臺灣博碩士論文加值系統

對於有性生殖之生物而言，性別決定是非常重要的。多數昆蟲性別決定機制之下游部分是相當保守，但上游基因卻不然。入侵紅火蟻(Solenopsis invicta) 是使用單基因座互補性別決定機制 (single-locus complementary sex determination, sl-CSD) 的真社會性膜翅目昆蟲。 因此，異型合子 (heterozygosity) 個體將發育成雌性，但半型合子 (hemizygosity) 或同型合子 (homozygosity) 個體則會發育為雄性。 我們實驗室過去的研究顯示入侵紅火蟻決定性別基因有別於已被深入研究之另一真社會昆蟲蜜蜂 (Apis mellifera)，我們將其命名為 sex determination locus (SDL)。 即便 目 前 尚 未 得 到 此 基 因 之 序 列 ， 此 基 因 已 被 證 實 位 於 一 高 度 變 異 區 域 (hypervariable region)。本研究欲確認另一同屬的螞蟻：熱帶火蟻 (Solenopsis geminata)是否亦是使用此新發現之性別決定基因，但由於目前為止尚未有雙套體雄蟲之紀錄，因此本研究將測試雌性個體是否全為異型合子。本研究記錄了與入侵紅火蟻 hypervariable region 同源區域附近的 10 個微衛星 (microsatellite) 基因座之基因型，並檢定其是否如同 CSD 機制一樣偏離哈溫平衡 (Hardy-Weinberg equilibrium)，結果顯示並未有顯著不同，後續的模擬資料顯示這是由於樣本數不足而造成。因此，這些微衛星資料被組成單倍群 (haplotype)， 並確認是否有任一區域是如同 CSD 機制預期之雌性個體均為異型合子，合乎預期之位置在一橫跨連續五個基因座的區域內被找到，且與隨機得到相同結果的機率有顯著不同。另外，我們亦比較了 hypervariable region 序列在此兩種火蟻間的區別。系統發生樹(phylogenic tree) 顯示在此區域有跨種多型性 (trans-species polymorphism) 之現象，而在平衡選汰 (balancing selection) 下此為一常見之模式。 CSD 機制必然與平衡選汰相關，因此綜合上述證據，我們可以間接了解此兩種全球入侵性螞蟻，即便分別已久，SDL 同源區域仍在其性別決定機制中扮演著重要的角色。

Sex determination is absolutely critical for sexual organisms. For most insect species the downstream components of the sex determination pathway are conserved, but the upstream genes and mechanisms are very diverse. The red imported fire ant (Solenopsis invicta, RIFA) is a eusocial Hymenoptera which uses the single-locus complementary sex determination (sl-CSD) mechanism whereby heterozygosity at a single sex locus results in females and hemizygosity (or homozygosity) at this locus yields males. Our lab has shown that the master trigger gene for RIFA sex determination, sex determination locus (SDL), is different from that of the honey bee (Apis mellifera). Although SDL has not been cloned in RIFA, it is characterized by a hypervariable region. I tested whether the tropical fire ant (Solenopsis geminata, TFA) also uses SDL, which I hypothesize is likely because these two species are congeneric. Conservation of function between these two species predicts that TFA SDL will always be heterozygous in females and homozygous in males. Diploid males were not available so this study focused on females. I genotyped 10 microsatellite loci in TFA that are putatively orthologous to RIFA loci near the hypervariable region. Absolute female heterozygosity would violate Hardy-Weinberg equilibrium, but analysis of these microsatellite data could not rejected the Hardy-Weinberg model, likely because of insufficient power given the large number of sex alleles. Nevertheless, by constructing 5-loci haplotypes, I never found any homozygous haplotypes in females, onsistent with the CSD model. In addition, the phylogenic tree of the hypervariable region and the nearby gene EGF-like revealed trans-species polymorphisms between TFA and RIFA indicating that the alleles may be older than these two species and suggesting that SDL is likely evolving under balancing selection. Together my results indicate that the homologous SDL locus could be important for sex determination in S. geminata.

口試委員會審定書……………………………………………………………………I
誌謝……………………………………………………………………………………II
中文摘要………………………………………………………………………………III
Abstract………………………………………………………………………………V
Table of contents……………………………………………………………………VII
List of Figures……………………………………………………………………X
List of Tables……………………………………………………………………XI
Abbreviation Table ………………………………………………………………XII
1. Introduction…………………………………………………………………………1
1.1 Sex determination systems…………………………………………………1
1.2 Core sex determination genes are conserved…………………………………2
1.3 Honeybees and the red imported fire ants (RIFA)………….………………….3
1.4 Tropical fire ant (Solenopsis geminata Fabricius, TFA)………4
1.5 Key questions and the hypothesis………………………………………………5
1.6 General research strategy……………………………………………………6
2. Materials and methods ……………………………………………………………9
2.1 Sample collection……………………………………………………………9
2.2 Ant husbandry……………………………………………………………………9
2.3 Microsatellite data analysis…………………………………………………10
2.3.1 The reference genome…………………………… 10
2.3.2 Primer design………………………………… 10
2.3.3 DNA extraction………………………………………………………11
2.3.4 Multiplex PCR……………………………………………………11
2.3.5 Capillary electrophoresis and microsatellite allele identification……………………12
2.3.6 Hardy-Weinberg test…………………………………………………………12
2.3.7 Simulation data for the Hardy-Weinburg test………………………13
2.3.8 The determination of phased data and heterozygosity power analysis………………14
2.4 SDL homologous sequence analysis…………………………………………………15
2.4.1 The homologous sequences of RIFA and outgroup species…………………15
2.4.2 DNA extraction……………………………………………………………………………16
2.4.3 Primer design………………………………………………………………………16
2.4.4 Selection of candidate neutral loci………………………………………………16
2.4.5 Cloning and sequencing……………………………………………………………17
2.4.5.1 Specific fragment PCR…………………………………………………………17
2.4.5.2 Transformation, culturing, and sequencing……………………………18
2.4.6 Analysis of nucleotide diversity and molecular evolution………………18
3. Results………………………………………………………………………………20
3.1 Microsatellite data and the Hardy-Weinburg test potentially suggest that the homologous hypervariable region could function in TFA sex determination……….20
3.1.1 Sequence data show that the SDL homologous region of TFA is similar to RIFA''s…………………………………………………………………20
3.1.2 Hardy-Weinberg equilibrium model could not be rejected based on single microsatellite locus analysis………………………………………………………20
3.1.3 The exact simulation p-value indicates that a sample size of 1.3 times the allele number is necessary to make the analysis significant……………………21
3.1.4 Analysis of heterozygosity of haplotype blocks potentially suggest that the region between THUMP to SDL6-4 as the candidate SDL locus……………21
3.2 Balancing selection is supported in the homologous sequence in the hypervariable region………………………………………………………………………………22
3.2.1 The description of the hypervariable region and the nearby region and genes…………………………………………………………………………22
3.2.2 Gene tree analyses support trans-species polymorphism (TSP) in the hypervariable region………………………………………………….23
4. Discussion……………………………………………………………………………26
4.1 Structural similarity between TFA and RIFA at the SDL homology candidate region……………………………………………………………………………26
4.2 Information from single microsatellite markers are insufficient to confirm or refute the sex locus…………………………………………………………………………26
4.3 The highly diverse haplotypes at the SDL region in TFA…………………………27
4.4 Balancing selection could be supported by the TSP phenomenon…………………28
5. References……………………………………………………………………………45
Appendix…………………………………………………………………………………50

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