Chromosome-level reference genome and resequencing of 322 accessions reveal evolution, genomic imprint and key agronomic traits in adzuki bean.

Adzuki bean (Vigna angularis) is an important legume crop cultivated in over 30 countries worldwide. We developed a high-quality chromosome-level reference genome of adzuki bean cultivar Jingnong6 by combining PacBio Sequel long-read sequencing with short-read and Hi-C technologies. The assembled genome covers 97.8% of the adzuki bean genome with a contig N50 of approximately 16 Mb and a total of 32 738 protein-coding genes. We also generated a comprehensive genome variation map of adzuki bean by whole-genome resequencing (WGRS) of 322 diverse adzuki beans accessions including both wild and cultivated. Furthermore, we have conducted comparative genomics and a genome-wide association study (GWAS) on key agricultural traits to investigate the evolution and domestication. GWAS identified several candidate genes, including VaCycA3;1, VaHB15, VaANR1 and VaBm, that exhibited significant associations with domestication traits. Furthermore, we conducted functional analyses on the roles of VaANR1 and VaBm in regulating seed coat colour. We provided evidence for the highest genetic diversity of wild adzuki (Vigna angularis var. nipponensis) in China with the presence of the most original wild adzuki bean, and the occurrence of domestication process facilitating transition from wild to cultigen. The present study elucidates the genetic basis of adzuki bean domestication traits and provides crucial genomic resources to support future breeding efforts in adzuki bean.

Targeted mutagenesis of flavonoid biosynthesis pathway genes reveals functional divergence in seed coat colour, oil content and fatty acid composition in Brassica napus L.

Yellow-seed is widely accepted as a good-quality trait in Brassica crops. Previous studies have shown that the flavonoid biosynthesis pathway is essential for the development of seed colour, but its function in Brassica napus, an important oil crop, is poorly understood. To systematically explore the gene functions of the flavonoid biosynthesis pathway in rapeseed, several representative TRANSPARENT TESTA (TT) genes, including three structural genes (BnaTT7, BnaTT18, BnaTT10), two regulatory genes (BnaTT1, BnaTT2) and a transporter (BnaTT12), were selected for targeted mutation by CRISPR/Cas9 in the present study. Seed coat colour, lignin content, seed quality and yield-related traits were investigated in these Bnatt mutants together with Bnatt8 generated previously. These Bnatt mutants produced seeds with an elevated seed oil content and decreased pigment and lignin accumulation in the seed coat without any serious defects in the yield-related traits. In addition, the fatty acid (FA) composition was also altered to different degrees, i.e., decreased oleic acid and increased linoleic acid and α-linolenic acid, in all Bnatt mutants except Bnatt18. Furthermore, gene expression analysis revealed that most of BnaTT mutations resulted in the down-regulation of key genes related to flavonoid and lignin synthesis, and the up-regulation of key genes related to lipid synthesis and oil body formation, which may contribute to the phenotype. Collectively, our study generated valuable resources for breeding programs, and more importantly demonstrated the functional divergence and overlap of flavonoid biosynthesis pathway genes in seed coat colour, oil content and FA composition of rapeseed.

A PAX3 insertion in the Celestial breed and certain feline breeding lines with dominant blue eyes.

During the last 60 years many inherited traits in domestic outbred cats were selected and retained giving birth to new breeds characterised by singular coat or morphological phenotypes. Among them, minimal white spotting associated with blue eyes was selected by feline breeders to create the Altai, Topaz, and Celestial breeds. Various established breeds also introduced this trait in their lineages. The trait, that was confirmed as autosomal dominant by breeding data, was first described in domestic cats from Kazakhstan and Russia, in British shorthair and British longhair from Russia, and in Maine Coon cats from the Netherlands, suggesting different founding effects. Using a genome-wide association study we identified a single region on chromosome C1 that was associated with the minimal white spotting and blue eyes phenotype (also called DBE by breeders for dominant blue eyes) in the French Celestial breed. Within that region we identified Paired Box 3 (PAX3) as the strongest candidate gene, since PAX3 is a key regulator of MITF (Melanocyte-Inducing Transcription Factor) and PAX3 variants have been previously identified in various species showing white spotting with or without blue eyes including the mouse and the horse. Whole genome sequencing of a Celestial cat revealed an endogenous retrovirus LTR (long terminal repeat) insertion within PAX3 intron 4 known to contain regulatory sequences (conserved non-coding element [CNE]) involved in PAX3 expression. The insertion is in the vicinity of CNE2 and CNE3. All 52 Celestial and Celestial-mixed cats with a DBE phenotype presented the insertion, that was absent in their 22 non-DBE littermates and in 87 non-DBE cats from various breeds. The outbred Celestial founder was also heterozygous for the insertion. Additionally, the variant was found in nine DBE Maine Coon cats related to the Celestial founder and four DBE Siberian cats with an uncertain origin. Segregation of the variant in the Celestial breed is consistent with dominant inheritance and does not appear to be associated with deafness. We propose that this NC_018730.3:g.206974029_206974030insN[395] variant represents the DBECEL (Celestial Dominant Blue Eyes) allele in the domestic cat.

