Effects of treated sample weight and DNA marker length on sediment eDNA based detection of a benthic invertebrate

Abstract

Environmental DNA (eDNA) from water samples has been used to monitor aquatic vertebrates such as fish, while very few studies have focused on benthic invertebrates, which inhabit sediment and are often used as indicators of water quality. Fundamental studies to establish a methodology to detect benthic invertebrates by eDNA from sediment samples are necessary. To clarify the effects of DNA marker length and sediment sample weight on the detection of a benthic invertebrate in sediments, we developed two DNA marker sequences (126 bp and 358 bp) from the mitochondrial DNA of the estuarine benthic amphipod Grandidierella japonica to analyze DNA extracted from different weights of sediment samples (0.25 g, 1 g, 2.5 g, and 5 g) collected from field sites and a lab aquarium. The DNA extraction efficiency was stable and high for the 1 g sample weight compared with the 0.25 g, 2.5 g, and 5 g sample weights. Based on quantitative PCR assays, the copy number of the short marker was 12.1 times as high than the long marker. Such 12.1 times difference in copy number caused the proportion of positive conventional PCR replicates of shorter marker was higher than that of longer marker. Additionally, the linear mixed-effects model analysis showed that the proportion of positive conventional PCR replicates was negatively related to the marker length and positively related to the sediment weight. This study indicated that the shorter marker and larger treated sample weight were suitable for qualitative analysis with conventional PCR.