Non-transgenic herbicide-resistant Chinese cabbage or Chinese cabbage type rape strain as well as cultivation method and application thereof

Abstract

The invention discloses a non-transgenic herbicide-resistant Chinese cabbage or Chinese cabbage type rape strain as well as a cultivation method and application thereof, and aims to solve the problem of weeds in the production practice of Chinese cabbage or Chinese cabbage type rape, a non-transgenic herbicide-resistant gene of a sibling species is transferred into Chinese cabbage or Chinese cabbage type rape through a distant hybridization technology; the non-transgenic herbicide-resistant Chinese cabbage or Chinese cabbage type rape germplasm is obtained; the non-transgenic herbicide-resistant Chinese cabbage or Chinese cabbage type rape germplasm is used as a herbicide-resistant character donor, and is hybridized with other varieties to obtain a new variety of Chinese cabbage or Chinese cabbage type rape with resistance to specific herbicides through screening. After the new non-transgenic herbicide-resistant cabbage or cabbage type rape variety is applied to production, the herbicide can be sprayed in the early stage, the growth of the cabbage or cabbage type rape is not affected while weed seedlings are killed in the early stage, the weed problem is effectively solved, and cost saving and efficiency increasing in the weeding link are achieved.

Description

Technical Field

[0001] This invention relates to the field of plant strain cultivation and breeding technology, specifically to non-GMO herbicide-resistant Chinese cabbage or Chinese cabbage-type rapeseed strains, their cultivation methods, and applications. Background Technology

[0002] Chinese cabbage belongs to the genus *Brassica* of the Brassicaceae family and is one of the species in the Yu's Triangle. The type harvested for oil extraction is called Chinese cabbage-type rapeseed; the type used as a vegetable is simply called Chinese cabbage. Chinese cabbage and Chinese cabbage-type rapeseed are the same species, sharing the same genome composition. In China, Chinese cabbage is further divided into large Chinese cabbage (*Brassica campestris* ssp. *pekinensis*) and small Chinese cabbage (*Brassica campestris* ssp. *chinensis*). Chinese scientist Li Jiawen believes that variations in small Chinese cabbage have resulted in varieties such as Chinese cabbage-type rapeseed (*B. campestris* ssp. *chinensis* var. *oleifera*) and common small Chinese cabbage (*B. campestris* ssp. *chinensis* var. *commons*). Liu Houli named the northern rapeseed, native to northern my country, *B. campestris* var. *oleifera*, and the southern rapeseed, native to southern my country, *B. chinensis* var. *oleifera*. He believed that the southern rapeseed evolved from Chinese cabbage and is a unique type of rapeseed in my country. Chinese cabbage is one of the most important vegetable species in my country and an important agricultural product for the Chinese people. From an evolutionary genetic perspective, the Chinese rapeseed type is the original species of Brassica napus L. (2n=38, AACC) and Brassica juncea L. (2n=36, AABB). The Chinese rapeseed type is one of the three major cultivated types of rapeseed in my country, with a long history of cultivation and abundant resources. In the early days, extracting oil from the seeds of Chinese rapeseed was an important method for obtaining rapeseed oil throughout my country. Later, with the introduction, improvement, and widespread cultivation of Brassica napus (also known as Chinese cabbage rapeseed), Chinese rapeseed is now mainly grown in high-altitude areas of provinces such as Qinghai, Gansu, Tibet, Sichuan, and Xinjiang, as well as in some southern regions with triple-cropping systems. In 2002, the planting area of ​​Chinese rapeseed accounted for approximately 15% of the national rapeseed planting area, reaching about 730,000 hectares in 2013. Although the planting scale of Chinese rapeseed is not large, it remains the preferred oilseed crop in high-altitude areas. Furthermore, Chinese rapeseed possesses excellent characteristics such as early maturity, yellow seeds, self-incompatibility, drought tolerance, tolerance to poor soil, short growing season, and strong cold resistance, making it an irreplaceable oilseed crop in some production areas.

[0003] Farmland weeds are a significant obstacle to crop production, with negative impacts manifesting in several ways: First, they compete for resources, such as nutrients, water, sunlight, and living space; second, they transmit diseases and pests, as some weeds carry viruses or fungi and serve as havens for pests; third, they induce diseases, as dense weed growth increases field humidity, promoting disease development, and some weeds release allelochemicals that inhibit crop growth; and fourth, they alter the quality of agricultural products, as weed seeds mixed with commercial crop seeds reduce the quality of produce. Ultimately, weed damage increases crop production costs and reduces yields and profitability. Therefore, efficient weed control in crop fields is a crucial aspect of current crop management. With continued urbanization and a persistent shortage of rural labor in my country, crop production will inevitably shift from traditional labor-intensive methods to simplified, large-scale, and mechanized production. The efficient use of chemical methods to remove weeds in crop fields will become an objective requirement, a development trend, and the optimal technological path for modern crop production in my country.

