Abstract. Icelandic sheep are characterized by a great diversity in horn phenotypes. Within their breed, they show a variability in terms of this trait to an extent rarely observed elsewhere. Previously, several genetic variants were published as markers for horn status (in terms of absence or presence of horns, including scurs) and horn traits (e.g., oval horns, horn length and polyceraty). The aim of this study was to genotype, for the first time, five of these genetic variants in Icelandic sheep with different horn phenotypes, as well as to analyze their inheritance. Phenotypic and pedigree data, as well as DNA samples from two Icelandic sheep farms, were used. Genetic variants were genotyped by published PCR-based methods in all samples (n=94) or in subsets. As in other sheep breeds with variable horn status, the inheritance of the presence or absence of horns was shown to be complex in Icelandic sheep, especially when sheep carry anything other than regularly formed horns. The 1.78 kb sized RXFP2 insertion on ovine chromosome 10 previously described to be associated with polledness in several sheep breeds was also found to be present in Icelandic sheep and showed some association but not a perfect segregation with the individuals' horn statuses. Missing associations were especially seen in sheep with scurs and oval horns. Regarding horn shape, there was no agreement with the studied variants described in Chinese breeds having comparable horn traits. However, matching tendencies were seen for the horn size variant that was found in the same study. All sheep with four or more horns carried the already published 4 bp deletion in HOXD1, as previously described for three other sheep breeds. Interestingly, for the first time, the deletion was also detected in phenotypically polled animals originating from multi-horned families. According to the results from animals genotyped simultaneously for the RXFP2 and the HOXD1 variants, polledness in sheep with a genetic disposition for polyceraty seems not to be controlled by the RXFP2 insertion. However, this and all other findings in Icelandic sheep need to be confirmed by analyzing a higher number of well-phenotyped animals.
In domestic goats, the polled intersex syndrome (PIS) refers to XX female-to-male sex reversal associated with the absence of horn growth (polled). The causal variant was previously reported as a 11.7 kb deletion at approximately 129 Mb on chromosome 1 that affects the transcription of both FOXL2 and several long non-coding RNAs. In the meantime the presence of different versions of the PIS deletion was postulated and trials to establish genetic testing with the existing molecular genetic information failed. Therefore, we revisited this variant by long-read whole-genome sequencing of two genetically female (XX) goats, a PIS-affected and a horned control. This revealed the presence of a more complex structural variant consisting of a deletion with a total length of 10 159 bp and an inversely inserted approximately 480 kb-sized duplicated segment of a region located approximately 21 Mb further downstream on chromosome 1 containing two genes, KCNJ15 and ERG. Publicly available short-read whole-genome sequencing data, Sanger sequencing of the breakpoints and FISH using BAC clones corresponding to both involved genome regions confirmed this structural variant. A diagnostic PCR was developed for simultaneous genotyping of carriers for this variant and determination of their genetic sex. We showed that the variant allele was present in all 334 genotyped polled goats of diverse breeds and that all analyzed 15 PIS-affected XX goats were homozygous. Our findings enable for the first time a precise genetic diagnosis for polledness and PIS in goats and add a further genomic feature to the complexity of the PIS phenomenon.
We investigated 4 European domestic shorthair kittens with skin lesions consistent with the dermatosparaxis type of the Ehlers-Danlos syndrome, a connective tissue disorder. The kittens were sired by the same tomcat but were born by 3 different mothers. The kittens had easily torn skin resulting in nonhealing skin wounds. Both clinically and histologically, the skin showed thin epidermis in addition to inflammatory changes. Changes in collagen fibers were visible in electron micrographs. The complete genome of an affected kitten was sequenced. A one base pair duplication leading to a frameshift in the candidate gene ADAMTS2 was identified, p.(Ser235fs*3). All 4 affected cats carried the frameshift duplication in a homozygous state. Genotypes at this variant showed perfect cosegregation with the autosomal recessive Ehlers-Danlos syndrome phenotype in the available family. The mutant allele did not occur in 48 unrelated control cats. ADAMTS2 loss-of-function variants cause autosomal recessive forms of Ehlers-Danlos syndrome in humans, mice, dogs, cattle, and sheep. The available evidence from our investigation together with the functional knowledge on ADAMTS2 in other species allows to classify the identified ADAMTS2 variant as pathogenic and most likely causative variant for the observed Ehlers-Danlos syndrome.
