Genome-wide association study for the extractable phenolic profile and coat color of common bean seeds (Phaseolus vulgaris L.).

BACKGROUND: A large variation in seed coat colors and seed phenolic metabolites is present in common bean (Phaseolus vulgaris L.). The study of the relationships between seed coat color phenotype and the phenolic profile is an important step in the elucidation of the gene network involved in the phenylpropanoid biosynthetic pathway. However, this relationship is still poorly understood in this species. RESULTS: A genome-wide association study (GWAS) was used to investigate the genomic regions associated with the synthesis of 10 flavonoids (5 anthocyanins and 5 flavonols) and with 10 seed coat color traits using a set of 308 common bean lines of the Spanish Diversity Panel (SDP) which have been genotyped with 11,763 SNP markers.. A total of 31 significant SNP-trait associations (QTNs) were identified, grouped in 20 chromosome regions: 6 for phenolic metabolites on chromosomes Pv01, Pv02, Pv04, Pv08, and Pv09, 13 for seed coat color on chromosomes Pv01, Pv02, Pv06, Pv07, and Pv10, and 1 including both types of traits located on chromosome Pv08. In all, 58 candidate genes underlying these regions have been proposed, 31 of them previously described in the phenylpropanoid pathway in common bean, and 27 of them newly proposed in this work based on the association study and their homology with Arabidopsis anthocyanin genes. CONCLUSIONS: Chromosome Pv08 was identified as the main chromosome involved in the phenylpropanoid pathway and in consequence in the common bean seed pigmentation, with three independent chromosome regions identified, Phe/C_Pv08(2.7) (expanding from 2.71 to 4.04 Mbp), C_Pv08(5.8) (5.89-6.59 Mbp), and Phe_Pv08(62.5) (62.58 to 63.28 Mbp). Candidate genes previously proposed by other authors for the color genes V and P were validated in this GWAS. Candidate genes have been tentatively proposed from this study for color genes B and Rk on Pv02, Asp on Pv07, and complex C on Pv08. These results help to clarify the complex network of genes involved in the genetic control of phenolic compounds and seed color in common bean and provide the opportunity for future validation studies.

Nile Tilapia: A Model for Studying Teleost Color Patterns.

The diverse color patterns of cichlid fishes play an important role in mate choice and speciation. Here we develop the Nile tilapia (Oreochromis niloticus) as a model system for studying the developmental genetics of cichlid color patterns. We identified 4 types of pigment cells: melanophores, xanthophores, iridophores and erythrophores, and characterized their first appearance in wild-type fish. We mutated 25 genes involved in melanogenesis, pteridine metabolism, and the carotenoid absorption and cleavage pathways. Among the 25 mutated genes, 13 genes had a phenotype in both the F0 and F2 generations. None of F1 heterozygotes had phenotype. By comparing the color pattern of our mutants with that of red tilapia (Oreochromis spp), a natural mutant produced during hybridization of tilapia species, we found that the pigmentation of the body and eye is controlled by different genes. Previously studied genes like mitf, kita/kitlga, pmel, tyrb, hps4, gch2, csf1ra, pax7b, and bco2b were proved to be of great significance for color patterning in tilapia. Our results suggested that tilapia, a fish with 4 types of pigment cells and a vertically barred wild-type color pattern, together with various natural and artificially induced color gene mutants, can serve as an excellent model system for study color patterning in vertebrates.

Comparative transcriptomics reveals candidate carotenoid color genes in an East African cichlid fish.

