Application of recessive nuclear male sterility gene BoaGMS1 in regulation of male development in Brassica campestris

Abstract

This invention discloses a recessive nuclear male sterility gene. BoaGMS1 Application in regulating male development in Chinese kale. This invention screens recessive nuclear male sterility mutants from Chinese kale breeding and isolates the Chinese kale male fertility gene using forward genetics. BoaGMS1 Further experiments confirmed that BoaGMS1 A gene mutation resulted in recessive nuclear male sterility in Chinese kale. Meanwhile, targeting... BoaGMS1 Cosegregating molecular markers developed for male-sterile lines can be used for identifying fertility alleles in plants, screening target plants in marker-assisted breeding, and determining seed purity. Furthermore, they can be used in the development of kale. BoaGMS1 Male-sterile lines can be applied to the breeding and production of new hybrid varieties of Chinese kale. Therefore, the male fertility gene for Chinese kale provided by this invention... BoaGMS1 BoaGMS1 BoaGMS1 BoaGMS1 BoaGMS1 BoaGMS1 BoaGMS It plays an important role in the utilization of heterosis and hybrid seed production of Chinese kale and even Brassica oleracea vegetables.

Description

Technical Field

[0001] This invention belongs to the field of plant biotechnology breeding, specifically involving recessive nuclear male sterility genes. BoaGMS1 Application in regulating male development of Chinese kale. Background Technology

[0002] Heterosis utilization is an effective way to improve crop yield and stress resistance. To date, heterosis has been widely applied in field crops such as corn and rice, as well as horticultural plants such as tomatoes, peppers, and cucumbers. Chinese kale, a vegetable crop belonging to the Brassicaceae family, exhibits significant heterosis in traits such as yield, quality, disease resistance, and stress resistance. Compared with conventional varieties, Chinese kale hybrids have advantages such as uniform emergence, uniform growth, and consistent harvest time. The breeding and promotion of Chinese kale hybrid varieties are beneficial to improving the marketability and economic benefits of Chinese kale sprouts. Therefore, breeding high-quality, high-yield, and highly marketable Chinese kale hybrid varieties is the main development direction of Chinese kale breeding.

[0003] Due to the small size of Chinese kale flowers, artificial emasculation and pollination for F1 hybrid seed production is impractical. Currently, the main methods for utilizing heterosis in Chinese kale are self-incompatible lines (SI) and cytoplasmic male sterility (CMS). However, the cost of propagating self-incompatible lines is high, limiting the acreage due to seed production volume. Furthermore, continuous self-pollination of parents can lead to viability degradation, resulting in the loss of some desirable traits and making it difficult to achieve 100% hybridization in hybrids, with a high likelihood of false hybrids in offspring. In addition, because Chinese kale lacks naturally occurring mutant CMS materials, the CMS lines used rely entirely on transfer from other closely related crops. However, most heterologous cytoplasmic CMS materials often suffer from unstable sterility, abnormal flower organ development, and poor fruit setting due to the influence of heterologous cytoplasm, leading to low efficiency in sterility line selection in practical applications. Moreover, it is often difficult to find a corresponding restorer line within a crop for CMS cytoplasm introduced heterologously, further limiting the utilization of sterility lines.

[0004] Genic male sterility (GMS) is a natural phenomenon widely found in higher plants, with the vast majority being recessive gene mutations. Recessive nuclear male sterility mutants are excellent male-sterile materials in crop hybrid production. During hybrid seed production, the paternal parent, containing wild-type alleles, can act as a "restoring factor" for the recessive alleles of the male-sterile mutant. Crossing the male-sterile line controlled by the recessive nuclear gene with a homozygous fertile plant yields the fertile F1 generation. With the development of molecular breeding techniques, especially the emergence of a new "seed production technology" (SPT), genogenic male sterility materials are receiving increasing attention and application from breeders.

