利用蚂蚁Vollenhovia Emeryi对近亲繁殖杂交的诱导和评价
Summary
在本议定书中, 介绍了进行近亲繁殖杂交的方法, 并对这些杂交的成功进行了描述, 为蚂蚁Vollenhovia emeryi。这些协议对于旨在了解膜翅目中性别决定系统的遗传基础的实验是很重要的。
Abstract
在 hymenopteran 模型生物蜜蜂中, 在蜜蜂中广泛研究了性别决定级联的遗传和分子成分。然而, 对于其他非模型 hymenopteran 类群中发现的性别决定机制, 如蚂蚁, 知之甚少。由于 hymenopteran 物种进化的生命周期的复杂性质, 在实验室中很难维持和进行实验性的交叉。在这里, 我们描述了进行近亲繁殖杂交的方法和评估这些杂交在蚂蚁Vollenhovia emeryi的成功。利用v. emeryi在实验室诱导近亲繁殖, 相对简单, 因为该物种具有独特的生物学特性。具体地说, 这个物种产生雄性雄性, 而雌性 reproductives 表现出翅膀多态性, 简化了遗传杂交中表型的识别。此外, 评估近亲繁殖的成功是直接的, 因为雄性可以连续生产的近亲繁殖十字架, 而正常的雄性只出现在一个明确定义的生殖季节在田间。我们的协议允许以v. emeryi为模型, 研究蚂蚁物种性别决定系统的遗传和分子基础。
Introduction
群居 Hymenopteran 类群, 如蚂蚁和蜜蜂, 进化出了一个 haplodiploid 的性别决定体系, 在这种系统中, 异型在一个或多个补充性决心 (CSD) 的个体成为女性, 而那些是同性恋者或 hemizygous成为男性 (图 1A)1。
基因和分子成分参与性别决定级联已被很好的研究在蜜蜂, api 蜜蜂, hymenopteran 模型生物2,3,4。最近的比较基因组调查显示, 蚂蚁和蜜蜂在性别决定途径中分享了许多假定的同系物, 如最初的性别决定基因, csd5。然而, 这些同系物的功能保护的证据仍然缺乏蚂蚁。
为了解决这一问题, 必须发展近亲繁殖线, 因为它们对遗传图谱和分子研究是必不可少的。然而 , 由于生命周期的复杂性质已经演变 , 很难在实验室中维持和进行实验叉。
在这里, 我们使用Vollenhovia emeryi作为一个模型, 以调查的遗传和分子基础的性别决定系统的蚂蚁6,7。本种的近亲繁殖系是在蚂蚁6中首次为与性别决定有关的性状的数量性状基因位点 (QTL) 的连锁映射而开发的。此外, 还研究了7的分子性别测定级联。这个物种进化出了一种不寻常的繁殖系统, 既使用了孤雌生殖和雄核发育 (图 1B)8,9。大多数新的皇后和男性是无性生产的母体和父系基因组, 分别。此外, 工人和一些皇后生产性8。这种生殖系统特别适合于遗传学研究, 因为近亲交配产生的杂交后代使用性生产的皇后和雄性的基因等同于传统的回交。由于性生产的皇后不同形态上的皇后从母体基因组10 (图 1B), 进行和评估近亲繁殖交叉是大大简化了使用这种方法。
本文建立了交叉试验实验菌落的确定方法, 利用全同胞对对近亲繁殖交叉口的应用, 并利用群体成员的基因分型和男性后代的解剖评价这些杂交的成功与否生殖器在 v. emeryi中被描述。
无论采用何种生殖系统, 近交交叉的应用往往是任何研究膜翅目性测定系统的必要的第一步。例如, 在Cardiocondyla obscurior, 在实验室里, 10 代全同胞交配后几乎完全没有双雄雄性, 这表明了惩教署所在地11的缺席。有可能预测惩教署基因座的数目, 从近亲交配产生的男性比例6,12,13。
Protocol
Representative Results
Discussion
本文论证了可用于诱导近亲繁殖杂交的协议, 并对emeryi中近亲繁殖的发生进行了评价。在实验中, 用于交叉的个体的基因分型是确保近亲繁殖杂交成功的必要条件。然而, 这些交叉试验的有效性显然是显而易见的, 因为双雄动物全年都可以生产, 而单倍体雄性只能在秋季在田间和实验室6生产。同胞交配皇后开始在交叉后立即产生雄性后代。在v. emeryi6<…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢日本东京 AntRoom 的代表 Taku 岛先生向我们提供了他的五 emeryi reproductives 的照片。该项目由日本促进科学学会 (16J00011) 为青年科学家研究金和资助青年科学家 (B) (16K18626) 提供资金。
Materials
| Plaster powder | N/A | N/A | Any brand can be used |
| Charcoal, Activated, Powder | Wako | 033-02117,037-02115 | |
| Slide glass | N/A | N/A | Any brand can be used |
| Dry Cricket diet | N/A | N/A | Any brand can be used |
| Brown shuger | N/A | N/A | Any brand can be used |
| Styrene Square-Shaped Case | AS ONE | Any size | Size varies by number of ants |
| Incbator | Any brand can be used | ||
| Aluminum block bath Dry thermo unit DTU-1B | TAITEC | 0014035-000 | |
| 1.5mL Hyper Microtube,Clear, Round bottom | WATSON | 131-715CS | |
| Ethanol (99.5) | Wako | 054-07225 | |
| Stereoscopic microscope | N/A | N/A | Any brand can be used |
| Forseps | DUMONT | 0108-5-PO | |
| Chelex 100 sodium form | SIGMA | 11139-85-8 | |
| Phosphate Buffer Saline (PBS) Tablets, pH7.4 | TaKaRa | T9181 | |
| Paraformaldehyde | Wako | 162-16065 | |
| -Cellstain- DAPI solution | Dojindo Molecular Technologies | D523 | |
| VECTASHIELD Hard・Set Mounting Medium with TRITC-Phalloidin | Vector Laboratories | H-1600 | |
| ABI 3100xl Genetic Analyzer | Applied Biosystems | Directly contact the constructor formore informations. | |
| Confocal laser scanning microscope Leica TCS SP8 | Leica | Directly contact the constructor formore informations. | |
| HC PL APO CS2 20x/0.75 IMM | Leica | Directly contact the constructor formore informations. | |
| HC PL APO CS2 63x/1.20 WATER | Leica | Directly contact the constructor formore informations. | |
| Leica HyDTM | Leica | Directly contact the constructor formore informations. | |
| Leica Application Suite X (LAS X) | Leica | Directly contact the constructor formore informations. |
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