Agouti Signaling Protein Gene Research Articles

VARIATION IN THE ASIP AND DUN GENES RESPONSIBLE FOR COAT COLOUR IN BOSNIAN MOUNTAIN HORSE

Within the Bosnian Mountain Horse Registry (BMH), some specific coat colours are permitted. Accurate determination of coat colours can be challenging as there are variations in coat colour shades and several dun dilution variants occur. The previously found single nucleotide polymorphisms (SNPs) within two coat colour loci, T-box 3 (TBX3) and Agouti Signalling Protein (ASIP), were genotyped in 313 BMH individuals. The obtained genotypes were then compared to the predicted phenotypes by using the observed coat colour types from the International Association of Bosnian Mountain Horse Breeders (IABMHB) database. It was found that the dark bay and black coat colours were the most representative coat colours both phenotypically and genotypically. The frequency of the dominant Dun (D) dilution allele is (0.09), and this frequency is higher than the previously predicted frequency recorded in the available BMH registers, as also demonstrated. Among the identified alleles, there was a discrepancy or inconsistency between the predicted coat colour based on genotypes and the observed coat colour in 73 horses (23%). The most frequent error concerned the misclassification of horses with genotypes aa and Aa at the ASIP gene, non-dun1/non-dun1 (nd1/nd1) and non-dun2/non-dun1 (nd1/nd2) at the TBX3 gene, which can be associated with the occurrence of slight dilution phenotypes in these individuals. In contrast to the Konik and Hucul breeds, no homozygosity of the D allele was found in the BMH. The D allele can be easily overlooked or not recognised in different phenotypic groups, such as dark bay and black horses. Therefore, the hypothesis that Dun dilution effects itself is not as strongly epistatic in the BMH as described in other horse breeds. This could be the result of an additional genetic modifier suppressing the phenotypic effect of the D allele. It also suggests that there has been a persistent selection pressure in favour of dark base colours, which may have contributed to the observed differences in BMH. Finally, a significant proportion (35%) of BMH individuals with genetically black coat colour were officially classified as dark bay. We hypothesise that the difficulty in visually distinguishing these two phenotypes is due to an independent locus upstream of the ASIP gene. This locus was recently identified as a factor that darkens the typical pigmentation in dark bay horses. The results of the study confirm the importance of molecular testing in accurately determining the coat colour of horses. This would help to avoid errors in coat colour descriptions in official breeding records and provide valuable information for selective breeding programmes aimed at producing specific and desired coat colours.

The Genetic Basis of Melanism in Abert's Squirrel (Sciurus aberti).

Melanism is widespread in different taxa and has been hypothesized to provide adaptive benefits in certain environments. Melanism is typically caused by mutations in one of two regulatory genes: the Melanocortin 1 Receptor (MC1R) or the Agouti Signaling Protein (ASIP). Melanism has repeatedly evolved among tree squirrels and their relatives (tribe Sciurini) in at least 12 different species based on our review of the literature. The causal mutations for melanism have been characterized in two species so far. This study examines Abert's Squirrel (Sciurus aberti), which has a melanistic morph whose genetic basis has not yet been established. We sequenced the MC1R and ASIP genes for five wild-type and seven melanistic S. aberti individuals to search for melanism-associated mutations. A novel single base pair mutation in the ASIP gene, unique to S. aberti, was found to be associated with melanism in the species, indicating that melanism in S. aberti evolved independently from other tree squirrels and thus represents an example of convergent evolution. The independent evolution of melanism in this species suggests that there is an adaptive advantage to the melanistic phenotype. The geographic range and habitat of S. aberti suggest possible benefits associated with thermoregulation, post-forest-fire camouflage, or other untested hypotheses.

Canine coat color E locus updates: Identification of a new MC1R variant causing 'sable' coat color in English Cocker Spaniels and a proposed update to the E locus dominance hierarchy.

