Genetic relationships between Japanese native and commercial breeds using 70 chicken autosomal SNP genotypes by the DigiTag2 assay.

Recently, single nucleotide polymorphisms (SNPs) have been used to identify genes or genomic regions responsible for economic traits, including genetic diseases in domestic animals, and to examine genetic diversity of populations. In this study, we genotyped 70 chicken autosomal SNPs using DigiTag2 assay to understand the genetic structure of the Japanese native chicken breeds Satsumadori and Ingie, and the relationship of these breeds with other established breeds, Rhode Island Red (RIR), commercial broiler and layer. Five breeds, each consisting of approximately 20 chickens, were subjected to the assay, revealing the following: Average expected heterozygosities of broiler, Satsumadori, RIR, layer and Ingie were 0.265, 0.254, 0.244, 0.179 and 0.176, respectively. Phylogenetic analysis using the concatenated 70 autosomal SNP genotypes distinguished all chickens and formed clusters of chickens belonging to the respective breeds. In addition, the 2-D scatter plot of the first two principal components was consistent with the phylogenic tree. Taken together with the pairwise F(st) distances, broiler and RIR were closely positioned near each other, while Ingie was positioned far from the other breeds. Structure analysis revealed that the probable number of genetic clusters (K) was six and four with maximum likelihood and ΔK values, respectively. The clustering with maximum likelihood revealed that, in addition to the clustering of the other five breeds, the Satsumadori was subdivided into two genetic clusters. The clustering with ΔK value indicated that the broiler and Rhode Island Red were assigned to the same genetic cluster.

A high-resolution comparative map of porcine chromosome 4 (SSC4).

We used the IMNpRH2(12,000-rad) RH and IMpRH(7,000-rad) panels to integrate 2019 transcriptome (RNA-seq)-generated contigs with markers from the porcine genetic and radiation hybrid (RH) maps and bacterial artificial chromosome finger-printed contigs, into 1) parallel framework maps (LOD ≥ 10) on both panels for swine chromosome (SSC) 4, and 2) a high-resolution comparative map of SSC4, thus and human chromosomes (HSA) 1 and 8. A total of 573 loci were anchored and ordered on SSC4 closing gaps identified in the porcine sequence assembly Sscrofa9. Alignment of the SSC4 RH with the genetic map identified five microsatellites incorrectly mapped around the centromeric region in the genetic map. Further alignment of the RH and comparative maps with the genome sequence identified four additional regions of discrepancy that are also suggestive of errors in assembly, three of which were resolved through conserved synteny with blocks on HSA1 and HSA8.

Molecular characterization of the DDX3Y gene and its homologs in cattle.

DDX3Y (also known as DBY) is a member of the DEAD box protein family, which is involved in ATP-dependent RNA unwinding, needed in a variety of cellular processes including splicing, ribosome biogenesis and RNA degradation. In the human, DDX3Y is located in the AZFa interval in the Y chromosome. Deletion of the AZFa region has been shown to disrupt spermatogenesis, causing subfertility and infertility in otherwise healthy men. Here, we report the characterization of the bovine (b) DDX3Y gene and its homologs DDX3X and PL10. We found 2 transcripts for the bDDX3Y (bDDX3Y-L and -S), which correspond to the long and short transcripts of the human DDX3Y and mouse Ddx3y gene. The 2 transcripts are identical except for a 3-bp (AGT) insertion at the position of nt 2025 and an expanded 3'UTR (nt 2155-2769) in bDDX3Y-L. The bDDX3Y-S encodes a peptide of 660 amino acids (aa), while the bDDX3Y-L encodes a peptide of 661 aa as the result of an additional serine (S) insertion at the position of aa 634. Both bDDX3Y isoforms contain the conserved DEAD-box motif. The bDDX3Y is composed of 17 exons. The homologous gene on the X chromosome, bDDX3X, is highly conserved to the Y-copy at mRNA (83%) and protein (88%) levels as well as in the genomic structure. The autosomal copy, bPL10, mapped on BTA15, is a processed pseudogene with a similarity of 88.1% to bDDX3Y and 93.7% to bDDX3X mRNA, suggesting that PL10 is a retroposon of DDX3X. RT-PCR analyses showed that bDDX3Y-L, -S, bDDX3X and bPL10 were all widely expressed with predominant expression in testis and brain. Testicular section in situ hybridization revealed that sense and anti-sense RNAs of bDDX3Y-L, -S, and bDDX3X were expressed in interstitial cells. These results together with the finding that the pseudogene bPL10 is transcriptionally active in this study provide a basis for further investigating the DDX3 gene function in spermatogenesis, male fertility and gene evolution in mammals.

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 CarthaGene 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.

Existence of Pink1 antisense RNAs in mouse and their localization.

