White and other fur colourations and hybridization in golden jackals (Canis aureus) in the Carpathian basin.

The golden jackal (Canis aureus) is a reoccurring species in the centre of the Carpathian basin, in Hungary. In total, 31 golden jackal tissue samples were collected, from 8 white-coated, 2 black-coated and one mottled animal across Hungary. Sequences and fragment length polymorphisms were studied for white colour (MC1R), and for black coat colouration (CBD103). In each white animal, the most widespread mutation causing white fur colour in dogs in homozygous form was detected. Three animals were found to carry the mutation in heterozygous form. The two black golden jackals were heterozygous for the 3 bp deletion in CBD103 that mutation for black coat colouration in dogs, and one of them also carried the mutation causing white fur. None of the white animals showed signs of hybridization, but both the black and the mottled coloured individuals were found to be hybrids based on genetic testing. Kinship was found three times, twice between white animals, and once between a white animal and an agouti animal carrying the mutation of white coat. Our results confirm the findings that golden jackal-dog hybrids may occur without human intervention, and the detected mutation causing white fur colour in golden jackals could possibly be due to an early hybridization event.

Mining the red deer genome (CerEla1.0) to develop X-and Y-chromosome-linked STR markers.

Microsatellites are widely applied in population and forensic genetics, wildlife studies and parentage testing in animal breeding, among others, and recently, high-throughput sequencing technologies have greatly facilitated the identification of microsatellite markers. In this study the genomic data of Cervus elaphus (CerEla1.0) was exploited, in order to identify microsatellite loci along the red deer genome and for designing the cognate primers. The bioinformatics pipeline identified 982,433 microsatellite motifs genome-wide, assorted along the chromosomes, from which 45,711 loci mapped to the X- and 1096 to the Y-chromosome. Primers were successfully designed for 170,873 loci, and validated with an independently developed autosomal tetranucleotide STR set. Ten X- and five Y-chromosome-linked microsatellites were selected and tested by two multiplex PCR setups on genomic DNA samples of 123 red deer stags. The average number of alleles per locus was 3.3, and the average gene diversity value of the markers was 0.270. The overall observed and expected heterozygosities were 0.755 and 0.832, respectively. Polymorphic Information Content (PIC) ranged between 0.469 and 0.909 per locus with a mean value of 0.813. Using the X- and Y-chromosome linked markers 19 different Y-chromosome and 72 X-chromosome lines were identified. Both the X- and the Y-haplotypes split to two distinct clades each. The Y-chromosome clades correlated strongly with the geographic origin of the haplotypes of the samples. Segregation and admixture of subpopulations were demonstrated by the use of the combination of nine autosomal and 16 sex chromosomal STRs concerning southwestern and northeastern Hungary. In conclusion, the approach demonstrated here is a very efficient method for developing microsatellite markers for species with available genomic sequence data, as well as for their use in individual identifications and in population genetics studies.

Population Genetic Structure of the Wild Boar (Sus scrofa) in the Carpathian Basin.

In the Carpathian Basin the wild boar (Sus scrofa) belongs among the most important game species both ecologically and economically, therefore knowing more about the basics of the genetics of the species is a key factor for accurate and sustainable management of its population. The aim of this study was to estimate the genetic diversity and to elucidate the genetic structure and location of wild boar populations in the Carpathian Basin. A total of 486 samples were collected and genotyped using 13 STR markers. The number of alleles varied between 4 and 14, at 9 of the 13 loci the observed heterozygosity was significantly different (p < 0.05) from the expected value, showing remarkable introgression in the population. The population was separated into two groups, with an Fst value of 0.03, suggesting the presence of two subpopulations. The first group included 147 individuals from the north-eastern part of Hungary, whereas the second group included 339 samples collected west and south of the first group. The two subpopulations' genetic indices are roughly similar. The lack of physical barriers between the two groups indicates that the genetic difference is most likely caused by the high reproduction rate and large home range of the wild boars, or by some genetic traces' having been preserved from both the last ice age and the period before the Hungarian water regulation.

