Piggy-BACing the human genome I: constructing a porcine BAC physical map through comparative genomics

Availability of the human genome sequence and high similarity between humans and pigs at the molecular level provides an opportunity to use a comparative mapping approach to piggy-BAC the human genome. In order to advance the pig genome sequencing initiative, sequence similarity between large-scale porcine BAC-end sequences (BESs) and human genome sequence was used to construct a comparatively-anchored porcine physical map that is a first step towards sequencing the pig genome. A total of 50,300 porcine BAC clones were end-sequenced, yielding 76,906 BESs after trimming with an average read length of 538 bp. To anchor the porcine BACs on the human genome, these BESs were subjected to BLAST analysis using the human draft sequence, revealing 31.5% significant hits (E < e(-5)). Both genic and non-genic regions of homology contributed to the alignments between the human and porcine genomes. Porcine BESs with unique homology matches within the human genome provided a source of markers spaced approximately 70 to 300 kb along each human chromosome. In order to evaluate the utility of piggy-BACing human genome sequences, and confirm predictions of orthology, 193 evenly spaced BESs with similarity to HSA3 and HSA21 were selected and then utilized for developing a high-resolution (1.22 Mb) comparative radiation hybrid map of SSC13 that represents a fusion of HSA3 and HSA21. Resulting RH mapping of SSC13 covers 99% and 97% of HSA3 and HSA21, respectively. Seven evolutionary conserved blocks were identified including six on HSA3 and a single syntenic block corresponding to HSA21. The strategy of piggy-BACing the human genome described in this study demonstrates that through a directed, targeted comparative genomics approach construction of a high-resolution anchored physical map of the pig genome can be achieved. This map supports the selection of BACs to construct a minimal tiling path for genome sequencing and targeted gap filling. Moreover, this approach is highly relevant to other genome sequencing projects.     

High-resolution comprehensive radiation hybrid maps of the porcine chromosomes 2p and 9p compared with the human chromosome 11

We are constructing high-resolution, chromosomal 'test' maps for the entire pig genome using a 12,000-rad WG-RH panel (IMNpRH2(12,000-rad))to provide a scaffold for the rapid assembly of the porcine genome sequence. Here we present an initial, comparative map of human chromosome (HSA) 11 with pig chromosomes (SSC) 2p and 9p. Two sets of RH mapping vectors were used to construct the RH framework (FW) maps for SSC2p and SSC9p. One set of 590 markers, including 131 microsatellites (MSs), 364 genes/ESTs, and 95 BAC end sequences (BESs) was typed on the IMNpRH2(12,000-rad) panel. A second set of 271 markers (28 MSs, 138 genes/ESTs, and 105 BESs) was typed on the IMpRH(7,000-rad) panel. The two data sets were merged into a single data-set of 655 markers of which 206 markers were typed on both panels. Two large linkage groups of 72 and 194 markers were assigned to SSC2p, and two linkage groups of 84 and 168 markers to SSC9p at a two-point LOD score of 10. A total of 126 and 114 FW markers were ordered with a likelihood ratio of 1000:1 to the SSC2p and SSC9p RH(12,000-rad) FW maps, respectively, with an accumulated map distance of 4046.5 cR(12,000 )and 1355.2 cR(7,000 )for SSC2p, and 4244.1 cR(12,000) and 1802.5 cR(7,000) for SSC9p. The kb/cR ratio in the IMNpRH2(12,000-rad) FW maps was 15.8 for SSC2p, and 15.4 for SSC9p, while the ratio in the IMpRH(7,000-rad) FW maps was 47.1 and 36.3, respectively, or an approximately 3.0-fold increase in map resolution in the IMNpRH(12,000-rad) panel over the IMpRH(7,000-rad) panel. The integrated IMNpRH(12,000-rad) andIMpRH(7,000-rad) maps as well as the genetic and BAC FPC maps provide an inclusive comparative map between SSC2p, SSC9p and HSA11 to close potential gaps between contigs prior to sequencing, and to identify regions where potential problems may arise in sequence assembly.     

