A successful strategy for comparative mapping with human ESTs: 65 new regional assignments in the pig.
ABSTRACT Large-scale sequencing of cDNAs from numerous tissues is currently being performed within the framework of the Human Genome Project. These expressed sequence tags (ESTs) are then mapped on a radiation hybrid panel to produce a high-resolution map of human genes. In this report, we estimate the efficiency of mapping these ESTs in the pig. A total of 344 human ESTs from Généthon were selected for amplification in other species by Zoo-PCR: 186 of these could be reproducibly amplified by use of pig DNA and the corresponding human primer pairs. One-hundred seven of these were tested on a porcine-rodent somatic cell hybrid panel, permitting regional localizations of 65 ESTs with agarose or single-strand conformation polymorphism analysis gels. The corresponding pig PCR products were sequenced: 60 ESTs matched significantly with the expected human sequences. Fifty-one of these localizations in the pig are in agreement with the comparative mapping data between humans and pigs based on heterologous chromosome painting. Seven ESTs that were localized in an unexpected region may indicate new chromosomal correspondences. This work significantly increases the number of genes mapped on the pig genome and demonstrates that this approach can be successfully applied to improve the gene density of mammalian genomic maps in chromosomal regions of interest, such as those in which QTL (Quantative Trait Loci) have been identified.
Article: Application of dissociation curve analysis to radiation hybrid panel marker scoring: generation of a map of river buffalo (B. bubalis) chromosome 20.[show abstract] [hide abstract]
ABSTRACT: Fluorescence of dyes bound to double-stranded PCR products has been utilized extensively in various real-time quantitative PCR applications, including post-amplification dissociation curve analysis, or differentiation of amplicon length or sequence composition. Despite the current era of whole-genome sequencing, mapping tools such as radiation hybrid DNA panels remain useful aids for sequence assembly, focused resequencing efforts, and for building physical maps of species that have not yet been sequenced. For placement of specific, individual genes or markers on a map, low-throughput methods remain commonplace. Typically, PCR amplification of DNA from each panel cell line is followed by gel electrophoresis and scoring of each clone for the presence or absence of PCR product. To improve sensitivity and efficiency of radiation hybrid panel analysis in comparison to gel-based methods, we adapted fluorescence-based real-time PCR and dissociation curve analysis for use as a novel scoring method. As proof of principle for this dissociation curve method, we generated new maps of river buffalo (Bubalus bubalis) chromosome 20 by both dissociation curve analysis and conventional marker scoring. We also obtained sequence data to augment dissociation curve results. Few genes have been previously mapped to buffalo chromosome 20, and sequence detail is limited, so 65 markers were screened from the orthologous chromosome of domestic cattle. Thirty bovine markers (46%) were suitable as cross-species markers for dissociation curve analysis in the buffalo radiation hybrid panel under a standard protocol, compared to 25 markers suitable for conventional typing. Computational analysis placed 27 markers on a chromosome map generated by the new method, while the gel-based approach produced only 20 mapped markers. Among 19 markers common to both maps, the marker order on the map was maintained perfectly. Dissociation curve analysis is reliable and efficient for radiation hybrid panel scoring, and is more sensitive and robust than conventional gel-based typing methods. Several markers could be scored only by the new method, and ambiguous scores were reduced. PCR-based dissociation curve analysis decreases both time and resources needed for construction of radiation hybrid panel marker maps and represents a significant improvement over gel-based methods in any species.BMC Genomics 12/2008; 9:544. · 4.07 Impact Factor
Article: Identification of differential gene expression in in vitro FSH treated pig granulosa cells using suppression subtractive hybridization.[show abstract] [hide abstract]
ABSTRACT: FSH, which binds to specific receptors on granulosa cells in mammals, plays a key role in folliculogenesis. Its biological activity involves stimulation of intercellular communication and upregulation of steroidogenesis, but the entire spectrum of the genes regulated by FSH has yet to be fully characterized. In order to find new regulated transcripts, however rare, we have used a Suppression Subtractive Hybridization approach (SSH) on pig granulosa cells in primary culture treated or not with FSH. Two SSH libraries were generated and 76 clones were sequenced after selection by differential screening. Sixty four different sequences were identified, including 3 novel sequences. Experiments demonstrated the presence of 25 regulated transcripts.A gene ontology analysis of these 25 genes revealed (1) catalytic; (2) transport; (3) signal transducer; (4) binding; (5) anti-oxidant and (6) structural activities. These findings may deepen our understanding of FSH's effects. Particularly, they suggest that FSH is involved in the modulation of peroxidase activity and remodelling of chromatin.Reproductive Biology and Endocrinology 02/2006; 4:35. · 2.05 Impact Factor
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ABSTRACT: Genetic linkage maps are necessary for mapping of mendelian traits and quantitative trait loci (QTLs). To identify the actual genes, which control these traits, a map based on gene-associated single nucleotide polymorphism (SNP) markers is highly valuable. In this study, the SNPs were genotyped in a large family material comprising more than 5,000 piglets derived from 12 Duroc boars crossed with 236 Danish Landrace/Danish Large White sows. The SNPs were identified in sequence alignments of 4,600 different amplicons obtained from the 12 boars and containing coding regions of genes derived from expressed sequence tags (ESTs) and genomic shotgun sequences. Linkage maps of all 18 porcine autosomes were constructed based on 456 gene-associated and six porcine EST-based SNPs. The total length of the averaged-sex whole porcine autosome was estimated to 1,711.8 cM resulting in an average SNP spacing of 3.94 cM. The female and male maps were estimated to 2,336.1 and 1,441.5 cM, respectively. The gene order was validated through comparisons to the cytogenetic and/or physical location of 203 genes, linkage to evenly spaced microsatellite markers as well as previously reported conserved synteny. A total of 330 previously unmapped genes and ESTs were mapped to the porcine autosome while ten genes were mapped to unexpected locations. The linkage map presented here shows high accuracy in gene order. The pedigree family network as well as the large amount of meiotic events provide good reliability and make this map suitable for QTL and association studies. In addition, the linkage to the RH-map of microsatellites makes it suitable for comparison to other QTL studies.BMC Genomics 04/2009; 10:134. · 4.07 Impact Factor