Large-Scale PCR-ASO assay:
Differential female broodstock success & larval cohort survival

          The mtDNA genome can be used directly to monitor differential female breeding success, or differential larval cohort survival in aquaculture cages.

          MtDNA genome re-sequencing of four females identifies four SNPs, and four SNP-specific ASOs are constructed (A, B, C, & D). A mass-spawning experiment mixes the four females with a large number of males (top). 96 larvae are chosen at random: all four PCR-ASO tests are run on each larva, one in each quadrant of a 384-well plate (bottom, left). The female with SNP A is the most successful spawner (56/96 larvae), while the female with SNP D is least successful (8/96 larvae), and the B & C females are equally successful (16 @).

          After on-growing, the tests are repeated on new fish. Among post-settlement fish, only 8/96 have the A SNP, whereas 56/96 have the D SNP, indicating a seven-fold survival advantage. C females have also gained a seven-fold advantage over B females (28 versus 4 /96). The data can be included in a production model to predict which female is optimal for choice as broodstock.

       In the diagrams above, the rtPCR detector actually uses only a single experimental fluorescent colour (plus a second for the Control), which a computer "re-colors" for the investigator's comfort. With the current generation of multiplex rt-PCR cyclers that can employ five simultaneous fluorescence colours (four "experimental" plus a control), the four tests can be run simultaneously, with a four-fold gain in throughput.