Method for creating new germplasm of high-glucoraphanin pine flower ball type broccoli

Abstract

The invention discloses a method for creating a new germplasm of pine flower ball type broccoli with high glucoraphanin content. According to the method, a fertility restoring line pRfo:: Rfo is used as a male parent and hybridized with a broccoli Ogura CMS commercial species of a moss type as a female parent, and the fertility of the broccoli Ogura CMS commercial species is restored; the method comprises the following steps: carrying out marker detection on a Bar transgenic element in an F1-generation plant of a fertility restored broccoli Ogura CMS commercial species to obtain an Rfo-containing F1 hybrid; the method comprises the following steps: hybridizing a high-glucoraphanin fertile parent serving as a female parent and an Rfo-containing F1 hybrid serving as a male parent, and screening to obtain an Rfo-free character excellent material; and hybridizing and screening by taking an Rfo-free material with excellent characters as a female parent and a DMP haploid induction line as a male parent to obtain the new germplasm of the high-glucoraphanin pine flower ball type broccoli. According to the method, the breeding time is greatly shortened, materials with target characters can be quickly and efficiently obtained, and the method has important significance on quickly realizing seed revitalization of domestic varieties.

Description

Technical Field

[0001] The invention belongs to the technical field of plant breeding, and in particular relates to a method for creating a new germplasm of high-glucoraphanin pine ball-type broccoli. Background Art

[0002] Broccoli (Brassica oleracea L.var.italica), also known as broccoli, is a variety of the Brassica oleracea species in the Brassica family. It is a favorite among consumers for its rich content of nutrients such as protein, vitamin C, and minerals, as well as its anti-cancer properties such as sulforaphane, earning it the nickname "the crown of vegetables." When consumed, the glucoraphanin in broccoli degrades into sulforaphane (also known as sulforaphane), an anti-cancer substance. Sulforaphane is reportedly effective in preventing cancer, cardiovascular and cerebrovascular diseases, and hypertension. Therefore, cultivating new varieties of broccoli high in glucoraphanin is crucial for meeting current consumer demand for healthy, high-quality vegetables.

[0003] Currently, all commercial broccoli varieties are of the Ogura CMS type, which cannot produce pollen. Consequently, their desirable traits cannot be reused through hybridization, limiting the innovation of germplasm resources. Therefore, the creation of Ogura CMS restorer lines is crucial for the sustainable utilization of these resources. By introducing the fertility restorer gene Rfo into the commercial Ogura CMS varieties of broccoli with desirable traits through Agrobacterium-mediated genetic transformation, fertility can be restored. These lines can then be used as breeding intermediates and hybridized with higher-generation inbred lines to be improved, enabling the rapid creation of new broccoli germplasm.

[0004] Using CRISPR / Cas9 technology, the DMP8 and DMP9 genes in broccoli are simultaneously knocked out, resulting in edited plants (haploid inducer lines). These plants are then used as male parents for hybridization with high-quality, lower-generation material to be purified, yielding haploids from which material meeting breeding objectives can be screened. The creation and utilization of this method can efficiently and rapidly advance broccoli germplasm innovation and breeding. Using the resulting transgenic fertility restorer lines as intermediate breeding material, introducing the superior traits of the commercial Ogura CMS variety into the breeding parent to be improved, and rapidly purifying the material using haploid inducer lines, combined with molecular marker-assisted selection, is one of the important approaches to developing new broccoli varieties with independent intellectual property rights. Summary of the Invention

[0005] In order to overcome the shortcomings and deficiencies of the prior art, the present invention aims to provide a method for creating a new germplasm of broccoli with high glucoraphanin content.

