Genetic Dissection of a Precocious Phenotype in Male Tiger Pufferfish (Takifugu rubripes) using Genotyping by Random Amplicon Sequencing, Direct (GRAS-Di)

Abstract

The novel non-targeted PCR-based genotyping system, namely Genotyping by Random Amplicon Sequencing, Direct (GRAS-Di), is characterized by the simplicity in library construction and robustness against DNA degradation and is expected to facilitate advancements in genetics, in both basic and applied sciences. In this study, we tested the utility of GRAS-Di for genetic analysis in a cultured population of the tiger pufferfish Takifugu rubripes. The genetic analyses included family structure analysis, genetic map construction, and quantitative trait locus (QTL) analysis for the male precocious phenotype using a population consisting of four full-sib families derived from a genetically precocious line. An average of 4.7 million raw reads were obtained from 198 fish. Trimmed reads were mapped onto a Fugu reference genome for genotyping, and 21,938 putative single-nucleotide polymorphisms (SNPs) were obtained. These 22 K SNPs accurately resolved the sibship and parent–offspring pairs. A fine-scale linkage map (total size: 1,949 cM; average interval: 1.75 cM) was constructed from 1,423 effective SNPs, for which the allele inheritance patterns were known. QTL analysis detected a significant locus for testes weight on Chr_14 and three suggestive loci on Chr_1, Chr_8, and Chr_19. The significant QTL was shared by body length and body weight. The effect of each QTL was small (phenotypic variation explained, PVE: 3.1–5.9%), suggesting that the precociousness seen in the cultured pufferfish is polygenic. Taken together, these results indicate that GRAS-Di is a practical genotyping tool for aquaculture species and applicable for molecular breeding programs, such as marker-assisted selection and genomic selection.

Genomic prediction for testes weight of the tiger pufferfish, Takifugu rubripes, using medium to low density SNPs

“…The genetic relationships among the parents were assessed as explained below prior to the mating to minimize inbreeding between full sibs. The F 1 generation was raised at NPIF in the conventional manner 17 , 25 . At 20 months old, 712 individuals were sampled; 500 (male = 246, female = 254) were euthanized with an overdoze of 2-phenoxy ethanol (> 600 mL/ton) and body size data (SL and BW) was collected.…”

“…In most individuals, the testes reach marketable size (> 100 g) in late January or February (when the fish is about 22 months old); however, some individuals have testes larger than 100 g in early December (at about 20 months old), a time when prices are also at their highest. As precociousness is a polygenic trait 23 , 25 then TW at harvest can potentially be improved by GS. To test this possibility, we raised an experimental population and applied GP for TW, SL, and BW using a genome-wide medium density SNP panel.…”

Effect of Three Types of Ion Beam Irradiation on Gerbera (Gerbera hybrida) In Vitro Shoots with Mutagenesis Efficiency

“…The obtained DNA was used as a template and the entire genome was amplified and sequenced using Genotyping by Random Amplicon Sequencing-Direct (GRAS-Di) technology using a next-generation genome sequencer (Illumina, Eurofins Genomics Inc., Tokyo, Japan). In contrast to conventional genotyping by sequencing, GRAS-Di technology has fewer biased analysis regions, genome-uniform genotyping, and genomic mutation detection and marketization, even in species without reference sequences such as gerbera [ 51 , 52 ]. The sequences obtained from the wild type and mutant were compared and sequences that differing and determined to be highly accurate were selected to determine DNA markers specific to the mutant.…”

A Survey of Genome-Wide Genetic Characterizations of Crossbred Dairy Cattle in Local Farms in Cambodia

“…During the last decade, several cost-effective approaches have been developed for analyzing genome-wide single nucleotide polymorphisms (SNPs) in many individuals with high genetic diversity, including restriction site-associated DNA sequencing (RAD-seq and ddRAD-seq) [ 6 , 7 ], and multiplexed inter-simple sequence repeat genotyping by sequencing (MIG-seq) [ 8 ]. Recently, attention has been paid to genotyping by random amplicon sequencing-direct (GRAS-Di) [ 9 ], as another cost-effective genome-wide genotyping method [ 10 , 11 ]. GRAS-Di performs genotyping for PCR amplicons which are retrieved throughout the genome using random primers.…”

“…GRAS-Di performs genotyping for PCR amplicons which are retrieved throughout the genome using random primers. GRAS-Di produces fewer missing data than RAD-seq [ 10 ] and more SNPs than MIG-seq [ 11 ]. Thus, GRAS-Di has been used widely, for example, to assess the population genetic structure of mangrove fish [ 9 ], the Tsushima leopard cat [ 12 ], and small apes [ 13 ].…”