Sequencing of 18S rRNA gene of Bdelloid rotifers and design of the primers for real-time PCR

Abstract

Three individuals of Bdelloid rotifer (J1, J2 and J3) were isolated from a MBR system in Nagasaki University and one individual of rotifer (J4) in the original seed sludge collected from a wastewater treatment plant for the MBR was isolated. The four rotifer species were able to proliferate in toxic Microcystis cell suspension. The partial sequence of 18S rRNA gene of each isolated rotifer was determined using In-fusion cloning and searched by BLAST. The gene of the four rotifers J1, J2, J3 and J4 showed the same sequence, then the consensus sequence was in the branch of Bdelloid rotifers in the phylogenetic tree. Furthermore, a specific Bdelloid forward primer 55F and reverse primer 395R for real-time PCR was designed based on the consensus sequence for quantitative researches on the Bdelloid rotifers population. We succeeded to quantify the population of a Bdelloid rotifer cultured in toxic Microcystis cell suspension using the new designed primer pairs.