The authors thank the three anonymous reviewers for their helpful comments on the manuscript. This work was partly supported by Grant-in-Aid for JSPS Fellows (26.11613).

注:本文是根据我们以前的工作30。一些部分转载来自日本和斯普林格日本植物学会许可。 1。访花观察<ol><li> 观测场的选择 <ol><li>搜索中，植物材料分布的地区，采用可靠的资源，如图画书，学术期刊等调整研究地点的数量，以适应研究目标（选择候选研究地点比如，一个广泛的位置花的游客在日本的比较）。 </li><li>检查所选择的候选群体的来自相关的出发点，如科研院所和住宿，位置和距离，如果需要长期的研究。 </li><li>通过在一个单位面积计数植物个体的数目大致估计每个种群规模。选择实验操纵大量人群S使用许多植物的个体。 </li><li>访花的预观察<ol><li>决定靶向个体在每个区域将在同一时间间隔期间观察到的，这取决于人口密度。选择5-10刚刚开的花或花蕾突破作为目标的花朵，并拒绝已经开通退化的。 </li><li>启动一整天的时间肉眼观察，因为花可以通过在不同的时间不同的传粉范围31,32参观。 </li><li>在记录片，记录访花的种类名称，每人花小时每个探视时间。 </li><li>密切观察访花是否触及花药和/或红斑;如果他们这样做，记录下参观者授粉。如果访花只能访问花草不要触摸生殖器官，并录制成游客。 </li><li>使用防虫网或手工制作的吸引，捕捉花香游客识别和保留它们作为标本（ 图1F）。用乙酸乙酯或手指压向胸部快速杀灭被困访客，并在软袋或在塑料管或壳体用100％乙醇保留它们。 </li><li>观察后，确定使用形态特征样本的具体名称，或在鉴定中的困难时，请专家为各分类群的帮助。 </li></ol></li></ol></li><li> 观察领域 <ol><li>选择根据自己的可及性，人口规模和花卉观众数量研究场所。选择的时间段，其中包括在开花季节花卉游客的代表性样本，并基于研究目的观察期（ 例如，通过访花探视频率波动的研究，长期的时间段应设置）。 </li><li>准备相应的录音设备，如数码摄像机（例如，对于夜间记录，应使用摄像机用红外线功能）。 </li><li>以同样的方式作为1.1.4选择目标的花朵。 </li><li>固定摄像机铝三脚架。设置目标的个人与前面的三脚架相机，约50厘米远。 </li><li>检查并拍摄前修改光对摄像机的屏幕显示的数量和材料的重点。 </li><li>开始在同一时间通过预观测资料估算适当的时区的目视观测和拍摄的视频（在本案中，观察期分别为平均六个多小时，大约从上午05时至下午13:00 ）。 </li><li>识别访客和传粉昆虫的种类名称，并记录他们的名字和他们的记录片材访问的次数。 </li><li>捕获并确定以同样的方式访客和授粉为1.1.4.5。 </li><li>重复步骤1.2.1至1.2.7各观测中ñ时期。设置基础上，研究目标和观测数据前的观察期。 </li></ol></li><li> 数据分析后观察 <ol><li>以同样的方式传粉观测如1.1.4.6后确定捕获试样的种类名称。 </li><li>检查视频剪辑，并注意以同样的方式作为用于目视物种名称和它们的访问时间。 </li><li>从视觉观察和录像数据计算每人每次花的游客小时花探视频率。使用基于基本统计并使用合适的软件，如R，SPSS和/或SAS 33-35（ 例如，方差双向分析（ANOVA适当的统计方法统计比较每个访问者的站点和岁之间的这些频率）与R中的软件杜克的诚实差异显著（HSD）的测试）。 </li></ol></li></ol> 2。套袋和保持架实验<ol><li> 筹备和套袋实验笼 <ol><li>对于套袋实验，编制袋有小网目尺寸（〜0.5-1毫米），并用它们来完全防止花探视（ 例如，分别在2011年和2012年用于装袋实验无纺布袋）。 </li><li>为保持架实验中，准备金属丝或塑料丝网板的直径，允许目标越小访问者通过，但不包括较大的，并连接这些网眼板，以形成一个笼子。