start-ver=1.4 cd-journal=joma no-vol=71 cd-vols= no-issue=4 article-no= start-page=405 end-page=416 dt-received= dt-revised= dt-accepted= dt-pub-year=2021 dt-pub=20211001 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Targeted genome modifications in cereal crops en-subtitle= kn-subtitle= en-abstract= kn-abstract=The recent advent of customizable endonucleases has led to remarkable advances in genetic engineering, as these molecular scissors allow for the targeted introduction of mutations or even precisely predefined genetic modifications into virtually any genomic target site of choice. Thanks to its unprecedented precision, efficiency, and functional versatility, this technology, commonly referred to as genome editing, has become an effective force not only in basic research devoted to the elucidation of gene function, but also for knowledgebased improvement of crop traits. Among the different platforms currently available for site-directed genome modifications, RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) endonucleases have proven to be the most powerful. This review provides an application-oriented overview of the development of customizable endonucleases, current approaches to cereal crop breeding, and future opportunities in this field. en-copyright= kn-copyright= en-aut-name=HisanoHiroshi en-aut-sei=Hisano en-aut-mei=Hiroshi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=AbeFumitaka en-aut-sei=Abe en-aut-mei=Fumitaka kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=HoffieRobert E. en-aut-sei=Hoffie en-aut-mei=Robert E. kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=KumlehnJochen en-aut-sei=Kumlehn en-aut-mei=Jochen kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= affil-num=1 en-affil=Institute of Plant Science and Resources, Okayama University kn-affil= affil-num=2 en-affil=Institute of Crop Science, National Agriculture and Food Research Organization kn-affil= affil-num=3 en-affil=Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) kn-affil= affil-num=4 en-affil=Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) kn-affil= en-keyword=barley kn-keyword=barley en-keyword= CRISPR kn-keyword= CRISPR en-keyword= maize kn-keyword= maize en-keyword=rice kn-keyword=rice en-keyword=TALEN kn-keyword=TALEN en-keyword=wheat kn-keyword=wheat en-keyword=zinc-finger nucleases kn-keyword=zinc-finger nucleases END start-ver=1.4 cd-journal=joma no-vol= cd-vols= no-issue= article-no= start-page=1 end-page=10 dt-received= dt-revised= dt-accepted= dt-pub-year=2021 dt-pub=20210829 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Regulation of germination by targeted mutagenesis of grain dormancy genes in barley en-subtitle= kn-subtitle= en-abstract= kn-abstract=High humidity during harvest season often causes pre-harvest sprouting in barley (Hordeum vulgare). Prolonged grain dormancy prevents pre-harvest sprouting; however, extended dormancy can interfere with malt production and uniform germination upon sowing. In this study, we used Cas9-induced targeted mutagenesis to create single and double mutants in QTL FOR SEED DORMANCY 1 (Qsd1) and Qsd2 in the same genetic background. We performed germination assays in independent qsd1 and qsd2 single mutants, as well as in two double mutants, which revealed a strong repression of germination in the mutants. These results demonstrated that normal early grain germination requires both Qsd1 and Qsd2 function. However, germination of qsd1 was promoted by treatment with 3% hydrogen peroxide, supporting the notion that the mutants exhibit delayed germination. Likewise, exposure to cold temperatures largely alleviated the block of germination in the single and double mutants. Notably, qsd1 mutants partially suppress the long dormancy phenotype of qsd2, while qsd2 mutant grains failed to germinate in the light, but not in the dark. Consistent with the delay in germination, abscisic acid accumulated in all mutants relative to the wild type, but abscisic acid levels cannot maintain long-term dormancy and only delay germination. Elucidation of mutant allele interactions, such as those shown in this study, are important for fine-tuning traits that will lead to the design of grain dormancy through combinations of mutant alleles. Thus, these mutants will provide the necessary germplasm to study grain dormancy and germination in barley. en-copyright= kn-copyright= en-aut-name=HisanoHiroshi en-aut-sei=Hisano en-aut-mei=Hiroshi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=HoffieRobert E. en-aut-sei=Hoffie en-aut-mei=Robert E. kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=AbeFumitaka en-aut-sei=Abe en-aut-mei=Fumitaka kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=MunemoriHiromi en-aut-sei=Munemori en-aut-mei=Hiromi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=MatsuuraTakakazu en-aut-sei=Matsuura