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.     

Evaluation of Compass as a comparative mapping tool for ESTs using horse radiation hybrid maps

Loci for 9322 equine expressed sequence tags (ESTs) were predicted using the Comparative Mapping by Annotation and Sequence Similarity (Compass) strategy in order to evaluate the programme's ability to make accurate locus predictions in species with comparative gene maps. Using human genome sequence information from Build 35 (May 2004) and published marker information from the radiation hybrid (RH) maps for equine chromosomes (ECA) 17 and X, 162 ESTs were predicted to locations on ECA17 and 328 ESTs to locations on ECAX by selection of the 'top blast hit'. The locations of 30 ESTs were assessed experimentally by RH mapping analysis to evaluate the accuracy of the Compass predictions. The data revealed that 53% (16 of 30) of the ESTs predicted on ECA17 and ECAX mapped to those chromosomes. Analysis of the results suggested the need to identify expressed orthologous sequences in order to generate more accurate predictions for ESTs. Locus predictions were reassessed with three modifications to the Compass strategy's orthologue selection parameters. Selection of the 'top gene hit' improved accuracy to 72% (21 of 29), while selection of the 'top expressed gene hit' improved accuracy to 86% (24 of 28). Using the default Compass parameters with the UniGene database improved prediction accuracy to 96% (22 of 23); however, this level of accuracy came with a substantial decrease in the total number of predictions. When used with optimized prediction parameters, the Compass strategy can be a practical in silico map location prediction tool for large EST sample sets from unsequenced animal genomes.     

Mapping of 443 porcine EST improves the comparative maps for SSC1 and SSC7 with the human genome

Numerous mapping studies of complex traits in the pig have resulted in quantitative trait loci (QTL) intervals of 10-20 cM. To improve the chances to identify the genes located in such intervals, increased expressed sequence tags (EST)-based marker density, coupled with comparative mapping with species whose genomes have been sequenced such as human and mouse, is the most efficient tool. In this study, we mapped 443 porcine EST with a radiation hybrid (RH) panel (384 had LOD > 6.0) and a somatic cell hybrid panel. Requiring no discrepancy between two-point and multipoint RH data allowed robust assignment of 309 EST, of which most were located on porcine chromosomes (SSC) 1, 4, 7, 8 and X. Moreover, we built framework maps for two chromosomes, SSC1 and SSC7, with mapped QTL in regions with known rearrangement between pig and human genomes. Using the Blast tool, we found orthologies between 407 of the 443 pig cDNA sequences and human genes, or to existing pig genes. Our porcine/human comparative mapping results reveal possible new homologies for SSC1, SSC3, SSC5, SSC6, SSC12 and SSC14 and add markers in synteny breakpoints for chromosome 7.     

A comparative map of chicken chromosome 24 and human chromosome 11

To improve the physical and comparative map of chicken chromosome 24 (GGA24; former linkage group E49C20W21) bacterial artificial chromosome (BAC) contigs were constructed around loci previously mapped on this chromosome by linkage analysis. The BAC clones were used for both sample sequencing and BAC end sequencing. Sequence tagged site (STS) markers derived from the BAC end sequences were used for chromosome walking. In total 191 BAC clones were isolated, covering almost 30% of GGA24, and 76 STS were developed (65 STS derived from BAC end sequences and 11 STS derived within genes). The partial sequences of the chicken BAC clones were compared with sequences present in the EMBL/GenBank databases, and revealed matches to 19 genes, expressed sequence tags (ESTs) and genomic clones located on human chromosome 11q22-q24 and mouse chromosome 9. Furthermore, 11 chicken orthologues of human genes located on HSA11q22-q24 were directly mapped within BAC contigs of GGA24. These results provide a better alignment of GGA24 with the corresponding regions in human and mouse and identify several intrachromosomal rearrangements between chicken and mammals.     

