Desenvolvimento de marcadores moleculares específicos para produção de leite em búfalos (Bubalus bubalis)

Abstract

The use of molecular markers is used in the genetic improvement of buffaloes to verify genes that are linked to traits of economic interest, and subsequently assist in the selection of superior individuals with greater productivity. Thus, the objective was to identify and develop specific molecular markers for genes related to milk production in Bubalus bubalis. With the Panther 16.0 program, function and pathway enrichment was carried out through the genomic sequence of B. bubalis for genes related to milk quality (CSN1S1, CSN1S2, CSN2, CSN3, LALBA; FABP4; LPL; PPARG; SDC; DGAT1) available in the Natinonal Center For Biotechnology Information (NCBI) database. With the String tool, a protein-protein interaction network was built. The identification and design of the primers was performed using Primer-Blast software, based on the download of sequences in FASTA format. The validation of the primers was performed in silico using the SMS - PCR Primer Stats program, to confirm the absence of hairpins and dimers. BLASTn was used to verify alignment, and the SMS PCR Products program, which searches for perfect anneal sites, and in the laboratory, using genetic material from the follicular bulb of the tail broom hairs of 46 buffaloes, 23 Murrah and 23 of the Mediterranean race. The result of the function and pathway enrichment analysis of the candidate genes for milk quality demonstrated that they are associated with complex biological activities, including lipid activity, lactose synthesis, hormonal regulation and lipid activity. The protein-protein interaction network demonstrates that there is an interconnection between the genes studied, but the main association occurs between the proteins in the casein cluster. In total, 100 primers were developed, of which only 41 passed all tests and are ready to be synthesized. The number of specific markers developed for each gene was CSN1S1 (7); CSN1S2 (7); CSN2 (4); CSN3(5); LALBA (4); FABP4 (3); LPL (3); PPARG (4); SDC (2); DGAT1 (2). The other primers were not efficient, and they did not show adequate standards for genotyping. Therefore, it can be concluded that through bioinformatics analysis it is possible to develop efficient primers, both for virtual PCR and for PCR in the laboratory.