Rice is a staple food crop for more than half of the world population. Severe losses of rice production was caused by various environmental conditions such as cold, heat and flooding annually. Rice is a highly sensitive to low temperature below 15-20 ℃ because of originating from tropical or subtropical climates. Especially, seedling of rice is easily damaged to low temperature and result in seedling yellowing, growth retardation, reduced tillering and yield losses at last. We used a recombinant inbreeding lines (RIL) population of 384 individuals derived from a cross between Hanareum 2, a highly cold sensitive variety and Unkwang, a cold tolerant variety for molecular mapping of QTLs related to cold tolerance. Seedling discoloration of each lines and parents caused by cold response were investigated in field condition after transplanting. And leaf samples of RIL population were collected for evaluation of chlorophyll content using 80% acetone extraction. The seedling of each lines and parents was subjected to low temperate by 5~13 ℃ during 14 days. The cold recovery score (CRS) of RILs was recorded after 4 days recovery period according to standard evaluation system (SES, IRRI). Total of eight QTLs were detected on chromosome 1, 7, 8, 10, 11 and 12 using cold tolerance traits, chlorophyll content, seedling discoloration and cold recovery score in 384 RILs. The qCRS12, which detected on chromosome 12 between two flanking markers id12002113, id12002563 (1.1 Mbp) showed 25 LOD score with 26% of phenotypic variation of cold recovery score in RILs population. The positive allele contributing to cold tolerance came from the cold tolerant parent Unkwang. The result may provide useful information for a marker-assisted breeding program to improve cold tolerant in rice.

The next-generation sequencing(NGS) technology is being used for more effective genetic mapping. In previous study, we obtained 60x coverage of sequence from Milyang23 and Gihobyeo on average comparing with Nipponbare reference genome. Also, we developed new derived cleaved amplified polymorphic sequence(dCAPS) markers based on the single nucleotide polymorphisms(SNPs) in coding region sequence(CDS) between these varieties. Totally, 1,726,798 SNPs between Milyang23 and Gihobyeo were detected. Among them, 146 SNP were selected for making dCAPS markers and located on genetic map with previously reported 219 PCR-based DNA markers. The map was applied to the detection of quantitative trait loci(QTLs) for stem internode diameters, culm length and panicle length within MGRIL population, and six QTLs with relatively high LOD score were found at three chromosomes; culm length and stem diameter including the first internode diameter, third and fourth internode diameter. This study showed that the NGS allowed the rapid discovery of a large number of SNPs for dCAPS marker. So, we tried to find out more single nucleotide polymorphisms(SNPs) which were located on the whole genome sequence, such as un-translated region(UTR), intron, Inter-region and coding region sequence(CDS) between Milyang23 and Gihobyeo varieties. And we collected phenotypic information about culm length, panicle length, four stem internode diameters and panicle number in rice MGRIL population for QTLs. Furthermore, results of QTL analysis described above will shows relevance of molecular markers in mapping genes for useful breeding.

Ramie (Boehmeria nivea L.) is a hardy perennial herbaceous plant of the Urticaceae family and has been grown as a fiber crop in several countries including Korea for many centuries. Ramie leaves also have been traditionally used as a major ingredient of a type of rice cake called ‘Song-pyun’ in the Southwest area of Korea, especially Yeong-Gwang province. Despite its economic importance, the molecular genetics of ramie have not been studied in detail yet. Genetic resources of ramie were widely collected from domestic local sites by Bioenergy Crop Research Center (RDA) and Yeong-Gwang Agricultural Technology Center. For the systematic and efficient management of the genetic resources, we developed SSR (simple sequence repeat) markers of ramie. To do this, we generated microsatellite-enriched genomic DNA libraries using magnetic bead hybridization selection method. 247 non-redundant contigs containing SSR motif were generated using nucleotide sequences of 376 clones from the libraries. Primer sets were designed from the flanking sequences of the repeat motif. Finally, we selected 10 SSR markers, possibly showing polymorphism among the genetic resources. Results on the genotype analysis of the ramie genetic resources using the SSR markers will be presented.

