RESUMO
Tibetan sheep, an indigenous breed, have a wide variety of phenotypes and a colorful coat, which make this breed an interesting model for evaluating the effects of coat-color gene mutations on this phenotypic trait. The agouti signaling protein (ASIP) gene is a positional candidate gene, as was inferred based on previous study. In our research, ASIP gene copy numbers in genomic DNA were detected using a novel approach, and the exon 2 g.100-104 mutation and copy number variation (CNV) of ASIP were associated with coat color in 256 sheep collected from eight populations with different coat colors by high-resolution melting curve assay. We found that the relative copy numbers of ASIP ranged from one to eight in Tibetan sheep. All of the g.100-104 genotypes in the populations were in Hardy-Weinberg equilibrium, and there was no relationship between the g.100-104 genotype and coat color (P > 0.05). The single ASIP CNV allele was found to be almost entirely associated with solid-black coat color; however, not all solid-black sheep displayed the putative single ASIP CNV genotype. From our study, we speculate that the ASIP CNV is under great selective pressure and the single ASIP CNV allows selection for black coat color in Tibetan sheep, but this does not explain all black phenotypes in Tibetan sheep.
Assuntos
Proteína Agouti Sinalizadora/genética , Proteína Agouti Sinalizadora/fisiologia , Cabelo/fisiologia , Pigmentação , Carneiro Doméstico/genética , Alelos , Animais , Cor , DNA/genética , Variações do Número de Cópias de DNA , Éxons , Genômica , Genótipo , Íntrons , Mutação , Fenótipo , Polimorfismo de Nucleotídeo Único , TibetRESUMO
Wool is produced via synthetic processes of wool follicles, which are embedded in the skin of sheep. The development of new-generation sequencing and RNA sequencing provides new approaches that may elucidate the molecular regulation mechanism of wool follicle development and facilitate enhanced selection for wool traits through gene-assisted selection or targeted gene manipulation. We performed de novo transcriptome sequencing of skin using the Illumina Hiseq 2000 sequencing system in sheep (Ovis aries). Transcriptome de novo assembly was carried out via short-read assembly programs, including SOAPdenovo and ESTScan. The protein function, clusters of orthologous group function, gene ontology function, metabolic pathway analysis, and protein coding region prediction of unigenes were annotated by BLASTx, BLAST2GO, and ESTScan. More than 26,266,670 clean reads were collected and assembled into 79,741 unigene sequences, with a final assembly length of 35,447,962 nucleotides. A total of 22,164 unigenes were annotated, accounting for 36.27% of the total number of unigenes, which were divided into 25 classes belonging to 218 signaling pathways. Among them, there were 17 signal paths related to hair follicle development. Based on mass sequencing data of sheepskin obtained by RNA-Seq, many unigenes were identified and annotated, which provides an excellent platform for future sheep genetic and functional genomic research. The data could be used for improving wool quality and as a model for human hair follicle development or disease prevention.
Assuntos
Carneiro Doméstico/genética , Pele/metabolismo , Transcriptoma , Lã/fisiologia , Animais , Mapeamento de Sequências Contíguas , Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Biblioteca Gênica , Genômica , Modelos Genéticos , Anotação de Sequência Molecular , Análise de Sequência de RNARESUMO
The desmoglein 4 (DSG4) gene is a potential candidate in the search for genes that may affect wool traits, because of its function. This study aimed to screen for polymorphisms in partial exon 16 and 3êUTR of the sheep desmoglein 4 DSG4 gene, and to test its possible association with wool length and crimp associated with fur. Overall, 326 sheep were scanned via single-strand conformational polymorphism assay, through three pairs of primers. The breeds included Tan, Han, and TanxHan from China, Polled Dorset from Australia, and Suffolk from Britain genotypes AA, BB, and AB for primer2 and genotypes DD, EE, and DE for primer3 were detected in native breeds. Six SNPs and 3-bp insertion/deletions were found in exon 16, of which 4 lead to amino acid substitutions. In addition, 1 SNP was found in 3êUTR. The DSG4 genotype was found to be strongly associated with all wool traits that were considered in this study (P < 0.01). Sheep with the genotype MM had a higher least square mean compared to sheep with the genotype WW or WM with respect to birth scapular wool length (P < 0.01), crimp number of birth scapular wool crimp (P < 0.01), crimp number of weaning scapular wool crimp (P < 0.01), and crimp number of weaning rump wool crimp (P < 0.01, P < 0.05). In conclusion, our study is the first to demonstrate that the DSG4 gene may be a candidate, or major gene, which influences important wool traits.
Assuntos
Desmogleínas/genética , Estudos de Associação Genética , Desequilíbrio de Ligação , Fenótipo , Característica Quantitativa Herdável , Ovinos/genética , Lã/crescimento & desenvolvimento , Animais , Frequência do Gene , Genótipo , Haplótipos , Polimorfismo Genético , Análise de Sequência de DNARESUMO
Variation in microsatellite or simple sequence repeat (SSR) loci has, until recently, relied heavily on the use of gel-based methods that can be both time consuming and difficult to genotype. Non gel-based systems are therefore important to increase simplicity and improve turn-around time without compromising assay sensitivity and accuracy. In this report, we assessed the latest of the non-gel-based methods, high-resolution melting (HRM) curve analysis. HRM is a technique that monitors exactly the decreasing fluorescence of intercalating dye in the process of dissociation of double-stranded DNA. The measurement immediately follows polymerase chain reaction in a one-step, closed-tube method. Four SSR loci of different complexity in sheep, namely MAF209, MCM140, CB226, and SRCRSP5, were assessed using the LightScanners System with LC Greens PLUS DNA binding dye. In order to improve the accuracy of genotyping, we applied internal oligo nucleotide calibrators while performing HRM. DNA polymorphisms were previously identified using capillary electrophoresis analysis (CE). The result showed that CE detected more genotypes than HRM in the same loci regardless of the level of polymorphism at the SSR loci. We demonstrate current limitations of the HRM method for the analysis of SSR loci.