Polymorphism identification in GDF9 gene and its association analysis with reproduction traits in Jinghai Yellow chicken.

GDF9 (growth differentiation factor 9) belongs to the transforming growth factor-ß (TGF-ß) superfamily and plays an irreplaceable role in female fertility. To reveal its genetic effects on productivity performance in chickens, 373 Jinghai Yellow chickens were chosen randomly to detect SNPs in GDF9 by PCR-SSCP and DNA sequencing methods. Eventually, four SNPs (g.2053G > A, g.2275T > C, g.2338C > T, g.2420T > C) in total had been detected. Amongst them, g.2420T > C was first found significantly associated with reproduction trait in chickens and heterozygous type C2T2 had higher average egg weight at 300 days of age (AEWD300) than T2T2 (p < 0.01). Least squares analysis showed that age at first laying (AFE) of H1 and H1H1 chickens were significantly earlier than that of H7 and H7H7 ones, respectively (p < 0.05). H1H5 hens showed higher AEWD300 than H4H7 ones (p < 0.05). For total egg number at 300 days of age (END300), mean of H5H5 was significantly higher than that of H4H4 (p < 0.05). Hence, the study suggested that hybrid vigor at g.2420T > C could be utilized in practice. H1H1, H1H5 and H5H5 could be the dominant diplotypes for chicken breeding. The study may contribute to the breeding progress of productive chickens and supply reference for oviparous animal production practice.

Genetic polymorphism of growth differentiation factor 9 (GDF9) gene related to fecundity in two Egyptian sheep breeds.

PURPOSE: This study explores polymorphisms in the growth differentiation factor 9 (GDF9) gene (exon 1) with respect to fertility in Egyptian sheep. METHODS: Blood samples were collected, and genomic DNA was extracted from 24 Saidi and 13 Ossimi ewes. A 710 bp portion of the GDF9 gene, was amplified using specific primers, and the sequence was analyzed to clarify the phylogenetic relationship of Egyptian breed sheep. In addition, the PCR-RFLP method using Pst1 or Msp1 restriction enzymes was used to mask polymorphisms of partial exon 1 of GDF9 gene to establish molecular markers for twinning. RESULTS: The lambing rate percentage and litter size showed significant difference between ewes, which produce single and twin lamb for each breed individually, whereas the coefficient of variation of the Saidi breed is greater than that of the Ossimi breed. The results suggested that the GDF9 gene shared a similarity in sequence compared to six accession numbers of Ovis aries found in GenBank. Molecular phylogenetic analyses were performed based on nucleotide sequences in order to examine the position of the Egyptian breeds among many other sheep breeds. The results indicate that accession number AF078545 of O. aries is closely related with Saidi and Ossimi ewes that produce single or twin lamb using the unweighted pair group method with arithmetic mean (UPGMA) analysis. Results showed that Msp1 enzyme digestion revealed polymorphic restriction pattern consisting of one band with 710 bp for ewes producing single lamb and two bands with 710 and 600 bp for ewes producing twin lamb in Saidi sheep breed. CONCLUSION: Sequence analysis and diversity of polymorphisms in the GDF9 gene (exon 1) have a novel base substitution (A-T) for detection of FecG mutations that serve as a molecular marker for twinning.

[Single base editing system mediates site-directed mutagenesis of genes GDF9 and FecB in Ouler Tibetan sheep].

In this study, a single base editing system was used to edit the FecB and GDF9 gene to achieve a targeted site mutation from A to G and from C to T in Ouler Tibetan sheep fibroblasts, and to test its editing efficiency. Firstly, we designed and synthesized sgRNA sequences targeting FecB and GDF9 genes of Ouler Tibetan sheep, followed by connection to epi-ABEmax and epi-BE4max plasmids to construct vectors and electrotransfer into Ouler Tibetan sheep fibroblasts. Finally, Sanger sequencing was performed to identify the target point mutation of FecB and GDF9 genes positive cells. T-A cloning was used to estimate the editing efficiency of the single base editing system. We obtained gRNA targeting FecB and GDF9 genes and constructed the vector aiming at mutating single base of FecB and GDF9 genes in Ouler Tibetan sheep. The editing efficiency for the target site of FecB gene was 39.13%, whereas the editing efficiency for the target sites (G260, G721 and G1184) of GDF9 gene were 10.52%, 26.67% and 8.00%, respectively. Achieving single base mutation in FecB and GDF9 genes may facilitate improving the reproduction traits of Ouler Tibetan sheep with multifetal lambs.