BACKGROUND: Carotenoids contribute significantly to animal body coloration, including the spectacular color pattern diversity among fishes. Fish, as other animals, derive carotenoids from their diet. Following uptake, transport and metabolic conversion, carotenoids allocated to body coloration are deposited in the chromatophore cells of the integument. The genes involved in these processes are largely unknown. Using RNA-Sequencing, we tested for differential gene expression between carotenoid-colored and white skin regions of a cichlid fish, Tropheus duboisi "Maswa", to identify genes associated with carotenoid-based integumentary coloration. To control for positional gene expression differences that were independent of the presence/absence of carotenoid coloration, we conducted the same analyses in a closely related population, in which both body regions are white. RESULTS: A larger number of genes (n = 50) showed higher expression in the yellow compared to the white skin tissue than vice versa (n = 9). Of particular interest was the elevated expression level of bco2a in the white skin samples, as the enzyme encoded by this gene catalyzes the cleavage of carotenoids into colorless derivatives. The set of genes with higher expression levels in the yellow region included genes involved in xanthophore formation (e.g., pax7 and sox10), intracellular pigment mobilization (e.g., tubb, vim, kif5b), as well as uptake (e.g., scarb1) and storage (e.g., plin6) of carotenoids, and metabolic conversion of lipids and retinoids (e.g., dgat2, pnpla2, akr1b1, dhrs). Triglyceride concentrations were similar in the yellow and white skin regions. Extracts of integumentary carotenoids contained zeaxanthin, lutein and beta-cryptoxanthin as well as unidentified carotenoid structures. CONCLUSION: Our results suggest a role of carotenoid cleavage by Bco2 in fish integumentary coloration, analogous to previous findings in birds. The elevated expression of genes in carotenoid-rich skin regions with functions in retinol and lipid metabolism supports hypotheses concerning analogies and shared mechanisms between these metabolic pathways. Overlaps in the sets of differentially expressed genes (including dgat2, bscl2, faxdc2 and retsatl) between the present study and previous, comparable studies in other fish species provide useful hints to potential carotenoid color candidate genes.

Detection of single nucleotide polymorphisms (SNP) in equine coat color genes using SNaPshotTM multiplex kit or pluronic F-108 tri-block copolymer and capillary electrophoresis.

Molecular methods for the detection of mammalian coat color phenotypes have expanded greatly within the past decade. Many phenotypes are associated with a single nucleotide polymorphism mutation in the genetic sequence. Traditionally, these mutations are detected through sequencing, hybridization assays or mini-sequencing. However, these techniques can be expensive and tedious. Previously, CE-SSCP using the F-108 polymer was able to distinguish SNPs for the melanocortin-1 receptor (mc1r) coat color gene in horses (Equus caballus) that differed by one nucleotide substitution. The objective of this study was to expand the detection of coat color SNPs in horses. The genes for the solute carrier family member 2 (slc45a2/matp), type III receptor protein-tyrosine kinase (kit) and mc1r genes using CE-SSCP and F-108 polymer were compared to mini-sequencing with the SNaPshotTM kit. The F-108 polymer reproducibly resolved homozygous and heterozygous individuals for the mc1r and kit markers, but was unable to resolve heterozygous individuals for slc45a2 at 38ºC. The need for temperatures <15ºC, the SNP position being close to the 5'-end, and conformational structures/free energy with similar values resulted in the inability to resolve the secondary structures. Despite this limitation, the CE-SSCP method could be used to provide a rapid phenotypic description for equine forensic investigations.

Utility of genetic variation in coat color genes to distinguish wild, domestic and hybrid South American camelids for forensic and judicial applications.

A molecular genetic protocol for distinguishing pure and hybrid South American camelids was developed to provide strong, quantifiable, and unbiased species identification. We detail the application of the approach in the context of a criminal case in the Andes Mountains of central Chile where the defendants were alleged to have illegally hunted three wild guanacos (Lama guanicoe), as opposed to hybrid domestic llama (Lama glama)/wild guanaco crosses, which are unregulated. We describe a workflow that differentiates among wild, domestic and hybrid South American camelids (Lama versus Vicugna) based on mitochondrial cytochrome b genetic variation (to distinguish between Lama and Vicugna), and MC1R and exon 4 variation of the ASIP gene (to differentiate wild from domestic species). Additionally, we infer the population origin and sex of each of the three individuals from a panel of 15 autosomal microsatellite loci and the presence or absence of the SRY gene. Our analyses strongly supported the inference that the confiscated carcasses corresponded with 2 male and 1 female guanacos that were hunted illegally. Statistical power analyses suggested that there was an extremely low probability of misidentifying domestic camelids as wild camelids (an estimated 0 % Type I error rate), or using more conservative approached a 1.17 % chance of misidentification of wild species as domestic camelids (Type II error). Our case report and methodological and analytical protocols demonstrate the power of genetic variation in coat color genes to identify hybrids between wild and domestic camelid species and highlight the utility of the approach to help combat illegal wildlife hunting and trafficking.

