Mitochondrial D-loop sequence variation among the 16 maternal lines of theLipizzan horse breed

Mitochondrial DNA from 49 Lipizzan horses representing 16 maternal lines fr om the original stud at Lipica was used for SSCP analysis and DNA sequencin g. The SSCP analysis of the 444 bp long fragment of the D-loop region exten ding from the tRNA(Pro) gene to the central conserved sequence block reveal ed three distinct groups of SSCP patterns. Both ends of the D-loop region ( 378 bp and 310 bp), which are considered as the most variable regions withi n the mammalian mitochondrial DNA, were sequenced. According to 49 polymorp hic sites identified within the both parts of the D-loop region, the 16 mat ernal lines were grouped into 13 distinct mitochondrial haplotypes. The min imal difference between two different haplotype DNA sequences was one nucle otide and the maximal 24 nucleotides. The inheritance of mitochondrial hapl otypes was stable and no sequence variation potentially attributable to mut ation within maternal line was observed. Considerable DNA sequence similari ty of Lipizzan mitochondrial haplotypes with the haplotypes from other bree ds was observed. Phylogenetic analysis of the sequence data revealed a dend rogram with three separated branches, supporting the historical data about the multiple origin of the Lipizzan breed.