Method for overcoming distant hybridization incompatibility of plants in the family rutaceae by using as-sjgfer1 and application

Abstract

The application discloses a method for overcoming distant hybridization incompatibility of Rutaceae by using AS-sjgFER1 and application thereof. The application adopts antisense oligonucleotide technology, and AS-sjgFER1 reagent is added dropwise to the stigma of Rutaceae, so that the distant hybridization incompatibility of Citrus morrisonicola and Chinese wax tree is overcome. The application has little harm to plants, has remarkable effect in overcoming distant hybridization incompatibility, has wide application range, and can be further used for overcoming distant hybridization incompatibility of other plants of Rutaceae, and has important value for breeding and seed production of Rutaceae.

Description

Technical Field

[0001] This invention relates to the field of Rutaceae plant breeding technology, specifically to a method and application of using AS-sjgFER1 to overcome incompatibility in distant hybridization of Rutaceae plants. Background Technology

[0002] Distant hybridization refers to hybridization between different species, genera, or more distantly related species in plant classification. Through distant hybridization, characteristics and traits of different species and genera can be combined, expanding genetic variation and creating new types of variations or even new species. However, for distantly related plants to hybridize, reproductive barriers before and after fertilization must be overcome. Prozygotic barriers refer to the complete inability of fertilization to occur after pollination of two fertile species, i.e., incompatibility exists. These barriers include two manifestations: pollen failing to germinate on the stigma or germinating abnormally, and pollen germinating normally but the pollen tube failing to grow properly in the style.

[0003] The genus *Citrus* in the Rutaceae family has undergone numerous intrageneric hybridizations during evolution, and most cultivated *Citrus* species are hybrids of two or more protozoan species. However, among Rutaceae species outside the *Citrus* genus, such as *Lampee* and *Murraya paniculata*, there are currently no reports of hybridization between them and other species, including those in the *Citrus* genus.

[0004] Citrus fruits, belonging to the Rutaceae family, are important fruit trees worldwide, holding a significant position in the national economy and possessing immense economic value. The citrus Huanglongbing (HLB) pathogen primarily infects Rutaceae plants, with cultivated Citrus, Citrus aurantium, and Citrus kumquat species serving as its hosts. Outside the Citrus genera, species like Wampee and Murraya paniculata exhibit strong resistance to HLB. Overcoming the barriers of distant hybridization and introducing HLB resistance into Citrus species would have significant research and economic value in helping citrus cultivation overcome the challenges posed by HLB.

[0005] Incompatibility between distant pollen and stigma is the first obstacle to distant hybridization, which has greatly limited the progress of distant hybridization breeding. Therefore, exploring efficient and widely applicable methods to overcome incompatibility in distant hybridization is an important part of distant hybridization breeding in the Rutaceae family. Summary of the Invention

[0006] To address the deficiencies and shortcomings of the existing technology, this invention proposes a reagent and method to overcome incompatibility in distant hybridization of Rutaceae plants. The reagent can overcome incompatibility in distant hybridization, is simple and convenient to operate, and is easy to promote.

[0007] To achieve the objectives of this invention, the technical solution adopted is as follows: A reagent that overcomes incompatibility in distant hybridization of Rutaceae plants using AS-sjgFER1, wherein the main component of the reagent is AS-sjgFER1, wherein AS-sjgFER1 is an antisense oligonucleotide AS-sjgFER1 targeting sjgFER1, and wherein AS-sjgFER1 has the base sequence shown in SEQ ID NO:1.

[0008] Furthermore, the reagent also contains Tween-20.

[0009] Furthermore, the concentration of AS-sjgFER1 in the reagent is 30~50 μmol / L.

[0010] Furthermore, the distant hybridization includes interspecific distant hybridization and intergeneric distant hybridization.

[0011] Furthermore, the volume percentage of Tween-20 in the reagent is 0.02%.

[0012] Another object of the present invention is to provide a method for overcoming incompatibility in distant hybridization of Rutaceae plants using the aforementioned reagent, the method comprising: During the initial flowering period of Rutaceae plants, select unopened large buds, apply the aforementioned reagent to their stigmas, and then proceed with pollination.

[0013] Furthermore, the reagent was applied twice to the stigma of the unopened large bud stage flowers.

[0014] Furthermore, the interval between the number of reagent drops is 30 minutes.

[0015] Furthermore, the amount added each time was 2.5 μL per column head.

