- 學位論文
銅鑼地區野生柑橘的形態特徵與RAPD分子標誌鑑定
Identification of wild citrus from Tongluo using morphological characteristics and RAPD markers
摘要
南庄橙(Citrus taiwanica Tan. & Shim.)為原生台灣的芸香科(Rutaceae)柑橘屬(Citrus)植物，相關研究甚少；由於野生族群遽減，已列入世界瀕危稀有植物，基於保持基因種源的多樣性與開發原生種的利用潛力，因此進行南庄橙的保存與研究。野生柑橘族群發現於1999年苗栗縣銅鑼鎮樟樹林地區，至2003年12月止，共發現13棵植株，分別編號並以衛星導航系統(GPS)定位記錄。將野生柑橘族群進行初步鑑定，於比對已知文獻及蠟葉標本後，認為此野生族群有可能為南庄橙。本研究由形態與DNA層次的分析為依據，推論與論證尋獲之野生柑橘族群的最可能「種」。 形態特徵選用台灣目前常見之柑橘栽培品種，以及與南庄橙血緣相近的種進行調查，包括：南庄橙、酸橙、柳橙、‘麻豆文旦’柚、‘Duncan’葡萄柚、椪柑、桶柑，均來自嘉義農業試驗分所之柑橘種原保存區。調查性狀葉的部分包含葉形、葉緣、質地、本葉長、本葉寬、翼葉長、翼葉寬，並計算其長寬比；花的部分為花色、花瓣數、花絲數目、花瓣長、花柱長、花絲長、花絲基部黏合程度；果實的部分有果皮色、果皮光滑程度、果皮果肉分離程度、果肉色、果高、果實直徑；種子的部分是種子形狀、合點顏色、胚色及胚的數目。根據以上性狀特徵，野生柑橘族群中僅1號株有多處不同於其他個體，且性狀與蠟葉標本及文獻記載都不符合，因此將之排除為南庄橙疑似種之外。其餘11株疑似株，族群內無顯著差異，為單一族群；進一步與其他柑橘種類比較，性狀表現不同於台灣現有的主要柑橘種類，與嘉義分所保存之南庄橙種原則有多處相似，為南庄橙的可能性極高。群集分析及主成分分析的結果，擬似種單株間性狀最接近，而與嘉義分所南庄橙及酸橙相似性最高，具有較近的親緣關係，但三者間可確實區分。 RAPD分析選用分類系統中重要的柑橘種類代表，包含形態特徵所有材料，並加入‘Tahiti’萊姆及佛手柑。使用51個引子進行PCR反應，共記錄277條清晰條帶。疑似種族群內，多型性主要發生在1號株與其他11株有不同產物，且1號株與‘麻豆文旦’柚有共同的專一性條帶。10個已知種與12株野生柑橘個體之聚類樹狀圖及主成分分析的結果，符合Swingle及Tanaka分類系統的分類群。1號株與‘麻豆文旦’柚親緣關係最近，與橙類或嘉義分所南庄橙的相似距離遠，親緣關係低於50%。其餘疑似株集中於橙類群，嘉義分所南庄橙與酸橙之間的遺傳相似度約60%，疑似株族群與酸橙的相似度為90%，其bootstrap值顯示兩者能確實分群。疑似株族群間遺傳基質的同質性高，其中最接近嘉義分所南庄橙為12號、13號及11號。 由形態特徵與分子標誌分析之結果，銅鑼鄉樟樹林地區之野生柑橘族群與酸橙及嘉義分所保存之南庄橙間具有極親密地親緣關係，其中又以酸橙的關係較為相近。憑藉形態特徵之翼葉寬度及大小及RAPD分析結果，可確實將酸橙與此野生柑橘族群區別，綜合地緣、本地歷史的相關因素後，判定此野生柑橘族群最可能為南庄橙或酸橙於台灣本島的新變種。
並列摘要
Citrus taiwanica Tan. & Shim., of which relevant researches are few, is one of the endemic Citrus in Taiwan. It is also the world endangered plant because of the severely scarce population. On the basis of keeping genetic diversity and developing the potential in endemic plant, we carry on the conservation and studied of C. taiwanica. Thirteen suspected individuals of C. taiwanica were found at the Chang-Shu-Lin region, in Tongluo township, Miaoli county in 1999. After numbering respectively and orienting each position with the global positioning satellite (GPS), 13 suspected individuals of C. taiwanica were compared with the specimen (in TAI) and description in related references. We supposed the wild population might be C. taiwanica. This study was based on the morphological characteristics and DNA level analysis with RAPD. Species which have close relationship to C. taiwanica, or which are universal cultivated in Taiwan were used in morphological investigation, including C. taiwanica, C. aurantium L., C. sinensis (L.) Osbeck, C. grandis (L.) Osbeck, C. paradisi Macf., C. reticulate Blanco, and C. tankan Hayata. All of them were planted in Citrus germplasm of Chiayi Agriculture Experiment Station (CAES). Investigational items are leaf shape, leaf margin, leaf texture, length of leaf, width of leaf, length of wing, width of wing, color of flower, petal number, filament number, length of petal, length of filament, length of stylus, coherence of filament, color of fruit rind, skin of fruit, coherence of rind, color of pulp, height of fruit, cross section diameter, seed shape, color of chalaza, color of embryo, and number of embryo. The results revealed suspected individual No.1 is different from others, so it is excluded the suspects of C. taiwanica. The rest of the suspects are very similar intra-population, and are different from all major cultivated species in Taiwan. They are the most related to C. aurantium and C. taiwanica from CEAS, whether these three were certainly separated. The same results are suggested in clustering analysis and principle component analysis. Species using in RAPD analysis include the three basic species (C. medica L., C. grandis, and C. reticulata). Besides samples in morphological investigation, C. aurantifolia Swingle and C. medica were added. Fifty-one primers produced 277 polymorphic bands. Among the suspected individuals, polymorphism was revealed on No.1, as well as several specific bands of no.1 were shared with C. grandis. Grouping resulted from dendrogram and principle coordinate analysis matches taxonomic definition both in Swingle system and Tanaka system. The suspected clone No. 1 was more similar with C. grandis, but away from orange-group or C. taiwanica from CAES. The genetic similarity distance was lower than 50%. The rest of the suspected clones, clustered in orange-group, were 90% similar with C. aurantium, but they indeed spilt into two groups with bootstrap value 100. C. taiwanica from CAES was 60% similar with C. aurantium. The suspects had narrow genetic base. The suspects of No.12, No. 13, and No.11 were the most similar with C. taiwanica. Both the results in morphological investigation and DNA markers showed the suspects of C. taiwanica in Tongluo township were very related to C. aurantium and C. taiwanica from CAES, especially to C. aurantium. As a result of clear differences on width and size of wings between the suspected clones and C. aurantium, combining geographical distribution and local history, we suggested the suspects should be C. taiwanica or a new variety of C. aurantium.