[0004] Chinese cabbage or Chinese cabbage-type rapeseed fields exhibit a diverse range of weed species, with broadleaf and grassy weeds growing together and causing equal damage. Currently, there are many types of herbicides and products available for controlling grassy weeds, making them relatively easy to control, while broadleaf weeds are much more difficult to manage. Adopting a method of "planting herbicide-resistant crops + spraying appropriate herbicides" can effectively control field weeds and is an excellent solution for modern crop production in my country. For Chinese cabbage or Chinese cabbage-type rapeseed, the lack of germplasm resources with specific resistance is the biggest obstacle to this solution. Summary of the Invention

[0005] The purpose of this invention is to provide non-GMO herbicide-resistant Chinese cabbage or Chinese cabbage-type rapeseed varieties, their breeding methods and applications, to solve the weed problem in the production of Chinese cabbage or Chinese cabbage-type rapeseed.

[0006] To achieve the above objectives, the technical solution provided by the present invention is as follows: The first aspect of this application provides a non-GMO herbicide-resistant Chinese cabbage or Chinese cabbage-type rapeseed line, the seeds of which are deposited at the China Center for Type Culture Collection (CCTCC) with accession number P202518 and accession date of September 3, 2025.

[0007] This strain is based on the Chinese cabbage genome and incorporates a segment of the rapeseed genome through distant hybridization.

[0008] That BRAHAS The gene sequence is shown in SEQ NO.5.

[0009] The second aspect of this application provides a method for cultivating non-GMO herbicide-resistant Chinese cabbage or Chinese cabbage-type rapeseed lines, comprising the following steps: S1: Using the non-GMO herbicide-resistant rapeseed variety Ning R101 as the male parent and a Chinese cabbage cultivar as the female parent, artificial pollination and hybridization were carried out to harvest F1 seeds of distant hybridization. S2: Sow the hybrid F1 seeds. When they grow to the three-leaf stage, use a herbicide as a resistance screening agent to spray the hybrid F1 seedlings at the three-leaf stage and screen out resistant F1 individual plants. S3: Transplant the resistant F1 single plants with soil to continue growing. In the following year, during the flowering period, use the resistant F1 single plants as the female parent and the Chinese cabbage cultivar as the male parent for artificial pollination and backcrossing to harvest BC1 generation seeds. S4: Sow BC1 generation seeds. When the seedlings reach the three-leaf stage, use herbicides as resistance screening agents to spray the BC1 generation seedlings at the three-leaf stage and screen out resistant BC1 individual plants. S5: Phenotypic and molecular identification of resistant BC1 single plants, and screening for resistant progeny single plants with gene mutations; S6: Transplanting single plants of the resistant offspring with gene mutations and then backcrossing them with the parental Chinese cabbage cultivar during the flowering period to obtain BC2 generation seeds; S7: Continue backcrossing the BC2 generation seeds with the Chinese cabbage cultivar parents for multiple generations to obtain the final resistant individual plants. The resistant individual plants are then screened for the optimal line based on self-pollination seed setting, natural seed setting, and hybridization resistance, and their seeds are harvested.

[0010] To optimize the above technical solution, the specific measures also include: In steps S2 and S4, the parent Chinese cabbage cultivar was sown simultaneously, with the three-leaf stage seedlings of the parent Chinese cabbage cultivar that were not sprayed with herbicides serving as a control.

[0011] The non-GMO herbicide-resistant rapeseed variety Ning R101 contains two mutation sites, one of which is located on chromosome C01. BnAHAS1 Its sequence is shown in SEQ NO.1, and the gene located on chromosome A01. BnAHAS3 Its sequence is shown in SEQ NO.2; correspondingly, its BnAHAS1 The protein exhibits the Trp-574-Leu mutation, the sequence of which is shown in SEQ NO.3. BnAHAS3 The protein undergoes the Trp-574-Leu mutation, the sequence of which is shown in SEQ NO.4. In step S5, the method for phenotypic and molecular identification of resistant BC1 single plants is as follows: [The following text appears to be a separate, unrelated section:] ...containing from... BnAHAS1 , BnAHAS3 Homologous gene fragments with mutated bases were separated to determine the mutated bases. Using the mutated bases as targets, the sequences were expanded forward and backward around them. Primers were designed to find the differences between the two homologous genes upstream and downstream, and PCR amplification was used to obtain fragments containing the mutated bases of each gene.

[0012] Furthermore, those capable of specifically amplifying [the virus] were screened. BnAHAS1 , BnAHAS3 Primers for the gene fragment are as follows: AAAGCTCGAGGCGTTTGCG; GTAAGTCTGGGAACAAACCAAAAGC.

[0013] Preferably, the Chinese cabbage cultivar is selected as Dwarf Yellow; the herbicide is selected as a sulfonylurea herbicide.