确保有不低于网直径大的差距。调整基于所述目标植物物种和个体数笼的尺寸和形状。 </li></ol></li><li> 套袋试验 <ol><li>选择每处理30个人，没有从食草动物或严重破坏环境。对于治疗，从各个工厂一花独放，或单株使用单独的花朵。 </li><li>袋ŧ他用胶带贴标签后，他们在目标领域的花（编号和字母; 图1G）。要小心，不要触摸塑料袋内的花药或魂飞魄散，以避免自花授粉的可能性。 </li><li>精心修复袋用软绳或线（ 图1d）花。 </li><li>小心地用软绳或线支持在必要时将袋或风，重压之下对倾斜或塌陷的个人支持设置单独的花朵直立。扭曲或缠绕细绳或金属丝轻轻围绕目标个体的茎，以免建立损坏。 </li><li>在套袋试验治疗<ol><li>对于"控制"的待遇，附加标签的目标鲜花和进行任何治疗。允许花卉游客自由参观。 </li><li>对于"异交"的待遇，涵盖了芽，直到他们的花，然后取出的袋装花花药。把一些polle从上留下烙印多个个体ñ粒。 </li><li>对于"自交"的待遇，涵盖了芽，直到花开放，并从同花的花粉把自己的上魂飞魄散。再次覆盖这些治疗花。 </li><li>对于"自动自我"治疗，盖上袋子，直到开花赛季结束的味蕾。 </li><li>对于"断芽"的待遇，确定已访问了破芽，观察小蜜蜂的出入口（ 图1b，C）。取出破芽的花药，以防止在同一花重复花粉沉积由小蜜蜂探视后，和袋这些芽迅速，以防止进一步的探访。 </li><li>对于"开花"的待遇，包芽，直到开场阶段，以避免在磨合萌芽阶段，不胜其烦。在此之后，取出包，并让参观者收集花蜜和花粉。 </li><li>对于"芽"的待遇，移除未开封的花蕾花药和人为异交他们。这些袋子迅速芽，防止花，不胜其烦。 </li></ol></li></ol></li><li> 凯奇实验 <ol><li>盖上笼准备目标植物的个体。手工调节的笼花朵的位置，以防止不同痕（ 图1E）之间的接触和花粉沉积。 </li><li>对于"笼子"的治疗，选择未开封的花蕾的个人和扣帽子对他们的选择鲜花的鉴定。凯奇与标记的芽个人从建立笼（ 即，花卉虫不能访问未开封的花蕾，可估计只网箱放置后参观昆虫的影响）之前拒绝的影响。 </li><li>牢固安装笼使用铁极，以防止通过笼子基极和地之间的访客条目地面。 </li></ol></li><li> 数据分析房颤之三套袋和保持架实验<ol><li>收集所有的标记花朵在开花季节（ 图1G）通过切割并从母体的个体中分离它们的端部。单独保留每个标记的样品，以防止其污染。 </li><li>检查存在或不存在的水果上的每个标记设置的flower.Record在设定果的情况下，每个果实的种子数目。 </li><li>计算（坐果比率，其定义为水果由花的数量除以数）和每成熟果实（种子集比，定义为通过数除以种子数种子数的每花果实数的比胚珠）使用所有记录的数字。 </li><li>统计学比较使用适当的方法和软件，如那些在1.3.3中列出的治疗之间的加工水果，种子组的比率（ 例如，单向ANOVA和Tukey的HSD或Fisher中的R软件33精确检验）。 </li><li>使用装袋实验的结果的材料的调查<ol><li>为了测试动物传粉的需要，在统计学上的"控制"和"自动自"治疗之间的比较结果。 </li><li>为了估计花粉限制的程度，比较"控制"和"异型杂交"治疗。 </li><li>为了测试自交亲和性，比较"异交"和"自交"治疗。 </li></ol></li><li>破芽授粉的影响评估<ol><li>比较"控制"和"芽"治疗，以确定破芽痕是否繁殖成熟，是否可以使用在破芽期进行控制的"控制"治疗的价值。 </li><li>比较"自动自"和"打破芽"处理，以确定是否花的繁殖成功的断裂与小蜜蜂授粉-bud阶段是统计学上比进行自花授粉（ 即，测试断裂芽授粉的存在）植物更高。在此之后，这两种治疗方法比较"控制"估计破芽授粉的授粉效率。 </li><li>比较"断芽"，"笼"和"花"处理，以估计破芽授粉繁殖的影响。 </li></ol></li></ol></li></ol>

The authors declare that they have no competing financial interests.

