Analysis of the Diversity of the AvBD Gene Region in Japanese Quail.

The avian ß-defensin (AvBD) gene region is an important component of the innate immune system, encoding a variety of antimicrobial peptides. The AvBD region forms a multigene cluster in a specific chromosomal region. Comparison of the AvBD region among various birds suggests the presence of defects, duplications, and pseudogenization at many loci. The AvBD region in certain galliform birds, namely chicken, turkey, and bobwhite quail, includes AvBD3, -6, and -7, with the latter exhibiting copy number variants (CNVs) in chickens. DNA for genomic analysis was extracted from the peripheral blood of 99 randomly selected quail (Coturnix japonica) from 6 inbred lines. Nine CjAvBD1 and 8 CjAvBD12 alleles were detected. Ten haplotypes, including three that were strain specific, were found in alleles from the quail AvBD1 (CjAvBD1) and -12 (CjAvBD12) loci. Next-generation sequencing was used to determine the nucleotide sequences of the CjAvBD gene region (56-70 kb) for 7 homozygous diplotypes of these 10 haplotypes. These 7 haplotypes contained between 12 and 16 CjAvBD genes and were composed of 11 common loci: CjAvBD1, -2, -4, -5, -8, -9, -10, -11, -12, -13, and -14, but lacked CjAvBD3 and -7. Furthermore, up to 5 CjAvBD101 (AvBD6 ortholog) CNVs were observed among the 7 haplotypes. In addition, we detected amino acid substitutions causing net charge mutations that could affect antimicrobial activity in CjAvBD4, -13, -14, and -101. These results suggest that the CjAvBD region is unique among the Galliformes and that its diversity results in potential functional variation in innate immunity.

Mapping of Calf Death in Japanese Black Cattle.

Weak calf syndrome (WCS) is a major cause of calf death in Japanese Black cattle. Among IARS disorders, the isoleucyl-tRNA synthetase c.235G>C mutation has been identified as one of the causes of WCS. However, calf deaths differing from those attributed to IARS disorder has been occurring. To identify other genes potentially responsible for these calf deaths, we constructed three populations of three bulls (Bull-1, -2 and -3) that did not carry the IARS mutation, and dead calves (18, 28, and 31 calves) and healthy cattle (18, 15, and 10 cattle) sired by these bulls. The populations were genotyped using the BovineSNP50 BeadChip, but homozygosity mapping did not detect any associated genomic regions with calf death. Linkage analysis performed using each population as a paternal half-sib family of Bull-1, Bull-2, and Bull-3 revealed that, in the Bull-1 population, calf death was mapped to the 8.94 Mb-14.53 Mb and 29.82 Mb-33.77 Mb regions of BTA29. The findings suggested that the incidence of calf death in calves sired by Bull-1 was a hereditary disease exhibiting a dominant, not recessive, inheritance pattern.

Genetic diversity of Japanese quail cathelicidins.

Japanese quail is a low-fat, meat-bird species exhibiting high disease resistance. Cathelicidins (CATHs) are host defense peptides conserved across numerous vertebrate species that play an important role in innate immunity. The activity of host defense peptides can be affected by amino acid substitutions. However, no polymorphisms in avian CATH genes have been reported to date. The aim of this study was to clarify the polymorphisms in CATHs in Japanese quail. DNA for genomic analyses was extracted from the peripheral blood of 99 randomly selected quail from 6 inbred lines. A total of 6, 4, 6, and 4 CjCATH1, -2, -3, and -B1 alleles were identified, respectively. Nine haplotypes, including 4 strain-specific haplotypes, were identified by combining alleles at the CjCATH1, -2, -3, and -B1 loci. In addition, 2 and 1 amino acid substitutions (I145F, Q148H, and P245H) predicted by PROVEAN and PolyPhen-2 to have deleterious effects were detected in CjCATH2 and -B1, respectively. Synthetic CjCATH2 and -B1 peptides exhibited greater antibacterial activity against Escherichia coli than chicken CATH2 and -B1, respectively. Furthermore, the CjCATHB1∗04 peptide exhibited less potent antimicrobial activity than other CjCATHB1 peptides examined. This is the first report of amino acid substitutions accompanied by changes in antibacterial activity in avian CATHs. These findings could be employed as indicators of improvements in innate immune response in poultry.

Contribution of mutation, recombination, and gene conversion to chicken MHC-B haplotype diversity.

