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

Buffalo DGAT1 was mainly investigated for the characterization of the gene itself and for the identification of the K232A polymorphism similarly to what has been accomplished in cattle, whereas no information has been reported so far at mRNA level. The importance of DGAT1 for lipid metabolism led us to investigate the transcript profiles of lactating buffaloes characterised by high (9.13 ± 0.23) and low (7.94 ± 0.29) for milk fat percentage, and to explore the genetic diversity at RNA and DNA level. A total of 336 positive clones for the DGAT1 cDNA were analysed 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 two groups of animals (P=0.0169). Apart from the correct transcript (17 exons long), the skipping of the exon 12 is the most significant in terms of clone’s distribution with 11.6% of difference between the two groups, whereas a totally different mRNA profile was found approximately in 12% of clones. The sequencing of genomic DNA allowed the identification of 10 polymorphic sites at intron level, which clarify, at least partially, the genetic events behind the production of complex mRNAs. Genetic diversity was found also at exon level. The SNP c.1053C>T represents the first example of polymorphism in a coding region for the DGAT1 in the Italian Mediterranean breed. In order 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 (in total 200 animals). The alleles were equally represented in the overall population, whereas the analysis of the two breeds showed inverted frequencies which resulted different (p<0.01), likely indicating diverse genetic structure of the two 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 transcript and genetic level for the buffalo DGAT1 and open the opportunity for further investigation on other genes involved in the milk fat metabolism for the river buffalo, including the future possibility to select alleles with quantitative and/or qualitative favourable effects.