[0005] Currently, male sterility genes have been identified in various plants. For example, Chinese patent CN116286851A discloses a recessive male sterility gene in maize. ms13-6060 This mutant is completely male-sterile and does not produce pollen grains, possessing significant theoretical and applied value in the utilization of heterosis in maize and in the process of sterile hybridization for seed production. Chinese patent CN113862278A discloses upland cotton... GhMS20 The application of this gene in creating a single dominant male-sterile line in cotton. This male-sterile cotton line can be used to produce hybrid seeds, develop new two-line hybrid cotton, perform recurrent selection, and create gene banks, which has very important applications in agricultural production. Chinese patent CN113088524A discloses a male-sterile gene for Chinese cabbage. Brams Application in the breeding of recessive nuclear male sterile lines of Chinese kale. However, there is currently no publicly available technology disclosing the application of male sterility genes in Chinese kale in the breeding of dominant and recessive male sterile lines of Chinese kale. Summary of the Invention

[0006] The purpose of this invention is to overcome the aforementioned defects and shortcomings in the existing technology and to provide a recessive nuclear male sterility gene for Chinese kale. BoaGMS1 Application in regulating male development of Chinese kale.

[0007] The second objective of this invention is to provide a recessive nuclear male sterility gene for Chinese kale. BoaGMS1 Application of mutants in regulating male development of Chinese kale.

[0008] The third objective of this invention is to provide a recessive nuclear male sterility gene for Chinese kale. BoaGMS1 Or the recessive nuclear male sterility gene in Chinese kale BoaGMS1 Application of mutants in the cultivation of male-sterile lines of Chinese kale.

[0009] The fourth objective of this invention is to provide a method for cultivating a recessive nuclear male-sterile line of Chinese kale.

[0010] The fifth objective of this invention is to provide the application of the recessive nuclear male-sterile line of Chinese kale obtained by the above-described method in Chinese kale breeding.

[0011] The sixth objective of this invention is to provide a method for identifying recessive nuclear male sterility genes in Chinese kale. BoaGMS1 Application of functional molecular markers in molecular-assisted selection breeding of Chinese kale.

[0012] The above-mentioned objective of this invention is achieved through the following technical solution: In their field breeding practice, the inventors' team discovered a male-sterile mutant through multi-generational tracking and systematic selection. This mutant plant exhibits significantly shortened filaments and abnormal anther development, specifically characterized by significantly smaller and thinner anthers, and a complete lack of pollen. Pollination of the mutant with wild-type (WT) kale pollen, combined with field plant phenotypic analysis, genetic segregation analysis, and cytological section observation, confirmed that the previously screened male-sterile mutant is a recessive nuclear male-sterile condition controlled by a single gene, specifically pollen-free abortion caused by abnormal development of the tapetum layer in the kale anthers. The gene was located and cloned, revealing high homology with a known male-sterile gene in the model plant Arabidopsis thaliana, and named [gene name missing]. BoaGMS1 The gDNA sequence of this gene is shown in SEQ ID No. 1, its CDS sequence is shown in SEQ ID No. 2, and the encoded amino acid sequence is shown in SEQ ID No. 3. BoaGMS1 It is specifically expressed and localized in the nucleus during the St7-St9 stage of the anther. BoaGMS1 After gene mutation, its expression pattern changes, which in turn affects anther development, but does not affect the subcellular localization of the protein.

[0013] To further validate candidate genes BoaGMS1 Functionally, a gene complementation verification vector was constructed, and it was heterologously transformed into Arabidopsis thaliana homologous gene T-DNA insertion mutants. The results showed that... BoaGMS1 The introduction of the gene effectively rescued the male sterility phenotype in Arabidopsis homologous mutants, thus confirming the candidate gene. BoaGMS1 This refers to the functional gene controlling the recessive nuclear male sterility trait. In plant breeding (especially nuclear male sterility GMS), recessive sterility (msms) has significant advantages over dominant sterility (Ms_) in terms of seed production safety, restorer line breadth, reproductive stability, genetic manipulation, and population improvement, making it the more mainstream choice for hybridization breeding. (The text then abruptly shifts to a seemingly unrelated topic: "Using recessive nuclear male sterile plants of Chinese kale...") boagms1 Test-cross hybridization was conducted with inbred lines of Chinese kale from different sources. The hybrid combinations showed relatively significant advantages over the control in terms of growth, yield, and quality. These are the male-sterile Chinese kale materials. boagms1 It has the potential to be cultivated into a high-quality sterile line.

