Precise identification of individuals and accurate parentage verification is critical to livestock breeding programs, facilitating efficient genetic improvement and management practices. Short tandem repeat (STR)-based genotyping in pigs, current genotyping methods are often limited by inadequate resolution, suboptimal throughput and susceptibility to cross-species amplification. This study addresses these limitations by developing and validating a robust, species-specific 21-plex STR typing system. Integrating 13 core loci recommended by International Society for Animal Genetics/Food and Agriculture Organization with eight additional polymorphic markers identified from high-throughput sequencing, the multiplex assay was optimized for simultaneous amplification using fluorescence-labeled primers and capillary electrophoresis. Comprehensive primer optimization and thermal cycling adjustments established uniform amplification conditions, achieving balanced peak heights and distinct genotyping profiles with a sensitivity threshold of 0.5 ng DNA input per reaction. Species-specificity testing demonstrated no cross-reactivity with sheep, cattle and dogs and only weak amplification for the IGF1 locus in cats without compromising genotyping accuracy. Validation using diverse pig populations confirmed the assay's high discriminatory power and reproducibility. The resulting assay is technically rigorous, scalable and cost-effective, making it suitable for broad application in pig genetic improvement programs, pedigree verification, meat traceability and germplasm conservation.

Palmoplantar keratoderma in humans is a condition defined by an abnormally thickened cornified skin layer on the hands and feet. In animals, the corresponding disease is commonly termed paw pad hyperkeratosis. It can be acquired due to repeated trauma, infections, cancer, or inflammatory dermatoses, or inherited due to pathogenic variants in genes involved in skin development. More than 60 different genes involved in the development of palmoplantar keratoderma have been described. Here, we investigated a female Labrador Retriever showing hyperkeratosis on all four paw pads and most digital pads. Histologically, the stratum corneum was expanded by predominantly orthokeratotic hyperkeratosis with occasional mild parakeratotic areas. DNA of the affected dog was isolated from EDTA-blood and whole genome sequencing was performed. Comparison of the whole genome sequencing data to 1664 unaffected control dogs revealed a private de novo heterozygous missense variant in the GJB6 gene which was not present in the parents. GJB6 encodes connexin 30, a subunit of the desmosome. In humans, pathogenic variants in this gene cause isolated deafness or Clouston syndrome, an autosomal dominant condition that is characterized by alopecia, nail dystrophy, and palmoplantar hyperkeratosis. The paw pad hyperkeratosis phenotype in the investigated dog shows similarities to Clouston syndrome and strongly suggests that the GJB6 missense variant is responsible for its condition. However, our investigation also highlights differences between human and dog that could provide deeper insights into the function of GJB6.

Plumage coloration is an economically important trait in poultry. Although PMEL mutations cause systemic pigmentation defects in vertebrates, their role in localized melanin deposition remains unclear. Wuhua yellow chickens, exhibiting black-spotted (BS) tails on a white background, provide a model system to address this knowledge gap. We integrated whole-genome sequencing of 47 roosters (24 solid white [SW] and 23 BS) with data from 100 black-tailed samples and transcriptomic profiling of follicles. Genome-wide association analysis identified 38 significant SNPs on chromosome 33 within PMEL and related genes, overlapping with selective sweep regions. Transcriptomics revealed 2702 differentially expressed genes, with PMEL significantly upregulated in BS follicles, which was confirmed by quantitative real-time PCR. Gene set enrichment analysis highlighted melanogenesis pathways (GO:0043473; normalized enrichment score = 1.81, false discovery rate = 0.037), with PMEL as a core gene. Kompetitive allele-specific PCR genotyping of the synonymous PMEL variant (chr33:7,093,765) showed a near-perfect genotype-phenotype concordance in males: SW were AA (476/476), BS were predominantly GA (98/115), and black-tailed breeds were GG (107/107). Critically, genotyping of 315 SW hens identified 14 G-allele carriers, confirming cryptic carriers and sex-dimorphic expression. The A allele was absent in diverse populations but present in commercial broilers (A = 0.105) according to the Global Chicken Reference Panel database. These results indicate that the G allele is strongly associated with melanin deposition, with the GA genotype linked to localized spotting and GG to systemic pigmentation. This study provides multi-omics insights into avian pigmentation and offers a kompetitive allele-specific PCR marker for precision breeding.