花观测和装袋实验在该研究中采用的以显示探视频率和植物的雌性繁殖成功，分别。在Dafni酒店（1992）38，因为它可以记录用于分析观众的定时和持续时间，并防止观察者偏压录像带方法是有效的。但是，在当时，该方法需要昂贵的设备，和观察时间是由电池寿命的限制。近来，装备用于产生视频记录的成本下降，并且可以在其他授粉研究可以采用这一技术方法。在这项研究中，探视频率...

植物授粉的相互作用是进化生物学和生态学研究最好的例子。花和授粉之间的互惠关系被认为促进了被子植物1,2-作为自然选择的结果的多样化，尽管其他生物和非生物因素也产生影响3,4,5。它也被认为花性状已经改变，以适应最有效的传粉者，并产生更多的果实和种子6。这些信念已建成但基于不同的指数，如授粉效果的大型研究，涉及各种不同的解释7。 已经广义传粉系统的开花植物是由各种类型的授粉8参观。在这里，花贵宾在定义为访问得到花香奖励，并传粉被定义为授粉花卉游客的动物物种。一些游客携带同种的花粉来参观花的柱头，可以划分为传粉者。其他游客还可能有一些种内的花粉;他们可能由于授粉和花之间的行为或形态不匹配进行授粉少。在植物繁殖的贡献相媲美，这些差异可能产生对花性状9不同程度的选择性压力，并可能导致开花植物的适应性分歧。因此，虽然授粉群落的组成和相对物种丰度是重要的10中，每个访问者的效果的准确评估也很关键，以确定所述自适应和/或植物的进化过程。 在这项研究中，传粉的效率，定义为每探视频率果实和种子生产，定量估计被确定11。该规范观察IES每个花卉游客的频率，估计在参观花繁殖产生影响。通过人类观察花卉访问的记录是传粉生态学一个经典方法。但是，这种方法强加观察员，谁被要求保持在植物的前面，并采取谨慎，长期测量一个很大的负担。近日，拍摄和记录的技术已经发展迅速，低成本的数码摄像机已经启用了引进录像来观察授粉12,13。这些方法可以方便的对花卉的游客基本信息的收集，并有助于培养对象植物的传粉生态学的理解。 然而，传粉的探视频率不一定相关，其授粉有效性7,14，并评价各授粉的定性影响是很重要的花健身。授粉有效性已经通过花粉在柱头15,16的数目，花粉管生长17,18和果实和/或种子产量19,20估算。装袋的实验中，进行了使用访客排他性袋，是用于测试的自相容性，自花授粉21,22，和无融合生殖23的存在下，典型的方法。此外，授粉效果在访问者组合一定授粉评价已经频繁的环境中进行的，而其他花的游客已被限制（即一个铁丝笼，净，或用网眼足够小袋子，以排除大授粉即在开花植物集）。例如，进行了小网袋套袋实验揭示蚂蚁或蓟马24,25的授粉能力。此外，使用一个金属丝笼或网状鸟排除实验已经示出的芦荟类群的有效传粉26-28。 这项研究的目的是:1）引入在以前的文章中使用的方法和2），以改善这些方法在花卉的游客，他们的访问频率等的研究一般使用，以及它们对植物健身效果石蒜荪变种。 荪是包含在石蒜属，这是广泛分布在日本和狭义韩国29和具有漏斗形红橙色的花朵（ 图1a）的品种之一。先前的研究表明，L.荪变种是血红多个昆虫物种，其中包括一名身份不明的小蜂种和较大的品种Amegilla弗洛雷亚 29访问。然而，这些游客探视频率和授粉效果尚未发现。首先进行的访花的授粉鉴定意见。通过探访小蜜蜂是表外实体RVED上还没有完全没有开通花（花蕾破; 图1b，C）。小蜜蜂赶紧搬来搬去在打破芽undehisced花药和使用他们的下颚采集花粉。该假设是小蜜蜂可能是在磨合蕾期授粉，因为花药和花的柱头之间的缝隙比蜜蜂的体长较小。因此，进行了套袋试验在磨合萌芽阶段，以测试小蜜蜂授粉能力，还考察L.的繁殖策略荪变种荪 。这些芽参观了小蜜蜂，这让蜜蜂的授粉能力的估计之后袋装。个人用未开封的芽笼。小网罩使用，通过它只有小蜜蜂可以传递，让整个flowe小蜜蜂授粉效率的估计环阶段。