Testing cryopreserved European eel sperm for hybridization (A. japonica × A. anguilla).

The objective of this study was to assess impact of cryopreserved European eel sperm and Japanese eel native sperm on early fertilization, hatch, survival, and malformation rates of larvae, as well as develop molecular techniques to distinguish different eel species. Eggs from Japanese eel females (Anguilla japonica) were artificially fertilized with sperm of Japanese eel males and cryopreserved sperm from European eel (A. anguilla, extender was modified Tanaka solution and methanol as cryoprotectant). There were no statistical differences (p > 0.05) among the measured parameters such as fertilization, hatch and survival after 10 days post-hatch rates due to large individual differences. The malformation rate of larvae compared to the hatching rate was higher in cryopreserved groups than in the control indicating that the methodology needs further refinement. Genetic analyses (PCR-RFLP, PCR-HRM) proved a clear result in the detection of paternal contribution in hybridization between the Japanese and the European eel and applied PCR-HRM method is a quick and cost effective tool to identify illegally imported A. anguilla at the glass eel stage, which can be transported from Europe to Asia.

The red deer Cervus elaphus genome CerEla1.0: sequencing, annotating, genes, and chromosomes.

We present here the de novo genome assembly CerEla1.0 for the red deer, Cervus elaphus, an emblematic member of the natural megafauna of the Northern Hemisphere. Humans spread the species in the South. Today, the red deer is also a farm-bred animal and is becoming a model animal in biomedical and population studies. Stag DNA was sequenced at 74× coverage by Illumina technology. The ALLPATHS-LG assembly of the reads resulted in 34.7 × 103 scaffolds, 26.1 × 103 of which were utilized in Cer.Ela1.0. The assembly spans 3.4 Gbp. For building the red deer pseudochromosomes, a pre-established genetic map was used for main anchor points. A nearly complete co-linearity was found between the mapmarker sequences of the deer genetic map and the order and orientation of the orthologous sequences in the syntenic bovine regions. Syntenies were also conserved at the in-scaffold level. The cM distances corresponded to 1.34 Mbp uniformly along the deer genome. Chromosomal rearrangements between deer and cattle were demonstrated. 2.8 × 106 SNPs, 365 × 103 indels and 19368 protein-coding genes were identified in CerEla1.0, along with positions for centromerons. CerEla1.0 demonstrates the utilization of dual references, i.e., when a target genome (here C. elaphus) already has a pre-established genetic map, and is combined with the well-established whole genome sequence of a closely related species (here Bos taurus). Genome-wide association studies (GWAS) that CerEla1.0 (NCBI, MKHE00000000) could serve for are discussed.

X- and Y-chromosome-specific variants of the amelogenin gene allow non-invasive sex diagnosis for the detection of pseudohermaphrodite goats.

The Polled Intersex Syndrome (PIS) is responsible for the absence of horns in homozygous and heterozygous goats causing a female-to-male sex reversal in the homozygous polled genotypic female (XX) goats. A simple and efficient non-invasive method was elaborated to detect the genotypic sex from hair and faecal samples using a pair of primers to amplify the X- and Y-linked alleles of the amelogenin gene. The PCR products were easily distinguishable using agarose gel electrophoresis: we detected an X-specific single band in samples originating from healthy phenotypic females and double (X- and Y-) bands in samples from males. The new PCR method is applicable for diagnosing the sex of PIS-affected animals already as newborn kids, in contrast with the phenotypic findings appearing only after puberty, and thus it may replace the cumbersome chromosome investigations.

Complete mitochondrial genome sequence of a Hungarian red deer (Cervus elaphus hippelaphus) from high-throughput sequencing data and its phylogenetic position within the family Cervidae.

