The culture of Nile tilapia (Oreochromis niloticus) is one of the most important aquaculture activitie s in Ecuador in specific and the world in general, where the cultiv ation of males is transcendental. The use of breedi ng with a sexual genotype YY guarantee the viability of male offspri ng in captivity, avoiding the use of invasive techn ologies for the environment, and possible interaction with human he alth. However the absence of rapid detection method s limits the application of this technology. Therefore, the pres ent study was sought optimizing a method for detect ing sexual genotypes using microsatellite markers. Genotyping was performed with UNH104, UNH898 and UNH995 microsatellites; of individuals with a known and se xual confirmed genotype, and their relationship was assessed by a test of independence chi-square, and finally the reliability as a diagnostic test was analyzed. Bein g the UNH898 marker which gave the highest dependency (p = 2.92 x 10 -28 ); showing two alleles (with a size of 264 and 296 bp) present mainly in males, forming a genotype to indi viduals YY (264/296) and XY (264/296 / X allele). T o differentiate males from females this marker showed sensitivity, specificity, PPV and NPV of 96.97%, 8 2.35%, 91.43% and 93.33% respectively, while differentiati ng YY from XY individuals; it was about 100.00%, 85 .29%, 85.71% and 100.00% respectively
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