The experimental population comprised of 101 Kuttanad ducks maintained at the University Poultry and Duck Farm, College of Veterinary and Animal Sciences, Mannuthy, Thrissur, Kerala from January to May 2022 under standard management conditions. Day-old female ducklings were wing banded, fed with starter feed (CP: 22-23%, ME: 2800 Kcal/kg) from 0 to 11 weeks of age and grower feed (CP: 18-19%, ME: 2600 Kcal/kg) from 12 to 16 weeks of age. They were vaccinated against Pasteurella and duck plague at 4, 8 weeks of age and at 12, 16 weeks of age respectively. Data on body weight was recorded individually at fortnightly intervals from hatch to 16 weeks of age.
Isolation of DNA and PCR amplification
One milliliter of blood was collected from the wing vein of each bird and transferred into vacutainer tubes containing ethylenediamine tetra acetic acid (EDTA) as anticoagulant. Genomic DNA extraction was carried out using the Quaigen DNeasy Blood and Tissue Kit (cat. Nos. 69504 and 69506), adhering to the manufacturer’s protocol with necessary modifications. The concentration, purity and quality of the extracted DNA were evaluated using a NanoDrop spectrophotometer (ThermoScientific, USA) and the resulting DNA was stored at -20°C until further use.
Oligonucleotide primers were designed from the
Anas platyrhynchos gene sequence (>NC_051799.1:c6089943-6085452
Anas platyrhynchos isolate Z2 breed Pekin duck chromosome 28, ZJU1.0, whole genome shotgun sequence) using Primer 3 (V.0.4.0) software (
http://bioinfo.ut.ee/primer30.4/) . The primers custom synthesized (Sigma-Aldrich) and selected for 366 bp fragment (exon 5 partial intron 4 and intron 3) were Forward: 5’CTCCTC CTGTCCAGTGGCTA3’ and Reverse: 5’CGATTATCGTG AGAGAGAAC3 and those for 227 bp fragment (exon 3 and partial intron 3) were Forward: 5’CGCTATTC TCCC AGTG CACG3’ and Reverse: 5’TGGAAACCCGCTG TTCATTG3’.
PCR amplification was carried out in a BIORAD thermal cycler using a reaction mixture composed of 1.5 µl of DNA template (50 ng/µl), 12.5 µl of 2X emerald green master mix, 1.5 µl of forward primer (10 pM/µl), 1.5 µl of reverse primer (10 pM/µl) and 8 µl of nuclease-free water resulting in a final volume of 25 µl. The PCR reaction conditions included an initial denaturation at 95°C for 3 min, followed by 35 cycles of denaturation at 95°C for 30s, annealing temperature of 61°C for 45 sec (exon 3 and partial intron 3) and 66.4°C for 1 min (exon 5 and partial intron 4), extension at 72°C for 1 min and a final extension at 72°C for 5 min. Subsequently, electrophoresis of PCR products was conducted in a 2% agarose gel along with a 100 bp ladder (Fermentas) using 1xTBE buffer at a constant voltage of 80 V for 50 min. The gels were stained with ethidium bromide and visualized under UV light. Gel documentation was performed with a Bio Rad gel documentation system (USA).
Genotyping of the dGH exon 5 and partial intron 4 (366 bp) locus
The exon 5 and partial intron 4 locus was genotyped by Single-Strand Conformation Polymorphism (SSCP) analysis. The PCR products of exon 5 and partial intron 4 (366 bp) locus were subjected to single-strand conformation polymorphism (SSCP) analysis. Eight microlitres of PCR products were mixed with 10 µl of denaturing dye (9.5 ml of deionized formamide, 0.4 ml of 0.5M EDTA, 2.5 mg of xylene-cyanole and 2.5 mg bromophenol blue) centrifuged, denatured at 95°C for 10 min and snap chilled immediately on ice for 10 min before conducting the PAGE. The SSCP analysis was done using vertical electrophoresis (Hoefer, USA). Denatured amplicons were loaded on 12% PAGE gel (6 ml of 30% Acrylamide / Bis-acrylamide (29:1), 10X TBE (1.5 ml), N, N, N2 , N2 , Tetra Methyl Ethylene Diamine (0.015 ml) and 10% Ammonium per sulphate (0.0832 ml) in nuclease-free water of 7.32 ml) in 1xTBE buffer with voltages of 9, 11 and 15 at respective running times of 90, 120 and 45 minutes for various PCR products. The gel was stained by a silver staining method (
Sanguinetti and Simpson, 1994). The PCR products from different SSCP patterns were sequenced in forward and reverse directions using a commercial service (SciGenom Labs Pvt. Ltd. Cochin). Nucleotide sequence alignments and comparisons were carried out using reference sequence (NC_051799. 1:c6089943-6085452) in GenBank using BLASTn and EMBOSS merger. The NCBI (National Centre for Biotechnology Information) Blast algorithm was used to search the NCBI GenBank database (
http://www.ncbi.nlm.nih.gov/) for homologous sequences.
Genotyping of the dGH exon 3 and partial intron 3 (227 bp) locus
The exon 3 and partial intron 3 region was genotyped using DNA pooling sequencing and High-Resolution Melt curve (HRM) analysis. For this, the amplicons of exon 3 and partial intron 3 (227 bp) were screened for identifying the SNPs using DNA pooling sequencing assay
(Bansal et al., 2002). A DNA pool containing PCR amplicons of four microliter of each sample was prepared and sequenced using the respective forward and reverse primers to detect the variations, if any, at nucleotide level by an automated sequencer at Agrigenome Labs Pvt. Ltd. Cochin using Sanger’s dideoxy chain termination method. The obtained sequences were aligned with other sequences in GenBank using Sequence Manipulation Suit (SMS), BLASTn (
http://www.ncbi.nlm.nih.gov/blast), Clustal Omega and EMBOSS merger (
http://emboss.bioinformatics.nl/cgi-bin/emboss/merger). The presence of SNP was confirmed only after comparing with the reference sequence using BLASTn (GenBankNC_051799.1:c6089943-6085452). The genotyping of the detected SNP was carried out using high resolution melt curve analysis (HRM) in Eco Real-Time PCR system (Illumina). The primers (HRME3F and HRME3R) were designed to amplify the regions flanking the SNP (139 bp product). The reaction consisted of 10 µL total reaction volume with 0.5 µL of genomic DNA (50 ng/ µl), 5 µL Sso Fast EVA green supermix, 0.5 µL of forward primer (10 pM/µl), 0.5 µL of reverse primer (10 pM/µl) and nuclease free water added to make up a final volume of 10µl. Thermal profile of reactions included 95°C for 10 min, 95°C for 30s, 65.4°C for 30s, 72°C for 30s followed by the melt curve analysis. The results were confirmed by sequencing the representative samples from each genotype after their detection through melting temperature (Tm) shift and the melt curve patterns.
Statistical analysis
Allelic and genotypic frequencies for each locus were calculated by the standard procedure (
Falconer and Mackay, 1996). The observed (H0) and expected (He) heterozygosity at the loci and chi-square test for Hardy-Weinberg equilibrium were estimated using Pop Gene 3.1 software. The following General Linear Model for fixed effects was used to analyze the association of
GH variants with body weight at fortnightly intervals using SPSS (V.21). Duncan multiple range test (
Kramer, 1957) was used to compare the means. The model used was:
yij = µ+gi+eij
Where,
y
ij= body weight measured on ij
th bird at different stages;
ì= Overall mean.
g
i= Fixed effect associated with i
th genotype (i = 1, 2,3);
e
ij= Random error.
Least squares means and their standard errors were computed for all the genotype effects using SPSS V.21.
