HvCO9 was characterized to elucidate the barley flowering control mechanisms and to investigate the functional diversification of the barley CONSTANS-like (CO-like) genes in flowering. HvCO9 was located on the same chromosome, 1HL, as Ppd-H2 (HvFT3), which is a positive regulator of short-day (SD) flowering. A phylogenetic analysis showed that HvCO9 was located on the same branch of the CO-like gene tree as rice Ghd7 and the barley and wheat VRN2 genes, which are all negative regulators of flowering. High level HvCO9 expressions were observed under SD conditions, whereas its expression levels were quite low under long-day (LD) conditions. HvCO9 expression correlated with HvFT1 and HvFT2 expression under SD conditions, although no clear effect of HvCO9 on HvFT3 expression, or vice versa, under SD conditions was observed. The over-expression of HvCO9 in rice plants produced a remarkable delay in flowering. In transgenic rice, the expression levels of the flowering-related Ehd1 gene, which is a target gene of Ghd7, and its downstream genes were suppressed, causing a delay in flowering. These results suggest that HvCO9 may act as a negative regulator of flowering under non-inductive SD conditions in barley; this activity is similar to that of rice Ghd7 under non-inductive LD conditions, but the functional targets of these genes may be different. Our results indicate that barley has developed its own pathways to control flowering by using homologous genes with modifications for the timing of expression. Further, it is hypothesized that each pathway may target different genes after gene duplication or species diversification.

Genomic selection is attracting attention in the field of crop breeding. To apply genomic selection effectively for autogamous (self-pollinating) crops, an efficient outcross system is desired. Since dominant male sterility is a powerful tool for easy and successive outcross of autogamous crops, we developed transgenic dominant male sterile rice (Oryza sativa L.) using the barnase gene that is expressed by the tapetum-specific promoter BoA9. Barnase-induced male sterile rice No. 10 (BMS10) was selected for its stable male sterility and normal growth characteristics. The BMS10 flowering habits, including heading date, flowering date, and daily flowering time of BMS10 tended to be delayed compared to wild type. When BMS10 and wild type were placed side-by-side and crossed under an open-pollinating condition, the seed-setting rate was <1.5%. When the clipping method was used to avoid the influence of late flowering habits, the seed-setting rate of BMS10 increased to a maximum of 86.4%. Although flowering synchronicity should be improved to increase the seed-setting rate, our results showed that this system can produce stable transgenic male sterility with normal female fertility in rice. The transgenic male sterile rice would promote a genomic selection-based breeding system in rice.

A loss of granule‐bound starch synthase I (GBSS I) activity results in starch granules that contain mostly amylopectin and little or no amylose, a phenotype described as waxy. Previously, two phenotypic classes of waxy alleles, wxa, associated with no detectable GBSS I, and wxb, associated with apparently inactive GBSS I in the endosperm, were reported in sorghum (Sorghum bicolor (L.) Moench). In this study, the waxy alleles in a sorghum core collection were investigated using DNA markers. Of the 337 sorghum accessions examined, 17 accessions that were confirmed to be waxy by a negative iodine staining result and 16 were found to be wxa. A novel waxy allele, wxc, was found in a Taiwanese landrace. This allele consists of a +1G to C mutation in the 5′ splice site at the intron 10–exon 11 boundary, a mutation that most likely resulted in the suppression of GBSS I gene expression. A DNA marker specific for wxc was produced to distinguish the wxc allele from other alleles, allowing the identification of heterozygous non‐waxy plants.

Although the advent of several new breeding techniques (nBts) is revolutionizing agricultural production processes, technical information necessary for their regulation is yet to be provided. Here, we show that high-throughput DNA sequencing is effective for the detection of unintended remaining foreign DNA segments in genome-edited rice. A simple k-mer detection method is presented and validated through a series of computer simulations and real data analyses. The data show that a short foreign DNA segment of 20 nucleotides can be detected and the probability that the segment is overlooked is 10 −3 or less if the average sequencing depth is 30 or more, while the number of false hits is less than 1 on average. This method was applied to real sequencing data, and the presence and absence of an external DNA segment were successfully proven. Additionally, our in-depth analyses also identified some weaknesses in current DNA sequencing technologies. Hence, for a rigorous safety assessment, the combination of k-mer detection and another method, such as Southern blot assay, is recommended. The results presented in this study will lay the foundation for the regulation of NBT products, where foreign DNA is utilized during their generation. Several current biotechnology techniques, such as genome editing, have been developed to produce novel crops and livestock in an effective manner 1-3. While their regulation has been initiated, they are currently not overseen in some areas 4-7 , and their status is, in many cases, still under debate, especially if foreign DNA sequences are used in the technologies and subsequently removed from the final product 8-10. Because current high-throughput sequencing technologies are promising for the detection of foreign DNA remnants in host genomes 11-16 , guidelines for large-scale sequence data submission have been developed for the safety assessment of genetically-modified organisms (GMOs) 17,18. However, although PCR-based methods have been widely used for the validation of genome-edited agricultural products (GEAPs) 19-21 , unlike GMOs, the number of reports focused on massive sequencing-based methods for GEAPs is still relatively limited 22,23. A study focused on the statistical aspect of detecting unauthorized DNA materials in GMOs was previously reported 24 , but a general theoretical framework for data analysis of foreign DNA-free GEAPs has yet to be established. Here, using rice as a model crop genome, we conducted a large-scale bioinformatics study of both simulated and real data and present fundamental information that will be necessary for regulations for new breeding techniques (NBTs). In particular, as low-cost DNA decoding of the sequencing-by-synthesis method (SBS) is anticipated to play a pivotal role for the detection of unintended remaining foreign DNA segments, sequence data generated by the HiSeq platform are the focus in this study. Strictly speaking, even a single base pair insertion of an exogenous nucleotide can create a GMO (e.g., legislation of the Cartagena act of ...