Identification of Genetic Relationship in Taiwan Avocado Cultivars by Leaf Volatile Constituent Analysis and SSR Markers

本試驗利用葉片揮發性化合物與簡單重複性序列，鑑別臺灣地區酪梨栽培種之親緣性。樣品採集自臺灣、薩爾瓦多及美國加里福尼亞州。揮發性化合物成分之試驗共有45個品種52個參試樣品，包含6個純墨西哥品系樣品，15個純瓜地馬拉品系樣品及3個純西印度品系樣品；簡單重複性序列分析共有65個參試樣品，包含6個純墨西哥品系樣品，15個純瓜地馬拉品系樣品及9個純西印度品系樣品。臺灣地區酪梨葉片採集自嘉義農業試驗分所及農民沈清德果園；薩爾瓦多樣品自薩國農業試驗所之種源保存區，共45個品種；另自美國加州大學取得11個純西印度品系由葉片萃取之去氧核醣核酸樣品。梨葉片揮發性化合物組成之分析方法，經比較頂空-氣密針、溶劑萃取、熱裂解及頂空-固相微萃取等樣品製備法後，選用頂空-固相微萃取聯用氣相層析質譜儀法分析。從單純品系樣品之氣相層析質譜儀分析結果中，12個品系專一化合物及10個數量級變異化合物，作為各酪梨品系之特徵指標化合物。數據分析以歐式距離計算，將指標化合物相對校正量普通對數值之相似性矩陣，以非加權配對算術平均法進行群聚分析。簡單重複性序列分析則選用14組引子，挑選81個具有多型性的條帶，以判別品系間的差異。數據分析以Jaccard index計算相似性矩陣，再以未加權平均連結法進行群聚分析。片揮發性化合物組成分析結果之群聚樹狀圖中，分群結果與酪梨品系的區別大致相符；臺灣地區酪梨栽培種樣品以此方法鑑別時，與純西印度品系樣品同一群。簡單重複性序列分析結果之群聚樹狀圖，將所有酪梨樣品區別為四大群；此方法鑑別臺灣地區酪梨栽培種樣品，其結果與純西印度品系樣品較接近。葉片揮發性化合物組成作為親緣關係之鑑別方法，具實驗重複性與再現性、操作簡單及分析快速等優點。葉片指標化合物之化學分析結果，經內標準品校正後之相對量資料可直接加入資料庫中合併分析，並直接加入群聚樹狀圖中。此分析模式未來可應用於其他物種親緣關係之檢測。Forty-five avocado species were selected from three pure races, known hybrid races and Taiwan cultivars in Taiwan, El Salvador and United States. Fifty-two leaf samples were used in volatile constituent tests, and sixty-five DNA samples were used in simple sequence repeat tests. Taiwan avocado leaf samples were collected from Chiayi Agricultural Experimental Station (CAES) and Shen’s orchard. Import leaf samples were supplied by Germplasm Reserves of Institute of Agricultural Experiments in El Salvador. Eleven DNA samples of pure West Indian race were provided by University of California, Riverside. In comparison with headspace-gas tight syringe direct injection, solvent extraction, pyrolyzer, headspace-solid phase microextraction were selected for sample preparation. GC-MS were used as the analyzing instrument. Twelve race-specific compounds and ten magnitude-varied characteristic compounds were selected as the indicators for further analysis. Similarity matrix of volatile constituents was calculated by Euclidean distance of base-10 logarithm value of indicator compounds calibrated abundance, and dendrogram was clustered based on unweighted pair group method arithmetic average (UPGMA). Similarity matrix was calculated by Jaccard index using 14 primers generated 81 polymorphic fragments. Three avocado pure race samples were separated into three major groups on the dendrogram of volatile constituents test results. Taiwan cultivar and pure West Indian race samples were clustered in the same group on volatile constituent dendrogram. On the dendrogram of simple sequence repeat test results, all test samples were divided into four major groups, Mexican, Guatemalan,.West Indian race related speciess, and Taiwan cultivars, repectively. Genetic distance of the Taiwan cultivar samples were closed to West Indian samples on the dendrogram of simple sequence repeat testing results. With the benefits of easy operation, fast analysis, good repeatability and reproducibility, volatile constituent analysis of avocado leaves could be used as a morphological taxonomy method. Calibrated abundance by internal standards of testing data of GC-MS could be merged into database, and generated a new dendrogram with the all data in the database. Experimental results proved that this is a feasible method for genetic relationship identification.中文摘要 ibtract iii寫表 x一章 前言 1二章 前人研究 5一節 酪梨簡介 5二節 形態分類 5三節 化學型分類 7四節 分子標誌 13三章 材料與方法 17一節 試驗材料 17二節 揮發性化合物分析 17三節 簡單重複性序列 21四章 結果與討論 27一節 樣品來源 27二節 樣品製備方法比較 29三節 以揮發性化合物組成鑑別酪梨品系 30四節 以簡單重複性序列鑑別酪梨品系 41五節 綜合討論 43五章 結語 45考文獻 4