Polymorphisms in the Leptin and SCD genes in Buffaloes of the Amazon

The objective this work was to evaluate genetically the buffaloes populations using polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) in the leptin and stearoyl-CoA desaturase (SCD) genes, and associate the genotypes with milk production. Sixty-nine samples of Bubalus bubalis 38 Murrah breed, 18 Mediterranean and 13 mixed-breed of the Murrah with Mediterranean were evaluated. Allele and genotype frequencies, the heterozygosity observed and expected, the inbreeding coefficients (FIS), the probabilities of the Hardy-Weinberg equilibrium, the F statistic for population differentiation and Shannon index were calculated using GENEPOP and GenALEx programs. The associations of the different genotypes with a role in milk production were tested by Analysis of Variance (ANOVA) and t-test. We reported the leptin and SCD allele contribution to genetic variability in buffaloes herds in the Brazilian Amazonian region. The A allele was more representative in leptin and SCD genes for all breed groups. No significant effects between genotypes and milk production were found in the present study, but there is an indicative that AA genotype in leptin gene affects milk production in Murrah breed.


Introduction
The total demand for animal food products is expected by FAO to more than double by 2030, driven by the growing middle-class population in developing countries (Briunsma, 2003). In Brazil, water buffalo (Bubalus bubalis) rearing has been developed on a large scale, with an annual increase of 1.1% (IBGE, 2019).
Production and marketing of buffalo milk and their dairy products are well differentiated and have grown increasingly to attract consumers seeking nutritional and functional quality (Araújo et al., 2012). After dairy cows, water buffaloes are the second most important species in the world in terms of milk production (Safari et al., 2018). The components of buffalo milk, such as fats and total solids, make a higher quality than cow milk (Rosales & Batalha, 2013). The mechanisms that regulate milk synthesis are controlled by several genes that can be considered as candidates for genetic variation in economically important traits in livestock selection strategies. Additionally, it would be useful to identify polymorphisms in these candidate genes, including those associated with quantitative trait for marker-assisted selection, used single nucleotide polymorphism (SNP), which can be applied in breeding programs in order to increase milk production (Naserkheil et al., 2019).
Among the various genes studied with reference to the production of milk in domestic animals leptin gene and stearoyl-CoA desaturase gene (SCD) were highlighted. The leptin gene is locally formed by the mammary gland (Bartha et al., 2005) and seems to have essential roles in gland growth (Hu et al. 2002) and lactogenesis (Feuermann et al., 2009). Whereas SCD gene is involved in the endogenous production of the cis-9, trans-11 isomers of CLA, which are generally found in ruminant milk and considered healthy in the human diet (Bhattacharya et al., 2006). Therefore, the objective of this study was to identify polymorphisms in leptin gene and SCD gene, and associate polymorphisms with the production of milk in Murrah, Mediterranean and mixed-breed herds in the Brazilian Amazonian region.

Methodology Experimental Animals
A total of 69 samples of B. bubalis, 38 of them being Murrah breed, 18 Mediterranean and 13 mixed-breed of the Murrah with Mediterranean were analyzed. The study complied with the Code of Ethics Protocol 002/2016 (Ethics Committee on Animal Use) and 22.1306023.00.00.

Phenotypes used
The total milk production was analyzed in each milking session within a 36 months period.

Laboratory procedures
A total of 5 mL of blood was collected from each animal and preserved in EDTA at 4°C to be analyzed in the laboratory.
Genomic DNA was extracted according to the protocol developed by Sambrook et al., (1989). Subsequently, the DNAs was quantified in 1.0% agarose gel and compared with increasing concentrations of DNA bacteriophage (50, 100 and 200ng/µL). The purity of DNA was evaluated in the Biomate 3 spectrophotometer (Thermoscientific, USA) within the range A260nm/A280nm and samples with rates equal to or higher than 1.8 were selected.
The polymerase chain reactions (PCR) were conducted in a final volume of 20µL. The table 1 showed the primers used in the studied. 4 and adjusted with ultrapure water to a volume of 15 µl. Subsequently heated to 37°C for 1 h for the leptin gene and 65°C for 5 h for the SCD gene. They were then visualized on Gelred stained 1.5% agarose gel (Biotium / USA) and further visualized in transilluminator under ultraviolet light.

Statistical analysis
The program GENEPOP (Raymond & Rousset, 1995) was used to determine the haplotype and genotype frequencies, the heterozygosity observed and expected, the inbreeding coefficients (FIS), the probabilities of the Hardy-Weinberg equilibrium and the estimates of the F statistic for population differentiation. The program GenALEx (Peakall & Smouse, 2012) was used to calculate the Shannon index. The associations of the different genotypes with milk production were tested via Analysis of Variance (ANOVA) and t-test using R software https://www.r-project.org/ and visualized with 'ggpubr' package (Kassambara, 2020). The level of significance established was 0.05.

Results and Discussion
For Leptin gene the PCR product were 522-bp covering intron 2 and exon 3. The A/G SNP was allocated in intron 2 and then genotyped by PCR-RFLP. Two different genotypes were detected as a result of the digestion of 522-bp fragment with BsaAI restriction enzyme and the genotypes designated as AA (522 bp    Source: Authors. Nasr et al. (2016) demonstrated that Egyptian buffaloes with AA genotype produced more milk, fat % and fat yields than animals with GG and AG genotypes. The same authors suggested that the A was the predominant allele and had a considerable consequence on rising milk yield. In the present study, the A allele is the most frequent in leptin gene for all breed groups. However, this allele not shows statistical significance association with milk production. Dubey et al., (2014) claim the infrequent allele is anticipated to be dispersed more in the form of heterozygotes than in homozygous state and it is improbable that the G allele will be totally eradicated from the population, except with targeted selection opposed to this allele. In our study, the GG genotype was not found in any of the breeds, which is expect since the GG genotype is associated with low milk production and our buffalo heard studied belongs to animal breeding program. Pauciullo et al., (2012) studied SCD gene in mediterranean buffaloes and found an over-dominance effect of the genotype AC associated with milk yield Buffaloes compared with AA and CC. Gu et al., (2019) demonstrated that the A to C substitution generated a higher binding affinity of the Sp1 transcription factor to the gene promoter. The stronger Sp1 binding produced a quantitative difference in gene expression, albeit not directly linked to desaturation activity of SCD in mammary gland. In our studies, we found the CC genotype only in the Mediterranean breed. Is necessary more studies with others population for demonstrated if this genotype can be used as an exclusive marker for the breed.
In the present study, the allele frequencies resulted in low genetic variability in Murrah, Mediterranean and mixedbreed, despite there being no deviation from the Hardy-Weinberg equilibrium. This low variability indicates that inbreeding has occurred.

Conclusion
In conclusion, the A allele was more representative in leptin and SCD genes in all breed groups. No significant effects between genotypes and milk production were found in the present study, but there is an indicative that AA genotype in leptin gene affects milk production in Murrah breed. Further studies with larger sample are necessary to prove this result.