Chicken ovarian follicles morphology and growth differentiation factor 9 gene expression in chicken ovarian follicles: review.

Ovary follicular development is a progressive system from the beginning of small cortical follicles to the ovulation of hierarchical follicles. The review was conducted to provide information on the indigenous chickens commonly used for egg production, chicken ovarian follicles morphology and expression of growth differentiation factor 9 (GDF9) gene in ovarian follicles and its relationship with egg production. The research databases used in the study include google scholar, Science Direct, PubMed, JSTOR and Cambridge Core. Google, Yahoo and Baidu search engines were used to search the information. In this study, the papers selected for use were original research articles and reviews to ensure that the information used was from research results. Besides, only recent English papers, 2010-2021, were used. The keywords used to search for articles were chicken ovarian follicles, ovarian morphology and GDF9 gene expression. The documents showed that pre-hierarchical follicles include many small and large white follicles, which are about 2-5mm in diameter and 5 to 6 small yellow follicles (SYF) that are about 5-10mm in diameter. Preovulatory follicles are about five to six in number and above 10mm in diameter, with the sizes from F6 to F1, with F1 as the largest follicle. Further, the studies revealed that GDF9 gene mRNA is expressed in the highest concentration in small yellow follicles and other studies reported that the expression of GDF9 gene has been found in follicles of the primary to preovulatory stages in chickens. This review concludes that the GDF9 gene expression is mainly throughout follicular growth and it stimulates the proliferation of pre-hierarchical granulosa cells. The increased egg production in chickens depends on progressive developmental stages and the growth of ovarian follicles.

Polymorphism Detection of GDF9 Gene and Its Association with Litter Size in Luzhong Mutton Sheep (Ovis aries).

Litter size is one of the most important economic traits in sheep. GDF9 and BMPR1B are major genes affecting the litter size of sheep. In this study, the whole coding region of GDF9 was sequenced and all the SNPs (single nucleotide polymorphisms) were determined in Luzhong mutton ewes. The FecB mutation was genotyped using the Sequenom MassARRAY®SNP assay technology. Then, the association analyses between polymorphic loci of GDF9 gene, FecB, and litter size were performed using a general linear model procedure. The results showed that eight SNPs were detected in GDF9 of Luzhong mutton sheep, including one novel mutation (g.41769606 T > G). The g.41768501A > G, g.41768485 G > A in GDF9 and FecB were significantly associated with litter size in Luzhong mutton ewes. The g.41768485 G > A is a missense mutation in the mature GDF9 protein region and is predicted to affect the tertiary structure of the protein. The results preliminarily demonstrated that GDF9 was a major gene affecting the fecundity of Luzhong mutton sheep and the two loci g.41768501A > G and g.41768485 G > A may be potential genetic markers for improving litter size.

Age-related decline in the expression of GDF9 and BMP15 genes in follicle fluid and granulosa cells derived from poor ovarian responders.