[0014] Therefore, this invention provides a recessive nuclear male sterility gene for Chinese kale. BoaGMS1 Application in regulating male development in Chinese kale, the aforementioned BoaGMS1 The amino acid sequence encoding the protein is shown in SEQ ID No. 3.

[0015] Furthermore, the development is described as recessive male sterility in Chinese kale.

[0016] Furthermore, the aforementioned BoaGMS1 The CDS sequence is shown in SEQ ID No. 2.

[0017] Furthermore, the aforementioned BoaGMS1 The gDNA sequence is shown in SEQ ID No. 1.

[0018] This invention also provides a male sterility gene for Chinese kale. BoaGMS1 A loss-of-function mutant, which in BoaGMS1 A GT base deletion occurs in the third exon of the gene at a position 1984 bp from the start codon, resulting in the amino acid sequence of the encoded protein as shown in SEQ ID No. 6. The CDS sequence of the mutant is shown in SEQ ID No. 5, and the gDNA sequence of the mutant gene is shown in SEQ ID No. 4, causing Chinese kale to exhibit recessive nuclear male sterility (i.e., abnormal development of the anther in the male organ and absence of pollen).

[0019] Therefore, this invention provides a recessive nuclear male sterility gene for Chinese kale. BoaGMS1 The application of mutants in regulating male development in Chinese kale, the aforementioned BoaGMS1 The amino acid sequence of the mutant protein is shown in SEQ ID No. 6.

[0020] This invention also provides a recessive nuclear male sterility gene for Chinese kale. BoaGMS1 Or the recessive nuclear male sterility gene in Chinese kale BoaGMS1 The application of mutants in the cultivation of male-sterile lines of Chinese kale, the aforementioned BoaGMS1 The amino acid sequence encoding the protein is shown in SEQ ID No. 3; BoaGMS1 The amino acid sequence of the mutant protein is shown in SEQ ID No. 6.

[0021] This invention also provides a method for cultivating a recessive nuclear male-sterile line of Chinese kale, which utilizes gene editing technology to target the recessive nuclear male-sterile gene in Chinese kale. BoaGMS1 Targeted editing was performed to introduce loss-of-function mutations, resulting in a recessive nuclear male-sterile line of Chinese kale. boagms1 ); the BoaGMS1 The amino acid sequence encoding the protein is shown in SEQ ID No. 3.

[0022] Furthermore, the method also includes the identification of recessive nuclear male sterile lines of Chinese kale using functional molecular marker pairs; the primer sequences of the functional molecular marker pairs are shown in SEQ ID No. 7 and SEQ ID No. 8.

[0023] Furthermore, the method also includes using a recessive nuclear male-sterile line of Chinese kale ( boagms1 Backcrossing and breeding were carried out into inbred lines of Brassica oleracea or other Brassica oleracea crops with different genetic backgrounds to obtain recessive nuclear male sterile lines of Brassica oleracea or other Brassica oleracea crops with different genetic backgrounds.

[0024] Because this gene can maintain the fertility stability of its sterile lines, it can be widely used to create hybrid combinations with Chinese kale inbred lines, thus extending its application to other male-sterile lines of Brassica vegetables.

[0025] The present invention also provides the application of the recessive nuclear male sterile line of Chinese kale obtained by the above method in Chinese kale breeding.

[0026] Furthermore, the breeding process involves using the recessive nuclear male-sterile line of Chinese kale obtained by the above method as the female parent, and crossing it with the male parent that has hybrid vigor to produce hybrid F1 generation.

