Method for overcoming distant hybridization incompatibility of rutaceous plants by using AS-sjgFER1 and application

Abstract

The invention discloses a method for overcoming distant hybridization incompatibility of rutaceous plants by using AS-sjgFER1 and application of the method. According to the method, an antisense oligonucleotide technology is adopted, and an AS-sjgFER1 reagent is dropwise added to stigmas of rutaceous plants, so that the incompatibility of distant hybridization of the four-season oranges and the clausena lansium is overcome. The method is small in damage to plants, remarkable in effect of overcoming the distant hybridization incompatibility and wide in application range, can be further used for overcoming the distant hybridization incompatibility of other rutaceous plants, and has important value for breeding and seed production of the rutaceous plants.

Description

Technical Field

[0001] The present invention relates to the technical field of Rutaceae plant breeding, and in particular to a method and application of overcoming incompatibility of Rutaceae plants by utilizing AS-sjgFER1. Background Art

[0002] Distant hybridization refers to the hybridization of plants between different species, genera, or even more distantly related species. Distant hybridization can combine the characteristics and properties of different species and genera, expanding genetic variation and creating new variants or even new species. However, hybridization between distantly related plants requires overcoming both pre- and post-fertilization reproductive barriers. Pre-fertilization barriers occur when two fertile species, after pollination, completely prevent fertilization, a phenomenon known as incompatibility. These barriers can manifest as either pollen failing to germinate or germinating abnormally on the stigma, or pollen germinating normally but with the pollen tube failing to grow properly within the style.

[0003] The genus Citrus in the Rutaceae family has undergone numerous intra-genus hybridizations during its evolution, and most cultivated Citrus species are hybrids of two or more original species. However, among Rutaceae species outside the genus Citrus, such as Kumquat and Murraya Osmanthus, there are currently no reports of hybridization between these species and other species, including those in the genus Citrus.

[0004] Citrus, a member of the Rutaceae family, is a major fruit tree worldwide, occupying a crucial position in the national economy and possessing enormous economic value. The citrus Huanglongbing disease pathogen primarily infects Rutaceae plants, with cultivated species of the Citrus, Poncirus, and Fortunella species serving as its hosts. Outside the citrus family, species such as Wampee and Murraya Fragrant have demonstrated strong resistance to Huanglongbing. Introducing Huanglongbing resistance into citrus species through the barrier of distant hybridization would have significant research and economic value in combating the disease in the citrus industry.

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

[0006] In order to solve the defects and deficiencies in the above-mentioned prior art, the present invention proposes a reagent and method for overcoming the incompatibility of distant hybridization in Rutaceae plants. The reagent can overcome the incompatibility of distant hybridization, is simple and convenient to operate, and is easy to promote.

[0007] In order to achieve the purpose of the present invention, the technical solution adopted by the present invention is:

[0008] A reagent utilizing AS-sjgFER1 to overcome the incompatibility of distant hybridization in Rutaceae plants. The main component of the reagent is AS-sjgFER1, which is an antisense oligonucleotide AS-sjgFER1 targeting sjgFER1 and has a base sequence as shown in SEQ ID NO: 1.

[0009] Furthermore, the reagent also contains Tween-20.

[0010] Furthermore, the concentration of AS-sjgFER1 in the reagent is 30-50 μmol / L.

[0011] Furthermore, the distant hybridization includes interspecific distant hybridization and intergeneric distant hybridization.

[0012] Furthermore, the volume percentage of Tween-20 in the reagent is 0.02%.

[0013] Another object of the present invention is to provide a method for overcoming incompatibility of distant hybridization of Rutaceae plants using the reagent, the method comprising:

[0014] During the initial flowering period of Rutaceae plants, unopened large-bud flowers are selected, and the reagent is dripped onto the stigma before pollination.

[0015] Furthermore, the reagent is dripped onto the stigma of the unopened large-bud stage flower twice.

[0016] Furthermore, the interval between the reagent addition times is 30 minutes.

[0017] Furthermore, the amount of each drop was 2.5 μL per column head.

