A high utility integrated map of the pig genome

Background

The domestic pig is being increasingly exploited as a system for modeling human disease. It also has substantial economic importance for meat-based protein production. Physical clone maps have underpinned large-scale genomic sequencing and enabled focused cloning efforts for many genomes. Comparative genetic maps indicate that there is more structural similarity between pig and human than, for example, mouse and human, and we have used this close relationship between human and pig as a way of facilitating map construction.

Results

Here we report the construction of the most highly continuous bacterial artificial chromosome (BAC) map of any mammalian genome, for the pig (Sus scrofa domestica) genome. The map provides a template for the generation and assembly of high-quality anchored sequence across the genome. The physical map integrates previous landmark maps with restriction fingerprints and BAC end sequences from over 260,000 BACs derived from 4 BAC libraries and takes advantage of alignments to the human genome to improve the continuity and local ordering of the clone contigs. We estimate that over 98% of the euchromatin of the 18 pig autosomes and the X chromosome along with localized coverage on Y is represented in 172 contigs, with chromosome 13 (218 Mb) represented by a single contig. The map is accessible through pre-Ensembl, where links to marker and sequence data can be found.

Conclusion

The map will enable immediate electronic positional cloning of genes, benefiting the pig research community and further facilitating use of the pig as an alternative animal model for human disease. The clone map and BAC end sequence data can also help to support the assembly of maps and genome sequences of other artiodactyls.     

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.     

Characterization of three maize bacterial artificial chromosome libraries toward anchoring of the physical map to the genetic map using high-density bacterial artificial chromosome filter hybridization

Three maize (Zea mays) bacterial artificial chromosome (BAC) libraries were constructed from inbred line B73. High-density filter sets from all three libraries, made using different restriction enzymes (HindIII, EcoRI, and MboI, respectively), were evaluated with a set of complex probes including the 185-bp knob repeat, ribosomal DNA, two telomere-associated repeat sequences, four centromere repeats, the mitochondrial genome, a multifragment chloroplast DNA probe, and bacteriophage lambda. The results indicate that the libraries are of high quality with low contamination by organellar and lambda-sequences. The use of libraries from multiple enzymes increased the chance of recovering each region of the genome. Ninety maize restriction fragment-length polymorphism core markers were hybridized to filters of the HindIII library, representing 6x coverage of the genome, to initiate development of a framework for anchoring BAC contigs to the intermated B73 x Mo17 genetic map and to mark the bin boundaries on the physical map. All of the clones used as hybridization probes detected at least three BACs. Twenty-two single-copy number core markers identified an average of 7.4 +/- 3.3 positive clones, consistent with the expectation of six clones. This information is integrated into fingerprinting data generated by the Arizona Genomics Institute to assemble the BAC contigs using fingerprint contig and contributed to the process of physical map construction.     

Human BAC ends

The Human BAC Ends database includes all non-redundant human BAC end sequences (BESs) generated by The Institute for Genomic Research (TIGR), the University of Washington (UW) and California Institute of Technology (CalTech). It incorporates the available BAC mapping data from different genome centers and the annotation results of each end sequence for the contents of repeats, ESTs and STS markers. For each BAC end the database integrates the sequence, the phred quality scores, the map and the annotation, and provides links to sites of the library information, the reports of GenBank, dbGSS and GDB, and other relevant data. The database is freely accessible via the web and supports sequence or clone searches and anonymous FTP. The relevant sites and resources are described at http://www.tigr.org/ tdb/humgen/bac_end_search/bac_end_intro.html     

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.     

The Physical and Genetic Framework of the Maize B73 Genome

Maize is a major cereal crop and an important model system for basic biological research. Knowledge gained from maize research can also be used to genetically improve its grass relatives such as sorghum, wheat, and rice. The primary objective of the Maize Genome Sequencing Consortium (MGSC) was to generate a reference genome sequence that was integrated with both the physical and genetic maps. Using a previously published integrated genetic and physical map, combined with in-coming maize genomic sequence, new sequence-based genetic markers, and an optical map, we dynamically picked a minimum tiling path (MTP) of 16,910 bacterial artificial chromosome (BAC) and fosmid clones that were used by the MGSC to sequence the maize genome. The final MTP resulted in a significantly improved physical map that reduced the number of contigs from 721 to 435, incorporated a total of 8,315 mapped markers, and ordered and oriented the majority of FPC contigs. The new integrated physical and genetic map covered 2,120 Mb (93%) of the 2,300-Mb genome, of which 405 contigs were anchored to the genetic map, totaling 2,103.4 Mb (99.2% of the 2,120 Mb physical map). More importantly, 336 contigs, comprising 94.0% of the physical map (∼1,993 Mb), were ordered and oriented. Finally we used all available physical, sequence, genetic, and optical data to generate a golden path (AGP) of chromosome-based pseudomolecules, herein referred to as the B73 Reference Genome Sequence version 1 (B73 RefGen_v1).     