There is a growing concern about the loss of animal genetic resources. The aim of this study was to analyze the genetic diversity and potential peculiarity of the endangered Kosovar sheep breed Balusha. For this purpose, a dataset consisting of medium-density SNP chip genotypes (39,879 SNPs) from 45 Balusha sheep was generated and compared with SNP chip genotypes from 29 individuals of a second Kosovar breed, Bardhoka. Publicly available SNP genotypes from 39 individuals of the relatively closely located sheep breeds Istrian Pramenka and Ruda were additionally included in the analyses. Analysis of heterozygosity, allelic richness and effective population size was used to assess the genetic diversity. Inbreeding was evaluated using two different methods (FIS, FROH). The standardized FST (di) and cross-population extended haplotype homozygosity (XPEHH) methods were used to detect signatures of selection. We observed the lowest heterozygosity (HO = 0.351) and effective population size (Ne5 = 25, Ne50 = 228) for the Balusha breed. The mean allelic richness levels (1.780–1.876) across all analyzed breeds were similar and also comparable with those in worldwide breeds. FROH estimates (0.023–0.077) were highest for the Balusha population, although evidence of decreased inbreeding was observed in FIS results for the Balusha breed. Two Gene Ontology (GO) TERMs were strongly enriched for Balusha, and involved genes belonging to the melanogenesis and T cell receptor signaling pathways, respectively. This could result from selection for the special coat color pattern of Balusha (black head) and resistance to certain infectious diseases. The analyzed diversity parameters highlight the urgency to preserve the local Kosovar Balusha sheep as it is clearly distinguished from other sheep of Southeastern Europe, has the lowest diversity level and may harbor valuable genetic variants, e.g., for resistance to infectious diseases.
Objective To sequence and analyze the whole genome of Japanese encephalitis virus (JEV) strain named 47 which was isolated from patient's cerebrospinal fluid sample in Heilongjiang province in 1950. Methods RNA was extracted from the recovery strain 47 and amplified with self-designed JEV genome sequencing primers. The differentiation analysis for nucleotides and coding amino acids and phylogenetic analysis were performed by the software of DNAStar, Modeltest, and Phylip. Results The whole genome of strain 47 has 10 977 nucleotides. An open reading frame from 95 to 10 391 including 10 296 nucleotides is capable of coding a 3432 amino acid pelyprotein. The nucleotide difference between strain 47 and 5 vaccine strains is 2.4%-4.4%, the amino acid difference between strain 47 and 5 vaccine strains is 0.3 %-1.1%. The best evolution model for the whole genome is GTR + I + G. Based on the phylogenetic analysis, strain 47 belongs to the geuotype Ⅲ JEV Conclusion Strain 47 is highly conserved on whole genome nucleotide and amino acid sequence. And it is belongs to the genotype Ⅲ JEV.
Key words:
Japanese encephalitis virus; Phylogeny; Gene amplification; Sequence analysis
Horns are the most obvious common feature of Bovidae. The naturally occurring absence of horns in these species, also known as polledness, is of surprisingly heterogeneous nature, although they are Mendelian traits. This review compares in detail the molecular differences among the causes of inherited polledness in the domestic ruminant species of cattle, yak, sheep, and goat based on the causal gene variants that have been discovered in recent years. The genetic causes for the lack of horns in small ruminants seem not only to be more complex, e.g., in sheep, breed-specific characteristics are still unexplained, but in goats, there is also the associated disorder of intersexuality—polled intersex syndrome (PIS). In connection with animal welfare and the associated discussion about a legal ban on the dehorning of all farm animals, naturally hornless animals and the causal genetic variants are of increasing research interest in the age of genome editing. However, the low acceptance of genetic engineering in livestock, especially in European societies, limits its use in food-producing animals. Therefore, genotype-based targeted selection of naturally occurring variants is still a widely used method for spreading this desired trait within and across populations, at least in cattle and sheep.
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