Recently, there has been considerable interest in genetic differentiation in the Cervidae family. A common tool used to determine genetic variation in different species, breeds and populations is mitochondrial DNA analysis, which can be used to estimate phylogenetic relationships among animal taxa and for molecular phylogenetic evolution analysis. With the development of sequencing technology, more and more mitochondrial sequences have been made available in public databases, including whole mitochondrial DNA sequences. These data have been used for phylogenetic analysis of animal species, and for studies of evolutionary processes. We determined the complete mitochondrial genome of a Central European red deer, Cervus elaphus hippelaphus, from Hungary by a next generation sequencing technology. The mitochondrial genome is 16 354 bp in length and contains 13 protein-coding genes, two rRNA genes, 22 tRNA genes and a control region, all of which are arranged similar as in other vertebrates. We made phylogenetic analyses with the new sequence and 76 available mitochondrial sequences of Cervidae, using Bos taurus mitochondrial sequence as outgroup. We used 'neighbor joining' and 'maximum likelihood' methods on whole mitochondrial genome sequences; the consensus phylogenetic trees supported monophyly of the family Cervidae; it was divided into two subfamilies, Cervinae and Capreolinae, and five tribes, Cervini, Muntiacini, Alceini, Odocoileini, and Capreolini. The evolutionary structure of the family Cervidae can be reconstructed by phylogenetic analysis based on whole mitochondrial genomes; which method could be used broadly in phylogenetic evolutionary analysis of animal taxa.

Genome sequencing and analysis of Mangalica, a fatty local pig of Hungary.

BACKGROUND: Mangalicas are fatty type local/rare pig breeds with an increasing presence in the niche pork market in Hungary and in other countries. To explore their genetic resources, we have analysed data from next-generation sequencing of an individual male from each of three Mangalica breeds along with a local male Duroc pig. Structural variations, such as SNPs, INDELs and CNVs, were identified and particular genes with SNP variations were analysed with special emphasis on functions related to fat metabolism in pigs. RESULTS: More than 60 Gb of sequence data were generated for each of the sequenced individuals, resulting in 11× to 19× autosomal median coverage. After stringent filtering, around six million SNPs, of which approximately 10% are novel compared to the dbSNP138 database, were identified in each animal. Several hundred thousands of INDELs and about 1,000 CNV gains were also identified. The functional annotation of genes with exonic, non-synonymous SNPs, which are common in all three Mangalicas but are absent in either the reference genome or the sequenced Duroc of this study, highlighted 52 genes in lipid metabolism processes. Further analysis revealed that 41 of these genes are associated with lipid metabolic or regulatory pathways, 49 are in fat-metabolism and fatness-phenotype QTLs and, with the exception of ACACA, ANKRD23, GM2A, KIT, MOGAT2, MTTP, FASN, SGMS1, SLC27A6 and RETSAT, have not previously been associated with fat-related phenotypes. CONCLUSIONS: Genome analysis of Mangalica breeds revealed that local/rare breeds could be a rich source of sequence variations not present in cosmopolitan/industrial breeds. The identified Mangalica variations may, therefore, be a very useful resource for future studies of agronomically important traits in pigs.

Constructing STR multiplexes for individual identification of Hungarian red deer.

Red deer is the most valuable game of the fauna in Hungary, and there is a strong need for genetic identification of individuals. For this purpose, 10 tetranucleotide STR markers were developed and amplified in two 5-plex systems. The study presented here includes the flanking region sequence analysis and the allele nomenclature of the 10 loci as well as the PCR optimization of the DeerPlex I and II. LD pairwise tests and cross-species similarity analyses showed the 10 loci to be independently inherited. Considerable levels of genetic differences between two subpopulations were recorded, and F(ST) was 0.034 using AMOVA. The average probability of identity (PI(ave)) was at the value of 2.6736 × 10(-15). This low value for PI(ave) nearly eliminates false identification. An illegal hunting case solved by DeerPlex is described herein. The calculated likelihood ratio (LR) illustrates the potential of the 10 red deer microsatellite markers for forensic investigations.

Introgression and isolation contributed to the development of Hungarian Mangalica pigs from a particular European ancient bloodline.

