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DEVELOPMENT OF A PCR BASED METHOD FOR FERTILITY ASSESSMENT OF MALE SEA LAMPREY
Ziping Zhang1, John R. Foster1, Michael J. Siefkes2
1 Department of Fisheries, Wildlife & Environmental Sciences, State University of New York, Cobleskill, NY 12043
2 Great Lakes Fishery Commission, 2100 Commonwealth Blvd., Suite 100, Ann Arbor, MI 48105
A cold water stand-alone aquatic incubator system was constructed and used to rear sea lamprey embryos at temperatures ranging from below room temperature to 5°C. The incubator system proved to be less expensive than manufactured systems and easy to assemble. Using the incubator system, sea lamprey embryonic samples were collected at each stage of development to analyze gene expression levels using next generation sequencing platform. About 14,000 transcripts were found throughout the embryonic developmental stages. Each stage was analyzed further to determine the amount of significant genes present. Statistical analysis of all the genes and embryonic stages were measured based on log2 calculations for the expressions fold of adjacent stages. After intensive analysis of verifying genetic expression, function, and embryonic importance, eight genes were identified with statistical significance and embryogenesis importance. A novel real-time extra long (XL) - PCR for DNA damage detection was developed. SYTO-82 was introduce to TaKaRa LA TaqTM hot start system as the fluoresce reporter. This XL-PCR system successfully quantified three extra-long fragments of both mtDNA and genome DNA from sea lamprey: 15030 bp LD-U11880, mtDNA (E=1.63, R2=0.9998); 11924bp SL-Contig254, Genome DNA (E=1.62, R2=0.9936); 10547bp SL-X14061 (E=1.64, R2=0.9985). In vitro- UV exposure results indicated that mtDNA was more sensitive than the other two genome DNA fragments. A PCR efficiency based real-time XL-PCR data analysis method was described, which can characterize the real extant of DNA damage.