Partial sequencing of the TOX and NCOA 2 genes in buffaloes

Buffaloes play an important role in food production and in the socioeconomic development of tropical regions. The characterization of genes permits the study of traits of this species and the development of animal production technology. This work partially studied the thymus high mobility group box protein (TOX) and the Nuclear Receptor Coactivator 2 (NCOA2) genes in female Murrah buffaloes using a PCR-sequencing technique. Six SNPs were identified in each gene. Two adjacent SNPs in TOX gene create/destroy a mature miRNA production site and are good candidates to be further studied. The homology of the regions of these genes with the cattle correspondents is very high (99 %).


INTRODUCTION
Buffaloes (Bubalis bubalis) play an important role in meat and milk production worldwide, especially in Asia, where 97,1 % of the bubaline population is located (FAO, 2013).Interestingly, most individuals of this species are found in developing or less-developed countries.Therefore, improvement of buffalo production will actively improve the socioeconomic status of these countries.The higher is the profit generated by the livestock production, the higher is the benefit for the human population.
Although cattle and buffaloes are related species, the reproduction of these species differs in some aspects and species-specific technologies need to be developed for a proper husbandry (Gimenes et al., 2011;Carvalho et al., 2013).Reproductive traits of buffaloes show low heritability (Seno et al., 2010) or are measured late in life (Galeazzi et al., 2010a,b).These facts make these traits candidates to be studied with molecular markers.
In a study on cattle, Fortes et al. (2011) showed that some genes that act as hypothalamic transcription factors, which include TOX and NCOA2, seem to be important for the development of puberty, since these transcription factors are used in the transcription of several genes.Hong et al. (1996) identified the NCOA2 gene in mice, this gene interacts with hormone binding domains of glucocorticoids such as estrogen and androgen.According to Hong et al. (1996), NCOA2 is the main component of the mechanism of RNA polymerase II transcription, acting as a transcriptional co-activator and forming a bridge between transcription and hormone-binding sites, i.e., this protein is required by transcription factors for the initiation of transcription.In another study, Hong et al. (1997) demonstrated the presence of the NCOA2 protein in the brain where it interacts with five steroid receptors and induces the hormone-dependent activation of mineralocorticoid, glucocorticoid and steroid receptors.
The thymus high mobility group box protein (TOX) was first isolated by Wilkinson et al. (2002) from thymus tissue of mice.This protein is part of the family of high mobility group proteins that bind to and induce sharp bends in DNA (Bianchi and Beltrame, 1998), thus regulating gene expression by altering the chromatin structure and modulating the formation of regulatory complexes of multiple proteins (Wilkinson et al., 1998).Wilkinson et al. (1998) showed that TOX participates in the development of thymus cells and causes changes in gene expression.Laz et al. (2007) described TOX gene, in mice, and concluded that transcription factor is expressed in liver and is regulated by growth.Wilkinson et al. (1998) demonstrated the presence of TOX in human brain and Fortes et (2011) detected this protein in the hypothalamus of cattle.
The characterization of genes is the initial step in the development of molecular markers.Therefore, the objective of the present study was to sequence partially the TOX and NCOA2 genes in buffaloes and to evaluate possible genetic variations in these genes in order to obtain information to develop genetic markers.

MATERIAL AND METHODS
Twenty unrelated female Murrah buffaloes (Bubalus bubalis) were used for this study.The animals were sourced from commercial farm located in Dourado, São Paulo, Brazil.Animals were raised on pasture with feed supplementation during the dry period from April to September.The farm participates in the milkrecording program of the Animal Science Department, São Paulo State University (Unesp), Jaboticabal.São Paulo, Brazil.
Deoxyribonucleic acid (DNA) was extracted from hair follicles by the phenol-chloroform-isoamyl alcohol method (Sambrook and Fritsch 1989).The primers used for amplification, the amplicon size, and the region amplified are shown in table I.
The reaction mixture contained 1.5 µL DNA (105 ng), 1,5 µL of each primer (15 pM), 7,5 µL GoTaq Colorless Master Mix, and 4 µL nuclease-free water in a final volume of 15 µL.Amplification was performed in a Master Cycler Gradient 5331 thermal cycler (Eppendorf ® , Germany, 2005) under the following conditions: denaturation at 95 ºC for 5 min, followed by 35 cycles of denaturation at 95 ºC for 1 min, specific annealing temperatures for each primer pair (according to de Camargo et al., 2014) for 1 min, and extension at 72 ºC for 1 min.A final extension step at 72 ºC for 5 min was included.
The PCR products were sequenced using both primers (forward and reverse) by the dideoxynucleotide chain termination reaction.Sequencing was performed in an automated ABI 3730 XL sequencer (Applied Biosystems) using the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems).
For identification of the polymorphisms, the sequences obtained were analyzed with the CodonCode Aligner program available at http://www.codoncode.com/aligner/download.htm.The Alibaba 2.1 TF Binding Prediction software available at http://www.generegulation.com/pub/programs/alibaba2/index.html was used for the identification of possible transcription factor-binding sites.The http://www.mirbase.org/search.shtmlsitewas used to identify possible microR-Table I. Primers used for amplification, amplified region, amplicon size, and annealing temperature of the primers (Os iniciadores utilizados para a amplificação, a região amplificada, em amplicons, e temperatura de emparelhamento dos iniciadores).

