Title

Citation

Random PCR-based genotyping by sequencing
1

technology GRAS-Di (genotyping by random ampliconsequencing, direct) reveals genetic structure of

Mol Ecol Resour. 19:1153-1163. (2019)mangrove fishes

Species

Goal of the study

F. amboinensis

GRAS-Di method is applied to ecological studies where a large number ofL. fulvus

SNPs is required, such as population structure analysis of marine fish, while

Z. dunckeri

high quality DNA samples are not always available

The identification of individuals in the Tsushima leopard population isdifficult using microsatellite marker analysis due to the population’s low
2

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

genetic diversity, so more DNA markers must be developed to design an
Animals (Basel). 10:1375. (2020)

Wild Tsushima Leopard Cat

appropriate conservation program.
To identify SNPs using genome-wide analysis with the GRAS-Di approachthat would clarify the genetic diversity and structure of the wild Tsushima
leopard cat.

East Asian origin of theEast Asian origin of the widespread alpine snow-bed
3 herb, Primula cuneifolia (Primulaceae), in the northernwidespread alpine snow-bed
J Biogeogr. 00: 1– 13. (2020)

Pacific region

herb, Primula cuneifolia(Primulaceae)(P. cuneifolia

To explore the molecular evidence for the recent northward migration of
alpine snow-bed species in the northern Pacific region.

and its sister species)

Divergence and introgression in small apes, the genus
4 Hylobates, revealed by reduced representation

Heredity (Edinb). 127:312-322. (2021)

sequencing

Gibbons in the genus

To uncover the divergence history and the contributions of hybridisation andHylobates

introgression to the evolution of Hylobates

Developped a high-density linkage map and identify quantitative trait loci
(QTLs) for recombinant inbred lines of Aegilops tauschii , a D-genome
GRAS-Di system facilitates high-density genetic map
5 construction and QTL identification in recombinant

Recombinant inbred lines of donor of bread wheat, using the recently developed genotyping by Random
Scientific Reports (2020)10:21455

inbred lines of the wheat progenitor Aegilops tauschii

the wheat progenitor

Amplicon Sequencing-Direct (GRAS-Di) system, which facilitates skimming

Aegilops tauschiiof the large and complicated genome and generates a large number ofgenetic markers. The deduced linkage groups based on the GRAS-Di
genetic markers corresponded to the chromosome number of Ae. tauschii .
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

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

Marine Biotechnology. 23:177-188 (2021)

Male Tiger Pufferfish

analyses included family structure analysis, genetic map construction, and(Takifugu rubripes )quantitative trait locus (QTL) analysis for the male precocious phenotype
using a population consisting of four full-sib families derived from agenetically precocious line.

Seed size and shape traits are important determinants of seed yield andA Major and Stable Quantitative Trait Locus qSS2 for
7 Seed Size and Shape Traits in a Soybean RIL
Population

QTL analysis of crown gall disease resistance in apple:
8 first plant R gene candidates effective against

Rhizobium rhizogenes (Ti)appearance quality in soybean [Glycine max (L.) Merr.]. Understanding the
Front. Genet., 16 April 2021,
Volume 12 - 2021

Soybean RIL Population

genetic architecture of these traits is important to enable their genetic
improvement through efficient and targeted selection in soybean breeding,and for the identification of underlying causal genes.

Identifing quantitative trait loci (QTLs) that control resistance to tumourTree Genetics & Genomes volume 17,
Article number: 25. (2021)

Apple tree crown gall

inducing Rhizobium isolates found in apple production areas in Japan.
Fine mapping Rrr1 , and determining the nuculeotide sequnence of its
candidate region, and predicting candidate gene.

F1 population of the
Biological and molecular characterization of linalool9 mediated field resistance against Xanthomonas citri
subsp. citri in citrus trees.