[0027] Given the mutant BoaGMS1 A 2 bp (CT) deletion in the third exon of a gene causes male sterility in Chinese kale. This invention develops a functional high-resolution melting curve (HRM) molecular marker targeting this mutation site. The primer sequences are shown in SEQ ID No. 7 and SEQ ID No. 8, respectively. This HRM molecular marker can rapidly, accurately, and efficiently identify... BoaGMS1 Different allelic genotypes of the gene offer advantages such as ease of operation, high throughput, and closed-tube detection to avoid cross-contamination, further establishing a technology system for assisted selection breeding of this gene. This invention provides important gene resources and breeding tools for utilizing heterosis in Chinese kale, significantly improving the breeding efficiency of high-quality Chinese kale hybrid combinations, and has significant application value in hybrid breeding of Chinese kale and Brassica oleracea crops.

[0028] Therefore, the present invention also provides a method for identifying recessive nuclear male sterility genes in Chinese kale. BoaGMS1 The application of functional molecular marker pairs in molecular-assisted selection breeding of Chinese kale, the primer sequences of the functional molecular marker pairs are shown in SEQ ID No. 7 and SEQ ID No. 8.

[0029] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a recessive nuclear male sterility gene. BoaGMS1Application in regulating male development in Chinese kale. This invention screens recessive nuclear male sterility mutants from Chinese kale breeding and isolates the Chinese kale male fertility gene using forward genetics. BoaGMS1 Further analysis, including genetic analysis, cytological section observation, sequence alignment, gene expression pattern detection, and heterologous complementation verification of Arabidopsis allele mutants, confirmed that... BoaGMS1 A gene mutation resulted in recessive nuclear male sterility in Chinese kale. Meanwhile, targeting... boagms1 Cosegregating molecular markers developed for male-sterile lines can be used for identifying fertility alleles in plants, screening target plants in marker-assisted breeding, and determining seed purity. Furthermore, they can be used in the development of kale. boagms1 Male-sterile lines can be applied to the breeding and production of new hybrid varieties of Chinese kale. Therefore, the male fertility gene for Chinese kale provided by this invention... BoaGMS1 It plays an important role in the utilization of heterosis and hybrid seed production of Chinese kale and even Brassica oleracea vegetables. Attached Figure Description

[0030] Figure 1 It is a recessive nuclear male sterile mutant of Chinese kale. boagms1 The anther and pollen phenotypes of wild-type plants. Among them, Figure 1 In the diagram, A represents the flower morphology of a fertile wild-type Chinese kale (WT) plant; B represents a male-sterile mutant. boagms1 Flower morphology; C represents the WT fertile plant (left) and the sterile mutant. boagms1 (Right) Morphology of stamens and pistils; D represents the WT fertile plant (left) and the sterile mutant. boagms1 (Right) Anther morphology; E shows I2-KI2 staining of pollen from WT fertile plants; F shows sterile mutant. boagms1 Pollen I2-KI2 staining. Note: AD plot, bar = 2000 μm; EF plot, bar = 500 μm.

[0031] Figure 2 This section compares the anther development process of the mutant and the wild-type. Among them, Figure 2 In the diagram, AB represents the tetrad stage; CF represents the uninucleate pollen stage; GH represents the binucleate pollen stage; and IJ represents the trinucleate pollen stage. BMs are binucleate microspores; DMs are degenerate microspores; E is the epidermis; En is the anther chamber wall; M is the mesonephaly; MP is mature pollen; Ms are microspores; PMC is pollen mother cells; T is the tapetum; and Tds are tetrads. Note: Bar is 50 μm.

[0032] Figure 3 for BoaGMS1Localization and cloning of genes. In the top figure, the dots represent SNPs, the horizontal axis represents their location on each chromosome, and the vertical axis represents the SNP index. A higher SI value indicates a better linkage. The middle figure (M1-M9) shows the nine developed localization molecular markers and the genotypic analysis of each recombinant; F represents the fertile phenotype, and S represents the sterile phenotype. The bottom figure illustrates candidate gene mutation sites.

[0033] Figure 4 for BoaGMS1 Analysis of relative gene expression levels in different tissues, organs, and anther development stages. Figure 4 In this context, A represents the fertile (WT) kale tissues. BoaGMS1 Gene expression analysis; B represents the anther developmental stages of fertile plants (WT) and sterile mutants (Mutant). BoaGMS1 Gene expression level analysis. Internal reference genes were used. BoaTublin Each sample was tested three times in total.

