The pinewood nematode, Bursaphelenchus xylophilus (Steinner & Buhrer) Nickle, has two different life stages according to several environmental factors: dispersal stage and propagative stages. The dispersal stage is closely related to the migration to other host pines, whereas the propagative stage is coupled to the direct cause of pine wilt. To establish expressed sequence tag (EST) database of two life cycles of B. xylophilus, subtractive EST libraries were constructed using suppressed subtractive hybridization (SSH). From 3,072 and 3,840 sequences of dispersal- and propagative-specific stage cDNA libraries, 1,927 and 2,604 clusters were generated, respectively, which were annotated by BLASTx and Gene ontology (GO). A total of 1,112 (57.7%) and 1,215 (46.7%) clusters from the dispersal- and propagative-specific stage cDNA libraries respectively had the matched BLASTx hits (E≤10-2), among which 913 (47.4%) and 960 (36.9%) were classified into three categories in Gene ontology. From GO database, some respective stage-specific genes were searched and estimated the relative transcripts level according to stages using the quantitative real-time PCR.

To successful molecular breeding, identification and functional characterization of breeding related genes and development of molecular breeding techniques using DNA markers are essential. Although the development of a useful marker is difficult in the aspect of time, cost and effort, many markers are being developed to be used in molecular breeding and developed markers have been used in many fields. Single nucleotide polymorphisms (SNPs) markers were widely used for genomic research and breeding, but has hardly been validated for screening functional genes in olive flounder. We identified single nucleotide polymorphisms (SNPs) from expressed sequence tag (EST) database in olive flounder; out of a total 4,327 ESTs, 693 contigs and 514 SNPs were detected in total EST, and these substitutions include 297 transitions and 217 transversions. As a result, 144 SNP markers were developed on the basis of 514 SNP to selection of useful gene region, and then applied to each of eight wild and culture olive flounder (total 16 samples). In our experimental result, only 32 markers had detected polymorphism in sample, also identified 21 transitions and 11 transversions, whereas indel was not detected in polymorphic SNPs. Heterozygosity of wild and cultured olive flounder using the 32 SNP markers is 0.34 and 0.29, respectively. In conclusion, we identified SNP and polymorphism in olive flounder using newly designed marker, it supports that developed markers are suitable for SNP detection and diversity analysis in olive flounder. The outcome of this study can be basic data for researches for immunity gene and characteristic with SNP.

Molecular markers are useful for selecting to include superior character genetic like as strong immune system and rapid growth in fish. The marker is also very important part of breeding technology in Olive flounder (Paralichthys olivaceus). Single nucleotide polymorphisms (SNPs) marker is already in use widely for genomic research and breeding. But this SNPs marker hardly has been validated for screening functional genes in Olive flounder. We study identify single nucleotide polymorphisms (SNPs) on Expressed sequence tag (EST) database, develop usable SNP marker and apply to wild sample and cultured of olive flounder. As a result, Out of total 4.327 ESTs, 693contigs and 514 SNP from total contigs were detected while these substitutions include 297 transitions and 217 transversions. 144 developed markers were applied in 16 samples (wild 8, culture 8), Out of total marker, only 32 markers had detected polymorphic in sample. Polymorphism of 32 markers was observed in the variety genes region involved in immunity and protein synthesis. And the 32 marker were identified 21 transitions, 11 transversions, and indel was not detected in polymorphic SNPs. The analysis on heterozygosity by sample showed 0.34 in wild sample and 0.29 in cultured sample. In conclusion, we was identified SNP and Polymorphism by designed new marker, it supports that development marker is suitable for SNP detection and diversity analysis in Olive flounder. The outcome of this study can be basic data for researches for immunity gene and characteristic with SNP.

This study was performed to analyze genetic relationship of the four major Cucurbitaceae crop. We used 120 Expressed Sequence Tag(EST)-Simple Sequence Repeat(SSR) primer sets of developed from watermelon and published in International Cucurbit Genomics Initiative (ICuGI) database. Among 120 EST-SSR primer, 51(49.17%) EST-SSR primer set successfully amplified and 49(40.8%) EST-SSR primer set showed polymorphisms among eight cultivars of Cucurbitaceae. In the first instance, amplified PCR products analysis was conducted by the agarose-gel electrophoresis then further analyzed by using Fragment Analyzer. A total 382 PCR band were producted by 49 EST-SSR primers in 24 plant panels, used the analysis of pairwise similarity and dendrogram construction. Assessment of the genetic relationships resulted in similarity index with range of 0.0103 to 0.8452. In dendrogram, 24 plant panels were formed three major groups (A, B, C) and 7 subgroups (A-1, A-2, B-1, B-2, B-3, C-1, C-2). Major group A was comprised of 2 subgroups, subgroup A-1 (6 watermelon cultivars, Citrullus lanatus var. vulgaris Schrad.) and subgroup A-2 (3 wild type watermelon, Citrullus lanatus var. citroides Mats. & Nakai). Major group B was comprised of 3 subgroups, subgroup B-1 (4 melon cultivars, Cucumis melo var. cantalupensis Naudin.), subgroup B-2 (2 oriental melon cultivars, Cucumis melo var. conomon Makino.) and subgroup B-3 (5 cucumber cultivars, Cucumis sativus L.). Major group C was comprised of 2 subgroups, subgroup C-1 [2 squash/ pumpkin cultivars, Cucurbita moschata (Duch. ex Lam.)/Duch. & Poir. and Cucurbita maxima Duch.] and subgroup C-2(2 squash/pumpkin cultivars, Cucurbita pepo L./Cucurbita ficifolia Bouche.)

