Trio-binning of a hinny refines the comparative organization of the horse and donkey X chromosomes and reveals novel species-specific features.

We generated single haplotype assemblies from a hinny hybrid which significantly improved the gapless contiguity for horse and donkey autosomal genomes and the X chromosomes. We added over 15 Mb of missing sequence to both X chromosomes, 60 Mb to donkey autosomes and corrected numerous errors in donkey and some in horse reference genomes. We resolved functionally important X-linked repeats: the DXZ4 macrosatellite and ampliconic Equine Testis Specific Transcript Y7 (ETSTY7). We pinpointed the location of the pseudoautosomal boundaries (PAB) and determined the size of the horse (1.8 Mb) and donkey (1.88 Mb) pseudoautosomal regions (PARs). We discovered distinct differences in horse and donkey PABs: a testis-expressed gene, XKR3Y, spans horse PAB with exons1-2 located in Y and exon3 in the X-Y PAR, whereas the donkey XKR3Y is Y-specific. DXZ4 had a similar ~ 8 kb monomer in both species with 10 copies in horse and 20 in donkey. We assigned hundreds of copies of ETSTY7, a sequence horizontally transferred from Parascaris and massively amplified in equids, to horse and donkey X chromosomes and three autosomes. The findings and products contribute to molecular studies of equid biology and advance research on X-linked conditions, sex chromosome regulation and evolution in equids.

Ancient segmentally duplicated LCORL retrocopies in equids.

LINE-1 is an active transposable element encoding proteins capable of inserting host gene retrocopies, resulting in retro-copy number variants (retroCNVs) between individuals. Here, we performed retroCNV discovery using 86 equids and identified 437 retrocopy insertions. Only 5 retroCNVs were shared between horses and other equids, indicating that the majority of retroCNVs inserted after the species diverged. A large number (17-35 copies) of segmentally duplicated Ligand Dependent Nuclear Receptor Corepressor Like (LCORL) retrocopies were present in all equids but absent from other extant perissodactyls. The majority of LCORL transcripts in horses and donkeys originate from the retrocopies. The initial LCORL retrotransposition occurred 18 million years ago (17-19 95% CI), which is coincident with the increase in body size, reduction in digit number, and changes in dentition that characterized equid evolution. Evolutionary conservation of the LCORL retrocopy segmental amplification in the Equidae family, high expression levels and the ancient timeline for LCORL retrotransposition support a functional role for this structural variant.

Case Report: Disorder of Sexual Development in a Chinese Crested Dog With XX/XY Leukocyte Chimerism and Mixed Cell Testicular Tumors.

A 10-year-old intact female Chinese Crested dog was presented for evaluation and further diagnostics due to persistent symptoms of vulvar swelling, vaginal discharge, and an 8-year history of acyclicity. At presentation, generalized hyperpigmentation and truncal alopecia were identified, with no aberrations of the female phenotype. Vaginal cytology confirmed the influence of estrogen at multiple veterinary visits, and hormonal screening of progesterone and anti-Mullerian hormone indicated gonadal presence. Based on findings from abdominal laparotomy and gonadectomy, the tissue was submitted for histopathology. Histopathologic evaluation identified the gonads to be abnormal testes containing multiple Sertoli and interstitial (Leydig) cell tumors. The histopathologic diagnosis of testes and concurrent normal external female phenotype in the patient lead to a diagnosis of a disorder of sexual development (DSD). Karyotype evaluation by conventional and molecular analysis revealed a two cell line chimeric pattern of 78,XX (80%) and 78,XY (20%) among blood leukocytes, as well as a positive PCR test for the Y-linked SRY gene. Cytogenetic analysis of skin fibroblasts revealed the presence of 78,XX cells exclusively, and PCR tests for the Y-linked SRY gene were negative in the hair and skin samples. These results are consistent with an XX/XY blood chimerism. This is one of the few case reports of a canine with the diagnosis of leukocyte chimerism with normal female phenotypic external genitalia. This case illustrates a distinct presentation for hormonally active Sertoli cell tumorigenesis and demonstrates surgery as a curative treatment option for clinically affected patients.