A Physical Map of Arabidopsis thaliana Chromosome 3 Represented by Two Contigs of CIC YAC, P1, TAC and BAC Clones

We have constructed a physical map of Arabidopsis thaliana chromosome3 by ordering the clones from CIC YAC, P1, TAC and BAC librariesusing the sequences of a variety of genetic and EST markersand terminal sequences of clones. The markers used were 112DNA markers, 145 YAC end sequences, and 156 end sequences ofP1, TAC and BAC clones. The entire genome of chromosome 3, exceptfor the centromeric and telomeric regions, was covered by twolarge contigs, 13.6 Mb and 9.2 Mb long. This physical map willfacilitate map-based cloning experiments as well as genome sequencingof chromosome 3. The map and end sequence information are availableon the KAOS (Kazusa Arabidopsis data Opening Site) web siteat http://www.kazusa.or.jp/arabi/.     

Expression analysis and physical mapping of low-molecular-weight glutenin loci in hexaploid wheat (Triticum aestivum L.).

Gliadins and glutenins are storage proteins important in determining the bread-, noodle-, and pasta-making quality of wheat. Glutenins consist of HMW and LMW subunits. The Glu-A3, Glu-B3, and Glu-D3 loci on the short arms of chromosomes 1A, 1B, and 1D, respectively, are the major loci for LMW glutenins. To construct physical maps of the Glu-3 loci, a set of 24 high-density filters representing a 3.1x genome coverage hexaploid wheat BAC library was screened by hybridization using a probe made of 3 LMW glutenin sequences. After 2 rounds of hybridization, a subset of 536 BAC clones were selected and fingerprinted. Three developing seed cDNA libraries were also constructed. A total of 5000-6000 ESTs were generated from each library, assembled into contigs and searched by homology for LMW glutenin sequences. In total, 90 full-length LMW glutenin sequences were found to cluster into 8 distinct groups representing at least 21 different LMW glutenin subunits. A set of 24 pairs of PCR primers was designed from these groups and used as markers on the BAC clones. The combined fingerprinting and marker data were used to build the physical maps using FPC software. A total of 91 contigs comprising 254 clones were obtained and 282 clones remained singletons.     

Anchoring of a large set of markers onto a BAC library for the development of a draft physical map of the grapevine genome

Five hundred and six EST-derived markers, 313 SSR markers and 26 BAC end-derived or SCAR markers were anchored by PCR on a subset of a Cabernet Sauvignon BAC library representing six genome equivalents pooled in three dimensions. In parallel, the 12,351 EST clusters of the grapevine UniGene set (build #11) from NCBI were used to design 12,125 primers pairs and perform electronic PCR on 67,543 nonredundant BAC-end sequences. This in silico experiment yielded 1,140 positive results concerning 638 different markers, among which 602 had not been already anchored by PCR. The data obtained will provide an easier access to the regulatory sequences surrounding important genes (represented by ESTs). In total, 1,731 islands of BAC clones (set of overlapping BAC clones containing at least one common marker) were obtained and 226 of them contained at least one genetically mapped anchor. These assigned islands are very useful because they will link the genetic map and the future fingerprint-based physical map and because they allowed us to indirectly place 93 ESTs on the genetic map. The islands containing two or more mapped SSR markers were also used to assess the quality of the integrated genetic map of the grapevine genome.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .Didier Lamoureux and Anne Bernole contributed equally to this work.     

Identification of 10 882 porcine microsatellite sequences and virtual mapping of 4528 of these sequences

A total of 10 882 porcine microsatellite repeats were identified in genomic shotgun sequences from the Sino-Danish Pig Genome Sequencing Consortium (http://www.piggenome.dk). Of these, 4528 microsatellites were placed on a pig-human comparative map by blast analysis of porcine sequences against the human genome (blast cut-off threshold =1 x 10(-5)). All microsatellite sequences placed on the comparative map are accessible at http://www.animalgenome.org/QTLdb/pig.html. These sequences increase the number of identified microsatellites in the porcine genome by several orders of magnitude. They are a new resource of microsatellite sequences for generating markers to be used in linkage studies and in fine mapping and positional cloning of quantitative trait loci.     