Genomic analysis links the American mink Royal pastel coat phenotype to retroviral element type 1 insertion in the HPS3 gene.

To date, only 10 of the more than 30 fur colours that had been observed in American mink (Neogale vison) have been linked to specific genes. The Royal pastel fur colour is part of a large family of brownish colours that are quite similar to one another, making breeding and selecting processes more difficult. Here we carried out whole-genome sequencing of five American minks with Royal pastel (b/b) phenotypes originating from two distinct mink populations. We identified an insertion of endogenous retroviral element type 1 (ERV1) into the first intron of the gene encoding the HPS3 protein, which regulates the trafficking of tyrosinase-containing vesicles to maturing melanosomes. With Cas9-targeted nanopore sequencing, we reconstructed the full-length sequence of the 11.7 Kb ERV1 insertion and observed hypermethylation that spread to the HPS3 gene promoter region. These findings highlight the role of HPS3 in the formation of melanosomes and melanin, as well as the genetic process regulating the intensity and spectrum of hair colour. Moreover, in mink breeding projects, these data are also useful for tracking economically important fur qualities.

Utilisation of indigenous knowledge to control gastrointestinal nematodes in Southern Africa.

Gastrointestinal nematodes (GIN) exacerbate the impact of droughts on the survival of cattle. The inadequacies of the conventional system make it increasingly important to explore indigenous knowledge (IK) to create drought-tolerant and GIN resilient herds. The objective of the study was to assess the indigenous strategies for controlling GIN during droughts. Face-to-face interviews with experts on IK were conducted to give insight into the importance, methods and ranking of GIN control. The experts identified 86 cattle that were used to test their assertions. The control methods used were identifying cattle that were susceptible to high GIN loads using predisposing factors, diagnosis of GIN burdens using faecal appearance, and treatment using phytotherapy. Experts ranked predisposing factors as the most critical control strategy and identified body condition, class, sex, coat colour, pregnancy status and lactation status as predisposing factors to high GIN burdens. Thin, older, dark-coloured cattle, as well as pregnant and lactating cows, were considered susceptible to GIN. However, pregnancy status, coat colour and sex were significantly associated with high GIN burdens. Cows were 2.6 times more likely to have high GIN burdens than bulls. Dark-coloured cattle were 3.5 times more likely to have high GIN burdens than light-coloured ones, and the likelihood of pregnant cows was 4.9 times higher than non-pregnant cows. A dark-coloured pregnant cow was extremely susceptible to high GIN burdens. In conclusion, knowledge of predisposing factors informs selection decisions when purchasing foundation stock. Cattle that are susceptible to high GIN loads are prioritised during droughts or culled where resources are scarce.

Does coat colour influence survival? A test in a cyclic population of snowshoe hares.

Some mammal species inhabiting high-latitude biomes have evolved a seasonal moulting pattern that improves camouflage via white coats in winter and brown coats in summer. In many high-latitude and high-altitude areas, the duration and depth of snow cover has been substantially reduced in the last five decades. This reduction in depth and duration of snow cover may create a mismatch between coat colour and colour of the background environment, and potentially reduce the survival rate of species that depend on crypsis. We used long-term (1977-2020) field data and capture-mark-recapture models to test the hypothesis that whiteness of the coat influences winter apparent survival in a cyclic population of snowshoe hares (Lepus americanus) at Kluane, Yukon, Canada. Whiteness of the snowshoe hare coat in autumn declined during this study, and snowshoe hares with a greater proportion of whiteness in their coats in autumn survived better during winter. However, whiteness of the coat in spring did not affect subsequent summer survival. These results are consistent with the hypothesis that the timing of coat colour change in autumn can reduce overwinter survival. Because declines in cyclic snowshoe hare populations are strongly affected by low winter survival, the timing of coat colour change may adversely affect snowshoe hare population dynamics as climate change continues.