The coat color phenotype 'sable' occurs in the English Cocker Spaniel dog breed. It closely resembles other canine color patterns known as domino/grizzle/pied (eA allele) and grizzle/domino (eG allele) determined by variants in the melanocortin 1 receptor gene (MC1R; 'extension' or E locus), a key multi-allele regulator of coat color. We examined genetic variation in MC1R, and found one new non-synonymous variant, c.250G>A (p.(Asp84Asn)), consistently associated with the English Cocker Spaniel 'sable' phenotype. We propose calling this newly identified allele eH and further show that the eA , eH and eG (previously known as EG ) alleles associate with similar phenotypes in dogs impacting genotypes regulated by beta-defensin 103 gene (CBD103; K locus) and agouti signaling protein gene (ASIP; A locus) in the absence of the EM and E alleles. This suggests that all three alleles are putative reduced-function variants of the MC1R gene. We propose the revised and updated E locus dominance hierarchy to be EM > E > eA /eH /eG > e1-3 .

Differential expression of ASIP transcripts reveals genetic mechanism underpinning black-tail independence from body plumage in yellow-bodied chickens.

The genetic foundation of chicken body plumage color has been extensively studied. However, little attention has been paid to the inheritance patterns and molecular mechanisms underlying the formation of distal feather colors (tail and wingtip). Differences in these colors are common; for example, the Chinese Huiyang Beard chicken has black tail feathers, but yellow body plumage. Here, the hybrid offspring of Huiyang Beard and White Leghorn chickens were used to study the inheritance patterns of tail-feather color. The expression levels of pigment genes in differently colored feather follicles were analyzed using quantitative real-time PCR. The results showed that genetic regulation of tail-feather color was independent of body-plumage color. The Dominant White locus inhibited eumelanin synthesis in tail feathers without affecting the formation of yellow body plumage, whereas the Silver locus had the opposite effect. The expression of agouti signaling protein (ASIP) gene class 1 transcripts was significantly lower in black tail-feather follicles than in yellow body follicles, whereas tyrosinase-related protein 1 (TYRP1) gene expression was significantly higher in black tail feathers. These differentially expressed genes were confirmed to exert an effect on eumelanin and pheomelanin formation in feathers, thus influencing the regulation of chicken tail-feather color. In conclusion, this study lays the foundation for further research on the genetic mechanisms of regional differences in feather color, contributing to a better understanding of plumage pigmentation in chickens.

OR07-01 Aberrant Expression Of Agouti Signaling Protein (ASIP) As A Cause of Monogenic Severe Childhood Obesity

Abstract Disclosure: K. Landgraf: None. E.T. Kempf: None. R. Stein: None. M. Hanschkow: None. Y. Tseng: None. T. Schöneberg: None. E. Zeggini: None. M. Blüher: None. A. Körner: None. Here we identified and characterized a heterozygous tandem duplication at the ASIP (agouti-signaling protein) gene locus causing ubiquitous, ectopic ASIP expression in a female index patient with extreme childhood obesity.From an adipose tissue sample of the patient, we observed pronounced and sustained differentiation potential of stroma-vascular fraction (SVF) cells into adipocytes in the patient compared to normal control cells. We further found reduced mitochondrial maximum respiration, spare capacity, and proton leak in the patient’s SVF cells as proxies for reduced energy expenditure. By transcriptome screen, we identified overexpression of one single gene, ASIP. We found and functionally confirmed a chromosomal rearrangement at the ASIP locus, that places ASIP under control of the ubiquitously active itchy E3 ubiquitin protein ligase (ITCH) promoter. The patient's phenotype of early-onset obesity, overgrowth, red hair, and hyperinsulinemia is concordant with that of mutant, so called agouti mice ubiquitously expressing the homolog nonagouti. Physiologically, ASIP antagonises the melanocortin 1 receptor (MC1R) in the skin. Due to its homology to the hypothalamic regulator of eating behaviour agouti-related protein (AGRP), we hypothesize that ectopic ASIP similarly influences eating behaviour via repressing melanocyte-stimulating hormone-mediated activation of MC4R and that the mutation might therefore represent a novel monogenic cause for severe childhood obesity. We show that the tandem duplication results in the ectopic generation of ASIP in patient-derived native stroma-vascular fraction (SVF) cells, adipocytes and blood cells as well as in induced pluripotent stem cells (iPSCs) generated from SVF cells of the patient, thus indicating ubiquitous ectopic ASIP expression. This overexpression persisted after differentiation of iPSCs into the three germ layers, mesoderm, ectoderm and endoderm, and after differentiation into hypothalamic-like neurons. Furthermore, we observed that ASIP causes a reduction of MC4R activity in vitro supporting the hypothesis that the obesity phenotype of our patient is caused by ectopic ASIP acting at the MC4R in the hypothalamus. Since the type of mutation escapes standard genetic screening algorithms, we rescreened the Leipzig childhood obesity cohort of 1745 patients and identified four additional patients with the identical mutation, ectopic ASIP expression and a similar phenotype. Taken together, our data indicate that ubiquitous ectopic ASIP expression is likely a monogenic cause of human obesity and might be potentially treatable with melanocortin 4 receptor agonists. Presentation: Thursday, June 15, 2023