[0014] The third aspect of this application provides an application of a non-GMO herbicide-resistant Chinese cabbage or Chinese cabbage-type rapeseed variety, which allows for the application of herbicides in the early stages of planting, killing weed seedlings in the early stages without affecting the growth of the Chinese cabbage or Chinese cabbage-type rapeseed itself. Compared with the prior art, the beneficial effects of the present invention are: This invention involves transferring the non-transgenic herbicide resistance trait, determined by the corresponding endogenous herbicide resistance gene in closely related species, into the genome of Chinese cabbage or Chinese cabbage-type rapeseed through distant hybridization. This allows the Chinese cabbage or Chinese cabbage-type rapeseed to acquire the non-transgenic herbicide resistance trait. After obtaining the new non-transgenic herbicide-resistant Chinese cabbage or Chinese cabbage-type rapeseed germplasm, it can be used to hybridize with other Chinese cabbage or Chinese cabbage-type rapeseed varieties to cultivate new non-transgenic herbicide-resistant Chinese cabbage or Chinese cabbage-type rapeseed varieties. Once these new non-transgenic herbicide-resistant Chinese cabbage or Chinese cabbage-type rapeseed varieties are introduced into actual production, the corresponding herbicide can be sprayed at specific times to kill associated weeds in the production field, solving the problem of weeds, especially broadleaf weeds, in Chinese cabbage or Chinese cabbage-type rapeseed production. This allows the crop to receive sufficient light, temperature, and nutrients, resulting in healthy and rapid growth, achieving cost reduction and efficiency improvement in Chinese cabbage or Chinese cabbage-type rapeseed production.

[0015] The method of this invention yields non-GMO herbicide-resistant Chinese cabbage or Chinese cabbage-type rapeseed, which possesses the characteristic of non-GMO resistance to specific herbicides. It can be used as a germplasm resource for breeding Chinese cabbage or Chinese cabbage-type rapeseed for the selection and breeding of new non-GMO herbicide-resistant Chinese cabbage or Chinese cabbage-type rapeseed varieties.

[0016] When non-GMO herbicide-resistant Chinese cabbage or Chinese cabbage-type rapeseed varieties are developed and applied in production, they have the following advantages compared to similar varieties without herbicide resistance: (1) A specific herbicide can be sprayed at an appropriate dose during the seedling stage (after the two-leaf stage) of Chinese cabbage or Chinese cabbage-type rapeseed to kill the associated weeds in the production field of this variety. This can save the labor cost of weeding for the control variety. Moreover, this method of weeding is low-cost, effective, and fast. (2) After timely weeding, the seedlings of Chinese cabbage or Chinese cabbage-type rapeseed can effectively absorb light, temperature and nutrients from the soil, without competitors, thus promoting healthy crop growth. (3) Crop resistance is generated by endogenous genes, does not involve transgenic technology and components, and has no concerns about ecological safety. (4) This type of Chinese cabbage or Chinese cabbage-type rapeseed crop has new resistance, which is a selling point for seed companies that operate these varieties in the market and gives them a competitive advantage in the market. Attached Figure Description

[0017] Figure 1 Herbicide resistance screening of parents and F1 generation. 1 represents the appearance of parent seedlings without bensulfuron-methyl spray; 2 represents the appearance of parent seedlings sprayed with bensulfuron-methyl 2 weeks ago; 3 and 4 represent the appearance of F1 seedlings of distant hybrids sprayed with bensulfuron-methyl 2 weeks ago (yellow boxes indicate the phenotype of pseudo-hybrid seedlings that are about to die).

[0018] Figure 2 Herbicide resistance screening of seedlings from the BC1 generation segregating population; red boxes indicate the phenotype of non-resistant individual plants, and white boxes indicate the phenotype of resistant individual plants.

[0019] Figure 3 : CDS region sequence alignment of the acetolactate synthase BnAHAS1 and BnAHAS3 genes in Brassica napus; the boxes indicate the base mutation positions of the BnAHAS1 and BnAHAS3 genes that can induce resistance to SU herbicides in rapeseed (calculated using the corresponding amino acid sequence of AHAS in the model plant Arabidopsis thaliana, referred to as the 574 site).

[0020] Figure 4 : Representative sequencing (reverse sequencing) peaks of materials with different genotypes at the 574 locus, with the dashed line representing the 574 locus.

[0021] Figure 5 Phenotypic characteristics of the receptor-sensitive parent dwarf yellow cabbage and herbicide-resistant Chinese cabbage line 47080 after two weeks of treatment with five different subclasses of AHAS inhibitor herbicides at the two-leaf stage.

[0022] Figure 6 Phenotype of non-GMO herbicide-resistant Chinese cabbage F1 hybrid after two weeks of treatment with 1X bensulfuron-methyl (a subclass of AHAS inhibitor herbicides) for weed control in field production.

[0023] Figure 7 : The sequence lists SEQ NO.1, SEQ NO.3 and SEQ NO.4 are highlighted in red. Detailed Implementation

[0024] The present invention will be further described in detail below through specific embodiments, but it should not be construed as limiting the scope of the subject matter of the present invention to the following embodiments. All technologies implemented based on the above content of the present invention fall within the scope of the present invention.

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the reagents, methods and equipment used are conventional reagents, methods and equipment in this technical field.