Linkage mapping of gene-associated SNPs to pig chromosome 11

Single nucleotide polymorphisms (SNPs) were discovered in porcine expressed sequence tags (ESTs) orthologous to genes from human chromosome 13 (HSA13) and predicted to be located on pig chromosome 11 (SSC11). The SNPs were identified as sequence variants in clusters of EST sequences from pig cDNA libraries constructed in the Sino-Danish pig genome project. In total, 312 human gene sequences from HSA13 were used for similarity searches in our pig EST database. Pig ESTs showing significant similarity with HSA13 genes were clustered and candidate SNPs were identified. Allele frequencies for 26 SNPs were estimated in a group of 80 unrelated pigs from Danish commercial pig breeds: Duroc, Hampshire, Landrace and Large White. Eighteen of the 26 SNPs genotyped in the PiGMaP Reference Families were mapped by linkage analysis to SSC11. The EST-based SNPs published here are new genetic markers useful for linkage and association studies in commercial and experimental pig populations. This study represents the first gene-associated SNP linkage map of pig chromosome 11 and adds new comparative mapping information between SSC11 and HSA13. Furthermore, our data facilitate future studies aimed at the identification of interesting regions on pig chromosome 11, positional cloning and fine mapping of quantitative trait loci in pig.     

Comparative mapping of Homo sapiens chromosome 4 (HSA4) and Sus scrofa chromosome 8 (SSC8) using orthologous genes representing different cytogenetic bands as landmarks.

The recently published draft sequence of the human genome will provide a basic reference for the comparative mapping of genomes among mammals. In this study, we selected 214 genes with complete coding sequences on Homo sapiens chromosome 4 (HSA4) to search for orthologs and expressed sequence tag (EST) sequences in eight other mammalian species (cattle, pig, sheep, goat, horse, dog, cat, and rabbit). In particular, 46 of these genes were used as landmarks for comparative mapping of HSA4 and Sus scrofa chromosome 8 (SSC8); most of HSA4 is homologous to SSC8, which is of particular interest because of its association with genes affecting the reproductive performance of pigs. As a reference framework, the 46 genes were selected to represent different cytogenetic bands on HSA4. Polymerase chain reaction (PCR) products amplified from pig DNA were directly sequenced and their orthologous status was confirmed by a BLAST search. These 46 genes, plus 11 microsatellite markers for SSC8, were typed against DNA from a pig-mouse radiation hybrid (RH) panel with 110 lines. RHMAP analysis assigned these 57 markers to 3 linkage groups in the porcine genome, 52 to SSC8, 4 to SSC15, and 1 to SSC17. By comparing the order and orientation of orthologous landmark genes on the porcine RH maps with those on the human sequence map, HSA4 was recognized as being split into nine conserved segments with respect to the porcine genome, seven with SSC8, one with SSC15, and one with SSC17. With 41 orthologous gene loci mapped, this report provides the largest functional gene map of SSC8, with 30 of these loci representing new single-gene assignments to SSC8.     

A first-generation EST RH comparative map of the porcine and human genome

We have constructed a first-generation EST radiation hybrid comparative map of the porcine genome by assigning 1058 markers to the IMpRH7000 panel. Chromosomal localization was determined with a 2pt LOD of 4.8 for 984 markers, using the IMpRH mapping tool. Annotated ESTs represent 46.2% or 489 of the markers. Marker distribution was not stochastic and ranged from 0.41 for SSC8 to 1.77 for SSC12, respectively. Two hundred fifty-one markers assigned to the physical map of the pig did not find a homologous sequence in V22 of the human genome assembly, indicative of gaps in the assembled human genome sequence. The comparative porcine/human map covers 3290 MB, or 98.3% of the presumed size of the human genome. However, 60 breakpoints were identified between chromosomes, as well as 90 micro-rearrangements within synteny groups. Six porcine chromosomes—SSC2, 5, 6, 7, 12, and 14—correspond to the three gene-richest human chromosomes, HSA17, 19, and 22, and show above average marker density. Porcine Chrs 1, 8, 11, and X display a low DNA/marker ratio and correspond to the 'genome deserts' on HSA 18, 4, 13, and X.     

