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Highlights • We developed an improved, robust method to eliminate repetitive genomic elements. • A > 50% increase in gene-derived reads was obtained in a diploid wheat cultivar. • Our method, applicable to any species, is superior to target-enrichment sequencing. Abstract Genome size and complexity often present major challenges to genome-based approaches in crop plants and other agricultural species. For instance, repetitive sequences comprise 80% to 90% of the genome of hexaploid wheat, which has a haploid genome size of approximately 17 Gb. In this study, we developed an improved design and procedure for short-read library preparation that uses a modified adaptor and duplex-specific nuclease (DSN) for the efficient elimination of highly repeated sequence elements within genomes. The improved adapter, which has a hairpin-like form for stability, was constructed from truncated sequences adjacent to the original Illumina TruSeq adapter and can be converted to a full-length adapter structure during PCR amplification. Using the hairpin-structured adaptor, we prepared randomly sheared genomic libraries from rice and diploid, tetraploid, and hexaploid wheat cultivars and evaluated the efficiency of DSN for the enzymatic depletion of repetitive elements. According to real-time quantitative PCR analysis, the relative abundances of 18S and 25S ribosomal DNA decreased respectively to 1.15% and 3.54% in rice and 1.70%–1.95% and 14.71%–20.01% in the three wheat cultivars. Whole-genome sequencing analysis of a diploid wheat cultivar, KU104-1, indicated that DSN treatment with the designed hairpin-structured adapter dramatically reduced highly repetitive elements, such as Ty1-Copia and Ty3-Gypsy retrotransposons and DNA transposons, within the genome, while sequencing reads derived from low-copy genes and protein coding sequences increased more than 50%. Our new procedure should be useful not only for wheat genomes but also for other agricultural plant species with relatively large and complex genomes. [ABSTRACT FROM AUTHOR]

Abstract Microalgae accumulate neutral lipids in oil bodies under sulfur-starvation conditions, and determination of the molecular basis of lipid accumulation under sulfur-starvation conditions will enable improvement of the production of biofuel. We analyzed two l -cysteine dependent mutants (Srp1 and Srp2) of the green alga Parachlorella kessleri. The wild-type cells accumulated lipids and developed oil bodies under sulfate-depleted conditions but not under sulfate-replete conditions. Srp1 and Srp2 showed suppressed proliferation and lipid and starch accumulation under sulfate-replete conditions but accumulated only lipids under sulfate-depleted conditions. Although the suppressed cell proliferation of the mutants was partially complemented in medium containing l -cysteine as a sulfur source, that of the wild type was completely complemented, and lipids did not accumulate in the wild type or the mutants. Our results suggest that the accumulation of lipids in P. kessleri under sulfate-depleted conditions results from a shortage of sulfate metabolites, such as l -cysteine, and that sulfate promotes starch accumulation. Highlights • Mutants of the green alga Parachlorella kessleri requires l -cysteine for proliferation. • The mutants exhibit not only lipid but also starch accumulation under sulfate-replete conditions. • A shortage of sulfate metabolites and sulfate promotes lipid accumulation and starch accumulation, respectively. [ABSTRACT FROM AUTHOR]

