Transcript analysis at DGAT1 reveals different mRNA profiles in river buffaloes with extreme phenotypes for milk fat.

Buffalo DGAT1 (diacylglycerol O-acyltransferase 1) was mainly investigated for the characterization of the gene itself and for the identification of the K232A polymorphism, similar to what has been accomplished in cattle, although no information has been reported so far at the mRNA level. The importance of DGAT1 for lipid metabolism led us to investigate the transcript profiles of lactating buffaloes characterized as high (9.13 ± 0.23) and low (7.94 ± 0.29) for milk fat percentage, and to explore the genetic diversity at the RNA and DNA level. A total of 336 positive clones for the DGAT1 cDNA were analyzed by PCR and chosen for sequencing according to the differences in length. The clone assembling revealed a very complex mRNA pattern with a total of 21 transcripts differently represented in the 2 groups of animals. Apart from the correct transcript (17 exons long), the skipping of exon 12 is the most significant in terms of distribution of clones with 11.6% difference between the 2 groups, whereas a totally different mRNA profile was found in approximately 12% of clones. The sequencing of genomic DNA allowed the identification of 10 polymorphic sites at the intron level, which clarify, at least partially, the genetic events behind the production of complex mRNA. Genetic diversity was found also at the exon level. The single nucleotide polymorphism c.1053C>T represents the first example of polymorphism in a coding region for the DGAT1 in the Italian Mediterranean breed. To establish whether this polymorphism is present in other buffalo breeds, a quick method based on PCR-RFLP was set up for allelic discrimination in the Italian Mediterranean and the Romanian Murrah (200 animals in total). The alleles were equally represented in the overall population, whereas the analysis of the 2 breeds showed different frequencies, likely indicating diverse genetic structure of the 2 breeds. The T allele might be considered as the ancestral condition of the DGAT1 gene, being present in the great part of the sequenced species. These data add knowledge at the transcript and genetic levels for the buffalo DGAT1 and open the opportunity for further investigation of other genes involved in milk fat metabolism for the river buffalo, including the future possibility of selecting alleles with quantitative or qualitative favorable effects (or both).