A genome-wide BAC end-sequence survey of sugarcane elucidates genome composition, and identifies BACs covering much of the euchromatin

BAC-end sequences (BESs) of hybrid sugarcane cultivar R570 are presented. A total of 66,990 informative BESs were obtained from 43,874 BAC clones. Similarity search using a variety of public databases revealed that 13.5 and 42.8 % of BESs match known gene-coding and repeat regions, respectively. That 11.7 % of BESs are still unmatched to any nucleotide sequences in the current public databases despite the fact that a close relative, sorghum, is fully sequenced, indicates that there may be many sugarcane-specific or lineage-specific sequences. We found 1,742 simple sequence repeat motifs in 1,585 BESs, spanning 27,383 bp in length. As simple sequence repeat markers derived from BESs have some advantages over randomly generated markers, these may be particularly useful for comparing BAC-based physical maps with genetic maps. BES and overgo hybridization information was used for anchoring sugarcane BAC clones to the sorghum genome sequence. While sorghum and sugarcane have extensive similarity in terms of genomic structure, only 2,789 BACs (6.4 %) could be confidently anchored to the sorghum genome at the stringent threshold of having both-end information (BESs or overgos) within 300 Kb. This relatively low rate of anchoring may have been caused in part by small- or large-scale genomic rearrangements in the Saccharum genus after two rounds of whole genome duplication since its divergence from the sorghum lineage about 7.8 million years ago. Limiting consideration to only low-copy matches, 1,245 BACs were placed to 1,503 locations, covering ~198 Mb of the sorghum genome or about 78 % of the estimated 252 Mb of euchromatin. BESs and their analyses presented here may provide an early profile of the sugarcane genome as well as a basis for BAC-by-BAC sequencing of much of the basic gene set of sugarcane.     

Improving the comparative map of porcine chromosome 10 with respect to human chromosomes 1, 9 and 10

ZOO-FISH mapping shows human chromosomes 1, 9 and 10 share regions of homology with pig chromosome 10 (SSC10). A more refined comparative map of SSC10 has been developed to help identify positional candidate genes for QTL on SSC10 from human genome sequence. Genes from relevant chromosomal regions of the public human genome sequence were used to BLAST porcine EST databases. Primers were designed from the matching porcine ESTs to assign them to porcine chromosomes using the INRA somatic cell hybrid panel (INRA-SCHP) and the INRA-University of Minnesota Radiation Hybrid Panel (IMpRH). Twenty-eight genes from HSA1, 9 and 10 were physically mapped: fifteen to SSC10 (ACO1, ATP5C1, BMI1, CYB5R1, DCTN3, DNAJA1, EPHX1, GALT, GDI2, HSPC177, OPRS1, NUDT2, PHYH, RGS2, VIM), eleven to SSC1 (ADFP, ALDHIB1, CLTA, CMG1, HARC, PLAA, STOML2, RRP40, TESK1, VCP and VLDLR) and two to SSC4 (ALDH9A1 and TNRC4). Two anonymous markers were also physically mapped to SSC10 (SWR1849 and S0070) to better connect the physical and linkage maps. These assignments have further refined the comparative map between SSC1, 4 and 10 and HSA1, 9 and 10.     

A new 4016-marker radiation hybrid map for porcine-human genome analysis

We constructed a 5000-rad comprehensive radiation hybrid (RH) map of the porcine (Sus scrofa) genome and compared the results with the human genome. Of 4475 typed markers, 4016 (89.7%) had LOD >5 compared with the markers used in our previous RH map by means of two-point analysis and were grouped onto the 19 porcine chromosomes (SSCs). All mapped markers had LOD >3 as determined by RHMAPPER analysis. The current map comprised 430 microsatellite (MS) framework markers, 914 other MS markers, and 2672 expressed sequence tags (ESTs). The whole-genome map was 8822.1 cR in length, giving an average marker density of 0.342 Mb/cR. The average retention frequency was 35.8%. Using BLAST searches of porcine ESTs against the RefSeq human nucleotide and amino acid sequences (release 22), we constructed high-resolution comparative maps of each SSC and each human chromosome (HSA). The average distance between ESTs in the human genome was 1.38 Mb. SSC contained 50 human chromosomal syntenic groups, and SSC11, SSC12, and SSC16 were only derived from the HSA13q, HSA17, and HSA5 regions, respectively. Among 38 porcine terminal regions, we found that at least 20 regions have been conserved between the porcine and human genomes; we also found four paralogous regions for the major histocompatibility complex (MHC) on SSC7, SSC2, SSC4, and SSC1. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Evolutionary breakpoints through a high-resolution comparative map between porcine chromosomes 2 and 16 and human chromosomes