BACKGROUND: Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) genes play important roles in folliculogenesis. Altered expression of the two have been found among patients with poor ovarian response (POR). In this prospective cohort study, we have determined the expression of the GDF9 and BMP15 genes in follicle fluid (FF) and granulosa cells (GCs) derived from poor ovarian responders grouped by age, and explored its correlation with the outcome of in vitro fertilization and embryo transfer (IVF-ET) treatment. METHODS: A total of 196 patients with POR were enrolled from a tertiary teaching hospital. The patients were diagnosed by the Bologna criteria and sub-divided into group A (< 35 year old), group B (35-40 year old), and group C (> 40 year old). A GnRH antagonist protocol was conducted for all patients, and FF and GCs were collected after oocyte retrieval. Expression of the GDF9 and BMP15 genes in the FF and GCs was determined with enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. RESULTS: Compared with group C, groups A and B had significantly more two pronuclei (2PN) oocytes and transplantable embryos, in addition with higher rates of implantation and clinical pregnancy (P <  0.05). The expression level of GDF9 and BMP15 genes in the FF and GCs differed significantly among the three groups (P <  0.05), showing a trend of decline along with age. The ratio of GDF9/BMP15 mRNA levels were similar among the three groups (P > 0.05). The relative levels of GDF9 and BMP15 proteins in GCs have correlated with the relative mRNA levels in GCs and protein concentrations in FF (P <  0.05). CONCLUSIONS: For poor ovarian responders, in particular those over 40, the expression of GDF9 and BMP15 is declined along with increased age and in accompany with poorer oocyte quality and IVF outcome, whilst the ratio of GDF9/BMP15 mRNA levels remained relatively constant. TRIAL REGISTRATION: Chinese Clinical Trial Registry Center ( ChiCTR1800016107 ). Registered on 11 May 2018.

A study on mutation points of GDF9 gene and their association with prolificacy in Egyptian small ruminants.

BACKGROUND: Genetic variants of the GDF9 gene were considered to be the potent gene markers for improving fecundity traits in Egyptian sheep and goats. Also, these favorable gene variants could be applied in the breeding program by gene-assisted selection (GAS), aiming towards the potential amelioration of reproduction and production in such small ruminants. The present investigation was designed to evaluate the genetic variants of the GDF9 gene on fecundity traits including the mean number of lambing "MNL" and mean number of twin production "MNTP" of Egyptian sheep and goats. RESULTS: This experiment involved 113 mothers, 83 of sheep and 30 of goats, at first, second, third, and fourth parity, and also 26 young females, 12 of sheep and 14 of goats at age of sexual maturation. T-ARMS-PCR analysis was performed on five mutation points (G1, G4, G6, G7, and G8). In sheep, the heterozygous mothers of G4 had significant elevation (P ≤ 0.05) of MNL and MNTP than wild-type homozygous ewes. However, the heterozygous mothers of G1 and G6 gave a reduction of MNL and MNTP as compared to mothers with wild-type genotypes. The ewes of G7 had heterozygous genotype (AG), and the ewes of G8 had wild type (CC). In goat, G4 and G7 were polymorphic, and G1, G6, and G8 were monomorphic type. Based on these findings, it must be selected the young sheep females of heterozygous in G4, and the young goat females of heterozygous in G4 and G7 for participating in a successful breeding program, because they will have potential high fecundity traits. CONCLUSION: The present results confirmed that the genetic variants of the GDF9 gene were considered to be the major gene markers for enhancement of the prolificacy in Egyptian sheep and goats and could be applied in a successful breeding program by gene-assisted selection (GAS) in small ruminants.

Two Novel Rare Strongly Linked Missense SNPs (P27R and A85G) Within the GDF9 Gene Were Significantly Associated With Litter Size in Shaanbei White Cashmere (SBWC) Goats.

