Complex transcriptomic pattern identified at river buffalo DGAT1

DGAT1 has been recognised as strong functional QTL for the milk fat content in cattle. In river 10 buffalo, DGAT1 has been investigated mainly for the characterization of the gene itself and for the 11 identification of the K232A polymorphism similarly to what has been done in cattle. No 12 investigation has been carried out at transcriptomic level so far. Aim of this study was to analyse the 13 transcript profile of DGAT1 in lactating buffaloes. 14 Milk samples were collected from 8 unrelated buffalos (at the 3rd lactation and 120 days from 15 calving), reared in Piedmont Region and belonging to one farm. Total RNA was isolated from milk 16 somatic cells using TRIzol. The reverse transcription was performed using an oligo dT18, whereas 17 the PCR reaction was accomplished using the following primers 5’-ATGGGCGACCGCGGCGG-18 3’ and 5’-TCAGGTGCCGGCTGCCGG-3’, corresponding to the nucleotides 1-17 (exon 1) and 19 complementary to the base pairs 1453-1470 (exon 17) of the buffalo DGAT1 cDNA (EMBL ID: 20 DQ120929). The PCR products were cloned into pGEM-vector, and transformed into JM109 21 competent cells. White recombinant clones were randomly chosen and screened by PCR. All 22 amplicons different in size (bp) were purified and sequenced in both directions. 23 A total of 147 positive recombinant clones were analysed. The sequence analysis showed a complex 24 mRNA pattern with a total of at least 6 different transcripts. The most represented mRNA was that 25 correctly assembled (in total 86 out of 147 clones, 58.5%), 1470bp long and coding for a functional 26 protein of 489aa (amino acids). The following mRNA population was deleted of the exon 12 27 (21.09%). Despite this deletion, the mature mRNA did not undergo any frame-shift and the 28 termination codon was kept as in the normal isoform. The putative protein 474 aa long is different 29 from other predicted DGAT1 isoforms derived from the buffalo genome project. Part of the other 30 transcripts were deleted of the last 66bp of the exon 8 (7.48%). This alternative splicing is 31 consequence of the incorrect identification of a splice donor site directly in the exon 8 and it is 32 responsible for a protein isoform 22 amino acids shorter. Minor transcripts are represented by 33 mRNAs with the insertion of the intron 13 (5.44%), populations deleted of the exon 16 (4.76%) and 34 transcripts characterised by the contemporary out-splicing of the exons 6 and 7 and the insertion of 35 the intron 13 (2.72%). The investigation at DNA level will likely clarify the variability found at 36 mRNA leve