PTEN-induced kinase 1 (PINK1), which is identified as the gene transactivated by the tumor suppressor PTEN, has been found to be one of the causative genes in Parkinson's disease (PD). In order to understand PD, rodent models containing affected Pink1 such as loss-of-function mutations have been exploited. Recently, natural antisense RNA of PINK1 has been demonstrated to be involved in the regulation of the PINK1 locus. However, no antisense RNAs of Pink1 except for human have been reported so far. Therefore, in the present study, while searching for the Pink1 antisense RNAs in mouse, we found that the antisense RNAs are transcribed from a mouse genomic region corresponding to the human region from which the antisense RNAs are produced. Further, we investigated the localization of the antisense RNAs in mouse brain using in situ hybridization; this demonstrated that the antisense RNAs were localized in the regions of brain where the Pink1 mRNA was found. In addition, the mRNA and antisense RNAs were found more densely in the hippocampus than in the other brain regions in newborn and 1-week-old mice, while those RNAs were found uniformly in the mouse brain regions of embryo day (E) 14, E17, and 8-weeks-old.

A 4,103 marker integrated physical and comparative map of the horse genome.

A comprehensive second-generation whole genome radiation hybrid (RH II), cytogenetic and comparative map of the horse genome (2n = 64) has been developed using the 5000rad horse x hamster radiation hybrid panel and fluorescence in situ hybridization (FISH). The map contains 4,103 markers (3,816 RH; 1,144 FISH) assigned to all 31 pairs of autosomes and the X chromosome. The RH maps of individual chromosomes are anchored and oriented using 857 cytogenetic markers. The overall resolution of the map is one marker per 775 kilobase pairs (kb), which represents a more than five-fold improvement over the first-generation map. The RH II incorporates 920 markers shared jointly with the two recently reported meiotic maps. Consequently the two maps were aligned with the RH II maps of individual autosomes and the X chromosome. Additionally, a comparative map of the horse genome was generated by connecting 1,904 loci on the horse map with genome sequences available for eight diverse vertebrates to highlight regions of evolutionarily conserved syntenies, linkages, and chromosomal breakpoints. The integrated map thus obtained presents the most comprehensive information on the physical and comparative organization of the equine genome and will assist future assemblies of whole genome BAC fingerprint maps and the genome sequence. It will also serve as a tool to identify genes governing health, disease and performance traits in horses and assist us in understanding the evolution of the equine genome in relation to other species.

Localization of sense and antisense transcripts of Prdx2 gene in mouse tissues.

Recently, it has been reported that antisense RNAs are transcribed from a large number of genes in various species including human and mouse. The Prdx2 gene, which is indicated to be involved in signal transduction related to platelet-derived growth factor as well as to protection from oxidizing agents, has been shown to produce sense and antisense transcripts. To obtain clues for possible roles of Prdx2 antisense transcripts, we have performed Northern blot analysis and in situ hybridization on tissues of 8-week-old C57BL/6J mice. The Northern blot analysis revealed that major parts of sense and antisense transcripts were poly(A-)-RNA. The analysis of the fractionated RNA of fibroblasts indicated that the poly(A-)-RNA would be localized in the cytoplasm of cells. The in situ hybridization demonstrated that the sense and antisense transcripts were localized in almost the same limited areas of brain, testis, and spleen. It also revealed that the sense and antisense transcripts coexisted in Purkinje cells. In thymus and stomach, the antisense transcripts were detected, but sense transcripts were not. When tissues of BALB/c mice were examined by in situ hybridization, the observations were essentially the same as those of C57BL/6J except that it appeared that the amounts of sense and antisense transcripts in testis of BALB/c were greater than those in C57BL/6J, and that the amounts of antisense transcripts in stomach of BALB/c were much smaller than those in C57BL/6J.

Genomic organization, expression and evolution of porcine CRSP1, 2, and 3.

Recently we identified and characterized porcine calcitonin receptor-stimulating peptide (CRSP) 1, CRSP2 and CRSP3 as members of the calcitonin/calcitonin gene-related peptide (CT/CGRP) family. In the present study, the genomic sequences and organization of CRSP1, 2, and 3 were determined, and the expression of the genes in the porcine brain was investigated using in situ hybridization. Analysis of 5'-upstream regions of the three CRSPs demonstrated that CRSP1 and CRSP2 have almost identical sequences (>98% similarity) and high sequence similarities including functional transcription binding sites with the corresponding region of human CALCA (CT/alpha CGRP), whereas CRSP3 retains less similarity with the above genes. RH mapping of CRSPs demonstrated that they resided in a region of swine chromosome 2 (SSC2). The arrangement of the genes in the region was found to be conserved in corresponding human and mouse regions. In situ hybridization demonstrated sense transcripts of the three genes in cerebrum, hippocampus, hypothalamus, pons/midbrain, and thalamus of 3-month-old pigs, and CRSP2 sense transcripts additionally in tractus opticus. The sense transcripts of alpha CGRP and CALCB (beta CGRP) were detected in cerebrum, hippocampus, and pons/midbrain of newborn mice, and to a lesser extent in pons/midbrain of 8-week-old mice. These results taken together with the chromosomal conservation and phylogenetic clustering of CT/CGRP family indicate that CRSP1, 2, and 3 may be functionally different from alpha CGRP and beta CGRP, though they are indicated to have a common progenitor gene.