[0016] Furthermore, the present invention also provides the application of the reagent in overcoming incompatibility in distant hybridization of Rutaceae plants.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention is applicable to distant hybridization incompatibility breeding of Rutaceae plants. By utilizing AS-sjgFER1 to suppress the expression of the FER1 gene in the stigma of Rutaceae plants during flowering, it effectively overcomes the intergeneric hybridization incompatibility barrier between Citrus aurantium and Cinnamomum camphora, promoting the germination of Cinnamomum camphora pollen on the stigma of Citrus aurantium during flowering, allowing the pollen to grow into the stigma of Citrus aurantium and complete the fertilization process. The method described in this invention is simple and convenient to operate, easy to promote, has minimal damage to plants, significantly overcomes distant hybridization incompatibility, and has a wide range of applications, including Citrus aurantium and other Citrus species, as well as Cinnamomum camphora. Furthermore, this method can be further used to overcome distant hybridization incompatibility in other Rutaceae plants, and has significant value for the breeding and seed production of Rutaceae plants. Attached Figure Description

[0018] The disclosure of this invention will become more readily understood with reference to the accompanying drawings. It will be readily understood by those skilled in the art that these drawings are merely illustrative of the technical solutions of this invention and are not intended to limit the scope of protection of this invention.

[0019] Figure 1 Four days after treating the stigmas of Citrus aurantiacus with AS-sjgFER1 and pollinating with yellow-skinned flowers, the results of aniline blue staining of the stigmas of Citrus aurantiacus were obtained.

[0020] Figure 2 The stigmas of the four-season orange tree, which were not treated with reagent, were stained with aniline blue 4 days after being pollinated with yellow-skinned pollen.

[0021] Figure 3 The stigmas of Citrus aurantiacus were treated with an aqueous solution containing 0.02% Tween-20 by volume, and aniline blue staining results were obtained 4 days after pollination with yellow-skinned pollen.

[0022] Figure 1-3 In this context, pt represents pollen tubes, and vb represents vascular bundles. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. All other embodiments obtained by those skilled in the art based on the embodiments in this application without inventive effort are within the scope of protection of this application.

[0024] The selective fertilization referred to in this invention, as described in *Modern Ecology*, is the phenomenon where a sperm nucleus with a specific genetic basis preferentially fertilizes an egg cell. Selective fertilization is mainly manifested in physiological, biochemical, and genetic characteristics, including incompatibility in distant hybridization and competition among multiple pollen sperm nuclei. In this invention, a distant hybridization between a four-season orange and a wampee is used as an example for illustration.

[0025] A reagent for overcoming incompatibility in distant hybridization of Rutaceae plants, the main component of which is the antisense oligonucleotide AS-sjgFER1 targeting sjgFER1, which is obtained through chemical synthesis, and the base sequence is shown in Table 1.

[0026] Table 1. AS-sjgFER1 base sequence

[0027] In the Rutaceae plant species described in this invention, the intergeneric hybridization incompatibility of Rutaceae plants is overcome, promoting germination, growth, entry into the stigma, and completion of the fertilization process. In this invention, the concentration of AS-sjgFER1 used varies slightly for different Rutaceae plant species and can be adjusted according to actual conditions.

[0028] For example, the concentration of AS-sjgFER1 used for Citrus aurantiacus is 30~50 μmol / L. At the same time, Tween-20 is added to the reagent to increase the adhesion and permeability of the stigma to the pollen. The volume percentage of Tween-20 in the reagent is 0.02%.

[0029] The reagent is to be applied twice to the stigma of Rutaceae plants during the bud stage in the morning, and pollination is carried out after the second application.

[0030] When adding the reagent, it is preferable to add it between 6:00 and 8:00 am on a sunny day. The interval between additions is 30 min, and the amount added each time is 2.5 μL per column head.

[0031] The following detailed description, in conjunction with embodiments, illustrates the reagents and methods for overcoming incompatibility in distant hybridization of Rutaceae plants provided by this invention. However, these should not be construed as limiting the scope of protection of this invention.

[0032] Example 1 The AS-sjgFER1 reagent was used to treat *Citrus aurantiacus* to overcome the incompatibility between *Citrus aurantiacus* and *Citrus reticulata* in distant hybridization. Specifically, before 8:00 AM, unopened flower buds of *Citrus aurantiacus* were selected, and flowers showing signs of opening were removed. AS-sjgFER1 was added twice. The AS-sjgFER1 solution contained 0.02% Tween-20 by volume and 30–50 μmol / L of AS-sjgFER1. The interval between the two treatments was 30 minutes.