A genome-wide association study of coat color in Chinese Rex rabbits.

Coat color is an important phenotypic characteristic of the domestic rabbit (Oryctolagus cuniculus) and has specific economic importance in the Rex rabbit industry. Coat color varies considerably among different populations of rabbits, and several causal genes for this variation have been thoroughly studied. Nevertheless, the candidate genes affecting coat color variation in Chinese Rex rabbits remained to be investigated. In this study, we collected blood samples from 250 Chinese Rex rabbits with six different coat colors. We performed genome sequencing using a restriction site-associated DNA sequencing approach. A total of 91,546 single nucleotide polymorphisms (SNPs), evenly distributed among 21 autosomes, were identified. Genome-wide association studies (GWAS) were performed using a mixed linear model, in which the individual polygenic effect was fitted as a random effect. We detected a total of 24 significant SNPs that were located within a genomic region on chromosome 4 (OCU4). After re-fitting the most significant SNP (OCU4:13,434,448, p = 1.31e-12) as a covariate, another near-significant SNP (OCU4:11,344,946, p = 7.03e-07) was still present. Hence, we conclude that the 2.1-Mb genomic region located between these two significant SNPs is significantly associated with coat color in Chinese Rex rabbits. The well-studied coat-color-associated agouti signaling protein (ASIP) gene is located within this region. Furthermore, low genetic differentiation was also observed among the six coat color varieties. In conclusion, our results confirmed that ASIP is a putative causal gene affecting coat color variation in Chinese Rex rabbits.

A unique single nucleotide polymorphism in Agouti Signalling Protein (ASIP) gene changes coat colour of Sri Lankan leopard (Panthera pardus kotiya) to dark black.

The Sri Lankan leopard (Panthera pardus kotiya) is an endangered subspecies restricted to isolated and fragmented populations in Sri Lanka. Among them, melanistic leopards have been recorded on a few occasions. Literature suggests the evolution of melanism several times in the Felidae family, with three species having distinct mutations. Nevertheless, the mutations or other variations in the remaining species, including Sri Lankan melanistic leopard, are unknown. We used reference-based assembled nuclear genomes of Sri Lankan wild type and melanistic leopards and de novo assembled mitogenomes of the same to investigate the genetic basis, adaptive significance, and evolutionary history of the Sri Lankan melanistic leopard. Interestingly, we identified a single nucleotide polymorphism in exon-4 Sri Lankan melanistic leopard, which may completely ablate Agouti Signalling Protein (ASIP) function. The wild type leopards in Sri Lanka did not carry this mutation, suggesting the cause for the occurrence of melanistic leopords in the population. Comparative analysis of existing genomic data in the literature suggests it as a P. p. kotiya specific mutation and a novel mutation in the ASIP-gene of the Felidae family, contributing to naturally occurring colour polymorphism. Our data suggested the coalescence time of Sri Lankan leopards at ~0.5 million years, sisters to the Panthera pardus lineage. The genetic diversity was low in Sri Lankan leopards. Further, the P. p. kotiya melanistic leopard is a different morphotype of the P. p. kotiya wildtype leopard resulting from the mutation in the ASIP-gene. The ability of black leopards to camouflage, along with the likelihood of recurrence and transfer to future generations, suggests that this rare mutation could be environment-adaptable.