This study reports a high-resolution comparative map between human chromosomes and porcine chromosomes 2 (SSC2) and 16 (SSC16), pointing out new homologies and evolutionary breakpoints. SSC2 is of particular interest because of the presence of several important QTLs. Among 226 porcine ESTs selected according to their expected localization, 151 were RH mapped and ordered on SSC2. This study confirmed the extensive conservation between SSC2 and HSA11 and HSA19 and refined the homology with HSA5 (three blocks defined). Furthermore the SSC2q pericentromeric region was shown to be homologous to another human chromosome (HSA1). A complex organization of these syntenies was demonstrated on SSC2q. Our strategy led us to improve also the SSC16 RH map by adding 45 markers. Two-color fluorescence in situ hybridization of markers representative of each synteny confirmed block order. Finally, 29 breakpoints were identified in both species, and porcine BACs containing two breakpoints were isolated.     

Assignment of 128 genes localized on human chromosome 14q to the IMpRH map

To provide a gene-based comparative map and to examine a porcine genome assembly using bacterial artificial chromosome-based sequence, we have attempted to assign 128 genes localized on human chromosome 14q (HSA14q) to a porcine 7000-rad radiation hybrid (IMpRH) map. This study, together with earlier studies, has demonstrated the following. (i) 126 genes were incorporated into two SSC7 RH linkage groups by C artha G ene analysis. (ii) In the remaining two genes, TOX4 linked to TCRA located in SSC7 by two-point analysis, whereas SIP1 showed no significant linkage with any gene/marker registered in the IMpRH Web Server. (iii) In the two groups, the gene clusters located from 19.9 to 36.5 Mb on HSA14q11.2-q13.3 and from 64.0 to 104.3 Mb on HSA14q23-q32.33 respectively were assigned to SSC7q21-q26. (iv) Comparison of the gene order between the present RH map and the latest porcine sequence assembly revealed some inconsistencies, and a redundant arrangement of 16 genes in the sequence assembly.     

Mapping four genes from human chromosome 4 to porcine chromosome 8 further develops the comparative map for an economically important chromosome of the swine genome

Because porcine chromosome (SSC) 8 has become the focal point of many efforts aimed at identifying quantitative trait loci affecting ovulation rate, genes distributed across human chromosome (HSA) 4 were physically mapped in the pig. A more refined comparative map of this region for these two species was produced. In this study, four genes were selected based on their location in the human genome, the availability of nucleotide sequence and their genomic organization. The genes selected were fibroblast growth factor basic (FGF2; HSA 4q25-27), gonadotropin releasing hormone receptor (GNRHR; HSA 4q13), phosphodiesterase 6 B (PDE6B; HSA 4p16.3) and aminopeptidase S (PEPS; HSA 4p11-q12). Genomic libraries were screened via PCR and clones were physically assigned using fluorescence in situ hybridization (FISH). These four genes from HSA 4 were physically mapped to SSC 8p2.3 (PDE6B), 8p1.1 (PEPS), 8q1.1-1.2 (GNRHR) and 8q2.2-2.4 (FGF2). These assignments provide additional benchmarks for the comparative map and help define the level of gene order conserved between HSA 4 and SSC 8.     

Improving the comparative map of SSC2p-q13 by sample sequencing of BAC clones

To improve the comparative map for pig chromosome 2 and increase the gene density on this chromosome, a porcine bacterial artificial chromosome (BAC) library was screened with 17 microsatellite markers and 18 genes previously assigned to pig chromosome 2. Fifty-one BAC clones located in the region of a maternally imprinted quantitative trait locus for backfat thickness (BFT) were identified. From these BACs 372 kb were sample sequenced. The average read length of a subclone was 442 basepair (bp). Contig assembly analysis showed that every bp was sequenced 1.28 times. Subsequently, sequences were compared with sequences in the nucleotide databases to identify homology with other mammalian sequences. Sequence identity was observed with sequences derived from 35 BACs. The average percentage identity with human sequences was 87.6%, with an average length of 143 bp. In total, sample sequencing of all BACs resulted in sequence identity with 29 human genes, 13 human expressed sequence tags (ESTs), 17 human genomic clones, one rat gene, one porcine gene and nine porcine ESTs. Eighteen genes located on human chromosome 11 and 19, and seven genes from other human locations, one rat gene and one porcine gene were assigned to pig chromosome 2 for the first time. The new genes were added to the radiation hybrid map at the same position as the locus from which the BAC that was sequenced was derived. In total 57 genes were placed on the radiation hybrid map of SSC2p-q13.     

