Sequence Heteroplasmy of D-Loop and rRNA Coding Regions in Mitochondrial DNA from Holstein Cows of Independent Maternal Lineages Jianming Wu, 1 Renotta K. Smith, 1 A. E. Freeman, 1 Donald C. Beitz, 1,3 Ben T. McDaniel, 2 and Gary L. Lindberg 1 Received 13 July 1999 —Final 6 January 2000 A mitochondrial DNA (mtDNA) fragment containing the D-loop, phenylalanine tRNA, valine tRNA, and 12S and 16 rRNA genes was cloned and sequenced from 36 cows of 18 maternal lineages to identify the polymorphic sites within those regions and to detect the existence of heteroplasmic mtDNA in cows. Seventeen variable sites were observed within the D-loop and rRNA coding regions of bovine mtDNA within a 2.5-kb span. The hypervariable sites in the D-loop and rRNA coding regions were identified at nucleotide positions 169, 216, and 1594. Heteroplasmic mtDNA (variable mtDNA within a tissue) existed extensively in cows and was detected within the above regions in 11 of 36 cows sequenced. The insertion, deletion, and nucleotide transversion polymorphisms were found only in homopolymer regions. Heteroplasmy was observed frequently and seemingly is persistent in cattle. Though heteroplasmy was demonstrated, most lineages and mtDNA sites showed no heteroplasmy. KEY WORDS: mitochondrial DNA; mitochondrial inheritance; Holstein cows; heteroplasmy. INTRODUCTION Bovine mitochondrial DNA (mtDNA) consists of 16,338 base pairs and codes for two rRNAs, 22 tRNAs, and 13 polypeptides. Although nucleotide sequences of 1 Department of Animal Science, Iowa State University, Ames, Iowa 50011. 2 Department of Animal Science, North Carolina State University, Raleigh, North Carolina 27695- 7621. 3 To whom correspondence should be addressed. Biochemical Genetics, Vol. 38, Nos. 9/10, 2000 323 0006-2928/00/1000-0323$18.00/0 © 2000 Plenum Publishing Corporation