Mothods Of SNPs Discovery In The Finless Porpoise (Neophocaena Phocaenoides)

This thesis includes four chapters:1) The applications of single nucleotide polymorphisms (SNPs) as novel genetic markers in ecology, evolution, and population biology were reviewed.2) A whole-genome shotgun library of the finless porpoise (Neophocaena phocaenoides) was constructed for isolating SNPs. Sixty-eight sets of primers were designed based on eighty-three sequences obtained, in which sixty-three sets successfully amplified target DNA fragments in 24 porpoises. Sequence variations were identified using denaturing high performance liquid chromatography (DHPLC). Twenty sets of primers were proved polymorphic, 18 of which were further confirmed by sequencing. Twenty-nine SNPs were discovered, including seven transversions, 21 transitions, and one insertion/deletion. Although approximately 10% false positive loci were found in DHPLC detection, genomic library construction in combination with DHPLC is applicable in high-throughput SNPs detection for non-model organisms.3) Denaturing high performance liquid chromatography (DHPLC) and direct sequencing in conjunction with DNA pooling were used as possible strategies for augmenting the efficiency, economy, and throughput of SNPs detection in the finless porpoise. Two identified SNPs were used to construct twelve DNA pools comprised of the previously genotyped individuals with a frequency of 0% to 50%. The SNPs can be detected out at a frequency no less than 12.5% when using DHPLC, whereas when using direct sequencing the frequency must be at least 20%. In conclusion, the sample number of DNA pooling for DHPLC and direct sequencing analysis should be no more than four in order to ensure the efficiency and accuracy of SNPs discovery and screening.4) Comparative anchor tagged sequences (CATS) were used to screen novel SNPs in the finless porpoise. Five loci were screened by using PCR, DHPLC, and direct sequencing. All the primer sets were designed based upon gene sequences from human, mouse and some other mammals. Seven SNPs were identified from four of the five sequenced regions, yielding a SNP on average every 370 base pairs (bp). This study may provide a reliable basis for acquiring more SNPs through CATS method in the finless porpoise and other wild animals.