[0018] In addition, the present invention also provides application of the reagent in overcoming incompatibility of distant hybridization of Rutaceae plants.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention is applicable to incompatibility breeding of distant hybridization of Rutaceae plants. AS-sjgFER1 is used to inhibit the expression of the FER1 gene in the stigma of Rutaceae plants during flowering, effectively overcoming the incompatibility barrier of intergeneric hybridization between Citrus grandis and Wampee, promoting the germination of Wampee pollen on the stigma of Citrus grandis during flowering, allowing the Wampee pollen to grow into the stigma of Citrus grandis and complete the fertilization process. The method of the present invention is simple and convenient to operate, easy to promote, has little damage to plants, is effective in overcoming incompatibility of distant hybridization, has a wide range of applications, and can be applied to Citrus grandis and Wampee plants; moreover, the method can be further used to overcome incompatibility of distant hybridization of other plants in the Rutaceae family, and has important value for the breeding and seed production of Rutaceae plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The disclosure of the present invention will become more easily understood with reference to the accompanying drawings. Those skilled in the art will readily appreciate that these drawings are merely used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention.

[0022] Figure 1 The stigmas of Four Seasons Orange were treated with AS-sjgFER1 and aniline blue dyed 4 days after pollination with yellow-peeled pollen.

[0023] Figure 2 This is the aniline blue staining result of the stigma of the Four Seasons Mandarin Orange, which was not treated with the drop reagent, 4 days after being pollinated with yellow peel pollen.

[0024] Figure 3 The stigmas of Four Seasons Mandarin Orange were treated with a 0.02% by volume Tween-20 aqueous solution. Four days after pollination with yellow peel pollen, the stigmas of Four Seasons Mandarin Orange were stained with aniline blue.

[0025] Figure 1-3 In the figure, pt is the pollen tube and vb is the vascular bundle. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are merely for explaining the present invention and are not intended to limit the present invention. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative work are within the scope of protection of this application.

[0027] Selective fertilization, as described in Modern Ecology, refers to the phenomenon of preferential fertilization between sperm nuclei with a specific genetic basis and egg cells. This phenomenon is primarily characterized by physiological, biochemical, and genetic characteristics, including incompatibility in distant hybridization and competition between sperm nuclei from multiple pollen. In the examples of the present invention, distant hybridization between Siji Mandarin and Huangpi is used as an example for illustration.

[0028] A reagent for overcoming incompatibility of distant hybridization in Rutaceae plants. The main component of the reagent is an antisense oligonucleotide AS-sjgFER1 targeting sjgFER1, which is synthesized by chemical synthesis. The base sequence is shown in Table 1.

[0029] Table 1 AS-sjgFER1 base sequence

[0030]

[0031] In the Rutaceae plant species described in the present invention, the incompatibility between genus hybridization of Rutaceae plants is overcome, promoting germination, growth, entry into the stigma, and completion of the fertilization process. In the present invention, the concentration of AS-sjgFER1 used for different Rutaceae plant species varies slightly and can be adjusted according to actual conditions.

[0032] For example, the concentration of AS-sjgFER1 used for four-season orange is 30-50 μmol / L, and Tween-20 is added to the reagent to increase the adhesion and permeability of the stigma to pollen. The volume percentage of Tween-20 in the reagent is 0.02%.

[0033] The method of using the reagent is to drip the reagent twice onto the stigma of the bud stage of the Rutaceae plant in the morning, and pollinate after the second drip.

[0034] When adding dropwise, the reagent is preferably added dropwise between 6:00 and 8:00 in the morning on a sunny day, with an addition interval of 30 minutes and a dropwise amount of 2.5 μL per column head.

[0035] The following describes in detail the reagents and methods of use for overcoming incompatibility in distant hybridization of Rutaceae plants provided by the present invention in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0036] Example 1

[0037] The AS-sjgFER1 reagent was used to treat Siji Mandarin oranges to overcome the incompatibility between Siji Mandarin oranges and Huangpi. Specifically, before 8:00 a.m., unopened buds of Siji Mandarin oranges were selected, and flowers showing signs of opening were removed. Two drops of AS-sjgFER1 were applied. The AS-sjgFER1 solution contained 0.02% Tween-20 by volume and a concentration of 30-50 μmol / L of AS-sjgFER1. The two drops were separated by 30 minutes.