[0034] Figure 5 Subcellular localization analysis of wild-type and mutant BoaGMS1 protein. Note: Subcellular localization of wild-type BoaGMS1-GFP (AD) and mutant Boagms1-GFP (EH) in tobacco leaf epidermal cells. NLS-RFP was used as a nuclear marker. Scale bar, 50 μm.

[0035] Figure 6 for pBoaGMS1:BoaGMS1 Heterologous replacement of Arabidopsis thaliana homologous mutants. Among them, Figure 6 In the diagram, A represents the morphology and fertility of the T-DNA insertion mutant plant; B represents the morphology and fertility of the replacement line plant; C represents the silique; and D represents the anther Alexandrite staining. The left side represents the wild type, the middle side represents the mutant, and the right side represents the replacement line. Scale bar, AC: 1 cm, D: 50 μm.

[0036] Figure 7 for BoaGMS1 HRM detection results of gene-specific molecular marker primer amplification products.

[0037] Figure 8 This diagram shows the field performance of test-cross combinations of male-sterile Chinese kale hybrids. Note: A, B, C, and D in the diagram represent F1 generation hybrid combinations of different Chinese kale inbred lines and male-sterile lines, respectively. Detailed Implementation

[0038] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0039] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0040] Example 1: Phenotypic identification and genetic analysis of recessive nuclear male sterile materials from Chinese kale The male sterility mutant material used in this invention boagms1 The inventors' team discovered a male-sterile mutant material through multi-generational tracking observation and systematic selection in field breeding practices.

[0041] Field phenotypic observation results Figure 1 As shown, the mutant and the wild-type plant showed no significant differences in overall morphology at different stages of growth. Figure 1 AB). Further detailed observation and comparison of floral organs revealed no significant differences between the two in the morphology and length of the pistil (stigma); however, the mutant plant exhibited significantly shortened filaments and abnormal anther development, specifically manifested as significantly smaller anthers overall and a relatively slender shape. Figure 1 CD). Furthermore, the mutant completely lacks pollen, exhibiting typical pollen-free male sterility characteristics. Figure 1 EF).

[0042] Pollination of the mutant with wild-type (WT) Chinese kale pollen resulted in fertile F1 generation plants, indicating that the male sterility trait controlled by the mutant gene is recessive. Further artificial pollination and bagging self-pollination were performed on individual F1 plants, and seeds were harvested to construct an F2 segregating population. Statistical analysis of fertility segregation in different populations was conducted, and the results are shown in Table 1. Regardless of whether the plants were grown in Shenzhen, Guangdong or Zhangye, Gansu, the segregation ratio of sterile to fertile plants in the F2 segregating population consistently met the chi-square test of 3:1. These results indicate that the male sterility trait of this mutant conforms to Mendelian single-gene recessive inheritance patterns.