Abstract Parachlorella kessleri (formerly Chlorella kessleri) can accumulate high levels of both starch and lipids that can be used for the production of bioethanol and biodiesel, respectively. We mutagenized P. kessleri by heavy-ion beam irradiation and obtained a biotechnologically promising mutant strain, PK4, for further improvement of lipid productivity in comparison with wild type [1]. PK4 reached two times the optical density and accumulated 2.6 times as much starch in complete urea-phosphate (UP) media compared to tris-acetate-phosphate (TAP) media under laboratory conditions. Similarly to wild type, PK4 accumulated only negligible amounts of lipids in complete UP media. PK4 accumulated more lipids (WT: 1.17 g L−1, PK4: 1.75 g L−1) and accumulated lipids faster than WT after dilution of the UP media (WT; 0.22 g L−1 day−1, PK4; 0.43 g L−1 day−1). The productivity of PK4 was analyzed in mass culture using a 150-L thin-layer photo bioreactor housed in a temperature and light-uncontrolled glass greenhouse. The PBR consisted of two glass plates (each 6 m long and 1 m wide) at an inclination of 1.6%, arranged in a meandering path and connected with a trough. The culture was diluted four-fold with water on day 7 post-inoculation to ensure prompt nutrient limitation in the UP media. Lipid accumulation in PK4 was significantly induced so that it accumulated 66% of lipid per dry weight. Under 150-L mass cultivation conditions, PK4 showed high biomass productivity (0.82 g L−1 day−1) and high lipid productivity (0.59 g L−1 day−1). In addition, the whole genome of PK4 was sequenced to clarify the genetic variation upon heavy-ion-beam irradiation by comparison with the reference genome of WT. As a result, genetic differentiation of PK4 was found at three genes encoding endo-1,4-β-mannanase, an ATP/ADP transporter, and an elicitor response protein. Highlights • PK4 is a heavy-ion beam irradiation mutant isolated from Parachlorella kessleri. • PK4 accumulated more lipids and faster than WT in diluted urea-phosphate (UP) media. • Its maximum lipid content was 66% per DW using a thin-layer photo bioreactor (150-L). • PK4 achieved high biomass and lipid productivity in the 150-L mass cultivation. • Genetic differentiation of PK4 was found at three genes such as endo-1,4-β-mannanase. [ABSTRACT FROM AUTHOR]

Acyl-CoA synthetase, which is one of the acid-thiol ligases (EC 6.2.1), plays key roles in metabolic and regulatory processes. This enzyme forms a carbon-sulfur bond in the presence of ATP and Mg2~, yielding acyl-CoA thioesters from the corresponding free acids and CoA. This enzyme belongs to the superfamily of adenylate-forming enzymes, whose three-dimensional structures are analogous to one another. We here discovered a new reaction while studying the short-chain acyl-CoA synthetase that we recently reported (Hashimoto, Y., Hosaka, H., Oinuma, K., Goda, M., Higashibata, H., and Kobayashi, M. (2005) J. Blot. Chern. 280, 8660-8667). When L-cysteine was used as a sub- strate instead of CoA, N-acyl-L-cysteine was surprisingly detected as a reaction product. This finding demonstrated that the enzyme formed a carbon-nitrogen bond (EC 6.3.1 acid-am- monia (or amide) ligase (amide synthase); EC 6.3.2 acid-amino acid ligase (peptide synthase)) comprising the amino group of the cysteine and the carboxyl group of the acid. N-Acyl-D-cysteine, N-acyl-DL-homocysteine, and N-acyl-L-cysteine methyl ester were also synthesized from the corresponding cysteine analog substrates by the enzyme. Furthermore, this unexpected enzyme activity was also observed for acetyl-CoA synthetase and firefly luciferase, indicating the generality of the new reac- tion in the superfamily of adenylate-forming enzymes. [ABSTRACT FROM AUTHOR]

Globally, the isolation of community-associated methicillin-resistant Staphylococcus aureus (MRSA) harbouring both the Panton–Valentine leucocidin (PVL) and toxic shock syndrome toxin 1 (TSST-1) genes is rare. However, we encountered an outbreak of the ST22-PT clone exhibiting this phenotype in Japan. Notably, the TSST-1 gene was duplicated in most of the strains. This study aimed to elucidate the mechanisms underlying this gene duplication. A total of 90 MRSA isolates were collected from the skin of outpatients in Fukuoka City, Japan, between 2017 and 2019. Whole-genome sequencing was performed on MRSA strains that were PVL and TSST-1 positive. A total of 43 (47.8%) strains produced TSST-1, 20 (22.2%) produced PVL, and 16 (17.8%) produced both. Fifteen isolates were classified as ST22/SCC mec type IVa (ST22-PT clone) and one as ST1/SCC mec type V (ST1-PT clone). Three distinct ST22-PT clones were identified: Fukuoka clone I (one PVL gene and one TSST-1 gene), Fukuoka clone II (addition of a TSST-1 gene to Fukuoka clone I), and Fukuoka clone III (marked by a chromosomal inversion in a large region from Fukuoka clone II). Fukuoka clone I may have integrated a novel pathogenicity island bearing the TSST-1 gene, leading to the emergence of Fukuoka clone II with a duplicated TSST-1 gene. This duplication subsequently instigated a chromosomal inversion in a large region owing to the homologous sequence surrounding TSST-1, giving rise to Fukuoka clone III. These findings provide crucial insights into the genetic evolution of MRSA. [Display omitted] [ABSTRACT FROM AUTHOR]