A structural variant at ASIP associated with the darkness of hair coat is found in multiple zebu cattle populations.

A structural variant (SV) in the agouti signaling protein gene (ASIP), namely ASIP-SV1, has been found to strongly correlate with the darkness of hair coat in specific regions of the body of bulls from the zebu (Bos indicus) Nellore breed. Here we visually analyzed the whole-genome sequence of zebu and taurine (Bos taurus) cattle to elucidate the extent of spread of ASIP-SV1 in different cattle populations. Of 216 sequences analyzed, 63 zebu (45.9%) and five taurine (6.3%) animals had at least one copy of ASIP-SV1. Four of the taurine animals presenting the SV were Romagnola cattle, a breed with history of zebu introgression. The remaining taurine animal was a Simmental, a breed frequently used in crossbreeding. These data provide evidence that ASIP-SV1 is commonly found in zebu populations, in addition to taurine animals with zebu admixture.

47 Investigating links between pigmentation genes and opioid effectiveness in horses

The pleiotropic effects of the melanocortin system show promise in overcoming limitations associated with large variations in opioid analgesic effectiveness observed in equine practice. Of particular interest is the melanocortin-1-receptor gene (MC1R), which influences melanin type through its epistatic interaction with the agouti signaling protein gene (ASIP), dictating the production of black or red pigment. MC1R has previously been associated with opioid effectiveness in other species; however, evidence of similar relationships in horses is lacking. As such, this study investigated if associations between opioid response and pigmentation genes were also observable in horses. Nociceptive response data were opportunistically collected during the performance of spinal fluid centesis (SFC), noting the pain response of individual horses after sedation. A target dosage range of 0.01–0.02 mg/kg body weight intravenously was used in the administration of the opioid hydromorphone, the primary drug of interest in this study. Genomic regions responsible for the bay (ASIP) and chestnut (MC1R) coat colors were extracted from genome sequence data, and opioid analgesic effectiveness was scored (1–3) based on pain response for each horse (n = 48). Haplotype and SNP association analyseswere performed using the SNPassoc (testing multiple models) and haplo.stats packages and generalized linear models within the statistical software R. Age, breed, sex, and dosage rates were recorded as covariates. Breeds with less than 2 horses were excluded from analyses. Six single-nucleotide polymorphisms (SNPs) within ASIP and one within MC1R were identified as significantly (P < 0.05) associated with opioid analgesic effectiveness during SFC. Haplotype analysis of the significant ASIP SNPs revealed that the most common ASIP haplotype was associated with lower opioid analgesic effectiveness, both in isolation and when looked at in conjunction with MC1R. The recessive MC1Re/e mutation resulting in the production of red pigment only was also significantly associated with decreased opioid analgesic effectiveness. The current study provides promising evidence for important links between pigmentation genes and opioid effectiveness in horses. However, given the complexity of opioid metabolism and the opportunistic nature of the current study, additional investigations are needed if precision medicine approaches are to become a reality in equine veterinary medicine.

Aberrant expression of agouti signaling protein (ASIP) as a cause of monogenic severe childhood obesity

Here we report a heterozygous tandem duplication at the ASIP (agouti signaling protein) gene locus causing ubiquitous, ectopic ASIP expression in a female patient with extreme childhood obesity. The mutation places ASIP under control of the ubiquitously active itchy E3 ubiquitin protein ligase promoter, driving the generation of ASIP in patient-derived native and induced pluripotent stem cells for all germ layers and hypothalamic-like neurons. The patient’s phenotype of early-onset obesity, overgrowth, red hair and hyperinsulinemia is concordant with that of mutant mice ubiquitously expressing the homolog nonagouti. ASIP represses melanocyte-stimulating hormone-mediated activation as a melanocortin receptor antagonist, which might affect eating behavior, energy expenditure, adipocyte differentiation and pigmentation, as observed in the index patient. As the type of mutation escapes standard genetic screening algorithms, we rescreened the Leipzig Childhood Obesity cohort of 1,745 patients and identified four additional patients with the identical mutation, ectopic ASIP expression and a similar phenotype. Taken together, our data indicate that ubiquitous ectopic ASIP expression is likely a monogenic cause of human obesity.

