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研究生:祖帝愛
研究生(外文):Diaiti Zure
論文名稱:以ISSR分子標記檢測宜蘭月桃(Alpinia x ilanensis)為日本月桃(A. japonica)與普來氏月桃(A. pricei)雜交之假說
論文名稱(外文):Testing a hypothetical hybrid, Alpinia x ilanensis, and its putative parents, A. japonica and A. pricei using Inter-Simple Sequence Repeats (ISSR) markers
指導教授:孫英玄
指導教授(外文):Ying-Hsuan Sun
口試委員:古新梅何政坤
口試委員(外文):Hsin-Mei KuCheng-Kuen Ho
口試日期:2015-07-27
學位類別:碩士
校院名稱:國立中興大學
系所名稱:國際農學研究所
學門:農業科學學門
學類:一般農業學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:英文
論文頁數:40
中文關鍵詞:NA
外文關鍵詞:AlpinahybridizationInter-Simple Sequence Repeats (ISSR)species-specific markersmorphological traits
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This study tested a hypothetical hybrid, Alpinia x ilanensis and its putative parents, A. japonica and A. pricei discovered in the Northeast coast of Taiwan. The hybrid and its putative parents were previously studies based on morphological and eco-geographical evidences. It was revealed that the hybrid has characteristics that are variable but wholly in the range of its putative parents with exception to its other qualitative characters, which differs from the parents but discerns the intermediate state of hybrid. However, the direction of hybridization in the hybrid and its putative parent was unknown and it was hypothesized as a natural hybrid between its parents. Based on this hypothesis, we tested the hybrid and its putative parents using Inter-Simple Sequence Repeat (ISSR) markers. Selection of the markers for this study was based on its advantages combining Simple Sequence Repeats (SSR) and Amplified Fragmented Length Polymorphism (AFLP). Also, because the advantage of available species-specific primers of the putative parents from previous studies.
To test the hypothesis 8 A. japonica, 12 A. pricei and 3 A. x ilanensis were collected near Jiaoxi Township, Ilan County. Genomic DNA of the taxa were extracted from leaves tissue using a modified CTAB method, quantified and amplified with nine ISSR primers (UBC 807, 808, 811, 812, 818, 824, 836 & 840) respectively. ISSR-PCR products were screened under UV-transilluminator for DNA bands. Photos of individual amplicons with their duplicates were taken computed for their binary and molecular weight data using PyElp v.1.3 software. The binary data were loaded into excel spread to generated a binary matrix, which was used to calculate the polymorphism and the genetic relationship using Jaccard similarity coefficient and unweight pair group method with arithmetic average (UPGMA).
We found ISSR primers UBC807, 808, 811, 836 and 840 to share similar band patterns between the hybrids and putative parents. However, the relationship can only be established between the hybrid and A. japonica but not between the hybrid and A. pricei although hybrid individuals clustered together in the phylogenetic analysis results based on the marker genotypes and distinct from the other 8 A. japonica. Introgression is unlikely due to the hybrid is unfertile and therefore cannot backcross with its parent. We assume the possibilities that the taxa were probably misidentified or the hybrid is an offspring of another Alpinia species. For this study, we used leaf morphological traits to derive our findings. We proposed further verification of the hypothetical hybrid and its putative parent using reproductive trait and other molecular markers.


Abstract i
Table of Contents iii
List of Tables v
List of Figures vi
Chapter 1 Introduction 1
Chapter 2 Literature review 3
2.1 Evolution of Alpinia Roxb. 3
2.2 Hybridization in plant species 5
2.3 Alpinia x ilanensis with its putative parents (A. japonica and A. pricei ) 8
2.4 Approaches used to detect hybridization 9
2.5 Molecular techniques and marker 11
2.6 Application of ISSR in plant studies 14
Chapter 3 Methodology 15
3.1 Plant materials 15
3.2 Genomic DNA extraction 17
3.3 DNA quantification and quality evaluation 17
3.4 PCR-ISSR amplification 18
3.5 Amplification mix and condition 18
3.6 Gel electrophoresis and visualization of DNA bands 19
3.7 Data analysis 19
Chapter 4 Results 21
4.1 Marker genotypes for the putative parents 21
4.2 Species-specific markers of putative parents species 22
4.3 Comparison of band patterns between putative parents and hybrid 27
4. 4 Genetic relationship of putative parents and hybrid 28
Chapter 5 Discussion 31
5. 1 Putative parent marker genotypes 32
5.2 Species-specific marker 32
5.3 Relationship between parents and hybrid 33
5.4 Testing the hypothesis 33
Chapter 6 Conclusion and future research 35
List of references 36


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