An 8.22 Mb Assembly and Annotation of the Alpaca (Vicugna pacos) Y Chromosome.

The unique evolutionary dynamics and complex structure make the Y chromosome the most diverse and least understood region in the mammalian genome, despite its undisputable role in sex determination, development, and male fertility. Here we present the first contig-level annotated draft assembly for the alpaca (Vicugna pacos) Y chromosome based on hybrid assembly of short- and long-read sequence data of flow-sorted Y. The latter was also used for cDNA selection providing Y-enriched testis transcriptome for annotation. The final assembly of 8.22 Mb comprised 4.5 Mb of male specific Y (MSY) and 3.7 Mb of the pseudoautosomal region. In MSY, we annotated 15 X-degenerate genes and two novel transcripts, but no transposed sequences. Two MSY genes, HSFY and RBMY, are multicopy. The pseudoautosomal boundary is located between SHROOM2 and HSFY. Comparative analysis shows that the small and cytogenetically distinct alpaca Y shares most of MSY sequences with the larger dromedary and Bactrian camel Y chromosomes. Most of alpaca X-degenerate genes are also shared with other mammalian MSYs, though WWC3Y is Y-specific only in alpaca/camels and the horse. The partial alpaca Y assembly is a starting point for further expansion and will have applications in the study of camelid populations and male biology.

Two Novel Cases of Autosomal Translocations in the Horse: Warmblood Family Segregating t(4;30) and a Cloned Arabian with a de novo t(12;25).

We report 2 novel autosomal translocations in the horse. In Case 1, a breeding stallion with a balanced t(4p;30) had produced normal foals and those with congenital abnormalities. Of his 9 phenotypically normal offspring, 4 had normal karyotypes, 4 had balanced t(4p;30), and 1 carried an unbalanced translocation with tertiary trisomy of 4p. We argue that unbalanced forms of t(4p;30) are more tolerated and result in viable congenital abnormalities, without causing embryonic death like all other known equine autosomal translocations. In Case 2, two stallions produced by somatic cell nuclear transfer from the same donor were karyotyped because of fertility issues. A balanced translocation t(12q;25) was found in one, but not in the other clone. The findings underscore the importance of routine cytogenetic screening of breeding animals and animals produced by assisted reproductive technologies. These cases will contribute to molecular studies of translocation breakpoints and their genetic consequences in the horse.

Characterization of A Homozygous Deletion of Steroid Hormone Biosynthesis Genes in Horse Chromosome 29 as A Risk Factor for Disorders of Sex Development and Reproduction.

Disorders of sex development (DSD) and reproduction are not uncommon among horses, though knowledge about their molecular causes is sparse. Here we characterized a ~200 kb homozygous deletion in chromosome 29 at 29.7-29.9 Mb. The region contains AKR1C genes which function as ketosteroid reductases in steroid hormone biosynthesis, including androgens and estrogens. Mutations in AKR1C genes are associated with human DSDs. Deletion boundaries, sequence properties and gene content were studied by PCR and whole genome sequencing of select deletion homozygotes and control animals. Deletion analysis by PCR in 940 horses, including 622 with DSDs and reproductive problems and 318 phenotypically normal controls, detected 67 deletion homozygotes of which 79% were developmentally or reproductively abnormal. Altogether, 8-9% of all abnormal horses were homozygous for the deletion, with the highest incidence (9.4%) among cryptorchids. The deletion was found in ~4% of our phenotypically normal cohort, ~1% of global warmblood horses and ponies, and ~7% of draught breeds of general horse population as retrieved from published data. Based on the abnormal phenotype of the carriers, the functionally relevant gene content, and the low incidence in general population, we consider the deletion in chromosome 29 as a risk factor for equine DSDs and reproductive disorders.

The Indian cobra reference genome and transcriptome enables comprehensive identification of venom toxins.

