• Mycobiology
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• v.46 no.4
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• pp.361-369
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• 2018

The rice blast fungus, Magnaporthe oryzae, is an important pathogen of rice plants. It is well known that genes encoded in the genome have different evolutionary histories that are related to their functions. Phylostratigraphy is a method that correlates the evolutionary origin of genes with evolutionary transitions. Here we applied phylostratigraphy to partition total gene content of M. oryzae into distinct classes (phylostrata), which we designated PS1 to PS7, based on estimation of their emergence time. Genes in individual phylostrata did not show significant biases in their global distribution among seven chromosomes, but at the local level, clustering of genes belonging to the same phylostratum was observed. Our phylostrata-wide analysis of genes revealed that genes in the same phylostratum tend to be similar in many physical and functional characteristics such as gene length and structure, GC contents, codon adaptation index, and level of transcription, which correlates with biological functions in evolutionary context. We also found that a significant proportion of genes in the genome are orphans, for which no orthologs can be detected in the database. Among them, we narrowed down to seven orphan genes having transcriptional and translational evidences, and showed that one of them is implicated in asexual reproduction and virulence, suggesting ongoing evolution in this fungus through lineage-specific genes. Our results provide genomic basis for linking functions of pathogenicity factors and gene emergence time.

• The Plant Pathology Journal
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• v.31 no.1
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• pp.12-24
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• 2015

Rice Blast is the most devastating disease causing major yield losses in every year worldwide. It had been proved that using resistant rice varieties would be the most effective way to control this disease. Molecular screening and genetic diversities of major rice blast resistance genes were determined in 192 rice germplasm accessions using simple sequence repeat (SSR) markers. The genetic frequencies of the 10 major rice blast resistance genes varied from 19.79% to 54.69%. Seven accessions IC337593, IC346002, IC346004, IC346813, IC356117, IC356422 and IC383441 had maximum eight blast resistance gene, while FR13B, Hourakani, Kala Rata 1-24, Lemont, Brown Gora, IR87756-20-2-2-3, IC282418, IC356419, PKSLGR-1 and PKSLGR-39 had seven blast resistance genes. Twenty accessions possessed six genes, 36 accessions had five genes, 41 accessions had four genes, 38 accessions had three genes, 26 accessions had two genes, 13 accessions had single R gene and only one accession IC438644 does not possess any one blast resistant gene. Out of 192 accessions only 17 accessions harboured 7 to 8 blast resistance genes.

• Proceedings of the Botanical Society of Korea Conference
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• 1985.08b
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• pp.59-71
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• 1985

In rice, limited efforts have been made to identify genes by the use of insertional mutagens, especially heterologous transposons such as the maize Ac/Ds. We constructed Ac and gene trap Ds vectors and introduced them into the rice genome by Agrobacterium-mediated transformation. In this report, rice plants that contained single and simple insertions of T-DNA were analyzed in order to evaluate the gene-tagging efficiency. The 3'end of Ds was examined for putative splicing donor sites. As observed in maize, three splice donor sites were identified at the 3'end of the Ds in rice. Nearly 80% of Ds elements wered excised from the original T-DNA sites, when Ac cDNA was expressed under a CaMV 35S promoter. Repetitive ratoon culturing was performed to induce new transpositions of Ds in new plants derived from cuttings. About 30% of the plants carried at least one Ds that underwent secondary transposition in the later cultures. 8% of transposed Ds elements expressed GUS in various tissues of rice panicles. With cloned DNA adjacent to Ds, the genomic complexities of the insertion sites were examined by Southern hybridization. Half of the Ds insertion sites showed simple hybriodization patterns which could be easily utilized to locate the Ds. Our data demonstrate that the Ac/Ds mediated gene trap system could prove an excellent tool for the analysis of functions of genes in rice. We discuss genetic strategies that could be employed in a largee scale mutagenesis using a heterologous Ac/Ds family in rice.

