杏中自体和（内）相容关系的确定结合手授、显微镜和遗传分析
Summary
本文提出了一种方法，将荧光显微镜的自（in）相容性与通过PCR分析识别S-基因型相结合的方法，确定杏（Prunus亚美尼亚卡L.）的授粉要求。
Abstract
罗萨塞的自不相容性是由一种主要由多环位点S控制的Game-phy-s不兼容系统（GSI）决定的。在杏中，确定自和（在）兼容性关系变得越来越重要，因为大量新的培养素的释放导致具有未知授粉要求的培养品种的增加。在这里，我们描述了一种方法，将通过手工授粉和显微镜确定自（in）相容性与通过PCR分析识别S-基因型的方法相结合。为了进行自我（in）相容性测定，从每个品种的气球阶段收集花在野外，在实验室手工授粉，固定，并沾上含艾琳蓝，以观察荧光显微镜下的花粉管行为。为了建立品种之间的不相容关系，从幼叶中提取了每个品种的DNA，通过PCR鉴定了S-等位物。这种方法允许建立不相容组，阐明品种之间的不相容关系，这为在设计新果园时选择合适的授粉器和在育种计划中选择合适的父母提供了有价值的信息。
Introduction
自我不相容是开花植物的一种策略，以防止自授，促进跨越1。在罗萨塞，这种机制是由一个游戏学自不相容系统（GSI）决定的，该系统主要由多环位点S22控制。在风格中，RNase基因编码S-stylar决定因素，RNase3，而决定S-花粉决定因素的F盒蛋白由SFB基因4编纂。自我不相容相互作用通过抑制花粉管生长的方式，防止卵管55，66的受精。
在过去二十年中，全世界都进行了品种更新， 7，,8.从不同的公共和私人育种项目引进了大量新的品种，导致杏培养品种增加，对未知授粉要求8。
使用不同的方法来确定杏的授粉要求。在现场，通过在笼子里的树木或被授精的花丛中进行对照授粉，然后记录水果集99、10、11、1210,11的百分比，12可以建立自（内）相容性。此外，在实验室中，通过半体内花卉培养和荧光显微镜下花粉管行为分析88、13、14、15、16、17，进行了控制授粉。,13,14,15,16,17最近，分子技术，如PCR分析和测序，允许在研究RNase和SFB基因18，19,19的基础上，对不相容关系进行表征。在杏中，有33个S-allee被报告（S1到S20，S S22到S30，S S52，S S53，S Sv，S Sx），包括一个与自我S兼容（Scc）12，18，20，21，22，23，24相关的等位基因。12,18,20,21,22,23,24截至目前，该物种已根据S-基因型,,8、9、17、25、26、279,17组，对该物种进行了25,26次不相容组。826具有相同S-等位基因的培养体是互不兼容的，而具有至少一个不同 S-alleleS的培养器，因此，在不同的不兼容组中分配，是相互兼容的。
为了确定杏培养的授粉要求，我们描述了一种方法，将荧光显微镜的自（in）相容性测定与杏仁培养体中PCR分析的S-基因型的识别相结合的方法。这种方法允许建立不兼容组，并阐明品种之间的不相容关系。
Protocol
1. 自（内）兼容性确定 在田里品尝花。有必要在气球阶段采集花（图1A），与杏子28的BBCH刻度第58阶段相对应，以避免不必要的先前授粉。 实验室的自检和交叉授粉 在气球阶段取出花的麻醉剂，放在一张纸上，在实验室温度下干燥。 24小时后，使用细网（0.26毫米）筛分花粉颗粒（图1…
Representative Results
杏的授粉研究要求在麻醉前一天在晚期气球阶段使用花（图1A）。这个阶段被认为是最有利的花粉和皮酸收集，因为花卉结构几乎成熟，但蚂蚁脱发尚未发生。这可以防止不想要的花粉的干扰，不仅来自同一朵花的花粉，而且来自其他花，因为封闭的花瓣会阻碍携带外部花粉的昆虫的到来。花粉颗粒很容易通过细网（图1B）从?…
Discussion
传统上，大多数商业杏欧洲品种是自我兼容的36。然而，在过去几十年中，在繁殖项目中，使用北美自我不兼容的培养品种作为父母，导致越来越多的新的自我不兼容的培养品种的释放，这些培养动物的授粉要求为77、8、37。8,37因此，确定杏培养素中的自兼容关系和相互兼容关系变得越来越重要。这在冬寒减?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
这项研究由欧洲大学欧洲区域发展基金、欧洲联盟（AGL2016-77267-R）和AGL2015-74071-JIN部长资助;国家教育研究所和阿格拉里亚和阿利门塔研究所（RFP2015-00015-00，RTA2017-00003-00）;戈比耶诺·德拉贡-欧洲社会基金、欧洲联盟（格鲁波·康鲁达多A12_17R）、生物潜水基金会和Agroseguro S.A.
Materials
| Agarose D1 Low EEO | Conda | 8010.22 | |
| BIOTAQ DNA Polymerase kit | Bioline | BIO-21060 | |
| Bright field microscope | Leica Microsystems | DM2500 | |
| CEQ System Software | Beckman Coulter | ||
| DNeasy Plant Mini Kit | QIAGEN | 69106 | |
| dNTP Set, 4 x 25 µmol | Bioline | BIO-39025 | |
| GenomeLab DNA Size Standard Kit – 400 | Beckman Coulter | 608098 | |
| GenomeLab GeXP Genetic Analysis System | Beckman Coulter | ||
| GenomeLab Separation Buffer | Beckman Coulter | 608012 | |
| GenomeLab Separation Gel LPA-1 | Beckman Coulter | 391438 | |
| HyperLadder 100bp | Bioline | BIO-33029 | |
| HyperLadder 1kb | Bioline | BIO-33025 | |
| Image Analysis System | Leica Microsystems | ||
| Molecular Imager VersaDoc MP 4000 system | Bio-Rad | 170-8640 | |
| NanoDrop One Spectrophotometer | Thermo Fisher Scientific | 13-400-518 | |
| pH-Meter BASIC 20 | Crison | ||
| Phusion High-Fidelity PCR Kit | Thermo Fisher Scientific | F553S | |
| Power Pack P 25 T | Biometra | ||
| Primer Forward | Isogen Life Science | ||
| Primer Reverse | Isogen Life Science | ||
| Quantity One Software | Bio-Rad | ||
| Stereoscopic microscope | Leica Microsystems | MZ-16 | |
| Sub-Cell GT | Bio-Rad | ||
| SYBR Safe DNA Gel Stain | Thermo Fisher Scientific | S33102 | |
| T100 Thermal Cycler | Bio-Rad | 1861096 | |
| Taq DNA Polymerase | QIAGEN | 201203 | |
| Vertical Stand Autoclave | JP Selecta |
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Tags
Self-compatibilityInter-compatibilityIncompatibilityApricotHand-pollinationMicroscopyGenetic AnalysisPollination RequirementsCultivar IdentificationIncompatibility GroupsSelf-incompatibilityFruit Tree CropsCherryPlumBBCH ScaleEmasculationPollen GerminationMicroscopyFixative SolutionEthanolPollen Germination MediumSodium SulfiteAutoclaving
Diesen Artikel zitieren
Herrera, S., Lora, J., Hormaza, J. I., Rodrigo, J. Determination of Self- and Inter-(in)compatibility Relationships in Apricot Combining Hand-Pollination, Microscopy and Genetic Analyses. J. Vis. Exp. (160), e60241, doi:10.3791/60241 (2020).