<table><tbody><tr><td>recording sheet</td><td>any</td><td>NA</td><td></td></tr><tr><td>insect net</td><td>any</td><td>NA</td><td></td></tr><tr><td>pooter</td><td>any</td><td>NA</td><td></td></tr><tr><td>ethyl acetate</td><td>any</td><td>NA</td><td></td></tr><tr><td>100% Ethanol</td><td>any</td><td>NA</td><td></td></tr><tr><td>plastic tube</td><td>any</td><td>NA</td><td></td></tr><tr><td>plastic case</td><td>any</td><td>NA</td><td></td></tr><tr><td>soft bag</td><td>any</td><td>NA</td><td></td></tr><tr><td>digital video camera(s)</td><td>any</td><td>NA</td><td></td></tr><tr><td>tripod(s)</td><td>any</td><td>NA</td><td></td></tr><tr><td>bags</td><td>any</td><td>NA</td><td></td></tr><tr><td>wire or plastic mesh boards</td><td>any</td><td>NA</td><td></td></tr><tr><td>iron wires</td><td>any</td><td>NA</td><td></td></tr><tr><td>labeling tape</td><td>any</td><td>NA</td><td></td></tr><tr><td>stick supporters</td><td>any</td><td>NA</td><td></td></tr><tr><td>soft strings or wire</td><td>any</td><td>NA</td><td></td></tr><tr><td>pincette(s)</td><td>any</td><td>NA</td><td></td></tr></tbody></table>

field experiments of pollination ecology: the case of lycoris sanguinea var. sanguinea

Plant-pollinator interactions have been studied for approximately one hundred years. During that time, many field methods have been developed to clarify the pollination effectiveness of each pollinator for visited flowers. Pollinator observations have been one of the most common methods to identify pollinators, and bagging and cage experiments have been conducted to show the effectiveness of specific pollinators. In a previous study of Lycoris sanguinea var. sanguinea, its effective pollinators, the visitation frequencies of each floral visitor, and its reproductive strategies were not identified. This study reports the observation that small bees visited flowers that were partially opened (breaking buds). To the best of our knowledge, this phenomenon has not been reported previously. Further, this study investigates the hypothesis that small bees can pollinate at that flowering stage. This study demonstrates the basic methods of field experiments in pollination ecology using L. sanguinea var. sanguinea. Pollinator observations and digital video showed the visitation frequencies of each floral visitor. Bagging and cage experiments revealed that these flowers could be pollinated fully and that breaking-bud pollination could be important for the pollination of this plant species. The advantages and disadvantages of each method are discussed, and recent developments, including laboratory experiments, are described.

被选定为授粉观察五个群体。在观测前阶段，在打开的鲜花和小蜜蜂破芽各种昆虫物种的探视得到了证实。花先生观察发现，大部分游客到所有五个研究点小蜂种Lasioglossum血吸虫个人。总探视记录表明该物种的探视比率在三个位点（ 图2）分别为90％以上。与此相反，第二最常客，Amegilla弗洛雷亚 ，的比率分别在这些领域中低于10％。这些蜂种也?...