[0033] After the second treatment, pollen was applied to the yellow-skinned flowers, and a pollination bag was used to prevent contamination from other pollen. Four days later, the pollination bag was removed, the style was picked and fixed with Carnoy's fixative, and the pollen germination was observed using aniline blue staining.

[0034] Figure 1 Four days after treating the stigmas of Citrus aurantiacus with AS-sjgFER1 and pollinating with yellow-skinned flowers, the results of aniline blue staining of the stigmas of Citrus aurantiacus were obtained.

[0035] Comparative Example 1 The procedure is basically the same as in Example 1, except that the unopened flower buds of the four-season orange are not treated with the reagent.

[0036] Figure 2The stigmas of the four-season orange tree, which were not treated with reagent, were stained with aniline blue 4 days after being pollinated with yellow-skinned pollen.

[0037] Comparative Example 2 The procedure was basically the same as in Example 1, except that an aqueous solution containing 0.02% Tween-20 by volume was used as a reagent to treat the unopened flower buds of the four-season orange.

[0038] Figure 3 The stigmas of Citrus aurantiacus were treated with an aqueous solution containing 0.02% Tween-20 by volume, and aniline blue staining results were obtained 4 days after pollination with yellow-skinned pollen.

[0039] pass Figure 1-3 The comparison shows that after treating the stigma of Citrus aurantiacus with the AS-sjgFER1 reagent of the present invention, the pollen of Citrus aurantiacus can germinate, grow and enter the ovary to complete the fertilization process, which can overcome the incompatibility barrier between Citrus aurantiacus and Citrus aurantiacus in distant hybridization.

[0040] The above description, in conjunction with specific / preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various substitutions or modifications can be made to these described embodiments without departing from the inventive concept, and all such substitutions or modifications should be considered within the scope of protection of the present invention.

Claims

1. A reagent that utilizes AS-sjgFER1 to overcome incompatibility in distant hybridization of Rutaceae plants, characterized in that, The main component of the reagent is AS-sjgFER1, which is an antisense oligonucleotide AS-sjgFER1 targeting sjgFER1. The base sequence of AS-sjgFER1 is shown in SEQ ID NO:

1.

2. The reagent according to claim 1, which utilizes AS-sjgFER1 to overcome incompatibility in distant hybridization of Rutaceae plants, is characterized in that... The reagent also contains Tween-20.

3. The reagent according to claim 1, which utilizes AS-sjgFER1 to overcome incompatibility in distant hybridization of Rutaceae plants, is characterized in that... The concentration of AS-sjgFER1 in the reagent is 30–50 μmol / L.

4. The reagent according to claim 2, which utilizes AS-sjgFER1 to overcome incompatibility in distant hybridization of Rutaceae plants, is characterized in that... The volume percentage of Tween-20 in the reagent is 0.02%.

5. A method for using the reagent described in any one of claims 1-4, which utilizes AS-sjgFER1 to overcome incompatibility in distant hybridization of Rutaceae plants, characterized in that... The process includes the following steps: during the initial flowering period of the Citrus aurantiaca plant, select unopened large buds, apply the reagent to their stigmas, and then perform yellow peel pollination.

6. The method of using the reagent according to claim 5, which utilizes AS-sjgFER1 to overcome incompatibility in distant hybridization of Rutaceae plants, is characterized in that... The reagent was applied twice to the stigma of unopened, large-budded flowers.

7. The method of using the reagent according to claim 6, which utilizes AS-sjgFER1 to overcome incompatibility in distant hybridization of Rutaceae plants, is characterized in that... The interval between the number of reagent drops is 30 minutes.

8. The method of using the reagent according to claim 6, which utilizes AS-sjgFER1 to overcome incompatibility in distant hybridization of Rutaceae plants, is characterized in that... The reagent is added in drops of 2.5 μL per column head each time.

9. The application of the reagent described in any one of claims 1-4, which utilizes AS-sjgFER1 to overcome incompatibility in distant hybridization of Rutaceae plants, in overcoming incompatibility in distant hybridization between Citrus reticulata and Citrus aurantium.

10. The application of the reagent described in any one of claims 5-8, which utilizes AS-sjgFER1 to overcome incompatibility in distant hybridization of Rutaceae plants, in overcoming incompatibility in distant hybridization between Citrus reticulata and Citrus aurantium.

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