Name: establishing pollination requirements in japanese plum by phenological monitoring, hand pollinations, fluorescence microscopy and molecular genotyping
Uploaded: 2020-11-09T15:30:00
Description: Watch this Scientific Journal Video about Establishing Pollination Requirements in Japanese Plum by Phenological Monitoring, Hand Pollinations, Fluorescence Microscopy and Molecular Genotyping at JoVE

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。

1. 田间手工授粉
<ol><li>
花粉提取
	<ol>
<li>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。</li>
		<li>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。</li>
		<li>通过?...

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?...

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。

<table>
	<tbody>
		<tr>
			<td>Acetic Acid Glacial</td>
			<td>Panreac</td>
			<td>131008.1611</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>iNtRON Biotechnology</td>
			<td>25999</td>
			<td></td>
		</tr>
		<tr>
			<td>Aniline blue</td>
			<td>Difco</td>
			<td>8504-88</td>
			<td></td>
		</tr>
		<tr>
			<td>Boric Acid (H3BO4)</td>
			<td>Panreac</td>
			<td>131015.1210</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Nitrate 4-hydrate (Ca(NO3)2&#183;4H2O)</td>
			<td>Panreac</td>
			<td>131231.1211</td>
			<td></td>
		</tr>
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			<td>Coverglass</td>
			<td>Deltalab</td>
			<td>D102460</td>
			<td>24 mm x 60 mm</td>
		</tr>
		<tr>
			<td>Digital Camera</td>
			<td>Imaging Developmet Systems</td>
			<td>UI-1490SE</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital Camera Software Suite</td>
			<td>Imaging Developmet Systems</td>
			<td>4.93.0.</td>
			<td></td>
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			<td>DNA Oligos</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td></td>
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			<td>dNTP Mix, 10 mM each</td>
			<td>ThermoSischer Scientific</td>
			<td>R0193</td>
			<td></td>
		</tr>
		<tr>
			<td>DreamTaq Green DNA polymerase</td>
			<td>ThermoFisher Scientific</td>
			<td>EP0713</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 96&#176;</td>
			<td>VWR-Chemicals</td>
			<td>83804.360</td>
			<td></td>
		</tr>
		<tr>
			<td>1Kb DNA Ladder (U.S. Patent No. 4.403.036) (500pb-12Kb)</td>
			<td>Invitrogen</td>
			<td>15615-016</td>
			<td>Size: 250&#181;g; Conc: 1.0 &#181;g/&#181;l</td>
		</tr>
		<tr>
			<td>Gel Documentation System</td>
			<td>Bio-Rad</td>
			<td>1708195</td>
			<td></td>
		</tr>
		<tr>
			<td>Hand Counter</td>
			<td>Tamaco</td>
			<td>TM-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Image Lab Software</td>
			<td>Bio-Rad</td>
			<td></td>
			<td>Image Analyse System for Gel Documentation System</td>
		</tr>
		<tr>
			<td>MetaPhor Agarose</td>
			<td>Lonza</td>
			<td>50180</td>
			<td></td>
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		<tr>
			<td>Microcentrifuge 5415 R</td>
			<td>Eppendorf</td>
			<td>Z605212</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope with UV epiflurescence</td>
			<td>Leica</td>
			<td>DM2500</td>
			<td>Exciter filter BP340-390, Barrier filter LP425</td>
		</tr>
		<tr>
			<td>Microslides</td>
			<td>Deltalab</td>
			<td>D100004</td>
			<td>26 mm x 76 mm</td>
		</tr>
		<tr>
			<td>Mini Electrophoresis System</td>
			<td>Fisherbrand</td>
			<td>14955170</td>
			<td></td>
		</tr>
		<tr>
			<td>Minicentrifuge</td>
			<td>ThermoFisher Scientific</td>
			<td>15334204</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 1000 Spectrophotometer</td>
			<td>ThermoFisher Scientific</td>
			<td>ND1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishes</td>
			<td>Deltalab</td>
			<td>200201</td>
			<td>55 mm x 14 mm</td>
		</tr>
		<tr>
			<td>Potassium Phosphate Tribasic (K3PO4&#183;1.5H2O)</td>
			<td>Panreac</td>
			<td>141513</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer forward &#39;Pru C2&#39;</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Primer forward Pru T2&#39;</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Primer reverse &#39;PCER&#39;</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RedSafe Nucleic Acid Staining Solution</td>
			<td>iNtRON Biotechnology</td>
			<td>21141</td>
			<td></td>
		</tr>
		<tr>
			<td>Saccharose</td>
			<td>Panreac</td>
			<td>131621.1211</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium sulphite anhydrous (Na2SO3)</td>
			<td>Panreac</td>
			<td>131717.1211</td>
			<td></td>
		</tr>
		<tr>
			<td>Speedtools plant DNA extraction Kit</td>
			<td>Biotools</td>
			<td>21272</td>
			<td></td>
		</tr>
		<tr>
			<td>TBE Buffer (10X)</td>
			<td>Panreac</td>
			<td>A0972,5000PE</td>
			<td></td>
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			<td>Thermal Cycler T100</td>
			<td>Bio-Rad</td>
			<td>1861096</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermomixer comfort</td>
			<td>Eppendorf</td>
			<td>T1317</td>
			<td></td>
		</tr>
		<tr>
			<td>Vertical Autoclave Presoclave II</td>
			<td>JP Selecta</td>
			<td>4001725</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Fisherbrand</td>
			<td>11746744</td>
			<td></td>
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establishing pollination requirements in japanese plum by phenological monitoring, hand pollinations, fluorescence microscopy and molecular genotyping

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。

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...