Growth differentiation factor 9 (GDF9) is a high-fertility candidate gene that plays a crucial role in early folliculogenesis in female mammals. In this study, direct sequencing was used to screen possible SNP loci in the goat GDF9 gene. Three SNP loci, p.proline27alanine (P27R), p.leucine61leucine (L61L), and p.alanine85glycine (A85G), were identified in Shaanbei white cashmere (SBWC) goats. Among the three SNPs, two rare missense SNP loci (P27R and A85G) were discovered to be strongly linked with each other (D' value = 0.926, r 2 value = 0.703). Both P27R and A85G loci had two genotypes: wild type and heterozygous type. A85G exerted a significant effect on litter size (P = 0.029) in SBWC goats, and the heterozygous genotype was superior in comparison with the wild type. The heterozygous genotype was also superior in P27R but no significant association was found. However, the combination genotypes of P27R and A85G were identified to have superior effects on litter size (P = 3.8E-15). This information suggested that these two SNPs influenced litter size in goats synergistically. Combining this information with our previous studies, we propose that the GDF9 gene is the principal high-fertility candidate gene and that the A85G locus is a promising SNP that affects litter size in goats. These results may fill a research gap regarding rare mutations as well as provide crucial molecular markers that could be useful in marker-assisted selection (MAS) goat rearing when selecting superior individuals.

Genetic Effects of Single Nucleotide Polymorphisms in the Goat GDF9 Gene on Prolificacy: True or False Positive?

Goat reproductive traits are complex quantitative traits controlled by polygenes and multipoint. To date, some high-fertility candidate genes in livestock have been unearthed and the growth differentiation factor 9 (GDF9) gene is one of them, which plays a crucial role in early folliculogenesis. According to the relevant previous studies and the National Center for Biotechnology Information Search database (NCBI), a total of 45 single nucleotide polymorphisms (SNPs) have been detected in the goat GDF9 gene, but which one or which ones have important effects on goat fecundity is still uncertain. Hence, in order to find effective molecular markers for goat genetic breeding and accelerate the goat improvement, this study summarized and classified the above 45 SNPs into four kinds, as well as compared and analyzed the same SNP effects and the different SNPs linkage effects on the reproductive traits in different goat breeds. Since there were many SNPs in the goat GDF9 gene, only 15 SNPs have been identified in more than 30 goat breeds worldwide and they showed different effects on the litter size. Therefore, this study mainly chose these 15 SNPs and discussed their relationship with goat productivity. Results showed that three non-synonymous SNPs A240V, Q320P, and V397I and three synonymous ones L61L, N121N, and L141L played a "true" role in the litter size trait in many goat breeds around the world. However, the regulatory mechanisms still need further research. These results provide an effective tool for follow-up research developing the goat molecular breeding strategies and improving the goat reproductive traits.

Two strongly linked single nucleotide polymorphisms (Q320P and V397I) in GDF9 gene are associated with litter size in cashmere goats.

Growth differentiation factor 9 (GDF9) gene is an effective intra-ovarian regulator; it plays a crucial role in early folliculogenesis in female mammals. The non synonymous mutations: g.3905A > C (also known as p.Gln320Pro/Q320P) and g.4135G > A (also know as p.Val397Ile/V397I), are two well-known and controversial single nucleotide polymorphisms (SNPs) within GDF9 gene in goats with different prolificacy, and so far, there were no studies on linkage between Q320P and V397I. Therefore, the aim of this work was to study whether Q320P and V397I mutations have a significant effect on litter size, in Shaanbei white cashmere goats (SBWC, n = 1511), and to explore the specific relationship between these two SNPs. The results showed that both of Q320P and V397I mutations exhibited three genotypes; the minor allele frequencies (MAF) of the SNPs were 0.286 and 0.477, respectively; and these two SNPs were in strong linkage disequilibrium (D' = 0.976, r2 = 0.348) in the studied goats. Moreover, association analyses revealed that Q320P was significantly associated with the first-born litter size in goats irrespective of the sample size (n = 1511; P = 0.008), while V397I significantly affected litter size until the sample size crossed 1300 (P = 0.015). Meanwhile, the diplotypes PP-II and QP-VI were observed to have a superior effect on litter size (P = 3.78 × 10-5) to that of the haplotypes (P = 1.12 × 10-7). Thus, the findings led us to assume that Q320P mutation was the major SNP affecting goat litter size. These findings can provide useful DNA markers for selecting superior individuals in marker-assisted selection (MAS) for breeding in relation to fecundity in goats.