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Summary

We present a methodology to establish the pollination requirements of apricot (Prunus armeniaca L.) cultivars combining the determination of self-(in)compatibility by fluorescence microscopy with the identification of the S-genotype by PCR analysis.

Abstract

Self-incompatibility in Rosaceae is determined by a Gametophytic Self-Incompatibility System (GSI) that is mainly controlled by the multiallelic locus S. In apricot, the determination of self- and inter-(in)compatibility relationships is increasingly important, since the release of an important number of new cultivars has resulted in the increase of cultivars with unknown pollination requirements. Here, we describe a methodology that combines the determination of self-(in)compatibility by hand-pollinations and microscopy with the identification of the S-genotype by PCR analysis. For self-(in)compatibility determination, flowers at balloon stage from each cultivar were collected in the field, hand-pollinated in the laboratory, fixed, and stained with aniline blue for the observation of pollen tube behavior under the fluorescence microscopy. For the establishment of incompatibility relationships between cultivars, DNA from each cultivar was extracted from young leaves and S-alleles were identified by PCR. This approach allows establishing incompatibility groups and elucidate incompatibility relationships between cultivars, which provides a valuable information to choose suitable pollinizers in the design of new orchards and to select appropriate parents in breeding programs.

Introduction

Self-incompatibility is a strategy of flowering plants to prevent self-pollination and promote outcrossing1. In Rosaceae, this mechanism is determined by a Gametophytic Self-Incompatibility System (GSI) that is mainly controlled by the multiallelic locus S2. In the style, the RNase gene encodes the S-stylar determinant, a RNase3, while a F-box protein, which determines the S-pollen determinant, is codified by the SFB gene4. The self-incompatibility interaction takes place through the inhibition of pollen tube growth along the style preventing the fertilization of the ovule5,6.

In apricot, a varietal renewal has taken place worldwide in the last two decades7,8. This introduction of an important number of new cultivars, from different public and private breeding programs, has resulted in the increase of apricot cultivars with unknown pollination requirements8.

Different methodologies have been used to determine pollination requirements in apricot. In the field, self-(in)compatibility may be established by controlled pollinations in caged trees or in emasculated flowers and subsequently recording the percentage of fruit set9,10,11,12. In addition, controlled pollinations have been carried out in the laboratory by semi-in vivo culture of flowers and analysis of the pollen tube behavior under fluorescence microscopy8,13,14,15,16,17. Recently, molecular techniques, such as PCR analysis and sequencing, have allowed the characterization of incompatibility relationships based on the study of the RNase and SFB genes18,19. In apricot, thirty-three S-alleles have been reported (S1 to S20, S22 to S30, S52, S53, Sv, Sx), including one allele related with self-compatibility (Sc)12,18,20,21,22,23,24. Up to now, 26 incompatibility groups have been stablished in this species according to the S-genotype8,9,17,25,26,27. Cultivars with the same S-alleles are inter-incompatible, whereas cultivars with at least one different S-allele and, consequently, allocated in different incompatible groups, are inter-compatible.

To define the pollination requirements of apricot cultivars, we describe a methodology that combines the determination of self-(in)compatibility by fluorescence microscopy with the identification of the S-genotype by PCR analysis in apricot cultivars. This approach allows establishing incompatibility groups and elucidate incompatibility relationships between cultivars.