Genomic analysis reveals a KIT-related chromosomal translocation associated with the white coat phenotype in yak.

White coat pigmentation is a striking phenotype of many domesticated species and has various genetic controls. The Tianzhu White yak, an indigenous breed with a complete white coat, has fascinated Tibetans for centuries. However, the genetic basis of this trait remains unknown. Here, we conducted population genomics analysis and genome-wide association study based on the whole-genome sequencing data of 38 white and 59 non-white-coated yak. The results revealed the presence of KIT-linked Cs alleles characterized by the translocations between chromosomes 6 and 29 in all-white yak. Furthermore, structural variations showed additional duplications of the Cs alleles in white yak compared with colour-sidedness cattle. Interestingly, the Cs alleles associated with the white coat phenotype in yak were found to have introgressed from taurine cattle. Our findings unveil the shared genetic control of the white coat phenotype and its evolution in closely related bovine species.

Canine coat pigmentation genetics: a review.

Our understanding of canine coat colour genetics and the associated health implications is developing rapidly. To date, there are 15 genes with known roles in canine coat colour phenotypes. Many coat phenotypes result from complex and/or epistatic genetic interactions among variants within and between loci, some of which remain unidentified. Some genes involved in canine pigmentation have been linked to aural, visual and neurological impairments. Consequently, coat pigmentation in the domestic dog retains considerable ethical and economic interest. In this paper we discuss coat colour phenotypes in the domestic dog, the genes and variants responsible for these phenotypes and any proven coat colour-associated health effects.

Identification of two new recessive MC1R alleles in red-coloured Evolèner cattle and other breeds.

Sequence variations in the melanocortin-1 receptor (MC1R) gene are associated with melanism in different animal species. Six functionally relevant alleles have been described in cattle to date. In a hypothesis-free approach we performed a genome-wide allelic association study with black, red and wild-coloured cattle of three Alpine cattle breeds (Eringer, Evolèner and Valdostana), revealing a single significant association signal close to the MC1R gene. We searched for candidate causative variants by sequencing the entire coding sequence and identified two novel protein-changing variants. We propose designating the mutant alleles at MC1R:c.424C>T as ev1 and at MC1R:c.263G>A as ev2 . Both affect conserved amino acid residues in functionally important transmembrane domains (p.Arg142Cys and p.Ser88Asn). Both alleles segregate predominantly in the Swiss Evolèner breed. They occur in other European cattle breeds such as Abondance and Rotes Höhenvieh as well. We observed almost perfect association between the MC1R genotypes and the coat colour phenotype in a cohort of 513 black, red and wild-coloured cattle. Animals carrying two copies of MC1R loss-of-function alleles or that were compound heterozygous for e, ev1 , or ev2 have a red to dark red (chestnut-like red) coat colour. These findings expand the spectrum of causal MC1R variants causing recessive red in cattle.

Signatures of selection are present in the genome of two close autochthonous cattle breeds raised in the North of Italy and mainly distinguished for their coat colours.

Autochthonous cattle breeds are genetic resources that, in many cases, have been fixed for inheritable exterior phenotypes useful to understand the genetic mechanisms affecting these breed-specific traits. Reggiana and Modenese are two closely related autochthonous cattle breeds mainly raised in the production area of the well-known Protected Designation of Origin Parmigiano-Reggiano cheese, in the North of Italy. These breeds can be mainly distinguished for their standard coat colour: solid red in Reggiana and solid white with pale shades of grey in Modenese. In this study we genotyped with the GeneSeek GGP Bovine 150k single nucleotide polymorphism (SNP) chip almost half of the extant cattle populations of Reggiana (n = 1109 and Modenese (n = 326) and used genome-wide information in comparative FST analyses to detect signatures of selection that diverge between these two autochthonous breeds. The two breeds could be clearly distinguished using multidimensional scaling plots and admixture analysis. Considering the top 0.0005% FST values, a total of 64 markers were detected in the single-marker analysis. The top FST value was detected for the melanocortin 1 receptor (MC1R) gene mutation, which determines the red coat colour of the Reggiana breed. Another coat colour gene, agouti signalling protein (ASIP), emerged amongst this list of top SNPs. These results were also confirmed with the window-based analyses, which included 0.5-Mb or 1-Mb genome regions. As variability affecting ASIP has been associated with white coat colour in sheep and goats, these results highlighted this gene as a strong candidate affecting coat colour in Modenese breed. This study demonstrates how population genomic approaches designed to take advantage from the diversity between local genetic resources could provide interesting hints to explain exterior traits not yet completely investigated in cattle.

