Identification of novel genes associated with litter size of indigenous sheep population in Xinjiang, China using specific-locus amplified fragment sequencing technology.

BACKGROUND: There are abundant sheep breed resources in the Xinjiang region of China attributing to its diverse ecological system, which include several high-litter size sheep populations. Previous studies have confirmed that the major high prolificacy gene cannot be used to detect high litter size. Our research team found a resource group in Pishan County, southern Xinjiang. It showed high fertility with an average litter size of two to four in one birth, excellent breast development, and a high survival rate of lambs. In the present study, we used this resource as an ideal sample for studying the genetic mechanisms of high prolificacy in sheep. METHODS: Indigenous sheep populations from Xinjiang, with different litter sizes, were selected for the research, and specific-locus amplified fragment sequencing (SLAF-seq) technology was used to comprehensively screen single nucleotide polymorphisms (SNPs) from the whole genome that may cause differences in litter size. Novel genes associated with litter size of sheep were detected using genome-wide association studies (GWAS), providing new clues revealing the regulation mechanism of sheep fecundity. Candidate genes related to ovulation and litter size were selected for verification using Kompetitive Allele Specific polymerase chain reaction (KASP) cluster analysis. RESULTS: We identified 685,300 SNPs using the SLAF-seq technique for subsequent genome-wide analysis. Subsequently, 155 SNPs were detected at the genome-wide level. Fourteen genes related to sheep reproduction were notated: COIL, SLK, FSHR, Plxna3, Ddx24, CXCL12, Pla2g7, ATP5F1A, KERA, GUCY1A1, LOC101107541, LOC101107119, LOC101107809, and BRAF. Based on literature reports, 30 loci of seven genes and candidate genes (CXCL12, FSHR, SLK, GUCY1A1, COIL, LOC101107541, and LOC101107119) related to ovulation and litter size were selected for verification using KASP cluster analysis. Among them, nine loci of three genes were successfully genotyped. Three loci of FSHR (GenBank ID: 443299, g. 75320741G>A site), GUCY1A1 (GenBank ID: 101110000, g. 43266624C>T site), and COIL (GenBank ID: 101123134, g. 7321466C>G site) were found to be significantly or extremely significantly associated with litter size. These three loci are expected to be used as molecular markers to determine differences in litter size in sheep.

Smad4 Feedback Enhances BMPR1B Transcription in Ovine Granulosa Cells.

BMPR1B is a type 1B receptor of the canonical bone morphogenetic protein (BMP)/Sma- and mad-related protein (Smad) signaling pathway and is well known as the first major gene associated with sheep prolificacy. However, little is known about the transcriptional regulation of the ovine BMPR1B gene. In this study, we identified the ovine BMPR1B gene promoter and demonstrated that its transcription was regulated by Smad4. In sheep ovarian follicles, three transcriptional variants of BMPR1B gene with distinct transcription start sites were identified using 5' RACE assay while variants II and III were more strongly expressed. Luciferase assay showed that the region -405 to -200 nt is the PII promoter region of variant II. Interestingly, two putative Smad4-binding elements (SBEs) were detected in this region. Luciferase and ChIP assay revealed that Smad4 enhances PII promoter activity of the ovine BMPR1B gene by directly interacting with SBE1 motif. Furthermore, in the ovine granulosa cells, Smad4 regulated BMPRIB expression, and BMPRIB-mediated granulosa cells apoptosis. Overall, our findings not only characterized the 5' regulatory region of the ovine BMPR1B gene, but also uncovered a feedback regulatory mechanism of the canonical BMP/Smad signaling pathway and provided an insight into the transcriptional regulation of BMPR1B gene and sheep prolificacy.

Association analysis of polymorphisms in six keratin genes with wool traits in sheep.

OBJECTIVE: The purpose of this study was to investigate the genetic effects of six keratin (KRT) genes on the wool traits of 418 Chinese Merino (Xinjiang type) (CMXT) individuals. METHODS: To explore the effects and association of six KRT genes on sheep wool traits, The polymerase chain reaction-based single-strand conformation polymorphism (PCR-SSCP), DNA sequencing, and the gene pyramiding effect methods were used. RESULTS: We report 20 mutation sites (single-nucleotide polymorphisms) within the six KRT genes, in which twelve induced silent mutations; five induced missense mutations and resulted in Ile→Thr, Glu→Asp, Gly→Ala, Ala→Ser, Se→His; two were nonsense mutations and one was a same-sense mutation. Association analysis showed that two genotypes of the KRT31 gene were significantly associated with fiber diameter (p<0.05); three genotypes of the KRT36 gene were significantly associated with wool fineness score and fiber diameter (p<0.05), three genotypes of the KRT38 gene were significantly associated with the number of crimps (p< 0.05); and three genotypes of the KRT85 gene were significantly associated with wool crimps score, body size, and fiber diameter (p<0.05). Analysis of the gene pyramiding effect between the different genotypes of the gene loci KRT36, KRT38, and KRT85, each genotype in a gene locus was combined with all the genotypes of another two gene loci and formed the different three loci combinations, indicated a total of 26 types of possible combined genotypes in the analyzed population. Compared with the other combined genotypes, the combinations CC-GG-II, CC-HH-IJ, CC-HH-JJ, DD-HH-JJ, CC-GH-IJ, and CC-GH-JJ at gene loci KRT36, KRT38, and KRT85, respectively, had a greater effect on wool traits (p<0.05). CONCLUSION: Our results indicate that the mutation loci of KRT31, KRT36, KRT38, and KRT85 genes, as well as the combinations at gene loci KRT36, KRT38, and KRT85 in CMXT have significant effects on wool traits, suggesting that these genes are important candidate genes for wool traits, which will contribute to sheep breeding and provide a molecular basis for improved wool quality in sheep.