[0026] This invention provides non-GMO herbicide-resistant Chinese cabbage or Chinese cabbage-type rapeseed varieties, their breeding methods, and applications. The specific implementation process is as follows: I. Acquisition of new non-GMO herbicide-resistant Chinese cabbage or Chinese cabbage-type rapeseed germplasm In 2018, the non-GMO herbicide-resistant rapeseed variety Ning R101 (variety registration number: GPD rapeseed (2018) 320256) was registered and began demonstration planting in rapeseed producing areas of Jiangsu Province. Ning R101 is the first non-GMO herbicide-resistant rapeseed variety, and its herbicide resistance comes from an endogenous gene located on chromosome C01. BnAHAS1 (See SEQ NO.1) and located on chromosome A01 BnAHAS3 One point mutation occurred in each gene (see SEQ NO. 2), specifically a single-base mutation from G to T at the +1676 and +1667 base positions in its CDS region (marked in red), resulting in a Trp-574-Leu mutation in the BnAHAS1 protein (see SEQ NO. 3, position 574 marked in red) (refer to the Arabidopsis AHAS position, the same below), and a Trp-574-Leu mutation occurred in the BnAHAS3 protein (see SEQ NO. 4, position 574 marked in red). Both mutations can induce resistance to sulfonylurea herbicides, and the coexistence of both mutant genes can achieve a resistance intensity of 6 times the recommended herbicide concentration.

[0027] Based on this, in the spring of 2018, during the rapeseed flowering period, the non-GMO herbicide-resistant rapeseed variety Ning R101 (variety registration number: GPD rapeseed (2018) 320256) was used as the male parent, and the Chinese cabbage cultivar Aijiaohuang (Li Ying et al., Yangtze River Vegetables, 2021(23): 10-12) was used as the female parent for artificial pollination and hybridization. The seed setting was relatively poor, with about 1-3 seeds per pod. The Chinese cabbage pods were harvested in time at maturity to obtain distant hybrid seeds of Brassica napus and Chinese cabbage. In the autumn of 2018, the recipient parent Aijiaohuang and the hybrid F1 seeds were planted individually in 50-cell seed trays filled with nutrient soil. After the F1 hybrid seeds germinated and continued to grow to the 3-leaf stage, bensulfuron-methyl (a common sulfonylurea herbicide, Kuanxing®, 10% bensulfuron-methyl wettable powder, produced by Jiangsu Kuida Agrochemical Co., Ltd.) was used as a resistance screening agent. A working solution was prepared at half the recommended concentration and sprayed directly onto the 3-leaf stage hybrid seedlings. The parent herbicide, Dwarf Yellow, was not sprayed and served as a control. The visual observation method for seedling damage was as follows: Approximately one week after herbicide spraying, the seedling phenotype was checked. Using the un-sprayed Dwarf Yellow parent seedlings as a control, individual plants with visibly yellowed central leaves were marked with bamboo sticks. Two weeks after herbicide spraying, the seedlings were observed again. If the central leaves of the previously marked seedlings were found to be withered, these were considered non-resistant and could be removed. If the previously yellowed central leaves recovered and turned green, the damage was considered minor and these were considered resistant. At this point, the resistant F1 seedlings can be transplanted with soil into 30 cm diameter pots to continue growing. During the 2019 flowering period, artificial pollination and backcrossing were continued using the resistant F1 as the female parent and the parent dwarf yellow as the male parent. The seed setting rate remained relatively poor, with approximately 2-3 seeds / siliques. Backcrossing BC1 seeds were harvested promptly at maturity. BC1 seeds were then planted individually in 50-cell seedling trays filled with nutrient soil. Each BC1 offspring population was planted with at least 50 plants. After germination, when the seedlings reached the 3-leaf stage, bensulfuron-methyl (a common sulfonylurea herbicide, 10% bensulfuron-methyl wettable powder, produced by Jiangsu Kuida Agrochemical Co., Ltd.) was used as a resistance screening agent. A working solution was prepared at half the recommended concentration and sprayed onto the three-leaf stage hybrid seedlings, without spraying the parent dwarf yellow as a control. Resistant seedlings were selected using the same visual observation method as the F1 generation (see...). Figure 1 ), and at the same time, in order to confirm that BC1 generation individual plants carry resistance genes derived from Brassica napus (see Figure 2 Molecular identification was also performed.

[0028] To identify the herbicide resistance gene in Brassica napus transferred to the recipient Brassica rapa genome at the molecular level, the applicants also developed molecular markers targeting the mutation sites of the mutant gene. However, BnAHAS The development and design of molecular markers for homologous genes is a large undertaking and quite challenging, and is described in detail below.

[0029] The genome of Brassica napus contains five relatively homologous genes. BnAHAS Genes, among which BnAHAS2-4 Located in subgenome A, while BnAHAS1 and BnAHAS5 It is located in the C subgenome. From a gene function perspective, BnAHAS4 and BnAHAS5 It is a pseudogene. BnAHAS1 (C1 chromosome) and BnAHAS3 (Chromosome A1) contains constitutively expressed highly homologous (98%) genes. BnAHAS2 (Chromosome A5) is a flower bud-specific gene, and... BnAHAS1 and BnAHAS3 The homology is low. Therefore, it can be concluded that... BnAHAS1 (C1 chromosome) and BnAHAS3 (Chromosome A1) The two genes showed high homology. Sequence alignment revealed that the main differences between the two homologous gene DNA sequences (downloaded from NCBI, gene numbers Z11524 and Z11526) were: 39 SNP differences, one consecutive two-base difference, and one difference in the presence or absence of a 9-base pair (see [link to relevant documentation]). Figure 3 Since the mutation sites of these two genes are also SNP mutations, it can be predicted that designing a method targeting the differences in SNP mutation sites will be very difficult.