Genomic Analyses Reveal Association of ASIP with a Recurrently evolving Adaptive Color Pattern in Frogs.

Traits shared among distantly related lineages are indicators of common evolutionary constraints, at the ecological, physiological, or molecular level. Here, we show that the vertebral stripe, a cryptic color pattern, has evolved hundreds of times in the evolutionary history of anurans (frogs and toads) and is favored in terrestrial habitats. Using a genome-wide association study, we demonstrate that variation near the Agouti signaling protein gene (ASIP) is responsible for the different vertebral stripe phenotypes in the African grass frog Ptychadena robeensis. RNAseq and real-time quantitative PCR revealed that differential expression of the gene and an adjacent long non-coding RNA is linked to patterning in this species. Surprisingly, and although the stripe phenotypes are shared with closely related species, we found that the P. robeensis alleles are private to the species and unlikely to evolve under long-term balancing selection, thus indicating that the vertebral stripe phenotypes result from parallel evolution within the group. Our findings demonstrate that this cryptic color pattern evolved rapidly and recurrently in terrestrial anurans, and therefore constitutes an ideal system to study repeated evolution.

The Knockout of the ASIP Gene Altered the Lipid Composition in Bovine Mammary Epithelial Cells via the Expression of Genes in the Lipid Metabolism Pathway.

Simple SummaryAccording to the FDA, a litre of milk contains about 33 g of total lipids, 95% of which are triacylglycerols made up of varying lengths and saturation of fatty acids. The increase in free fatty acids in milk may lead to changes in the flavour of milk and milk products. Bovine mammary epithelial cells (bMECs) function to synthesize and secrete milk and are good in vitro models for cells in milk quality research. In this research, we report for the first time the effect of the ASIP gene knockout in bovine mammary epithelial cells using CRISPR/Cas9 technology. We found that ASIP knockout could down-regulate the expression of PPARγ, FASN, and SCD, thus affecting the saturation of fatty acids in milk and upregulating the expression of FABP4, ELOVL6, and ACSL1, increasing the synthesis of medium-long chain fatty acids. These results may allow the selection of potential targets in future molecular breeding efforts for dairy cows.Agouti signalling protein (ASIP) is a coat colour-related protein and also is a protein-related to lipid metabolism, which had first been found in agoutis. According to our previous study, ASIP is a candidate gene that affects the lipid metabolism in bovine adipocytes. However, its effect on milk lipid has not been reported yet. This study focused on the effect of the ASIP gene on the lipid metabolism of mammary epithelial cells in cattle. The ASIP gene was knocked out in bMECs by using CRISPR/Cas9 technology. The result of transcriptome sequencing showed that the differentially expressed genes associated with lipid metabolism were mainly enriched in the fatty acids metabolism pathways. Furthermore, the contents of intracellular triglycerides were significantly increased (p < 0.05), and cholesterol tended to rise (p > 0.05) in bMECs with the knockout of the ASIP gene. Fatty acid assays showed a significant alteration in medium and long-chain fatty acid content. Saturated and polyunsaturated fatty acids were significantly up-regulated (p < 0.05), and monounsaturated fatty acids were significantly decreased in the ASIP knockout bMECs (p < 0.05). The Q-PCR analysis showed that knockout of ASIP resulted in a significant reduction of gene expressions like PPARγ, FASN, SCD, and a significant up-regulation of genes like FABP4, ELOVL6, ACSL1, HACD4 prompted increased mid-to long-chain fatty acid synthesis. Overall, ASIP plays a pivotal role in regulating lipid metabolism in bMECs, which could further influence the component of lipid in milk.

