A new set of highly efficient primers for COI amplification in rotifers

Abstract

Rotifers are a small-sized but key group of freshwater zooplankters with high species richness, linking primary producers to higher consumers in aquatic food webs. DNA barcoding has been widely used in exploring its biodiversity, cryptic speciation and phylogeny. However, the inefficiency of universal primers to amplify COI of rotifers hinders our understanding of their species richness and genetic diversity. Here, we develop a new pair of primers, 30 F and 885 R, to amplify the COI gene of rotifers. We used 22 species to test their PCR success rate and found that the new pair of primers was more efficient (86%) than two pairs of universal primers, namely, dgLCO and dgHCO (32%), and Folmer primers (59%). The new primers will allow the barcoding of groups that were so far difficult to sequence and will contribute to clarify species diversity and phylogeny of rotifers.

Single Cell Analysis Reveals a New Microsporidia-Host Association in a Freshwater Lake

“…On the Microsporidia positive SAGs, rotifers were characterized by amplification of the COI using the 30F and 885R primers set according to Zhang et al [14] with some modifications (see Supplementary Material). The rotifer affiliation analysis revealed that this potential new species of Crispospora is able to parasite the genus Kellicottia (Fig.…”

Application of COI Primers 30F/885R in Rotifers to Regional Species Diversity in (Sub)Tropical China

“…DNA from a single species was used as a template. A 760-bp segment of COI was amplified using the primers 30F and 885R [31]. The total amplification volume of primers 30F/885R was 30 μL, including 15 μL 2 × HieffTM PCR Master Mix (With Dye), 11 μL ddH2O, 0.5 μL of forward and reverse primers (100 μM), 3 μL DNA, respectively.…”

“…The total amplification volume of primers 30F/885R was 30 μL, including 15 μL 2 × HieffTM PCR Master Mix (With Dye), 11 μL ddH2O, 0.5 μL of forward and reverse primers (100 μM), 3 μL DNA, respectively. The amplification started with initial denaturation 2 min at 98 °C, then six cycles of (95 °C for 30 s, 54 °C for 40 s (−0.5 °C/each cycle), 72 °C for 30 s), and 36 cycles of (95 °C for 30 s, 51 °C for 40 s, 72 °C for 30 s) and final extension of 72 °C for 2 min [31].…”

“…DNA from a single species was used as a template. A 760-bp segment of COI was amplified using the primers 30F and 885R [31] The PCR products were detected in 1.0% agarose gel. The amplified products with clear and bright target bands were selected and sent to Tianyi Huiyuan Gene Technology Company for purification and sequencing.…”

“…Elías-Gutiérrez et al [30] used the Zplank primers to amply rotifer COI and obtained 11 BINs (Barcode Index Numbers) of rotifers, with a sequencing success rate of 100%. Recently, Zhang et al [31] used a metagenomics method to assemble nine mitochondrial genomes from Brachionus and Keratella, with which they designed a new pair of primers just for rotifer COI: 30F/885R. The pair of primers performed efficiently (86%), much higher than dgLCO/dgHCO (32%) and Folmer primers (59%).…”