Tree Physiology, Volume 41, Issue 11,
November 2021, Pages 2171–2188,

resistant Ponkan mandarin

Quantitative trait locus (QTL) analysis revealed that linalool accumulationand susceptible ‘Harehime’and Xcc (citrus canker disease, caused by the bacterial pathogen(‘E-647’ × ‘Miyagawa-

Xanthomonas citri subsp. Citri) susceptibility QTLs overlapped.

wase’) cultivar
The seedlings of hybrid
Analysis of the possible cytogenetic mechanism for10

overcoming hybrid lethality in an interspecific crossbetween Nicotiana suaveolens and Nicotiana

parents (N. suaveolens andScientific Reports volume 11, Article

N. tabacum), two lethal

number: 7812 (2021)

hybrid seedlings, and one

tabacum .

hybrid seedling that

Finding hybrids that overcome lethality, either spontaneously or by artificial
culture, lack the distal part of the Q chromosome, a region that includes thegene responsible for lethality.

overcame lethality.
Detection of an identical QTL for indices of smut disease incidence with a
Detection of a major QTL related to smut disease
11 resistance inherited from a Japanese wild sugarcane
using GRAS-Di technology.

Breeding Science. 2021 Volume 71 Issue
3 Pages 365-374

markedly high LOD score at the end of linkage group 8 of ‘Yaenoushie’. This
Japanese wild sugarcane

QTL could be used for marker-assisted selection (MAS) to significantly
improve smut disease resistance without negative effects on otheragronomic traits.
The genomic mutation in the flower color, petal shape, and male sterile

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

Plants. 10:1480. (2021)

GerberaMutagenesis Efficiency

mutants were confirmed by GRAS-Di technology. As a result, 1 marker forflower color mutants, 6 markers for petal slender mutants, and 31 markers
for male sterile mutants were identified.
The objective was to develop an artificial method for overcoming self-

Artificial Control of the Prunus Self-incompatibility
13 System Using Antisense Oligonucleotides against
Pollen Genes

The Horticulture Journal/Volume 91(2022) Issue 4, Pages 437-447

incompatibility (SI) via the knockdown of SFB (S haplotype–specific F-box)
Prunusor MGST(M-locus encoded glutathione S-transferase-like) genes in pollen.
After the self-pollination, GRAS-Di analyses were performed to determine
kinship relationships.
To address how far speciation has proceeded along the speciation

14

Genomic Evidence for Speciation with Gene Flow in
Broadcast Spawning Marine Invertebrates

Mol Biol Evol. 38:4683-4699.

Abalone (H. discus, H.
madaka, and H. gigantea)

continuum, the patterns of genomic divergence in the Western Pacificabalones were examined. using nuclear SNP loci obtained with GRAS-Di
and whole-genome sequencing, as well as with mitochondrial genome
sequencing.

To validate genome-wide association scan for sex determining locus of Ayu,
15

A Y-linked anti-Müllerian hormone type-II receptor isthe sex-determining gene in ayu, Plecoglossus altivelis

linkage analysis of genetic sex was performed, using Ayu F1 full-sib families
PLoS Genet. 17: e1009705.

Ayu (Plecoglossus altivelis)

captured from a wild population in the Nagaragawa River. Two F1 full-sib
family (88 siblings, two male parents and two female parents) were used forlinkage analysis using GRAS-Di analysis.
To investigate the potential value of genomic selection for 10 traits related

Genomic prediction of 10 metamorphic traits of
16 captive-bred Japanese eels (Anguilla japonica ) using

Aquaculture. 548: 737671. (2022)

the GRAS-Di genotyping method

Japanese eel (Anguillajaponica)

to the timing of metamorphosis and the body size at that time, 1689 glass
eels were used. To identify and genotype SNP markers, which were used toassign parentage and as a basis for genomic selection, GRAS-Di sequencing
was performed.
To obtain the genome-wide genotype data from 98 full-sibling progenies in

17

Repeated translocation of a supergene underlying rapid
sex chromosome turnover in Takifugu pufferfish

Proc Natl Acad Sci U S A. 119:e2121469119. (2022)

Takifugu snyderi

T. snyderi family, GRAS-Di technology was used. Then, linkage map
construction and mapping of the sex-determining locus in T. snyderi were
performed.
In order to assess the genetic diversity of the captive population of
Tsushima Leopard Cat (TLC), the authors modified the draft genome using

Comparison of the Genetic Diversity of the Captive and
18 Wild Populations of the Tsushima Leopard Cat Using a

Animals. 12:1464. (2022)

GRAS-Di Analysis

Tsushima leopard cat

short-read and long-read sequencing data and compared the genetic(Prionailurus bengalensis

diversity of captive and wild populations of TLC by GRAS-Di analysis.

euptilurus)

The results showed that the captive population had almost the same
genetic diversity as the wild population and managed to remain in goodcondition.
To examine genetic variations among wood mice inhabiting the islands ofAncient rivers shaped the current genetic diversity of
19 the wood mouse (Apodemus speciosus ) on the islands