• Journal of Plant Biology
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• v.38 no.4
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• pp.365-371
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• 1995

Binary vectors, pBI-ActR1, pBI-ActF1 and pBSH-ActR1, were constructed using pGA642, the replication origin of pTi12 and the rice actin promoter. The sizes of pBI-ActR1, pBI-ActF1 and pBSH-ActR1 were 12.9 kb, 13.2 kb and 11.95 kb, respectively. These vectors containing a rice actin promoter followed by a GUS structural gene could induce stronly the expression of GUS gene in transformed rice cells. Rice explants from 3-4 day old seedlings after germinatin were cocultured with A. tumefaceins harboring pBI-ActR1, pBI-ActF1 or pBSH-ActR1, and then GUS expression in the explants was assayed. Transformation of rice explants by these binary vectors was tissue-specific, such that the meristematic regions of shoot apex, root and hypocotyl were transformed by these binary vectors.

• Proceedings of the Botanical Society of Korea Conference
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• 1996.07a
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• pp.61-74
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• 1996

Human and monogastric animals can not synthesize 10 out of the 20 amino asids and therefor need to obtain these from their diet. The plant seed is a major source of dietary protein. It is particular important in their study to increase nutritional quality of the seed storage proteins. The low contents of lysine, asparagine and threonenein various cereal seeds and of cystein and methionine. In legume seeds is due to the low proportions of these amino acids in the major storage proteins, we have tried to apply the three strategies; (1) mutagenesis and selection of specific amino acid analogue resistance, (2) cloning and expression study of lysine biosynthesis related gene, (3) transfomation of lysine rich soybean glycinin gene. The 5-methyltryptophan (5MT) resistant cell lines, SAR1, SAR2 and SAR3 were selected from anther derived callus of rice (Oryza sativa L. "Sasanishiki"). Among these selected cell lines, two (SAR1 and SAR3) were able to grow stably at 200 mg/L of 5MT. Analysis of the freed amino acids in callus shows that 5MT resistant cells (SAR3) accumulated free tryptophan at least up to 50 times higher than those that of the higher than of SAS. These results indicated that the 5MT resistant cell lines are useful in studies of amino acid biosynthesis. Tr75, a rice (Oryza sativa L., var. Sasanishiki) mutant resistant to 5MT was segregated from the progenies of its initial mutant line, TR1. The 5MT resistant of TR75 was inherited in the M8 generations as a single dominant nuclear gene. The content of free amino acids in the TR75 homozygous seeds increased approximately 1.5 to 2.0 fold compared to wild-type seeds. Especially, the contents of tryptophan, phenylalanine and aspartic acid were 5.0, 5.3 and 2.7 times higher than those of wild-type seeds, respectively. The content of lysine is significantly low in rice. The lysine is synthesized by a complex pathway that is predominantly regulated by feedback inhibition of several enzymes including asparginase, aspatate kinase, dihydrodipicolinat synthase, etc. For understanding the regulation mechanism of lysine synthesis in rice, we try to clone the lysine biosynthetic metabolism related gene, DHPS and asparaginase, from rice. We have isolated a rice DHPS genomic clone which contains an ORF of 1044 nucleotides (347 amino acids, Mr. 38, 381 daltons), an intron of 587 nucleotides and 5'and 3'-flanking regions by screening of rice genomic DNA library. Deduced amino acid sequence of mature peptide domain of GDHPS clone is highly conserved in monocot and dicot plants whereas that of transit peptide domain is extremely different depending on plant specie. Southern blot analysis indicated that GDHPS is located two copy gene in rice genome. The transcripts of a rice GDHPS were expressed in leaves and roots but not detected in callus tissues. The transcription level of GDHPS is much higher in leaves indicating enormous chloroplast development than roots. Genomic DNA clones for asparaginase genes were screened from the rice genomic library by using plaque hybridization technique. Twelve different genomic clones were isolated from first and second screening, and 8 of 12 clones were analyzed by restriction patterns and identified by Southern Blotting, Restriction enzyme digestion patterns and Southern blot analysis of 8 clones show the different pattern for asparaginase gene. Genomic Southern blot analysis from rice were done. It is estimated that rice has at least 2-3 copy of asparaginase gene. One of 8 positive clones was subcloned into the pBluescript SK(+) vector, and was constructed the physical map. For transformation of lysine rich storage protein into tobacco, soybean glycinin genes are transformed into tobacco. To examine whether glycinin could be stably accumulated in endosperm tissue, the glycinin cDNA was transcriptionally fused to an endosperm-specific promotor of the rice storage protein glutelin gene and then introduced into tobacco genomic via Agrobacterium-mediated transformation. Consequently the glycinin gene was expressed in a seed-and developmentally-specific manner in transgenic tobacco seeds. Glycinin were targeted to vacuole-derived protein bodies in the endosperm tissue and highly accumulated in the matrix region of many transgenic plant (1-4% of total seed proteins). Synthesized glycinin was processed into mature form, and assembled into a hexamer in a similar manner as the glycinin in soybean seed. Modified glycinin, in which 4 contiguous methionine residues were inserted at the variable regions corresponding to the C - teminal regions of the acidic and basic polypeptides, were also found to be accumulated similarly as in the normal glycinin. There was no apparent difference in the expression level, processing and targeting to protein bodies, or accumulation level between normal and modified glycinin. glycinin.

