Radiation hybrid map assignments of 11 ESTs obtained from a 28-day-old swine embryo cDNA library to the IMpRH map

In order to improve the map resolution and to locate more genes on the porcine radiation hybrid map, expressed sequence tags (ESTs) were isolated from a 28-day-old normal pig embryo cDNA library. The ESTs were sequenced from the 5'-end and similarities were checked with sequences registered in the NCBI DNA database (http://www.ncbi.nlm.nih.gov/blast/). The ESTs sequences which have high identity scores (>80%) against human genes or ESTs were further sequenced from the 3' untranslated region. The ESTs which were sequenced successfully were used to design primers for PCR analysis of the radiation hybrid panel. Eleven ESTs were physically mapped to porcine chromosomes 2, 4, 8, 10, 13, 14 and X. The localizations are in agreement with the comparative mapping data between human and pig. The results will provide unique information to the comparative map of human and pig.     

Comparative mapping in the pig: localization of 214 expressed sequence tags

In total, 214 ESTs (Expressed Sequence Tags) were assigned to the porcine gene map by using somatic cell hybrid mapping, radiation hybrid mapping, and FISH. The ESTs were isolated from a porcine small intestine cDNA library on the basis of significant sequence identity with human annotated genes. In total, 390 primer pairs were designed primarily in the 3' UTR of the sequences. Overall, 58.6% of the ESTs were successfully mapped by this approach. In total, 191 of the localizations are in agreement with the human comparative map, strongly indicating that these represent true orthologous genes. The remaining 23 ESTs provide new comparative mapping data, which should be considered as preliminary until confirmed by other studies. Our mapping efforts provide a significant contribution to the porcine map as well as to the comparative map for human and pig.     

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.     

Construction of a 1.2-Mb BAC/PAC contig of the porcine gene RYR1 region on SSC 6q1.2 and comparative analysis with HSA 19q13.13

We screened a porcine bacterial artificial chromosome (BAC) and a P1 derived artificial chromosome (PAC) library to construct a sequence-ready approximately 1.2-Mb BAC/PAC contig of the ryanodine receptor-1 gene (RYR1) region on porcine chromosome (SSC) 6q1.2. This genomic segment is of special interest because it harbors the locus for stress susceptibility in pigs and a putative quantitative trait locus for muscle growth. Detailed physical mapping of this gene-rich region allowed us to assign to this contig 17 porcine genes orthologous to known human chromosome 19 genes. Apart from the relatively well-characterized porcine gene RYR1, the other 16 genes represent novel chromosomal assignments and 14 genes have been cloned for the first time in pig. Comparative analysis of the porcine BAC/PAC contig with the human chromosome (HSA) 19q13.13 map revealed a completely conserved gene order of this segment between pig and human. A detailed porcine-human-mouse comparative map of this region was constructed.     

A physical map of the 20q12-q13.1 region associated with type 2 diabetes

Several recent genetic studies have suggested linkage of Type 2 diabetes (non-insulin-dependent diabetes mellitus) susceptibility to a region of chromosome 20q12-q13.1. To facilitate the identification and cloning of a diabetes susceptibility gene(s) in this region, we have constructed correlated radiation hybrid and YAC/BAC contig physical maps of the region. A high-resolution radiation hybrid map encompassing 9.5 Mb between the PLC and the CEBPB genes was constructed using 68 markers: 25 polymorphic markers, 15 known genes, 21 ESTs, and 7 random genomic sequences. The physical order of the polymorphic markers within this radiation hybrid map is consistent with published genetic maps. A YAC/BAC contig that gives continuous coverage between PLC and CEBPB was also constructed. This contig was constructed from 24 YACs, 34 BACs, and 1 P1 phage clone onto which 71 markers were mapped: 23 polymorphic markers, 12 genes, 24 ESTs, and 12 random genomic sequences. The radiation hybrid map and YAC/BAC physical map enable precise mapping of newly identified transcribed sequences and polymorphic markers that will aid in linkage and linkage disequilibrium studies and facilitate identification and cloning of candidate Type 2 diabetes susceptibility genes residing in 20q12-q13.1.     

