Ionizing radiation directly and indirectly affects gene expression within the plant genome. To access the physiological response of rice to different types of ionizing radiation, rice seeds were exposed to gamma-ray and ion beam radiation. Exposure to ionizing radiation dramatically decreased the shoot length compared with non-irradiated plants. Fluorescence-activated-cell-sorting (FACs) was used to measure DNA contents. There were significant correlations of dose-dependent between irradiated plant and non-irradiated plant. The radicals induced by the ionizing radiation in the plant could be observed by electron spin resonance (ESR). It was confirmed that the number of free radicals in cell was greatly increased all irradiated plants than non-irradiated plant. A significant positive correlation was shown between ionizing radiation dose and signal intensity. In order to determine the Genetic diversity, AFLP analysis was conducted with the irradiated plant and non-irradiated plant. Based on band patterns, the cluster analysis was conducted to evaluate the genetic variation by using the UPGMA (Unweighted Pair Grouping Method of Averages). Genetic diversity of irradiated plants by low dose ion beam was the closest non-irradiated plant and irradiated by high dose gamma-ray was the furthest from non-irradiated. We describe the detailed methods of ionizing irradiation and discuss its applications in genetic research as well as plant breeding.

Ionizing radiation affects gene expression from plant genomes. To monitor the genome-wide transcriptional changes induced by three types of ionizing radiation, we used the rice RNA sequencing to identify genes that are up- or down-regulated by gamma rays (GAs), proton (PRs) and ion beams (IBs). The Oryza sativa jacalin-like lectin domain containing proteins (OsJAC1) gene was highly induced by GAs, PRs and IBs. OsJAC1 was selected based on the expression patterns of a genome-wide dataset of RNA sequencing. Many jacalin-related lectin genes have been shown to be associated with disease resistance, biotic and abiotic stress signaling. Therefore, we studied its expression pattern in response to different abiotic stress and phytohormone treatments. The expression patterns of OsJAC1 under two different abiotic stress conditions (salt and heat stress) and phytohormones (salicylic acid and methyl jasmonate) were examined. The transcripts of OsJAC1 were significantly induced in response to abiotic stress conditions, including salt and heat treatments. In addition, it was induced in response to the salicylic acid and methyl jasmonate treatments, respectively. To investigate the sub-cellular localization of OsJAC1, the gene was expressed as a fusion protein tagged with GFP, in tobacco leaf epidermis and examined under confocal microscope. The OsJAC1 was clearly localized at the nucleus. These results provide critical insights into the molecular functions of the rice jacalin-like lectin domain containing proteins as receptors of external signals.

‘Tocomi-1’, a new japonica rice cultivar derived from a 200 Gy gamma ray irradiation with high tocopherol content and red pericarp. The local adaptability test of MRXII-1001-1 was carried out from 2012 to 2014 and it was named as ‘Tocomi-1’ in 2014. This variety is medium matured with heading date of August 12 in honam plain area of Korea. This variety is about 80 cm tall culm length and 106 spikelets per panicle. Its 1,000 grain-weight of rice seeds is 25.4 g. The yield potential of this variety is about 5.15 MT/ha in local adaptability test for three years. This variety exhibited greater seed longevity than the Donganbyeo, indicating a crucial role for tocopherols in maintaining viability during quiescence, and displayed faster seedling growth during the early growth stage. Tocopherol contents was 50% higher than the Donganbyeo. To study the molecular mechanism underlying vitamin E biosynthesis, we examined the expression patterns of seven rice genes encoding vitamin E biosynthetic enzymes. Accumulation levels of the OsVTE2 transcript and OsVTE2 protein in the ‘Tocomi-1’ were significantly higher than in the Donganbyeo. Sequence analysis revealed that the ‘Tocomi-1’ harbored a point mutation in the OsVTE2 promoter region, which resulted in the generation of MYB transcription factor—binding cis-element. These results help identify the promoter regions that regulate OsVTE2 transcription, and offer insights into the regulation of tocopherol content in ‘Tocomi-1’.

