Method for breeding brassica napus lines with cytoplasmic restorer independent of the source of recovery
By using testcrosses and molecular marker detection, the genetic instability and long breeding cycle of radish cytoplasmic restorer lines of Brassica napus were solved, enabling the breeding of genetically stable radish cytoplasmic restorer lines, thus improving breeding efficiency and reducing costs.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- CHENGDU ACAD OF AGRI & FORESTRY SCI
- Filing Date
- 2023-12-27
- Publication Date
- 2026-05-19
AI Technical Summary
In Brassica napus, the restorer gene for radish cytoplasmic male sterility lines is difficult to find or is genetically unstable, leading to long breeding cycles and poor fruit set in radish cytoplasmic restorer lines.
By testcrossing and backcrossing genetically stable Brassica napus and radish cytoplasmic male sterile lines, pollination and ploidy determination were carried out using a double haploid induction line of Brassica napus that does not contain the radish cytoplasmic restorer gene. Combined with the detection of special molecular markers, homozygous fertile lines were screened out to form a Brassica napus-radish cytoplasmic restorer line that does not depend on the restorer source.
Genetic stability of the Brassica napus-radish cytoplasmic restorer line was achieved, shortening the breeding cycle, improving efficiency, and reducing costs.
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Figure CN117837492B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of crop breeding technology, specifically to a method for breeding a cytoplasmic restorer line of Brassica napus-radish that does not rely on a restorer source. Background Technology
[0002] Ogu CMS is a cytoplasmic male sterility system established by Ogura based on the naturally occurring male sterile source, radish (Raphanus), and named after it. Bannerot used a continuous backcrossing method to introduce the nucleus of Brassica napus cells into radish oguCMS, thus cultivating the Brassica napus ogu CMS male sterile line. Radish cytoplasmic male sterility lines are stable and unaffected by temperature and light sensitivity, achieving 100% sterility. Because the restorer gene in this sterile cytoplasm originates from radish, and there are no homologous or allelic restorer genes in Brassica napus, almost all Brassica napus lines serve as maintainer lines. Breeding radish cytoplasmic male sterility lines in Brassica napus is relatively easy; new sterile lines can be obtained by backcrossing a genetically stable Brassica napus line with a radish cytoplasmic male sterility line for 5-6 generations. Similarly, this type of sterile cytoplasmic gene has been successfully transferred to Chinese cabbage, non-heading Chinese cabbage, cauliflower, and broccoli, and has been widely used in hybrid production. This technical challenge has also become a problem for broccoli germplasm. However, restorer lines for radish cytoplasmic sterility are extremely difficult to find in the Brassica genus, and transferring the restorer gene from radish to Brassica napus is very difficult. Therefore, European countries generally use the method of blending hybrids for transitional use, that is, blending 20% conventional variety seeds into sterile hybrids to solve the problem of normal seed setting. The French National Institute for Agricultural Research (INRA) and Pioneer Hibred jointly developed a patent: by transferring the Rfo gene into ogu CMS and combining it with molecular marker technology, they successively cultivated double-low restorer lines and selected a true Brassica napus-radish cytoplasmic sterile hybrid, thus realizing the commercial production of Brassica napus oguCMS hybrids. In recent years, it has become the main type of rapeseed hybrid vigor in countries and regions such as Europe and Canada, accounting for more than 40% of the rapeseed planting area in these regions. However, in my country, due to the limitation of patents on restorer genes for radish cytoplasmic male sterility lines, there is very little commercial breeding application of radish cytoplasmic male sterility lines.
[0003] Studies on different cytoplasmic male sterility (CMS) systems in plants have shown that the cytoplasmic male sterility phenotype is caused by dominant mutations in the mitochondrial genome. Therefore, different CMS-related genes have been used to distinguish different cytoplasms. Bonhomme et al. found that orf138 is associated with male sterility in ogu CMS; Liu et al. found that orf224 is a specific gene for pol CMS; Landgren et al.'s research demonstrated that orf263 is specific for TURE CMS and orf222 is a key gene for nap CMS; Heng et al. believed that orf288 can cause male sterility in hua CMS. With the latest advances in mitochondrial genome sequencing, a large number of mitochondrial markers have been developed to identify different CMS types in rapeseed. Based on comparative sequencing analysis of mitochondrial genome sequences in Brassica plants, the mitotic type of hau CMS differs from other mitotic types. Recently, mitochondrial sequence-specific (MSS) markers have also been developed to distinguish different CMS mitotic types in Brassica. The combination of traditional testcross and cytological studies with molecular marker development has been used to identify and characterize different sterile lines in Brassica crops.