Watch this Scientific Journal Video about Field Experiments of Pollination Ecology: The Case of Lycoris sanguinea var. sanguinea at JoVE.com

Field Experiments of Pollination Ecology: The Case of Lycoris sanguinea var. sanguinea

以显示一个给定植物物种的授粉有效性的田间试验的多种方法已被开发出来。这项研究表明了传粉生态学使用血红石蒜变种的血红的案例研究和小说授粉机制，打破芽授粉现场实验的基本方法。

传粉生态学的田间试验:为例<em&gt;血红石蒜</em&gt;变种。<em&gt;荪</em

Plant-pollinator interactions have been studied for approximately one hundred years. During that time, many field methods have been developed to clarify ...

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Field Experiments of Pollination Ecology: The Case of Lycoris sanguinea var. sanguinea

11.4K 次观看.  Chiba University. 以显示一个给定植物物种的授粉有效性的田间试验的多种方法已被开发出来。这项研究表明了传粉生态学使用血红石蒜变种的血红的案例研究和小说授粉机制，打破芽授粉现场实验的基本方法。 

视频: Field Experiments of Pollination Ecology: The Case of Lycoris sanguinea var. sanguinea

Methods for Performing Crosses in Setaria viridis, a New Model System for the Grasses

Setaria viridis is an emerging model system for C4 grasses. It is closely related to the bioenergy feed stock switchgrass and the grain crop foxtail millet. Recently, the 510 Mb genome of foxtail millet, S. italica, has been sequenced 1,2 and a 25x coverage genome sequence of the weedy relative S. viridis is in progress. S. viridis has a number of characteristics that make it a potentially excellent model genetic system including a rapid generation time, small stature, simple growth requirements, prolific seed production 3 and developed systems for both transient and stable transformation 4. However, the genetics of S. viridis is largely unexplored, in part, due to the lack of detailed methods for performing crosses. To date, no standard protocol has been adopted that will permit rapid production of seeds from controlled crosses.
The protocol presented here is optimized for performing genetic crosses in S. viridis, accession A10.1. We have employed a simple heat treatment with warm water for emasculation after pruning the panicle to retain 20-30 florets and labeling of flowers to eliminate seeds resulting from newly developed flowers after emasculation. After testing a series of heat treatments at permissive temperatures and varying the duration of dipping, we have established an optimum temperature and time range of 48 &deg;C for 3-6 min. By using this method, a minimum of 15 crosses can be performed by a single worker per day and an average of 3-5 outcross progeny per panicle can be recovered. Therefore, an average of 45-75 outcross progeny can be produced by one person in a single day. Broad implementation of this technique will facilitate the development of recombinant inbred line populations of S. viridis X S. viridis or S. viridis X S. italica, mapping mutations through bulk segregant analysis and creating higher order mutants for genetic analysis.

Methods for Performing Crosses in Setaria viridis, a New Model System for the Grasses

We have developed a methodology for performing crosses in Setaria viridis (S. viridis). The method involves pruning the panicle prior to a hot water treatment to kill viable pollen. Crosses are performed following a well-controlled growth regime and typically result in the recovery of 1 to 7 cross-pollinated seed/s per panicle.

方法中进行杂交<em&gt;狗尾草</em&gt;，新模型体系的草

Setaria viridis is an emerging model system for C4 grasses. It is closely related to the bioenergy feed stock switchgrass and the grain crop foxtail ...

科研

环境科学

In Vitro Rearing of Solitary Bees: A Tool for Assessing Larval Risk Factors

Although solitary bees provide crucial pollination services for wild and managed crops, this species-rich group has been largely overlooked in pesticide regulation studies. The risk of exposure to fungicide residues is likely to be especially high if the spray occurs on, or near host plants while the bees are collecting pollen to provision their nests. For species of Osmia that consume pollen from a select group of plants (oligolecty), the inability to use pollen from non-host plants can increase their risk factor for fungicide-related toxicity. This manuscript describes protocols used to successfully rear oligolectic mason bees, Osmia ribifloris sensu lato, from egg to prepupal stage within cell culture plates under standardized laboratory conditions. The in vitro-reared bees are subsequently used to investigate the effects of fungicide exposure and pollen source on bee fitness. Based on a 2 &#215; 2 fully crossed factorial design, the experiment examines the main and interactive effects of fungicide exposure and pollen source on larval fitness, quantified by prepupal biomass, larval developmental time, and survivorship. A major advantage of this technique is that using in vitro-reared bees reduces natural background variability and allows the simultaneous manipulation of multiple experimental parameters. The described protocol presents a versatile tool for hypotheses testing involving the suite of factors affecting bee health. For conservation efforts to be met with significant, lasting success, such insights into the complex interplay of physiological and environmental factors driving bee declines will prove to be critical.

