PCR-SSCP and Sequencing Analysis For Studying Leptin Gene Polymorphism and Its Association with Reproductive Status of Egyptian Buffalo

LEPTIN gene is one of the potential genes involves intricately in metabolism and growth of animals. Leptin has myriad effect on tissues and endocrine system that ultimately lead to the whole body energy metabolism and has major impact on performance and wellbeing of livestock species. Owing to have key biological features, leptin is one of the most significant candidate gene markers for Marker assisted selection (MAS) studies. This study was performed to identify leptin gene polymorphism and its relation to reproductive status in Egyptian buffaloes. Total of 150 buffaloes were subjected to ultrasound examination for evaluation of reproductive status. Blood samples were collected from all animals and were stored at -20 degrees C till DNA isolation. Amplification of specific fragment 422bp (intron 2) of leptin gene was performed by polymorphism chain reaction (PCR). Single strand confirmation polymorphism (SSCP) was performed on PCR amplicons. Purified PCR amplicons of different electrophoresis patterns were sequenced in both directions using two primers. Sequences results were analyzed using NCBI BLASTn and BioEdit Sequence Alignment software. Two variants(AA and BB) of leptin gene were detected. Genotype AA represented by 64% in fertile buffalo, while its distribution among infertile one was 36%. Genotype BB, was equally distributed by 50% in both fertile and infertile animals. Sequence analysis of both normal and polymorphic one showed different singlenucleotide polymorphisms (SNPs) in leptin gene, but statistically these SNPs had no correlation with reproductive status (fertile or infertile) of examined buffalo.