Genomic diversity and breed composition of Vietnamese smallholder dairy cows.

Vietnamese smallholder dairy cows (VDC) are the result of crossbreeding between different zebu (ZEB) and taurine dairy breeds through many undefined generations. Thus, the predominant breed composition of VDC is currently unknown. This study aimed to evaluate the level of genetic diversity and breed composition of VDC. The SNP data of 344 animals from 32 farms located across four dairy regions of Vietnam were collected and merged with genomic reference data, which included three ZEB breeds: Red Sindhi, Sahiwal and Brahman, three taurine breeds: Holstein (HOL), Jersey (JER) and Brown Swiss (BSW), and a composite breed: Chinese Yellow cattle. Diversity and admixture analyses were applied to the merged data set. The VDC were not excessively inbred, as indicated by very low inbreeding coefficients (Wright's FIS ranged from -0.017 to 0.003). The genetic fractions in the test herds suggested that the VDC are primarily composed of HOL (85.0%); however, JER (6.0%), BSW 5.3%) and ZEB (4.5%) had also contributed. Furthermore, major genotype groupings in the test herds were pure HOL (48%), B3:15/16HOL_1/16ZEB (22%) and B2:7/8HOL_1/8ZEB (12%). The genetic makeup of the VDC is mainly components of various dairy breeds but also has a small percentage of ZEB; thus, the VDC could be a good genetic base for selecting high milk-producing cows with some degree of adaptation to tropical conditions.

Ancient hybridization patterns between bighorn and thinhorn sheep.

Whole-genome sequencing has advanced the study of species evolution, including the detection of genealogical discordant events such as ancient hybridization and incomplete lineage sorting (ILS). The evolutionary history of bighorn (Ovis canadensis) and thinhorn (Ovis dalli) sheep present an ideal system to investigate evolutionary discordance due to their recent and rapid radiation and putative secondary contact between bighorn and thinhorn sheep subspecies, specifically the dark pelage Stone sheep (O. dalli stonei) and predominately white Dall sheep (O. dalli dalli), during the last ice age. Here, we used multiple genomes of bighorn and thinhorn sheep, together with snow (O. nivicola) and the domestic sheep (O. aries) as outgroups, to assess their phylogenomic history, potential introgression patterns and their adaptive consequences. Among the Pachyceriforms (snow, bighorn and thinhorn sheep) a consistent monophyletic species tree was retrieved; however, many genealogical discordance patterns were observed. Alternative phylogenies frequently placed Stone and bighorn as sister clades. This relationship occurred more often and was less divergent than that between Dall and bighorn. We also observed many blocks containing introgression signal between Stone and bighorn genomes in which coat colour genes were present. Introgression signals observed between Dall and bighorn were more random and less frequent, and therefore probably due to ILS or intermediary secondary contact. These results strongly suggest that Stone sheep originated from a complex series of events, characterized by multiple, ancient periods of secondary contact with bighorn sheep.

Exploiting within-breed variability in the autochthonous Reggiana breed identified several candidate genes affecting pigmentation-related traits, stature and udder defects in cattle.

Autochthonous cattle breeds constitute important reservoirs of genetic diversity. Reggiana is an Italian local cattle breed reared in the north of Italy for the production of a mono-breed Parmigiano-Reggiano cheese. Reggiana cattle usually have a classical solid red coat colour and pale muzzle. As part of the strategies designed for the sustainable conservation of this genetic resource, we investigated at the genome-wise level the within-breed detected variability of three pigmentation-related traits (intensity of red coat colour, based on three classes - light/diluted, normal and dark; spotted patterns/piebaldism that sometime emerge in the breed; muzzle colour - pink/pale, grey and black), stature, presence/absence and number of supernumerary teats and teat length. A total of 1776 Reggiana cattle (about two-thirds of the extant breed population) were genotyped with the GeneSeek GGP Bovine 150k SNP array and single-marker and haplotype-based GWASs were carried out. The results indicated that two main groups of genetic factors affect the intensity of red coat colour: darkening genes (including EDN3 and a few other genes) and diluting genes (including PMEL and a few other genes). Muzzle colour was mainly determined by MC1R gene markers. Piebaldism was mainly associated with KIT gene markers. Stature was associated with BTA6 markers upstream of the NCAPG-LCORL genes. Teat defects were associated with TBX3/TBX5, MCC and LGR5 genes. Overall, the identified genomic regions not only can be directly used in selection plans in the Reggiana breed, but also contribute to clarifying the genetic mechanisms involved in determining exterior traits in cattle.