In Vitro Rearing of Solitary Bees: A Tool for Assessing Larval Risk Factors

Fungicide sprays on flowering plants may expose solitary bees to high concentrations of pollen-borne fungicide residues. Using laboratory-based experiments involving in vitro-reared bee larvae, this study investigates the interactive effects of consuming fungicide-treated pollen derived from host and non-host plants.

体外孤蜂饲养: 一种评估幼虫危险因素的工具

Although solitary bees provide crucial pollination services for wild and managed crops, this species-rich group has been largely overlooked in pesticide ...

Comparative Karyotype Analysis of Lycoris aurea Herb Populations Using Fluorochrome Banding and 45S and 5S rDNA-FISH

To understand karyotype variation in eight populations, detailed karyotypes were meticulously established using chromosomal measurements, fluorescence bands, and rDNA FISH signals. The number of 45S rDNA sites varies from one to five pairs per population, with the most common number per karyotype being four pairs. The 45S rDNA locus is predominantly located in the short arms and terminal regions of chromosomes, while the 5S rDNA locus is found mainly in the short arm and the terminal or proximal regions. Populations HBWF, HNXN, HBBD, and HNZX showed a similar distribution of 45S rDNA sites, as did GXTL, HBFC, and SCLS, indicating a close relationship between populations with similar 45S rDNA site distributions. The karyotypes of all studied populations are symmetrical, comprising stable and metastable centromeres or exclusively stable centromeres. Scatter plots of MCA and CVCL effectively distinguish their karyotypic structures. The analysis includes six quantitative parameters (x, 2n, TCL, MCA, CVCL, CVCI). Additionally, the results indicate that PCoA based on these six parameters is a robust method for determining biological karyotype relationships among the eight populations. The chromosome number in Lycoris populations is x = 6-8. Based on the current study and literature, genomic differentiation of these populations is discussed in terms of genome size, heterochromatin, 45S and 5S rDNA sites, and karyotype asymmetry.

This protocol investigates the heterochromatin distribution in eight populations of Lycoris aurea (L&#8242;Her) Herb, using combined PI and DAPI chromosome staining, fluorescence in situ hybridization (FISH) with 18S-5.8-26S rRNA gene regions (45S rDNA regions), and 5S ribosomal DNA probes.

To understand karyotype variation in eight populations, detailed karyotypes were meticulously established using chromosomal measurements, fluorescence ...

遗传学

Evaluating the Effect of Pesticides on the Larvae of the Solitary Bees

Current ecological risk assessments of pesticides on pollinators have primarily considered only laboratory conditions. For the larvae of solitary bees, ingestion of provisions contaminated with pesticides may increase the mortality rate of the larvae, decrease the collection rate and the population of adult solitary bees in the next year from a demographic perspective. But there are limited studies on the effects of pesticides on the larvae of solitary bees. Therefore, understanding how pesticides influence the larvae of solitary bees should be considered an integral part of pesticide ecological risk assessment. This study presents a method to expose the larvae of solitary bee, Osmia excavata, to lethal or sublethal doses of pesticide, tracking larval weight gain, developmental duration, eclosion ability, and food consumption efficiency conversion of ingested food. To demonstrate the effectiveness of this method, the larvae of O. excavata were fed with provisions containing acute lethal and sublethal doses of chlorpyrifos. Then, the above indexes of the treated larvae were investigated. This technique helps to predict and mitigate the risk of pesticides to pollinators.

