利用RAPD與ISSR標誌分析芥藍的遺傳歧異度

Genetic Diversity of Chinese Kale (Brassica oleracea L. var. alboglabra) by RAPD and ISSR Markers

梁婷雅(Ting-Ya Liang)

指導教授 ： 曹幸之

共同指導教授 ： 陳榮芳

國立臺灣大學/生物資源暨農學院/園藝學研究所/碩士(2007年)

https://doi.org/10.6342/NTU.2007.02874

摘要

芥藍（Brassica oleracea var. alboglabra）為中國特有蔬菜，比其他甘藍類蔬菜較為耐熱及早熟。在台灣，栽培品種依用途有葉用一次採收、腋芽多而分次採收及嫩花苔分次採收。依採種方式以開放受粉（OP）品種為主。由於芥藍多以外形特徵命名，使得許多芥藍品種具有相同名稱，但不能確定這些品種是否相同，或不同品種間有多大的遺傳差異。本研究利用RAPD （random amplified polymorphic DNA）與ISSR （inter simple sequence repeat）兩種分子標誌探討所蒐集 44個芥藍品種的遺傳歧異度，同時調查各品種葉形、株形、葉色及抽苔性等主要外形特徵。白格林群（6個品種）葉黃綠、葉色最淺，葉綠素計讀值（SPAD）約 30；黑格林、黑芥藍及白花芥藍群（20個品種）葉色深綠、SPAD值 55- 67；及黃花芥藍（10個品種）葉色及SPAD值介於兩群間。

首先比較芥藍品種內的整齊度，利用 30個RAPD引子分析五個OP品種及一個F1品種，各八個單株的品種內及品種間遺傳變異。參試品種中 36 黃花嫩葉芥藍為黃花品種，其他為白花品種，總共產生 113個分子標誌，其中單型性標誌 52個，多型性標誌 61個（佔 54%），平均每個引子可產生 3.8個標誌。參試樣品間遺傳相似度介於 0.31- 1.0相似度介於 0.31- 1，平均相似度為 0.78， 37.翠寶芥藍（F1）整齊度高，各OP品種內有差異。經UPGMA群叢分析與PCO（principal coordinate analysis）分析結果顯示，黃花品種與白花品種分開，F1翠寶與其他OP品種分開，但各品種內單株仍可自行叢聚。

材料先將 44個芥藍品種及四個甘藍類變種，每品種以 12株DNA混合為一樣品。以 29個RAPD引子分析，共得到 186個分子標誌，其中單型性標誌 27個，多型性標誌 159個（85%），特異性標誌有 43個，平均每個引子可產生 6.4個標誌，品種間相似度介於 0.17- 0.96，平均相似度為 0.70。以 9.印度芥藍與其他各品種平均相似度最低，甚至遠於其他甘藍變種。 38.正宗黃花芥藍與其他芥藍相似度 0.54- 0.74。除了這兩個品種， 42種芥藍中，共得到 135個標誌，其中 82個標誌具有多型性（60.7%）。將芥藍依外形分成白花群、白格林群及黃花群三群。白花群之特有標誌有 8個，群內平均相似度為 0.78，白格林群內平均相似度為 0.76，有 1個特有標誌。黃花群內平均相似度為 0.70，有 10個特有標誌。UPGMA群叢分析與PCO分析都將芥藍分為三群，分別為白花品種、白格林品種及黃花品種，與形態分類相符。其中黃花品種間差異較大，白格林品種與大部分白花品種群內差異小。

包括芥藍與甘藍類變種的相同 48個品種，以 33個ISSR引子進行分析，共得到 213個分子標誌，單型性標誌 24個，多型性標誌 189個（88.7%），特異標誌 19個，相似度介於 0.24- 0.96，平均相似度為 0.67。除 9.印度芥藍及 38.正宗黃花芥藍外， 42種芥藍中，共得到 179個標誌，58個標誌為單型性， 121個標誌具有多型性（67.6%）。其中 3個標誌為白花品種特有， 2個標誌為白格林品種特有， 5個標誌為黃花品種特有。白花群與白格林群較黃花群有較高群內相似度。經UPGMA群叢分析與PCO分析結果顯示，所搜集的芥藍品種具有相當遺傳歧異度，大致分為三群，群內仍有遺傳差異。兩種分子標誌分析結果與外表性狀─花色與葉色分群相符，同名的黑格林或白格林親緣相近。