Selection in the Finnhorse, a native all-around horse breed.

Selection by breeders modifies the morphology, behaviour and performance of domesticated species. Here, we examined signs of selection in Finnhorse, the only native horse breed in Finland. We first searched divergent genomic regions between Finnhorses and other breeds, as well as between different breeding sections of the Finnhorse with data from Illumina Equine SNP70 BeadChip, and then studied several of the detected regions in more detail. We found altogether 35 common outlier SNPs between Finnhorses and other breeds using two different selection tests. Many of the SNPs were located close to genes affecting coat colour, performance, size, sugar metabolism, immune response and olfaction. We selected genes affecting coat colour (KIT, MITF, PMEL), performance (MSTN) and locomotion (DMRT3) for a more detailed examination. In addition, we looked for, and found, associations with height at withers and SNPs located close to gene LCORL. Among the four breeding sections of Finnhorses (harness trotters, riding horses, draught horses and pony-sized horses), a single SNP located close to the DMRT3 gene was significantly differentiated and only between harness trotters and pony-sized horses.

Targeted mutagenesis of BnTT8 homologs controls yellow seed coat development for effective oil production in Brassica napus L.

Yellow seed is a desirable trait with great potential for improving seed quality in Brassica crops. Unfortunately, no natural or induced yellow seed germplasms have been found in Brassica napus, an important oil crop, which likely reflects its genome complexity and the difficulty of the simultaneous random mutagenesis of multiple gene copies with functional redundancy. Here, we demonstrate the first application of CRISPR/Cas9 for creating yellow-seeded mutants in rapeseed. The targeted mutations of the BnTT8 gene were stably transmitted to successive generations, and a range of homozygous mutants with loss-of-function alleles of the target genes were obtained for phenotyping. The yellow-seeded phenotype could be recovered only in targeted mutants of both BnTT8 functional copies, indicating that the redundant roles of BnA09.TT8 and BnC09.TT8b are vital for seed colour. The BnTT8 double mutants produced seeds with elevated seed oil and protein content and altered fatty acid (FA) composition without any serious defects in the yield-related traits, making it a valuable resource for rapeseed breeding programmes. Chemical staining and histological analysis showed that the targeted mutations of BnTT8 completely blocked the proanthocyanidin (PA)-specific deposition in the seed coat. Further, transcriptomic profiling revealed that the targeted mutations of BnTT8 resulted in the broad suppression of phenylpropanoid/flavonoid biosynthesis genes, which indicated a much more complex molecular mechanism underlying seed colour formation in rapeseed than in Arabidopsis and other Brassica species. In addition, gene expression analysis revealed the possible mechanism through which BnTT8 altered the oil content and fatty acid composition in seeds.

A genome-wide scan study identifies a single nucleotide substitution in the tyrosinase gene associated with white coat colour in a red deer (Cervus elaphus) population.

BACKGROUND: Red deer with very pale coat colour are observed sporadically. In the red deer (Cervus elaphus) population of Reinhardswald in Germany, about 5% of animals have a white coat colour that is not associated with albinism. In order to facilitate the conservation of the animals, it should be determined whether and to what extent brown animals carry the white gene. For this purpose, samples of one white hind and her brown calf were available for whole genome sequencing to identify the single nucleotide polymorphism(s) responsible for the white phenotype. Subsequently, samples from 194 brown and 11 white animals were genotyped. RESULTS: Based on a list of colour genes of the International Federation of Pigment Cell Societies, a non-synonymous mutation with exchange of a glycine residue at position 291 of the tyrosinase protein by arginine was identified as the cause of dilution of the coat colour. A gene test led to exactly matching genotypes in all examined animals. The study showed that 14% of the brown animals carry the white gene. This provides a simple and reliable way of conservation for the white animals. However, results could not be transferred to another, unrelated red deer population with white animals. Although no brown animals with a white tyrosinase genotype were detected, the cause for the white colouring in this population was different. CONCLUSIONS: A gene test for the conservation of white red deer is available for the population of the Reinhardswald. While mutations in the tyrosinase are commonly associated with oculocutaneous albinism type 1, the amino acid exchange at position 291 was found to be associated with coat colour dilution in Cervus elaphus.

A genome-wide scan study identifies a single nucleotide substitution in MC1R gene associated with white coat colour in fallow deer (Dama dama).

