DIRECT SEQUENCING OF GENOMIC DNA FOR CHARACTERIZATION OF A SATELLITE DNA IN 5 SPECIES OF EASTERN PACIFIC ABALONE

A tandemly repeated satellite DNA of 290-291 base pairs (bp) was ident ified by SalI digestion of genomic DNA of five species of Eastern Paci fic (California) abalone (genus, Haliotis). Following cloning and sequ encing of one repeat unit from one species, the consensus sequences of this satellite were determined for five species by directly sequencin g genomic DNA using satellite-specific primers. Phylogenetic trees of the consensus satellite sequences had the same topology as trees const ructed for two abalone sperm acrosomal proteins. In 12 randomly picked clones of the Red abalone (H. rufescens) San satellite, 16 positions varied, the variation being spread throughout the sequence. GenBank da tabase searches found no significant similarities between this satelli te and known sequences. Southern analysis showed that all 290-bp SalI repeats were excised from genomic DNA by Sau3A1 digestion. The tandem arrangement of satellite repeats was confirmed by sequencing through t he Sail site into the next repeat using genomic DNA as template, time- dependent appearance of DNA ladders with an approximate 300-bp spacing in SalI digests of genomic DNA, and ladders of bands with an approxim ate 300-bp spacing generated by polymerase chain reaction (PCR) using genomic DNA as template. In the Red abalone, the 290-bp SalI satellite represents approximately 0.5% of total DNA, equivalent to approximate ly 28,000 copies per haploid genome. The species-specific consensus se quence of this satellite, obtained directly using genomic DNA as the s equencing template, provides a molecular marker that could be used for identification of hybrid parentage, taxonomy, population identificati on, and forensic studies.