Zoological Lett. 8:9 (2022).

of the Seto Inland Sea, Japan

Wood mouse (Apodemus

the Seto Inland Sea, the autohrs used the GRAS-Di method.

speciosus)

They tested the hypothesis that ancient rivers could explain the genetic
relationships among the wood mice on these islands.
To reveal genetic relatedness and diversity of 62 cultivars and breeding

Genetic relationship and diversity of cultivars and20

breeding lines of tetraploid Italian ryegrass (Lolium

multiflorum Lam .) and its hybrids with Lolium-Festucalines of tetraploid Italian ryegrass (Lolium multiflorum Lam.; 39 accessions)
Grassland Science. 69:65-78. (2023)

Italian ryegrass (Lolium

and its interspecific hybrids, Festulolium (18 accessions), and hybrid

multiflorum Lam.)ryegrass (Lolium × hybridum Hausskn.; 5 accessions), 2,824 genome-wide

complex based on genome-wide allele frequency

allele frequencies were obtained by GRAS-Di method using bulk genomic
DNA testing.
In order to reveal the genetic variation and the milk production in
Cambodian crossbred dairy cattle and provide information for improving theefficiency of future cattle crossbreeding strategies to establish an adequate

21

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

dairy strain, the autors conducted a genome-wide survey of two dairy farmAnimals. 12:2072. (2022)

Cattle/Cow

populations, using GRAS-Di analysis, in the suburb area of Phnom Penhcity, Cambodia.
Then, they performed STRUCTURE analyses of the two farm populations tounderstand their genetic characteristics, in comparison with a pureJapanese Holstein-Friesian cow, as a reference.

To present an example of a species in an intermediate stage of speciation,
the giant water bug Appasus japonicus (Heteroptera, Belostomatidae) wasinvestigated using mating experiments and phylogenetic analyses of the
Genome-wide molecular phylogenetic analyses and22

mating experiments which reveal the evolutionary
history and an intermediate stage of speciation of a

mtDNA COI and 16S rRNA regions, and nuclear DNA SSR and its genomeMol Ecol. 30:5179-5195. (2021)

Giant water bug (Appasus

wide SNPs (11,241 SNPs).

japonicus)

The results of our phylogenetic analyses based on their mtDNA data set andgiant water bug

the genome-wide SNPs data set strongly supported the paraphyly of theJapanese populations. suggesting that their ancestral lineage which being
distributed in the Japanese Archipelago subsequently migrated to theEurasian Continent.
To investigate the genetic loci associated with maturity time as a trait thatdefines the ecotype under natural linkage disequilibrium (LD) conditions.
Using F2 segregating populations, the authors developed a high-density

Genetic basis of maturity time is independent from that23of flowering time and contributes to ecotype
differentiation in common buckwheat (Fagopyrum

BMC Plant Biol. 22:353. (2022)

esculentum Moench).

Common buckwheat

genetic linkage map by GRAS-Di analysis via re-estimation of co-dominant(Fagopyrum esculentum

marker genotypes.

Moench)Using this map, they detected major QTLs for maturity time. These QTLs
were located at loci different from those of the flowering time QTLs,
suggesting that the photoperiod responses of maturity time and flowering
time would have different mechanisms.

Genetic diversity and structure of diploid Italian24

ryegrass (Lolium multiflorum Lam.) cultivars andbreeding materials in Japan based on genome-wide

In order to reveal the genetic diversity of cultivars and breeding materials of
Grassland Science. 68:263-276 (2022)

Italian ryegrass (Lolium

diploid Italian ryegrass in Japan, the authors investigated genetic diversity

multiflorum Lam.)among and within 89 accessions by using genome-wide allele frequencies

allele frequency

obtained by GRAS-Di.
To establish a chromosome-scale de novo assembly of E. grandiflorum
genome sequences, the authors performed four genomic and genetic
approaches: (1) PacBio Sequel deep sequencing, (2) error correction of theassembly by Illumina short reads, (3) scaffolding by chromatin conformation

Chromosome-scale genome assembly of Eustoma
25 grandiflorum , the first complete genome sequence inthe genus Eustoma

capture sequencing (Hi-C), and (4) genetic linkage maps derived from an F2
G3 Genes|Genomes|Genetics, 13:
jkac329 (2023)

Eustoma grandiflorum

mapping population.
To assess the genome structure divergence and phylogenetic relations, theassembled genome and predicted genes were compared with those of otherspecies, including those in the order Gentianales,
This is the first report on the construction of reference genome sequencesin E. grandiflorum as well as in the genus Eustoma, leading to insights into
the genome structure of the genus Eustoma and the family Gentianaceae.

