Mb sequences with 29.8 Mb of nonoverlapping sequences to public databases. BAC clones corresponding to telomere and centromere regions were confirmed by BAC-fluorescence in situ hybridization (FISH) on a pachytene chromosome. The genetically centromeric region at 54.6 cM was covered by a minimum tiling path spanning 2.1 Mb with no physical gaps. The precise position of the centromere was revealed by using three overlapping BAC/PACs for ~150 kb. In addition, FISH results revealed uneven chromatin condensation around the centromeric region at the pachytene stage. This map is of use for positional cloning and further characterization of the rice functional genomics. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. Chia-Hsiung Cheng and Mei-Chu Chung have equal contributions.     

Integration of chicken cytogenetic and genetic maps: 18 new polymorphic markers isolated from BAC and PAC clones

As an approach to integrate the chicken genetic and cytogenetic maps, bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC) clones were localized by fluorescence in situ hybridization (FISH) on chromosomes and by genetic mapping on the East Lansing and Compton reference families. Some of the clones used in this study were previously selected for the presence of potentially polymorphic (CA)n repeats and a microsatellite marker was developed when possible for genetic mapping. For other clones, a single strand conformational polymorphism (SSCP) was developed and used for this purpose. Between the two approaches, 18 markers linking the cytogenetic and genetic maps, seven on macrochromosomes and 11 on microchromosomes, were generated. Our results enabled the assignment and orientation of a linkage group to chromosome 3, together with the assignment of linkage groups to eight different microchromosomes, a fraction of the genome lacking mapping data and for which the degree of coverage by the genetic map was not well estimated previously.     

Autistic disorder and chromosome 15q11-q13: construction and analysis of a BAC/PAC contig

Autistic disorder (AD) is a neurodevelopmental disorder that affects approximately 2-10/10,000 individuals. Chromosome 15q11-q13 has been implicated in the genetic etiology of AD based on (1) cytogenetic abnormalities; (2) increased recombination frequency in this region in AD versus non-AD families; (3) suggested linkage with markers D15S156, D15S219, and D15S217; and (4) evidence for significant association with polymorphisms in the gamma-aminobutyric acid receptor subunit B3 gene (GABRB3). To isolate the putative 15q11-q13 candidate AD gene, a genomic contig and physical map of the approximately 1.2-Mb region from the GABA receptor gene cluster to the OCA2 locus was generated. Twenty-one bacterial artificial chromosome (BAC) clones, 32 P1-derived artificial chromosome (PAC) clones, and 2 P1 clones have been isolated using the markers D15S540, GABRB3, GABRA5, GABRG3, D15S822, and D15S217, as well as 34 novel markers developed from the end sequences of BAC/PAC clones. In contrast to previous findings, the markers D15S822 and D15S975 have been localized within the GABRG3 gene, which we have shown to be approximately 250 kb in size. NotI and numerous EagI restriction enzyme cut sites were identified in this region. The BAC/PAC genomic contig can be utilized for the study of genomic structure and the identification and characterization of genes and their methylation status in this autism candidate gene region on human chromosome 15q11-q13.     

