Jae-Han Son

Development of expressed sequence tag derived-simple sequence repeats in the genus Lilium

Although lily is the second largest flower crop in cutting flower commodity, only six simple sequence repeats SSRs have been reported. Thus, we developed expressed sequence tag derived-SSRs (EST-SSRs) for the Lilium genus. Among 2,235 unique ESTs, 754 ESTs contained SSR motifs, among which 165 ESTs were amenable to primer design. Among these 165 EST-SSRs, 131 EST-SSRs showed amplification in at least one Lilium species, and 76 EST-SSRs showed amplification in at least nine species. Of the 76 EST-SSRs, 47 showed amplification in all Lilium species analyzed. Using 10 breeding lines, we selected 21 EST-SSRs that had the highest number of alleles and polymorphism information content. The polymorphism information content values of these selected EST-SSRs ranged from 0.49 to 0.94 with an average of 0.76, which are higher than other plant species. The phylogenetic dendrogram derived from the amplification profiles of the 21 high polymorphic EST-SSRs was congruent with the genetic background of the 84 selected lily accessions and hybrids, which are available in commerce. Thus, the developed EST-SSRs will be very useful in germplasm management, genetic diversity analysis, cultivar finger printing, and molecular breeding in the lily.

Species relationships among Allium species by ISSR analysis

The genus Allium includes several important condiment and ornamental vegetable species. The botanical status of many more than 600 species in the genus Allium has not been clearly classified. We used inter simple sequence repeat (ISSR) technique to analyze the phylogenetic relationships among 24 individual accessions and species that represent cultivated Allium species and their ancestral species. Nucleotide sequence analysis was used to rule out homoplasy, the possibility that unrelated ISSR-bands migrated to the same positions during gel electrophoresis. To avoid spurious results, PCR reactions were conducted in duplicate on independent thermocyclers and only reproducible bands were used for analysis. We detected 153 polymorphic bands from 20 ISSR primers. The ISSR polymorphism profiles were used to group most of the Allium species and accessions according to previous classification. However, A. chinense, previously included in the Rhizirideum section, was distinct from other species in the phylogenetic dendrogram. A. wakegi, the Korean endemic ‘Jjokpa’ and hybrid between A. cepa and A. fistulosum, was grouped in the Cepa section. A. monanthum, another Korean endemic ‘Dalae,’ was grouped in the Rhizirideum section. Because phylogenetic analysis of these three Korean endemic species has not been reported previously, the current report may allow these species to be included in future phylogenetic studies.

Sequence diversification of 45S rRNA ITS, trnH-psbA spacer, and matK genic regions in several Allium species

Allium is a very diverse genus with over 600 species distributed worldwide. Haplotype analyses of 45S rRNA ITS, trnH-psbA spacer, and matK gene sequences in 9 Allium species were carried out, subsequent to which phylogenetic relations of the nine species were also analyzed. Of the three genes, the nuclear 45S rRNA ITS sequences showed the highest variation with one haplotype in each species. The other two chloroplast genes revealed that more than one haplotype was present in each species, and each haplotype was present in several of the species. In the matK gene, EcoRI restriction revealed heteroplasmy in which the functional gene retains the EcoRI recognition site while the nonfunctional, pseudogene does not. Phylogenetic patterns were not consistent among the haplotypes of the 45 rRNA ITS, trnH-psbA spacer, and matK genic regions. This phylogenetic incongruency might be due to the presence of multiple haplotypes in each of the chloroplast genes. However, the inconsistency of the phylogenetic relationships, based on the 45S rRNA ITS sequences makes a strong case for further analysis.

Isolation of cold-responsive genes from garlic, Allium sativum

Cold stress discourages development of the full genetic potential of plants and results in serious adverse effects on plant growth and limits agricultural productivity. Garlic (Allium sativum) requires low temperatures for the induction of flowering and bulb development. However, low or freezing temperatures can often cause physiological damage. Cold-responsive genes were isolated from a Korean garlic variety through systematic analyses using differential display (DD)-PCR, reverse transcription (RT)-PCR, and quantitative (Q)-PCR. Of the 2470 transcripts observed, 76 transcripts were up-regulated and 20 transcripts were down-regulated in response to cold temperatures (4°C) as determined by DD-PCR analysis. The differentially-expressed genes were further narrowed to 15 up-regulated and four down-regulated genes through subsequent RT-PCR and Q-PCR analyses. Of the 15 up-regulated genes, 11 genes were homologous to previously-reported abiotic-stress regulated genes in other species, and four genes did not match any known genes. Of the four down-regulated genes, three genes matched function-annotated genes and one gene did not match any genein the BLAST analysis. While most of the up-regulated poly-peptides were hydrophilic, the down-regulated polypeptides were neutral or hydrophobic. Of the 19 differentially-regulated genes, four genes showed different expression profiles in different tissues upon cold stress. Genetic manipulation of the CR genes obtained may provide molecular tool for overcoming frost damage of the garlic plants during hibernation.

Sequence diversity of a domesticated transposase gene, MUG1, in Oryza species.