[0043] Table 1 boagms1 Statistics on F2 segregation of mutants

[0044] Example 2: Observation of cytological sections of Chinese kale anthers To investigate mutants boagms1 The causes of male sterility (no pollen) were first determined by comparing the total fertility (WT) with that of semi-thin sections of anthers at various developmental stages. boagms1 (Mutant) Anther development process, results as follows Figure 2 As shown, the differences between the two occurred in the mid-to-late developmental stages. During the tetrad stage, there was no significant difference between the wild type and the mutant; both microspore mother cells could complete meiosis to become tetrads. However, the innermost tapetal cells of the anther wall were enlarged and had denser cytoplasm, with significantly deeper staining than the outer three layers of wall cells. Figure 2A–B). By the early uninucleate pollen stage, the microspores of both the wild type and the mutant had freed from the tetrad and small central vacuoles had appeared. Figure 2 C–D). Differences emerged between the two types of tapetum: in the wild type, the tapetum continued to swell and developed numerous small vacuoles, indicating a period of vigorous secretion where lipids and proteins necessary for microspore development were transported into the spore chamber. Figure 2 C); The mutant tapetum cells were lightly stained and showed large vacuolar structures (C). Figure 2 D). In the subsequent late uninucleate pollination stage, the tapetum of the wild type becomes thinner and darker in color than in the previous stage, with reduced vacuolation, while the microspores increase in size and the central vacuole enlarges (D). Figure 2 E). The mutant tapetum vacuoles showed a significantly abnormally large volume, while the vacuoles in the microspores shrank or even disappeared. Figure 2 F). During the binucleate pollen stage, the wild-type tapetum significantly degenerates and thins, vacuoles almost disappear, and microspores become binucleate pollen grains, accumulating large amounts of nutrients such as starch within the cells. Figure 2 G); however, in the mutant, the tapetum is thinner, the vacuoles still occupy a large volume, the microspores do not accumulate nutrients, the cell contents disappear abnormally, and the pollen grains shrivel. Figure 2 H). By the trinuclear pollen stage, the tapetum of the wild type has completely degraded, and the pollen grains are mature and plump. Figure 2 I); while in the mutant, the anther cavity completely collapsed, and the pollen grains were more severely wrinkled (I); Figure 2 J). In summary, the semi-thin section analysis suggests that pollen abortion in SS may be caused by abnormal tapetum function in the early stages of mononuclear pollen development, leading to a delay in the degradation process.

[0045] Example 3: Male sterility gene in Chinese kale BoaGMS1 Gene localization and cloning After confirming that the mutant phenotype is controlled by a single recessive nuclear gene, preliminary localization was performed using the SIMM (Simultaneous Identification of Multiple Mutations) gene mapping method. Thirty plants with sterile phenotypes and 30 plants with fertile phenotypes were randomly selected from the F2 segregating population. DNA was extracted from each, and the samples were mixed in equal volumes and resequencing using Illumina HiSeq 2000, yielding 18 Gb of data with an average coverage of 30x. Candidate regions for the mutants were obtained by eliminating SNP sites in the control material where the proportion of non-mutated sites did not match the sequencing depth. The candidate regions were further screened by calculating the mutation frequency and the Euclidean distance (ED) of the mutation sites. SNP sites with an SNP index close to 1 on different chromosomes were considered predicted candidate sites, with chromosome 9 being particularly prominent. Figure 3 ).

[0046] Linkage analysis of segregating populations confirmed that the SNP site in this region was closely linked to the sterility gene. Further molecular markers were developed within this region, and map-based cloning was used to perform localization analysis on over 4900 F2 segregating populations. Ultimately, the gene was located in a region of approximately 60.48 kb between markers M4 and M7 on the long arm of chromosome 9 of Chinese kale. Figure 3 Genome sequence analysis of this region predicted the presence of three ORFs. Further sequencing and alignment analysis of the promoters and coding regions of these three ORFs from fertile and sterile plants revealed no difference between ORF1 and ORF2 sequences. However, the mutant ORF3 exhibited a two-base (CT) deletion in the third exon, resulting in a frameshift mutation and a corresponding 48 bp extension at the 3' end. Homology comparison of the gene's protein sequence indicated that ORF3 is highly homologous to a known male sterility gene in the model plant Arabidopsis thaliana, encoding a transcription factor with transcriptional activation activity. This gene is tentatively named... BoaGMS1 ( Brassica oleracea var. alboglabra L. Genic Male Sterility 1)( Figure 3 ).

[0047] Example 4 BoaGMS1 Gene expression pattern analysis Wild-type fertile plants (WT) and male-sterile mutants were extracted separately. boagms1 RNA from anthers at nine different developmental stages (St6-St14), as well as from roots, stems, leaves, and pistils of fertile plants, was reverse transcribed into cDNA and used to select candidate genes. BoaGMS1 Perform qRT-PCR analysis.

[0048] The results are as follows Figure 4 As shown, it indicates BoaGMS1 It is specifically expressed during the anther development stages St7-St9, with the highest expression level observed during the anther development stage St8. Figure 4 A). Compared to the wild type, the anther development of male-sterile plants at various stages... BoaGMS1 Gene expression levels were significantly higher than in wild-type fertile plants, and gene expression was delayed ( Figure 4 B). The above results indicate that... BoaGMS1 After gene mutation, its expression pattern changes, which in turn affects the development of anthers.