Molecular cytogenetic maps of sorghum linkage groups 2 and 8

To integrate genetic, physical, and cytological perspectives of the Sorghum bicolor genome, we selected 40 landed bacterial artificial chromosome (BAC) clones that contain different linkage map markers, 21 from linkage group 2 (LG-02) and 19 from linkage group 8 (LG-08). Multi-BAC probe cocktails were constructed for each chromosome from the landed BACs, which were also preevaluated for FISH signal quality, relative position, and collective chromosome coverage. Comparison to the corresponding linkage map revealed full concordance of locus order between cytological and prior segregation analyses. The pericentromeric heterochromatin constituted a large quasi-uniform block in each bivalent and was especially large in the bivalent corresponding to LG-08. Centromere positions in LG-02 and LG-08 were progressively delimited using FISH to identify landed BACs for which the FISH signals visibly flanked the centromere. Alignment of linkage and cytological maps revealed that pericentromeric heterochromatin of these sorghum chromosomes is largely devoid of recombination, which is mostly relegated to the more distal regions, which are largely euchromatic. This suggests that the sorghum genome is thus even more amenable to physical mapping of genes and positional cloning than the C-value alone might suggest. As a prelude to positional cloning of the fertility restorer, Rf1, FISH of BAC clones flanking the Rf1 locus was used to delimit the chromosomal position of the gene. FISH of BACs that contain the most proximal linkage markers enabled localization of Rf1 to a approximately 0.4-Mbp euchromatic region of LG-08. Cytogenetic analyses of Rf1 and other trait loci will aid in assessing the feasibility of positional cloning and help formulate strategies required for cloning this and other agriculturally critical genes.     

A cytogenetically based physical map of chromosome 1B in common wheat.

We have constructed a cytogenetically based physical map of chromosome 1B in common wheat by utilizing a total of 18 homozygous deletion stocks. It was possible to divide chromosome 1B into 17 subregions. Nineteen genetic markers are physically mapped to nine subregions of chromosome 1B. Comparison of the cytological map of chromosome 1B with an RFLP-based genetic linkage map of Triticum tauschii revealed that the linear order of the genetic markers was maintained between chromosome 1B of hexaploid wheat and 1D of T. tauschii. Striking differences were observed between the physical and genetic maps in relation to the relative distances between the genetic markers. The genetic markers clustered in the middle of the genetic map were physically located in the distal regions of both arms of chromosome 1B. It is unclear whether the increased recombination in the distal regions of chromosome 1B is due to specific regions of increased recombination or a more broadly distributed increase in recombination in the distal regions of Triticeae chromosomes.     

Development of Chromosome-specific Cytogenetic Markers and Merging of Linkage Fragments in Papaya

Carica papaya L. is a tropical and sub-tropical fruit-tree crop with a small genome and nine pairs of chromosomes. The transgenic cultivar ‘SunUp’ has been sequenced and three high-density genetic maps are available for mapping agronomically and economically-important traits. However, the small size and similar morphology of papaya chromosomes hinder their identification and few cytological resources are available for integration of genetic and cytogenetic information. Fluorescence in situ hybridization (FISH) was performed on mitotic metaphase chromosomes using BAC clones harboring mapped simple sequence repeat (SSR) markers as probes. A total of 104 BAC clones covering all 12 linkage groups (LGs) were tested and 12 of them, that gave a single specific signal, were chosen as representative of the 12 LGs of the SSR genetic map. This set of chromosome-specific DNA markers acted as a foundation for papaya chromosome karyotyping and re-assigning orientation of LGs. Chromosome-specific markers allowed us to assign the minor LGs 10, 11, and 12 to major LGs 8, 9, and 7, respectively. We thus reduced the number of LGs in the genetic map to nine, corresponding to the haploid number of papaya chromosomes. We also tested the relative order of DNA markers on minor LGs 10 and 11 to place them on top of LGs 8 and 9 in the correct orientation. Ribosomal DNAs (rDNAs), a set of major cytogenetic markers, were positioned on specific papaya chromosomes. The 25S rDNA showed strong signals at the constriction site of a single pair of chromosomes identified as LG 2 by LG 2-specific BAC clone. The 5S rDNA showed strong signals on two pairs of chromosomes that are syntenic with LG 4- and LG 5-specific BAC clones. This integrated map will facilitate genome assembly, quantitative trait locus (QTL) mapping, and the study of cytological, physical and genetic distance relationships between papaya chromosomes.     