Phylogeographic study using nuclear genome sequences of &lt;i&gt;Asip&lt;/i&gt; to infer the origins of ventral fur color variation in the house mouse &lt;i&gt;Mus musculus&lt;/i&gt;

While the house mouse (Mus musculus), widely distributed in Eurasia, is known to have substantial coat color variation between and within local populations, in both primary and secondary distribution areas, including the Japanese archipelago, the evolutionary history of the color variation is poorly understood. To address the ventral fur color variation, we quantified the lightness of museum skin specimens, and found that the southern subspecies, M. m. castaneus (CAS), has high and low lightness in dry and rainy geographic regions, respectively. The northern subspecies, M. m. musculus (MUS), has low and high levels of lightness in the high and middle latitudes of northern Eurasia, respectively. We examined sequence variation of the agouti signaling protein gene (Asip), which is known to be responsible for the ventral fur color. We performed phylogenetic analyses with 196 haplotype sequences of Asip (~180 kb) generated by phasing the whole-genome data of 98 wild mice reported previously. Network and phylogenetic tree construction revealed clustering of haplotypes representing the two subspecies, MUS and CAS. A number of subclusters with geographic affinities appeared within the subspecies clusters, in which the essential results were consistent with those reconstructed with whole mitochondrial genome data, indicating that the phased haplotype genome sequences of the nuclear genome can be a useful tool for tracing the dispersal of geographical lineages. The results of phylogeographic analysis showed that CAS mice with darker ventral fur possessed similar Asip haplotypes across the geographic distribution, suggesting that these haplotypes are major causes of the historical introduction of Asip haplotypes for darker ventral fur in mice from northern India to the peripheral areas, including the Japanese archipelago. Similarly, MUS in East Asia, which has a white abdomen, formed an Asip haplogroup with that from northern Iran, also with a white abdomen.

Factors affecting gene expression associated with the skin color of black-bone chicken in Thailand

The objective of this study was to investigate the effect of breed, sex, and age on the gene expression level of melanocortin 1 receptor (MC1R), DOPA chrome tautomerase (DCT), tyrosinase-related protein 1 (TYRP1), tyrosinase (TYR), and agouti signaling protein (ASIP) genes in Thai commercial chicken lines. All chicken have received Newscastle vaccination, and no antibiotics or any drugs were used in this study. Four chicken breeds including Black-Chinese, KU-Phuparn, Sri Mok, and Pradu Hang Dam were used in this study. These breeds can be classified by their skin color into 3 group including black (Black Chinese and KU-Phuparn), light black (Sri Mok), and yellowish white (Pradu Hang Dam). One hundred chickens per breed were used in this study. Breast skin tissue was randomly collected from 8 chickens (4 males, 4 females) per breed at 4, 8, 12, and 16 wk of age. The mRNA expression was analyzed using qRT-PCR and the gene expression level was calculated as 2−ΔΔCT. From the results, breed significantly (P < 0.01) affected the expression level for the 5 genes evaluated. Birds with the black skin color had greater TYRP1 and TYR gene expression when compared to chickens with light black and yellowish-white skin color, respectively. Whereas, chickens with yellowish-white skin color had greater ASIP gene expression when compared to chickens having the other skin colors. Sex significantly affected DCT, TYRP1, and TYR gene expression where the gene expression in males was greater when compared to females (P < 0.05). Age affected all gene expression levels (P < 0.01). At 4 wk of age, MC1R, DCT, TYRP1, and TYR gene expression was the highest and decreased as bird age increased (P < 0.05); however, ASIP gene expression was greatest at 8 wk of age. After 8 wk of age all gene expression for the genes evaluated in this study decreased as age increased. In addition, an interaction between breed and sex (P < 0.05) impacted DCT and ASIP gene expression. The results from this study showed that all genes evaluated can be used as candidate markers to further improve the blackness of the chicken's skin because the most desired skin color is black in the Thai black-bone chicken population.

