Determination of Self-(In)compatibility and Inter-(In)compatibility Relationships in Citrus Using Manual Pollination, Microscopy, and S-Genotype Analyses

Summary

This protocol provides a rapid method for determining pollen compatibility and incompatibility in citrus cultivars.

Abstract

Citrus uses S-RNase-based self-incompatibility to reject self-pollen and, therefore, requires nearby pollinizer trees for successful pollination and fertilization. However, identifying suitable varieties to serve as pollinizers is a time-consuming process. To solve this problem, we have developed a rapid method for identifying pollination-compatible citrus cultivars that utilizes agarose gel electrophoresis and aniline blue staining. Pollen compatibility is determined based on the identification of S genotypes by extracting total DNA and performing PCR-based genotyping assays with specific primers. Additionally, styles are collected 3-4 days after manual pollination, and aniline blue staining is performed. Finally, the growth status of the pollen tubes is observed with a fluorescence microscope. Pollen compatibility and incompatibility can be established by observing whether the pollen tube growth is normal or suppressed, respectively. Due to its simplicity and cost-effectiveness, this method is an effective tool for determining the pollen compatibility and incompatibility of different citrus varieties to establish incompatibility groups and incompatibility relationships between different cultivars. This method provides information essential for the successful selection of suitable pollinizer trees and, thus, facilitates the establishment of new orchards and the selection of appropriate parents for breeding programs.

Introduction

Self-incompatibility (SI) is a genetically controlled mechanism present in approximately 40% of angiosperm species. In this process, the pistil rejects pollen from a plant with the same SI genotype and, thus, prevents self-fertilization^1,2. Ma jia pummelo is a local variety in Jinagsu province, China, with the excellent qualities of large, pink fruit, a rich juice content, a sweet and sour taste, and a thick peel³. Although SI promotes outcrossing, it negatively impacts the yield and quality of fruits⁴ and necessitates suitable pollinizer trees with distinct SI genotypes for reliable fruit-setting rates and high yields. At present, there are two main types of SI, sporophytic self-incompatibility (SSI), represented by Brassicaceae, and gametophytic self-incompatibility (GSI), represented by Rosaceae, Papaveraceae, Rutaceae, and Solanaceae^5,6,7,8.

Citrus is one of the most important fruit crops in the world. The S-RNase-based GSI system is found in many citrus accessions and negatively influences the fruit-setting rate⁹. In this system, SI is controlled by the S locus, a single polymorphic locus with two complex alleles that carry pistil S determinants and pollen S determinants⁷. The female determinant is the S ribonuclease (S-RNase), and the male determinant is the S locus F-box (SLF)⁷. The cells of the pistil secrete S-RNase proteins. The non-self S-RNases are recognized by the SLF proteins, which leads to the ubiquitination and degradation of the non-self S-RNases by the 26S proteasome pathway. In contrast, the self S-RNases are able to accumulate and inhibit pollen tube (PT) growth because they evade the SLF proteins and, therefore, are prevented from ubiquitinzation^10,11,12,13.

Here, we report an in vivo technique that is useful for identifying S-genotypes and degrees of pollen compatibility and incompatibility. The protocol involves extracting total DNA from leaves and predicting the S genotype using S-specific primers. Moreover, aniline blue staining and fluorescence microscopy followed by hand pollination provide evidence for the degree of compatibility and incompatibility. The semi in vivo pollination procedure, which involves the manual pollination of flowers in the laboratory^14,15, has also been adapted to assess the degrees of self-compatibility and incompatibility. However, we have also used field pollination followed by the bagging of flowers to avoid contamination from undesired pollen to allow the pollen tubes to develop in natural conditions. This protocol is simple and straightforward and provides the information necessary for the successful selection of suitable pollinizer trees.