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.

Assignment of 115 genes from HSA9 and HSA14 to SSC1q by RH mapping to generate a dense human-pig comparative map.

A large number of significant QTL for economically important traits including average daily gain have been located on SSC1q, which, as shown by chromosome painting, corresponds to four human chromosomes (HSA9, 14, 15 and 18). To provide a comprehensive comparative map for efficient selection of candidate genes, 81 and 34 genes localized on HSA9 and HSA14 respectively were mapped to SSC1q using a porcine 7000-rad radiation hybrid panel (IMpRH). This study, together with the cytogenetic map (http://www2.toulouse.inra.fr/lgc/pig/cyto/genmar/htm/1GM.HTM), demonstrates that SSC1q2.1-q2.13 corresponds to the region ranging from 44.6 to 63.2 Mb on HSA14q21.1-q23.1, the region from 86.5 to 86.8 Mb on HSA15q24-q25, the region from 0.9 to 27.2 Mb on HSA9p24.3-p21, the region from 35.1 to 38.0 Mb on HSA9p13, the region from 70.3 to 79.3 Mb on HSA9q13-q21 and the region from 96.4 to 140.0 Mb on HSA9q22.3-q34. The conserved synteny between HSA9 and SSC1q is interrupted by at least six sites, and the synteny between HSA14 and SSC1q is interrupted by at least one site.

Molecular characterization of the bovine chromodomain Y-like genes.

The human chromodomain protein, Y-like (CDYL) gene family consists of three members, one on the Y chromosome (CDY) and two on autosomes (CDYL and CDYL2). Studies in the human and mouse showed that genes in the CDYL family are abundantly expressed in testis and play an important role in spermatogenesis. In this study, we have characterized the bovine CDYL (bCDYL) and CDYL2 (bCDYL2) genes. We found that bCDYL and bCDYL2 are very similar to the human orthologues at both mRNA (79% and 85%) and protein (89% and 93%) levels. However, the similarity between the bCDYL and bCDYL2 proteins is low (41%). The bCDYL gene is composed of nine exons, and the bCDYL2 has seven exons. The bCDYL and bCDYL2 genes were mapped by radiation hybrid mapping to bovine chromosomes (BTA) 24 and 18 respectively. The bCDYL gene has four transcript variants that produce four protein isoforms. RT-PCR expression analysis in 12 bovine tissues showed that bCDYL variant 2 was expressed in the testis only, bCDYL variants 1, 3 and 4 were expressed predominantly in the testis and at very low or undetectable levels in the remaining tissues and bCDYL2 was expressed ubiquitously. Examination of bovine testis with in situ hybridization revealed that the bCDYL and bCDYL2 transcripts were found mainly in spermatids, though the amounts of transcripts varied among genes/variants. In addition, antisense transcripts were detected in bCDYL variants 2/3 and 4, as well as in the bCDYL2 gene.

Chromosomal assignments of eight expressed sequence tags for unknown genes showing early embryonic death-associated changes of expression patterns in the fetal placenta of the cow carrying somatic nuclear-derived cloned embryo.

Eight expressed sequence tags for unknown novel genes showing early embryonic death-associated changes of expression patterns in the fetal placenta of the cow carrying somatic nuclear-derived cloned embryo were assigned to bovine chromosomes using deoxyribonucleic acids (DNAs) of bovine/murine somatic cell hybrid panel.

Somatic cell hybrid mapping of expressed sequence tags for genes showing early embryonic death-associated changes of expression patterns in the fetal placenta of the cow carrying somatic nuclear-derived cloned embryo.

We previously detected 368 expressed sequence tags showing early embryonic death-associated changes of expression patterns in the fetal placenta of the cow carrying somatic nuclear-derived cloned embryo. In the present study 7 (presumed expressed sequence tags for HYPC, SPTBN1 and TNNC2, and four expressed sequence tags for unknown novel genes) out of the 368 expressed sequence tags were mapped to bovine chromosomes by analyzing deoxyribonucleic acids of bovine/murine somatic cell hybrid panel with polymerase chain reaction using primers specific for those bovine genes.

Genomic structure of swine taste receptor family 1 member 3, TAS1R3, and its expression in tissues.

