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The Enrichment of Specific Hair Follicle-Associated Cell Populations in Plucked Hairs Offers an Opportunity to Study Gene Expression Underlying Hair Traits
Gene expression differences can assist in characterizing important underlying genetic mechanisms between different phenotypic traits. However, when population-dense tissues are studied, the signals from scarce populations are diluted. Therefore, appropriately choosing a sample collection method that enriches a particular type of effector cells might yield more specific results. To address this issue, we performed a polyA-selected RNA-seq experiment of domestic horse (Equus ferus caballus) plucked-hair samples and skin biopsies. Then, we layered the horse gene abundance results against cell type-specific marker genes generated from a scRNA-seq supported with spatial mapping of laboratory mouse (Mus musculus) skin to identify the captured populations. The hair-plucking and skin-biopsy sample-collection methods yielded comparable quality and quantity of RNA-seq results. Keratin-related genes, such as KRT84 and KRT75, were among the genes that showed higher abundance in plucked hairs, while genes involved in cellular processes and enzymatic activities, such as MGST1, had higher abundance in skin biopsies. We found an enrichment of hair-follicle keratinocytes in plucked hairs, but detected an enrichment of other populations, including epidermis keratinocytes, in skin biopsies. In mammalian models, biopsies are often the method of choice for a plethora of gene expression studies and to our knowledge, this is a novel study that compares the cell-type enrichment between the non-invasive hair-plucking and the invasive skin-biopsy sample-collection methods. Here, we show that the non-invasive and ethically uncontroversial plucked-hair method is recommended depending on the research question. In conclusion, our study will allow downstream -omics approaches to better understand integumentary conditions in both health and disease in horses as well as other mammals.
Charting the equine miRNA landscape: An integrated pipeline and browser for annotating, quantifying, and visualizing expression
MicroRNAs (miRNAs) are essential regulators of gene expression, yet few comprehensive databases exist for miRNA expression in non-model species, limiting our ability to characterize their roles in gene regulation, development, and disease. Similarly, isomiRs - length and sequence isoforms of canonical miRNAs with potentially altered regulatory targets and functions - have received even less attention in non-model species, including the horse, leaving a critical gap in our understanding of their biological significance. To address these challenges, we developed an open-source, containerized pipeline for identifying and quantifying miRNAs and isomiRs (FARmiR: Framework for Analysis and Refinement of miRNAs), and an associated interactive browser (AIMEE: Animal IsomiR and MiRNA Expression Explorer). AIMEE was developed to make miRNA expression data more accessible and user-friendly, a feature often lacking from other expression atlases. These tools were developed using equine data but can be readily extended to other species. Using these tools, we aggregated 461 small RNA-seq datasets, spanning 61 distinct tissues, integrating data from public repositories, an American Quarter Horse cohort, and the Functional Annotation of ANimal Genome (FAANG) consortium Thoroughbred samples, predicting 5,781 miRNAs and isomiRs. This work represents the largest systematically curated atlas of equine miRNA expression to date, providing a valuable resource that will enhance our understanding of miRNA and isomiR functions in tissue-specific regulation and ultimately improve biomarker discovery, functional genomics, and precision veterinary medicine.
Studies of mitochondrial DNA D-loop sequence variation may support the Polish Primitive Horse (Konik) conservation programme
The protection and balanced development of all existing maternal lines is one of the primary goals of the Polish Primitive Horse (PPH or Konik) conservation programme. However, previous studies have indica- ted that managing PPH conservative breeding may encounter challenges because of the numerous existing pedigree errors. We therefore attempted to check whether mtDNA markers may prove useful in correcting PPH pedigrees and improving the breed conservation programme. A 510 bp mtDNA D-loop fragment was sequenced for 396 samples representing all sixteen officially recognised maternal lines. These samples were derived from different time points in the PPH breeding history. Our analysis confirmed the presence of nineteen mtDNA haplotypes. A comparison of the molecular and pedigree data showed that the frequency of particular haplotypes was highly uneven. Although for the majority of the maternal lines we were able to identify a potential 'founder haplotype', only four of them (Dzina I, Popielica, Geneza and Bona) turned out to be 'genetically pure'. Our study confirmed that an mtDNA analysis is a useful method for assessing PPH maternal genetic diversity and illustrating the breed's history. Our findings suggest that PPH conservation programmes would benefit from revising the official pedigrees using molecular data alongside breeding records.