engleski

Development of new genome-derived simple sequence repeats (SSRs) for Anisakis simplex s.s. and Anisakis pegreffii

Microsatellites, also known as short sequence repeats (SSRs), are tandemly repeated nucleotide motifs (1-6 bp) found in nuclear genomes of many taxa. Great variations in the number of basic repeated units are characteristic for these loci, resulting in high heterozygosity and the presence of multiple alleles making them the markers of choice for various population and evolutionary genetic studies. We have used available genomic resources of Anisakis simplex hosted at WormBase ParaSite to conduct in silico screening for SSRs and develop first marker set for this nematode, the cause of zoonotic fish-borne anisakiasis. Genus Anisakis is comprised of nine species with low level of morphological differentiation at species level. It is generally assumed that A. simplex senusu stricto (s.s.) is more pathogenic to humans than other species, emphasizing the importance of correct species identification and possible occurrence of hybridisation between A. simplex s.s. and A. pegreffii in sympatric areas of parasitation. In total, 23, 574 microsatellites were identified in non-coding and coding regions, covering 0.31% of A. simplex genome inspected. When considering repeats from perfect and compound loci together, trinucleotides were most abundant followed by mono- and dinucleotides in all regions alike. Potentially informative non-coding loci were randomly selected, tested and optimised for multiplex PCR. Twenty-six primer-pairs gave a unique PCR product of expected size in A. simplex and A. pegreffii, three in A. ziphidarium and none in A. typica, reflecting their phylogenetic divergence. Twenty loci were further chosen to assess the relationship between individuals showing mito-nuclear signatures of both A. simplex and A. pegreffii, inferred through PCR-RFLP analyses of the ITS region and sequencing of the cytochrome oxidase 2 (cox2) mtDNA, in allopatric area of the Adriatic Sea. Using our panel of microsatellite loci, we inferred general admixture of all tested populations, except in North Sea-originating "pure" A. simplex s.s. Estimates of ancestry and interclass heterozygosity place most recombinant genotypes with parental class of A. pegreffii, indicating that observed mito-nuclear mosaicism is likely retention of ancestral polymorphism ; also questioning if pure A. simplex was ever recorded in the Adriatic Sea.

Anisakis simplex complex ; simple sequence repeats ; recombinant genotypes ; genetic structure ; Adriatic sea

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