To define whole genome-level of structural variation by ionization energies and radiation doses in plant, the seeds of Ilpum rice cultivar were acutely irradiated with gamma rays (100Gy, 200Gy, and 400Gy) and ion-beams (20Gy, 40Gy, and 80Gy), respectively. Six M1 rice plants were re-sequenced by Hi-Seq2500 with Ilpum cultivar as control. The average sequencing coverage of the individuals was 10.6X, and the average mapping rate to the rice reference genome (IRGSP-1.0) sequence was 96.95%. The individual plants were irradiated with gamma-400Gy and ion-50Gy had highest variation of SNP with 471,837 and 469,147, respectively. The number of insertion/deletion was 77,500 and 77,106, the synonymous and frame-shift were 7,859 and 7,763 in above two individuals. Although high genome variation shown between Ilpum cultivar and irradiated individuals, there were non-correlation between number of variation and radiation doses. However, five individuals, except ion-20Gy, showed 33 common variant blocks (CVBs) spanning 6 Mb in whole rice genome (1.6%). The CVBs were distributed on 12 rice chromosomes, Chromosome 6 had biggest CVB (5 blocks, 1.3Mb), whereas chromosome 9 had smallest CVB (0.01Mb). Total five hundred fifty one genes were in CVBs which can regard radiation sensitive genes or may be regarded as radiation hot spots in rice genome. This study will contribute to the improvement of the radiation mutation breeding research in genetic and genomic aspect.

Exposure to ionizing radiation is regarded as a kind of abiotic stresses that can change the expression of genes in living organisms. This study aimed on investigating the variations in gene expressions induced by two different types of irradiations with different doses, which were low linear energy transfer (LET) gamma rays (100, 200, and 400 Gy) and high LET ion-beams (20, 40, and 80 Gy) on rice. RNA sequencing was carried out using the Illumina HiSeq-2500 platform. The average amount of reads were 4.8 Gb per individual, and 5 to 8% of the reads were removed after quality control. More than 90% of the RNA-seq reads were mapped to the rice reference genome sequence (IRGSP-1.0). A total of 247 differentially expressed genes (DEGs) were identified by comparison of the gene expression levels between the wildtype and the irradiated individuals. The 247 DEGs were divided into five modules and 27 intra-modular hub genes were found using the weighted correlation network analysis (WGCNA) method. The MEturquiose module had the most number of genes with 75 related to carbohydrate and small molecule metabolic processes. The co-expression network reconstructed using ARACNE (algorithm for reconstruction of accurate cellular networks) showed specific up- or down-regulation of the genes in each module according to the types and doses of radiation. This study will contribute to understanding the gene expression responses to ionizing irradiation.

There is a growing number of plant genomes that are being sequenced, but most of these available assemblies do not cover the entire genome mainly due to the highly repetitive sequences found in most plant genomes. Nevertheless, these repeats, although a challenge in assembly algorithms, provide relevant information about a genome’s history that could help explain its structure and complexity. Here, we cytogenetically mapped previously and presently characterized major repeats of Panax ginseng genome, including several LTR retrotransposons (PgDel2, PgDel3, PgTat1, PgTat2, PgTork) and one tandem repeat, PgTR Fluorescence in situ hybridization (FISH) results showed differential accumulation of Ty3/gypsy LTR retrotransposons into different chromosomal regions or subgenomes, suggesting a non-random preferential amplification of retrotransposons in these regions and an allopolyploid origin of P. ginseng. In silico analysis based on 1x whole genome sequence reads suggests that PgTR is the most abundant tandem repeat in ginseng, which was further corroborated by FISH analysis. More importantly, its unique distribution pattern among the 24 ginseng chromosomes, coupled with the non-random distribution of LTR retrotransposons and rDNA arrays, allowed us to discriminate and characterize each individual ginseng chromosome. These different newly characterized cytogenetic markers allowed reorganization of previously reported ginseng karyotype with better resolution, demonstrating the irutility in ginseng chromosome identification. These information give us insight about the genomic structure of P. ginseng, and should be useful for future comparative cytogenetics studies among closely related species to unravel its genomic history. This work was supported by the Next-Generation BioGreen21 Program (No. PJ008202), Rural Development Administration, Republic of Korea.