Crops are exposed to various environmental stresses. These have been affecting the growth of crops, resulting in the severe loss of agronomic production in many countries. Therefore, development of new varieties of resistant crops is required to assure the desired productivity of crops in stress conditions. In this study, a putatively stress-related gene BrTSR53 was isolated from Brassica rapa. The BrTSR53 is 481 bp long and contains ORF region of 234 bp. The expression of BrTSR53 was determined by quantitative real-time PCR analysis. After 3 hr, the highest quantities of mRNA were revealed in cold and salt stress treatments. In drought stress treatments, there was the highest expression after 36 hr. Therefore, it was confirmed that the ORF in BrTSR53 should be a gene that confer increased resistance to B. rapa growing in different stress conditions. The ORF region of BrTSR53 gene was cloned into an expression vector, pYES-DEST52, and a new protein with molecular weight of 13 kDa was detected by western blot analysis. Also, stress tolerance tests showed that BrTSR53-ORF transgenic yeast exhibited increased resistance to the salt stresses compared with the control. In conclusion, the present data predicts that novel ORF in BrTSR53 can serve as an important genetic resource for abiotic stress resistance.

To identify genes that play critical roles during male gametogenesis in Arabidopsis, we have isolated several pollen morphological mutants from a mutagenized seed pool generated with a T-DNA activation vector. In this study, we have focused on a mutant plant producing ~50% abnormal pollen grains including high levels of collapsed pollen at maturity. The pollen developmental analysis showed that the mutant pollen phenotype was first observed at tricellular stage. Interestingly, the mutation was only maintained as a heterozygote due to the severely reduced genetic transmission through both sexes. TAIL PCR analysis led to the identification of the responsible gene which encodes a conserved oligomeric golgi complex component-related protein (COGCC). RT-PCR analysis showed predominant expression of the gene in reproductive organs including developing spores. The gene identity was confirmed by the result that mutant plants harboring a T-DNA containing corresponding wild type gene produced less level of mutant pollen grains. Furthermore, confocal laser scanning microscopy using mature pollen expressing COGCC-RFP driven under the native promoter showed small punctate signals, which are likely to be from the Golgi complex. Further experiments for co-localization of the COGCC-RFP with the Golgi markers are underway.

We recently reported rice promoters that are active in late stages of pollen development. However, rice promoters that allow manipulation of gene expression at earlier stages of pollen development are still very limited to date. In this study, we have chosen 10 putative microspore promoters, OsMSP1 through OsMSP10, based on publicly available transcriptomic datasets in rice (Oryza sativa L.). Sequence analysis of these promoter regions revealed some cis regulatory elements involved in pollen-specific expression. We also examined promoter activities using the promoter-GUS reporter constructs in both transgenic rice and Arabidopsis. In rice, all of the 10 promoters directed GUS signals from the microspore stage throughout the all stages of pollen development. In addition, while GUS signals from 4 promoters, OsMSP2, OsMSP7, OsMSP9 and OsMSP10, seem to be expressed preferentially during pollen development, those from other six promoters were observed in vegetative tissues such as leaves, stems, and roots of seedlings. Similarly, in Arabidopsis, all of the 10 promoters directed GUS signals during pollen development. In detail, 8 promoters, OsMSP1 ~ OsMSP8 directed GUS signals from the microspore stage, whereas 2 promoters, OsMSP9 and OsMSP10, exhibited GUS signals from tricellular stage. Furthermore, seven promoters, except for OsMSP1, OsMSP2 and OsMSP10, showed GUS signals in shoot apical region or root tissues of seedlings. Furthermore, we verified microspore activity of four promoters, OsMSP1, OsMSP2, OsMSP3 and OsMSP6, by complementation analysis of the sidecar pollen (scp) mutant which displays microspore-specific defects. Currently, further analyses are underway for GUS expression of T2 generation in transgenic rice and scp complementation with remaining promoters.