Integrating genetic linkage maps with pachytene chromosome structure in maize

Genetic linkage maps reveal the order of markers based on the frequency of recombination between markers during meiosis. Because the rate of recombination varies along chromosomes, it has been difficult to relate linkage maps to chromosome structure. Here we use cytological maps of crossing over based on recombination nodules (RNs) to predict the physical position of genetic markers on each of the 10 chromosomes of maize. This is possible because (1). all 10 maize chromosomes can be individually identified from spreads of synaptonemal complexes, (2). each RN corresponds to one crossover, and (3). the frequency of RNs on defined chromosomal segments can be converted to centimorgan values. We tested our predictions for chromosome 9 using seven genetically mapped, single-copy markers that were independently mapped on pachytene chromosomes using in situ hybridization. The correlation between predicted and observed locations was very strong (r(2) = 0.996), indicating a virtual 1:1 correspondence. Thus, this new, high-resolution, cytogenetic map enables one to predict the chromosomal location of any genetically mapped marker in maize with a high degree of accuracy. This novel approach can be applied to other organisms as well.     

Construction of a bacterial artificial chromosome (BAC) library for potato molecular cytogenetics research.

Lack of reliable techniques for chromosome identification is the major obstacle for cytogenetics research in plant species with large numbers of small chromosomes. To promote molecular cytogenetics research of potato (Solanum tuberosum, 2n = 4x = 48) we developed a bacterial artificial chromosome (BAC) library of a diploid potato species S. bulbocastanum. The library consists of 23,808 clones with an average insert size of 155 kb, and represents approximately 3.7 equivalents to the potato genome. The majority of the clones in the BAC library generated distinct signals on specific potato chromosomes using fluorescence in situ hybridization (FISH). The hybridization signals provide excellent cytological markers to tag individual potato chromosomes. We also demonstrated that the BAC clones can be mapped to specific positions on meiotic pachytene chromosomes. The excellent resolution of pachytene FISH can be used to construct a physical map of potato by mapping molecular marker-targeted BAC clones on pachytene chromosomes.     

Anchoring of rice BAC clones to the rice genetic map in silico

A wealth of molecular resources have been developed for rice genomics, including dense genetic maps, expressed sequence tags (ESTs), yeast artificial chromosome maps, bacterial artificial chromosome (BAC) libraries and BAC end sequence databases. Integration of genetic and physical maps involves labor-intensive empirical experiments. To accelerate the integration of the bacterial clone resources with the genetic map for the International Rice Genome Sequencing Project, we cleaned and filtered the available EST and BAC end sequences for repetitive sequences and then searched all available rice genetic markers with our filtered databases. We identified 418 genetic markers that aligned with at least one BAC end sequence with >95% sequence identity, providing a set of large insert clones with an average separation of 1 Mb that can serve as nucleation points for the sequencing phase of the International Rice Genome Sequencing Project.     

Generation of a 5.5-Mb BAC/PAC contig of pig chromosome 6q1.2 and its integration with existing RH, genetic and comparative maps

We generated a sequence-ready BAC/PAC contig spanning approximately 5.5 Mb on porcine chromosome 6q1.2, which represents a very gene-rich genome region. STS content mapping was used as the main strategy for the assembly of the contig and a total of 6 microsatellite markers, 53 gene-related STS and 116 STS corresponding to BAC and PAC end sequences were analyzed. The contig comprises 316 BAC and PAC clones covering the region between the genes GPI and LIPE. The correct contig assembly was verified by RH-mapping of STS markers and comparative mapping of BAC/PAC end sequences using BLAST searches. The use of microsatellite primer pairs allowed the integration of the physical maps with the genetic map of this region. Comparative mapping of the porcine BAC/PAC contig with respect to the gene-rich region on the human chromosome 19q13.1 map revealed a completely conserved gene order of this segment, however, physical distances differ somewhat between HSA19q13.1 and SSC6q1.2. Three major differences in DNA content between human and pig are found in two large intergenic regions and in one region of a clustered gene family, respectively. While there is a complete conservation of gene order between pig and human, the comparative analysis with respect to the rodent species mouse and rat shows one breakpoint where a genome segment is inverted.