en-aut-mei=Takakazu kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=EndoMasaki en-aut-sei=Endo en-aut-mei=Masaki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=MikamiMasafumi en-aut-sei=Mikami en-aut-mei=Masafumi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= en-aut-name=NakamuraShingo en-aut-sei=Nakamura en-aut-mei=Shingo kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=8 ORCID= en-aut-name=KumlehnJochen en-aut-sei=Kumlehn en-aut-mei=Jochen kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=9 ORCID= en-aut-name=SatoKazuhiro en-aut-sei=Sato en-aut-mei=Kazuhiro kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=10 ORCID= affil-num=1 en-affil=Institute of Plant Science and Resources, Okayama University kn-affil= affil-num=2 en-affil=Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben kn-affil= affil-num=3 en-affil=Institute of Crop Science, NARO kn-affil= affil-num=4 en-affil=Institute of Plant Science and Resources, Okayama University kn-affil= affil-num=5 en-affil=Institute of Plant Science and Resources, Okayama University kn-affil= affil-num=6 en-affil=Institute of Agrobiological Sciences, NARO kn-affil= affil-num=7 en-affil=Institute of Agrobiological Sciences, NARO kn-affil= affil-num=8 en-affil=Institute of Crop Science, NARO kn-affil= affil-num=9 en-affil=Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben kn-affil= affil-num=10 en-affil=Institute of Plant Science and Resources, Okayama University kn-affil= en-keyword=Hordeum vulgare kn-keyword=Hordeum vulgare en-keyword=seed dormancy kn-keyword=seed dormancy en-keyword=targeted genome modification kn-keyword=targeted genome modification en-keyword=CRISPR kn-keyword=CRISPR en-keyword=Cas9 nuclease kn-keyword=Cas9 nuclease en-keyword=pre-harvest sprouting kn-keyword=pre-harvest sprouting END start-ver=1.4 cd-journal=joma no-vol=12 cd-vols= no-issue= article-no= start-page=715545 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2021 dt-pub=20210819 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Identification of a Novel Quinvirus in the Family Betaflexiviridae That Infects Winter Wheat en-subtitle= kn-subtitle= en-abstract= kn-abstract=Yellow mosaic disease in winter wheat is usually attributed to the infection by bymoviruses or furoviruses; however, there is still limited information on whether other viral agents are also associated with this disease. To investigate the wheat viromes associated with yellow mosaic disease, we carried out de novo RNA sequencing (RNA-seq) analyses of symptomatic and asymptomatic wheat-leaf samples obtained from a field in Hokkaido, Japan, in 2018 and 2019. The analyses revealed the infection by a novel betaflexivirus, which tentatively named wheat virus Q (WVQ), together with wheat yellow mosaic virus (WYMV, a bymovirus) and northern cereal mosaic virus (a cytorhabdovirus). Basic local alignment search tool (BLAST) analyses showed that the WVQ strains (of which there are at least three) were related to the members of the genus Foveavirus in the subfamily Quinvirinae (family Betaflexiviridae). In the phylogenetic tree, they form a clade distant from that of the foveaviruses, suggesting that WVQ is a member of a novel genus in the Quinvirinae. Laboratory tests confirmed that WVQ, like WYMV, is potentially transmitted through the soil to wheat plants. WVQ was also found to infect rye plants grown in the same field. Moreover, WVQ-derived small interfering RNAs accumulated in the infected wheat plants, indicating that WVQ infection induces antiviral RNA silencing responses. Given its common coexistence with WYMV, the impact of WVQ infection on yellow mosaic disease in the field warrants detailed investigation. en-copyright= kn-copyright= en-aut-name=KondoHideki en-aut-sei=Kondo en-aut-mei=Hideki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=YoshidaNaoto en-aut-sei=Yoshida en-aut-mei=Naoto kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=FujitaMiki en-aut-sei=Fujita en-aut-mei=Miki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=MaruyamaKazuyuki en-aut-sei=Maruyama en-aut-mei=Kazuyuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=HyodoKiwamu en-aut-sei=Hyodo en-aut-mei=Kiwamu kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=HisanoHiroshi en-aut-sei=Hisano en-aut-mei=Hiroshi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=TamadaTetsuo en-aut-sei=Tamada en-aut-mei=Tetsuo kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= en-aut-name=AndikaIda Bagus en-aut-sei=Andika en-aut-mei=Ida Bagus kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=8 ORCID= en-aut-name=SuzukiNobuhiro en-aut-sei=Suzuki en-aut-mei=Nobuhiro kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=9 ORCID= affil-num=1 en-affil=Institute of Plant Science and Resources (IPSR), Okayama University kn-affil= affil-num=2 en-affil=Agricultural Research