[0030] The following approaches were used in the primer design process: One approach involves designing primers based on the base differences at the SNP mutation site. The SNP is used as the 3' end of the designed primer for amplification. However, because there is only one base difference at the 3' end, even with adjustments to parameters such as annealing temperature, template concentration, and extension time, specific amplification cannot be achieved during PCR amplification. BnAHAS1 and BnAHAS3 The gene fragment was not the cause of this approach, which therefore failed.

[0031] Second, CAPS primers were designed to differentiate the mutation site by incorporating it into the restriction enzyme cleavage site. Specific sequences of restriction endonucleases were searched for around the mutation site, and it was found that the sequence containing the mutated base, "GCAATG," is precisely the recognition site of the restriction endonuclease BsrDI. However, comparison... BnAHAS1 and BnAHAS3 Gene sequencing revealed that the two genes had identical base sequences within the first 100 bp and last 150 bp surrounding the mutated base. This meant that even if the mutation site could be recognized by restriction endonucleases, the identical sequences of the two homologous genes prevented them from being separated, thus rendering the proposed approach ineffective.

[0032] Third, homologous gene fragments containing mutated bases are first separated, and then the mutated bases are identified. Based on this idea, the mutated base is used as the target, and the sequence is expanded forward and backward from it. Primers are designed to identify the differences between the two homologous genes upstream and downstream, hoping to obtain fragments containing the mutated bases of each gene through PCR amplification. The applicant designed primers using SNPs from upstream and downstream sequences, using the difference SNP between the two homologous genes as the last base at the 3' end of the primer. At the same time, they tried to artificially change the penultimate base at the 3' end to create stronger PCR amplification differences. After testing hundreds of upstream and downstream primer combinations and sequencing the amplification products, they finally screened out primers that could specifically amplify the mutated bases. BnAHAS1 , BnAHAS3 Primers for two gene fragments containing mutated base sites. The two primers are: 0531-4-F:AAAGCTCGAGGCGTTTGCG; 0531-8-R: GTAAGTCTGGGAACAAACCAAAAGC.

[0033] Using this pair of primers, specific amplification can be achieved. BnAHAS3 The target gene fragment can then be sequenced using first-generation sequencing to determine whether the mutation site contains the sensitive wild-type G (reverse sequencing shows C), the resistant homozygous mutant T (reverse sequencing shows A), or the resistant heterozygous mutant G / T (reverse sequencing shows C / A) (see...). Figure 4 This successful primer pair involved a significant amount of work and a substantial investment of effort from the applicant.

[0034] Phenotypic and molecular identification after herbicide application allows for the clear selection of BC1 generation herbicide-equipped organisms. BnAHAS1 -574 or BnAHAS3 The resistant progeny of the -574 mutation was transplanted into large flowerpots and backcrossed with the parent dwarf yellow plant during the flowering period to obtain BC2 generation seeds.

[0035] Starting with the BC2 generation, backcrossing was continuously carried out until the BC5 generation. After selecting resistant BC5 generation individual plants, they were self-pollinated individually during the flowering period, and the siliques were harvested. The siliques had poor seed set, with approximately 1-3 seeds per silique. The following year, seeds of the BC5F2 generation were harvested individually again. These BC5F2 generation seeds were then sown individually and identified using both phenotypic and molecular methods following herbicide application. Those exhibiting resistance were then selected. BnAHAS3 Single plants with the mutated gene were transplanted, along with several plants that showed the closest phenotype, into the breeding field for close proximity to planting. During the flowering period, these plants were covered with seed mulch and introduced with native bees to assist pollination and fruit setting. A large quantity of seeds were harvested at maturity, and in 2022, 14 seeds carrying the mutated gene were obtained. BnAHAS3 Mutant genes II. Identification of the genetic stability of non-GMO herbicide-resistant Chinese cabbage or Chinese cabbage-type rapeseed germplasm The above 14 with BnAHAS3 New Chinese cabbage lines with mutated genes and a genetic background of dwarf yellow (hereinafter referred to as "new Chinese cabbage lines") were numbered. Seeds of each number were sown individually in 50-cell trays, with at least 100 plants per number. The original parent, dwarf yellow, was sown in the same manner and meticulously managed until the three-leaf stage. Herbicide resistance was then tested, with dwarf yellow serving as a control. The test was conducted as follows: a working solution of bensulfuron-methyl (Kuanxing®, 10% bensulfuron-methyl wettable powder, produced by Jiangsu Kuida Agrochemical Co., Ltd.) at half the recommended concentration was prepared. Controls included spraying and no spraying. All new Chinese cabbage lines were sprayed. Approximately one week after herbicide application, the seedling phenotype was examined. Using the untreated parental dwarf yellow seedlings as a control, individual plants with noticeably yellowed central leaves were marked with bamboo sticks. Two weeks after herbicide application, the seedlings were observed again. If the central leaves of the previously marked seedlings were found to be withered, these were considered non-resistant and removed. If the previously yellowed central leaves recovered and turned green, the damage was considered minor and these were identified as resistant. Based on the resistance identification results, the resistance ratio of each line was recorded, and the resistant plants from each line were transplanted into the field. From this point on, the phenotype of each numbered plant was carefully observed, and growth consistency and characteristics were recorded. During the flowering period, five numbered plants were tied together with rope and covered with an 80-mesh nylon net, with approximately 100 bumblebees placed inside to assist pollination. After flowering, the net was removed, allowing the siliques to mature naturally. During the greening stage, samples were taken from the pods, the number of seeds per pod was counted, and the self-pollination and fruit setting of each number were compared. For other uncovered individual plants within each number, which underwent open pollination and fruit setting under natural conditions, samples were also taken from the pods during the greening stage, the number of seeds per pod was counted, and the natural fruit setting of each number was compared.