The Korea brassica genome project: a glimpse of the brassica genome based on comparative genome analysis with Arabidopsis

A complete genome sequence provides unlimited information in the sequenced organism as well as in related taxa. According to the guidance of the Multinational Brassica Genome Project (MBGP), the Korea Brassica Genome Project (KBGP) is sequencing chromosome 1 (cytogenetically oriented chromosome #1) of Brassica rapa. We have selected 48 seed BACs on chromosome 1 using EST genetic markers and FISH analyses. Among them, 30 BAC clones have been sequenced and 18 are on the way. Comparative genome analyses of the EST sequences and sequenced BAC clones from Brassica chromosome 1 revealed their homeologous partner regions on the Arabidopsis genome and a syntenic comparative map between Brassica chromosome 1 and Arabidopsis chromosomes. In silico chromosome walking and clone validation have been successfully applied to extending sequence contigs based on the comparative map and BAC end sequences. In addition, we have defined the (peri)centromeric heterochromatin blocks with centromeric tandem repeats, rDNA and centromeric retrotransposons. In-depth sequence analyses of five homeologous BAC clones and an Arabidopsis chromosomal region reveal overall co-linearity, with 82% sequence similarity. The data indicate that the Brassica genome has undergone triplication and subsequent gene losses after the divergence of Arabidopsis and Brassica. Based on in-depth comparative genome analyses, we propose a comparative genomics approach for conquering the Brassica genome. In 2005 we intend to construct an integrated physical map, including sequence information from 500 BAC clones and integration of fingerprinting data and end sequence data of more than 100 000 BAC clones. The sequences have been submitted to GenBank with accession numbers: 10 204 BAC ends of the KBrH library (CW978640-CW988843); KBrH138P04, AC155338; KBrH117N09, AC155337; KBrH097M21, AC155348; KBrH093K03, AC155347; KBrH081N08, AC155346; KBrH080L24, AC155345; KBrH077A05, AC155343; KBrH020D15, AC155340; KBrH015H17, AC155339; KBrH001H24, AC155335; KBrH080A08, AC155344; KBrH004D11, AC155341; KBrH117M18, AC146875; KBrH052O08, AC155342.     

Whole‐genome profiling and shotgun sequencing delivers an anchored,gene‐decorated,physical map assembly of bread wheat chromosome 6A

Bread wheat (Triticum aestivum L.) is the most important staple food crop for 35% of the world's population. International efforts are underway to facilitate an increase in wheat production, of which the International Wheat Genome Sequencing Consortium (IWGSC) plays an important role. As part of this effort, we have developed a sequence‐based physical map of wheat chromosome 6A using whole‐genome profiling (WGP^?). The bacterial artificial chromosome (BAC) contig assembly tools fingerprinted contig (fpc ) and linear topological contig (ltc ) were used and their contig assemblies were compared. A detailed investigation of the contigs structure revealed that ltc created a highly robust assembly compared with those formed by fpc . The ltc assemblies contained 1217 contigs for the short arm and 1113 contigs for the long arm, with an L₅₀ of 1 Mb. To facilitate in silico anchoring, WGP^? tags underlying BAC contigs were extended by wheat and wheat progenitor genome sequence information. Sequence data were used for in silico anchoring against genetic markers with known sequences, of which almost 79% of the physical map could be anchored. Moreover, the assigned sequence information led to the ‘decoration’ of the respective physical map with 3359 anchored genes. Thus, this robust and genetically anchored physical map will serve as a framework for the sequencing of wheat chromosome 6A, and is of immediate use for map‐based isolation of agronomically important genes/quantitative trait loci located on this chromosome.     

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.     

Genetic mapping and BAC assignment of EST-derived SSR markers shows non-uniform distribution of genes in the barley genome

A set of 111,090 barley expressed sequence tags (ESTs) was searched for the presence of microsatellite motifs [simple sequence repeat (SSRs)] and yielded 2,823 non-redundant SSR-containing ESTs (SSR–ESTs). From this, a set of 754 primer pairs was designed of which 525 primer pairs yielded an amplicon and as a result, 185 EST-derived microsatellite loci (EST–SSRs) were placed onto a genetic map of barley. The markers show a uniform distribution along all seven linkage groups ranging from 21 (7H) to 35 (3H) markers. Polymorphism information content values ranged from of 0.24 to 0.78 (average 0.48). To further investigate the physical distribution of the EST–SSRs in the barley genome, a bacterial artificial chromosomes (BAC) library was screened. Out of 129 markers tested, BAC addresses were obtained for 127 EST–SSR markers. Twenty-seven BACs, forming eight contigs, were hit by two or three EST–SSRs each. This unexpectedly high incidence of EST–SSRs physically linked at the sub-megabase level provides additional evidence of an uneven distribution of genes and the segmentation of the barley genome in gene-rich and gene-poor regions.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.Primer sequences for developed SSR markers are available upon request from the corresponding author (A. Graner).     

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.