Different mutational function of low‐ and high‐linear energy transfer heavy‐ion irradiation demonstrated by whole‐genome resequencing of Arabidopsis mutants

Heavy‐ion irradiation is a powerful mutagen that possesses high linear energy transfer (LET). Several studies have indicated that the value of LET affects DNA lesion formation in several ways, including the efficiency and the density of double‐stranded break induction along the particle path. We assumed that the mutation type can be altered by selecting an appropriate LET value. Here, we quantitatively demonstrate differences in the mutation type induced by irradiation with two representative ions, Ar ions (LET: 290 keV μm^?1) and C ions (LET: 30.0 keV μm^?1), by whole‐genome resequencing of the Arabidopsis mutants produced by these irradiations. Ar ions caused chromosomal rearrangements or large deletions (≥100 bp) more frequently than C ions, with 10.2 and 2.3 per mutant genome under Ar‐ and C‐ion irradiation, respectively. Conversely, C ions induced more single‐base substitutions and small indels (<100 bp) than Ar ions, with 28.1 and 56.9 per mutant genome under Ar‐ and C‐ion irradiation, respectively. Moreover, the rearrangements induced by Ar‐ion irradiation were more complex than those induced by C‐ion irradiation, and tended to accompany single base substitutions or small indels located close by. In conjunction with the detection of causative genes through high‐throughput sequencing, selective irradiation by beams with different effects will be a powerful tool for forward genetics as well as studies on chromosomal rearrangements.     

Comprehensive identification of mutations induced by heavy‐ion beam irradiation in Arabidopsis thaliana

Heavy‐ion beams are widely used for mutation breeding and molecular biology. Although the mutagenic effects of heavy‐ion beam irradiation have been characterized by sequence analysis of some restricted chromosomal regions or loci, there have been no evaluations at the whole‐genome level or of the detailed genomic rearrangements in the mutant genomes. In this study, using array comparative genomic hybridization (array‐CGH) and resequencing, we comprehensively characterized the mutations in Arabidopsis thaliana genomes irradiated with Ar or Fe ions. We subsequently used this information to investigate the mutagenic effects of the heavy‐ion beams. Array‐CGH demonstrated that the average number of deleted areas per genome were 1.9 and 3.7 following Ar‐ion and Fe‐ion irradiation, respectively, with deletion sizes ranging from 149 to 602 180 bp; 81% of the deletions were accompanied by genomic rearrangements. To provide a further detailed analysis, the genomes of the mutants induced by Ar‐ion beam irradiation were resequenced, and total mutations, including base substitutions, duplications, in/dels, inversions, and translocations, were detected using three algorithms. All three resequenced mutants had genomic rearrangements. Of the 22 DNA fragments that contributed to the rearrangements, 19 fragments were responsible for the intrachromosomal rearrangements, and multiple rearrangements were formed in the localized regions of the chromosomes. The interchromosomal rearrangements were detected in the multiply rearranged regions. These results indicate that the heavy‐ion beams led to clustered DNA damage in the chromosome, and that they have great potential to induce complicated intrachromosomal rearrangements. Heavy‐ion beams will prove useful as unique mutagens for plant breeding and the establishment of mutant lines.     

Whole‐exome sequencing identifies a novel mutation of GPD1L (R189X) associated with familial conduction disease and sudden death

Cardiac conduction disease (CCD) is a serious disorder and the leading cause of mortality worldwide. It is characterized by arrhythmia, syncope or even sudden cardiac death caused by the dysfunction of cardiac voltage‐gated channel. Previous study has demonstrated that mutations in genes encoding voltage‐gated channel and related proteins were the crucial genetic lesion of CCD. In this study, we employed whole‐exome sequencing to explore the potential causative genes in a Chinese family with ventricular tachycardia and syncope. A novel nonsense mutation (c.565C>T/p.R189X) of glycerol‐3‐phosphate dehydrogenase‐like (GPD1L) was identified and co‐segregated with the affected family members. GPD1L is a crucial interacting protein of SCN5A, a gene encoded sodium channel α‐subunit Na_v1.5 and mainly associated with Brugada syndrome (BrS). The novel mutation (c.565C>T/p.R189X) may result in a premature stop codon at position 189 in exon 4 of the GPD1L gene and lead to functional haploinsufficiency of GPD1L due to mRNA carrying this mutation will be degraded by nonsense‐mediated mRNA decay, which has been confirmed by Western blot in HEK293 cells transfected HIS‐GPD1L plasmid. The levels of GPD1L decreasing may disturb the function of Na_v1.5 and induce arrhythmia and syncope in the end. In conclusion, our study not only further supported the important role of GPD1L in CCD, but also expanded the spectrum of GPD1L mutations and will contribute to the genetic diagnosis and counselling of families with CCD.     