[0036] During the flowering period, a test was also conducted to determine the hybridization and seed setting. Single plants of each line that were not covered by the mulberry tree were used as one of the parents. Two other Chinese cabbage varieties, namely Dwarf Elf (Li Ying et al., Yangtze Vegetables, 2021(23):10-12) and Baiyuqing No. 1 (Plant Variety Rights No.: CNA20221008013, Jiangsu Academy of Agricultural Sciences), and two Chinese cabbage-type rapeseed varieties, namely Beiyou No. 4 (Registration No.: GPD Rapeseed (2018)630001) and Yangyou No. 3 (Registration No.: Suzhong Shenzi No. 287), were used as the second parents for pairwise reciprocal hybridization. After hybridization and pollination, the plants were covered with non-woven bags. After 3 weeks, the bags were removed, and the pods were cut off when the loquats turned yellow. The number of seeds in each pod was counted to obtain the F1 seeds of each pair of hybrids. The following year, each F1 seed was planted individually in 50-cell trays, with at least 10 seedlings planted for each number. The resistant parents (i.e., the new Chinese cabbage lines) and non-resistant parents of each combination served as controls. Herbicide resistance was tested at the three-leaf stage using the same method described above, with all parents and F1 seedlings sprayed. Seedling growth was checked after 3 weeks, and the resistance percentages of all parents and F1 seedlings were recorded.

[0037] Based on the comprehensive analysis of data on self-pollination, natural fruit setting, and resistance of F1 hybrids, strain number 47080 was selected as the optimal strain. After harvesting its seeds, they were sent to the China Center for Type Culture Collection (CCTCC, Wuhan University) for preservation, with the preservation number P202518 and the preservation date of September 3, 2025.

[0038] Molecular tests were performed on 47080 strains, including the following aspects: (1) Detection of the natural characteristics of Chinese cabbage or Chinese cabbage-type rapeseed. BRAHAS The presence of genes. This was determined by downloading the *Brassica rapa* reference genome from the website http: / / brassicadb.cn / # / and utilizing... BnAHAS The gene sequence was BLASTed on the Chinese cabbage reference genome to obtain the Chinese cabbage genome. BRAHAS Gene sequence (SEQ NO. 5). BRAHAS Gene sequence and rapeseed BnAHAS 1 and BnAHAS3 Gene sequences were compared, and three specific primers for the gene sequences were designed. PCR amplification was performed using genomic DNA from strain 47080 as a template. The results showed that no amplification was achieved. BRAHAS Gene sequence, but can only be amplified BnAHAS3 Gene sequences indicate that through distant hybridization, Brassica napus... BnAHAS3 The genome segment containing the gene is related to cabbage. BRAHASHomologous recombination occurred in the genomic region containing the gene sequence. (2) Whole-genome resequencing was performed on 47080 strains, and the sequencing reads were compared with the reference genome (Dwarf Yellow) to generate a BAM format alignment file. The original Chinese cabbage genome was then viewed in the IGV software. BRAHAS The location of the gene was discovered. BnAHAS3 The mutated gene was replaced. These results indicate that line 47080 invaded the Chinese cabbage genome through distant hybridization, introducing genes containing... BnAHAS3 The mutant gene fragment of Brassica napus gives it resistance to ALS inhibitor herbicides. Combined with the fact that the line has Chinese cabbage characteristics in its overall phenotype and genetics throughout the entire growth period, it also indicates that the genome of line 47080 is based on the Chinese cabbage genome, with some Brassica napus genome fragments infiltrated through distant hybridization.