Genome duplication is an abundance phenomenon and in plant kingdom and consequently formed paralogous region. Korean ginseng (Panax ginseng C.A. Meyer) has a possibility of tetraploid by comparing chromosome numbers of relative species. During development of EST-SSR markers in Korean ginseng, most of primer sets have produced multiple bands in gel electrophoresis. In this study, for identifying origin of multiple bands, five EST-SSR markers showing multi-band were selected and two bands around expected size were sequenced. Sequence comparison classified the multiple bands into individual loci. Two bands can be identified by SNP or InDel variation with number of SSR units. Sequencing result represented that paralogous loci with high similarity were existence caused by recent duplication. One clear band were amplified with newly designed locus specific primer picked from SNP variation. SNP and InDel polymorphism between paralgous loci were useful for identifying each locus. This study will provide better understanding of ginseng genome and will be helpful for development of DNA markers.

Ginseng (Panax ginseng C.A. Meyer) is the most famous medicinal herb in East Asia. Although medicinal components and their functions have been widely investigated, ginseng has been regarded as an underdeveloped crop in genetics and genomics research areas. This study was conducted to elucidate the structure and evolution of the ginseng genome by analyses of expressed sequence tags (ESTs) and bacterial artificial chromosome (BAC) sequences. The EST analysis based on the calculation of synonymous substitutions per synonymous site (Ks) in paralog and ortholog pairs revealed that two rounds of polyploidy events occurred in the common ancestor of ginseng and American ginseng (P. quinquefolius L.) and subsequent divergence of the two species. The sequence analysis of repeat-rich BAC clones characterized the major component of the ginseng genome, long terminal repeat retrotransposons (LTR-RTs). The LTR-RTs were classified into five main families, of which three (PgDel, PgTat, and PgAthila) belonged to Ty3/gypsy and the other two (PgTork and PgOryco) to Ty1/Copia. High abundance of the LTR-RTs were revealed by whole genome shotgun (WGS) read mapping and fluorescence in situ hybridization (FISH) analysis. Particularly, the most abundant PgDel family have played major roles in expanding heterochromatic regions as well as remodeling euchromatic regions. Biased intensity of the PgDel2 FISH signals on half the total chromosomes demonstrates the allopolyploid feature of ginseng genome. Insertion time estimation of each LTR-RT implied that LTR-RTs have proliferated after the recent polyploidization of ginseng. These results suggest that the ginseng genome of the present day has been expanded and evolved by two rounds of polyploidization and accumulation of LTR-RTs.

Next generation sequencing (NGS) approaches can also be useful tool for characterization of organelle genomes. We generated chloroplast (CP) genome sequences of two Korean ginseng cultivars, Chunpoong and Yunpoong, based on reference-guided assembly using whole genome NGS data. We used 0.5x of P. ginseng genome NGS reads to assemble CP genome. Of the NGS reads used, about 6% were mapped to the reference CP genome with mean coverage of 94x due to high copy number of CP genome in plant cell. CP genomes of the two cultivars were predicted to be 156,248 bp and 156,355 bp in length and showed about 0.1% differences at nucleotide level, compared to reference CP genome sequenced from P. ginseng (Acc.no. NC_006290), whereas difference between CP genomes of the two cultivars is very rare. In this study, we developed the molecular marker to perform taxon identification and also to elucidate phylogenetic relationship among Korean ginseng cultivars. Now, we are analyzing the CP genomes of other P. ginseng cultivars together with other Panax species including American ginseng and Panax related species.

family in the Brassica genome sequences by computational approach. The MITE family showed a total of 264bp length including 36bp terminal inverted repeats and remained 2bp (TA) targets it eduplication by its insertion. By searching the genome database of Brassica species, 516, 227, and 15 members were identified from 470Mbp of Brassica oleraceae, 154Mbp of B.rapa and 15Mbp of B.napus, respectively, indicating that there are approximately 692, 760, 1235 copies in B.oleracea, B.rapa and B.napus genomes,respectively. A total of 225 relatively intact MITE members, 146,68, and 11 members, which showed >80% sequence similarity and sequence coverage were identified and retrieved for MITE analysis from B.oleracea, B.rapa and B.napus genomes, respectively. Out of 225 MITE family members 159 having full structure of MITE and 66 having the truncated end either in right TIR or left TIR. Insertion polymorphism due to insertion or non-insertion of MITEs showed high level of polymorphism among accessions intra and inter species of Brassica. The new MITE would provide abetter tool for study molecular breeding in Brassica species and also helpful to understand their contribution in evolution and diversification of the highly duplicated Brassica genome.