Large porcine F2 crosses are a valuable resource for discovering QTL and genetic variants for relevant traits. Past studies have been largely limited to SNPs and short insertions and deletions. Structural variants (SVs) are becoming a major area of interest in this respect. Here we present results from a genome‐wide association study with SVs imputed from medium‐density SNP array to the whole genome sequence level that were used to investigate the genetic relationship between important production traits and metabolic enzyme activity in an F2 cross based on the breeds Meishan, Piétrain, and European wild boar. Genetic and phenotypic correlations between the two trait classes were high. We were able to pinpoint common genetic loci to a QTL on SSC7, encompassing numerous large intron deletions in the PRIM2 gene as well as in HMGCLL1, BMP5, TRERF1, COL21A1, LRRC1, and UBR2. The most pronounced genetic associations were observed for the content of NADP‐malate dehydrogenase in the tissue. Hence, we propose that the content and activity of malate dehydrogenase is directly connected to important pig production traits, and we present a comprehensive list of large intronic deletions as promising candidates for causality. The variants were validated in independent pig populations, where the majority of the discovered SVs were present, indicating that they are not only relevant to the breeds investigated here.

Sexual reproduction is a hallmark of most eukaryotes, yet hybridization can lead to alternative reproductive strategies, such as hybridogenesis. During hybridogenesis, found in the European water frog (Pelophylax esculentus) complex, one of the parental genomes is eliminated during gametogenesis, and the other one propagates clonally to gametes. We analyzed diploid hybrid males from populations common for hybrids and Pelophylax lessonae (L-E systems) from the eastern part of their distribution range. All but one hybrid produced spermatocytes and spermatids with Pelophylax ridibundus chromosomes, suggesting premeiotic elimination of P. lessonae genome and endoreplication of the P. ridibundus genome. We also observed spermatocytes with 13 or 26 univalents, indicating alterations in genome elimination and endoreplication. By comparing spermatocytes with germ cell genome composition, we suggest that genome elimination and endoreplication do not occur in adult males. Further, we examined introgressions using mitochondrial and nuclear genetic markers and comparative genomic hybridization. Most hybrids carried P. ridibundus and P. lessonae haplogroups, but five had Pelophylax cf. bedriagae mitochondrial DNA. In one hybrid, we revealed introgression of P. ridibundus chromosomal segments on P. lessonae chromosome. Although L-E systems are generally considered as stable, our results indicate variability in hybrid male gametogenesis, which may be related to introgressive hybridization.

The myostatin protein is a potent negative regulator of skeletal muscle growth encoded by the MSTN gene. MSTN loss‐of‐function variants lead to a particular cattle phenotype characterized by an increase in skeletal muscle mass, known as “double muscling” or “double muscled”. However, most of the MSTN causal variants that have been linked to this phenotype lack experimental validation. This is the case, for example, for the five missense MSTN variants reported to be causal according to the Online Mendelian Inheritance in Animals. RNA splicing plays a major role in regulating gene expression; therefore, exploring the effects of variants on RNA splicing may provide relevant information on their functional impact. Here, we have set up a full‐length gene assay (FLGA) to functionally assess MSTN splicing variants, and we have used it to test the five missense variants plus a well‐described deep intronic splicing variant as a positive control. We also evaluated the performances of SpliceAI and Pangolin, two deep learning‐based splice predictors, to identify potential splicing effects of these six variants. Our FLGA system performed well and showed that none of the missense variants has an effect on splicing, unlike the positive control. For each variant, splicing program predictions were perfectly concordant with the effect observed in the FLGA. We have produced a relevant and powerful assay to analyze MSTN splicing variants in cattle. SpliceAI and Pangolin may be efficiently used to screen large datasets of MSTN variants and sort the best candidates prior to experimental validation using an FLGA.

Intramuscular fat (IMF) content is an important factor for meat quality evaluation, which varies between species, and mainly involves the number and size of intramuscular adipocytes. However, the potential mechanism has not been well elucidated. In this study, the whole transcriptomic data of longissimus dorsi muscle from Laiwu pigs (LW, fatty type) and Duroc×Landrace×Yorkshire pigs (DLY, lean type) were collected and an in-depth analysis was conducted. Through differentially expressed analysis between species, 1899 mRNAs, 10 microRNAs (miRNAs), and 247 circular RNAs were identified. An integrated analysis of differentially expressed mRNAs and miRNAs generated two network modules that potentially regulate IMF formation in LW pigs, one consisted of 13 mRNAs (such as ESR1 and COQ3) and one miRNA gene (ssc-miR-874), and another included 31 mRNAs (such as SCML4 and PDE7B) and three miRNAs (ssc-miR-125a, ssc-miR-1343, and ssc-miR-204). In addition, a competitive endogenous RNA network including 13 circular RNAs, three miRNAs, and 31 genes was also delineated, which is probably involved in differential regulatory patterns of IMF deposition between LW and DLY pigs. The function of the SCML4 gene was verified with the 3T3-L1 cell line invitro, and, when SCML4 was decreased, the expression of key proteins associated with fat formation (PPARγ and FABP4) was significantly inhibited. In summary, the study provided the novel insight of a competitive endogenous RNA regulatory network for IMF content in pig, and our results indicated that SCML4 is likely to be a regulatory gene promoting IMF formation.