Association of single nucleotide polymorphism in NLRC3, NLRC5, HIP1, and LRP8 genes with fecal egg counts in goats naturally infected with Haemonchus contortus.

Haemonchus contortus is a common, intractably pathogenic and economically important gastrointestinal nematode for goat producers worldwide, especially in tropical and subtropical regions. The objective of this study is to identify single nucleotide polymorphisms (SNPs) of 12 candidate goat genes mainly related to the innate immune response associated with fecal egg counts (FECs) of Haemonchus contortus in goat as an indicator of the level of parasite infection. Phenotypic data including FEC and blood traits were recorded in 189 native goats from China and 191 ones from Bangladesh, respectively. Bangladeshi goats had significantly (P < 0.01) lower FEC compared to that of Chinese goats, suggesting higher susceptible and infection rates in Chinese goat populations. FEC was significantly positive correlated with body weight (r = 0.64, P < 0.01) and hemoglobin (r = 0.49, P < 0.01) value, but negative with pack cell volume (r = - 0.63, P < 0.05) in goats. Genotyping of SNPs was performed using a matrix-assisted laser desorption ionization time of flight mass spectrometry assay and a generalized linear model was used to evaluate the association between each SNP and goat FEC trait. Eleven novel SNPs in the NLRC3, NLRC5, HIP1, and LRP8, out of 46 variants from these 12 genes, were significantly associated with FEC of goats with a nominal significance level of P < 0.05. Of these 11 SNPs, linkage disequilibrium were revealed among SNPs in LRP8 (r2 = 0.87 to 1), between SNPs in NLRC3, NLRC5, and HIP1 (r2 = 0.96 to 0.99), respectively. Further, haplotypes within NLRC3, NLRC5, and HIP1 were significantly associated (P < 0.001) with FEC. In artificial challenge trail, quantitative real-time PCR exposed that the relative expression of mRNA was higher in the resistant group for NLRC3 (P < 0.01), LRP8 and HIP1 (P < 0.001) but lower in the resistant group for NLRC5 (P < 0.0001), compared to the susceptible group. The possible SNP markers and genes identified in this study could be potentially used in marker-assisted selection for breeding local goats breeds resistant to gastrointestinal nematode parasite particularly for Haemonchus contortus, and then for improving health and productivity of goat.

A genome-wide single nucleotide polymorphism scan reveals genetic markers associated with fertility rate in Chinese Jing Hong chicken.

The function of the sperm storage tubules is directly correlated with the fertility of laying hens. However, little is known about the molecular mechanisms regulating the fertility traits in chicken. To identify genetic markers associated with reproductive traits, we calculated fertility rate at 61 to 69 wk (51 D) of Jing Hong chickens parent generation as the phenotype and the genotype were detected by the chicken 600K Affymetrix Axiom High Density single nucleotide polymorphisms (SNP)-array. The genome-wide association study using 190 Jing Hong hens showed that the 20 SNP in chromosomes 3 and 13 were significantly associated with fertility rate. To verify these results, a total of 1900 Jing Hong laying hens from 2 populations (P1 and P2) were further genotyped by polymerase chain reaction-restriction fragments length polymorphisms method. The association analysis results revealed that 12 polymorphisms (AX-75769978, AX-76582632, AX-75730546, AX-75730496, AX-75730588, AX-76530282, AX-76530329, AX-76529310, AX-75769906, AX-75755394, AX-80813697 and AX-76582809) out of 20 showed highly significant effects (P < 0.0001) on fertility rate in P1, P2 and P1+P2. Six haplotypes (TTAA, TTGG, TTAG, CTAA, CTGG, and CTAG) were inferred based on significant loci (AX-75730546 and AX-76530282) also showed significant association with fertility rate, where haplotype CTAG was shown to be markedly associated with the significantly highest (P < 0.0001) fertility rate (in P1, 86.42 ± 0.59; P2, 85.98 ± 0.59 and P1+P2, 86.16 ± 0.42) followed by other haplotypes for the irrespective of population studied. Collectively, we report for the first time that 12 SNP in the chromosomes 3 and 13 were significantly associated with fertility rate during the later stage of egg production, which could be used as the potential genetic markers that would be able to facilitate in the selection and improvement of fertility rate through chicken breeding.

Association of single nucleotide polymorphism at long non-coding RNA 8138.1 with duration of fertility in egg-laying hens.