Institute, HOKUREN Federation of Agricultural Cooperatives kn-affil= affil-num=3 en-affil=Institute of Plant Science and Resources (IPSR), Okayama University kn-affil= affil-num=4 en-affil=Institute of Plant Science and Resources (IPSR), Okayama University kn-affil= affil-num=5 en-affil=Institute of Plant Science and Resources (IPSR), Okayama University kn-affil= affil-num=6 en-affil=Institute of Plant Science and Resources (IPSR), Okayama University kn-affil= affil-num=7 en-affil=Institute of Plant Science and Resources (IPSR), Okayama University kn-affil= affil-num=8 en-affil=College of Plant Health and Medicine, Qingdao Agricultural University kn-affil= affil-num=9 en-affil=Institute of Plant Science and Resources (IPSR), Okayama University kn-affil= en-keyword=Betaflexiviridae kn-keyword=Betaflexiviridae en-keyword=quinvirus kn-keyword=quinvirus en-keyword=bymovirus kn-keyword=bymovirus en-keyword=yellow mosaic disease kn-keyword=yellow mosaic disease en-keyword=wheat kn-keyword=wheat en-keyword=virome kn-keyword=virome en-keyword=soil borne kn-keyword=soil borne en-keyword=variants kn-keyword=variants END start-ver=1.4 cd-journal=joma no-vol=1 cd-vols= no-issue=2 article-no= start-page=100053 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2020 dt-pub=20200613 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Protocol for Genome Editing to Produce Multiple Mutants in Wheat en-subtitle= kn-subtitle= en-abstract= kn-abstract=Here, we describe a protocol for producing multiple recessive mutants via genome editing in hexaploid wheat (Triticum aestivum) cv. Fielder. Using Agrobacterium-delivered CRISPR/Cas9 and three sub-genome-specific primer sets, all possible combinations of single, double, and triple transgene-free mutants can be generated. The technique for acceleration of generation advancement with embryo culture reduces time for mutant production. The mutants produced by this protocol can be used for the analysis of gene function and crop improvement. For complete details on the use and execution of this protocol, please refer to Abe et?al. (2019). en-copyright= kn-copyright= en-aut-name=AbeFumitaka en-aut-sei=Abe en-aut-mei=Fumitaka kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=IshidaYuji en-aut-sei=Ishida en-aut-mei=Yuji kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=HisanoHiroshi en-aut-sei=Hisano en-aut-mei=Hiroshi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=EndoMasaki en-aut-sei=Endo en-aut-mei=Masaki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=KomariToshihiko en-aut-sei=Komari en-aut-mei=Toshihiko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=TokiSeiichi en-aut-sei=Toki en-aut-mei=Seiichi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=SatoKazuhiro en-aut-sei=Sato en-aut-mei=Kazuhiro kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= affil-num=1 en-affil=Division of Basic Research, Institute of Crop Science, NARO kn-affil= affil-num=2 en-affil=Plant Innovation Center, Japan Tobacco Inc. kn-affil= affil-num=3 en-affil=Institute of Plant Science and Resources, Okayama University kn-affil= affil-num=4 en-affil=Division of Applied Genetics, Institute of Agrobiological Sciences, NARO kn-affil= affil-num=5 en-affil=Plant Innovation Center, Japan Tobacco Inc. kn-affil= affil-num=6 en-affil=Division of Applied Genetics, Institute of Agrobiological Sciences, NARO kn-affil= affil-num=7 en-affil=Institute of Plant Science and Resources, Okayama University kn-affil= END start-ver=1.4 cd-journal=joma no-vol=11 cd-vols= no-issue= article-no= start-page=509 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2020 dt-pub=20200407 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Virome Analysis of Aphid Populations That Infest the Barley Field: The Discovery of Two Novel Groups of Nege/Kita-Like Viruses and Other Novel RNA Viruses en-subtitle= kn-subtitle= en-abstract= kn-abstract=Aphids (order Hemiptera) are important insect pests of crops and are also vectors of many plant viruses. However, little is known about aphid-infecting viruses, particularly their diversity and relationship to plant viruses. To investigate the aphid viromes, we performed deep sequencing analyses of the aphid transcriptomes from infested barley plants in a field in Japan. We discovered virus-like sequences related to nege/kita-, flavi-, tombus-, phenui-, mononega-, narna-, chryso-, partiti-, and luteoviruses. Using RT-PCR and sequence analyses, we determined almost complete sequences of seven nege/kitavirus-like virus genomes; one of which was a variant of the Wuhan house centipede virus (WHCV-1). The other six seem to belong to four novel viruses distantly related to Wuhan insect virus 9 (WhIV-9) or Hubei nege-like virus 4 (HVLV-4). We designated the four viruses as barley aphid RNA virus 1 to 4 (BARV-1 to -4). Moreover, some nege/kitavirus-like