The present protocol explains a method to feed pesticide-contaminated provisions to the larvae of the solitary bees, Osmia excavata. The procedure examines the ecotoxicity of the pesticide to the larvae of the solitary bees.

评估农药对孤独蜜蜂幼虫的影响

Current ecological risk assessments of pesticides on pollinators have primarily considered only laboratory conditions. For the larvae of solitary bees, ...

生物化学

Establishing Pollination Requirements in Japanese Plum by Phenological Monitoring, Hand Pollinations, Fluorescence Microscopy and Molecular Genotyping

The Japanese plum cultivars commonly grown are interspecific hybrids derived from crosses between the original Prunus salicina with other Prunus species. Most hybrids exhibit gametophytic self-incompatibility, which is controlled by a single and highly polymorphic S-locus that contains multiple alleles. Most cultivated hybrids are self-incompatible and need pollen from a compatible donor to fertilize their flowers. Establishing pollination requirements in Japanese plum is becoming increasingly important due to the high number of new cultivars with unknown pollination requirements. In this work, a methodology for the determination of pollination requirements in Japanese plum-type hybrids is described. Self-(in)compatibility is determined by hand-pollinations in both the field and in the laboratory, followed by monitoring pollen tube elongation with fluorescence microscopy, and also monitoring fruit maturation in the field. Selection of pollinizer cultivars is assessed by combining the identification of S-genotypes by PCR analysis with the monitoring of flowering time in the field. Knowing the pollination requirements of cultivars facilitates the selection of cultivars for the design of new orchards and allows the early detection of productivity problems related with pollination deficiency in established orchards.

A methodology for the determination of pollination requirements in Japanese plum-type hybrids is described, which combines field- and laboratory-pollinations and observations of pollen tubes under the fluorescence microscopy with the identification of S-genotypes by PCR and the monitoring of flowering for the selection of pollinizers.

通过物候监测、手授粉、荧光显微镜和分子基因分型确定日本李子的授粉要求

The Japanese plum cultivars commonly grown are interspecific hybrids derived from crosses between the original Prunus salicina with other Prunus species. ...

发育生物学

Deploying Community Scientists to Conduct Nondestructive Genetic Sampling of Rare Butterfly Populations

Global insect declines continue to accelerate. Effective genetic sampling is critically needed to advance the understanding of many taxa and address existing knowledge gaps. This protocol represents a demonstrated method for nondestructively sampling rare butterflies for population genetic structure or DNA barcoding analyses. It uses the chorion of hatched butterfly ovae to yield sufficiently high quantity and quality DNA for successful gene sequencing to confirm species identity and quantify genetic variation. It may be particularly useful when other tissue sampling techniques are impractical or unavailable. While developed for a lepidopteran, it nonetheless could easily be adapted for use with other insect species. It was specifically designed with ease of use as a goal to help maximize broad implementation by individuals of varying experience and skill levels, such as community scientists, conservation practitioners, and students, and for use over large geographic areas to facilitate broad population sampling. The data generated can help inform taxonomic and listing decisions, conservation and management actions, and enhance basic ecological research.

Here, we present a straightforward protocol for noninvasive genetic sampling of butterfly populations based on the field collection of residual egg debris. It can be used to confirm species identity and quantify genetic variation. This protocol can be easily adapted to broader groups for community science involvement.

部署社区科学家对稀有蝴蝶种群进行无损基因采样

Global insect declines continue to accelerate. Effective genetic sampling is critically needed to advance the understanding of many taxa and address ...

生物学

Determination of Self-(In)compatibility and Inter-(In)compatibility Relationships in Citrus Using Manual Pollination, Microscopy, and S-Genotype Analyses