[0038] After the second treatment, the yellow-peeled flowers were pollinated and covered with pollination bags to prevent contamination from other pollen. Four days later, the pollination bags were removed, the styles were removed and fixed with Carnoy's fixative, and the pollen germination was observed using aniline blue staining.

[0039] Figure 1 The stigmas of Four Seasons Orange were treated with AS-sjgFER1 and aniline blue dyed 4 days after pollination with yellow-peeled pollen.

[0040] Comparative Example 1

[0041] The method is basically the same as Example 1, except that the unopened flower buds of the four-season orange are not treated by dripping the reagent.

[0042] Figure 2 This is the aniline blue staining result of the stigma of the Four Seasons Mandarin Orange, which was not treated with the drop reagent, 4 days after being pollinated with yellow peel pollen.

[0043] Comparative Example 2

[0044] The method is basically the same as Example 1, except that a 0.02% by volume Tween-20 aqueous solution is used as a reagent to drip the unopened flower buds of the four-season orange.

[0045] Figure 3 The stigmas of Four Seasons Mandarin Orange were treated with a 0.02% by volume Tween-20 aqueous solution. Four days after pollination with yellow peel pollen, the stigmas of Four Seasons Mandarin Orange were stained with aniline blue.

[0046] pass Figure 1-3 By comparison, it can be seen that after the stigma of Four Seasons Orange is treated with the AS-sjgFER1 reagent of the present invention, the pollen of Wampee can germinate, grow and enter the ovary to complete the fertilization process, which can overcome the incompatibility barrier of distant hybridization between Four Seasons Orange and Wampee.

[0047] The above is a further detailed description of the present invention in conjunction with specific / preferred embodiments, and the specific implementation of the present invention should not be considered to be limited to these descriptions. Those skilled in the art of the art to which the present invention belongs may make several substitutions or modifications to the described embodiments without departing from the scope of the present invention, and such substitutions or modifications should be considered to fall within the scope of protection of the present invention.

Claims

1. A reagent for overcoming incompatibility of distant hybridization in Rutaceae plants using AS-sjgFER1, characterized in that: The main component of the reagent is AS-sjgFER1, which is an antisense oligonucleotide AS-sjgFER1 targeting sjgFER1. The AS-sjgFER1 has a base sequence as shown in SEQ ID NO:

1.

2. The reagent for overcoming incompatibility of distant hybridization of Rutaceae plants using AS-sjgFER1 according to claim 1, characterized in that: The reagent also contains Tween-20.

3. The reagent for overcoming incompatibility of distant hybridization of Rutaceae plants using AS-sjgFER1 according to claim 1, characterized in that: The concentration of AS-sjgFER1 in the reagent is 30-50 μmol / L.

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

5. A method for using the reagent for overcoming incompatibility of Rutaceae plants by using AS-sjgFER1 as claimed in any one of claims 1 to 4, characterized in that: The method comprises the following steps: selecting unopened large-bud flowers of Rutaceae plants at the beginning of flowering, dripping the reagent onto the stigma of the flowers and then pollinating the flowers.

6. The method for using the reagent for overcoming incompatibility of distant hybridization of Rutaceae plants using AS-sjgFER1 according to claim 5, characterized in that: The reagent was dripped onto the stigma of the unopened large-bud stage flower twice.

7. The method for using the reagent for overcoming incompatibility of distant hybridization of Rutaceae plants using AS-sjgFER1 according to claim 6, characterized in that: The interval between the addition of the reagent is 30 minutes.

8. The method for using the reagent for overcoming incompatibility of distant hybridization of Rutaceae plants using AS-sjgFER1 according to claim 6, characterized in that: The amount of the reagent added each time was 2.5 μL per column head.

9. Use of the reagent for overcoming incompatibility of Rutaceae plants by using AS-sjgFER1 as claimed in any one of claims 1 to 4 in distant hybridization of Rutaceae plants.

10. Use of the method for overcoming the incompatibility of Rutaceae plants in distant hybridization using the reagent AS-sjgFER1 as claimed in any one of claims 5 to 8 in distant hybridization of Rutaceae plants.

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