The Mhc is a highly conserved gene region especially interesting to geneticists because of the rapid evolution of gene families found within it. High levels of Mhc genetic diversity often exist within populations. The chicken Mhc is the focus of considerable interest because of the strong, reproducible infectious disease associations found with particular Mhc-B haplotypes. Sequence data for Mhc-B haplotypes have been lacking thereby hampering efforts to systematically resolve which genes within the Mhc-B region contribute to well-defined Mhc-B-associated disease responses. To better understand the genetic factors that generate and maintain genomic diversity in the Mhc-B region, we determined the complete genomic sequence for 14 Mhc-B haplotypes across a region of 59 kb that encompasses 14 gene loci ranging from BG1 to BF2. We compared the sequences using alignment, phylogenetic, and genome profiling methods. We identified gene structural changes, synonymous and non-synonymous polymorphisms, insertions and deletions, and allelic gene rearrangements or exchanges that contribute to haplotype diversity. Mhc-B haplotype diversity appears to be generated by a number of mutational events. We found evidence that some Mhc-B haplotypes are derived by whole- and partial-allelic gene conversion and homologous reciprocal recombination, in addition to nucleotide mutations. These data provide a framework for further analyses of disease associations found among these 14 haplotypes and additional haplotypes segregating and evolving in wild and domesticated populations of chickens.

Robertsonian translocation as a result of telomere shortening during replicative senescence and immortalization of bovine oviduct epithelial cells.

We investigated chromosome (Chr) aberrations in the process of replicative senescence and immortalization of cultured bovine oviduct epithelial cells (BOEC) before and after transfecting vectors SV40 large T or human telomerase reverse transcriptase (hTERT). We found that a gradual increase in the number of metacentric chromosomes occurred during replicative senescence but not immortalization of BOEC. The accumulation of metacentric chromosomes was concomitant with decreases in the number of acrocentric autosomes, strongly suggesting that Robertsonian (Rb) translocation frequently occurred in cultured BOEC. The process was also correlated with an accumulation of extremely shortened telomeres (<4 kb). The maximum number of metacentric chromosomes reached a plateau (8.75 +/- 0.53) in the senescent BOEC (approximately 48 population doublings), and the value was stably maintained in all immortalized lines. These results suggest that not all autosomes may be involved in Rb translocation. Fluorescence in situ hybridization analysis using probes specific for Chr1, Chr29, telomeres, and x-chromosomes of bovine confirmed the presence of t(1;29) with other unidentified fused chromosomes. There was no evidence for duplication of sex chromosomes. Because no detectable fluorescence in situ hybridization signals at the centromere for telomeres were indicative of no direct integration of telomere sequences in the Rb translocated chromosomes, these results raise a possibility that Rb translocation between certain autosomes of bovine cells is partly but critically dependent upon a physical state of telomere attrition. The cells and cell lines established in this study could provide a promising system for further studies on the mechanisms of chromosomal translocation because of centromeric fusion in bovine cells.

Characterization of the cathelicidin cluster in the Japanese quail (Coturnix japonica).

The Japanese quail has several advantages as a low-fat meat bird with high immunity against diseases. Cathelicidins (CATHs) are antimicrobial peptides that play an important role in innate immunity. The aim of this study was to characterize the CATH cluster in the Japanese quail (Coturnix japonica). The Japanese quail CATH (CjCATH) cluster, contains four CATH genes, as in the chicken. The coding sequences of CjCATHs exhibited >85.3% identity to chicken CATHs. The predicted amino acid sequences of the four CjCATH genes contained the cathelin-like domain characteristic of CATH proteins. Polymorphisms were detected in the open reading frames (ORFs) of all CjCATH sequences. Two amino acid substitutions were observed in the antimicrobial region of the mature peptide of CjCATH2, and predicted to influence peptide function. CjCATH1 is expressed in lung, heart, bone marrow and bursa of Fabricius (BF). CjCATH2 is expressed in bone marrow. CjCATH3 is expressed in lung, heart, bone marrow, BF, tongue and duodenum. CjCATHB1 is expressed in bone marrow and BF. This study is the first to characterize CATH genes in the Japanese quail, and identifies novel antimicrobial peptide sequences belonging to the cathelicidin family, which may play a role in immunity in this species.

The major histocompatibility complex (Mhc) class IIB region has greater genomic structural flexibility and diversity in the quail than the chicken.

