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Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
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发育生物学

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

Published: November 09, 2020
doi:
10.3791/61897
, ,
1Unidad de Hortofruticultura,Centro de Investigación y Tecnología Agroalimentaria de Aragón (CITA), 2(CITA-Universidad de Zaragoza),Instituto Agroalimentario de Aragón – IA2, 3Departamento de Hortofruticultura, Centro de Investigaciones Científicas y Tecnológicas de Extremadura (CICYTEX),Instituto de Investigaciones Agrarias Finca La Orden

Summary

本文介绍了一种测定日本李子型杂交种授粉需求的方法，该方法将田间和实验室授粉以及荧光显微镜下花粉管的观察与通过PCR鉴定 S基因型和监测开花以选择授粉者相结合。

Abstract

通常种植的日本李子品种是种间杂交种，来源于原始 水杨 李与其他 李 子物种之间的杂交。大多数杂交种表现出配子体自不相容性，其由包含多个等位基因的单个高度多态性的 S-位点控制。大多数栽培的杂交种是自不相容的，需要来自相容供体的花粉来施肥。由于大量新品种的授粉需求未知，在日本李子中建立授粉要求变得越来越重要。本文介绍了日本李子型杂交种授粉需求测定方法。自（不）相容性是通过在田间和实验室中手工授粉来确定的，然后用荧光显微镜监测花粉管伸长率，以及监测果实在田间的成熟度。通过将PCR分析对 S基因型的鉴定与田间开花时间监测相结合，对授粉品种的选择进行评估。了解品种的授粉要求有助于选择品种用于新果园的设计，并允许早期发现与已建立果园授粉不足相关的生产力问题。

Introduction

日本李子（Prunus salicina Lindl.）原产于中国1.在19世纪，这种作物从日本引入美国，在那里它与其他北美二倍体李子2杂交。在20世纪，其中一些杂交种被传播到世界各地的温带地区。如今，术语”日本李子”是指广泛的种间杂交种，这些杂交种来自原始水杨假单胞菌与多达15个其他二倍体李子属之间的杂交种。

与蔷薇科的其他物种一样，日本李子表现出配子体自不相容性（GSI），其由包含多个等位基因的单个高度多态性的S位点控制6。S-位点包含两个基因，它们编码在雌蕊中表达的核糖核酸酶（S-RNase）和在花粉粒7中表达的F-box蛋白（SFB）。在自不相容反应中，当花粉粒（单倍体）中表达的S-等位基因与雌蕊（二倍体）中表达的两个中的一个相同时，由于S-RNase8的作用使花粉管RNA降解，跨样式的花粉管的生长被阻止。由于该过程阻止了胚珠中雌性配子体的受精，因此GSI促进了品种之间的异交。

虽然一些日本李子品种是自相容的，但目前种植的大多数品种都是自相容的，需要来自互相容捐赠者的花粉来施肥3。在杏仁9、杏10、11、12和甜樱桃13等李属的核果品种中，可以通过不同的方法确定品种的授粉需求。自（不）相容性可以通过在田间自花授粉和随后监测坐果来确定，或者通过在实验室的受控条件下进行半体内自花授粉和在显微镜下观察花粉管来确定14，15，16，17，18.品种间的不相容关系可以通过在田间或实验室使用潜在传粉品种的花粉进行异花授粉来确定，并通过PCR分析鉴定每个品种的S-等位基因14，15，16，19，20，21，22.在甜樱桃或杏仁等物种中，也可以通过鉴定与自相容性相关的特定S等位基因来评估自身（in）相容性，如甜樱桃13中的S4′或杏仁23中的Sf。

来自主要生产国的几个李子育种计划正在释放一些新的品种2，14，其中许多具有未知的授粉要求。本文介绍了日本李子型杂交种授粉需求测定方法。自（不）相容性由田间和实验室的自花授粉决定，然后在荧光显微镜下观察花粉管。授粉品种的选择结合了通过PCR分析鉴定S基因型和监测田间开花时间。

Protocol

1. 田间手工授粉 花粉提取 为了获得花粉，在阶段D24收集花蕾，根据BBCH量表25，26上的第57阶段。注意：与其他 李 子相比，日本李子需要更多的花蕾，因为它们的花药产生的花粉较少。 使用塑料网（2 mm x 2 mm孔径）取出花药，并将其在室温下放在纸上24小时，直到花药开裂。 通过?…