• Korean Journal of Breeding Science
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• v.41 no.3
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• pp.244-251
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• 2009

This study was carried out to identify useful single gene and gene combination resistant to K1, K2, K3 and 24 bacterial blight(BB) isolates (including K3a, HB01009) breaking down Xa3 gene. Xa3, Xa4, xa5 and Xa7 genes were resistant to K1, K2, K3 of bacterial blight pathogen. Against 24 BB isolates breaking down Xa3 gene, Xa1, Xa2, xa8, Xa10, Xa11, xa13 genes were susceptible, whereas Xa4 gene was moderately resistant and xa5 and Xa21 genes were resistant. IRBB7 having Xa7 gene showed resistance responding to 24 BB isolates, whereas IRBB107 carrying Xa7 gene was susceptible to 10 BB isolates and moderately resistant to 14 BB isolates. Near-isogenic lines (NILs) of Toyonishiki and IR24, both possessing Xa7 gene, showed different resistance response against 24 BB isolates according to genetic background. Xa3+xa5, Xa4+xa5, Xa4+xa13, Xa4+Xa21, xa5+xa13, xa5+Xa21, xa13+Xa21, Xa4+xa5+xa13, Xa4+xa5+Xa21, Xa4+xa13+Xa21, xa5+xa13+Xa21, and Xa4+xa5+xa13+Xa21 were resistant to K1, K2, K3 and 24 isolates breaking down Xa3 gene. When Xa3 and xa13 genes were combined with xa5, Xa4, Xa21, resistance response was enhanced compared with single gene lines containg only Xa3 or xa13. Similarly, when Xa4 gene was combined with xa5 and Xa21, resistance response was improved by the gene combination effect.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.56 no.4
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• pp.329-341
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• 2011

The objective of this study was to determine the genetic diversities of major rice blast resistance genes among 84 accessions of aromatic rice germplasm. Eighty four accessions were characterized by a dominant 11 set of PCR-based SNP and CAPS marker, which showed the broad spectrum resistance and closest linkage to seven major rice blast resistance (R) genes, Pia, Pib, Pii, Pi5 (Pi3), Pita (Pita-2), and Pi9 (t). The allele specific PCR markers assay genotype of SCAR and STS markers was applied to estimate the presence or absence of PCR amplicons detected with a pair of PCR markers. One indica accession, Basmati (IT211194), showed the positive amplicons of five major rice blast resistance genes, Pia, Pi5 (Pi3), Pib, Pi-ta (Pi-ta2), and Pik-5 (Pish). Among 48 accessions of the PCR amplicons detected with yca72 marker, only five accessions were identified to Pia gene on chromosome 11. The Pib gene was estimated with the NSb marker and was detected in 65 of 84 accessions. This study showed that nine of 84 accessions contained the Pii gene and owned Pi5 (Pi3) in 42 of 84 accessions by JJ817 and JJ113-T markers, which is coclosest with Pii on chromosome 9. Only six accessions were detected two alleles of the Pita or Pita-2 genes. Three of accessions were identified as the Pi9 (t) gene locus.