BACKGROUND: The quail and chicken major histocompatibility complex (Mhc) genomic regions have a similar overall organization but differ markedly in that the quail has an expanded number of duplicated class I, class IIB, natural killer (NK)-receptor-like, lectin-like and BG genes. Therefore, the elucidation of genetic factors that contribute to the greater Mhc diversity in the quail would help to establish it as a model experimental animal in the investigation of avian Mhc associated diseases. AIMS AND APPROACHES: The main aim here was to characterize the genetic and genomic features of the transcribed major quail MhcIIB (CojaIIB) region that is located between the Tapasin and BRD2 genes, and to compare our findings to the available information for the chicken MhcIIB (BLB). We used four approaches in the study of the quail MhcIIB region, (1) haplotype analyses with polymorphic loci, (2) cloning and sequencing of the RT-PCR CojaIIB products from individuals with different haplotypes, (3) genomic sequencing of the CojaIIB region from the individuals with the different haplotypes, and (4) phylogenetic and duplication analysis to explain the variability of the region between the quail and the chicken. RESULTS: Our results show that the Tapasin-BRD2 segment of the quail Mhc is highly variable in length and in gene transcription intensity and content. Haplotypic sequences were found to vary in length between 4 to 11 kb. Tapasin-BRD2 segments contain one or two major transcribed CojaIIBs that were probably generated by segmental duplications involving c-type lectin-like genes and NK receptor-like genes, gene fusions between two CojaIIBs and transpositions between the major and minor CojaIIB segments. The relative evolutionary speed for generating the MhcIIBs genomic structures from the ancestral BLB2 was estimated to be two times faster in the quail than in the chicken after their separation from a common ancestor. Four types of genomic rearrangement elements (GRE), composed of simple tandem repeats (STR), were identified in the MhcIIB genomic segment located between the Tapasin-BRD2 genes. The GREs have many more STR numbers in the quail than in the chicken that displays strong linkage disequilibrium. CONCLUSION: This study suggests that the Mhc classIIB region has a flexible genomic structure generated by rearrangement elements and rapid SNP accumulation probably as a consequence of the quail adapting to environmental conditions and pathogens during its migratory history after its divergence from the chicken.

Characterization and expression of non-polymorphic liver expressed antimicrobial peptide 2: LEAP-2 in the Japanese quail, Coturnix japonica.

Liver-expressed antimicrobial peptide 2 (LEAP-2) is a cationic peptide that plays an important role in innate immunity for host defense. The aim of this study was to characterize the LEAP-2 gene in the Japanese quail (Coturnix japonica). Japanese quail LEAP-2 (CjLEAP-2) was identified from the Japanese quail draft genome database by a local BLAST analysis using chicken LEAP-2 (GgLEAP-2). The exon-intron structure of CjLEAP-2, analyzed from three quails, is composed of three exons, as is the chicken LEAP-2 homolog (GgLEAP-2). An analysis of the coding sequence revealed that CjLEAP-2 is 231 bp long, like GgLEAP-2, and 93% identical to GgLEAP-2 at the nucleic acid level. The predicted amino acid sequence of CjLEAP-2 contained the liver-expressed antimicrobial peptide 2-precursor domain and four cysteine residues characteristic of the LEAP-2 protein. The amino acid sequence of the mature peptide of CjLEAP-2 was 100% identical to that of GgLEAP-2. We confirmed that CjLEAP-2 was transcribed in at least seven tissues, including the digestive system. Additionally, the mature peptide region of CjLEAP-2 exhibited no polymorphisms in 99 quails from six strains. Taken together, these findings indicate that CjLEAP-2 is non-polymorphic and therefore, it likely plays an important role in the innate immunity of quail as it does in chicken.

Basic characterization of avian ß-defensin genes in the Japanese quail, Coturnix japonica.

In this study, we identified a cluster of 14 avian ß-defensins (AvBD; approximately 66 kbp) in the Japanese quail, Coturnix japonica. Except for AvBD12 (CjAvBD12) and -13, the CjAvBDs coding sequences exhibited greater than 78.0% similarity to the respective orthologous chicken AvBD genes (GgAvBD). The putative amino acid sequence encoded by each CjAvBD contained six cysteine residues and the GXC (X1-2) motif considered essential for the ß-defensin family. Each CjAvBDs also formed a sub-group with the respective orthologous genes of various bird species in a phylogenetic tree analysis. Synteny between the CjAvBD cluster and GgAvBD cluster was confirmed. The CjAvBD cluster was mapped on the long-arm end of chromosome 3 by linkage analysis based on single nucleotide polymorphisms (SNPs) of CjAvBD1 and CjAvBD12 (approximately 46 kbp), as well as GgAvBD cluster. We also confirmed that CjAvBD1, -4, -5, -9, and -10 are transcribed in 20 tissues, including immune and digestive tissues. However, our experimental data indicated that the CjAvBD cluster lacks the AvBD3 and -7 loci, whereas the CjAvBD101α, -101ß, and -101θ loci arose from gene duplication of the AvBD6 orthologous locus in the CjAvBD cluster after differentiation between Coturnix - Gallus.