Comparative mapping of human chromosome 10 to pig chromosomes 10 and 14

Identification of predictive markers in QTL regions that impact production traits in commercial populations of swine is dependent on construction of dense comparative maps with human and mouse genomes. Chromosomal painting in swine suggests that large genomic blocks are conserved between pig and human, while mapping of individual genes reveals that gene order can be quite divergent. High-resolution comparative maps in regions affecting traits of interest are necessary for selection of positional candidate genes to evaluate nucleotide variation causing phenotypic differences. The objective of this study was to construct an ordered comparative map of human chromosome 10 and pig chromosomes 10 and 14. As a large portion of both pig chromosomes are represented by HSA10, genes at regularly spaced intervals along this chromosome were targeted for placement in the porcine genome. A total of 29 genes from human chromosome 10 were mapped to porcine chromosomes 10 (SSC10) and 14 (SSC14) averaging about 5 Mb distance of human DNA per marker. Eighteen genes were assigned by linkage in the MARC mapping population, five genes were physically assigned with the IMpRH mapping panel and seven genes were assigned on both maps. Seventeen genes from human 10p mapped to SSC10, and 12 genes from human 10q mapped to SSC14. Comparative maps of mammalian species indicate that chromosomal segments are conserved across several species and represent syntenic blocks with distinct breakpoints. Development of comparative maps containing several species should reveal conserved syntenic blocks that will allow us to better define QTL regions in livestock.     

A high-resolution comparative RH map of porcine Chromosome (SSC) 2

A high-resolution comparative map was constructed for porcine Chromosome (SSC) 2, where a QTL for back fat thickness (BFT) is located. A radiation hybrid (RH) map containing 33 genes and 25 microsatellite markers was constructed for this chromosome with a 3000-rad porcine RH panel. In total, 16 genes from human Chromosome (HSA) 11p, HSA19p, and HSA5q were newly assigned to SSC2. One linkage group was observed at LOD 3.0, and five linkage groups at LOD 4.0. Comparison of the porcine RH map with homologous human gene orders identified four conserved segments between SSC2 and HSA11, HSA19, and HSA5. Concerning HSA11, a rearrangement of gene order is observed. The segment HSA11p15.4-q13 is inverted on SSC2 when compared with the distal tip of SSC2p, which is homologous to HSA11p15.5. The boundaries of the conserved segments between human and pig were defined more precisely. This high-resolution comparative map will be a valuable tool for further fine mapping of the QTL area. Received: 10 November 2000 / Accepted: 23 January 2001     

Comparative mapping between human chromosome 3 and porcine chromosome 13.

Zoo-FISH and somatic cell hybrid panels have earlier demonstrated extended synteny conservation between human chromosome 3 (HSA3) and pig chromosome 13 (SSC13). In the present study, eight human genes viz., ADCY5, CASR, COL7A1, COL8A1, ITIH1, RHO, SIAT1 and XPC, spread along the length of HSA3, were chosen for expanding the comparative map between the two chromosomes. Using human and rat cDNAs, or human- and porcine-specific PCR products as probes, 8 porcine lambda clones were isolated. After subcloning and partial sequence determination, identity of the clones with regards to the specific genes was established. The eight type 1 markers thus obtained were biotin labeled and FISH mapped to pig metaphase spreads. All lambda clones localized to SSC13. In combination with the hitherto published mapping data of coding sequences on SSC13, a preliminary comparative status depicting the relative organization of this chromosome with respect to HSA3 was developed. The comparative map thus obtained bears significance in searching for candidate genes of economically important traits mapped to SSC13.     