Citrus uses S-RNase-based self-incompatibility to reject self-pollen and, therefore, requires nearby pollinizer trees for successful pollination and fertilization. However, identifying suitable varieties to serve as pollinizers is a time-consuming process. To solve this problem, we have developed a rapid method for identifying pollination-compatible citrus cultivars that utilizes agarose gel electrophoresis and aniline blue staining. Pollen compatibility is determined based on the identification of S genotypes by extracting total DNA and performing PCR-based genotyping assays with specific primers. Additionally, styles are collected 3-4 days after manual pollination, and aniline blue staining is performed. Finally, the growth status of the pollen tubes is observed with a fluorescence microscope. Pollen compatibility and incompatibility can be established by observing whether the pollen tube growth is normal or suppressed, respectively. Due to its simplicity and cost-effectiveness, this method is an effective tool for determining the pollen compatibility and incompatibility of different citrus varieties to establish incompatibility groups and incompatibility relationships between different cultivars. This method provides information essential for the successful selection of suitable pollinizer trees and, thus, facilitates the establishment of new orchards and the selection of appropriate parents for breeding programs.

Determination of Self-Incompatibility and Inter-Incompatibility Relationships in Citrus Using Manual Pollination, Microscopy, and S-Genotype Analyses

This protocol provides a rapid method for determining pollen compatibility and incompatibility in citrus cultivars.

使用手动授粉、显微镜和 S 基因型分析测定柑橘中的自（内）相容性和相互（内）相容关系

Citrus uses S-RNase-based self-incompatibility to reject self-pollen and, therefore, requires nearby pollinizer trees for successful pollination and ...

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白蝴蝶的人工饲养和重金属耐受性

Rearing the Cabbage White Butterfly (Pieris rapae) in Controlled Conditions: A Case Study with Heavy Metal Tolerance

The cabbage white butterfly (Pieris rapae) is an important system for applied pest control research and basic research in behavioral and nutritional ecology. Cabbage whites can be easily reared in controlled conditions on an artificial diet, making them a model organism of the butterfly world. In this paper, a manipulation of heavy metal exposure is used to illustrate basic methods for rearing this species. The general protocol illustrates how butterflies can be caught in the field, induced to lay eggs in greenhouse cages, and transferred as larvae to artificial diets. The methods show how butterflies can be marked, measured, and studied for a variety of research questions. The representative results give an idea of how artificial diets that vary in components can be used to assess butterfly performance relative to a control diet. More specifically, butterflies were most tolerant to nickel and least tolerant to copper, with a tolerance of zinc somewhere in the middle. Possible explanations for these results are discussed, including nickel hyper-accumulation in some mustard host plants and recent evidence in insects that copper may be more toxic than previously appreciated. Finally, the discussion first reviews variations to the protocol and directions for troubleshooting these methods, before considering how future research might further optimize the artificial diet used in this study. Overall, by providing a detailed video overview of the rearing and measurement of cabbage whites on artificial diets, this protocol provides a resource for using this system across a wide range of studies.

作者聚焦：调查卷心菜蝴蝶对城市污染物的耐受性 

This paper presents a detailed protocol for rearing the cabbage white butterfly in controlled lab conditions with an artificial diet, which allows precise manipulations of early-life nutrition and toxin exposure. The representative results show how heavy metal toxicity can be assayed with this protocol.

在受控条件下饲养卷心菜白蝴蝶（Pieris rapae）：具有重金属耐受性的案例研究

The cabbage white butterfly (Pieris rapae) is an important system for applied pest control research and basic research in behavioral and nutritional ...

传粉生态学的田间试验:为例

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方法中进行杂交

体外孤蜂饲养: 一种评估幼虫危险因素的工具

Comparative Karyotype Analysis of Lycoris aurea Herb Populations Using Fluorochrome Banding and 45S and 5S rDNA-FISH

评估农药对孤独蜜蜂幼虫的影响

通过物候监测、手授粉、荧光显微镜和分子基因分型确定日本李子的授粉要求

部署社区科学家对稀有蝴蝶种群进行无损基因采样

使用手动授粉、显微镜和 S 基因型分析测定柑橘中的自（内）相容性和相互（内）相容关系

在受控条件下饲养卷心菜白蝴蝶（Pieris rapae）：具有重金属耐受性的案例研究

在受控条件下饲养卷心菜白蝴蝶（Pieris rapae）：具有重金属耐受性的案例研究

在受控条件下饲养卷心菜白蝴蝶（Pieris rapae）：具有重金属耐受性的案例研究