Protocol

1. Preparation for aniline blue staining

1. Prepare the following reagents and tools for the experiment: a pollinator brush, tweezers, a pencil, sulfate paper, a pollination bag, zip lock bags, paper clips, formaldehyde, glacial acetic acid, absolute ethanol, centrifuge tubes, forceps, glue droppers, glass slides, coverslips, scalpels, and polyethylene glycol.
2. Prepare in vitro germination medium containing 0.02% MgSO₄, 0.01% KNO₃, 0.03% Ca(NO₃)₂, 0.01% H₃BO₃, 20% PEG-4000, and 15% sucrose. Adjust the pH to 6.0-6.2 with KOH. Use a magnetic stirrer as PEG-4,000 is difficult to dissolve.
3. Prepare Carnoy's fixative solution, which is absolute ethanol and glacial acetic acid mixed at a ratio of 3:1. Prepare FAA fixation solution, which is 40% formaldehyde, 80% ethanol, and glacial acetic acid (1:8:1). Prepare 4 M sodium hydroxide (NaOH) and aniline blue solution, which is 0.1% aniline blue in 0.1 M K₃PO_4. Use an amber bottle to store the aniline blue solution because it is light-sensitive.

2. Pollen collection

1. Know the flowering period of the experimental trees (Ma jia pummelo in this study) in advance. Collect mature unopened flowers from the beginning of the flowering period to the peak flowering stage, and put them in a zip lock bag. The flowers can be stored at 4 °C in the refrigerator for 24 h.
2. Take the flowers to the lab. Use tweezers to collect the anthers, and place them in a Petri dish containing filter paper. Collect anthers from 20 to 30 flowers.
3. Place the Petri dish containing the anthers in a 28 °C oven for 24 h until the pollen grains dry. For the detailed flower organization, see Hu et al.⁹.
4. Put the dried pollen into a 1.5 mL centrifuge tube. Save the pollen in an air-tight zip lock bag containing color-changing silica gel (desiccant). Close the bag, and label the bag with the name of the pollen variety and the date of storage. The dried pollen can be stored in a −20 °C refrigerator for 96 weeks¹⁶.

3. In vitro pollen germination test

1. Pour 300 µL of liquid medium into a cell culture dish or the cap of a 2 mL centrifuge tube, and sprinkle the pollen evenly with the help of a pollination brush. Incubate the pollen at 28 °C in a humidified dark environment for 12 h.
2. Remove the top 1 mm of the tip of a 1,000 µL pipette tip. Use the pipette to absorb the pollen with a small amount of culture solution and move it to the center of the microscope slide. Cover it with a coverslip. Observe the specimen with an inverted microscope using a 10x objective.
3. Perform three independent replicates using approximately the same pollen density for each replicate¹⁷. Visually manage the amount of pollen, making sure that whole of the Petri dish is covered with pollen and that each Petri dish has an almost equal amount of pollen.
4. The germinated pollen produces a pollen tube with a length approximately twice its diameter. Calculate the germination rate from 20 visual fields, which gives the percentage of germinated pollen in all pollen fields.

4. Pollination

1. Choose a sunny day without wind for pollination. Select 10 fully developed buds about to open, peel back the petals carefully, and take care not to bruise them.
2. Use a pollination brush to spread a sufficient amount of viable pollen onto the surface of the stigma, and take care not to damage the pistil. For self-pollination, use the pollen from the same plant/cultivar. For cross-pollination, use the pollen from a plant with a different genotype.
3. Cover the pollinated flowers with a sulfate paper bag. Use a paper clip to seal the bag and to prevent pollination by genotypically distinct pollens.
4. Write the name of the species and the number and time of pollination on the label. Hang the label on the branches near the pollinated flowers.

5. Sampling, fixation, and preservation

1. Remove the pollination bags approximately 3-4 days after pollination, and collect the pollinated flowers in zip lock bags.
2. Immediately remove the petals, receptacles, and ovaries from the flowers, and immerse the stigmas fused to styles in a centrifuge tube containing a freshly prepared fixative solution. Incubate the stigmas and styles in the fixative solution overnight at 4 °C.
3. The next day, discard the fixative solution, and wash the stigmas and styles two to three times in 95% ethanol.
4. Transfer the styles to a 70% ethanol solution. Ensure that the sample is completely immersed in the solution. The styles at this stage can be stored at 4 °C for 1-2 months.