Efficient Production of Doubled Haploids as Isogenic
26 Line Founders via Double Heat Shock Treatment inWillow Gudgeon (Gnathopogon caerulescens )Aquaculture Research.

Willow Gudgeon

2023:9783381 (2023)(Gnathopogon caerulescens) GRAS-Di was used.

To confirm the zygosity of some hatchlings treated with Double Heat Shock,To present evidence of an ongoing speciation event in the Japanese
Phylogeography of Kisaura Ross (Trichoptera:
Philopotamidae) of the Japanese Archipelago and the
27 character displacement evolution observed in asecondary contact area between geneticallyArchipelago, the authors conducted molecular phylogenetic analyses based
Zoological Journal of the Linnean Society,197:176–188. (2023)

Kisaura Ross (Trichoptera:
Philopotamidae)

differentiated intra-specific lineages

on the mtDNA COI region (658 bp) and genome-wide nDNA single
nucleotide polymorphisms (SNPs) (16 254 loci) using the K. nozakii species
complex and a sister-species, Kisaura borealis.
It was revealed that the Kisaura nozakii species complex is composed ofmultiple allopatrically differentiated genetic lineages.
The authors report the chromosome-level genome assembly of kawakawa E.
affinis, the first available chromosome-level assembly within the Pelagiaria
clade.

Chromosome-Scale Genome Assembly and
28 Transcriptome Assembly of Kawakawa Euthynnus

Front Genet. 2021 12:739781

Euthynnus affinis

affinis ; A Tuna-Like Species

Two types of linkage evidence: diploid, based on full-sib family linkage
analyses, and haploid, based on linkage analyses of interspecific hybrids ofE. affinis female and T. orientalis male, were obtained.
Based on a genome coordinate of each marker in the linkage maps using
GRAS-Di, they anchored and oriented scaffolds into pseudochromosomes.
GRAS-Di was used to identify SNPs in a panel of 96 individuals
representing the Turkish national hazelnut collection.

GRAS-Di SNP-based molecular characterization and29

fingerprinting of a Turkish Corylus avellana core set
provide insights into the cultivation and breeding of

Mol Genet Genomics. 298:413–426 (2023)

Hazelnut (Corylus avellanaL.)

hazelnut in Turkey

The resulting 7609 high-quality SNPs were physically mapped to the
Tombul cultivar reference genome and used for population structure anddiversity analyses. These analyses revealed that cultivars are not lessdiverse than wild accessions and that 44% of the panel had admixed
ancestry.
To unveil potential polymorphism between closely related Japanese ricecultivars, the authors used a GRAS-Di method to develop genetic linkagemaps.
The average genetic distance between markers showed a clear decrease in

High-Density Linkage Maps from Japanese Rice
30 japonica Recombinant Inbred Lines Using Genotyping

Plants. 12:929. (2023)

Riceby Random Amplicon Sequencing-Direct (GRAS-Di)

the four RIL populations (190 for RIL71, 96 for RIL98, 95 for RIL16, and 94
for RIL91) when markers of both linkage maps (maps by GRAS-Di and thoseby GoldenGate SNP assay) were integrated.
Genetic studies using these markers successfully localized five QTLs
associated with heading date on chromosomes 3, 6, and 7, and which
previously were identified as Hd1, Hd2, Hd6, Hd16, and Hd17.
To identify the genetic factors of culm strength of Omachi using RILs
between Omachi and Koshihikari, QTL analysis were performed.

Identification of Novel Quantitative Trait Loci for Culm
31 Thickness of Rice Derived from Strong-Culm Landracein Japan, Omachi

Using 1,904 DNA markers obtained from a GoldenGate® Assay and GRAS-Di
Rice 16:4 (2023)

Rice® analysis, three QTLs for the culm diameter of the 5th internode on
chromosomes 3 and 7 (qCD3, qCD7-1, qCD7-2) were identified.
The results suggest that the Omachi alleles of qCD3, qCD7-1 and qCD7-2
are useful for breeding to increase lodging resistance and yield.
(As of July/2023)