Further occurrences of melanism in a northern, peripheral, population of Bobcat (&lt;i&gt;Lynx rufus&lt;/i&gt;)

Although melanism is understood to occur commonly among some felids, it is reported to be most frequent among cat species that occur in humid, tropical, and densely vegetated habitats. Previously, a single record of a melanistic Bobcat (Lynx rufus) from eastern Canada (New Brunswick) appeared to be a northern outlier, with all other reports of melanism in this species restricted to the warm, humid, climate of southern peninsular Florida. Here, I document a further five occurrences of melanism in Bobcat from New Brunswick and review evidence that a mutation in an agouti-signalling protein gene may be responsible for melanism in New Brunswick Bobcats.

Variants at the ASIP locus contribute to coat color darkening in Nellore cattle

BackgroundNellore cattle (Bos indicus) are well-known for their adaptation to warm and humid environments. Hair length and coat color may impact heat tolerance. The Nellore breed has been strongly selected for white coat, but bulls generally exhibit darker hair ranging from light grey to black on the head, neck, hump, and knees. Given the potential contribution of coat color variation to the adaptation of cattle populations to tropical and sub-tropical environments, our aim was to map positional and functional candidate genetic variants associated with darkness of hair coat (DHC) in Nellore bulls.ResultsWe performed a genome-wide association study (GWAS) for DHC using data from 432 Nellore bulls that were genotyped for more than 777 k single nucleotide polymorphism (SNP) markers. A single major association signal was detected in the vicinity of the agouti signaling protein gene (ASIP). The analysis of whole-genome sequence (WGS) data from 21 bulls revealed functional variants that are associated with DHC, including a structural rearrangement involving ASIP (ASIP-SV1). We further characterized this structural variant using Oxford Nanopore sequencing data from 13 Australian Brahman heifers, which share ancestry with Nellore cattle; we found that this variant originates from a 1155-bp deletion followed by an insertion of a transposable element of more than 150 bp that may impact the recruitment of ASIP non-coding exons.ConclusionsOur results indicate that the variant ASIP sequence causes darker coat pigmentation on specific parts of the body, most likely through a decreased expression of ASIP and consequently an increased production of eumelanin.

Estimating the copy number of the agouti signaling protein (ASIP) gene in goat breeds with different color patterns

The agouti signaling protein (ASIP) gene has a crucial role in pigmentation by encoding a protein that binds the melanocortin 1 receptor and stimulates the synthesis of pheomelanin rather than eumelanin. Several studies have suggested that an increased copy number of the ASIP gene might explain the white pigmentation of certain goat breeds, as previously demonstrated in sheep. In the current work, we have identified the segregation of the ASIP CNV in Murciano-Granadina (black or brown coat), Malagueña (brown, blond or white coat) and Saanen (white coat) goats with available Illumina Goat SNP50 BeadChip (Illumina Inc., San Diego, CA) data by using the PennCNV v1.0.5 and QuantiSNP v2 tools. This result shows that the ASIP CNV segregates in non-white breeds. To gain new insights into this issue, we have estimated the copy number of the ASIP gene in 83 goats from 8 breeds with different coloration patterns using a real-time quantitative PCR approach. Our results showed an increased ASIP copy number not only in Saanen (3.50 ± 0.23 copies relative to the calibrator) and white Malagueña (3.51 ± 0.51 copies) goats, but also in the Murciano-Granadina breed (3.33 ± 0.58 copies) as well as in blond/brown individuals from the Malagueña (3.58 ± 0.73 copies) breed. The number of ASIP copies was not significantly different in these four caprine populations (P > 0.05). Moreover, we did not observe a trend towards increased ASIP copy number in breeds with predominantly white colors, such as Maltese (2.85 ± 0.28 copies), Jonica (2.82 ± 0.39 copies) and Blanca de Rasquera (2.37 ± 0.33 copies). Our results, combined with recent findings demonstrating the high structural complexity of the ASIP locus, indicate that additional functional and expression studies should be performed in order to fully understand the role of ASIP structural variation in goat pigmentation.

Genomic Analysis Revealed a Convergent Evolution of LINE-1 in Coat Color: A Case Study in Water Buffaloes (Bubalus bubalis).

