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標題: 霧社血斑天牛族群結構及雄蟲後交尾生殖策略
Genetic structure and male post-mating strategy of Aeolesthes oenochrous (Coleoptera: Cerambycidae)
作者: 丘建賢
Chiu, Chien-Hsien
Contributors: 葉文斌
關鍵字: 保育類昆蟲;粒線體全長;遺傳多樣性;後交尾優勢;多形性指紋分析;異質體
Conservation;complete mitochondrial genome;genetic diversity;post-mating advantage;AFLP;heteroplasmy
日期: 2012
Issue Date: 2013-11-18 11:47:31 (UTC+8)
Publisher: 昆蟲學系所
摘要: 霧社血斑天牛Aeolesthes oenochrous (Fairmaire)主食山櫻花(Prumus campamulata),被列為保育類昆蟲。近年因觀光產業廣植山櫻花,此天牛的族群也隨著增長,也因而引起是否需要保育的質疑。同時,在血斑天牛的交尾行為中,雄蟲在交尾後會有守護雌蟲的行為,顯示出後交尾雄蟲較具生殖優勢的可能性。本研究希望藉由各類DNA資料的建立,分析遺傳變異,以評估此物種保育的需求性,並探討後交尾雄蟲具優勢的可能性。霧社血斑天牛的粒線體全長為15,747 bp,為典型的昆蟲粒線體結構,其中COI與ND1基因擁有非典型的起始密碼子,分別為AAT及TTG。針對無法直接定序的A+T-rich片段選殖了48個分子複製發現有兩個poly-T的序列共有35個基因型,似有異質體(heteroplasmy)的現象;藉由poly-T內側設計引子直接定序發現,異質體的現象可能是由A+T-rich片段上的兩個poly-T結構所產生的DNA滑動(DNA slippage)而導致。同時進一步利用粒線體基因COI和核基因ITS2探討霧社血斑天牛的族群結構,在七個族群83隻個體中,有19個COI基因單型,而ITS2序列,除了一段微衛星序列在個體內及個體間有5-9個不同的重複數外,其餘556 bp皆無變異。從COI網狀分析及親緣分析的結果來看,此天牛在台灣分成兩個基因單型群,分別為西部基因單型群及廣泛分佈基因單型群,因此推測血斑天牛在更新世冰期至少擁有兩個以上的避難所;而COI基因型歧異度(0.779)及ITS2觀測異質度(0.7647)均高的桃園族群為可能避難所之一;族群變動分析的結果則顯示,西部基因單型群在過去可能有族群擴張的現象。在雄蟲後交尾適存度的研究中,我們採用解析能力較高的AFLP (amplified fragment length polymorphism)技術來探討子代的父系親本,共有九隻母蟲順利配對,但僅5組配對結果較為可信;經由魏可遜配對組符號等級檢定(Wilcoxon matched-pairs signed-ranks test)的無母數分析結果顯示,三組的子代具後交尾雄蟲優勢,證實後交尾雄蟲在生殖適存度上擁有較高優勢的可能性。
Wushe blood-spotted longhorned beetle, Aeolesthes oenochrous (Fairmaire), feeding on domestic cherry trees (Prumus campamulata), is one of the endangered species in Taiwan. In the past decade, the beetle’s habitat and population size have increased by enhancing plantation of cherry trees for tourism; thereby whether the species should be protected was indeed questioned. Meanwhile, male of Wushe blood-spotted longhorned beetle showed guarding behavior after mating which indicated the possibility of higher reproductive fitness in post-mating male. The study herein established the DNA data of A. oenochrous for analyzing genetic diversity, and estimated the conserving essentiality of the beetle. Moreover, the reproductive fitness of post-mating male was examined by AFLP (amplified fragment length polymorphism) method. The mitogenome of A. oenochrous, 15,747 bp in length, showed a typical insect gene organization. However, atypical start codons of AAT and TTG are present in two protein coding genes of COI and ND1, respectively. Pseudo-heteroplasmy was spotted in A+T-rich region, based on 35 haplotypes acquired from 48 A+T-rich clones. In fact, the pseudo heteroplasmy was caused by DNA slippage on two poly-T stretches found in A+T-rich region; using new paired primers enclosing inner sides of these two stretches would overcome this slippage problem. Moreover, mitochondrial DNA, COI (cytochrome oxidase I) and nuclear gene, ITS2 (internal transcribed spacer 2) were used to elucidate the genetic structure of A. oenochrous in Taiwan. Eighty-three individuals within seven populations contained 19 COI haplotypes, and no variation was found in 556 bp of ITS2 sequences except for a 5-9 repeat segments of microsatellite. According to the network and phylogenetic analyses of COI, A. oenochrous population in Taiwan involves western and widespread haplogroups, which might indicate that at least two refugia existed in Taiwan during late Pleistocene. And Taoyuan could be one of the refugia since it showed the highest value in both COI haplotype diversity (0.779) and ITS2 heterozygosity (0.7647). Moreover, the western haplogroup experienced a recent population expansion based on the demographic analyses. In the study of reproductive fitness of post-mating male, AFLP method was applied to infer the offspring paternity. A total of 9 females were mated successfully, but only 5 of the mating results are available for further analyses. By using nonparametric Wilcoxon matched-pairs signed-ranks test in statistical analysis, three mating pairs showed that the post-mating male fertilized more eggs than the former male, which supports the possibility of higher reproductive fitness in post-mating males.
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