關鍵字

芥藍 ； DNA 分子標誌 ； RAPD ； ISSR ； 遺傳歧異度

並列摘要

Chinese kale (Brassica oleracea var. alboglabra), an old cultivated leafy vegetable from China is more heat tolerant and takes shorter time to harvest than other Brassica oleracea L. crops. It is grown in Taiwan for its tender leaves with once-over harvest or multiple harvest for tender stalks with inflorescence. There are many Chinese kale cultivars mostly open-pollinated with almost synonymous names just after their leaf color, leaf shape or flower color. It not only causes ambiguity in their identification but also there is little knowledge on their genetic diversity. This study uses both RAPD（random amplified polymorphic DNA）and ISSR（inter simple sequence repeat）markers to assess the genetic distances among different types and genetic similarity within the same type of Chinese kale in a total of 44 accessions each was bulked by 12 individual plants. Morphological characterization is also observed. The group of ‘Bai-gelin’ (6 cultivars) has light colored leaves with lowest Chlorophyll meter readings (about 30 unit, by SPAD) ; the group of white flowered ‘He-gelin’ (26 cultivars) has dark green leaves with high SPAD values (55-67 unit), and the group of yellow flowered (10 cvs.) has intermediate leaf color and SPAD values.
To assess the genetic variation within a cultivar, five OPs and one F1, each with eight plants were studied by 30 RAPD primers. Among 48 samples, 113 markers, of which 61 polymorphic (52%) were generated. Each primer produced 3.8 markers in average. The Jacckard’s similarity coefficient ranges between 0.31- 1.0 with an average of 0.78. The F1 ‘Cui bao’ has the greatest similarity and genetic variations exist in each of the five OP cultivars. The UPGMA dendrogram and PCO （principal coordinate）analysis depict separate clustering of the only one yellow flowered cultivar from the white flowered ones, and the F1 is distinct from the remaining 4 cultivars.
Forty four Chinese kale cultivars each bulked from 12 plants were studied together with kale, collard, cabbage and broccoli in RAPD analysis, 186 markers were generated by 29 primers. Among these, 159 markers ( 85%) were polymorphic and 43 markers are cultivar specific and each primer produced in average of 3.8 markers. The genetic similarity ranges between 0.17- 0.96 with an average of 0.70. One cultigen, ‘Indian kale’ ( 9) has the least similarity, even beyond the range of other botanical varieties of B. o. and cv. ‘True yellow flowered’ (38) has similarity coefficient ranging 0.54- 0.74. Excluding these two, 42 Chinese kale accessions generated 135 markers with 82 (60.7) being polymorphic. Three groups by morphology are characterized as ‘white flowered’, light colored leaf type, and ‘yellow flowered’ There are 8, 1, and 10 group-specific markers , respectively with these three groups. The UPGMA dendrogram and PCO analysis has the same result of grouping and more genetic variation exists in the yellow flowered group than in other two groups.
ISSR analysis was conducted in the same 44 Chinese kale cultivars and 4 varieties of B. o. L. , 213 markers were amplified with 189 polymorphic ones (85%) by 33 ISSR primers. There are 19 cultivar-specific bands and the genetic similarity coefficient ranges between 0.24-0.96 with an average of 0.67. Among 42 Chinese kale accessions (excluding ‘Indian kale’ and ‘True yellow flowered kale’), there are 179 markers and 121 being polymorphic (67.6%). There are 3 unique bands in ‘white flowered’ group, 2 in ‘light colored leaf type’, and 5 in ‘yellow flowered’ group. More genetic relatedness appears in groups of ‘White flowered’ and “light leaf colored’ than in ‘yellow-flowered’ Both UPGMA dendrogram and PCO plot showed similar grouping as in RAPD analysis. There is genetic divergence among as well as within the three groups based on both molecular markers and the grouping goes in accordance with the morphological traits- flower color and leaf color.

並列關鍵字

Chinese kale ； DNA marker ； RAPD ； ISSR ； Genetic diversity

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