Visible pigmentation phenotypes can be used to explore the regulation of gene expression and the evolution of coat color patterns in animals. Here, we performed whole-genome and RNA sequencing and applied genome-wide association study, comparative population genomics and biological experiments to show that the 2,809-bp-long LINE-1 insertion in the ASIP (agouti signaling protein) gene is the causative mutation for the white coat phenotype in swamp buffalo (Bubalus bubalis). This LINE-1 insertion (3′ truncated and containing only 5′ UTR) functions as a strong proximal promoter that leads to a 10-fold increase in the transcription of ASIP in white buffalo skin. The 165 bp of 5′ UTR transcribed from the LINE-1 is spliced into the first coding exon of ASIP, resulting in a chimeric transcript. The increased expression of ASIP prevents melanocyte maturation, leading to the absence of pigment in white buffalo skin and hairs. Phylogenetic analyses indicate that the white buffalo-specific ASIP allele originated from a recent genetic transposition event in swamp buffalo. Interestingly, as a similar LINE-1 insertion has been identified in the cattle ASIP gene, we discuss the convergent mechanism of coat color evolution in the Bovini tribe.

Phylogeography and population history of the least weasel (Mustela nivalis) in the Palearctic based on multilocus analysis

The least weasel (Mustela nivalis) is one of the most widely distributed carnivorans. While previous studies have identified distinct western and eastern mitochondrial DNA (mtDNA) lineages of the species in the western Palearctic, their broader distributions across the Palearctic have remained unknown. To address the broad-scale phylogeographical structure, we expanded the sampling to populations in Eastern Europe, the Urals, the Russian Far East, and Japan, and analyzed the mtDNA control region and cytochrome b, the final intron of the zinc finger protein on Y chromosome (ZFY), and the autosomal agouti signaling protein gene (ASIP). The mtDNA data analysis exposed the previous western lineage (Clade I) but poorly supported assemblage extending across Palearctic, whereas the previous eastern lineage (Clade II) was reconfirmed and limited in the south western part of the Palearctic. The ZFY phylogeny showed a distinctive split that corresponding to the mtDNA lineage split, although less phylogeographical structure was seen in the ASIP variation. Our data concur with the previous inference of the Black Sea–Caspian Sea area having an ancestral character. The Urals region harbored high mitochondrial diversity, with an estimated coalescent time of around 100,000 years, suggesting this could have been a cryptic refugium. Based on the coalescent-based demographic reconstructions, the expansion of Clade I across the Palearctic was remarkably rapid, while Clade II was relatively stable for a longer time. It seems that Clade II has maintained a constant population size in the temperate region, and the expansive Clade I represents adaptation to the cold regions.

Multiple origins of melanism in two species of North American tree squirrel (Sciurus)

BackgroundWhile our understanding of the genetic basis of convergent evolution has improved there are still many uncertainties. Here we investigate the repeated evolution of dark colouration (melanism) in eastern fox squirrels (Sciurus niger; hereafter “fox squirrels”) and eastern gray squirrels (S. carolinensis; hereafter “gray squirrels”).ResultsWe show that convergent evolution of melanism has arisen by independent genetic mechanisms in two populations of the fox squirrel. In a western population, melanism is associated with a 24 bp deletion in the melanocortin-1-receptor gene (MC1RΔ24 allele), whereas in a south-eastern population, melanism is associated with a point substitution in the agouti signalling protein gene causing a Gly121Cys mutation. The MC1R∆24 allele is also associated with melanism in gray squirrels, and, remarkably, all the MC1R∆24 haplotypes are identical in the two species. Evolutionary analyses show that the MC1R∆24 haplotype is more closely related to other MC1R haplotypes in the fox squirrel than in the gray squirrel. Modelling supports the possibility of gene flow between the two species.ConclusionsThe presence of the MC1R∆24 allele and melanism in gray squirrels is likely due to introgression from fox squirrels, although we cannot completely rule out alternative hypotheses including introgression from gray squirrels to fox squirrels, or an ancestral polymorphism. Convergent melanism in these two species of tree squirrels has evolved by at least two and probably three different evolutionary routes.