Random PCR‐based genotyping by sequencing technology GRAS‐Di (genotyping by random amplicon sequencing, direct) reveals genetic structure of mangrove fishes

Abstract

While various technologies for high‐throughput genotyping have been developed for ecological studies, simple methods tolerant to low‐quality DNA samples are still limited. In this study, we tested the availability of a random PCR‐based genotyping‐by‐sequencing technology, genotyping by random amplicon sequencing, direct (GRAS‐Di). We focused on population genetic analysis of estuarine mangrove fishes, including two resident species, the Amboina cardinalfish (Fibramia amboinensis, Bleeker, 1853) and the Duncker's river garfish (Zenarchopterus dunckeri, Mohr, 1926), and a marine migrant, the blacktail snapper (Lutjanus fulvus, Forster, 1801). Collections were from the Ryukyu Islands, southern Japan. PCR amplicons derived from ~130 individuals were pooled and sequenced in a single lane on a HiSeq2500 platform, and an average of three million reads was obtained per individual. Consensus contigs were assembled for each species and used for genotyping of single nucleotide polymorphisms by mapping trimmed reads onto the contigs. After quality filtering steps, 4,000–9,000 putative single nucleotide polymorphisms were detected for each species. Although DNA fragmentation can diminish genotyping performance when analysed on next‐generation sequencing technology, the effect was small. Genetic differentiation and a clear pattern of isolation‐by‐distance was observed in F. amboinensis and Z. dunckeri by means of principal component analysis, FST and the admixture analysis. By contrast, L. fulvus comprised a genetically homogeneous population with directional recent gene flow. These genetic differentiation patterns reflect patterns of estuary use through life history. These results showed the power of GRAS‐Di for fine‐grained genetic analysis using field samples, including mangrove fishes.

East Asian origin of the widespread alpine snow‐bed herb, Primula cuneifolia (Primulaceae), in the northern Pacific region

“…nipponica from four different populations (Table S1.1 in Appendix S1). The DNA samples were used for genotyping by sequencing (GBS) by genotyping by random amplicon sequencing‐direct (GRAS‐Di; Hosoya et al., 2019), which was performed at the Bioengineering Laboratory (Sagamihara, Japan). Briefly, DNA samples were directly amplified using 63 random primers (Table S2.2 in Appendix S2), in which each individual was tagged with independent adapter sequences.…”

“…All amplicons were sequenced using Illumina NextSeq. Compared to other GBS methods such as RADseq, GRAS‐Di does not require a large amount of high‐quality DNA (Davey et al., 2011; Hosoya et al., 2019). Following the methodology described in a previous study (Fujii et al., 1999), Primula farinosa L. (formerly P. modesta Bisset & S. Moore) was included as an outgroup.…”

More affordable and effective noninvasive single nucleotide polymorphism genotyping using high‐throughput amplicon sequencing

“…However, these techniques require specialized equipment and are typically prohibitively expensive for the large sample sizes needed for wildlife population monitoring (Andrews, Barba, Russello, & Waits, 2018; Carroll et al., 2018). A potential cost‐effective alternative involves amplifying many SNPs together in multiplex PCR followed by high‐throughput sequencing (Campbell, Harmon, & Narum, 2015; Hosoya, Hirase, Kikuchi, & Nanjo, 2019; Meek & Larson, 2019; Suyama & Matsuki, 2015). Such genotyping by amplicon sequencing is highly flexible with respect to the number of samples and SNPs processed, affordable, amenable to high throughput, and requires no proprietary kits for library preparation and only standard laboratory equipment.…”

Genetic Diversity and Genetic Structure of the Wild Tsushima Leopard Cat from Genome-Wide Analysis

“…Further, the advance of molecular genetics technology through next-generation sequencing (NGS) has made it possible to obtain extensive nucleotide sequences, enabling rapid and large-scale development of DNA markers. Moreover, more economical and efficient approaches using NGS have been developed, such as restriction site-associated DNA sequencing (RADSeq) [ 14 ], double digest RADSeq (ddRADSeq) [ 15 ], MIG-Seq (multiplexed ISSR genotyping by sequencing) [ 16 ], genotyping-in-thousands by sequencing (GT-Seq) [ 17 , 18 ], and genotyping by random amplicon sequencing, direct (GRAS-Di) [ 19 , 20 ]. These approaches enable simultaneous sequencing and genotyping of thousands of SNP markers, and they have been used in various species, including endangered species [ 18 , 21 ].…”

“…These approaches enable simultaneous sequencing and genotyping of thousands of SNP markers, and they have been used in various species, including endangered species [ 18 , 21 ]. Although GRAS-Di has few applications thus far, GRAS-Di is useful in characterizing wildlife because it can function with the available low-quality DNA in a polymerase chain reaction (PCR)-based method and does not require a reference genome, similar to MIG-seq [ 19 , 20 ]. GRAS-Di is a technology that produces a sequence library by two PCRs using random primers and adapter sequences, and detects SNPs using NGS.…”