In this study, we report that the development of a multiplex PCR method using species-specific primers for the simultaneous detection of Poaceae family members, including adlay, barley, maize, rice and wheat, based on the sequence polymorphism of the DNA-directed RNA polymerase beta'' chain (rpoC2) genes Species-specific primers were constructed with common forward primer and each reverse primers which have differences on the basis of sequences. Each primer pairs could amplify PCR products of 443 bp for rice, 346 bp for barley, 278 bp for adlay, 221 bp for wheat and 96 for maize, respectively, from the five chloroplast DNAs. The series of template DNA concentrations were identified by the sensitivity of multiplex PCR. The band of products were clearly amplified from the DNA concentration range of 0.01 to 10 ng/μL. In addition, the species-specific primers were examined for the detection of seven commercial flour mixed products. The combination of the sensitivity of a multiplex PCR with the specificity of the primers for the detection of species would allow to be applied in analyses of processed foods.

Rice flour is used in many food products. However, dough made from rice lacks extensibility and elasticity, whereas that of wheat is suitable for many food products including breads. We have produced marker-free transgenic rice plants containing a wheat TaGlu-Ax1 gene encoding the HMG-GS from the Korean wheat cultivar ‘Jokyeong’ using the Agrobacteriummediated co-transformation method. The TaGlu-Bx7-own promoter was inserted into a binary vector for seed-specific expression of the TaGlu-Ax1 gene. Two expression cassettes comprised of separate DNA fragments containing only TaGlu-Ax1 and hygromycin phosphotransferase II (HPTII) resistance genes were introduced separately to the Agrobacterium tumefaciens EHA105 strain for co-infection. Each EHA105 strain harboring TaGlu-Ax1 or HPTII was infected to rice calli at a 3:1 ratio of TaGlu-Ax1 and HPTII, respectively. Then, among 210 hygromycin-resistant T0 plants, we obtained 20 transgenic lines with both TaGlu-Ax1 and HPTII genes inserted into the rice genome. We reconfirmed integration of the TaGlu-Ax1 gene into the rice genome by Southern blot analysis. Transcripts and proteins of the wheat TaGlu-Ax1 were stably expressed in the rice T1 seeds. Finally, the marker-free plants harboring only the TaGlu-Ax1 gene were successfully screened at the T1 generation.

Ratio of functional changes from orthologous genes is being widely used for detecting the signature for natural selection between species. However, one to one orthologous genes allows only for the analysis due to methodological limitation. A number of genes have in-paralogs as a result of gene expansion in crops. Here, we report a new approach for detecting accelerated evolution, which includes in-paralogs as well as one to one orthologs. This new approach can detect many novel accelerated genes among rice in-paralogs, which have not been investigated yet.

Fusarium crown root rot (FCRR) is a severe fungal disease caused by Fusarium oxysporum f. sp. radicis-lycopersici (FORL) in tomato. Resistance to FORL is conferred by single dominant locus Frl on chromosome 9, but its precise genomic location is not clearly determined. In this study, detailed location of Frl was assessed by using a set of molecular markers physically anchored on Chr.9 and F2 and RIL population derived from FORL-resistant inbred AV107-4 (S.lycopersicum) x susceptible L3708 (S. pimpinellifolium). Bioassay of the two populations with a FORL strain isolated from Korea resulted in single dominant heritance of the resistance. Two SCAR and 11 CAPS markers encompassing 3.6Mb~72Mb of Chr.9 were developed from the Tomato-EXPEN 2000 map and SolCAP SNP-array analysis. These markers were genotyped on 345 F2 plants. A high level of cosegregation with the resistance were observed for 5 markers which were mapped at a large physical interval of 5.1Mb (T1212) to 46.4Mb (SSR237), indicating that genetic recombination was highly suppressed in this region. Cosegregation of these markers with Frl was confirmed by using 126 RILs. The results implied that, in contrast with the previously reported long arm, Frl is present on a pericentromeric region of short arm of Chr. 9, in which crossing-over is severely suppressed. The marker set was further tested on 12 FORL-resistance or susceptibility commercial cultivars. Unlike the biparental populations, frequent linkage break was observed for T1212 and D4 in commercial cultivars. T1212 and D4 showed 50% and 100% match with the phenotype, respectively. D4, a CAPS, was converted to a high resolution melting (HRM) marker and tested on 55 breeding lines from private seed companies (Fig.3). All breeding lines showed the HRM genotype for resistance allele, indicating that D4 can be useful for selecting FORL-resistance tomato plants.