Multi‐genic pattern found in rare type of hypopituitarism: a whole‐exome sequencing study of Han Chinese with pituitary stalk interruption syndrome

Pituitary stalk interruption syndrome (PSIS) is a rare type of hypopituitarism manifesting various degrees of pituitary hormone deficiency. Although mutations have been identified in some familial cases, the underpinning mechanisms of sporadic patients with PSIS who are in a vast majority remain elusive, necessitating a comprehensive study using systemic approaches. We postulate that other genetic mechanisms may be responsible for the sporadic PSIS. To test this hypothesis, we conducted a study in 24 patients with PSIS of Han Chinese with no family history using whole‐exome sequencing (WES) and bioinformatic analysis. We identified a group of heterozygous mutations in 92% (22 of 24) of the patients, and these genes are mostly associated with Notch, Shh, Wnt signalling pathways. Importantly, 83% (20 of 24) of the patients had more than one mutation in those pathways suggesting synergy of compound mutations underpin the pathogenesis of sporadic PSIS.     

Comparison of thermoluminescence characteristics in γ‐ray and C5+ ion beam‐irradiated LiCaAlF6:Ce phosphor

We compare the thermoluminescence (TL) behavior of Ce³⁺ ion‐activated LiCaAlF₆ exposed to γ‐rays and a carbon ion beam. The reported phosphor is synthesized using an in‐house precipitation method with varying concentrations of activator ion and is characterized by X‐ray diffraction (XRD) and TL. Rietveld refinement is performed to study the structural statistics. The TL glow curve consists of a prominent glow peak at 232°C with three shoulders at 115, 159 and 333°C when exposed to γ‐rays from a ⁶⁰Co source. When exposed to a C⁵⁺ ion beam, the TL glow curve consists of five peaks with peak temperatures near 156, 221, 250, 287 and 330°C, and is found to vary slightly with changing fluence. Glow curve convolution deconvolution (GCCD) functions are applied to the TL curves for complete analysis of the glow curve structure and TL traps. The order of kinetics (b), activation energy (E) and frequency factor are determined using Chen's peak shape method and theoretical curves are drawn using GCCD functions. A track interaction model (TIM) is used to explain the sublinearity/saturation at higher fluences. Ion beam parameters are analyzed using Monte‐Carlo simulation‐based SRIM‐2013 code. Copyright © 2016 John Wiley & Sons, Ltd.     

Whole exome sequencing identified a novel DAG1 mutation in a patient with rare,mild and late age of onset muscular dystrophy‐dystroglycanopathy

Muscular dystrophy‐dystroglycanopathy (limb‐girdle), type C, 9 (MDDGC9) is the rarest type of autosomal recessive muscular dystrophies. MDDGC9 is manifested with an early onset in childhood. Patients with MDDGC9 usually identified with defective glycosylation of DAG1, hence it is known as “dystroglycanopathies”. Here, we report a Chinese pedigree presented with mild MDDGC9. The proband is a 64 years old Chinese man. In this family, both the proband and proband's younger brother have been suffering from mild and late onset MDDGC9. Muscle biopsy showed that the left deltoid muscle with an advanced stage of dystrophic change. Immunohistochemistry staining of dystrophin, α‐sarcoglycan, β‐sarcoglycan and dysferlin are normal. Molecular genetic analysis of the proband has been done with whole exome sequencing. A homozygous novel missense mutation (c.2326C>T; p.R776C) in the exon 3 of the DAG1 gene has been identified in the proband. Sanger sequencing revealed that this missense mutation is co‐segregated well among the affected and unaffected (carrier) family members. This mutation is not detected in 200 normal healthy control individuals. This novel homozygous missense mutation (c.2326C>T) causes substitution of arginine by cystine at the position of 776 (p.R776C) which is evolutionarily highly conserved. Immunoblotting studies revealed that a significant reduction of α‐dystroglycan expression in the muscle tissue. The novelty of our study is that it is a first report of DAG1 associated muscular dystrophy‐dystroglycanopathy (limb‐girdle), type C, 9 (MDDGC9) with mild and late age of onset. In Chinese population this is the first report of DAG1 associated MDDGC9.     