A PCR amplification method without DNA extraction.

To develop a simple and inexpensive method for direct PCR amplification of animal DNA from tissues, we optimized different components and their concentration in lysis buffer systems. Finally, we acquired the optimized buffer system composed of 10 mmol tris(hydroxymethyl)aminomethane (Tris)-Cl (pH 8.0), 2 mmol ethylene diamine tetraacetic (EDTA) (pH 8.0), 0.2 mol NaCl and 200 µg/mL Proteinase K. Interestingly, the optimized buffer is also very effective when working with common human sample types, including blood, buccal cells and hair. The direct PCR method requires fewer reagents (Tris-Cl, EDTA, Protease K and NaCl) and less incubation time (only 35 min). The cost of treating every sample is less than $0.02, and all steps can be completed on a thermal cycler in a 96-well format. So, the proposed method will significantly improve high-throughput PCR-based molecular assays in animal systems and in common human sample types.

Evaluación de métodos de corrección para efectos ambientales para peso al destete en corderos Suffolk / Evaluation of adjustment methods for environmental effects for weaning weight in Suffolk lambs

Data of weaning weights (WW) from 2 172 Suffolk lambs with complete genealogical information, obtained from a flock in central Mexico from 1992 to 2004, were analyzed using mixed linear models with direct and maternal effects, to generate correction factors and to compare different methods for the adjustment of sex, type of birth and age of the mother effects. The methods compared were: analysis of weaning weights with a complete mixed model (PDMOD), analysis of WW preadjusted to 68 days and for sex and age of the mother-type of birth with factors developed from this population (PD68PRE), analysis of WW adjusted to 68 days with a complete model (PD68MOD) and analysis of WW preadjusted to 68 days and for sex, age of the mother and type of birth with factors developed for the Suffolk population of the United States of America (PD68USA). The effects of year of birth, sex, type of birth, age of the mother, year of birth x age of the mother interaction and the linear and quadratic effects of weaning age were all significant (P < 0.01). The inclusion of all the effects in the model gave slightly smaller residual coefficients of variation with reductions < 0.51 %, compared to preadjusted data with correction factors generated either in the flock or adapted from those suggested for the Suffolk population of the United States of America. Small differences between methods in the ranking of the animals according to the genetic evaluations based on empirical BLUP's for direct and maternal genetic effects were found, with Spearman's correlation values > 0.96.

Datos de pesos al destete (PD) de 2 172 corderos Suffolk con información genealógica completa, obtenidos de 1992 a 2004 en un rebaño en el centro de México, fueron analizados usando modelos lineales mixtos con efectos directos y maternos, para generar factores de corrección y comparar diferentes métodos de ajuste para los efectos de sexo, tipo de nacimiento y edad de la madre. Los métodos comparados fueron: análisis de PD con un modelo completo (PDMOD), análisis de PD preajustados a 68 días y para sexo, edad de la madre-tipo de nacimiento con factores desarrollados a partir de esta población (PD68PRE), análisis de PD ajustados a 68 días con un modelo completo (PD68MOD) y análisis de PD preajustados a 68 días y para sexo, edad de la madre y tipo de nacimiento con factores desarrollados para la población Suffolk de los Estados Unidos de América (PD68USA). Los efectos de año de nacimiento, sexo, tipo de nacimiento, edad de la madre, interacción año de nacimiento x edad de la madre y edad al destete lineal y cuadrática fueron significativos (P < 0.01). La inclusión de todos los efectos en el modelo dio coeficientes de variación residual ligeramente menores, con reducciones < 0.51%, en comparación con el uso de datos preajustados con factores de corrección generados en el propio rebaño o adaptados de los recomendados para la población Suffolk de los Estados Unidos de América. Las diferencias entre los métodos en la jerarquización de los animales de acuerdo con las evaluaciones genéticas con los BLUP empíricos de los efectos directos y maternos fueron pequeñas, con valores de correlaciones de Spearman > 0.96.