[0039] III. Identification of resistance to specific herbicides in non-GMO herbicide-resistant Chinese cabbage or Chinese cabbage-type rapeseed germplasm. Given that the herbicide resistance of strain 47080 is based on the mutated BnAHAS gene, and that AHAS enzymes are the target enzymes of plant AHAS inhibitor herbicides, and that this class of herbicides has five subclasses, in addition to sulfonylureas (SU), imidazolinones (IMI), triazolopyrimidines (TP), pyrimidine oxybenzoates (PTB), and sulfonamide carbonyl diazolinones (SCT). Representative commercial herbicides from each of the five subcategories were selected: bensulfuron-methyl (Kuanxing®, 10% bensulfuron-methyl wettable powder, produced by Jiangsu Kuida Agrochemical Co., Ltd.) as the SU category representative; imidacloprid (5% imidacloprid aqueous solution, produced by Shandong Xianda Agrochemical Co., Ltd.) as the IMI category representative; diflubenzuron (50g / L suspension concentrate, produced by Shandong Lvba Chemical Co., Ltd.) as the TP category representative; bispyribac-sodium (10% suspension concentrate, produced by Hunan Nongda Haite Agrochemical Co., Ltd.) as the PTB category representative; and flufensulfuron-methyl (70% water-dispersible granules, produced by Jiangsu Ruibang Agrochemical Co., Ltd.) as the SCT category representative. The 47080 plant line was treated with the field recommended concentration (1X) of each commercial herbicide at the two-leaf stage, with the recipient parent, Dwarf Yellow, used as a control. Phenotypic results were observed two weeks after herbicide application. Figure 5 .

[0040] The parental strain, Dwarf Yellow, turned yellow and died after treatment with the five herbicides mentioned above, specifically manifested as yellowing of functional leaves and complete withering of the central leaves. In contrast, strain 47080 maintained green leaves two weeks after treatment with all five herbicides. Plants treated with SU, IMI, and SCT subclasses of herbicides were completely unaffected and maintained normal growth; TP herbicide treatment resulted in slight but not significant malformation of the central leaves, with little impact on plant growth; PTB herbicide treatment caused slight yellowing of functional leaves and significant malformation of the central leaves. This indicates that strain 47080 has achieved resistance to some (3 / 5) AHAS inhibitor herbicides at a level suitable for practical production applications.

[0041] IV. Resistance Transformation of Non-GMO Herbicide-Resistant Chinese Cabbage or Chinese Cabbage-Type Rapeseed Germplasm To verify the heritability of the resistance trait in line 47080, two Chinese cabbage varieties (for vegetable use) – Dwarf Elf (Li Ying et al., Yangtze Vegetables, 2021(23): 10-12) and Baiyuqing No. 1 (Plant Variety Rights No.: CNA20221008013, Jiangsu Academy of Agricultural Sciences) and two Chinese cabbage-type rapeseed varieties – Beiyou No. 4 (Registration No.: GPD Rapeseed (2018) 630001) and Yangyou No. 3 (Registration No.: Suzhongshenzi No. 287) – were selected as non-resistance recipients. Line 47080 was used as the resistance donor for hybridization, resulting in four F1 hybrids. After harvesting the F1 seeds, resistance was identified according to the above-mentioned herbicide resistance identification method. The results showed that all F1s were resistant to spraying with at least one times the recommended concentration of bensulfuron-methyl herbicide (Kuanxing®, 10% bensulfuron-methyl wettable powder, produced by Jiangsu Kuida Agricultural Chemical Co., Ltd.). F1 seedlings were transplanted to the field, and backcrossed with non-resistant parents during flowering. BC1 backcross seeds were harvested at maturity. Simultaneously, F1 individual plants underwent bud-removal self-pollination during flowering, and F2 seeds were harvested at maturity. Seeds from the segregating generations (BC1 and F2) of each combination were sown individually in 50-cell trays, and herbicide resistance was assessed at the three-leaf stage using the aforementioned method. The results showed that all BC1 generations exhibited a 1:1 segregation of herbicide resistance, and all F2 generations showed a 3:1 segregation. This indicates that the resistance trait conforms to a single dominant gene inheritance pattern, consistent with actual genetics. The results of this experiment demonstrate that the herbicide resistance trait of the 47080 line is heritable, and its inheritance pattern conforms to a single dominant gene inheritance pattern, allowing for the transfer of resistance according to classical genetics. Furthermore, the herbicide resistance trait of the 47080 line can be utilized for breeding using traditional methods.

[0042] Table 1. Resistance performance of different generations of hybrids

[0043] V. Field weeding patterns for non-GMO herbicide-resistant Chinese cabbage or Chinese cabbage-type rapeseed new lines Based on the preceding experiments, a weed control model for Chinese cabbage or Chinese cabbage-type rapeseed production, namely "planting resistant varieties + spraying specific herbicides," has been summarized. In production, the main producers plant resistant varieties, and sowing and field management follow traditional methods. After the seedlings emerge and grow to the three-leaf stage, the corresponding herbicide can be sprayed. This kills weeds if they are present, and prevents weed germination if they are absent. Simultaneously, the seedlings of Chinese cabbage or Chinese cabbage-type rapeseed are largely unharmed, allowing the resistant crop seedlings to grow healthily without the adverse effects of weeds. The F1 seeds obtained in Section 4 were planted in the production field, and 1X bensulfuron-methyl (Kuanxing®, 10% bensulfuron-methyl wettable powder, produced by Jiangsu Kuida Agricultural Chemical Co., Ltd.) was sprayed at the three-leaf stage. Two weeks later, the field was found to be virtually weed-free, and the crop seedlings were growing well (see...). Figure 6 ).

