Identification of suitable reference genes for normalization of quantitative real-time PCR-based gene expression in chicken (Gallus gallus).

The recent availability of genome information greatly facilitates the fundamental research on chicken. In different organs, gene expression patterns can provide clues to understanding the biological functions. For rapid and accurate quantification of gene expression, quantitative real-time PCR (qPCR) has become one of the most widely used methods. However, the success of qPCR data normalization depends on the use of a suitable reference gene and a single reference gene is not universally suitable for all the experiments. Therefore, reference gene validation is a crucial step for different organ tissues of chicken where suitable reference genes for qPCR analysis in varieties of tissues have not been investigated exhaustively so far. In this study, we have selected 30 Gallus gallus candidate reference genes from NCBI, amplified and studied their expression profiles by qPCR in different organ tissues (breast muscle, thigh muscle, heart, liver, spleen, gizzard, and bursa) of chicken. The result showed that, for breast muscle HSP10 and RPL23, thigh muscle RPL14 and RPL13, liver ALB and HSP70, spleen ALB and GAPDH, heart CYCS and TUBA8B, gizzard RPL5 and 18S rRNA, and bursa EEF1A1 and PGK2 are most stable genes respectively. The results also showed that for different organ tissues, individual or a combination of reference genes should be selected for data normalization. In this study, we have identified and validated 30 reference genes in seven different organ tissues to provide accurate transcript normalization and quantification, which can be useful for gene expression studies in other avian species.

Molecular Characterization of Mx1 Gene in Native Indian Breeds of Chicken.

The genetic polymorphism of Mx1 gene was explored in Indian chicken breeds. PCR-RFLP analysis in 102 bp fragment of partial intron 13 and partial exon 14 of Mx1 gene revealed two genotypes viz. RS and SS with two alleles viz. R and S both in Naked Neck and Tellicherry breeds of chicken. The homozygous genotype RR was not identified. When deduced amino acid sequences were compared, the asparagine amino acid was found to be substituted in "R" allele for serine in "S" allele. PCR-SSCP analysis of 284 bp fragment in 5'-UTR and partial promoter region revealed three genotypes viz. CC, CG, and CH with three different alleles viz. C, G, and H in Naked Neck breed of chicken and five genotypes viz. DI, JK, KK, KL, and KM with six different alleles viz. D, I, J, K, L, and M in Tellicherry breed of chicken. The homozygous genotypes viz. GG and HH in Naked Neck and DD, II, JJ, LL, and MM in Tellicherry chicken was not identified. The nucleotide substitution rate estimated to be in the range of 0.004-0.011. The identified genetic variation can be helpful for better insight to disease resistance property of the Mx1 gene.

Expression Profiling of Activin type IIB Receptor During Ontogeny in Broiler and Indigenous Chicken.

Augmenting the meat production is among the primary breeding objective of genetic selection programs in poultry production. However, the knowledge about the expression of genes regulating muscle growth at the molecular level is inadequate. Activin type IIB receptor (ACTRIIB) has been reported to play vital role in the negative regulation of muscle growth by binding to multiple members of transforming growth factor-ß superfamily. The present investigation was carried out to comprehend the trend of ACTRIIB messenger RNA in pectoralis major muscle during embryonic (E5-20) and post embryonic age (days 1, 14, 28, and 42) in both Control Broiler (CB) and Aseel by using Real-time PCR. The expression profile of ACTRIIB gene displayed a similar trend in CB and Aseel, however Aseel showed significantly (P < 0.001) higher transcription throughout the period. The fold change in expression of ACTRIIB in Aseel relative to CB varied from 3.94 to 14.72 folds and 3.28 to 7.14 folds during embryonic and post embryonic age, respectively. ACTRIIB exhibited its peak on E7, E11, and E16 during embryonic age, which coincides with the formation of primary and secondary muscle fibers in both lines. While at the time of post-embryonic age, ACTRIIB showed highest transcription on day 1 and lowest transcription on day 28 in both CB and Aseel. Within each line, the expression of ACTRIIB differed significantly (P < 0.001) between days in the course of embryonic and post-embryonic period. ACTRIIB gene expression had significant (P < 0.05) effect on all carcass traits except neck weight. Our results suggest that Aseel expressed higher levels of ACTRIIB transcript than CB. The study inferred that expression pattern of ACTRIIB was analogous in both CB and Aseel, which might imply that molecular mechanisms underlying muscle development and regulation are comparable in nature.

Assessment of genetic relatedness of crossbred chicken populations using microsatellite markers.

To measure genetic relatedness between populations, for breeding purposes, we analyzed 170 birds from six crossbred populations of three pure lines of White Leghorn chickens, using 14 microsatellite markers. All the microsatellites were polymorphic, with 2-6 alleles. The mean number of alleles per locus was 3.21. The effective number of alleles varied from 1.14 to 3.94. The observed heterozygosity varied from 0.133 to 1.00, with a mean of 0.748. The F (IS) values were mostly negative, with an average of -0.345. The mean F (ST) value was 0.056. The Nm values ranged from 1.91 to 42.17. The highest genetic identity was observed between IWI x IWK and IWK x IWI. The relation between any two groups of crosses was more than 85%. The results suggest that the crossbred populations were very closely related.

