Polimorfismos na região promotora, Éxon I e Éxon II do gene do receptor da melatonina associados às características reprodutivas em búfalas na Amazônia

Abstract

The buffaloes production in the Pará state have is a high representative activity to the regional economy and production of meat, milk and dairy products. Therefore, it is necessary to develop new technologies that increase the enjoyment of the production and reproduction of buffaloes in the Amazon region. Thus, the objectives of this research thesis was to characterize the promoter region of the melatonin receptor gene 1 (MTRN1A) and identify polymorphisms in exons I and II of that gene and link them to reproductive traits that have economic importance in buffaloes in the region Amazon. Were collected 400 buffaloes samples and 60 animals were used to characterize the promoter region, 140 to detect the polymorphism (SNPs) in exon II and 77 to detect the polymorphism in exon I. DNA extraction was peformed by phenol method. Then were selected different primers to make Polymerase Chain Reaction (PCR). The promoter region was sequenced MTRN1A a total of 1621 base pairs by the Sanger method, polymorphism in exon II was detected by PCR-RFLP (Restriction Fragment Length Polymorphism) with HpaI enzyme and the polymorphism exon I was found by sequencing DNA by Sanger method. Allele and genotype frequencies of all SNPs were evaluated, the inbreeding coefficient (Fis) and the likelihood of Hardy-Weinberg equilibrium. SNPs in the promoter region and exon I have been associated with reproductive traits of buffaloes by ANOVA or Student's t test and / or chisquare test. The significance level was 0,05. 26 SNPs were detected in the promoter region at positions -1511 (C → T), -1465 (G → T), -1422 (A → G), -1411 (G → A), -1395 (G → T), - 1298 (A → G) -1295 (G → A) -1242 (C → A) -1150 (C → T), -1 147 (G → C) -1136 (A → G) -911 ( G → A), -909 (A → G) -724 (C → G) -656 (A → C) -649 (C → T), -644 (G → A), -511 (A → C) -481 (G → A), -425 (C → T) -395 (G → A), -383 (G → T), -254 (C → T) -206 (T → C) , -133 (T → G) and -94 (C → T) and a deletion unit (ACAA) at position -1483. Of the total detected SNPs, 75% of the wild alleles tiveral frequencies greater than 0,5. For the characteristic interval between calving (IEP), only five SNPs (-1298, -1136, -911, -724 and - 656) were significantly associated (P <0.05) and three SNPs (-1395, -724 and -94) were significantly associated (P <0.05) with the characteristic age at first calving (IPP) and none for the characteristic concentration of deliveries (P> 0.05). A total of 11 SNPs was strongly associated with binding factors in gene regulation. The SNP in exon II by PCR-RFLP (HpaI) at position 306 (T → C), the most frequent allele T in Upland animals (0,529) and C in lowland animals, the two populations showed coefficients Fis inbreeding (0,040 and 0,091, respectively) and strong deviations from Hardy-Weinberg equilibrium (P> 0.05). The mutation occurs at codon 106 and is the sinonímio type without changing the messenger RNA handles structure. The SNP in exon I was detected at position 62 (T → C) and of the non sinonímio, exchanging of Leucine to Proline. The mutant allele was the most frequent C (0,623) inbreeding coefficient Fis = 0.397 and Hardy-Weinberg deviations (P <0.05). None of the genotypes were associated with calving interval and age at first birth (P> 0.05). Therefore, SNPs are strong candidates for selection, but it would be interesting to evaluate them in other herds in the Amazon region and / or in other regions of Brazil and / or other countries to effect them as excellent molecular markers for reproductive traits of buffalo.