A previous genome-wide transcriptional analysis identified long non-coding RNA 8138.1 (lncRNA8138.1) as a candidate gene related to hen duration of the fertility (DF) trait. LncRNA8138.1 gene response to growth factor and reproductive system development suggests it has a vital role in reproduction. In this study, we investigated the lncRNA8138.1 gene sequence in a population of egg-laying hens. The sequence analysis of the lncRNA8138.1 gene containing about 1.6 k nucleotides (nt) was observed with four single nucleotide polymorphisms (SNPs) and 7 nt indel including r.4937159A > G; r.4937219T > C; r.4937258G > C; r.4937318C > G and g.4937319_4937325delinsTGTGTGG. Next, the genomic DNAs from laying hen populations were subjected to polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) to detect a region of 457 bp carrying lncRNA8138.1 r.4937159A > G substitution. Further inspection of the region containing r.4937159A > G mutation revealed three genotypes viz., AA, AG, and GG were observed with respective frequencies of 0.106, 0.607, and 0.287 in laying hen population 1 (P1) (n = 1, 042) and respective frequencies of 0.176, 0.708, and 0.116 in laying hen population 2 (P2) (n = 826). Moreover, to further examining the frequencies of r.4937159A > G genotypes in P1 and P2, and their additive and dominance effects; r.4937159A > G locus was significantly associated with DF-trait in both P1 and P2 (EN: the number of eggs, FN: the number of fertile eggs after a single AI), and DN (the number of days post-insemination until last fertile egg). In testing for additive and dominance effects, additive effect was significant (P < 0.05) in both P1 and P2 for DF-trait, and the dominance effect was significant (P < 0.05) for EN and FN traits, suggesting that r.4937159A > G polymorphism is a potential biomarker for DF-trait. However, the identified novel r.4937159A > G mutation and others require further investigation to confirm phenotypic causality and potential genetic relationships with reproductive traits. Overall, our findings suggest the significance of genetic variation in long non-coding RNAs may assist in future breeding programs to improve selection for prolonged DF-trait.

Novel Polymorphisms in RAPGEF6 Gene Associated with Egg-Laying Rate in Chinese Jing Hong Chicken using Genome-Wide SNP Scan.

The improvement of egg production is of vital importance in the chicken industry to maintain optimum output throughout the laying period. Because of the elongation of the egg-laying cycle, a drop in egg-laying rates in the late laying period has provoked great concern in the poultry industry. In this study, we calculated the egg-laying rate at weeks 61-69 (60 days) of Jing Hong chickens parent generation as the phenotype, and the genotype were detected by the chicken 600K Affymetrix Axiom High Density (HD) Single Nucleotide Polymorphisms (SNP)-array. The Genome-Wide Association Study (GWAS) result showed that the egg production trait is significantly associated with five SNPs (AX-75745366, AX-75745380, AX-75745340, AX-75745388, and AX-75745341), which are in the rap guanine nucleotide exchange factor 6 (RAPGEF6) gene on chicken chromosome 13. A total of 1676 Chinese commercial Jing Hong laying hens-including two populations, P1 population (858 hens) and P2 population (818 hens)-were genotyped using the Polymerase Chain Reaction-Restriction Fragments Length Polymorphisms (PCR-RFLP) method for the association analysis of egg-laying rates for the verification of the GWAS results. Genotypic and allelic frequencies of five SNPs were inconsistent with Hardy-Weinberg equilibrium, and the average population genetics parameters considering all the SNP values; i.e., gene homozygosity (Ho), gene heterozygosity (He), the effective number of alleles (Ne), and the polymorphism information content (PIC) were 0.75, 0.25, 1.40, and 0.20 in P1; 0.71, 0.29, 1.46, and 0.24 in P2; and 0.73, 0.27, 1.43, and 0.22 in P1 + P2 populations, respectively. The association analysis results revealed that out of the five polymorphisms, three of them (AX-75745366, AX-75745340, and AX-75745341; Patent applying No: 201810428916.5) had highly significant effects on egg-laying rates according to the GWAS results. Population-specific association analyses also showed similar significant association effects with this trait. Four haplotypes (AAGG, AAAG, AGGG, and AGAG) were inferred based on significant loci (AX-75745340 and AX-75745341) and also showed significant associations with the egg-laying rate, where haplotype AAGG had the highest egg-laying rate, with the exception of the egg-laying rate in P1 population, followed by other haplotypes. Furthermore, genotypes TT, AA, and GG showed the highest egg-laying rate compared to the corresponding genotypes at AX-75745366, AX-75745340, and AX-75745341 SNP loci in P1+P2, respectively. A similar result was found in the population-specific analysis except for the P1 population, in which TC genotype showed the highest egg-laying rate. No significant association was found in the egg-laying rate during the 60 days laying period for the SNPs (AX-75745380 and AX-75745388) in any group of population (p ≥ 0.05). Collectively, we report for the first time that 3 SNPs in the RAPGEF6 gene were significantly associated with the egg-laying rate during the later stage of egg production, which could be used as the potential candidate molecular genetic markers that would be able to facilitate in the selection and improvement of egg production traits through chicken breeding.