Microsatellite variability and its relationship with growth, egg production, and immunocompetence traits in chickens.

Variability of microsatellites and a possible relationship with growth, egg production, and immunocompetence traits were estimated for six crossbred chicken populations of White Leghorn. Nine microsatellite markers were explored; an association study used the least square maximum-likelihood method on 170 birds of six genetic groups. Seven microsatellites were polymorphic, with two to four alleles. The polymorphism information content (PIC) of five markers was more than 52%. Microsatellites MCW0041, ADL0210, and MCW0110 were significantly (P < 0.05) associated with egg production traits. Genotype 33 of MCW0041 had the highest egg production, up to 64 and 72 weeks of age. Genotypes 11 and 13 of this marker produced the lowest number of eggs. The heterozygous genotype 34 of ADL0210 had the highest egg production, up to 52, 64, and 72 weeks of age. Homozygote 11 of MCW0110 produced the highest number of eggs, up to 28 weeks of age. MCW0041 was significantly (P < 0.05) associated with body weight at 28 and 40 weeks of age. No microsatellite was significantly associated with egg weight at any age, with age at sexual maturity, or with immune response to sheep RBC.

Cloning and characterization of DGAT1 gene of Riverine buffalo.

The present study was carried out to characterize the DGAT1 gene of Riverine buffalo. Total RNA was extracted from the mammary tissue of buffalo and DGAT1cDNA were synthesized by RT-PCR, then cloned using pDRIVE cloning vector and sequenced. The sequencing revealed that the size of DGAT1 gene was 1470 bp with GC content of 62.30%. The gene encoded for 489 amino acid precursors and that it possessed 32 amino acids signal peptide. The similarity of buffalo DGAT1 mRNA sequence with that of cattle, pig, monkey, human, mice and rat were determined as 98.4, 90.7, 85.4, 85.0, 77.4 and 77.1%, respectively. Phylogenetic tree constructed from the derived DGAT1 protein sequences of 15 different species illustrated a unique branches for mammals, fly, nematode and plants. Among mammals, cattle and buffalo grouped together, whereas swine formed another group in the same branch. Four motifs were predicted in buffalo DGAT1 peptide sequence, one N-linked glycosylation site (246th position), two putative tyrosine phosphorylation site (316 and 261), one putative diacylglycerol binding site (382-392 amino acid position) and a conserved domain MBOAT (membrane bound acyl transferase from 150 to 474 amino acids) with a histidine as an active residue.

Cloning and characterization of alpha(s2)-casein gene of Riverine buffalo.

The present study was carried out to characterize the alpha(s2)-casein gene in Riverine buffalo. Total RNA was extracted from the mammary tissue of buffalo and alpha(s2)-casein cDNA were synthesized by RT-PCR, then cloned using pDRIVE-cloning vector and sequenced. The sequencing revealed that the size of alpha(s2)-casein was 669 bp with GC content of 41.11%. The gene encoded for 222 amino acid precursors and that it possessed 15 amino acids signal peptide. The similarity of buffalo alpha(s2)-casein mRNA sequence with that of cattle, sheep, goat, pig and camel were estimated as 97.9, 93.6, 93.4, 73.5 and 73.0%, respectively. In the phylogenetic trees, constructed from the data of the alpha(s2)-casein mRNA sequences as well as protein sequences, it has been observed that the cattle and buffalo were in the same group whereas sheep and goat formed another group. The camel and swine were placed in two separate groups.

DNA polymorphism of leptin gene in Bos indicus and Bos taurus cattle

Leptin plays an important role in the regulation of feed intake, energy metabolism, growth and reproduction of cattle. We used the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique to screen for DNA polymorphisms of the leptin gene in 403 cattle belonging to various breeds of Bos indicus (Hariana, Sahiwal, Gir and Nimari cattle), Bos taurus (Holstein Friesian (HF) and Jersey cattle) as well as Bos taurus x Bos indicus crossbreds (½ HF x ½ Hariana). In all the cattle we amplified two regions of the leptin gene, a 522 bp fragment comprising the partial intron 2 and exon 3 and another 94 bp fragment consisting of part of exon 2. Digestion of 522 bp PCR products with the BsaAI restriction enzyme revealed three genotypes in all the breeds of cattle studied. This is the first report of the presence of leptin gene polymorphism in purebred Bos indicus cattle of Indian origin (indicine cattle). Almost similar gene and genotype frequencies were observed in all the breed groups, while the frequency of mutant homozygotes (AA) was very low (0.03 to 0.07). On digestion of the 94 bp fragment with the Kpn2I restriction enzyme, three genotypes were observed in HF, Jersey and crossbred cattle. The CC genotype had the highest frequency (0.68) in crossbreds whereas the frequency of CT heterozygotes was highest (0.69) in HF cattle. This mutation was absent in all the breeds of indicine cattle. The results suggest that the BsaAI-RFLP mutation has occurred far back in evolution before the divergence of taurine and indicine cattle while the Kpn2I mutation has occurred recently as indicated by the fact that this mutation was only detected in taurine cattle.