A radiation hybrid comparative map of ovine chromosome 1 aligned to the virtual sheep genome

Ovis aries chromosome one (OAR1) is the largest submetacentric chromosome in the sheep genome and is homologous to regions on human chromosomes 1, 2, 3 and 21. Using the USUoRH5000 ovine whole-genome radiation hybrid (RH) panel, we have constructed a RH map of OAR1 comprising 102 framework and 75 placed/binned markers across five linkage groups spanning 3759.43 cR5000, with an average marker density of 21.2 cR5000/marker. The alignment of our OAR1 RH map shows good concordance with the recently developed virtual sheep genome, with fewer than 1.86% discrepancies. A comparative map of OAR1 was constructed by examining the location of RH-mapped orthologues in sheep within the genomes of cow, human, horse and dog. Analysis of the comparative map indicates that conserved syntenies within the five ovine RH linkage groups underwent internal chromosomal rearrangements which, in general, reflect the evolutionary distances between sheep and each of these four species. The ovine RH map presented here integrates all available mapping data and includes new genomic information for ovine chromosome 1.     

An integrated comparative map of the porcine X chromosome

The objectives of this study were to assign both microsatellite and gene-based markers on porcine chromosome X to two radiation hybrid (RH) panels and to develop a more extensive integrated map of SSC-X. Thirty-five microsatellite and 20 gene-based markers were assigned to T43RH, and 16 previously unreported microsatellite and 15 gene-based markers were added to IMpRH map. Of these, 30 microsatellite and 12 gene-based markers were common to both RH maps. Twenty-two gene-based markers were submitted to BLASTN analysis for identification of orthologues of genes on HSA-X. Single nucleotide polymorphisms (SNPs) were detected for 12 gene-based markers, and nine of these were placed on the genetic map. A total of 92 known loci are present on at least one porcine chromosome X map. Thirty-seven loci are present on all three maps; 31 loci are found on only one map. Location of 33 gene-based markers on the comprehensive map translates into an integrated comparative map that supports conservation of gene order between SSC-X and HSA-X. This integrated map will be valuable for selection of candidate genes for porcine quantitative trait loci (QTLs) that map to SSC-X.     

A high-resolution radiation hybrid map of bovine chromosome 5q1.3-q2.5 compared with human chromosome 12q

In this study we present a comprehensive 3000-rad radiation hybrid map on bovine chromosome 5 (BTA5) of a region between 12.8 and 74.0 cM according to the linkage map, which contains a quantitative trait loci for ovulation rate. We mapped 28 gene-associated sequence tagged site markers derived from sequences of bovine BAC clones and 10 microsatellite markers to the BTA5 region. In comparison with HSA12q, four blocks of conserved synteny were apparent showing three chromosomal breakpoints and two inversions in this segment of BTA5. Therefore, we have improved breakpoint resolution in the human-bovine comparative map, which enhances the determination of candidate genes underlying traits of interest mapped to BTA5.     

A high-resolution comparative radiation hybrid map of equine chromosome 4q12-q22

In this study, we present a comprehensive 5000-rad radiation hybrid map of a 40-cM region on equine chromosome 4 (ECA4) that contains quantitative trait loci for equine osteochondrosis. We mapped 29 gene-associated sequence tagged site markers using primers designed from equine expressed sequence tags or BAC clones in the ECA4q12-q22 region. Three blocks of conserved synteny, showing two chromosomal breakpoints, were identified in the segment of ECA4q12-q22. Markers from other segments of HSA7q mapped to ECA13p and ECA4p, and a region of HSA7p was homologous to ECA13p. Therefore, we have improved the resolution of the human-equine comparative map, which allows the identification of candidate genes underlying traits of interest.     

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.     

A radiation hybrid map of bovine chromosome 24 and comparative mapping with human chromosome 18

We present herein a bovine chromosome 24 (BTA24) radiation hybrid (RH) map using 40 markers scored on a panel of 90 RHs. Of these markers, 29 loci were ordered with odds of at least 1000:1 in a framework map. An average retention frequency of 17.4% was observed, with relatively higher frequencies near the centromere. The length of the comprehensive map was 640 centiray5000 (cR5000) with an average marker interval of approximately 17.3 cR5000. The observed locus order is generally consistent with currently published bovine linkage and physical maps. Nineteen markers were either Type I loci or closely associated with expressed sequences and thus could be used to compare the BTA24 RH map with human mapping information. All genes located on BTA24 were located on human chromosome 18, and previously reported regions of conserved synteny were extended. The comparative data revealed the presence of at least six conserved regions between these chromosomes.     