Using heavy-ion beam mutagenesis of Triticum monococcum strain KU104-1, we identified a mutant that shows extra early-flowering; it was named extra early-flowering 3 (exe3 ). Here, we carried out expression analyses of clock-related genes, clock downstream genes and photoperiod pathway genes, and found that the clock component gene PHYTOCLOCK 1/LUX ARRHYTHMO ( PCL1/LUX ) was not expressed in exe3 mutant plants. A PCR analysis of DNA markers indicated that the exe3 mutant had a deletion of wheat PCL1/LUX ( WPCL1 ), and that the WPCL1 deletion was correlated with the mutant phenotype in the segregation line. We confirmed that the original strain KU104-1 carried a mutation that produced a null allele of a flowering repressor gene VERNALIZATION 2 ( VRN2 ). As a result, the exe3 mutant has both WPCL1 and VRN2 loss-of-function mutations. Analysis of plant development in a growth chamber showed that vernalization treatment accelerated flowering time in the exe3 mutant under short day (SD) as well as long day (LD) conditions, and the early-flowering phenotype was correlated with the earlier up-regulation of VRN1 . The deletion of WPCL1 affects the SD-specific expression patterns of some clock-related genes, clock downstream genes and photoperiod pathway genes, suggesting that the exe3 mutant causes a disordered SD response. The present study indicates that VRN1 expression is associated with the biological clock and the VRN1 up-regulation is not influenced by the presence or absence of VRN2 . [ABSTRACT FROM AUTHOR]

Highlights: [•] A broad spectrum of phenotypes was produced by heavy-ion beam irradiation. [•] Lipid production fatty acid profiles and starch contents were altered. [•] Heavy-ion beam irradiation shows potential in the breeding of microalgae. [ABSTRACT FROM AUTHOR]

Abstract: Heavy-ion beams are known to cause great damage to cellular components and are particularly renowned for their ability to generate DNA double-strand breaks (DSBs). To gain insight into the mutagenic effect of carbon-ion beams and how such damage is repaired by the cell, Neurospora crassa mutants deficient in one of three components involved in the repair of DSBs, nonhomologous end-joining (NHEJ), homologous recombination repair (HR), and the Mre11-Rad50-Xrs2 (MRX) complex, were irradiated with a carbon-ion beam and killing effect, mutation frequencies, and the type of mutation incurred by survivors were analysed. The sensitivity of the NHEJ-deficient strain (mus-52) was higher than that of the wild-type and the HR-deficient (mei-3) strains at low doses of radiation, but was little changed as the level increased. As a result both the wild-type and HR-deficient strains were more sensitive than the NHEJ-deficient strain at high radiation levels. In addition, the frequency of forward mutation at the adenine-3 (ad-3) loci of the NHEJ-deficient mutant was lower than that of the wild-type strain at all levels, while the mutation frequency of the HR-deficient strain was consistently ∼3-fold higher than the wild-type. From the comparison of mutation type of each strain, deletions were frequently observed in wild-type strain, whilst base substitution and deletion in the mus-52 and mei-3 strains. These mutations resulting from carbon-ion-beam irradiation depend on the mechanism invoked to cope with DSBs. Furthermore, in wild-type cells, these mechanisms likely compete to repair DSBs. [Copyright &y& Elsevier]

Abstract: Linear energy transfer (LET) is an important parameter to be considered in heavy-ion mutagenesis. However, in plants, no quantitative data are available on the molecular nature of the mutations induced with high-LET radiation above 101–124keVμm−1. In this study, we irradiated dry seeds of Arabidopsis thaliana with Ar and C ions with an LET of 290keVμm−1. We analyzed the DNA alterations caused by the higher-LET radiation. Mutants were identified from the M2 pools. In total, 14 and 13 mutated genes, including bin2, egy1, gl1, gl2, hy1, hy3–5, ttg1, and var2, were identified in the plants derived from Ar- and C-ions irradiation, respectively. In the mutants from both irradiations, deletion was the most frequent type of mutation; 13 of the 14 mutated genes from the Ar ion-irradiated plants and 11 of the 13 mutated genes from the C ion-irradiated plants harbored deletions. Analysis of junction regions generated by the 2 types of irradiation suggested that alternative non-homologous end-joining was the predominant pathway of repair of break points. Among the deletions, the proportion of large deletions (>100bp) was about 54% for Ar-ion irradiation and about 64% for C-ion irradiation. Both current results and previously reported data revealed that the proportions of the large deletions induced by 290-keVμm−1 radiations were higher than those of the large deletions induced by lower-LET radiations (6% for 22.5–30.0keVμm−1 and 27% for 101–124keVμm−1). Therefore, the 290keVμm−1 heavy-ion beams can effectively induce large deletions and will prove useful as novel mutagens for plant breeding and analysis of gene functions, particularly tandemly arrayed genes. [Copyright &y& Elsevier]