Recently, proteome analysis is becoming a powerful tool for the functional characterization of plants. Due to the availability of vast nucleotide sequence information and based on the progress achieved in sensitive and rapid protein identification by mass spectrometry, proteome approaches open up now perspectives to analyze the complex functions of model crop species at different level. In this study, we have N-terminal sequencing data for the 100 embryo and 53 seed proteins of rice separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) were collected and systematically organized for a protein sequence data-file. An attempt was made to link the embryo proteins of rice to DNA sequences for understanding their functions. One hundred proteins of the 700 spots were detected in the embryo using 2-DE gels whereas we used micro sequenced. Of these, 28% of the embryo proteins were matched to DNA sequences with known functions, but 72% of the proteins were identified to be unknown functions as previously reported by Woo et al.,. In addition, twenty-four spots of protein with 100% of homology and nine with over 80% were matched to ESTs (expressed sequence tags) after expanding the amino acid sequences of the protein spots by Database searches using the available EST databases of rice at the NCBI (http://www/ncbi.nlm.nih.gov/) and DDBJ (http://www.ddbj.nig.ac.jp/). Also, a total of 53 proteins out of 700 protein spots separated on the 2-DE gels were analyzed by the peptide mass fingerprinting method (MALDI-TOF/MS). High-quality mass spectra suitable for peptide mass fingerprinting were obtained from 41 spots. Using the ESI-Q-TOF/MS, however, we were able to identify 53 seed proteins of rice, including 12 proteins not registered in database. The rapid expansion of DNA sequence databases to the utilization of EST now provides the whole or partial gene sequences of model organisms, and the recent advances in protein micro-characterization by mass spectrometry allow the possibility of linking these DNA sequences to the proteins in functional complexes. Proteome Database of rice is updated, and is available on the World Wide Web at http://gene64.rda.affrc.go. This work shows that the proteome analysis could be a useful strategy to link the sequence information to the functional genomics.

지금까지 양구슬냉이의 유전정보는 거의 연구되지 않았으므로 우리는 양구슬냉이의 잎으로부터 cDNA library를 제작하고 발현유전자의 종류와 기능별 분류를 조사하였다. 그 결과를 요약하면 다음과 같다. 1. cDNA library에서 1334개 의 클론들을 얻었고 삽입된 단편들의 염기서열의 평균길이는 736bp였다. 우리는 1269개의 high-quality expressed sequence tags (ESTs) 서열을 얻었다. 이러한 EST의 클러스터 분석결과 고유 염기서열(unigene)을 가진 유전자의 수는 851개를 나타냈다. 2. Unigene 476개는 GeneBank에 기능이 알려진 유전자들과 고도의 상동성을 나타내었다. 다른 375개의 unigene들은 기능이 알려지지 않은 것들이었다. 나머지 63개는 NCBI데이터베이스에 어떤 유전자와도 상동성을 보이지 않았고 이러한 유전자들은 아마도 양구슬냉이의 잎에서 발현되는 새로운 유전자일 것으로 보인다. 3. 데이터베이스에서 상동성을 나타낸 EST들을 기능별 주석에 따라서 17개의 카테고리로 분류하였다. 대표적으로 가장 분포도가 높은 카테고리는 결합 기능 또는 보조인자 요구의 단백질(27%), 대사(11%), 세포 소기관 위치(11%), 세포수송과 수송기관 그리고 수송 경로(7%), 에너지(6%), 대사와 단백질 기능의 조절(6%) 등이 있다. 이러한 우리의 연구 결과는 양구슬냉이의 유용한 유전적 자원과 전반적인 mRNA 발현 정보를 제공해 줌으로써 대체 에너지 작물로 떠오르는 양구슬냉이의 다양한 분자적 연구에 기여할 것으로 사료된다.

Rye (Secale cereale L.) chromatins have been used to introduce agronomically important traits into wheat (Triticum aestivum L.). Wheat-rye translocations in the form of 1RS.1AL, 1RS.1BL, 2BS.2RL have been developed for an important genetic source of disease and pest resistance. The long arm of rye chromosome 2 (2RL) has valuable genes that confer resistance to pests such as biotype L of Hessian fly, powdery mildew, leaf and stem rust. Here, we report the generation and analysis of expressed sequence tags from Hessian fly infested wheat-rye translocation. RNAs were isolated from young seedlings infested by Hessian fly. cDNA library was constructed using Clontech cDNA library construction kit. Random sequencing of candidate clones were performed. The EST clones might be useful to clone target gene sequences and would provide clues on molecular interaction between wheat and Hessian fly.