Effect of a single polymorphism in the Japanese quail NK-lysin gene on antimicrobial activity.

NK-lysins are cationic peptides that play important roles in host protection, and are an important constituent of innate immunity. We identified nine single-nucleotide polymorphisms (SNPs) in the NK-lysin open reading frame (ORF) from 32 Japanese quails in six strains: A, B, ND, K, P, and Y. The G to A substitution at nucleotide position 272 in the ORF resulted in a Gly (G) to Asp (D) amino acid substitution (Cj31G and Cj31D alleles). The Cj31D allele was detected in P (frequency 0.76) and Y (frequency 0.03) strains. We compared the antimicrobial activities of four synthetic peptides from the helix 2-loop-helix 3 region of avian NK-lysins against Escherichia coli: Cj31G and Cj31D from quail and Gg29N and Gg29D from chicken. The antimicrobial activities of the four peptides decreased in the following order: Gg29N > Cj31G > Gg29D > Cj31D (P < 0.05). Although there were no differences in the predicted secondary structure of the Cj31G and Cj31D, the net charge of the Cj31G was higher than that of Cj31D. These data indicated that the antimicrobial activity of CjNKL is influenced by net charge, similar to that which has been observed in chicken.

IARS mutation causes prenatal death in Japanese Black cattle.

Isoleucyl-tRNA synthetase (IARS) c.235G > C (p.V79L) is a causative mutation for a recessive disease called IARS disorder in Japanese black cattle. The disease is involved in weak calf syndrome and is characterized by low birth weight, weakness and poor suckling. The gestation period is often slightly extended, implying that intrauterine growth is retarded. In a previous analysis of 2597 artificial insemination (AI) procedures, we suggested that the IARS mutation might contribute toward an increase in the incidence of prenatal death. In this study, we extended this analysis to better clarify the association between the IARS mutation and prenatal death. The IARS genotypes of 92 animals resulting from crosses between carrier (G/C) × G/C were 27 normal (G/G), 55 G/C and 10 affected animals (C/C) (expected numbers: 23, 46 and 23, respectively). Compared to the expected numbers, there were significantly fewer affected animals in this population (P < 0.05), suggesting that more than half of the affected embryos died prenatally. When the number of AI procedures examined was increased to 11 580, the frequency of re-insemination after G/C × G/C insemination was significantly higher at 61-140 days (P < 0.001). The findings suggested that the homozygous IARS mutation not only causes calf death, but also embryonic or fetal death.

Complete mitochondrial genomes of the tooth of a poached Bornean banteng (Bos javanicus lowi; Cetartiodactyla, Bovidae).

Here we report the complete mitochondrial genome of the Bornean banteng Bos javanicus lowi (Cetartiodactyla, Bovidae), which was determined using next-generation sequencing. The mitochondrial genome is 16,344 bp in length containing 13 protein-coding genes, 21 tRNAs and 2 rRNAs. It shows the typical pattern of bovine mitochondrial arrangement. Phylogenetic tree analysis of complete mtDNA sequences showed that Bornean banteng is more closely related to gaur than to other banteng subspecies. Divergence dating indicated that Bornean banteng and gaur diverged from their common ancestor approximately 5.03 million years ago. These results suggest that Bornean banteng might be a distinct species in need of conservation.

Basic characterization of avian NK-lysin (NKL) from the Japanese quail, Coturnix japonica.

We identified an antimicrobial cationic peptide that was expressed in the natural killer cells and cytotoxic T-lymphocytes of Japanese quail. The gene, designated CjNKL, was located downstream of AEBP1L and POLD2 in a region syntenic with the chicken genome. CjNKL comprised four exons, as does chicken GgNKL. The coding sequence in CjNKL was 411 bp long and exon 3 of CjNKL lacked 9 bp when compared to chicken GgNKL, but CjNKL and GgNKL were 81% identical at the nucleic acid level. The saposin like type-B domain of CjNKL contained the six essential cysteines, one proline, 15 cationic amino acids residues, and an antibacterial region that are characteristic of NKL proteins. The 5' flanking region of CjNKL contained positive regulatory elements, an activator protein-1 binding site and two nuclear factor (NF)-κB binding sites, and a negative regulatory element, CAAT/enhancer binding protein ß (C/EBPß) binding site. However, the number of NF-κB sites and C/EBPß sites within CjNKL are fewer than the number within GgNKL. Additionally, we confirmed that CjNKL was transcribed in at least 18 tissues, including immune and digestive tissues. These data indicated that transcriptional activation of CjNKL differed slightly from those of GgNKL.

