Isolation And Primary Characterization Of Sika Deer (Cervus Nippon) Microsatellites

Microsatellite DNAs play an important role in population genetic diversity analysis, genomic mapping, QTL allocation, disease clinics and pathology, paternity testing, individualization etc. The sika deer is an important economic species and endangered species. Microsatellites are of great significance in the conservation genetics, QTL allocation, and population management etc. of the sika deer. However, there have been very few studies on the microsatellite isolation from sika deer genome. This paper is filling this blank. Genomic DNA was digested with Mbo I, and isolated through electrophoresis of 1.5% agarose gel. Fragments of 1000bp⁴00bp was recovered and purified. A part of the recovered DNA was used to construct a partial genomic library, and the other part was hybridized with (CA) ₁₅ probe labeled with Biotin. A microsatellite enriched library was then constructed after an enrichment process using magnetic bead (Dynabeads^? M-280 Streptavidin) and PCR amplifications. Several oligo probes namely (CA) ₁₅, (TTC) ₁₅ etc. were applied to detect the microsatellite bearing clones from the two libraries. 58 positive clones were randomly picked up and subjected to sequencing. Sequences revealed 28 microsatellites being isolated, occupying 43.75% of the positive clones. Major types of motif of microsatellites isolated were included. Software Primer premier 5.0 and DNAstar were used to design PCR primers for 19 microsatellites, and corresponding PCR system and cycling programs were also established. 10 loci were effectively amplified. The primary assessment of them using parameters such as number and size range of alleles, allelic frequency, observed heterozygosity indicated that 7 loci were polymorphic, and potentially applicable to sika deer genetic analysis as powerful tools after systemaitic genetic characterization.