様々な野菜可食部組織を用いたDNAフィンガープリント法の開発(育種・遺伝資源)  [in Japanese] Development of DNA Fingerprinting Technique Using Edible Tissues in Vegetables(Breeding & Germplasm Resources)  [in Japanese]

33種類の野菜可食部から, 微量サンプル抽出法で得られたタンパク質の混入度(A260/A280)は, いずれも1.8以上で, PCRに適する値が得られた.しかし, リーフレタス, ゴボウから得られたDNA溶液は褐色を呈し, PCR阻害がみられた.しかし, このような場合, 最終濃度10mMの2-メルカプトエタノールをDNA溶液に添加し, Nucleon PhytoPure Kitで精製することで, 着色がなくなり, PCR阻害もみられなくなった.増幅可能となったDNAを用いて, 6プライマー組合せについて蛍光AFLP分析を行ったところ, 各材料で総数65〜249のピークがそれぞれ得られた.野菜の種類ごとに適したプライマー組合せを選択することで, 品種識別を効率的に行う手段になり得るものと考えられる.さらに, 他殖性作物における品種識別の可能性を検討するために, ネギ16品種・系統それぞれにおいて16個体を混合して作製したDNAサンプルを用いたAFLP分析を行った.その結果として得られたDNAフィンガープリントを比較することにより, 用いた品種・系統を相互に識別することが可能であった.

To develop a DNA fingerprinting technique suitable for edible tissues of vegetables, the reliable DNA extraction methods followed by fluorescent AFLP were proposed for each material. DNA was extracted from edible tissues of 33 vegetables using the miniprep DNA isolation method. In all materials, the DNA concentration and purity (A260/280 ratios of more than 1.8, A230/260 ratios of less than 2.95) estimated by UV spectrophotometry were thought to be sufficient for the next PCR step, but the browned DNA solution extracted from leaf lettuce or edible burdock inhibited PCR. The subsequent PCR was successful after purification of the crude DNA solutions, including 2-Mercaptethanol at a final concentration of 10mM, using Nucleon PhytoPure Kit (Amersham Biosciences Corp.). A total of 65 to 249 peaks were obtained by fluorescent AFLP analysis using six primer combinations in 33 vegetables. The technique developed in this study seems to have potential for cultivar identification in various vegetables. Furthermore, a similar AFLP analysis using bulked DNA samples from 16 plants each in three cultivars and 13 strains of Japanese bunching onion (Allium fistulosum L.) was conducted to examine the possibility of cultivar identification in outcrossing species. The DNA fingerprint obtained in this study allowed these cultivars and strains to be distinguished from each other.