[0006] The present invention is achieved by providing a method for creating a new germplasm of high-glucoraphanin pine-shaped broccoli, the method comprising the following steps:

[0007] (1) The radish Rfo gene and its 2 kb promoter were transferred into broccoli Ogura CMS commercial variety 1 by Agrobacterium-mediated genetic transformation to obtain the fertility restorer line pRfo::Rfo;

[0008] (2) using the fertility restorer line pRfo::Rfo as the male parent and hybridizing with the moss-type broccoli Ogura CMS commercial variety 2 as the female parent to restore the fertility of the broccoli Ogura CMS commercial variety 2;

[0009] (3) Marker detection of the Bar transgenic element in F1 plants of the restored fertility broccoli Ogura CMS commercial variety 2 was performed to obtain F1 hybrids containing Rfo;

[0010] (4) hybridizing a fertile parent with high glucoraphanin content as the female parent and the F1 hybrid containing Rfo as the male parent, and screening to obtain excellent materials with the trait of not containing Rfo;

[0011] (5) The excellent material with no Rfo trait was used as the female parent and the DMP haploid induced line was used as the male parent to perform hybridization and screening to obtain a new germplasm of high glucoraphanin-containing broccoli.

[0012] Preferably, in step (1), the broccoli Ogura CMS commercial variety 1 is the broccoli Ogura CMS commercial variety "Excellent Cold Resistance".

[0013] Preferably, in step (2), the broccoli Ogura CMS product variety 2 is the broccoli Ogura CMS product variety "Mitheus".

[0014] Preferably, in step (4), the high glucoraphanin fertile parent is QA1.

[0015] Preferably, in step (5), the screening criteria are: double haploids with pedicel length between commercial species 1 and commercial species 2, fine buds, and high glucoraphanin content.

[0016] Compared with the shortcomings and deficiencies of the prior art, the present invention has the following beneficial effects:

[0017] (1) The present invention utilizes the developed Ogura CMS fertility restorer line pRfo::Rfo as the male parent to restore the fertility of the commercial moss broccoli variety "Mitheus" (Ogura CMS type). Compared with distant hybridization using Brassica napus plants containing the restorer gene Rfo, this method significantly shortens breeding time and saves manpower and material resources. Furthermore, it avoids the problems encountered in distant hybridization, such as the difficulty in eliminating the donor gene background and the low transmission rate of the Rfo gene.

[0018] (2) The present invention utilizes the created haploid induction line (DMP-edited plants) to rapidly purify the F1 material in one generation, which greatly shortens the breeding time compared with the traditional breeding method that requires 7 to 8 generations of self-pollination purification. Compared with the breeding method of obtaining haploids by microspore culture that is limited by the genotype material, it is simple and efficient.

[0019] (3) The use of molecular markers to assist breeding during the germplasm creation process of the present invention can quickly and efficiently obtain materials with target traits, thereby improving breeding efficiency.

[0020] (3) The present invention provides a method for efficiently creating a new germplasm of broccoli with high glucoraphanin content and medium pedicel length, which is distinctive and innovative, and has important significance for the rapid revitalization of the seed industry of domestic varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 A flowchart of the steps of the method in an embodiment of the present invention;

[0022] Figure 2 This is the Bar-labeled agarose gel electrophoresis diagram of the Rfo fertility restorer gene transfection, where M is a marker. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present 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 only used to explain the present invention and are not intended to limit the present invention.

[0024] The embodiment of the present invention discloses a method for creating a new germplasm of high-glucoraphanin pine ball broccoli, combining Figure 1 It is understood that the method comprises the following steps:

[0025] 1. The radish Rfo gene and its 2kb promoter were transferred into the broccoli Ogura CMS commercial variety "Cold-resistant Excellent" through Agrobacterium-mediated genetic transformation to obtain the fertility restorer line pRfo::Rfo;

[0026] In step 1, the commercial broccoli variety Ogura CMS, "Excellent Cold Resistance," was selected and bred by Sakata Seed Co., Ltd., Japan. The genetic transformation method and the process of obtaining the fertility restorer line pRfo::Rfo are detailed in the reference: Xing Miaomiao, Xu Yuanyuan, Lu Yuyu, Yan Jiyong, Zeng Aisong. Obtaining the Ogura CMS restorer line of broccoli by genetic transformation of Rfo. Chinese Journal of Agricultural Sciences. 2023, 56(15): 2966-2976.