Abstract: To investigate the nature of plant chromosomes irradiated by heavy-ion beams, the effects of nitrogen (N) and neon (Ne) ion beams on hexaploid wheat chromosomes were compared with those of X-ray. Chromosome aberrations, such as short, ring and dicentric chromosomes appeared in high frequency. The average numbers of chromosome breaks at LD-50 by irradiation with X-ray, N and Ne ion beams were 32, 20 and 20, respectively. These values may be underestimated because chromosome rearrangement without change in chromosome morphology was not counted. Thus, we subsequently used a wheat line with a pair of extra chromosomes from an alien species (Leymus racemosus) and observed the fate of the irradiated marker chromosomes by genomic in situ hybridization. This analysis revealed that 50Gy of neon beam induced about eight times more breaks than those induced by X-ray. This result suggests that heavy-ion beams induce chromosome rearrangement in high frequency rather than loss of gene function. This suggests further that most of the novel mutations produced by ion beam irradiation, which have been used in plant breeding, may not be caused by ordinary gene disruption but by chromosome rearrangements. [Copyright &y& Elsevier]

Abstract: A positive selection system for gene disruption using a sucrose-sensitive transgenic rhizobium was established and used for the molecular characterization of mutations induced by ion beam irradiations. Single nucleotide substitutions, insertions, and deletions were found to occur in the sucrose sensitivity gene, sacB, when the reporter line was irradiated with highly accelerated carbon and iron ion beams. In all of the insertion lines, fragments of essentially the same sequence and of approximately 1188bp in size were identified in the sacB regions. In the deletion lines, iron ions showed a tendency to induce larger deletions than carbon ions, suggesting that higher LET beams cause larger deletions. We found also that ion beams, particularly “heavier” ion beams, can produce single gene disruptions and may present an effective alternative to transgenic approaches. [Copyright &y& Elsevier]

• The properties of ion beam-induced mutations in rice were revealed by whole-exome sequencing. • The average mutation frequency in the M1 generation of ion beam-irradiated rice was estimated to be 2.6 × 10−7 per base. • The identification of a small number of high-impact mutation facilitates detection of candidate genes responsible for phenotypes of mutants. Ion beams are physical mutagens used for plant and microbe breeding that cause mutations via a mechanism distinct from those of chemical mutagens or gamma rays. We utilized whole-exome sequencing of rice DNA in order to understand the properties of ion beam-induced mutations in a genome-wide manner. DNA libraries were constructed from selected carbon-ion-beam-induced rice mutants by capturing with a custom probes covering 66.3 M bases of nearly all exons and miRNAs predicted in the genome. A total of 56 mutations, including 24 single nucleotide variations, 23 deletions, and 5 insertions, were detected in five mutant rice lines (two dwarf and three early-heading-date mutants). The mutations were distributed among all 12 chromosomes, and the average mutation frequency in the M1 generation was estimated to be 2.7 × 10−7 per base. Many single base insertions and deletions were associated with homopolymeric repeats, whereas larger deletions up to seven base pairs were observed at polynucleotide repeats in the DNA sequences of the mutation sites. Of the 56 mutations, six were classified as high-impact mutations that caused a frame shift or loss of exons. A gene that was functionally related to the phenotype of the mutant was disrupted by a high-impact mutation in four of the five lines tested, suggesting that whole-exome sequencing of ion-beam-irradiated mutants could facilitate the detection of candidate genes responsible for the mutant phenotypes. [ABSTRACT FROM AUTHOR]