This protocol provides a rapid method for determining pollen compatibility and incompatibility in citrus cultivars.
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.
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-fertilization1,2. 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 Solanaceae5,6,7,8.
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 ubiquitinzation10,11,12,13.
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 laboratory14,15, 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.
1. Preparation for aniline blue staining
2. Pollen collection
3. In vitro pollen germination test
4. Pollination
5. Sampling, fixation, and preservation
6. Aniline blue staining
7. Fluorescence microscopy
8. PCR-based S genotype identification
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.
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 apricot22,23. 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.
The authors have nothing to disclose.
This project was financially supported by the National Natural Science Foundation of China (32122075, 32072523).
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