The development of novel genome-SSRs, multiplex PCR panels, and allelic ladders for parentage identification in Tachypleus tridentatus

Abstract

Tachypleus tridentatus, one of the oldest animal species on Earth, with significant biological and ecological value, its population sizes have declined due to overfishing. Stock enhancement technology has been used to protect T. tridentatus, however, its efficiency needs to be addressed. In the present study, a parentage identification system was developed. We identified 235,336 SSRs and 182,394 primer pairs were designed. Out of 321 synthesized primers, 14 highly polymorphic primers were selected to reveal the genetic diversity of 110 T. tridentatus. SSRs were amplified with the 14 developed primers, which were fluorescently labeled, and the resulting PCR products were analyzed using the ABI DNA Analyzer 3730xl. The number of alleles per locus, the polymorphism information content, the observed heterozygosity, and the expected heterozygosity ranged from 7 to 48, 0.581 to 0.963, 0.636 to 0.909, and 0.645 to 0.969, respectively. The cumulative exclusion probability with both unknown parents, with only unknown parents, or with both known parents was >99.9999% for the 14 loci. Furthermore, to facilitate data exchange across laboratories and platforms, multiplex PCR detection, SSR fragment size ladders, and parentage identification were employed. Out of the 14 primers, 6 optimal primers were selected for multiplex PCR validation, and two sets of triplex systems were established. Three thousand clones for 160 alleles of the six primers were selected for plasmid extraction and sequencing, and 600 correct plasmids were obtained for new T. tridentatus SSR fragment size ladder construction. Two pairs of parents with 12 offspring each were used for parentage identification by the multiplex PCR systems and ladders, the cumulative average exclusion probability of these 6 primers reached 99.9999% among parents and their offsprings, and was 0% in nonparent-offspring group. The parentage identification system constructed in this study can serve as a tool for monitoring the survival rates of T. tridentatus in stock enhancement programs.