Identification of a single SNP that affects the LH-beta promoter activity in the Moroccan prolific D'man breed

Common Moroccan breeds D'man, Sardi, and Timahdite have been found to differ regarding litter size. D'man is known for its high prolificacy ( 2-7 lambs), whereas Timahdite and Sardi are normally prolific ( 1-2 lambs). Here, we aimed to identify genetic variants in the LH-beta promoter and to study the promoter activity amongst these breeds using sequencing for the former and the in vitro luciferase assay for the latter. Sequence analysis revealed 16 genetic variants among these common breeds. Luciferase assay analysis demonstrated a higher promoter activity in D'man compared to the Sardi/Timahdite breeds. A small region of 541 bp was further characterized as possessing a high promoter activity. This region contains 2 palindromic sequences and 7 variants. Based on in silico analysis, only 2 variants at position -559 and -568 were found to be informative. These 2 variants were localized within a region rich in transcription factor binding sites including GATA-1/GATA-2, E4/th1, CP2, and c-Ets. Using site-directed mutagenesis, only the variant at position -559 A/G was found to substantially influence the promoter activity. Taken together, differences in the level of LH-beta transcription among highly and minimally prolific Moroccan breeds were demonstrated and a novel single variant was identified that could explain this difference.