[0027] 2. Using the fertility restorer line pRfo::Rfo as the male parent and the moss-type broccoli Ogura CMS commercial variety "Mixiusi" as the female parent, hybridization is performed to restore the fertility of the broccoli Ogura CMS commercial variety "Mixiusi".

[0028] In the embodiment of the present invention, the commercial broccoli variety Ogura CMS "Mitheus" was bred by Seminis Seed Company of the United States.

[0029] In this embodiment, during the flowering period of March to April 2022, the developed Ogura CMS fertility restorer line pRfo::Rfo was used as the male parent and hybridized with the moss-type Ogura CMS commercial broccoli variety "Mixiusi" as the female parent to restore the fertility of "Mixiusi." The seeds were harvested in June and sown in August.

[0030] 3. Marker detection of the Bar transgenic element was performed in the F1 generation plants of the Ogura CMS commercial variety "Mitheus", which restores fertility, to obtain F1 hybrids containing Rfo.

[0031] In step 3, the genomic DNA of the hybrid F1 material is extracted using the CTAB method. The specific steps are as follows:

[0032] (1) Take a young leaf about the size of a thumb cap, place it in a 2 mL round-bottom centrifuge tube, and add 5-6 2.5 mm zirconium seeds;

[0033] (2) Add 500 μL of CTAB lysis buffer preheated at 65°C to the centrifuge tube and crush the tissue using a tissue crusher for 3 to 5 minutes;

[0034] (3) Carefully place the centrifuge tube in a 65°C water bath for 15 minutes, gently shaking once every 5 minutes; cool to room temperature, add 500 μL of chloroform / isoamyl alcohol (24:1), and mix for 3 minutes;

[0035] (4) Centrifugation at 12000 rpm for 15 min;

[0036] (5) Pipette the supernatant into a new conical centrifuge tube, add an equal volume of pre-chilled isopropanol, mix well, and let stand at -20°C for 1 h;

[0037] (6) Centrifuge at 12000 rpm for 15 min, discard the supernatant, add 500 μL of 75% alcohol, and shake gently;

[0038] (7) Centrifuge at 12000 rpm for 10 min, discard the supernatant, and air-dry at room temperature;

[0039] (8) Add 200 μL sterile ddH2O and mix well.

[0040] In step 3, the genomic DNA of the hybrid F1 material was used as a template and the primers designed as shown in Table 1 were used to perform PCR amplification on the transgenic screening marker Bar, and the target band was analyzed by agarose gel electrophoresis.

[0041] Table 1 Bar marker primers

[0042]

[0043] The PCR system was a 10 μL system, 0.2 μL of each upstream and downstream primer (concentration 10 μmol / μL), about 40 ng of template DNA, 5 μL of 2×TsingKe mix, and supplemented with ddH2O.

[0044] The PCR reaction program was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 40 s, for a total of 30 cycles; and finally extension at 72°C for 5 min.

[0045] The PCR product of the transgenic screening marker Bar was analyzed by 1% agarose gel electrophoresis for about 30 minutes. The gel imager was used to take pictures and analyze the bands ( Figure 2 ).

[0046] The resulting F1 plants were tested for Bar (transgenic element) markers, and fertile F1 materials were selected for their Rfo content, high glucoraphanin content, and traits similar to the Mitheus type (fine buds and long pedicels). These intermediate breeding materials were transplanted into greenhouses and vernalized under low temperatures during the winter, awaiting bolting and flowering the following year.

[0047] 4. Using the high glucoraphanin fertile parent QA1 as the female parent and the F1 hybrid containing Rfo as the male parent, hybridization is performed to screen and obtain excellent materials with the trait of not containing Rfo.

[0048] In step 4, the high glucoraphanin fertile parent QA1 is a high-generation inbred line that was imported from Japan in 1984 by the applicant of the present invention, and purified and preserved by self-pollination of resource B242.

[0049] In the embodiment of the present invention, during the flowering period from March to April 2023, the high glucoraphanin fertile parent QA1 was used as the female parent and the F1 containing Rfo obtained above was used as the male parent for hybridization to screen and obtain excellent materials with the trait of not containing Rfo.

