Abstract
本論文的目標在尋找遺傳標誌以建立縐葉菸草的遺傳圖。 random amplified polymorphic DNA (RAPD) 方法是應用在生物系統遺傳分析上 的一個很好的工具。 RAPD 標誌的多形性，是由於兩個引子之間染色體的 改變或者是鹼基的改變使得引子無法正確結合而產生。 我們以 Nicotiana plumbaginifolia (43C) 為母本，N. longiflora (30C) 為父 本，交配產生 F1 植株及 F1 自交所產生的 99 個 F2 植株所萃取的 genomic DNA 分析其 RAPD 標誌多形性。 從 400 個 GC 含量在 50%~80% 之間的逢機引子， 我們篩選出 30 個引子，利用 PCR 反應，得 到 85 個遺傳標誌。平均每個引子產生 2.87 個遺傳標誌，這些遺傳標誌 的 DNA 片段範圍在 200 bp 到 2600 bp 之間，其片段大小在 400 bp 到 1200 bp 者佔最大部分。經由卡方分析發現，有 52 個遺傳標誌符合 孟德爾遺傳分離律。利用 MAPMAKER 軟體分析這些個遺傳標誌間的連鎖機 率， 其中 47 個遺傳標誌被分成 16 個連鎖群 (groups)，再利用 "map" 指令計算相鄰遺傳標誌間的距離， 整個連鎖圖總共有 449.1 centimorgans。 he objective of this work is to find genetic markers to establish a genetic map for Nicotiana plumbaginifolia. Applicaton of the random amplified polymorphic DNA (RAPD) assay has recently provided a powerful tool for genetic analysis in biological system. DNA polymorphism is the result of either chromosomal changes in the amplified regions or base changes that alter primer binding. In this thesis, we attempted to analyze RAPD markers derived from genomic DNA extracted from 99 individuals of the progeny of F2 intercross ( F1 = N. plumbaginifolia x N. longiflora). A total of 86 genetic markers was obtained by polymerase chain reaction (PCR) with 30 out of the 400 primers with G+C content of 50%-80%. In average, a polymorphism revealing primer gave 2.87 markers in this study. The size of the amplified fragment ranged from 200 bp to 2600 bp, mainly within 400 bp to 1200 bp. Fifty-two of the genetic markers matched the Mendel(s segregation rule as analyzed by chi-square test at 1% significance level. Data analys is using the computer software MAPMAKER resulted in a set of sixteen linkage groups. Of all the RAPD markers analyzed, 47 RAPD loci were assigned to sixteen linkage groups. A basic genetic map was established using the "map" command to resolve the distance between the adjacent markers. The total lenth of the map was 449.1 centimorgans.