Determination of Self- and Inter-(in)compatibility Relationships in Apricot Combining Hand-Pollination, Microscopy and Genetic Analyses

This method can help determine pollination requirements in different species and establish incompatibility relationships between cultivars. The main advantage of this approach is that it allows evaluating several incompatibility under laboratory conditions. Avoiding the numerous problems that can occur in experiments performed under field conditions.

This allows identification along with dedication of a number of cultivar to the corresponding incompatibility groups. Determining the inter-compatibility relationship between cultivars. This approach can also be useful to determine self-incompatibility and incompatibility relationship in other fruit tree crops like cherry or plum.

Begin by sampling the flowers in the field. Collect the flowers at balloon stage, which corresponds to Stage 58 on the BBCH scale for apricot to avoid previous pollination. In the laboratory, remove the anthers of the flowers and place them on a piece of paper to dry at room temperature.

After 24 hours, sieve the pollen grains with a fine 26 millimeter mesh. Emasculate a group of 30 flowers at the same balloon developmental stage for each self-and cross-pollination and place the pistols on florist foam in water 24 hours after emasculation and pollinate the pistols using a paint brush with pollen from flowers of the same cultivar. Pollinate another set of pistols of each cultivar with pollen from flowers of a compatible pollinizer as a control.

After 72 hours, remove the pistols from the wet florist foam and fix the pistols in a fixative solution of 3:1 ethanol to acetic acid for at least 24 hours at 4 degrees Celsius. Then discard the fixative and add 75%ethanol, making sure that samples are completely submerged in the solution. Samples can be stored in the ethanol at 4 degrees Celsius until use.

Scatter the pollen grains of the same cultivars used for the control pollinations in solidified pollen germination medium and observe them under the microscope after 24 hours. Prepare the pistols for microscopy by washing them 3 times with distilled water for one hour per wash. After the last wash, leave them in 5%sodium sulfite at 4 degrees Celsius for 24 hours.

Then autoclave them at one kilogram per centimeter squared for 10 minutes in sodium sulfite to soften the tissue. Place the autoclaved pistols over a glass slide and use a scalpel to remove the trichomes around the ovary. Then squash the pistol with a coverglass.

Apply a drop of aniline blue over the preparations to stain callous depositions during pollen tube growth and observe the pollen tubes along the style with a microscope according to manuscript directions. Use a commercial kit to extract genomic DNA from young leaves collected in the spring. Then set up PCR by combining reagents according to manuscript directions.

Vortexing the reaction mix and distributing it among the wells in the PCR plate. Add 1 microliter of the DNA dilution into each well and run PCR using the thermo-cycling program outlined in the manuscript. Once the reaction is complete, analyze the results using capillary electrophoresis or agarose gel electrophoresis.

For capillary electrophoresis add 1 microliter of the PCR product into the well of the reader plate along with 1 drop of mineral oil to prevent water evaporation. Then prepare the separation plate by adding separation buffer. Use the commercial software for the gene analyzer to create a new sample plate and save the sample names for all wells on the plate.

Then select the method of analysis and insert the two plates into the gene analyzer. Fill the capillary array with distilled water. Load the linear polyacrylamide gel and click run.

If analyzing the PCR results with gel electrophoresis, prepare a 1%agarose gel by dissolving agarose in electrophoresis running buffer according to manuscript directions. Then add 4 microliters of nucleic acid stain to the gel and mix gently. Set up a gel tray with a comb and slowing pour the gel in the middle taking care to avoid bubbles.

Leave the gel at room temperature until completely solidified, about 30-45 minutes. Then put into the electrophoresis chamber. Remove the comb and cover it with 1x TAE buffer.

Load the gel with the DNA ladder followed by the PCR products mixed with loading dye. Then run the gel at 90 volts for 60-90 minutes until the blue dye line is at approximately 75%of the gel lane. Once the run is complete, visualize the gel using a transilluminator.

Pollen tube growth in self-and cross-pollinations was observed by fluorescence microscopy to determine self(in)compatibility for each cultivar. Cultivars were considered at self-incompatible when pollen tube growth was arrested and self-compatible when at least one pollen tube reached the base of the style. 5 primers pair combinations were used to identify S-alleles using PCR in combination with capillary or gel electrophoresis.

First, the S-alleles were identified by the amplification of the first S-RNase intron using the SRc primer pair. Then the second intron of the RNase was amplified with three primer sets to distinguish S6, S9, S1, and S7 alleles. And the V2 and HVB variable regions of the SFB gene were amplified with the AprFBC8 primer set to identify the Sc and S8 alleles.

Once the S-alleles had been identified, different incompatibility groups with the genotypes that are inter-incompatible can be established. It's important to remember to use control in capillary electrophoresis, since they use an automatic fragment analysis system that can report as more differences in fragment size causing inaccurate allele identification. The combination of microscopy observations and genetic analysis has resulted a very useful approach to determine self-incompatibility in apricot and establish the incompatibility relationships between cultivars.

Self-incompatibility is now the link to locals in apricot. The determination of factors seem to be involved. Future effort has to be focused on the genetic edification of this cultivar in apricot.

The combination of this documentary approach is result in valuable information for the appropriate selection of cultivars in commercial orchards and pattern genotypes in apricot breeding programs. A similar approach can be used in other woody perennial crops.