Primer sequences
Primer number Amplicon size (bp) Amplified region/gene Annealing temperature (ºC) NA (miRNA).The creation of possible miRNA sites for the suborder Ruminantia was investigated.
The sequences were edited according to the Gen-Bank recommendations and deposited in its website.

RESULTS AND DISCUSSION
All primer pairs amplified fragments of the predicted size in DNA extracted from buffaloes, except for primer 1 of the TOX gene which did not amplify any fragment.Since the primers were designed based on the bovine sequence, this lack of amplification may be due to differences in the DNA sequences between these two species.The sequences generated were deposited in GenBank under the accession numbers KF418275 (NCOA2 gene) and KF418277 (TOX gene).The regions of the TOX and NCOA2 genes amplified in buffaloes showed 99 % homology with the respective sequences of these genes in the cattle genome (NC_007312.5),demonstrating that these genes are highly conserved in the two species.
Six single-nucleotide polymorphisms (SNPs) were identified in the TOX gene of buffaloes.Two SNPs were located in exon 5 (g.1812G/T and g.1884C/T) and four in intron 5 (g.2267G/A, g.2292A/G, g.2367A/G, and g.2368C/G).The polymorphisms in exon 5 do not cause amino acid substitutions and are silent mutations in the codon of an arginine (g.1812G/T) and of a serine (g.1884C/T).We also investigated whether the SNPs created/destroyed mature miRNA production sites in ruminants.The adjacent SNPs g.2637A/G and g.2638C/G created a production site of bta-miR-2285m with an A/C combination.The bta-miR-2285 family has over 40 members (Lawless et al., 2013) with production sites spanning the entire bovine genome (Guduric-Fuchs et al., 2012).Micro-RNAs are non-coding RNAs that regulate the expression of other genes in the organism.Therefore, these SNPs are candidates for subsequent association studies.
In addition, six interspecific SNPs (comparing the sequence of cattle and buffaloes) were identified in the coding regions of the TOX gene.Three of these SNPs were located in exon 3 (positions 692, 722 and 836) and three in exon 4 (positions 1176, 1245 and 1254).These positions are indicated in the GenBank sequence KF418277.However, all are silent mutations and encode the same amino acids in both species.
Six SNPs were identified in the NCOA2 gene of buffaloes.Two SNPs are located in intron 1 (g.454A/G and g.733T/C), one in intron 2 (g.1109G/A), two in exon 3 (g.1366T/Cand g.1390T/C), and one in intron 3 (g.1631A/T).The polymorphisms in exon 3 did not cause amino acid substitutions and were silent mutations in the codon of a threonine (g.1366T/C) and of an aspartic acid (g.1390T/C).
Investigation of interspecific SNPs in the NCOA2 gene of buffaloes and cattle identified three changes in the promotor region (positions 13, 19 and 24) and three changes in exon regions, one in exon 1 (position 251) and two in exon 4 (position 1914 and 1929).These positions are indicated in the GenBank sequence KF418275.The change in the promotor region at position 13 crea-ted a binding site for the transcription factor Sp1 and the change at position 24 created two binding sites for the transcription factors USF and NF-1.If these transcription factors were used, this interspecific mutation could indicate possible differences in the expression of this gene between these species.The interspecific SNPs located in exons did not cause any amino acid substitution.Some polymorphisms in these regions of NCOA2 gene were associated with reproductive traits in zebu cattle (de Camargo et al., 2014).The association shows that these genes influence reproduction in ruminants and may be useful in the future to genetic evaluation.
The present results show that the TOX and NOCA2 genes are highly conserved in cattle and buffaloes.However, differences were observed in buffaloes that permit the development of molecular markers for these regions and future association studies with productive traits of interest.Differences were also observed between cattle and buffaloes, suggesting differential expression of these genes in the two species.Studies have shown low expression of the NCOA2 gene in subfertile mice (Konno et al., 2010).In addition, NCOA2 gene is known to be involved in the energy metabolism of cattle (Pryce et al., 2012), providing further important evidence for its study as a candidate gene.
Further studies characterizing additional regions of the TOX and NCOA2 genes and evaluating the association of the SNPs found with reproductive performance would be useful to develop molecular markers for this species.

CONCLUSION
The regions studied in the TOX and NCOA2 genes are highly conserved between cattle and buffaloes.However, some genetic variations were observed among buffaloes and they could be used for the development of molecular markers in association studies.Differences were also detected between cattle and buffaloes and they may indicate different expression mechanisms of the same genes between the species.