BACKGROUND: Mangalica breeds are indigenous to Hungary and their breeding history dates back to about 200-250 years ago. They are fat-type pigs and have a rare curly hair phenotype. The aim of our study was to establish the relationships between these unique breeds and other European breeds. RESULTS: Based on a core sequence of 382 bp present in 2713 mitochondrial D-loop sequences from pigs belonging to 38 local breeds from nine countries, five cosmopolitan breeds and wild boars from 14 countries, we identified 164 haplotypes. More than half of the 2713 sequences belonged to either four haplotypes characteristic of continental European breeds or two haplotypes characteristic of British/cosmopolitan breeds; each haplotype is present in more than 100 individuals. Most Mangalica individuals belonged either to one of these common continental European haplotypes or to two Mangalica-specific haplotypes that were absent in all other breeds. In addition, we identified the ancestral mitochondrial D-loop signature present in these 2713 sequences and found that ~ 80% carried the European ancient signatures, ANC-Aside and ANC-Cside or their closely related signatures, while most of the remaining sequences carried a modern Asian signature, ANC-Easia. Mangalica individuals carried the ANC-Aside signature, but not the ANC-Cside or ANC-Easia signatures. CONCLUSIONS: In all the Mangalica individuals, a unique ancient European signature was found in the mitochondrial DNA D-loop region, but they belonged almost exclusively to either certain very abundant European or two Mangalica-specific D-loop haplotypes. This indicates that the present-day Mangalica population in Hungary evolved either by introgression of other European breeds and wild boars or via total isolation after the divergence of European ancient porcine bloodlines.

The effect of LacI autoregulation on the performance of the lactose utilization system in Escherichia coli.

The lactose operon of Escherichia coli is a paradigm system for quantitative understanding of gene regulation in prokaryotes. Yet, none of the many mathematical models built so far to study the dynamics of this system considered the fact that the Lac repressor regulates its own transcription by forming a transcriptional roadblock at the O3 operator site. Here we study the effect of autoregulation on intracellular LacI levels and also show that cAMP-CRP binding does not affect the efficiency of autoregulation. We built a mathematical model to study the role of LacI autoregulation in the lactose utilization system. Previously, it has been argued that negative autoregulation can significantly reduce noise as well as increase the speed of response. We show that the particular molecular mechanism, a transcriptional roadblock, used to achieve self-repression in the lac system does neither. Instead, LacI autoregulation balances two opposing states, one that allows quicker response to smaller pulses of external lactose, and the other that minimizes production costs in the absence of lactose.

Antler development and coupled osteoporosis in the skeleton of red deer Cervus elaphus: expression dynamics for regulatory and effector genes.

Antlers of deer display the fastest and most robust bone development in the animal kingdom. Deposition of the minerals in the cartilage preceding ossification is a specific feature of the developing antler. We have cloned 28 genes which are upregulated in the cartilaginous section (called mineralized cartilage) of the developing ("velvet") antler of red deer stags, compared to their levels in the fetal cartilage. Fifteen of these genes were further characterized by their expression pattern along the tissue zones (i.e., antler mesenchyme, precartilage, cartilage, bone), and by in situ hybridization of the gene activities at the cellular level. Expression dynamics of genes col1A1, col1A2, col3A1, ibsp, mgp, sparc, runx2, and osteocalcin were monitored and compared in the ossified part of the velvet antler and in the skeleton (in ribs and vertebrae). Expression levels of these genes in the ossified part of the velvet antler exceeded the skeletal levels 10-30-fold or more. Gene expression and comparative sequence analyses of cDNAs and the cognate 5' cis-regulatory regions in deer, cattle, and human suggested that the genes runx2 and osx have a master regulatory role. GC-MS metabolite analyses of glucose, phosphate, ethanolamine-phosphate, and hydroxyproline utilizations confirmed the high activity of mineralization genes in governing the flow of the minerals from the skeleton to the antler bone. Gene expression patterns and quantitative metabolite data for the robust bone development in the antler are discussed in an integrated manner. We also discuss the potential implication of our findings on the deer genes in human osteoporosis research.

Identifying novel genes involved in both deer physiological and human pathological osteoporosis.

