Development And Application Of Single Nucleotide Polymorphism Markers In The Pacific Abalone, Haliotis Discus Hannai Ino

Single nucleotide polymorphism (SNP) markers were discovered from expressed sequences (ESTs) of the Pacific abalone (Haliotis discus hannai). An allele-specific polymerase chain reaction (AS-PCR) was developed and optimized for SNPs genotype scoring. Gene-associated SNP markers were exploited for a mapping family and analyzed in 123 progenies. The method of locating functional genes on genetic linkage maps was also discussed in the paper.About 150 EST-contigs containing four or more sequences were obtained by assembling 5800 ESTs and 302 SNPs were discovered from 86 EST-contigs. The numbers of A/G (C/T), A/C (G/T), A/T and C/G SNPs were 147, 90, 21 and 16, respectively. Fifty-two contigs had a positive match with functional ESTs in the public database after BLASTx analysis (E-value <=1E-5), among which there were 220 SNPs but all were synonymous cSNPs. Roughly estimated, the SNP frequency was not less than 1% in the genome of H. discus.Twenty-eight SNPs were validated by PCR direct sequencing (PCR-DS) of pooled DNA. PCR-DS was simple and accurate, could detect several loci by one reaction and discover new SNPs sometimes. The principle for AS-primer design was confirmed by evaluating the impacts of primer-template mismatch on PCR amplifications. The AS-PCR was suitable for small-scale SNPs genotyping as the specificity of detection was enhanced by adding an additional strong mismatch in the penultimate base of the 3'end of AS-primers.The PCR products of the parents of a mapping family from forty-three pairs of EST-derived primers were directly sequenced in both directions. Eighty-six SNPs were found among which 82 were new SNPs and most were in introns. In most cases the SNP genotype of one parent was homozygous (AA) and the genotype of another parent was heterozygous (AB), which were theoretically segregable in the progeny. The cases were fewer that SNP genotypes of both parents were homozygous or heterozygous.Eleven sets of AS-primers of nine genes were designed for genotyping 11 SNPs in 123 progenies, of which 5 were maternal markers and 6 were paternal markers. Chi-square test indicated that 2 loci showed significant segregation distortion (P < 0.05) from Mendelian ratio. The other 9 loci can probably be located on the genetic linkage map being constructed. Besides, the maternal and the paternal SNP markers from two adjacent loci of one gene could be used as one marker in genomic mapping.