Protocol

1. Self-(in)compatibility determination

  1. Sample the flowers in the field. It is necessary to collect the flowers at balloon stage (Figure 1A), corresponding to stage 58 on the BBCH scale for apricot28, to avoid unwanted previous pollination.
  2. Self- and cross-pollinations in the laboratory
    1. Remove the anthers of the flowers at balloon stage and place them on a piece of paper to dry at laboratory temperature.
    2. After 24 h, sieve the pollen grains by using a fine mesh (0.26 mm) (Figure 1B).
    3. Emasculate a group of 30 flowers at the same balloon stage for each self-pollination and cross-pollination and place the pistils on florist foam in water at laboratory temperature (Figure 1C).
    4. Hand pollinate the pistils with the help of a paintbrush with pollen from flowers of the same cultivar 24 h after emasculation. In addition, pollinate another set of pistils of each cultivar with pollen from flowers of a compatible pollinizer as control (Figure 1D).
    5. After 72 h, fix the pistils in a fixative solution of ethanol/acetic acid (3:1) for at least 24 h at 4 °C29. Then discard the fixative and add 75% ethanol ensuring that the samples are completely submerged in the solution. Samples can be conserved in this solution at 4 °C until use8,17,30,31,32.
  3. Evaluating pollen viability through in vitro pollen germination
    1. To prepare the germination medium, weight 25 g of sucrose, 0.075 g of boric acid (H3BO3) and 0.075 g of calcium nitrate (Ca(NO3)2)33.
    2. Add the components of the medium in 250 mL of distilled water and dissolve completely.
    3. Solidify the medium adding 2 g of agarose and mix by swirling.
    4. Check the pH of the medium using a pH meter and adjust the value to 7.0 with NaOH or HCl solution.
    5. Autoclave the mixture to sterilize the medium.
    6. After autoclaving, cool down the medium and distribute it into Petri dishes in a sterile laminar flow hood.
    7. Scatter the pollen grains of the same cultivars used for the controlled pollinations in the solidified pollen germination medium and observe them under the microscope after 24 h6.
      NOTE: To sterilize the laminar flow hood, clean the surface with 70% ethanol and switch on the UV lamp during 10 min.
    8. Store the Petri dishes in a refrigerator at 4 °C until use.
  4. Microscopy observations
    1. Wash the pistils three times for 1 h with distilled water and leave them in 5% sodium sulphite at 4 °C. After 24 h, autoclave them at 1 kg/cm2 during 10 min in sodium sulphite to soften the tissues34.
    2. Place the autoclaved pistils over a glass slide and, with the help of a scalpel, remove the trichomes around the ovary to get a better visualization of the pollen tubes. Then, squash the pistils with a cover glass.
    3. Prepare 0.1% (v/v) aniline blue stain: mix 0.1 mL of aniline blue in 100 mL of 0.1 N potassium phosphate tribasic (K3PO4). Apply a drop of aniline blue over the preparations to stain callose depositions during pollen tube growth.
    4. Observe the pollen tubes along the style by a microscope with UV epifluorescence using 340-380 bandpass and 425 longpass filters.

2. DNA extraction

  1. Sample 2-3 leaves in the field in spring. It is recommended to sample the leaves at young stages since DNA obtained is of higher quality and lower levels of phenolic compounds compared to old leaves.
  2. Extract Genomic DNA following the steps described in a commercially available kit (see Table of Materials).
  3. Analyze the quantity and quality of DNA concentrations using UV-vis spectrophotometer (260 nm).

3. S-allele identification

  1. Setting up of the PCR Reactions
    1. Prepare a 50 ng/μL dilution in distilled water of each DNA extraction sample.
    2. Thaw out the PCR reagents slowly and keep them on ice. Leave the DNA polymerase in the freezer until needed.
    3. Prepare the amplification reactions using the different combinations of primers. Create the PCR reaction mix by combining the components in Table 1. Vortex the PCR reaction mix well and distribute the volume indicated for the different combinations of primers to each well of the PCR plate. Then, add 1 μL of the DNA dilution in each well.
    4. Place the PCR plate in the thermocycler and run the corresponding PCR program shown in Table 1.
  2. Analyze the amplified fragments. There are mainly two different ways to analyze the PCR amplified fragments: capillary electrophoresis (CE) with fluorescent-labelled primers or as visualize amplicons of agarose gel electrophoresis with not-labelled primers.
    1. Capillary Electrophoresis
      1. To prepare the loading buffer, mix 35 μL of deionized formamide with 0.45 μL of labeled sizing standard. Vortex the reagent to mix well, and then dispense 35.5 μL into the well of the reader plate.
      2. Add 1 μL of the PCR product into the well. In addition, add a drop of mineral oil to prevent water evaporation.
      3. Prepare the separation plate adding separation buffer.
      4. Use the commercial software included with the gene analyzer (see Table of Materials). Create a new sample plate and save the sample names for all wells on the plate.
      5. Select the method of analysis. In this case, denature the samples at 90 °C for 120 s, inject at 2.0 kV for 30 s, and separate at 6.0 kV for 35 min.
      6. Insert the two plates into the gene analyzer. Fill the capillary array with distilled water.
      7. Load the patented linear polyacrylamide (LPA) gel. Finally, click Run.
    2. Gel Electrophoresis
      1. Prepare a 1% agarose gel adding 1.5 g of molecular biology grade agarose in 150 mL of 1x TAE (Tris-acetate-EDTA) electrophoresis running buffer (40 mM Tris, 20 mM acetic acid, and 1 mM EDTA at pH 8.0). Dissolve the agarose by microwave heating for 2-3 min.
      2. To visualize the DNA, add 4 μL of a nucleic acid stain (see Table of Materials) and mix gently.
      3. Add a gel comb, with sufficient wells for ladders, controls and samples, into a gel tray. Then, pour slowly the mix into the middle of the gel tray and avoid bubbles.
      4. Let the gel cool down for 30-45 min at room temperature until the gel has completely solidified. Introduce the gel in the electrophoresis chamber, remove the gel comb and fill the chamber with enough 1x TAE buffer to cover the gel.
        NOTE: Check the placement of the gel. The wells should be placed close to the negative pole since negatively charged DNA migrates towards the cathode.
      5. Add 5 μL of loading buffer (0.1% (v/v) bromophenol blue) to the PCR products and mix well.
      6. To estimate the size of the bands, load 5 μL of DNA molecular weight ladder (see Table of Materials).
      7. Load the samples into the additional wells of the gel.
      8. Once all the samples and the DNA molecular weight ladder are loaded, run the gel at 90 V for 1-1.5 h, until the blue dye line is approximately at 75% the length of the gel.
      9. Visualize the bands in a transilluminator for nucleic acids.