6. Aniline blue staining

1. Wash the style samples stored in 70% ethanol with distilled water three to four times. Immerse in 4 M NaOH solution, seal, and incubate in a 65 °C water bath for 60 min. During this step, the color of the style changes from yellow-white to orange-red.
2. Soak the styles in distilled water for 30 min. Discard the distilled water, and wash the styles with distilled water three to four times or until the color of the style becomes yellow.
3. Place the sample in a 10 mL tube, add the aniline blue until the sample has been dipped, and dye for 12 h in the dark.
4. Observe the pollen tube growth with a fluorescence microscope.

7. Fluorescence microscopy

1. Before observing the specimens, place the slide on a flat table, and add two to three drops of polyethylene glycol to the surface of the slide.
2. Wash the style to be observed with distilled water. Use a scalpel to divide it into two halves along the longitudinal axis. Place one half of the style on the glass slide, and cover with a coverslip.
3. Place the slide on the stage of the microscope above the aperture, and visualize using a 10x objective. Use the DAPI filter (excitation: 325-375 nm; emission: 435-485 nm). Observe five styles for each type of pollination. Observe the pollen tube growth.

8. PCR-based S genotype identification

1. Extract the genomic DNA from the stigma sample using the CTAB method¹⁸.
   1. Put the collected leaves into a 2 mL centrifuge tube, and snap-freeze in liquid nitrogen. Prepare HCl:EDTA:NaCl:H₂O buffer at a ratio of 1:1:3:5 and a chloroform isoamyl alcohol mixture at a ratio of 24:1
   2. Add 10 mL of the prepared buffer, 0.2 g of CTAB, and 200 µL of mercaptoethanol to a 50 mL centrifuge tube, and put them in a water bath at 65 °C for 5 min until the solution becomes clear and transparent.
   3. Put the blades into a mortar, add the frozen samples, add liquid nitrogen, and grind. Put the ground sample into a 2 mL centrifuge tube, add 600 µL of CTAB mixture, put it in a water bath at 65 °C for 60 min, and mix it upside down every 30 min.
   4. Add 700 µL of the chloroform isoamyl alcohol mixture (24:1), and mix upside down for 10 min. Centrifuge at 24 °C at 12,000 x g for 10 min, pipette the supernatant, and transfer to a 1.5 mL centrifuge tube.
   5. Add 60 µL of 5 M NaCl solution and 1 mL of absolute ethanol, and mix upside down. Freeze at −20 °C for 30 min, and centrifuge at 24 °C and 9,000 x g for 5 min.
   6. Discard the supernatant, add 1 mL of 70% ethanol solution, and leave at room temperature for 1-2 h. Centrifuge at 24 °C, 9,000 x g for 5 min, discard the supernatant, aspirate the excess ethanol solution, and air-dry for 5 min.
   7. Add 100 µL of sterile water to dissolve, measure the DNA concentration with a spectrophotometer, and freeze in a -4 °C freezer for long-term storage.
   8. Configure the RT-PCR reaction system. Prepare the following reaction mix for 10 µL containing 5 µL of 2x PCRMix, 0.25 µL each of forward and reverse primer, 1 µL of DNA (100 ng/ µL), and 3.5 µL of H₂O.
2. Set up the PCR program as per Table 1. The PCR program for all isoforms was 95 °C for 5 min 32x (95 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min) and 72 °C for 5 min. Separate the products on 1.5% TAE-agarose gels and photograph⁹. Verify the specified S genotype using genomic DNA.

Representative Results

For the experiments done here, mature flowers were selected, the anthers were collected, dried in an oven, and the pollen was germinated at 28°C for 12 h. The pollen viability and germination rates were quantified as shown in Figure 1.

