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To develop new varieties of perennial plants, it generally needs a lengthy and laborious procedure. Here, we attempted to use ion beam irradiation mutagenesis that is considered one of the laborsaving tools to promote breeding of perennial plants. We evaluated the biological effect of five ion beam sources including C, Ne, Ar, Si and Fe and neutron on Japanese gentian and apple using the National Institute of Radiological Sciences (NIRS)-Heavy Ion Medical accelerator in Chiba (HIMAC) and Neutron exposure Accelerator System for Biological Effect experiments (NASBEE). As the results, the biological effects were observed by less than 10 Gy irradiation of ion beams in in vitro cultured gentian plants, whereas apple trees were less sensitive to ion beam irradiation after grafting. Growth of in vitro cultured gentians was repressed by 3 Gy of neutron irradiation, while that of grafted apple tree was not affected by 4 Gy irradiation. During in vitro proliferation, seven pink-flowered lines from original blue flower were obtained in gentian by C and Ne ion beam irradiation, in total. Genomic and RT-PCR analyses of these lines revealed that the mutations were definitely caused by the deletion of flavonoid 3’,5’-hydroxylase (F3’5’H) genomic region. These results provide useful information for the mutagenesis and breeding of gentian and apple tree in the future.

Carnations carrying a recessive I gene show accumulation of the yellow pigment chalcononaringenin 2′-glucoside (Ch2′G) in their flowers, whereas those with a dominant I gene do accumulation the red pigment, anthocyanin. Although this metabolic alternative at the I gene could explain yellow and red flower phenotypes, it does not explain the development of orange flower phenotypes which result from the simultaneous accumulation of both Ch2′G and anthocyanin. The carnation whole genome sequencing project recently revealed that two chalcone isomerase genes are present, one that is consistent with the I gene (Dca60979) and another (Dca60978) that had not been characterized. Here, we demonstrate that Dca60979 shows a high level of gene expression and strong enzyme activity in plants with a red flower phenotype; however, functional Dca60979 transcripts are not detected in plants with an orange flower phenotype because of a dTdic1 insertion event. Dca60978 was expressed at a low level and showed a low level of enzyme activity in plants, which could catalyze a part of chalcone to naringenin to advance anthocyanin synthesis but the other part remained to be catalyzed chalcone to Ch2′G by chalcone 2′-glucosyltransferase, resulting in accumulation of anthocyanin and Ch2′G simultaneously to give orange color.

Additional file 1: Table S1. Sanger sequencing analysis of the PCR amplicons of the DNA from GST1 genome-edited lines and the WT control. Table S2. Next-generation sequencing analysis of the PCR amplicons of the DNA and RNA from GST1 genome-edited lines and the WT control. Table S3. Primers used in this study. Table S4. Glutathione S-transferase proteins used for constructing the phylogenetic tree in Fig. 1a.

Additional file 2: Figure S1. Flower color characteristics in GST1 genome-edited gentian lines #13, #23, and #29. (A) Flower color phenotypes of GST1 genome-edited lines #13, #23, and #29. (B) L*, a*, and b* color values at the surface of fresh petals were measured with the CM-3600A spectrophotometer (Konica Minolta, Tokyo, Japan). The chroma values and hue angles were also calculated. Figure S2 Sequence of the third exon of the modified GST1 for the complementation assay. The highlighted nucleotides (i.e., shaded, bold, or boxed) are the same as those in Fig. 2b. The substituted nucleotides for the transient expression assay are indicated in red. These substitutions did not change the encoded amino acids.

Superatoms have been investigated due to their possible substitution for other elements. The solution-phase synthesis of superatoms has attracted attention to realize the availability of superatoms. However, the previous approach is basically limited to the formation of a single cluster. Here, superatoms are investigated and the number of valence electrons in these superatoms is changed by designing the number of gallium atoms present. Based on the dendrimer template method, clusters consisting of 3, 12, 13, and other numbers of atoms have been synthesized. The halogen-like superatomic nature of Ga13 is structurally and electrochemically observed as completely different to the other clusters. The gallium clusters of 13 and 3 atoms, which can fill the 2P and 1P superatomic orbitals, respectively, exhibit different reactivities. The 3-atom gallium cluster is suggested as being reduced to Ga3 H2 - due to the lower shift of energy levels in the unoccupied orbitals. The results for these gallium clusters provide candidates for superatoms.

The main factors affecting the outcome of Trueperella pyogenes (T. pyogenes) mastitis were examined through a survey of diagnostic data and interviews relating to the occurrence of T. pyogenes mastitis in 83 quarters from 82 Holstein cows between August 2012 and April 2014. Ultimately, one cow was sold during the examination, and 82 quarters from 81 cows were used for analysis on prognosis. T. pyogenes mastitis occurred year round in both lactating and dry cows. The incidence of T. pyogenes mastitis did not significantly differ by month or show seasonality in either lactating or dry cows. Therefore, the occurrence of T. pyogenes mastitis also differed from that of summer mastitis. The 1-month survival rate of infected cows was 64.6% (53/82), and the recovery rate of quarters with T. pyogenes mastitis was 14.6% (12/82). Bivariate logistic regression analysis was performed with survival and culling of infected cows as objective variables and with recovery and non-recovery of quarters with T. pyogenes mastitis as objective variables. The severe cases were significantly culled (odds ratio, 16.30) compared to mild cases, and the status of quarters didn't recover (odds ratio, 6.50). The results suggest that mild to moderate symptom severity at the time of onset are the main factors affecting outcomes in cows and recovery of quarters infected with T. pyogenes mastitis. Further, high level of NAGase activity also suggested the potential use as an indicator of culling of cows with T. pyogenes mastitis.

Figure S1. Schematic representation of the flavonoid biosynthesis pathway in torenia. ANS, anthocyanidin synthase; C4H, cinnamate 4-hydroxylase; CHI, chalcone isomerase; CHS, chalcone synthase; 4CL, 4-coumarate: CoA ligase; DFR, dihydroflavonol 4-reductase; DHK, dihydrokaempferol; DHM, dihydromyricetin; DHQ, dihydroquercetin; F3H, flavanone 3-hydroxylase (target gene in this study); F3′H, flavonoid 3′ -hydroxylase; F3′,5′H, flavonoid 3′, 5′ -hydroxylase; FNSII, flavone synthase II; GT, glucosyltransferase; MT, methyltransferase; PAL, phenylalanine ammonia lyase. Figure S2. Sequence chromatograms of the TfF3H gene in different transgenic torenia lines. PCR products were subjected to Sanger sequencing using the TfF3HU7 primer. The region of the sequence chromatogram including the target site is enlarged. Transgenic line numbers are shown on each chromatogram. WT indicates the wild-type sequence (cv. Crown Violet). Table S1. Primers used for multiplex amplicon sequencing by NGS. (PDF 1803 kb)