Zinc (Zn) deficiency is one of the important abiotic factors limiting rice productivity world-wide and also a widespread nutritional disorder affecting human health. Zinc is one of the most important essential micronutrient for human About thirty percentage world’s population doesn’t still get enough zinc through their diets. As a staple food of over half world’s population, rice should take the responsibility to provide much more zinc in the future. We analyzed the transcriptome profiles for rice grain from high zinc content and low zinc content lines at the early milky stage using the Illumina Sequencing method. The analysis results for the sequencing data indicated that many transcripts showed different expressions between high zinc content and low zinc content in early milky stage of rice and RT-qPCR analyses confirmed the expression patterns of selected transcripts. Functional analysis of the differentially expressed transcripts indicated that genes have functional annotation and their functions are mainly involved in oxidation-reduction, metabolic, transport , transcript regulation, defense response and photosynthetic processes. Based on the functional annotation of the differentially expressed genes, the possible process that regulates these differentially expressed transcripts in rice grain responding to Zinc at the early milky stage was further analyzed. The functional classification of those genes indicated their connection with various metabolic pathways, Zinc transport, signal transduction, transcriptional regulation, and other processes related to growth and development in early milky stage of rice. Using Illumina sequencing technology, the differences between the transcriptomes of high zinc content and low zinc content lines the early milky stage was described here for the first time. The candidate transcripts may provide genetic resources that may be useful in the improvement of Zinc concentration of rice. The model proposed here is based on differences in expression and transcription between two rice lines. In addition, the model may support future studies on the molecular mechanisms underlying plant responses to Zinc.

BADH1 and BADH2 are two homology genes, encoding betaine aldehyde dehydrogenase in rice. In the present study, we scanned BADHs sequences of 295 rice cultivars, and 10 wild rice accessions to determine the polymorphisms, gene functions and domestication of these two genes. A total of 16 alleles for BADH1 and 10 alleles for BADH2 were detected in transcription region of cultivars and wild species. Association study showed that BADH1 has significant correlation with salt tolerance in rice during germination stage, the SNP P11483(T/A)ishighlycorrelatedwithsalttoleranceindex(STI)(P<10-4). While, BADH2 was only responsible for rice fragrance, of which two BADH2 alleles (P23036, P25390) explain 97% of aroma variation in our germplasm. It indicated that there are no overlapping functions between the two homology genes. In addition, a large LD block was detected in BADH2 region, however, no large LD blocks in a 4-Mb region of BADH1. Only BADH2 region shown significant bias Tajima’s D value from the balance. Extended haplotype homozygosity study revealed fragrant accessions had a large LD block that extended around the mutation site (P23036) of BADH2, while both of the BADH1 alleles (SNP P11483(T/A)) did not show large extended LDblock. All these results suggested that BADH2 was identified as a domesticated gene during rice evolution, while BADH1 was not selected by human beings.