植物抗病虫SSH文库的EST分析

抑制差减杂交技术广泛应用于植物抗病虫(真菌、细菌和线虫)机理研究。本文在归纳出植物抗病虫SSH文库中ESTs的总体情况之基础上,重点分析了表达频率高的抗病、防御和信号转导基因,并展望了SSH的发展前景,有利于认识植物抗病虫分子机理的普遍规律和推广应用该技术。     

A refined radiation hybrid map of the telomeric region of bovine chromosome 18q25-q26 compared with human chromosome 19q13

Genome-wide scans have mapped economically important quantitative trait loci (QTL) for mastitis susceptibility in dairy cattle at the telomeric end of bovine chromosome 18 (BTA18). In order to increase the density of markers in this chromosomal region and to improve breakpoint resolution in the human-bovine comparative map, this study describes the chromosomal assignment of seven newly developed gene-associated markers and five microsatellites and eight previously mapped sequence tagged site markers near these QTL. The orientation of KCNJ14, BAX, CD37, NKG7, LIM2, PRKCG, TNNT1, MGC2705, RPL28, EPN1, ZNF582, ZIM2, STK13, ZNF132 and SLC27A5 on the 3000-rad radiation hybrid (RH) map of BTA18 is homologous to the organization found on the corresponding 10 Mbp of human chromosome 19q (HSA19q). The resulting bovine RH map with a length of 20.9 cR spans over about 11 cM on the bovine linkage map. The location of KCNJ14 and SLC27A5 flanking the RH map on BTA18q25-26 has been confirmed by fluorescence in situ hybridization. The data of this refined human-bovine comparative map should improve selection of candidate genes for mastitis susceptibility in dairy cattle.     

Identification and physical mapping of genes expressed in the corpus luteum in cattle

A representational difference analysis was performed to identify genes expressed in the corpus luteum of cattle. The corpus luteum is an ovarian structure that is essential for the establishment and maintenance of pregnancy. Knowledge of gene expression and function of corpus luteum will be important to improve fertility in humans and domestic animals. Housekeeping genes were removed from the corpus luteum representation (tester) using skeletal muscle as the subtracting agent (driver). A total of 80 clones of the final subtraction product were analysed by sequencing and 11 new bovine gene sequences were identified (pBTCL1-11). The sequences were mapped to segments of 10 different chromosomes using a somatic cell hybrid panel and a radiation hybrid panel. With one exception the locations are in agreement with published comparative maps of cattle and man. Expression in corpus luteum was verified by RT-PCR for all the 11 clones.     

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.     

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.     

Characterization and Genetic Mapping of Simple Sequence Repeats in the Rice Genome

We searched partial sequences of over 22,706 rice cDNA and 1220genomic DNA clones to find and characterize simple sequencerepeats (SSRs) in the rice genome. The most frequently foundrepeated SSR motif in both cDNA and genomic DNA sequences wasd(CCG/CGG)n. The second most frequently found SSR was d(AG/CT)n.In contrast with mammalian genomes, in which d(AC/GT)n sequencesare the most abundant, d(AC/GT)n sequences were not frequentlyobserved in rice. Sequences containing d(CCG/CGG)n, d(AG/CT)nrepeats, and other SSRs were chosen for polymorphism detection.It was predicted that 17 of 20 SSRs in cDNA sequences were locatedin 5'-untranslated regions near initiation codons. Twenty-twoloci can be mapped on our RFLP linkage map by these SSRs. Sixmarkers were tested with 16 japonica rice varieties as templatesfor PCR. Two markers exhibited amplified fragment length polymorphismamong these rice varieties, implying that SSRs are polymorphicamong rice varieties which have similar genetic backgrounds.Even these polymorphic SSRs are located within or around geneswhich code ubiquitous proteins.