Comparative mapping of a region on chromosome 10 containing QTL for reproduction in swine

Several quantitative trait loci (QTL) for important reproductive traits (age of puberty, ovulation rate, nipple number and plasma FSH) have been identified on the long arm of porcine chromosome 10. Bi-directional chromosome painting has shown that this region is homologous to human chromosome 10p. Because few microsatellite or type I markers have been placed on SSC10, we wanted to increase the density of known ESTs mapped in this region of the porcine genome. Genes were chosen for their position on human chromosome 10, sequence availability from the TIGR pig gene indices, and their potential as a candidate gene. The PCR primers were designed to amplify across introns or 3'-UTR to maximize single nucleotide polymorphism (SNP) discovery. Parents of the mapping population (one sire and seven dams) were amplified and sequenced to find informative markers. The SNPs were genotyped using primer extension and mass spectrometry. These amplification products were also used to probe a BAC library (RPCI-44, Roswell Park Cancer Institute) for positive clones and screened for microsatellites. Six genes from human chromosome 10p (AKR1C2, PRKCQ, ITIH2, ATP5C1, PIP5K2A and GAD2) were mapped in the MARC swine mapping population. Gene order was conserved within these markers from centromere to telomere of porcine chromosome 10q, as compared with human chromosome 10p. Four of these genes (PIP5K2A, ITIH2, GAD2 and AKR1C2), which map under QTL, are potential candidate genes. Identification of porcine homologues near important QTL and development of a comparative map for this chromosome will allow further fine- mapping and positional cloning of candidate genes affecting reproductive traits.     

Piggy-BACing the human genome II. A high-resolution, physically anchored, comparative map of the porcine autosomes

Using the INRA-Minnesota porcine radiation hybrid panel, we have constructed a human-pig comparative map composed of 2274 loci, including 206 ESTs and 2068 BAC-end sequences, assigned to 34 linkage groups. The average spacing between comparative anchor loci is 1.15 Mb based on human genome sequence coordinates. A total of 51 conserved synteny groups that include 173 conserved segments were identified. This radiation hybrid map has the highest resolution of any porcine map to date and its integration with the porcine linkage map (reported here) will greatly facilitate the positional cloning of genes influencing complex traits of both agricultural and biomedical interest. Additionally, this map will provide a framework for anchoring contigs generated through BAC fingerprinting efforts and assist in the selection of a BAC minimal tiling path and assembly of the first sequence-ready map of the porcine genome.     

Comparative mapping between humans and pigs: localization of 58 anchorage markers (TOASTs) by use of porcine somatic cell and radiation hybrid panels

To increase the number of Type I markers that are directly informative for comparative mapping, 58 anchorage markers, TOASTs (Traced Orthologous Amplified Sequence Tags), were mapped in pig. With specific consensus primers, 76 TOASTs were tested in pig: 50 were regionally localized in pig on a somatic cell hybrid panel (SCHP), and 51 were mapped on the whole genome, INRA/University of Minnesota porcine Radiation Hybrid panel (IMpRH). Comparison of marker positions on RH and cytogenetic maps indicated general concordance except for two chromosomal regions. For RH mapping, all markers, apart from one, were significantly linked (LOD > 4.8) to a marker of the first-generation radiation hybrid map. Localization of new markers on the initial map is necessary for drawing a framework map as shown for Chromosome Sscr 14. The addition of four TOASTs has enabled us to propose an improved map, using a threshold likelihood ratio of 1000/1. At the whole-genome level, this work significantly increased (by 50%) the number of precisely mapped genes on the porcine RH map and confirmed that the IMpRH panel is a valuable tool for high-resolution gene mapping in pig. Porcine PCR products were sequenced and compared with human sequences to verify their identity. Most of the localizations made it possible to either confirm or refine the previous comparative data between humans and pigs obtained through heterologous chromosomal painting or gene mapping. Moreover, the use of TOASTs in mapping studies appears to be a complement to other strategies using CATS, human ESTs, or heterologous FISH with BACs which had already been applied to improve the gene density of comparative genomic maps for mammals. Received: 15 March 2000 / Accepted: 27 July 2000     

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.     

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.     

