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|標題:||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
Testing a hypothetical hybrid, Alpinia x ilanensis, and its putative parents, A. japonica and A. pricei using Inter-Simple Sequence Repeats (ISSR) markers
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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.
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