BACKGROUND: The coat colour of fallow deer is highly variable and even white animals can regularly be observed in game farming and in the wild. Affected animals do not show complete albinism but rather some residual pigmentation resembling a very pale beige dilution of coat colour. The eyes and claws of the animals are pigmented. To facilitate the conservation and management of such animals, it would be helpful to know the responsible gene and causative variant. We collected 102 samples from 22 white animals and from 80 animals with wildtype coat colour. The samples came from 12 different wild flocks or game conservations located in different regions of Germany, at the border to Luxembourg and in Poland. The genomes of one white hind and her brown calf were sequenced. RESULTS: Based on a list of colour genes of the International Federation of Pigment Cell Societies ( http://www.ifpcs.org/albinism/ ), a variant in the MC1R gene (NM_174108.2:c.143 T > C) resulting in an amino acid exchange from leucine to proline at position 48 of the MC1R receptor protein (NP_776533.1:p.L48P) was identified as a likely cause of coat colour dilution. A gene test revealed that all animals of the white phenotype were of genotype CC whereas all pigmented animals were of genotype TT or TC. The study showed that 14% of the pigmented (brown or dark pigmented) animals carried the white allele. CONCLUSIONS: A genome-wide scan study led to a molecular test to determine the coat colour of fallow deer. Identification of the MC1R gene provides a deeper insight into the mechanism of dilution. The gene marker is now available for the conservation of white fallow deer in wild and farmed animals.

Feline chimerism revealed by DNA profiling.

In dogs and cats, unusual coat colour phenotypes may result from various phenomena, including chimerism. In the domestic cat, the tortoiseshell coat colour that combines red and non-red hairs is the most obvious way to identify chimeras in males. Several cases of tortoiseshell males have been reported, some of which were diagnosed as chimeras without any molecular confirmation. Here, we report the case of a female feline chimera identified thanks to its coat colour and confirmed through DNA profiling and a coat colour test. We ruled out the hypothesis of mosaicism and aneuploidy. All the data were consistent with a natural case of female chimerism.

A de novo germline mutation of KIT in a white-spotted Brown Swiss cow.

White-spotting coat colour phenotypes in cattle are either fixed characteristics of specific cattle breeds or occur sporadically owing to germline genetic variation of solid-coloured parents. A Brown Swiss cow showing a piebald pattern resembling colour-sidedness was referred for genetic evaluation. Both parents were normal solid-brown-coloured cattle. The cow was tested negative for the three known DNA variants in KIT, MITF and TWIST2 associated with different depigmentation phenotypes in Brown Swiss cattle. Whole-genome sequencing of the cow was performed and a heterozygous variant affecting the coding sequence of the bovine KIT gene was identified on chromosome 6. The variant is a 40 bp deletion in exon 9, NM_001166484.1:c.1390_1429del, and leads to a frameshift that is predicted to produce a novel 50 amino acid-long C-terminus replacing almost 50% of the wt KIT protein, including the functionally important intracellular tyrosine kinase domain (NP_001159956.1:p.(Asn464AlafsTer50)). Interestingly, among three available offspring, two solid-coloured daughters were genotyped as homozygous wt whereas a single son showing a slightly milder but still obvious depigmentation phenotype inherited a copy of the novel variant allele. The genetic findings provide strong evidence that the identified loss-of-function KIT variant most likely represents a de novo germline mutation that is causative owing to haploinsufficiency.

A deletion spanning the promoter and first exon of the hair cycle-specific ASIP transcript isoform in black and tan rabbits.

Black and tan animals have tan-coloured ventral body surfaces separated by sharp boundaries from black-coloured dorsal body surfaces. In the at mouse mutant, a retroviral 6 kb insertion located in the hair cycle-specific promoter of the murine Asip gene encoding agouti signalling protein causes the black and tan phenotype. In rabbits, three ASIP alleles are thought to exist, including an at allele causing a black and tan coat colour that closely resembles the mouse black and tan phenotype. The goal of our study was to identify the functional genetic variant causing the rabbit at allele. We performed a WGS-based comparative analysis of the ASIP gene in one black and tan and three wt agouti-coloured rabbits. The analysis identified 75 at -associated variants including an 11 kb deletion. The deletion is located in the region of the hair cycle-specific ASIP promoter and thus in a region homologous to the site of the retroviral insertion causing the at allele in mice. We observed perfect association of the genotypes at this deletion with the coat colour phenotype in 49 rabbits. The comparative analysis and the previous knowledge about the regulation of ASIP expression suggest that the 11 kb deletion is the most likely causative variant for the black and tan phenotype in rabbits.