MUG1 is a MULE transposon-related domesticated gene in plants. We assessed the sequence diversity, neutrality, expression, and phylogenetics of the MUG1 gene among Oryza ssp. We found MUG1 expression in all tissues analyzed, with different levels in O. sativa. There were 408 variation sites in the 3886 bp of MUG1 locus. The nucleotide diversity of the MUG1 was higher than functionally known genes in rice. The nucleotide diversity (π) in the domains was lower than the average nucleotide diversity in whole coding region. The π values in nonsynonymous sites were lower than those of synonymous sites. Tajima D and Fu and Li D* values were mostly negative values, suggesting purifying selection in MUG1 sequences of Oryza ssp. Genome-specific variation and phylogenetic analyses show a general grouping of MUG1 sequences congruent with Oryza ssp. biogeography; however, our MUG1 phylogenetic results, in combination with separate B and D genome studies, might suggest an early divergence of the Oryza ssp. by continental drift of Gondwanaland. O. longistaminata MUG1 divergence from other AA diploids suggests that it might not be a direct ancestor of the African rice species.

Effect of Film Layers and Storing Period on the Fermentation Quality of Whole Crop Barley Silage

Dept. Animal bio, Chanbuk National University, Jeonju, 561-756ABSTRACTThis study was undertaken to characterize feed value and silage quality according to storage period and film layers for whole-crop barley silage. The crude protein(CP) content increased in all silage during the storage periods compared to those before silage,this content slightly increased over the prolonged storage period but it was not significant(p>0.05). Depending on the film layers of silage, 6 layers were higher than 4 layers. The neutral detergent fiber(NDF) and acid detergent fiber(ADF) contents also increased in all silage during storage periods compared to those before silage(P<0.05), but they were maintained at similar levels during the storage period. Depending on the film layers of silage, 6 layers were higher than 4 layers. The total digestible nutrients(TDN) content decreased in all silage during the storage periods. However, it was maintained at a similar level for the duration of each storage period. Depending on the film layers of silage, 6 layers were lower than 4 layers. The pH value was decreased during the prolonged storage period and depending on the film layers, 6 layers were lower than 4 layers.In the organic acid contents during the prolonged storage period, lactic acid increased, acetic acid was lower, and butyric acid was significantly higher(p<0.05). Depending on the film layers, 6 layers showed higher levels of lactic acid and lower levels of butyric acid(p<0.05). Therefore, these results showed that 6 layer wrapping was advantageous for long term storage of whole crop barley silage, while also indicating that it is desirable to use 4 layer wrapping within a six month period.(Key words :Whole crop barley, Storing period, Film layers, Fermentation quality)

Sequence variation in ITS spacers and 5.8S rDNA and relationship of E, St, P, Ns, Xm, and H genomes in the genera of Agropyron, Elytrigia, Leymus, Pascopyrum, Psathyrostachys, and Hordeum

Understanding species evolution and improvement requires information of their genome origin and differentiation. Among the species in the family Gramineae, genome identities of Agropyron-Elytrigia-Leymus group are still ambiguous. In order to delineate the genome relationship, nucleotide sequence analysis in the rDNA ITS regions was carried out among the species in the genera Elytrigia, Agropyron, Psathyrostachys, Leymus, and Psacopyrum containing E, St, P, Ns, and Xm genomes. The ITS-1 and ITS-2 showed a narrow range of variation in length except for the presence of a pentanucleotide, TGGGG, in/del in some haplotypes, whereas higher numbers of nucleotide substitutions were observed in most genera. There were 187 variable sites in the ITS-1, 5.8S, and ITS-2 regions, in which a few genome specific mutations were observed. While the level of variation was similar between ITS-1 and ITS-2, the rate of transition mutation versus transversion mutations was different among the ITS-1, 5.8S, and ITS-2 segments. GC contents of the ITS regions ranged between 55–65% between genomes and the haplotypes of P and H genomes were slightly higher than others. In phylogenetic analysis, the ITS haplotypes were classified into two groups; one containing H, Ns, NsXm genomes, and another containing P, St, and E genomes, which are congruous to the genome affinities from other studies. Among the four genomes in Pascopyrum smithii (2n=8x=56, StStNsNsHHXmXm), the haplotypes of H and St genomes were identified with the reference diploid species, but the haplotypes having Ns and Xm genomes were not found in the present analysis.

Comparative Pathogenicity of Fusarium graminearum Isolates from Wheat Kernels in Korea

Fusarium head blight (FHB) caused by Fusarium species is a major disease of wheat and barley around the world. FHB causes yield reductions and contamination of grains with trichothecene mycotoxins including; nivalenol (NIV), deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-ADON), and 15-acetylde-oxynivalenol (15-ADON). The objectives of this study were to identify strains of F. graminearum isolated in Korea from 2012-harvested wheat grain and to test the pathogenicity of these NIV- and DON-producing isolates. Three hundred and four samples of wheat grain, harvested in 2012 in Chungnam, Chungbuk, Gyeongnam, Jeonbuk, Jeonnam, and Gangwon provinces were collected. We recovered 44 isolates from the 304 samples, based on the PCR amplification of internal transcribed spacer (ITS) rRNA region and sequencing. Our findings indicate that F. asiaticum was the predominant (95% of all isolates) species in Korea. We recovered both F. asiaticum and F. graminearum from samples collected in Chungnam province. Of the 44 isolates recovered, 36 isolates had a NIV genotype while 8 isolates belonged to the DON genotype (3-ADON and 15-ADON). In order to characterize the pathogenicity of the strains collected, disease severity was assessed visually on various greenhouse-grown wheat cultivars inoculated using both NIV- and DON-producing isolates. Our results suggest that Korean F. graminearum isolates from wheat belong to F. asiaticum producing NIV, and both F. graminearum and F. asiaticum are not significantly different on virulence in wheat cultivars.