Representative Results

每个日本李子花蕾包含一个花序，有1-3朵花。与其他核果物种一样，每朵花由四个漩涡组成：腕骨，雄蕊，花瓣和萼片，它们在花的底部融合形成一个杯子。花的结构比其他核果小，雌蕊短而脆弱，被含有少量花粉颗粒的雄蕊包围。在盛开时，每个花序的花朵在短茎上分开，显示白色花瓣在花开前几天形成一个被绿色萼片包围的气球（D期，57 BBCH）（图1A）。花在花开时完全…

Discussion

本文描述的日本李子品种授粉要求的方法要求通过在田间或实验室中控制授粉来确定每个品种的自（不）相容性，并随后用荧光显微镜观察花粉管的生长。不相容关系是通过分子基因分型表征 S-等位基因建立的。最后，通过监测物候学进行授粉媒介的选择，以检测每年开花时重合的品种。

由于大量授粉要求未知的新品种，并且其中大多数是自相容的，因此在日本新的?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

该研究由国家农业和食品技术研究所（RFP2015-00015-00和RTA2017-00003-00）资助;Gobierno de Aragón——欧洲社会基金，欧洲联盟（Grupo Consolidado A12-17R）和Jente de Extremadura——Fondo Europeo de desarrollo Regional （FEDER），Regional de Investigación（IB16181），Grupo de Investigación（AGA001，GR18196）。B.I. Guerrero得到了墨西哥国家科学与技术委员会（CONACYT，471839）的奖学金支持。

Materials

Acetic Acid Glacial Panreac 131008.1611
Agar iNtRON Biotechnology 25999
Aniline blue Difco 8504-88
Boric Acid (H3BO4) Panreac 131015.1210
Calcium Nitrate 4-hydrate (Ca(NO3)2·4H2O) Panreac 131231.1211
Coverglass Deltalab D102460 24 mm x 60 mm
Digital Camera Imaging Developmet Systems UI-1490SE
Digital Camera Software Suite Imaging Developmet Systems 4.93.0.
DNA Oligos ThermoFisher Scientific
dNTP Mix, 10 mM each ThermoSischer Scientific R0193
DreamTaq Green DNA polymerase ThermoFisher Scientific EP0713
Ethanol 96° VWR-Chemicals 83804.360
1Kb DNA Ladder (U.S. Patent No. 4.403.036) (500pb-12Kb) Invitrogen 15615-016 Size: 250µg; Conc: 1.0 µg/µl
Gel Documentation System Bio-Rad 1708195
Hand Counter Tamaco TM-4
Image Lab Software Bio-Rad Image Analyse System for Gel Documentation System
MetaPhor Agarose Lonza 50180
Microcentrifuge 5415 R Eppendorf Z605212
Microscope with UV epiflurescence Leica DM2500 Exciter filter BP340-390, Barrier filter LP425
Microslides Deltalab D100004 26 mm x 76 mm
Mini Electrophoresis System Fisherbrand 14955170
Minicentrifuge ThermoFisher Scientific 15334204
NanoDrop 1000 Spectrophotometer ThermoFisher Scientific ND1000
Petri Dishes Deltalab 200201 55 mm x 14 mm
Potassium Phosphate Tribasic (K3PO4·1.5H2O) Panreac 141513
Primer forward 'Pru C2' ThermoFisher Scientific
Primer forward Pru T2' ThermoFisher Scientific
Primer reverse 'PCER' ThermoFisher Scientific
RedSafe Nucleic Acid Staining Solution iNtRON Biotechnology 21141
Saccharose Panreac 131621.1211
Sodium sulphite anhydrous (Na2SO3) Panreac 131717.1211
Speedtools plant DNA extraction Kit Biotools 21272
TBE Buffer (10X) Panreac A0972,5000PE
Thermal Cycler T100 Bio-Rad 1861096
Thermomixer comfort Eppendorf T1317
Vertical Autoclave Presoclave II JP Selecta 4001725
Vortex Fisherbrand 11746744
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Tags

Pollination RequirementsJapanese PlumPhenological MonitoringHand PollinationsFluorescence MicroscopyMolecular GenotypingSelf-incompatible CultivarsPollen ExtractionBBCH ScaleFruit SetSelf-pollinationCross-pollinationPollen GerminationFixative SolutionMicroscopy
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Guerrero, B. I., Guerra, M. E., Rodrigo, J. Establishing Pollination Requirements in Japanese Plum by Phenological Monitoring, Hand Pollinations, Fluorescence Microscopy and Molecular Genotyping. J. Vis. Exp. (165), e61897, doi:10.3791/61897 (2020).
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