[0044] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent substitutions, and improvements made by those skilled in the art to the above embodiments without departing from the scope of the technical solution of the present invention, based on the technical essence of the present invention, shall still fall within the protection scope of the technical solution of the present invention.

Claims

1. A non-GMO herbicide-resistant Chinese cabbage or Chinese cabbage-type rapeseed variety, characterized in that: Its seeds are deposited at the China Center for Type Culture Collection (CCTCC) with accession number P202518 and deposit date of September 3, 2025.

2. The non-GMO herbicide-resistant Chinese cabbage or Chinese cabbage-type rapeseed variety according to claim 1, characterized in that: This strain is based on the Chinese cabbage genome and incorporates a segment of the rapeseed genome through distant hybridization.

3. The non-GMO herbicide-resistant Chinese cabbage or Chinese cabbage-type rapeseed variety according to claim 1, characterized in that: Its BrAHAS gene sequence is shown in SEQ NO.

5.

4. The method for cultivating non-GMO herbicide-resistant Chinese cabbage or Chinese cabbage-type rapeseed varieties according to any one of claims 1 to 3, characterized in that, Includes the following steps: S1: Using the non-GMO herbicide-resistant rapeseed variety Ning R101 as the male parent and a Chinese cabbage cultivar as the female parent, artificial pollination and hybridization were carried out to harvest F1 seeds of distant hybridization. S2: Sow the hybrid F1 seeds. When they grow to the three-leaf stage, use a herbicide as a resistance screening agent to spray the hybrid F1 seedlings at the three-leaf stage and screen out resistant F1 individual plants. S3: Transplant the resistant F1 single plants with soil to continue growing. In the following year, during the flowering period, use the resistant F1 single plants as the female parent and the Chinese cabbage cultivar as the male parent for artificial pollination and backcrossing to harvest BC1 generation seeds. S4: Sow BC1 generation seeds. When the seedlings reach the three-leaf stage, use herbicides as resistance screening agents to spray the BC1 generation seedlings at the three-leaf stage and screen out resistant BC1 individual plants. S5: Phenotypic and molecular identification of resistant BC1 single plants, and screening for resistant progeny single plants with gene mutations; S6: Transplanting single plants of the resistant offspring with gene mutations and then backcrossing them with the parental Chinese cabbage cultivar during the flowering period to obtain BC2 generation seeds; S7: Continue backcrossing the BC2 generation seeds with the Chinese cabbage cultivar parents for multiple generations to obtain the final resistant individual plants. The resistant individual plants are then screened for the optimal line based on self-pollination seed setting, natural seed setting, and hybridization resistance, and their seeds are harvested.

5. The method for cultivating non-GMO herbicide-resistant Chinese cabbage or Chinese cabbage-type rapeseed varieties according to claim 4, characterized in that: In steps S2 and S4, the parent Chinese cabbage cultivar was sown simultaneously, with the three-leaf stage seedlings of the parent Chinese cabbage cultivar that were not sprayed with herbicides serving as a control.

6. The method for cultivating non-GMO herbicide-resistant Chinese cabbage or Chinese cabbage-type rapeseed varieties according to claim 4, characterized in that: The non-GMO herbicide-resistant rapeseed variety Ning R101 contains two mutation sites: the gene BnAHAS1 located on chromosome C01, with the sequence shown in SEQ NO.1, and the gene BnAHAS3 located on chromosome A01, with the sequence shown in SEQ NO.

2. Correspondingly, the BnAHAS1 protein exhibits a Trp-574-Leu mutation, with the sequence shown in SEQ NO.3, and the BnAHAS3 protein exhibits a Trp-574-Leu mutation, with the sequence shown in SEQ NO.

4.

7. The method for cultivating non-GMO herbicide-resistant Chinese cabbage or Chinese cabbage-type rapeseed varieties according to claim 6, characterized in that: In step S5, the method for phenotypic and molecular identification of resistant BC1 single plants is as follows: homologous gene fragments containing mutant bases from BnAHAS1 and BnAHAS3 are separated to determine the mutant bases. Using the mutant bases as targets, the sequences are expanded forward and backward around them. Primers are designed to find the difference points between the two homologous genes upstream and downstream, and fragments containing mutant bases of each gene are obtained by PCR amplification.

8. The method for cultivating non-GMO herbicide-resistant Chinese cabbage or Chinese cabbage-type rapeseed varieties according to claim 7, characterized in that: Primers capable of specifically amplifying the BnAHAS1 and BnAHAS3 gene fragments were selected and are listed below: AAAGCTCGAGGCGTTTGCG; GTAAGTCTGGGAACAAACCAAAAGC.

9. The application of the non-GMO herbicide-resistant Chinese cabbage or Chinese cabbage-type rapeseed varieties as described in claim 1, characterized in that: Herbicides can be sprayed in the early stages of planting to kill weed seedlings in the early stages without affecting the growth of Chinese cabbage or Chinese cabbage-type rapeseed.

10. The application of the non-GMO herbicide-resistant Chinese cabbage or Chinese cabbage-type rapeseed varieties according to claim 9, characterized in that: The selected Chinese cabbage variety is Dwarf Yellow; the selected herbicide is a sulfonylurea herbicide.

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