Mate choice screening in captive solitary carnivores: The role of male behavior and cues on mate preference and paternity in females of a model species, American mink (Neovison vison).

Mate choice studies suggest that choosy females benefit from increased fecundity, litter size, and offspring survival. Thus, providing females with the opportunity to choose among potential mates, deemed genetically suitable based on studbook data, might improve breeding management in production and zoo animals and thereby the sustainability of captive populations. Investigating mate preference via odor from potential mates before animal transfer is a proposed strategy for incorporating mate choice into breeding management. In this study, we test whether olfactory cues and signals from males can be used to assess and measure female mate preference in American mink. Eighteen females were subjected to a 4-day stimulus test in which females showed a preference for one of two males' urine and feces. Subsequently, each female was subjected to a 10-day mate preference test involving the same two males of the first test. Paternity tests revealed that 13 females had offspring, which could be assigned to only one male, suggesting that these females performed a mate choice. In nine of these females preference during the stimulus test was directed toward the male that fathered their offspring. Our results suggest that even though there was a preference difference in scent stimulus trials from potential mates this preference was not predictive of eventual mate preference or paternity. Other factors such as aspects of male behavior seem to play a role, when the mates are introduced. Our study supports that mate preference and mate choice are complex matters influenced by multiple cues and signals.

A large insertion in intron 2 of the TYRP1 gene associated with American Palomino phenotype in American mink.

A number of American mink phenotypes display a range of brownish colours. One of these phenotypes, namely American Palomino (b (P) b (P) ) (AP) has been found to be associated with the tyrosinase-related protein 1 (TYRP1) gene by genotyping microsatellite markers in one sire family. Trials for amplifying the genomic DNA and cDNA at the beginning of intron 2 of AP TYRP1 revealed the presence of a large insertion of approximately eight kb. The insertion most likely disrupts different elements necessary for the splicing of intron 2 of the TYRP1 gene. In AP RNAseq data indicate, however, the presence of the wild-type (wt) transcript at very low levels and Western blot reveals three products when using an antibody raised against middle part of the TYRP1 protein. One individual from another brown mink phenotype-commercially named Dawn-was also investigated at the molecular level by long-range PCR and the same size insertion appears to be present. By this we suggest that certain modifiers of TYRP1 would induce different brown colour degradation, which results in at least two different phases of brown.

Comparison of American mink embryonic stem and induced pluripotent stem cell transcriptomes.

BACKGROUND: Recently fibroblasts of many mammalian species have been reprogrammed to pluripotent state using overexpression of several transcription factors. This technology allows production of induced pluripotent stem (iPS) cells with properties similar to embryonic stem (ES) cells. The completeness of reprogramming process is well studied in such species as mouse and human but there is not enough data on other species. We produced American mink (Neovison vison) ES and iPS cells and compared these cells using transcriptome analysis. RESULTS: We report the generation of 10 mink ES and 22 iPS cell lines. The majority of the analyzed cell lines had normal diploid chromosome number. The only ES cell line with XX chromosome set had both X-chromosomes in active state that is characteristic of pluripotent cells. The pluripotency of ES and iPS cell lines was confirmed by formation of teratomas with cell types representing all three germ layers. Transcriptome analysis of mink embryonic fibroblasts (EF), two ES and two iPS cell lines allowed us to identify 11831 assembled contigs which were annotated. These led to a number of 6891 unique genes. Of these 3201 were differentially expressed between mink EF and ES cells. We analyzed expression levels of these genes in iPS cell lines. This allowed us to show that 80% of genes were correctly reprogrammed in iPS cells, whereas approximately 6% had an intermediate expression pattern, about 7% were not reprogrammed and about 5% had a "novel" expression pattern. We observed expression of pluripotency marker genes such as Oct4, Sox2 and Rex1 in ES and iPS cell lines with notable exception of Nanog. CONCLUSIONS: We had produced and characterized American mink ES and iPS cells. These cells were pluripotent by a number of criteria and iPS cells exhibited effective reprogramming. Interestingly, we had showed lack of Nanog expression and consider it as a species-specific feature.

An American mink (Neovison vison) transcriptome.

HiSeq2000 Illumina pair-end sequenced transcript data originating from a pool of four different tissues of a wild-type American mink yielded approximately 90 Gb of raw data. Subsequently, unique contigs were assembled by a combined approach using velvet and phrap. Of these assembled contigs, about 136 000 match the dog genome and nearly 30 000 contigs match the human transcriptome at more than 17 000 unique gene locations. Gene annotation for these contigs was performed employing custom-made scripts run in combination with comparative sequence similarity search and alignment in the dog and human genome using blast algorithms. Transcripts representing five genes known to be associated with pigmentation were reliably aligned against large mink genomic contigs derived from BAC clones. Sequence comparison between transcript and genomic data revealed seven SNPs. In this study, we generated and annotated mink transcript sequences representing more than 16 000 known genes. This is the first comprehensive transcriptome for the American mink genome, which will facilitate further development in mink expression profiling studies and provide a good annotation basis in the perspectives of a whole genome sequencing project. The project was deposited at EMBL database with the accession number PRJEB1260.

New insights into the melanophilin (MLPH) gene controlling coat color phenotypes in American mink.