A high-resolution comparative map between pig chromosome 17 and human chromosomes 4, 8, and 20: identification of synteny breakpoints

We report on the construction of a high-resolution comparative map of porcine chromosome 17 (SSC17) focusing on evolutionary breakpoints with human chromosomes. The comparative map shows high homology with human chromosome 20 but suggests more limited homologies with other human chromosomes. SSC17 is of particular interest in studies of chromosomal organization due to the presence of QTLs that affect meat quality and carcass composition. A total of 158 pig ESTs available in databases or developed by the Sino-Danish Pig Genome Sequencing Consortium were mapped using the INRA-University of Minnesota porcine radiation hybrid panel. The high-resolution map was further anchored by fluorescence in situ hybridization. This study confirmed the extensive conservation between SSC17 and HSA20 and enabled the gene order to be determined. The homology of the SSC17 pericentromeric region was extended to other human chromosomes (HSA4, HSA8) and the chromosomal breakpoint boundaries were accurately defined. In total 15 breakpoints were identified.     

Conserved synteny and gene order difference between human chromosome 12 and pig chromosome 5.

A comparative map of human chromosome 12 (HSA 12) and pig chromosome 5 (SSC 5) was constructed using ten pig expressed sequence tags (ESTs). These ESTs were isolated from primary granulosa cell cultures by differential display (EST b10b), or from a granulosa cDNA library (VIIIE1, DRIM, N*9, RIIID2 and RVIC1) or from a small intestine cDNA library (ATPSB, ITGB7, MYH9, and STAT2). Also used were two Traced Orthologous Amplified Sequence Tags (TOASTs) (LALBA, TRA1), one microsatellite-associated gene (IGF1) and finally five human YACs selected for their cytogenetic position, with a view to increasing the number of informative markers for the comparison. Large-insert clones were obtained by screening a pig bacterial artificial chromosome (BAC) library with specific primers for each EST and TOAST and for IGF1. These BACs were used as probes for fluorescent in situ hybridisation (FISH) both on porcine and human metaphases. In addition, the human YACs were FISH mapped on pig chromosomes. This allowed us to refine and, in some cases, to correct the previous mapping obtained with a somatic cell hybrid panel. While these data confirm chromosome painting results showing that the distal part of SSC 5p arm is conserved on HSA 22, while the rest of the chromosome corresponds to HSA 12, they also demonstrate gene-order differences between human and pig. In addition, it was also possible to determine the position of the synteny breakpoint.     

Assignment of 117 genes from HSA5 to the porcine IMpRH map and generation of a dense human-pig comparative map

Twenty-two and eight significant quantitative trait loci for economically important traits have been located on porcine chromosomes (SSC) 2q and SSC16 respectively, both of which have been shown to correspond to human chromosome 5 (HSA5) by chromosome painting. To provide a comprehensive comparative map for efficient selection of candidate genes, we assigned 117 genes from HSA5 using a porcine radiation hybrid (IMpRH) panel. Sixty-six genes were assigned to SSC2 and 48 to SSC16. One gene was suggested to link to SSC2 markers and another to SSC6. One gene did not link to any gene, expressed sequence tag or marker in the map, including those in the present investigation. This study demonstrated the following: (1) SSC2q21-q28 corresponds to the region ranging from 74.0 to 148.2 Mb on HSA5q13-q32 and the region from 176.0 to 179.3 Mb on HSA5q35; (2) SSC16 corresponds to the region from 1.4 to 68.7 Mb on HSA5p-q13 and to the region from 150.4 to 169.1 Mb on HSA5q32-q35 and (3) the conserved synteny between HSA5 and SSC2q21-q28 is interrupted by at least two sites and the synteny between HSA5 and SSC16 is also interrupted by at least two sites.     

Regional localisations of VIM, HSD3b, ACTA1 and PGM1 in pigs

The comparative map between human and pig has progressed rapidly over the past 2 years. Nevertheless, some points still need to be clarified, particularly the correspondences between human chromosome 10 (HSA10) and porcine chromosome 10 (SSC10) and between human chromosome 1 (HSA1) and porcine chromosomes. The gene codings for vimentin (VIM) carried by HSA10 and three genes carried by HSA1 (hydroxy delta 5 steroid dehydrogenase 3 beta: HSD3B; alpha actin 1: ACTA1; and phosphoglucomutase 1: PGM1) were selected and the regional localisations on pig chromosomes were determined using a well-characterised somatic cell hybrid panel.