A chloroplast variation map generated using whole genome re‐sequencing of Korean landrace rice reveals phylogenetic relationships among Oryza sativa subspecies

Although the overall structure of the chloroplast genome is generally conserved, several sequence variations have been identified that are valuable for plant population and evolutionary studies. Here, we constructed a chloroplast variation map of 30 landrace rice strains of Korean origin, using the Oryza rufipogon chloroplast genome (GenBank: NC_017835 ) as a reference. Differential distribution of single‐nucleotide polymorphisms and INDELs across the rice chloroplast genome is suggestive of a region‐specific variation. Population structure clustering revealed the existence of two clear subgroups (indica and japonica) and an admixture group (aus). Phylogenetic analysis of the 30 landrace rice strains and six rice chloroplast references suggested and supported independent evolution of O. sativa indica and japonica. Interestingly, two aus type accessions, which were thought to be indica type, shared a closer relationship with the japonica type. One hypothesis is that ‘Korean aus’ was intentionally introduced and may have obtained japonica chloroplasts during cultivation. We also calculated the nucleotide diversity of 30 accessions and compared the results to six rice chloroplast references. These data demonstrated that although nucleotide diversity is low in all strains tested, aus and indica have a higher nucleotide diversity than japonica.     

A loss‐of‐function allele of OsHMA3 associated with high cadmium accumulation in shoots and grain of Japonica rice cultivars

Excessive cadmium (Cd) accumulation in rice poses a risk to food safety. OsHMA3 plays an important role in restricting Cd translocation from roots to shoots. A non‐functional allele of OsHMA3 has been reported in some Indica rice cultivars with high Cd accumulation, but it is not known if OsHMA3 allelic variation is associated with Cd accumulation in Japonica cultivars. In this study, we identified a Japonica cultivar with consistently high Cd accumulation in shoots and grain in both field and greenhouse experiments. The cultivar possesses an OsHMA3 allele with a predicted amino acid mutation at the 380^th position from Ser to Arg. The haplotype had no Cd transport activity when the gene was expressed in yeast, and the allele did not complement a known nonfunctional allele of OsHMA3 in F₁ test. The allele is present only in temperate Japonica cultivars among diversity panels of 1483 rice cultivars. Different cultivars possessing this allele showed greatly increased root‐to‐shoot Cd translocation and a shift in root Cd speciation from Cd―S to Cd―O bonding determined by synchrotron X‐ray absorption spectroscopy. Our study has identified a new loss‐of‐function allele of OsHMA3 in Japonica rice cultivars leading to high Cd accumulation in shoots and grain.     

FLOURY ENDOSPERM11‐2 encodes plastid HSP70‐2 involved with the temperature‐dependent chalkiness of rice (Oryza sativa L.) grains

