Trinucleotide microsatellite loci for a social wasp, Polistes
Trinucleotide microsatellite loci for a social wasp, Polistes
复制标题
社交黄蜂 Polistes 的三核苷酸微卫星位点
作者：
J. Strassmann;K. Barefield;C. R. Solís;C. Hughes;D. Queller
它们建造敞开的纸巢，在其中抚养后代。雌性之间缺乏明显的形态种姓和社会行为的巨大可塑性，加上易于观察单独有油漆标记的成年个体，使其成为行为研究的模型属（Reeve 1991；Turillazzi & WestEberhard 1996）。所有物种都是真社会性的，但处于相对初级的水平：群体相对较小（Reeve 1991），工蚁与蚁后并不总是不同（Haggard & Gamboa 1980），社会关系往往以冲突为特征（West-Eberhard 1969；Strassmann 1981a；Noonan 1981），某些物种的蚁后必须充当起搏器，用身体操纵工蚁工作。 （里夫和甘博亚，1983）。 Polistes 已成为各种主题的模型系统，包括统治等级制度 (WestEberhard 1969)、群体生活的优势 (Strassmann et al. 1988; Strassmann & Queller 1989; Strassmann 1991)、亲属选择 (Noonan 1981; Strassmann 1981b; Queller & Strassmann 1988)、亲属识别（Gamboa et al. 1987）、篡夺（Klahn 1988）以及女王的社会控制（Reeve & Gamboa 1983）。迄今为止，很难将行为相互作用的详细研究与遗传相关性的研究结合起来，因为很难获得单个相互作用物对与可用遗传标记的相关性的精确估计（例如 Strassmann 等人，1989）。显然，需要一组更具多态性的标记。微卫星位点具有高度多态性、共显性，并且可以从多种 DNA 来源进行基因分型，包括酒精保存的组织和女性受精囊中的精子（Evans 1993；Queller 等人 1993；Peters 等人 1995）。在这里，我们提出了来自马蜂的 18 个新微卫星位点和来自环马蜂的 8 个新位点。所有这些基因座都包含三核苷酸重复区域，其比二核苷酸重复区域更容易明确评分。为了获得这些位点，我们在带有平均 500 bp 插入片段（未富集微卫星）的质粒中构建了非常大的部分基因组文库（Hughes & Queller 1993；参见 Strassmann 等人 1996，了解我们方法和具体方案的基本原理）。我们分别用含有 10 或 12 个 AAT、AAC、AAG、CAT 或 TAG 重复（10 个可能的三核苷酸重复中的 5 个）的合成寡核苷酸探测复制膜。我们发现了数百个潜在的积极因素。重新检测插入片段的 Southern 印迹表明大多数这些序列含有重复序列。我们对大约 80 个 P.bellicosus 克隆的部分或全部进行了测序。其中，59 个包含至少一个重复区域。我们为含有不间断重复序列的克隆设计了聚合酶链式反应 (PCR) 引物，其中重复长度为 5 个或更多，我们可以读取侧翼区域（表 1）。我们没有围绕所有重复序列设计 PCR 引物，原因包括侧翼的性质、缺乏足够的侧翼序列，或者因为它们是二核苷酸重复序列，而不是我们想要的三核苷酸重复序列。我们评估了 3-24 个无关个体的这些引物的杂合性。基因组 DNA 按照 Hughes & Queller (1993) 中的描述或使用 Strassmann 等人中的 Strassmann.1 方案进行制备。 （1996）。 PCR 在油下进行，体积为 10 μL，由 2 μL 稀释的基因组 DNA（约 1 纳克）、2 μL 引物混合物 (2.5 μM)、0.1 μL 10 mM dNTP 混合物、1 μL 10× 缓冲液（Taq 提供）、4.08 μL dH2O、0.62 μl 25 mM MgCl2、0.05 μL 组成。 Taq 聚合酶（5 单位/μL，Promega）、0.15 μL 35S dATP (12.5 μCi/μL)。在 95 °C 初始变性 5 分钟后，我们进行了 30-35 个循环，其中包括 92 °C 60 秒变性、60 秒退火（针对所用引物优化的温度：参见表 1）和 72 °C 45 秒延伸。此后，在 72°C 下再延长 5 分钟以完成延伸。 PCR 产物在 6% 变性丙烯酰胺凝胶上进行电泳（Strassmann 等人，1996）。尽管杂合度从 0.05 到 1 不等，但所有 18 个微卫星位点均被证明在 P.bellicosus 中具有多态性（表 1）。 P. 环状体的 8 个基因座中有 7 个被证明是多态性的，杂合性范围为 0.17 至 1（表 1）。我们还发布了 Polistes permanentis 和另一种社会黄蜂 Parachartergus 的其他微卫星 PRIMER Note
that build open-faced paper nests in which offspring are reared. The lack of distinct morphological castes and great plasticity in social behaviour among females, combined with the ease of observing individually paintmarked adults, has made this a model genus for behavioural studies (Reeve 1991; Turillazzi & WestEberhard 1996). All species are eusocial, but at a relatively rudimentary level: colonies are relatively small (Reeve 1991), workers are not consistently different from queens (Haggard & Gamboa 1980), social relations are often characterized by strife (West-Eberhard 1969; Strassmann 1981a; Noonan 1981), and the queen in some species must act as the pacemaker, physically manipulating workers into working (Reeve & Gamboa 1983). Polistes has served as a model system for a wide variety of topics including dominance hierarchies (WestEberhard 1969), advantages of group living (Strassmann et al. 1988; Strassmann & Queller 1989; Strassmann 1991), kin selection (Noonan 1981; Strassmann 1981b; Queller & Strassmann 1988), kin recognition (Gamboa et al. 1987), usurpation (Klahn 1988), and social control by queens (Reeve & Gamboa 1983). To date, it has been difficult to combine detailed studies of behavioural interactions with studies of genetic relatedness because of the difficulty in obtaining precise estimates of relatedness for individual pairs of interactants with the available genetic markers (e.g. Strassmann et al. 1989). Clearly, a more polymorphic set of markers is needed. Microsatellite loci are highly polymorphic, codominant, and can be genotyped from many sources of DNA including alcohol-preserved tissues and sperm in a female’s spermatheca (Evans 1993; Queller et al. 1993; Peters et al. 1995). Here we present 18 new microsatellite loci derived from Polistes bellicosus and eight new loci from Polistes annularis. All of these loci contain trinucleotide repeat regions which are much easier to score unambiguously than are dinucleotide repeats. To obtain these loci we constructed very large partial genomic libraries in plasmids with inserts (not enriched for microsatellites) averaging 500 bp (Hughes & Queller 1993; see Strassmann et al. 1996, for the rationale of our approach and specific protocols). We separately probed replicate membranes with synthesized oligonucleotides containing 10 or 12 repeats of either AAT, AAC, AAG, CAT or TAG, five of the 10 possible trinucleotide repeats. We picked up hundreds of potential positives. Reprobing a Southern blot of the inserts indicated that most of these sequences contained repeats. We sequenced part or all of about 80 clones of P. bellicosus. Of these, 59 contained at least one repeat region. We designed polymerase chain reaction (PCR) primers for clones containing uninterrupted repeats, five or more repeats long for which we could read the flanking regions (Table 1). We did not design PCR primers around all repeats for reasons including the nature of the flanks, absence of sufficient flanking sequence, or because they were dinucleotide repeats, not the trinucleotides we were after. We evaluated these primers for heterozygosity on 3–24 unrelated individuals. Genomic DNA was prepared as described in Hughes & Queller (1993) or using protocol Strassmann.1 in Strassmann et al. (1996). PCR was carried out under oil in a 10 μL volume made up of 2 μL diluted genomic DNA (about a nanogram), 2 μL of primer mix (2.5 μM), 0.1 μL 10 mM dNTP mix, 1 μL 10× buffer (provided with Taq), 4.08 μL dH2O, 0.62 μl 25 mM MgCl2, 0.05 μL Taq polymerase (5 units/μL, Promega), 0.15 μL 35S dATP (12.5 μCi/μL). After an initial denaturing for 5 min at 95 °C, we carried out 30–35 cycles of 60-s denaturing at 92 °C, 60-s annealing (at a temperature optimized for the primers used: see Table 1) and 45-s extension at 72 °C. After that, 5 extra minutes at 72 °C allowed for the completion of the extension. PCR products were run on 6% denaturing acrylamide gels (Strassmann et al. 1996). All of the 18 microsatellite loci proved to be polymorphic in P. bellicosus though heterozygosity varied from 0.05 to 1 (Table 1). Seven of the eight loci from P. annularis proved to be polymorphic, with heterozygosities ranging from 0.17 to 1 (Table 1). We have published other microsatellites for Polistes annularis and for another social wasp, Parachartergus PRIMER NOTE