Watch this Scientific Journal Video about Establishing Pollination Requirements in Japanese Plum by Phenological Monitoring, Hand Pollinations, Fluorescence Microscopy and Molecular Genotyping at JoVE

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

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。

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. ...

This protocol is becoming increasingly important due to the high number of new self-incompatible Japanese plum cultivars with unknown pollination requirements. The main advantage of this technique is that it allows for the determination of the pollination requirements of cultivars by combining two complimentary approaches in field and laboratory conditions. This approach can also be applied to determine pollination requirements and other self-incompatible fruit tree species like apricot or sweet cherry.For pollen extraction, collect flower buds at balloon stage according to stage 57 on the BBCH scale. Remove the anthers using a plastic mesh and place them on paper at room temperature for 24 hours until anther dehiscence. Sieve the pollen grains through a fine mesh and conserve them in a 10 millimeter glass tube with a cap at four degrees Celsius until use.When 10 to 20%of the flowers are open, select and label several branches, leaving 1, 000 to 1, 500 flowers per pollination treatment. On the next day, when more flowers are open, pollinate each flower using a paintbrush with pollen from the same cultivar for self-pollination and pollen from other cultivars as cross-pollination control. Pollinate every day until all flowers are open.Record weekly counts of flowers and developing fruits from anthesis to harvest to characterize the fruit drop pattern and quantify the final fruit set in each pollination treatment. Collect 50 to 100 flowers at balloon stage according to stage 57 on the BBCH scale. Emasculate 30 flowers per treatment in the laboratory for self and cross-pollination.Make a fresh cut on the base of each flower pedicle under water and place it on a piece of wet florist foam. Use one piece of foam for each pollination treatment. After 24 hours, hand pollinate each pistil with the pollen collected previously using a fine paintbrush.Pollinate one set of pistils with pollen from the same cultivar and the other set with pollen from another compatible cultivar as control. Fix these pollinated pistils in a fixative solution of ethanol and acetic acid for at least 24 hours at four degrees Celsius. Spread the pollen of each cultivar previously used as a pollen donor in the pollinations in two Petri dishes and incubate them at 25 degrees Celsius for 24 hours.To observe the pollen grains, add two to three drops of aniline blue solution to each Petri dish and observe them under a UV epifluorescence microscope after five minutes. Count viable and non-viable pollen grains in the three fields per Petri dish with each field containing about 100 to 200 pollen grains in two Petri dishes for each cultivar. To observe the pollen tubes, squash softened pistils in a drop of 1%aniline blue solution under a cover glass on a slide.Observe the pollen tube growth along the style under a microscope with UV epifluorescence using exciter filter BP340-390 and barrier filter LP425. Collect three to four young leaves from each cultivar in the field and extract DNA from the leaves. Set up a master mix for each pair of primers in 1.5 milliliter micro tubes.Add all reagents and mix thoroughly. Aliquot 16 microliters of this master mix into each 200 microliter PCR tube containing four microliters of DNA template or control. Mix them gently, close the caps and centrifuge at 2, 000 times G for 30 seconds.Place the tube in the thermocycler and run the PCR program as mentioned in the text manuscript. For estimating the fragment size by gel electrophoresis, load the first and last wells with two microliters of the DNA ladder and three microliters of the PCR product in the other wells. Close the chamber and run the gel at 100 volts for 30 minutes.Observe the gel under UV light using a gel imaging system. Use the DNA ladder to estimate the size of the amplified fragments and compare it with the positive and negative controls. Monitor the phenology of different trees of each cultivar over different years of flowering.Establish the length of the flowering period from the first to the last open flowers. Full bloom is considered when at least 50%of flowers are at stage F according to stage 65 on the BBCH. The pattern of fruit drop of hand pollinated flowers after emasculation or supplementary pollination showed that most flowers dropped two to three weeks after pollination, but 4%of self-pollinated flowers remained in the tree, indicating that this cultivar is self-compatible.Viable pollen grains showed a pollen tube longer than its length after 24 hours in the culture medium and non-viable pollen grains did not germinate. During pollination, pollen grains were transferred to the stigma. Each germinating pollen grain produced a pollen tube, which grew through the pistil structures.In self-incompatible cultivars, the pollen tube stopped growing in the upper third of the style. However, in self-compatible cultivars, the pollen tube could grow through the style, reach the ovary and fertilize an ovule. PCR analysis was carried out using primers from the conserved regions of sRNAs of sweet cherry and Japanese plum, making it possible to determine the size of S-alleles in each cultivar.The length of the flowering period for a total of four years was calculated. Flowering in orchard conditions allowed the determination of which cultivars are coincident at flowering time each year. When attempting this protocol, make sure to use a higher number of flowers of Japanese plum for hand pollinations since the percentage of fruit set is very low compared to other Prunus species.It is not possible to determine self-incompatibility by molecular approaches. Further work of S-allele sequencing is required to assess if some S-alleles are associated with self-compatibility. The combination of field and laboratory approaches has resulted in valuable information for the design of new commercial orchards and can be used in other fruit tree species.

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