Snakebite envenoming is a serious and neglected tropical disease that kills ~100,000 people annually. High-quality, genome-enabled comprehensive characterization of toxin genes will facilitate development of effective humanized recombinant antivenom. We report a de novo near-chromosomal genome assembly of Naja naja, the Indian cobra, a highly venomous, medically important snake. Our assembly has a scaffold N50 of 223.35 Mb, with 19 scaffolds containing 95% of the genome. Of the 23,248 predicted protein-coding genes, 12,346 venom-gland-expressed genes constitute the 'venom-ome' and this included 139 genes from 33 toxin families. Among the 139 toxin genes were 19 'venom-ome-specific toxins' (VSTs) that showed venom-gland-specific expression, and these probably encode the minimal core venom effector proteins. Synthetic venom reconstituted through recombinant VST expression will aid in the rapid development of safe and effective synthetic antivenom. Additionally, our genome could serve as a reference for snake genomes, support evolutionary studies and enable venom-driven drug discovery.

Chromosome-Level Alpaca Reference Genome VicPac3.1 Improves Genomic Insight Into the Biology of New World Camelids.

The development of high-quality chromosomally assigned reference genomes constitutes a key feature for understanding genome architecture of a species and is critical for the discovery of the genetic blueprints of traits of biological significance. South American camelids serve people in extreme environments and are important fiber and companion animals worldwide. Despite this, the alpaca reference genome lags far behind those available for other domestic species. Here we produced a chromosome-level improved reference assembly for the alpaca genome using the DNA of the same female Huacaya alpaca as in previous assemblies. We generated 190X Illumina short-read, 8X Pacific Biosciences long-read and 60X Dovetail Chicago® chromatin interaction scaffolding data for the assembly, used testis and skin RNAseq data for annotation, and cytogenetic map data for chromosomal assignments. The new assembly VicPac3.1 contains 90% of the alpaca genome in just 103 scaffolds and 76% of all scaffolds are mapped to the 36 pairs of the alpaca autosomes and the X chromosome. Preliminary annotation of the assembly predicted 22,462 coding genes and 29,337 isoforms. Comparative analysis of selected regions of the alpaca genome, such as the major histocompatibility complex (MHC), the region involved in the Minute Chromosome Syndrome (MCS) and candidate genes for high-altitude adaptations, reveal unique features of the alpaca genome. The alpaca reference genome VicPac3.1 presents a significant improvement in completeness, contiguity and accuracy over VicPac2 and is an important tool for the advancement of genomics research in all New World camelids.

Range-Wide Snow Leopard Phylogeography Supports Three Subspecies.

The snow leopard, Panthera uncia, is an elusive high-altitude specialist that inhabits vast, inaccessible habitat across Asia. We conducted the first range-wide genetic assessment of snow leopards based on noninvasive scat surveys. Thirty-three microsatellites were genotyped and a total of 683 bp of mitochondrial DNA sequenced in 70 individuals. Snow leopards exhibited low genetic diversity at microsatellites (AN = 5.8, HO = 0.433, HE = 0.568), virtually no mtDNA variation, and underwent a bottleneck in the Holocene (∼8000 years ago) coinciding with increased temperatures, precipitation, and upward treeline shift in the Tibetan Plateau. Multiple analyses supported 3 primary genetic clusters: (1) Northern (the Altai region), (2) Central (core Himalaya and Tibetan Plateau), and (3) Western (Tian Shan, Pamir, trans-Himalaya regions). Accordingly, we recognize 3 subspecies, Panthera uncia irbis (Northern group), Panthera uncia uncia (Western group), and Panthera uncia uncioides (Central group) based upon genetic distinctness, low levels of admixture, unambiguous population assignment, and geographic separation. The patterns of variation were consistent with desert-basin "barrier effects" of the Gobi isolating the northern subspecies (Mongolia), and the trans-Himalaya dividing the central (Qinghai, Tibet, Bhutan, and Nepal) and western subspecies (India, Pakistan, Tajikistan, and Kyrgyzstan). Hierarchical Bayesian clustering analysis revealed additional subdivision into a minimum of 6 proposed management units: western Mongolia, southern Mongolia, Tian Shan, Pamir-Himalaya, Tibet-Himalaya, and Qinghai, with spatial autocorrelation suggesting potential connectivity by dispersing individuals up to ∼400 km. We provide a foundation for global conservation of snow leopard subspecies, and set the stage for in-depth landscape genetics and genomic studies.