sequences were found by searches on the transcriptome shotgun assembly (TSA) libraries of arthropods and plants. Phylogenetic analyses showed that BARV-1 forms a clade with WHCV-1 and HVLV-4, whereas BARV-2 to -4 clustered with WhIV-9 and an aphid virus, Aphis glycines virus 3. Both virus groups (tentatively designated as Centivirus and Aphiglyvirus, respectively), together with arthropod virus-like TSAs, fill the phylogenetic gaps between the negeviruses and kitaviruses lineages. We also characterized the flavi/jingmen-like and tombus-like virus sequences as well as other RNA viruses, including six putative novel viruses, designated as barley aphid RNA viruses 5 to 10. Interestingly, we also discovered that some aphid-associated viruses, including nege/kita-like viruses, were present in different aphid species, raising a speculation that these viruses might be distributed across different aphid species with plants being the reservoirs. This study provides novel information on the diversity and spread of nege/kitavirus-related viruses and other RNA viruses that are associated with aphids. en-copyright= kn-copyright= en-aut-name=KondoHideki en-aut-sei=Kondo en-aut-mei=Hideki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=FujitaMiki en-aut-sei=Fujita en-aut-mei=Miki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=HisanoHiroshi en-aut-sei=Hisano en-aut-mei=Hiroshi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=HyodoKiwamu en-aut-sei=Hyodo en-aut-mei=Kiwamu kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=AndikaIda Bagus en-aut-sei=Andika en-aut-mei=Ida Bagus kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=SuzukiNobuhiro en-aut-sei=Suzuki en-aut-mei=Nobuhiro kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= affil-num=1 en-affil=Institute of Plant Science and Resources (IPSR), Okayama University kn-affil= affil-num=2 en-affil=Institute of Plant Science and Resources (IPSR), Okayama University kn-affil= affil-num=3 en-affil=Institute of Plant Science and Resources (IPSR), Okayama University kn-affil= affil-num=4 en-affil=Institute of Plant Science and Resources (IPSR), Okayama University kn-affil= affil-num=5 en-affil=College of Plant Health and Medicine, Qingdao Agricultural University kn-affil= affil-num=6 en-affil=Institute of Plant Science and Resources (IPSR), Okayama University kn-affil= en-keyword=negevirus kn-keyword=negevirus en-keyword=kitavirus kn-keyword=kitavirus en-keyword=aphid kn-keyword=aphid en-keyword=virome kn-keyword=virome en-keyword=RNA seq kn-keyword=RNA seq en-keyword=barley kn-keyword=barley en-keyword=diversity kn-keyword=diversity en-keyword=horizontal transmission kn-keyword=horizontal transmission END start-ver=1.4 cd-journal=joma no-vol=9 cd-vols= no-issue= article-no= start-page=3745 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2019 dt-pub=201936 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Imaging Amyloplasts in the Developing Endosperm of Barley and Rice en-subtitle= kn-subtitle= en-abstract= kn-abstract=Amyloplasts are plant-specific organelles responsible for starch biosynthesis and storage. Inside amyloplasts, starch forms insoluble particles, referred to as starch grains (SGs). SG morphology differs between species and SG morphology is particularly diverse in the endosperm of Poaceae plants, such as rice (Oryza sativa) and barley (Hordeum vulgare), which form compound SGs and simple SGs, respectively. SG morphology has been extensively imaged, but the comparative imaging of amyloplast morphology has been limited. In this study, SG-containing amyloplasts in the developing endosperm were visualized using stable transgenic barley and rice lines expressing amyloplast stroma-targeted green fluorescent protein fused to the transit peptide (TP) of granule-bound starch synthase I (TP-GFP). The TP-GFP barley and rice plants had elongated amyloplasts containing multiple SGs, with constrictions between the SGs. In barley, some amyloplasts were connected by narrow protrusions extending from their surfaces. Transgenic rice lines producing amyloplast membrane-localized SUBSTANDARD STARCH GRAIN6 (SSG6)-GFP were used to demonstrate that the developing amyloplasts contained multiple compound SGs. TP-GFP barley can be used to visualize the chloroplasts in leaves and other plastids in pollen and root in addition to the endosperm, therefore it provides as a useful tool to observe diverse plastids. en-copyright= kn-copyright= en-aut-name=MatsushimaRyo en-aut-sei=Matsushima en-aut-mei=Ryo kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=HisanoHiroshi en-aut-sei=Hisano en-aut-mei=Hiroshi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= affil-num=1 en-affil= Institute of Plant Science and Resources, Okayama University kn-affil= affil-num=2 en-affil= Institute of Plant Science and Resources, Okayama University kn-affil= END