The mutation causing the Silverblue color type (pp) is one of the most used recessive mutations within American mink (Neovison vison) fur farming, since it is involved in some of the popular color types such as Violet and Saphire which originate from a combination of recessive mutations. In the present study, the genomic and mRNA sequences of the melanophilin (MLPH) gene were studied in Violet, Silverblue and wild-type (wt) mink animals. Although breeding schemes and previous literature indicates that the Violet (aammpp) phenotype is a triple recessive color type involving the same locus as the Silverblue (pp) color type, our findings indicate different genotypes at the MLPH locus. Upon comparison at genomic level, we identified two deletions of the entire intron 7 and of the 5' end of intron 8 in the sequence of the Silverblue MLPH gene. When investigating the mRNA, the Silverblue animals completely lack exon 8, which encodes 65 residues, of which 47 define the Myosin Va (MYO5A) binding domain. This may cause the incorrect anchoring of the MLPH protein to MYO5A in Silverblue animals, resulting in an improper pigmentation as seen in diluted phenotypes. Additionally, in the MLPH mRNA of wt, Violet and Silverblue phenotypes, part of intron 8 is retained resulting in a truncated MLPH protein, which is 359 residues long in wt and Violet and 284 residues long in Silverblue. Subsequently, our findings point out that the missing actin-binding domain, in neither of the 3 analyzed phenotypes affects the transport of melanosomes or the consequent final pigmentation. Moreover, the loss of the major part of the MYO5A domain in the Silverblue MLPH protein seems to be the responsible for the dilute phenotype. Based on our genomic DNA data, genetic tests for selecting Silverblue and Violet carrier animals can be performed in American mink.

A re-assigned American mink (Neovison vison) map optimal for genome-wide studies.

Our previously published second generation genetic map for the American mink (Neovison vison) has been used and redesigned in its best for genome-wide studies with maximum of efficiency. A number of 114 selected markers, including 33 newly developed microsatellite markers from the CHORI-231 mink Bacterial Artificial Chromosome (BAC) library, have been genotyped in a two generation population composed of 1200 individuals. The outcome reassigns the position of some markers on the chromosomes and it produces a more reliable map with a convenient distance between markers. A total of 104 markers mapped to 14 linkage groups corresponding to the mink autosomes. Six markers are unlinked and four markers are allocated to the X chromosome by homology but no linkage was detected. The sex-average linkage map spans 1192 centiMorgans (cM) with an average intermarker distance of 11.4cM and 1648cM when the ends of the linkage groups and the autosomal unlinked markers are added. Sex-specific genetic linkage maps were also generated. The male sex-specific map had a total length of 1014.6cM between the linked markers and an average inter-marker interval of 9.7cM. The female map has a corresponding length of 1378.6cM and an average inter-marker interval of 13.3cM. The study is complemented with additional anchorage for most of the chromosomes of the map by BAC in situ hybridization with clones containing microsatellites strategically selected from the various parts of the genome. This map provides an improved tool for genetic mapping and comparative genomics in mink, also useful for the future assembly of the mink genome sequence when this will be taken forward.

Construction of an American mink bacterial artificial chromosome (BAC) library and sequencing candidate genes important for the fur industry.

BACKGROUND: Bacterial artificial chromosome (BAC) libraries continue to be invaluable tools for the genomic analysis of complex organisms. Complemented by the newly and fast growing deep sequencing technologies, they provide an excellent source of information in genomics projects. RESULTS: Here, we report the construction and characterization of the CHORI-231 BAC library constructed from a Danish-farmed, male American mink (Neovison vison). The library contains approximately 165,888 clones with an average insert size of 170 kb, representing approximately 10-fold coverage. High-density filters, each consisting of 18,432 clones spotted in duplicate, have been produced for hybridization screening and are publicly available. Overgo probes derived from expressed sequence tags (ESTs), representing 21 candidate genes for traits important for the mink industry, were used to screen the BAC library. These included candidate genes for coat coloring, hair growth and length, coarseness, and some receptors potentially involved in viral diseases in mink. The extensive screening yielded positive results for 19 of these genes. Thirty-five clones corresponding to 19 genes were sequenced using 454 Roche, and large contigs (184 kb in average) were assembled. Knowing the complete sequences of these candidate genes will enable confirmation of the association with a phenotype and the finding of causative mutations for the targeted phenotypes.Additionally, 1577 BAC clones were end sequenced; 2505 BAC end sequences (80% of BACs) were obtained. An excess of 2 Mb has been analyzed, thus giving a snapshot of the mink genome. CONCLUSIONS: The availability of the CHORI-321 American mink BAC library will aid in identification of genes and genomic regions of interest. We have demonstrated how the library can be used to identify specific genes of interest, develop genetic markers, and for BAC end sequencing and deep sequencing of selected clones. To our knowledge, this is the first report of 454 sequencing of selected BAC clones in mammals and re-assures the suitability of this technique for obtaining the sequence information of genes of interest in small genomics projects. The BAC end sequences described in this paper have been deposited in the GenBank data library [HN339419-HN341884, HN604664-HN604702]. The 454 produced contigs derived from selected clones are deposited with reference numbers [GenBank: JF288166-JF288183 &JF310744].

Molecular characterization of the Himalayan mink.

A rare color variant of the American mink (Neovison vison), discovered on a ranch in Nova Scotia and referred to as the "marbled" variety, carries a distinctive pigment distribution pattern resembling that found in some other species, e.g., the Siamese cat and the Himalayan mouse. We tested the hypothesis that the color pattern in question-light-colored body with dark-colored points (ears, face, tail, and feet)-is due to a mutation in the melanin-producing enzyme tyrosinase (TYR) that results in temperature-sensitive pigment production. Our study shows that marbled mink carry a mutation in exon 4 of the TYR gene (c.1835C > G) which results in an amino acid substitution (p.H420Q). The location of this substitution corresponds to the amino acid position that is also mutated in the TYR protein of the Himalayan mouse. Thus, the marbled variant is more aptly referred to as the Himalayan mink.