A Third MLPH Variant Causing Coat Color Dilution in Dogs.

Altered melanosome transport in melanocytes, resulting from variants in the melanophilin (MLPH) gene, are associated with inherited forms of coat color dilution in many species. In dogs, the MLPH gene corresponds to the D locus and two variants, c.-22G > A (d1) and c.705G > C (d2), leading to the dilution of coat color, as described. Here, we describe the independent investigations of dogs whose coat color dilution could not be explained by known variants, and who report a third MLPH variant, (c.667_668insC) (d3), which leads to a frameshift and premature stop codon (p.His223Profs*41). The d3 allele is found at low frequency in multiple dog breeds, as well as in wolves, wolf-dog hybrids, and indigenous dogs. Canids in which the d3 allele contributed to the grey (dilute) phenotype were d1/d3 compound heterozygotes or d3 homozygotes, and all non-dilute related dogs had one or two D alleles, consistent with a recessive inheritance. Similar to other loci responsible for coat colors in dogs, this, alongside likely additional allelic heterogeneity at the D locus, or other loci, must be considered when performing and interpreting genetic testing.

Sex-biased expression between guppies varying in the presence of ornamental coloration.

Sex-biased gene expression provides a means to achieve sexual dimorphism across a genome largely shared by both sexes. Trinidadian guppies are ideal to examine questions of sex-bias as they exhibit sexual dimorphism in ornamental coloration with male only expression. Here we use RNA-sequencing to quantify whole transcriptome gene expression differences, with a focus on differential expression of color genes between the sexes. We determine whether males express genes positively correlated with coloration at higher levels than females. We find that all the differentially expressed color genes were more highly expressed by males. Males also expressed all known black melanin synthesis genes at higher levels than females, regardless of whether the gene was significantly differentially expressed in the analysis. These differences correlated with the visual color differences between sexes at the stage sampled, as all males had ornamental black coloration apparent. We propose that sexual dimorphism in ornamental coloration is caused by male-biased expression of color genes.

Biological characteristics of mouse skin melanocytes.

The objective of this research was to evaluate the optimal passage number according to the biological characteristics of mouse skin melanocytes from different passages. Skin punch biopsies harvested from the dorsal region of 2-day old mice were used to establish melanocyte cultures. The cells from passage 4, 7, 10 and 13 were collected and evaluated for their melanogenic activity. Histochemical staining for tyrosinase (TYR) activity and immunostaining for the melanocyte specific markers including S-100 antigen, TYR, tyrosinase related protein 1 (TYRP1), tyrosinase related protein 2 (TYRP2) and micropthalmia associated transcription factor (MITF) confirmed purity and melanogenic capacity of melanocytes from different passages, with better melanogenic activity of passage 10 and 13 cells being observed. Treatment of passage 13 melanocytes with α-melanocyte stimulating hormone (α-MSH) showed increased expression of MITF, TYR and TYRP2 mRNA. However, considering the TYR mRNA dramatically high expression which is the characteristics of melanoma cells, melanocytes from passage 10 was the optimal passage number for the further research. Our results demonstrate culture of pure populations of mouse melanocytes to at least 10 passages and illustrate the potential utility of passage 10 cells for studies of intrinsic and extrinsic regulation of genes controlling pigmentation and coat color in mouse.