Citrus was manually pollinated, and the pollen compatibility and incompatibility were assessed using aniline blue staining and fluorescence microscopy. The compatible pollen could germinate on the surface of the stigma and produce a normal pollen tube that could grow and ultimately lead to fertilization in the ovary. In contrast, incompatible pollen tubes grew through approximately two-thirds of the style and then stopped growing (Figure 2).

To identify the S genotype, total DNA was extracted from the plant. Specific primers were designed based on the sequence of the S locus that were useful for amplifying part of the S locus in the PCR reactions. The amplification products were analyzed using agarose gel electrophoresis. The amplified bands were detected (between 500-1,000 bp). The corresponding S genotype was identified (Figure 3). By this method, we have identified the S genotypes of 63 pummelo germplasm resources⁷. Our group has identified 21 S-haplotypes in different citrus species using this method¹⁹ (Table 2).

Figure 1: Different rates of pollen germination. Germination and growth of the pollen. (A) The viable pollen has a higher germination rate, and a normal pollen tube can be grown. (B) The non-viable or less viable pollen has a much lower germination rate, and few pollen tubes can grow. Scale bars = 100 µm. Please click here to view a larger version of this figure.

Figure 2: Fluorescence microscopy images of pollen tubes in pistils after pollination. (A) Self-compatible pistil with numerous growing pollen tubes. (B) Self-incompatible pistil with pollen tube growth arrested within the style. Abbreviations: Pt = pollen tube; vb = vascular bundle. Scale bars = 1 mm. Please click here to view a larger version of this figure.

Figure 3: Specific amplification of the S-RNase gene from Ma jia pummelo. After PCR amplification and gel electrophoresis, it was found that the two amplified bands S₁₀ and S₁₆ were the brightest. These data indicate that the genotype of Ma jia pummelo was S₁₀ and S₁₆. Please click here to view a larger version of this figure.

Table 1: The reaction system used for the PCR-based S genotype identification. Please click here to download this Table.

Table 2: List of the primers for 21 S genotypes in citrus identified by our group. Please click here to download this Table.

Discussion

In fruit crops, both parthenocarpy and SI are important traits because they pave the way for seedless fruits - a trait that is highly appreciated by consumers. Self-incompatibility promotes the rejection of self-pollen and, thus, prevents inbreeding²⁰. Among citrus, pummelo is a self-incompatible variety⁷. Almost 40% of all angiosperm species exhibit SI²¹. This trait prevents fruit setting, lowers the yield, and brings huge economic losses to growers. To solve this problem, farmers include pollinizer trees throughout their orchards. However, the selection of suitable pollinizer trees is a challenging task that requires time-consuming laboratory experiments. To solve these issues, we have developed a rapid method for identifying SI genotypes and determining the pollen compatibilities and incompatibilities of different citrus varieties to facilitate the accurate selection of pollinizer trees. Moreover, the pollen viability and germination rates can also be determined using the in vitro method described in this protocol.

There are some reports on the determination of SI genotypes and self-(in)compatibility and inter-(in)compatibility using a combination of different methods in Japanese plum and apricot^22,23. The development of S-specific primers relies on the identification of the S genotype. In citrus, the transcriptome analysis of stigma and pollen from 64 pummelo accessions has identified nine S-RNases specifically expressed in the styles and one variant of the S-RNase. Another 12 pairs of S-specific primers were developed later in citrus by Liang et al.⁷ and Wei et al.¹⁹. However, relative to pear and apple, fewer S genotypes have been identified in citrus⁴. The identification of PCR-based S genotypes is a critical step, as it provides a basis for compatible/incompatible combinations. There are also some limitations to this protocol. The S genotypes of some citrus varieties cannot be identified using this method. This finding indicates that further expansion of the S genotype library in citrus is required. Additionally, the S-specific primers cannot distinguish the S genotypes of cultivars with highly similar S sequences and, thus, nonspecifically amplify similar S sequences.