• Proceedings of the Korean Society of Crop Science Conference
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• 2017.06a
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• pp.265-265
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• 2017

This study was carried out to develop rice variety integrated with rice bacterial blight resistance gene and to know the information of major agronomic traits of developed variety. Advanced backcross Lines 21 having Xa3 and Xa21 gene cross from japonica cultivar Hwanggeumnuri and indica variety IRBB21. Days after seeding and culm length of ABLs21 were 108 days (Aug. 16) and 76 cm, respectively. Ripened grain rates was 87.4 %, which was similar to the parents. 1000 grain weight of brown rice of ABLs21 was 21.4g, which was lower than the donor parent. Milled rice yield of ABLs21 was 532 kg/10a, which was smaller than recurrent parent and higher than the donor parent. Grain length/width ratio of brown rice was form of japonica with short-ellipse and glossiness of cooked rice has japonica trait. Head rice rate showed a large difference compare to the donor parent and similar to the recurrent parent. ABLs21 would be useful genetic resources for resistance breeding program against bacterial blight.

• Korean Journal of Breeding Science
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• v.42 no.2
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• pp.144-150
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• 2010

The objective of this study was to map gene/QTL for photoblastism in a weedy rice (photoblastic rice: PBR) using DNA markers. Light-induced effect on germination of seeds was compared among three accessions (Oryza sativa L.), PBR, Milyang 23 and Ilpum. Results showed that PBR seeds started to show photoblastism during seed development, different from Ilpum and Milyang 23. Frequency distribution of germination in the F4 lines from crosses between Ilpum and PBR and, Milyang 23 and PBR revealed bimodal distributions suggesting that photoblastism was controlled by a few genes. Bulked segregant analysis using $F_4$ populations derived from the above two crosses was conducted to identify gene/QTL for photoblastism. Two QTL were identified on chromosomes 1 and 12 explaining 11.2 and 12.8% of the phenotypic variance, respectively. Two QTL were further mapped between two SSR markers, RM8260 and RM246 on chromosome 1, and between RM270 and 1103 on chromosome 12. It is noteworthy that two QTL for photoblastism were colocalized with the QTL for seed dormancy reported in the previous QTL studies. The clustering of two genes for photoblastism and dormancy possibly indicates that these regions constitute rice phytochrome gene clusters related to germination. Because PBR has a low degree of dormancy, a pleiotropic effect of a single gene controlling dormancy and photoblastism can be ruled out. The linked markers will provide the foundation for positional cloning of the gene.

• Journal of Plant Biotechnology
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• v.37 no.4
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• pp.483-493
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• 2010

Knock-out of a gene by insertional mutagenesis is a direct way to address its function through the mutant phenotype. Among ca. 15,000 gene-trapped Ds insertion lines of rice, we identified one line from selected sensitive lines in highly salt stress. We conducted gene tagging by TAIL-PCR, and DNA gel blot analysis from salt sensitive mutant. A gene encoding an oligopeptide transporter (OPT family) homologue was disrupted by the insertion of a Ds transposon into the OsOPT10 gene that was located shot arm of chromosome 8. The OsOPT10 gene (NP_001062118.) has 6 exons and encodes a protein (752 aa) containing the OPT family domain. RT-PCR analysis showed that the expression of OsOPT10 gene was rapidly and strongly induced by stresses such as high-salinity (250 mM), osmotic, drought, $100\;{\mu}M$ ABA. The subcellular localization assay indicated that OsOPT10 was localized specifically in the plasma membrane. Overexpression of OsOPT10 in Arabidopsis thaliana and rice conferred tolerance of transgenic plants to salt stress. Further we found expression levels of some stress related genes were inhibited in OsOPT10 transgenic plants. These results suggested that OsOPT10 might play crucial but differential roles in plant responses to various abiotic stresses.