Aroma in rice (Oryza sativa L.) results from the loss of function of the betaine aldehyde dehydrogenase (Badh2) gene on chromosome 8. An 8-bp deletion in exon 7 of Badh2 was reported to be the main allele functionally associated with fragrance. The discovery of new functional alleles will provide additional genetic resources to improve fragrant rice. In this study, we sequenced the Badh2 gene in 30 rice accessions and filtered the Badh2 polymorphisms from whole genome re-sequence data of 295 rice accessions. Seven alleles were detected from the sequence data. Six of the seven were known alleles and one was a novel allele (badh2-E12). The novel allele was a 3-bp deletion in exon 12. Five functional markers, targeting six of the seven alleles, were identified. Fourteen accessions were selected to test the utility of these markers. The five molecular markers reliably distinguishing this fragrant rice from other fragrant or non-fragrant rice accessions. Analysis of two F2 rice population validated the genetic markers FME12-3 and FME14I as functional markers. These two markers co-segregated with the fragrance phenotype. These markers will be used in a Badh2 diversity study and to breed improved fragrant rice accessions via marker-assisted selection.

Pre-harvest sprouting (PHS) results in lower yields for rice and other crops, especially in rainy season before harvest. By using gene based functional studies to reveal the mechanism of PHS related pathways can be a good way in breeding for more PHS resistant accessions. Orthologous genes, which are homologous genes that diverged after a speciation event, generally maintain a similar function in different species to that of the ancestral gene in which they evolved from. Applied with a McDonald-Kreitman Test (MKT), we examined more than 10,000 orthologous genes between rice (Oryza sativa) and Brachypodium (outgroup) based on different phenotypic groups in order to find some fast evolutionary genes in rice PHS. Three groups which represented the PHS susceptible (group 1), PHS medium (group 2) and PHS resistant (group 3) were separated based on the phenotype and each group was examined with the outgroup for MKT. Total 60 fast evolutionary genes that have a positive selection with FDR ≤ 0.05 were found in the three groups, and 19, 5 and 8 genes were specific existed in group 1, 2 and 3, respectively. Annotation of these genes were conducted and the predicted functions were investigated, leading that one Ethylene receptor-like gene that may related to PHS based on the previous studies, which need to be validated later, however. In addition, network analysis of these characterized genes were also investigated, which could reveal the connection of genes between each other. Moreover, the association study between the candidate gene ethylene receptor and the PHS phenotype was performed and indicated that this gene is significantly correlated with PHS in rice. All these above indicated that with this orthologous based method, we can find some important candidate genes that may play an important role in some traits.

Although the overall structure of the chloroplast genome is generally conserved, a number of sequence variations have been identified, which 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 (SNPs) 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 to six rice chloroplast references, which shown a higher diversity in the indica and aus groups than in the japonica group in lower level substitution diversity.

We report the C language implementation of a program that merges SNP data from all samples and estimates various statistical quantities related to SNP. The software combines the SNP information from different samples according to the SNP position in the nucleotide sequence. The combined SNP information is converted into HapMap format that can be used as an input for genome-wide association analysis for quantitative traits. The software additionally provides estimates of the minor allele frequency, the heterozygosity ratio, and the In/Del frequency. The software is prepared as a stand-alone program and is downloadable from http://info.kongju.ac.kr/snpmerge/.

Iron is an essential mineral found in every cell of the human body to make the oxygen-carrying proteins hemoglobin and myoglobin. More than 2 billion people face Fe deficiency. Rice can be a potentially valuable source to supplement that mineral since it is staple food for two-thirds of the world’s population. To bring the nutritional level of the milled product up to that of the whole grain (brown), rice should be enriched with thiamin, niacin and iron. Also iron has important role that absorption from the photosynthetic cells proceeds, chlorophyll synthesis and the growth process of the plant. Orthologous genes, which are homologous genes that diverged after a speciation event, generally maintain a similar function in different species. We applied a McDonald-Kreitman Test (MKT) to examine more than 10,000 orthologous genes between rice (Oryza sativa) and Brachypodium (outgroup) based on different phenotypic groups. This analysis was undertaken to find fast evolutionary genes in rice iron uptake. Three groups were separated based on the phenotype and each group was examined with the outgroup for MKT. Fast evolutionary genes that have a positive selection with FDR ≤ 0.05 were detected at each groups. Annotation of these genes were conducted and the predicted functions were also discussed here. And also, the association study between the candidate gene related to iron uptake phenotype was performed. These results support that using this orthologous based method, we may find some important candidate genes underlying the ion uptake in rice.