Altogether, due to its cost-effectiveness and ease of use, this method is an effective tool for determining pollen compatibility and incompatibility for different citrus varieties. This protocol can be used for the selection of suitable pollinizer trees and in breeding research programs. It can be applied to several species from the Rutaceae family (e.g., Citrus trifoliata and Fortunella japonica) for the selection of pollinizer trees.

Materials

Name	Company	Catalog Number	Comments
absolute ethanol	Sinopharm Chemical ReagentCo., Ltd	10009218
Aniline blue	Sinopharm Chemical Reagent Co.,Ltd
Boric acid, H3BO3	Sinopharm Chemical ReagentCo., Ltd	10004818
Brown bottle	Labgic Technology Co., Ltd
Calcium nitrate tetrahydrate, Ca(NO3 )2	Sinopharm Chemical ReagentCo., Ltd	80029062
Centrifugal tube	Labgic Technology Co., Ltd
centrifuge tubes	Labgic Technology Co., Ltd
CTAB	GEN-VIEW SCIENTIFIC INC	57-09-0(CAS)
Dropping	Jiangsu Songchang Medical Equipment Co., Ltd
Ethylenediaminetetraacetic acid, EDTA	Sinopharm Chemical Reagent Co.,Ltd	10009617
Forceps	LUXIANZI Biotechnology Co., Ltd
formaldehyde	Sinopharm Chemical ReagentCo., Ltd	10010018
Fully automatic sample fast grinder	Shanghai Jingxin Industrial Development Co., Ltd	Tissuelyser-96
glacial acetic acid	Sinopharm Chemical ReagentCo., Ltd	10000218
Grinding Tube	Shanghai Jingxin Industrial Development Co., Ltd
Isoamyl alcohol	Sinopharm Chemical Reagent Co.,Ltd	10003218
Isopropyl alcohol	Sinopharm Chemical Reagent Co.,Ltd	80109218
label	M&G Chenguang Stationery Co., Ltd.
Leica DMi8	Shanghai Leica Co.,Ltd	21903797
Magnesium sulfate heptahydrate, MgSO4	Sinopharm Chemical ReagentCo., Ltd	10013018
MICROSCOPE Cover glass	Zhejiang Shitai Industrial Co., Ltd
NaCl	Sinopharm Chemical Reagent Co.,Ltd	10019318
paper clips	M&G Chenguang Stationery Co., Ltd.
pencil	M&G Chenguang Stationery Co., Ltd.
pollinator brush	Shanghai Yimei Plastics Co., Ltd
Polyethylene glycol, PEG 6000	Beijing Dingguo Changsheng Biotechnology Co., Ltd	DH229-1
Polyethylene glycol, PEG-4000	Guangzhou saiguo biotech Co., Ltd	1521GR500
Potassium hydroxide, KOH	Sinopharm Chemical ReagentCo., Ltd	10017008
Potassium nitrate, KNO3	Sinopharm Chemical ReagentCo., Ltd	10017218
Scalpel	Jiangsu Songchang Medical Equipment Co., Ltd
Slide	Zhejiang Shitai Industrial Co., Ltd
Sodium hydroxide, NAOH	Sinopharm Chemical Reagent Co.,Ltd	10019718
Sucrose	Sinopharm Chemical ReagentCo., Ltd	10021418
sulfate paper	Taizhou Jinnong Mesh Factory
Thermostat water bath	Shanghai Jinghong Experimental Equipment Co., Ltd	L-909193
Trichloromethane	Sinopharm Chemical Reagent Co.,Ltd	10006818
Tripotassium phosphate tribasic trihydrate, K3PO4	Shanghai Lingfeng Chemical Reagent Co.,Ltd	20032318
Tris-HCl	GEN-VIEW SCIENTIFIC INC	1185-53-1
zip lock bags	M&G Chenguang Stationery Co., Ltd.
β-Mercaptoethanol	GEN-VIEW SCIENTIFIC INC	60-24-2(CAS)