Osteoporosis attacks 10% of the population worldwide. Humans or even the model animals of the disease cannot recover from porous bone. Regeneration in skeletal elements is the unique feature of our newly investigated osteoporosis model, the red deer (Cervus elaphus) stag. Cyclic physiological osteoporosis is a consequence of the annual antler cycle. This phenomenon raises the possibility to identify genes involved in the regulation of bone mineral density on the basis of comparative genomics between deer and human. We compare gene expression activity of osteoporotic and regenerating rib bone samples versus autumn dwell control in red deer by microarray hybridization. Identified genes were tested on human femoral bone tissue from non-osteoporotic controls and patients affected with age-related osteoporosis. Expression data were evaluated by Principal Components Analysis and Canonical Variates Analysis. Separation of patients into a normal and an affected group based on ten formerly known osteoporosis reference genes was significantly improved by expanding the data with newly identified genes. These genes include IGSF4, FABP3, FABP4, FKBP2, TIMP2, TMSB4X, TRIB, and members of the Wnt signaling. This study supports that extensive comparative genomic analyses, here deer and human, provide a novel approach to identify new targets for human diagnostics and therapy.

Plaque-based competitive hybridization.

The authors have developed a simple, cost-saving experimental design, plaque-based competitive hybridization (PBCH), for genome-wide identification of genes differentially expressed in different tissues. PBCH offers advantages in comparison with other methods used in comparative genomics by combining the principles of differential hybridization with the subtractive hybridization. PBCH is particularly advantageous when libraries with few differences are to be analyzed. The authors demonstrate the use of PBCH by identifying 3 genes, up-regulated in the developing velvet antler of red deer (Cervus elaphus): ApoD, C011A2, and S100a1. The fidelity and sensitivity of PBCH is also shown: 1 specific clone among a library sample of 15,000 can be recognized. Possibilities for further utilizations are discussed.

Gene expression dynamics in deer antler: mesenchymal differentiation toward chondrogenesis.

Annual re-growth of deer antler represents a unique example of complete organ regeneration. Because antler mesenchymal cells retain their embryonic capacity to develop into cartilage or bone, studying antler development provides a natural system to follow gene expression changes during mesenchymal differentiation toward chondrogenic/osteogenic lineage. To identify novel genes involved either in early events of mesenchymal cell specialization or in robust bone development, we have introduced a 3 K heterologous microarray set-up (deer cDNA versus mouse template). Fifteen genes were differentially expressed; genes for housekeeping, regulatory functions (components of different signaling pathways, including FGF, TGFbeta, Wnt), and genes encoding members of the Polycomb group were represented. Expression dynamics for genes are visualized by an expression logo. The expression profile of the gene C21orf70 of unknown function is described along with the effects when over-expressed; furthermore the nuclear localization of the cognate protein is shown. In this report, we demonstrate the particular advantage of the velvet antler model in bone research for: (1) identification of mesenchymal and precartilaginous genes and (2) targeting genes upregulated in robust cartilage development.

Identification of differentially expressed genes in the developing antler of red deer Cervus elaphus.

Understanding the molecular mechanisms underlying bone development is a fundamental and fascinating problem in developmental biology, with significant medical implications. Here, we have identified the expression patterns for 36 genes that were characteristic or dominant in the consecutive cell differentiation zones (mesenchyme, precartilage, cartilage) of the tip section of the developing velvet antler of red deer Cervus elaphus. Two major functional groups of these genes clearly outlined: six genes linked to high metabolic demand and other five to tumor biology. Our study demonstrates the advantages of the antler as a source of mesenchymal markers, for distinguishing precartilage and cartilage by different gene expression patterns and for identifying genes involved in the robust bone development, a striking feature of the growing antler. Putative roles for "antler" genes that encode alpha-tropomyosine (tpm1), transgelin (tagln), annexin 2 (anxa2), phosphatidylethanolamine-binding protein (pebp) and apolipoprotein D (apoD) in intense but still controlled tissue proliferation are discussed.