The frequent occurrence of chalky rice (Oryza sativa L.) grains becomes a serious problem as a result of climate change. The molecular mechanism underlying chalkiness is largely unknown, however. In this study, the temperature‐sensitive floury endosperm11‐2 (flo11‐2) mutant was isolated from ion beam‐irradiated rice of 1116 lines. The flo11‐2 mutant showed significantly higher chalkiness than the wild type grown under a mean temperature of 28°C, but similar levels of chalkiness to the wild type grown under a mean temperature of 24°C. Whole‐exome sequencing of the flo11‐2 mutant showed three causal gene candidates, including Os12g0244100, which encodes the plastid‐localized 70‐kDa heat shock protein 2 (cpHSP70‐2). The cpHSP70‐2 of the flo11‐2 mutant has an amino acid substitution on the 259th aspartic acid with valine (D259V) in the conserved Motif 5 of the ATPase domain. Transgenic flo11‐2 mutants that express the wild‐type cpHSP70‐2 showed significantly lower chalkiness than the flo11‐2 mutant. Moreover, the accumulation level of cpHSP70‐2 was negatively correlated with the chalky ratio, indicating that cpHSP70‐2 is a causal gene for the chalkiness of the flo11‐2 mutant. The intrinsic ATPase activity of recombinant cpHSP70‐2 was lower by 23% at V_max for the flo11‐2 mutant than for the wild type. The growth of DnaK‐defective Escherichia coli cells complemented with DnaK with the D201V mutation (equivalent to the D259V mutation) was severely reduced at 37°C, but not in the wild‐type DnaK. The results indicate that the lowered cpHSP70‐2 function is involved with the chalkiness of the flo11‐2 mutant.     

Semi‐dominant mutation in the cysteine‐rich receptor‐like kinase gene,ALS1, conducts constitutive defence response in rice

• Plants have evolved a sophisticated two‐branch defence system to prevent the growth and spread of pathogen infection. The novel Cys‐rich repeat (CRR) containing receptor‐like kinases, known as CRKs, were reported to mediate defence resistance in plants. For rice, there are only two reports of CRKs. A semi‐dominant lesion mimic mutant als1 (apoptosis leaf and sheath 1) in rice was identified to demonstrate spontaneous lesions on the leaf blade and sheath.
• A map‐based cloning strategy was used for fine mapping and cloning of ALS1, which was confirmed to be a typical CRK in rice. Functional studies of ALS1 were conducted, including phylogenetic analysis, expression analysis, subcellular location and blast resistance identification.
• Most pathogenesis‐related (PR) genes and other defence‐related genes were activated and up‐regulated to a high degree. ALS1 was expressed mainly in the leaf blade and sheath, in which further study revealed that ALS1 was present in the vascular bundles. ALS1 was located in the cell membrane of rice protoplasts, and its mutation did not change its subcellular location. Jasmonic acid (JA) and salicylic acid (SA) accumulation were observed in als1, and enhanced blast resistance was also observed.
• The mutation of ALS1 caused a constitutively activated defence response in als1. The results of our study imply that ALS1 participates in a defence response resembling the common SA‐, JA‐ and NH1‐mediated defence responses in rice.

     

Ethylene‐dependent aerenchyma formation in adventitious roots is regulated differently in rice and maize

In roots of gramineous plants, lysigenous aerenchyma is created by the death and lysis of cortical cells. Rice (Oryza sativa) constitutively forms aerenchyma under aerobic conditions, and its formation is further induced under oxygen‐deficient conditions. However, maize (Zea mays) develops aerenchyma only under oxygen‐deficient conditions. Ethylene is involved in lysigenous aerenchyma formation. Here, we investigated how ethylene‐dependent aerenchyma formation is differently regulated between rice and maize. For this purpose, in rice, we used the reduced culm number1 (rcn1) mutant, in which ethylene biosynthesis is suppressed. Ethylene is converted from 1‐aminocyclopropane‐1‐carboxylic acid (ACC) by the action of ACC oxidase (ACO). We found that OsACO5 was highly expressed in the wild type, but not in rcn1, under aerobic conditions, suggesting that OsACO5 contributes to aerenchyma formation in aerated rice roots. By contrast, the ACO genes in maize roots were weakly expressed under aerobic conditions, and thus ACC treatment did not effectively induce ethylene production or aerenchyma formation, unlike in rice. Aerenchyma formation in rice roots after the initiation of oxygen‐deficient conditions was faster and greater than that in maize. These results suggest that the difference in aerenchyma formation in rice and maize is due to their different mechanisms for regulating ethylene biosynthesis.     

