Totally, 26 collections, 17 from Korea and 9 from China, were investigated for their sequences of 5S rDNA, especially the non-transcribed spacers (NTSs). Sequences of 5S rDNA were isolated by PCR using the primers, 5s-rRNA1 and 5s-rRNA2. Genomic DNA PCR produced single amplification of 300, 330, or 350 base pair fragments. Sequence analysis revealed that all inserts contained the part of 5S rDNA gene sequence and the full length of the NTS region. Three different sizes of the fragments were confirmed due to different size of NTS and their length were 300bp, 330bp and 350bp, respectively. Among 17 Korean foxtail millets tested, 14 collections showed single 300bp amplification. Longest fragment amplification, 350bp, was obtained only from the foxtail millet from China origin, even though 2 of them include 300bp fragment. CLUSTALW multiple alignments of 26 foxtail millets clearly revealed 4 areas with certain degree of sequence heterogeneity (region I, II, III, IV). Among 4 boxed areas, foxtail millet genotypes from China have distinct insertion especially in region III. Five of them have extra insertion of sequence and their additional sequences were either 45 or 48 base pair. Three Korean foxtail millets have 32 bp insertion. Other 8 Korean collections have short insert sequences (6 to 8 bp), 3 with 8 bp and 5 with 6 bp. In addition to insert, deletion sequences were also confirmed as major deletion was observed in region II of Chinese collection. The size of deletion was 7 bp long. According to phylogenic tree constructed using MEGA4 program, clear grouping was not revealed. To obtain more convincing results various collections from many countries should be obtained and analyzed to distinguish different germplasm from different origin.
Twenty two common millet (Panicum miliaceum L.) varieties collected from Korea, China and Russia were investigated for their phylogenetic relationship using 5S ribosomal DNA sequences with a hope to provide the basic information on their exact origin. Sequences of 5S rDNA were isolated by PCR. The primers, 5s-rRNA1 and 5s-rRNA2, were designed to isolate the complete NTS. Genomic DNA amplification produced two fragments with different length, 900 bp and 400 bp fragments, confirming the presence of two types of 5S rDNA repeats that differed from each other in the length of the NTS region. Amplified DNAs of 400 bp fragment were subcloned and used for further investigation. The obtained NTS sequences ranged from 200 to 300 bp and homology of sequences among plant materials was much higher than long repeat. CLUSTALW multiple aligment of 5S rDNA sequences from 22 different common millets revealed the clear difference by their origin. And critically different areas with insert or deletion were also confirmed. Those sequence difference seems to be used for discrimination of cultivars from different origin and use as molecular markers for origin identification. In phylogenic tree construction, the clear classification was shown where the genotypes from China and Russia is positioned together and stay away from domestic genotypes.
We examined the morphometric characteristics and fluctuating asymmetry of diploid and triploid marine medaka, Oryzias dancena. We used morphometric parameters the truss and classical dimensions. Significant differences in all the classical and truss dimensions of the diploid and triploid fish. All the dimensions of the triploid fish were greater than those of the diploid fish. The triploid marine medaka shows sexual dimorphism in these characters, and the sexual dimorphism of the triploid marine medaka is similar to that of the diploid marine medaka. Thus, the growth of triploid marine medaka is faster than that of the diploid fish, and it displays clear sexual dimorphism, with male fish having longer dorsal and anal fins than female fish. we examined fluctuating asymmetry of eye diameter, maxilla length, operculum length, number of pectoral fin ray and number of pelvic fin ray. In all experimental groups, Eye diameter and maxilla length showed no significant difference between left side and right side (P>0.05). Trends of operculum length in triploid male group and pectoral fin ray's number in diploid male group showed similar trend with trends of operculum length in triploid female group and pectoral fin ray’s number in diploid female group. However, trends of operculum length in diploid male group and pectoral fin ray's number in triploid male group showed opposite trend with trends of operculum length in diploid female group and pectoral fin ray’s number in triploid female group. Trend of pelvic fin ray's number in all groups showed similar trend with trend of pectoral fin ray’s number in all groups.
For the development of SSR marker system in Vicia villosa Roth, an enriched library was constructed by using a modified biotin-streptavidin capture method and the selected clones were sequenced with GS-FLX(454). Of 37,794 sequenced reads, we found that 8,474 reads (22.4%) were redundant, leaving 29,320 unique ones (77.6%). Among the unique clones, 17,174 reads (58.6%) were having microsatellite repeating motifs. Sequence analysis of all SSR-containing reads revealed a predominance of the di-nucleotide SSRs (62.5%). The tri-nucleotide and the tetra-nucleotide SSRs were 5.7% and 22.5%, respectively. As the di-nucleotide type, the AG/GA class of repeat motif was most frequently identified (55.0% of the total di-nucleotide SSRs), followed by the CT/TC class (19.5%), and the TA/AT class (12.1%). Among the tri-nucleotide SSRs, the AGT/GTA/TAG class of repeat motifs was predominant (22.2%), followed by the ACT/CTA/TAC class (17.8%). Among the tetra-nucleotide SSRs, the CTTT/TTTC/TTCT/TCTT class of repeat motifs was predominant (31.2%), followed by the AAAG/AAGA/AGAA/ GAAA class (19.9%). Finally, we designed 779 primer pairs from the flanking sequences of SSR containing reads. We are undertaking the analysis of polymorphisms using the diverse collected accessions of Vicia villosa Roth now. This newly developed SSR marker set shall provide a very useful tool for implementing molecular diversity assessment and population structure studies of Vicia villosa Roth onward.
Blast resistance of one hundred and thirty-one rice cultivars bred in Korea was tested with thirty Korean isolates and twenty-two Philippines isolates using three screening methods. In the blast nursery conducted in Korea and in the Philippines, average disease index of rice cultivars were 4.6 and 2.2, respectively. Seventy-nine cultivars showed different resistance reaction in Korea and in the Philippines, and 19 cultivars showed the same resistant reaction in two locations. In the seedling test, Korean blast isolates displayed different levels of virulence. 93-093, a Korean isolate, was compatible with 90 cultivars whereas 97-057 showed a compatible reaction with 13 cultivars. Twenty-three cultivars showed high level of resistance against Korean and Philippines isolates but Chucheongbyeo, Heugnambyeo, and Manmibyeo showed susceptible reaction to all blast isolates. Through the sequential planting test in Korea and in the Philippines, Palgongbyeo and Seomjinbyeo displayed durable resistance, and Nagdongbyeo and Gihobyeo showed high level of disease infection over the planting time. These results indicate that blast isolates collected in two countries have different genetic background and number of compatible isolates should be considered in definition the durability of rice cultivar to rice blast.