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. 2017 Jul;7(3):204.
doi: 10.1007/s13205-017-0837-z. Epub 2017 Jun 30.

qPCR and HRM-based diagnosis of SNPs on growth differentiation factor 9 (GDF9), a gene associated with sheep (Ovis aries) prolificacy

Raquel Anahí Escobar-Chaparro  1   2 , Gabriel Guillén  3 , Luis Uribe Espejo-Galicia  2 , Víctor Manuel Meza-Villalvazo  2 , Julián Mario Peña-Castro  2 , José Abad-Zavaleta  4
Affiliations

qPCR and HRM-based diagnosis of SNPs on growth differentiation factor 9 (GDF9), a gene associated with sheep (Ovis aries) prolificacy

Raquel Anahí Escobar-Chaparro et al. 3 Biotech. 2017 Jul.

Abstract

Prolificacy is a desirable trait for genetic improvement of sheep flocks, since it holds the potential to improve productivity. Animals carrying single-nucleotide polymorphisms (SNPs) in genes associated with this trait can be identified and employed to increase prolificacy in flocks. In this study, we report a diagnostic method based on quantitative PCR and high-resolution melting curves to detect different SNPs in the prolificacy-associated gene growth differentiation factor 9 (GDF9). The diagnostic method was validated using artificial sequences representing known SNPs in GDF9, then applied to a real flock comprising four breeds and admixed animals (n = 306). Five different SNPs were identified in this flock, as was a low or null frequency of occurrence of SNPs positively associated with prolificacy. This indicates a need to implement a breeding strategy for recovering or reintroducing such SNPs. Our method provides a genotyping strategy for identifying individuals with SNPs of interest for prolificacy, which will help producers plan a breeding strategy for this trait. This method can be adapted and expanded for the diagnosis of other traits of interest.

Keywords: Animal breeding; Fertility; Genomic DNA; Genotyping.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
qPCR-HRM analysis for G1 single-nucleotide polymorphism (SNP) using synthetic controls and two different intercalating dyes. Normalized melting curves (NMC, left) and differential curves (DC, right) are shown using intercalating dye. a SYBR-Green or b EVA-Green. Colors correspond to a different SNP as indicated. Two independent experimental replicates are presented. RFU relative fluorescence units
Fig. 2
Fig. 2
Genotyping of sheep GDF9 single-nucleotide polymorphisms (SNPs) by qPCR-HRM and validation by sequencing. a–d Normalized melting curves and differential curves produced using SYBR-Green. Colors correspond to different SNPs as indicated in the legend. Two independent experimental replicates are presented for controls, and for sheep gDNA, all analyzed in the same plate. e–k Electropherograms of gDNA results sequencing from representative individuals to validate the indicated SNP
Fig. 3
Fig. 3
Allelic combinations of GDF9 SNP G4 and G6 on individuals of different sheep breeds
See this image and copyright information in PMC

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