Three representative inter and intra‐subspecific crosses reveal the genetic architecture of reproductive isolation in rice

Systematic characterization of genetic and molecular mechanisms in the formation of hybrid sterility is of fundamental importance in understanding reproductive isolation and speciation. Using ultra‐high‐density genetic maps, 43 single‐locus quantitative trait loci (QTLs) and 223 digenic interactions for embryo‐sac, pollen, and spikelet fertility are depicted from three crosses between representative varieties of japonica and two varietal groups of indica, which provide an extensive archive for investigating the genetic basis of reproductive isolation in rice. Ten newly detected single‐locus QTLs for inter‐ and intra‐subspecific fertility are identified. Three loci for embryo‐sac fertility are detected in both Nip × ZS97 and Nip × MH63 crosses, whereas QTLs for pollen fertility are not in common between the two crosses thus leading to fertility variation. Five loci responsible for fertility and segregation distortion are observed in the ZS97 × MH63 cross. The importance of two‐locus interactions on fertility are quantified in the whole genome, which identify that three types of interaction contribute to fertility reduction in the hybrid. These results construct the genetic architecture with respect to various forms of reproductive barriers in rice, which have significant implications in utilization of inter‐subspecific heterosis along with improvement in the fertility of indica–indica hybrids at single‐ and multi‐locus level.     

Evolutionary force of AT‐rich repeats to trap genomic and episomal DNAs into the rice genome: lessons from endogenous pararetrovirus

In plant genomes, the incorporation of DNA segments is not a common method of artificial gene transfer. Nevertheless, various segments of pararetroviruses have been found in plant genomes in recent decades. The rice genome contains a number of segments of endogenous rice tungro bacilliform virus‐like sequences (ERTBVs), many of which are present between AT dinucleotide repeats (ATrs). Comparison of genomic sequences between two closely related rice subspecies, japonica and indica, allowed us to verify the preferential insertion of ERTBVs into ATrs. In addition to ERTBVs, the comparative analyses showed that ATrs occasionally incorporate repeat sequences including transposable elements, and a wide range of other sequences. Besides the known genomic sequences, the insertion sequences also represented DNAs of unclear origins together with ERTBVs, suggesting that ATrs have integrated episomal DNAs that would have been suspended in the nucleus. Such insertion DNAs might be trapped by ATrs in the genome in a host‐dependent manner. Conversely, other simple mono‐ and dinucleotide sequence repeats (SSR) were less frequently involved in insertion events relative to ATrs. Therefore, ATrs could be regarded as hot spots of double‐strand breaks that induce non‐homologous end joining. The insertions within ATrs occasionally generated new gene‐related sequences or involved structural modifications of existing genes. Likewise, in a comparison between Arabidopsis thaliana and Arabidopsis lyrata, the insertions preferred ATrs to other SSRs. Therefore ATrs in plant genomes could be considered as genomic dumping sites that have trapped various DNA molecules and may have exerted a powerful evolutionary force.     

Molecular characterization and targeted quantitative profiling of the sphingolipidome in rice

Recent advances in comprehensive metabolite profiling techniques, the foundation of metabolomics, is facilitating our understanding of the functions, regulation and complex networks of various metabolites in organisms. Here, we report a quantitative metabolomics technique for complex plant sphingolipids, composed of various polar head groups as well as structural isomers of hydrophobic ceramide moieties. Rice (Oryza sativa L.) was used as an experimental model of monocotyledonous plants and has been demonstrated to possess a highly complex sphingolipidome including hundreds of molecular species with a wide range of abundance. We established a high‐throughput scheme for lipid preparation and mass spectrometry‐based characterization of complex sphingolipid structures, which provided basic information to create a comprehensive theoretical library for targeted quantitative profiling of complex sphingolipids in rice. The established sphingolipidomic approach combined with multivariate analyses of the large dataset obtained clearly showed that different classes of rice sphingolipids, particularly including subclasses of glycosylinositol phosphoceramide with various sugar‐chain head groups, are distributed with distinct quantitative profiles in various rice tissues, indicating tissue‐dependent metabolism and biological functions of the lipid classes and subclasses. The sphingolipidomic analysis also highlighted that disruption of a lipid‐associated gene causes a typical sphingolipidomic change in a gene‐dependent manner. These results clearly support the utility of the sphingolipidomic approach in application to wide screening of sphingolipid‐metabolic phenotypes as well as deeper investigation of metabolism and biological functions of complex sphingolipid species in plants.