Results

Pollination studies in apricot require the use of flowers at the late balloon stage one day before anthesis (Figure 1A). This stage is considered the most favorable for both pollen and pistil collection, since floral structures are nearly mature, but anther dehiscence has not yet occurred. This prevents the interference of undesired pollen, not only of pollen from the same flower but also from other flowers, since the closed petals impede the arrival of insects carrying exte...

Discussion

Traditionally, most commercial apricot European cultivars were self-compatible36. Nevertheless, the use of North American self-incompatible cultivars as parents in breeding programs in the last decades has resulted in the release of an increasing number of new self-incompatible cultivars with unknown pollination requirements7,8,37. Thus, the determination of self- and inter-(in)compatibility relationships...

Disclosures

The authors have nothing to disclose.

Acknowledgements

This research was funded by Ministerio de Ciencia, Innovación y Universidades-European Regional Development Fund, European Union (AGL2016-77267-R, and AGL2015-74071-JIN); Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (RFP2015-00015-00, RTA2017-00003-00); Gobierno de Aragón-European Social Fund, European Union (Grupo Consolidado A12_17R), Fundación Biodiversidad, and Agroseguro S.A.

Materials

NameCompanyCatalog NumberComments
Agarose D1 Low EEOConda8010.22
BIOTAQ DNA Polymerase kitBiolineBIO-21060
Bright field microscopeLeica MicrosystemsDM2500
CEQ System SoftwareBeckman Coulter
DNeasy Plant Mini KitQIAGEN69106
dNTP Set, 4 x 25 µmolBiolineBIO-39025
GenomeLab DNA Size Standard Kit - 400Beckman Coulter608098
GenomeLab GeXP Genetic Analysis SystemBeckman Coulter
GenomeLab Separation BufferBeckman Coulter608012
GenomeLab Separation Gel LPA-1Beckman Coulter391438
HyperLadder 100bpBiolineBIO-33029
HyperLadder 1kbBiolineBIO-33025
Image Analysis SystemLeica Microsystems
Molecular Imager VersaDoc MP 4000 system Bio-Rad170-8640
NanoDrop One SpectrophotometerThermo Fisher Scientific13-400-518
pH-Meter BASIC 20Crison
Phusion High-Fidelity PCR KitThermo Fisher ScientificF553S
Power Pack P 25 TBiometra
Primer ForwardIsogen Life Science
Primer ReverseIsogen Life Science
Quantity One SoftwareBio-Rad
Stereoscopic microscopeLeica MicrosystemsMZ-16
Sub-Cell GTBio-Rad
SYBR Safe DNA Gel StainThermo Fisher ScientificS33102
T100 Thermal CyclerBio-Rad1861096
Taq DNA PolymeraseQIAGEN201203
Vertical Stand AutoclaveJP Selecta

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