[0049] Example 5: Subcellular localization analysis of BoaGMS1 protein To clarify the subcellular localization of the BoaGMS1 protein, it was cloned from both wild-type and mutant materials. BoaGMS1 The cDNA sequence of the gene. The stop codon will be removed. BoaGMS1 and Boagms1The CDS sequence of the gene was fused with the green fluorescent protein (GFP) gene to construct a transient expression vector. After verification by sequencing, the vector was transformed into Agrobacterium GV1301. Using the nuclear localization signal protein NLS-RFP as a nuclear marker control, Agrobacterium carrying both the recombinant plasmid and the control plasmid were co-injected into leaves of *Nicotiana benthamiana* for transient co-expression. The injected tobacco plants were cultured under normal conditions for 48 hours, and the distribution of fluorescence signals was observed using a laser confocal microscope.

[0050] The results are as follows Figure 5 As shown, the fluorescence signals of both wild-type BoaGMS1-GFP and mutant Boagms1-GFP fusion proteins completely overlap with the NLS-RFP-labeled nuclear locations, indicating that both are localized in the nucleus. This demonstrates that the 16 amino acids added to the C-terminus of the mutant Boagms1 protein due to sequence variation do not alter its nuclear localization characteristics and do not affect the protein's subcellular localization.

[0051] Example 6 Candidate Genes BoaGMS1 Back-up verification To further validate candidate genes BoaGMS1 Functionally, a gene complementation verification vector was constructed. This vector contained a promoter of Arabidopsis homologous genes. BoaGMS1 Genome fragments were cloned into the pCAM1300 vector to obtain pAtMS1:BoaGMS1-pCAM1300 Complementary expression vectors were used, and their heterologous transformation was performed on Arabidopsis thaliana homologous gene T-DNA insertion mutants. Positive complemented transgenic plants were obtained through screening and named [the following]. BoaGMS1-COM By using potassium iodide staining and stereomicroscopic observation, the... BoaGMS1-COM A comparative analysis was conducted on the pollen fertility and plant growth of complementary transgenic plants.

[0052] The results are as follows Figure 6 As shown, BoaGMS1-COM The pollen quantity and anther staining (Alexandrite staining) of the plants were basically consistent with the wild type, both showing fertility, while the mutants exhibited the typical sterility characteristic of having no pollen. Furthermore, BoaGMS1-COM The plant is growing well and the pods are developing well, and fertility has been fully restored. Figure 6 The above complementary experimental results show that... BoaGMS1 The introduction of the gene effectively rescued the male sterility phenotype in Arabidopsis homologous mutants, thus confirming the candidate gene. BoaGMS1 These are the functional genes that control this male infertility trait.

[0053] Example 7 BoaGMS1 Development and application of gene functional molecular markers Given the mutant BoaGMS1A 2 bp (CT) deletion exists in the third exon of the gene, which is the molecular basis for male sterility in Chinese kale. Primers were designed targeting this specific site, and a functional molecular marker based on high-resolution melting (HRM) technology was developed, named BoaGMS1-HRM. The primer sequence of this marker is as follows: BoaGMS1-HRM-F: 5'-GTGTTCAACATAACGAGGAAG-3' (SEQ ID No. 7); BoaGMS1-HRM-R: 5'-CTCAAATAAACACACGTGAGG-3' (SEQ ID No. 8).

[0054] The genomic DNA of wild-type, mutant, and F1 hybrid progeny materials was amplified by PCR using the above primers. The amplification products were analyzed by high-resolution melting curve analysis using a Biorad CFX96 real-time quantitative PCR instrument. The genotypes of the samples were identified by collecting the fluorescence signals of the melting curves and drawing melting maps.