Taste receptor family 1 member 3, TAS1R3, is shown to be involved in sweet and umami tastes in mouse, and the nucleotide sequence of the gene has been reported in rat, gorilla, and human. Pigs are frequently used as models for human diseases, and are also considered to be source animals for xenotransplantation to humans due to their anatomical and physiological similarities to humans. Therefore, in the present study, the genomic structure of the swine TAS1R3 gene was determined, and TAS1R3 expression was studied in various swine tissues. The gene was shown to reside on swine chromosome 6q22-->q23, from which three types of mRNAs were generated: 3,752 bp derived from six exons in tongue, 3,704 bp from six exons and 3,630 bp from seven exons in testis. The 6 exons/5 introns were structurally similar to those of humans and mice, but the 7 exons/6 introns structure of TAS1R3 was first observed in swine. High expressions of TAS1R3 were revealed in tongue, kidney, and testis by real-time PCR. The expression profile of the tissues except for kidney was similar to that of mouse. When in situ hybridization using an RNA probe for TAS1R3 was performed on swine tongue and testis tissues, TAS1R3 expressions were revealed in tongue circumvallate papillae, fungiform papillae, mucosal epithelium, follicular B lymphocytes, lymphocytes in submucosal tissues of lingual tonsil, and spermatogenic cells. Using peripheral mature B lymphocytes, the expression of TAS1R3 in B lymphocytes was further confirmed by real-time PCR and sequencing of the real-time PCR product.

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.

Assignment of 204 genes localized on HSA17 to a porcine RH (IMpRH) map to generate a dense comparative map between pig and human/mouse.

Bi- and uni-directional chromosome painting (ZOO-FISH) and gene mapping have revealed correspondences between human chromosome (HSA) 17 and porcine chromosome (SSC) 12 harboring economically important quantitative trait loci. In the present study, we have assigned 204 genes localized on HSA17 to SSC12 to generate a comprehensive comparative map between HSA17 and SSC12. Two hundred fifty-five primer pairs were designed using porcine sequences orthologous with human genes. Of the 255 primer pairs, 208 (81.6%) were used to assign the corresponding genes to porcine chromosomes using the INRA-Minnesota 7000-rad porcine x Chinese hamster whole genome radiation hybrid (IMpRH) panel. Two hundred three genes were integrated into the SSC12 IMpRH linkage maps; and one gene, PPARBP, was found to link to THRA1 located in SSC12 but not incorporated into the linkage maps. Three genes (GIT1, SLC25A11, and HT008) were suggested to link to SSC12 markers, and the remaining gene (RPL26) did not link to any genes/expressed sequence tags/markers registered, including those in the present study. A comparison of the gene orders among SSC12, HSA17, and mouse chromosome 11 indicates that intra-chromosomal rearrangements occurred frequently in this ancestral mammalian chromosome during speciation.

Assignment of 101 genes localized in HSA10 to a swine RH (IMpRH) map to generate a dense human-swine comparative map.

Economically important traits such as growth and backfat in pigs have been shown to be influenced by genes in swine chromosome (SSC) 10q12-->qter corresponding to human chromosome (HSA) 10p. However, since gene information in the swine chromosomal region was limited, we attempted to generate a dense comparative map between SSC10 and HSA10 by mapping the 115 genes of HSA10 to a swine RH map (IMpRH map). In the mapping ten genes were assigned to SSC10, 88 to SSC14, and one to SSC3. One gene was suggested to link to SSC3, and another to SSC9. The correspondences between HSA10 and SSC10 and between HSA10 and SSC14 were essentially consistent with the observations obtained from bi/uni-directional chromosome painting or other results. This study further indicated that a large number of intrachromosomal rearrangements occurred in the synteny-conserved regions following species separation.

Analysis of recessive lethality on swine chromosome 6 in a Göttingen miniature resource family.

Previously, we reported recessive gene(s) that terminate fetal development on swine chromosome (SSC) 6 between SW855 and SW122. The affected alleles originated from a Göttingen miniature pig used for construction of a Göttingen miniature pig x Meishan resource population. However, it is not known when the gene(s) are activated during fetal development, which is one of the important factors in selecting candidate genes responsible for fetal death. In the present study, a second swine population consisting of 159 progeny was produced by mating pigs carrying the deleterious allele(s). This population allowed us to narrow the genetic region harbouring the affected gene(s) and to demonstrate that the region was confined between RYR1 and SW782 (5.7 cM on the National Institute of Animal Industry (NIAI) map and 100 cR on the INRA/University of Minnesota porcine radiation hybrid panel map). In order to determine when the affected gene(s) are activated and in turn terminate fetal development, embryos produced in the second population were collected at several development stages and genotyped for markers in the region. Genes in the homozygous state affected embryo development between 9 and 11 days post-coitus.