[0050] 5. Using the excellent material with no Rfo trait as the female parent and the DMP haploid induced line as the male parent, hybridization and screening were performed to obtain a new germplasm of high glucoraphanin-rich broccoli.

[0051] In step 5, during the flowering period of March to April 2023, the excellent material obtained in the previous year was used as the maternal parent, and a haploid induction line (DMP-edited plant) (the backbone parent H-6 was used as the transgenic recipient material, and the DMP8 and DMP9 genes were edited using CRISPR / Cas9 technology to create a haploid induction line. Reference: Zhao X, Yuan K, Liu Y, Zhang N, Yang L, Zhang Y, Wang Y, Ji J, Fang Z, Han F, Lv H. In vivo maternal haploid induction based on genome editing of DMP in Brassica oleracea. Plant Biotechnol J.2022,20(12):2242-2244.) was used as the male parent for hybridization, and double haploids with pedicel length between the tight-head type "cold-resistant excellent" and the moss type "Mitheus", fine-grained buds, and high glucoraphanin content were selected, thus obtaining a new high-glucoraphanin loose-head broccoli germplasm.

[0052] The present invention further measures the glucoraphanin content in broccoli curds. Specifically, curds of different varieties were sampled in early December. Curds from five individual plants with uniform maturity were pooled and vacuum freeze-dried. The dried samples were then ground using a grinder and divided into three equal portions, sealed and stored at -80°C. The glucoraphanin content was then measured (three biological replicates).

[0053] Weigh 0.05 g of lyophilized powder from the flower curd into a 10 mL centrifuge tube, add 5 mL of 90% methanol solution, shake well, and let stand for 1 hour. Transfer 1 mL of the crude extract to an activated purification column, wash with 1 mL of 90% methanol solution and 1 mL of ultrapure water, then add 100 µL of 0.1 mM sulfatase and incubate at 25°C overnight. Wash the column with 1 mL of ultrapure water, collect the filtrate, and filter (0.45 mm pore size) to obtain the isolated and purified desulfurized glucosinolate sample. The glucoraphanin content in the flower curd was determined by HPLC using ONPG as an internal standard. The results are shown in Table 2.

[0054] Table 2 Glucoraphanin content in different materials

[0055]

[0056] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for creating a new germplasm of broccoli with high glucoraphanin content, characterized in that: The method comprises the following steps: (1) The radish Rfo gene and its 2 kb promoter were transferred into broccoli Ogura CMS commercial variety 1 by Agrobacterium-mediated genetic transformation to obtain the fertility restorer line pRfo::Rfo; (2) using the fertility restorer line pRfo::Rfo as the male parent and hybridizing with the moss-type broccoli Ogura CMS commercial variety 2 as the female parent to restore the fertility of the broccoli Ogura CMS commercial variety 2; (3) Marker detection of the Bar transgenic element in F1 plants of the restored fertility broccoli Ogura CMS commercial variety 2 was performed to obtain F1 hybrids containing Rfo; (4) hybridizing a fertile parent with high glucoraphanin content as the female parent and the F1 hybrid containing Rfo as the male parent, and screening to obtain excellent materials with the trait of not containing Rfo; (5) The excellent material with no Rfo trait was used as the female parent and the DMP haploid induced line was used as the male parent to perform hybridization and screening to obtain a new germplasm of high glucoraphanin-containing broccoli.

2. The method according to claim 1, wherein In step (1), the broccoli Ogura CMS commercial variety 1 is the broccoli Ogura CMS commercial variety "Excellent Cold Resistance".

3. The method according to claim 1, wherein In step (2), the broccoli Ogura CMS product variety 2 is the broccoli Ogura CMS product variety "Mitheus".

4. The method according to claim 1, wherein In step (4), the high glucoraphanin fertile parent QA1.

5. The method according to claim 1, wherein In step (5), the screening criteria are: double haploids with pedicel length between commercial species 1 and commercial species 2, fine buds, and high glucoraphanin content.

Images