[0055] The results are as follows Figure 7 As shown, based on the differences in peak shape of the melting curves, the tested materials can be clearly distinguished into three genotypes: wild-type homozygous (red curve), mutant homozygous (blue curve), and heterozygous (gray curve). This HRM molecular marker can rapidly, accurately, and efficiently identify... BoaGMS1 Different allelic genotypes of the gene offer advantages such as ease of operation, high throughput, and closed-tube detection to avoid cross-contamination. The successful development of this functional molecular marker provides a reliable technical means for assisted selection breeding of male sterility in Chinese kale, and can be used for early screening and identification of male sterile lines, significantly improving breeding selection efficiency.

[0056] Example 8 Field performance of hybrid test combinations of Chinese kale nuclear male sterile materials Using sterile plants boagms1 Test crosses were performed with Chinese kale inbred lines from different genetic backgrounds.

[0057] The results are as follows Figure 8 As shown, the results indicate that the male-sterile kale material exhibits excellent combining ability, not only enabling it to be widely hybridized with kale inbred lines from different backgrounds for seed production, but also demonstrating a very high seed set rate after small-scale bee pollination of the sterile plants, significantly improving the seed production efficiency of the kale male-sterile lines. Furthermore, after continuous trials in Guangdong, Ningxia, and other regions, the hybrids have shown good commercial traits and field production performance, with some hybrid combinations exhibiting relatively significant advantages over the control in terms of growth, yield, and quality. These results demonstrate that the male-sterile kale material... boagms1 It has the potential to be cultivated into a high-quality sterile line.

Claims

1. Recessive nuclear male sterility gene in Chinese kale BoaGMS1 Its application in regulating male development in Chinese kale is characterized by, The BoaGMS1 The amino acid sequence encoding the protein is shown in SEQ ID No.

3.

2. The application according to claim 1, characterized in that, The BoaGMS1 The CDS sequence is shown in SEQ ID No.

2.

3. Recessive nuclear male sterility gene in Chinese kale BoaGMS1 The application of mutants in regulating male development in Chinese kale, characterized by: The BoaGMS1 The amino acid sequence of the mutant protein is shown in SEQ ID No.

6.

4. Recessive nuclear male sterility gene in Chinese kale BoaGMS1 Or the recessive nuclear male sterility gene in Chinese kale BoaGMS1 The application of mutants in the cultivation of recessive nuclear male-sterile lines of Chinese kale, characterized by: The BoaGMS1 The amino acid sequence encoding the protein is shown in SEQ ID No. 3; BoaGMS1 The amino acid sequence of the mutant protein is shown in SEQ ID No.

6.

5. A method for cultivating a recessive nuclear male-sterile line of Chinese kale, characterized in that, To utilize gene editing technology to target the recessive nuclear male sterility gene in Chinese kale BoaGMS1 Targeted editing was performed to introduce loss-of-function mutations, resulting in a recessive nuclear male-sterile line of Chinese kale; BoaGMS1 The amino acid sequence encoding the protein is shown in SEQ ID No.

3.

6. The method according to claim 5, characterized in that, The method further includes the identification of recessive nuclear male sterile lines of Chinese kale using functional molecular marker pairs; the primer sequences of the functional molecular marker pairs are shown in SEQ ID No. 7 and SEQ ID No.

8.

7. The method according to claim 5, characterized in that, The method also includes backcrossing and transferring the recessive nuclear male sterile line of Chinese kale into inbred lines of Chinese kale or cabbage and other Brassica vegetables with different genetic backgrounds to obtain recessive nuclear male sterile line materials of Chinese kale or cabbage and other Brassica crops with different genetic backgrounds.

8. The application of the recessive nuclear male sterile line of Chinese kale obtained by the method described in any one of claims 5 to 7 in Chinese kale breeding.

9. The application according to claim 8, characterized in that, The breeding method involves using a recessive nuclear male-sterile line of Chinese kale obtained by any of the methods described in claims 5 to 7 as the female parent, and crossing it with a male parent that has hybrid vigor to produce hybrid F1 generation.

10. A method for identifying recessive nuclear male sterility genes in Chinese kale BoaGMS1 The application of functional molecular markers in molecular-assisted selection breeding of Chinese kale is characterized by... The primer sequences of the functional molecular marker pairs are shown in SEQ ID No. 7 and SEQ ID No. 8.

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