A human-horse comparative map based on equine BAC end sequences

In an effort to increase the density of sequence-based markers for the horse genome we generated 9473 BAC end sequences (BESs) from the CHORI-241 BAC library with an average read length of 677 bp. BLASTN searches with the BESs revealed 4036 meaningful hits (E 相似文献   

De novo sequencing of hazelnut bacterial artificial chromosomes (BACs) using multiplex Illumina sequencing and targeted marker development for eastern filbert blight resistance

Bacterial artificial chromosome (BAC) libraries are widely used in map-based cloning of plant genes. Eastern filbert blight (EFB), caused by the pyrenomycete Anisogramma anomala (Peck) E. Müller, is a devastating disease of European hazelnut (Corylus avellana L.) in the Pacific Northwest. A dominant allele at a single locus from the obsolete pollenizer “Gasaway” confers complete resistance. Our map-based cloning efforts use a BAC library for “Jefferson” hazelnut, which is heterozygous for resistance. Screening the library with primer pairs designed from RAPD markers closely linked to the EFB resistance locus identified 38 BACs. We sequenced 28 of these BACs using Illumina technology, by multiplexing with barcoded adapters. De novo sequence assembly using the programs Velvet and SOPRA and further alignment using CodonCode Aligner generated contigs whose length ranged from 393 to 108,194 bp. The number of contigs per BAC ranged from 1 to 19, and estimated coverage of assembled BACs ranged from 64 % to 100 %. Preliminary analysis of the sequences identified 779 simple sequence repeats (SSRs), from which we developed 23 markers. Of these, 17 were assigned to linkage group 6 adjacent to the disease resistance locus, five were placed on other linkage groups, and one could not be assigned to a linkage group. The BAC sequences and new SSR markers will be useful for our efforts at map-based cloning of the disease resistance gene.     

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.     

Radiation hybrid comparative mapping between human chromosome 17 and porcine chromosome 12 demonstrates conservation of gene order

A comparative study of human chromosome 17 (HSA17) and pig chromosome 12 (SSC12) was conducted using both somatic cell hybrid panel (SCHP) and radiation hybrid (RH) panel analysis. Sequences from an expressed sequence tag (EST) project in pig reproduction were examined and six genes and ESTs originally believed to map to HSA17 were selected for this study. The genes/ESTs were TATA box binding protein-associated factor (TAF2N/RBP56), alpha-2-plasmin inhibitor (SERPINF2/PLI), H3 histone family 3B (H3F3B), aminopeptidase puromycin sensitive (NPEPPS), an expressed sequence tag (ESTMI015) and P311 protein (P311). The SCHP analysis mapped five genes/ESTs (TAF2N, H3F3B, SERPINF2, NPEPPS and ESTMI015) to SSC12q11-q15 and SSC12p11-p15 with 100% concordance, and assigned P311 to SSC2 (1/2q24)-q29 with 100% concordance. Radiation hybrid analysis of all six genes confirmed the SCHP mapping results, with average retention frequency of 25%. Recent human sequence data demonstrated that P311 is actually located on HSA5q. As HSA5q and SSC2q show conserved syntenic regions predicted from bi-directional painting, our P311 mapping data is consistent with these results. An expanded comparative SSC12 RH map integrating the five new type I markers and 23 previously mapped loci was established using a LOD score threshold of 4.8. The gene order of the five genes/ESTs on the SSC12 framework RH map (H3F3B-ESTMI015-NPEPPS-TAF2N-SERPINF2) is identical to the HSA17 GB4 map but with inversion of the map as conventionally drawn.     

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.     

Physical assignments of human chromosome 13 genes on pig chromosome 11 demonstrate extensive synteny and gene order conservation between pig and human

Previous mapping between the human and pig genomes suggested extensive conservation of human chromosome 13 (HSA13) to pig chromosome 11 (SSC11). The objectives of this study were comparative gene mapping of pig homologs of HSA13 genes and examining gene order within this conserved synteny group by physical assignment of each locus. A detailed HSA13 to SSC11 comparison was chosen since the comparative gene map is not well developed for these chromosomes and a rearranged gene order within conserved synteny groups was observed from the comparison between HSA13 and bovine chromosome 12 (BTA12). Heterologous primers for PCR were designed and used to amplify pig homologous fragments. The pig fragments were sequenced to confirm the homology. Six pig STSs (FLT1, ESD, RB1, HTR2A, EDNRB, and F10) were physically mapped using a somatic cell hybrid panel to SSC11, and fluorescent in situ hybridization (FISH) mapping was also applied to improve map resolution and determine gene order. Results from this study increase the comparative information available on SSC11 and suggest a conserved gene order on SSC11 and HSA13, in contrast to human:bovine comparisons of this syntenic group.