Primary analysis of DNA polymorphisms in the TRIM region (MHC subregion) of the Japanese quail, Coturnix japonica.

Based on sequences of two cosmid clones from Japanese quail (Coturnix japonica, Coja), we confirmed that the syntenic cluster, GNB2L1∼BTN1∼BTN2, is located in the quail TRIM subregion of the quail major histocompatibility complex (MHC Coja) region. These cosmids also included four CjBG loci and one CjLEC locus; therefore, the quail TRIM subregion was thought to be adjacent to the BG/LEC subregion. We then identified three polymorphic markers - CjHEP21, CjTRIM39.2 and CjBTN2 - in the TRIM subregion that may be useful for the functional analysis of the MHC-Coja region. We examined MHC-Coja sequences from 321 individual quails sampled from 11 inbred strains, and we found eight alleles for each of the three genes - CjHEP21, CjTRIM39.2 and CjBTN2. These polymorphisms represent the first avian DNA markers in the TRIM subregion. Additionally, we discovered a quail-specific VNTR (variable number of long tandem repeats, 133-137 bp) in intron 7 of CjBTN2. We identified 25 haplotypes in the sample of 321 quail; these haplotypes comprised combinations of all 24 alleles of the three polymorphic genes. We suggest that there are two recombination hotspots, one between each pair of adjacent loci. All strains, except AMRP, contained multiple haplotypes; the AMRP strain contained a single, apparently fixed haplotype.

Basic characterization of 90 kDa heat shock protein genes HSP90AA1, HSP90AB1, HSP90B1 and TRAP1 expressed in Japanese quail (Coturnix japonica).

In the current study, we describe four novel members of the 90 kDa heat shock protein (HSP90) family expressed in Japanese quail, Coturnix japonica. The coding regions of the genes, CjHSP90AA1, CjHSP90AB1, CjHSP90B1 and CjTRAP1, exhibited more than 94% similarity to their related genes in chicken. The putative proteins encoded by these quail genes contained motifs considered essential for HSP90 gene function. In addition, the predicted proteins were more similar to HSP90AA1, HSP90AB1, HSP90B1 and TRAP1 proteins expressed in vertebrates than they were to other members of the HSP90 family. Exon numbers of CjHSP90AA1 (11), CjHSP90AB1 (12) or CjTRAP1 (18) are the same as the chicken and mammalian orthologs. Furthermore, gene order in the regions surrounding CjHSP90AB1 and CjTRAP1 has been preserved, providing evidence that the genomic regions were orthologous to HSP90-containing regions in the chicken genome. The promoter regions of the genes also contained conserved motifs identified in related genes of chicken. However, the nucleotide sequences of the 5'-flanking region of these genes were highly polymorphic. We also found that CjHSP90AA1 exhibited a robust response to heat shock treatment. Taken together, the data suggest that CjHSP90AA1, CjHSP90AB1, CjHSP90B1 and CjTRAP1 encode orthologs of HSP90AA1, HSP90AB1, HSP90B1 and TRAP1, respectively.

Comparative genomic analysis of two avian (quail and chicken) MHC regions.

We mapped two different quail Mhc haplotypes and sequenced one of them (haplotype A) for comparative genomic analysis with a previously sequenced haplotype of the chicken Mhc. The quail haplotype A spans 180 kb of genomic sequence, encoding a total of 41 genes compared with only 19 genes within the 92-kb chicken Mhc. Except for two gene families (B30 and tRNA), both species have the same basic set of gene family members that were previously described in the chicken "minimal essential" Mhc. The two Mhc regions have a similar overall organization but differ markedly in that the quail has an expanded number of duplicated genes with 7 class I, 10 class IIB, 4 NK, 6 lectin, and 8 B-G genes. Comparisons between the quail and chicken Mhc class I and class II gene sequences by phylogenetic analysis showed that they were more closely related within species than between species, suggesting that the quail Mhc genes were duplicated after the separation of these two species from their common ancestor. The proteins encoded by the NK and class I genes are known to interact as ligands and receptors, but unlike in the quail and the chicken, the genes encoding these proteins in mammals are found on different chromosomes. The finding of NK-like genes in the quail Mhc strongly suggests an evolutionary connection between the NK C-type lectin-like superfamily and the Mhc, providing support for future studies on the NK, lectin, class I, and class II interaction in birds.