In rice (Oryza sativa L.), there is a diversity in flowering time that is strictly genetically regulated by plenty of genes. The floral transition from vegetative to reproductive development is a very important step in the life cycle of a flowering plant. Orthologous genes, which are homologous genes that diverged after a speciation event, generally maintain a similar function in different species. with a McDonald-Kreitman Test (MKT), we examined more than 10,000 orthologous genes between rice (Oryza sativa) and Brachypodium (outgroup), based on different phenotypic groups, to find some fast evolutionary genes of rice flowering time. Three groups with early flowering time (group 1), midium flowering time (group 2) and late flowering time (group 3) were separated and each group was examined for McDonald-Kreitman Test (MKT). Total 70 fast evolutionary genes under a positive selection were found in the three groups, and 14, 42 and 14 genes were specific existed in group 1, 2 and 3, respectively. Annotation of these genes were conducted and the predicted functions were also surveyed. In addition, network analysis of these characterized genes were also investigated to infer related pathways. And also, the association study between the one early flowering factor and the flowering time phenotype was performed and indicated that this gene is significantly correlated with flowering time in rice. These results suggest that using this orthologous based method, we could find some important candidate genes underlying flowering time regulations.

Salt is the major factor limiting crop productivity in saline soils. Development of genetic basis of high salt-tolerant rice is necessary to satisfy urgent needs in rice breeding. In this study, 295 rice accessions from a Korean authentic core set were used to identify the evolution associated genes regarding salt tolerance. By using McDonald-Kreitman Test (MKT), we detected orthologous genes in rice (Oryza sativa) using Brachypodium as an outgroup to investigate fast evolved genes that express differentially based on distinct phenotypic groups. Three groups which represented the salt sensitive (group 1), salt medium tolerant (group 2) and salt tolerant (group 3) were separated and each group was examined with the outgroup in neutral and non-neutral polymorphism together with the divergence levels. Total 53 fast evolutionary genes that have a positive selection with FDR ≤ 0.05 were found in the three groups. Among them, 15, 31 and 7 genes were included exclusively in group 1, 2 and 3, respectively. Annotation of these genes showing the predicted functions were checked. Two genes were found to be related to high salt tolerance based on the previous studies. Besides, association study of the candidate gene alleles and salt tolerance phenotype was carried out, indicating that these genes were correlated with salt tolerance. All these result support that using this type of evolution study, we may find some important candidate genes which are related to important traits in rice, such as the salt tolerance, providing important information for future gene based molecular breeding and functional analysis in rice.

Rice (Oryza sativa L.) is one of the most important staple crops in the world, providing main energy source for more than half of the world’s population. It is even closely associated with economic and political stability in many developing countries of Asia and Africa. These days, moreover, amount of land suitable for the agriculture is shrinking due to a variety factors, such as rapid climate changes and industrializations, while rice eating human populations keeps growing. To meet the nutritional and socio-economic demands worldwide, dedicated efforts in developing superior rice varieties need to be reinforced, accumulating and combining beneficial alleles as much as possible from rice germplams. Here, we propose a pipeline for genome assisted breeding where new genomic technologies including GWAS, omics and evolutionary studies together with follow-up breeding programs are integrated. Once pinpointing candidates genes, the integrated genomics approach allows informed choice of parents for the following breeding program based on the haplotype information, in addition to providing precise molecular marker information. We also conducted proof-of-concept analysis, using various agriculturally important phenotypes for rice improvements.