[0004] CMS is generally believed to be associated with mitochondrial genome rearrangements, which in many cases is attributed to the generation of new open reading frames (ORFs). Some experimental evidence confirms the correlation between CMS-associated ORFs and the occurrence of CMS. This communication pathway from mitochondria to the nucleus is defined as mitochondrial retrograde regulation (MRR), primarily confirmed in yeast and animals. Some ABC model genes associated with floral organ development, namely nuclear MADS-box TF genes, have been shown to be targets of MRR-regulated allologous transformation of floral organs. Furthermore, in some CMS systems, mitochondria also significantly influence several other nuclear genes. Plant mitochondrial DNA and mutations associated with cytoplasmic male sterility and abnormal growth phenotypes are also relevant. Compared to variations in mtDNA size, the number of mitochondrial genes in plants is relatively conserved, with approximately 60-70 genes found in mitosis across different terrestrial plant species. Most mitochondrial DNA is non-protein-coding. However, most mitochondrial-coding genes are crucial for mitochondrial function and normal plant development. The phenotype of cytoplasmic male sterility is controlled by a combination of mitochondrial CMS genes encoded in the nuclear genome and nuclear fertility restoration genes (Rf genes). Brown et al. have identified the Rf gene of ogu CMS and named it Rfo or orf687. This gene (Rfo) can alter the posttranscriptional expression of orf138 and can directly or indirectly reduce the content of ORF138 protein. Dahan et al. found that Rfo encodes a 687-amino acid pentapeptide repeat (PPR) protein, named ORF687; the functions of PPR proteins include RNA cleavage, RNA instability, or translational repression, therefore the CMS-RF system is important for understanding the interaction between the plant nuclear and mitochondrial genomes. Yamagishi found at least nine orf138 species distributed in wild and cultivated radishes, and two or more Rf genes are present in radishes. In recent years, although the CMS gene orf138 and the Rf gene Rfo in the Ogura CMS system of radish have been repeatedly identified, the exact functional relationship between orf138 and Rfo remains unclear, and the physical location of the Ogura CMS restorer gene Rfo in the Brassica napus genome still needs further investigation. Currently, the radish cytoplasmic male sterility restorer genes in Brassica napus all originate from radish, obtained through distant hybridization with radish or by transferring offspring from distant hybridization, but the restorer genes all originate from radish. However, the radish cytoplasmic male sterility restorer gene in Brassica napus has no homologous gene or allele in Brassica napus, so this gene is easily lost during breeding, making it difficult to obtain genetically stable Brassica napus restorer lines. Furthermore, it is difficult to obtain 100% restored hybrid F1 when crossbred with sterile lines. Therefore, using this sterile system to breed Brassica napus hybrids is risky. Summary of the Invention
[0005] To address the aforementioned shortcomings in the existing technology, this invention provides a method for breeding a radish cytoplasmic restorer line of Brassica napus that does not rely on a restorer source. This invention has a short improvement cycle, high efficiency, and low cost, effectively solving the problems of long breeding cycle, genetic instability, and negative effects of short siliques and poor fruit set of radish cytoplasmic restorer lines and radish restorer sources.
[0006] To achieve the above objectives, the technical solution adopted by this invention to solve its technical problem is: to provide a method for breeding a Brassica napus radish cytoplasmic restorer line that does not depend on a restorer source, specifically including the following steps:
[0007] S1. Test cross or back cross multiple generations of genetically stable Brassica napus and Brassica napus-radish cytoplasmic male sterile lines to obtain radish cytoplasmic hybrid F1 generation or stable new radish cytoplasmic male sterile lines;
[0008] S2. Use a rapeseed double haploid inducing line without radish cytoplasmic restorer gene to pollinate the radish cytoplasmic hybrid F1 generation or a stable new radish cytoplasmic male sterile line obtained in step S1. Determine the ploidy of the offspring, select tetraploid fertile single plants for bagging and self-pollination, and then test cross the tetraploid fertile single plants with the genetically stable radish cytoplasmic male sterile line to obtain the test cross offspring. Perform special molecular marker detection on the tetraploid fertile single plants.
[0009] S3. Retain fertile single plants in the testcross progeny obtained in step S2 that can amplify specific bands of molecular markers, and continue self-pollination until homozygous fertile lines are selected. Then, test cross with the cytoplasmic male sterile line of Brassica napus and radish. The fertile lines in the testcross progeny that are all fertile are the stable cytoplasmic restorer lines of Brassica napus and radish.
[0010] Furthermore, in step S2, the method for breeding rapeseed double haploid inducible lines that do not contain the radish cytoplasmic restorer gene includes the following steps:
[0011] S2-1. The F1 generation seeds from the cross between two rapeseed parent materials are subjected to artificial chromosome doubling to obtain doubled F1 generation plants. Then, the F1 generation plants are self-pollinated to obtain F2 generation. The fertile offspring of the F2 generation plants are selected for self-pollination to obtain F3 generation. The homozygosity of the F3 generation plants is identified. The plants that show consistent molecular marker maps among the individual plants and are all hybrid offspring of the two parents are selected as homozygous early generation stable lines.
[0012] S2-2. Perform reciprocal crosses between the early generation stable lines obtained in step S1 and 10 conventional homozygous stable lines of rapeseed. Select the early generation stable lines that show segregation in the F1 generation and stable lines in the F2 generation to obtain early generation stable lines with parthenogenesis inheritance.
[0013] S2-3. Cross the early stable line with parthenogenesis inheritance obtained in step S2 with rapeseed with dominant traits to obtain hybrid F1 generation seeds. Artificially double the chromosomes of the hybrid F1 generation seeds to obtain doubled F1 plants with dominant traits. Then, eliminate abnormally doubled plants, aneuploid plants, and doubled plants without dominant traits from the F1 plants. Select hexaploid or octoploid rapeseed plants with stable ploidy inheritance, good fruit setting, parthenogenesis inheritance characteristics, and dominant traits.
[0014] In steps S2-4 and S2-3, the dominant trait in polyploid plants with stable ploidy inheritance, parthenogenesis, and dominant traits can eliminate hybrid plants produced in the testcross offspring. If a plant with a dominant trait or an aneuploid plant appears in the testcross offspring, it indicates that the plant was produced by crossing a polyploid plant with the maternal parent, and the plant should be removed. If the testcross offspring are completely sterile, have normal ploidy (i.e., diploid or tetraploid rapeseed), and do not carry a dominant trait, it indicates that the paternal gene corresponding to the testcross offspring has not entered the testcross offspring, and the dominant polyploid plant is the rapeseed double haploid induction line.
[0015] Furthermore, the specific methods for artificial chromosome doubling in steps S2-1 and S2-3 include the following steps:
[0016] (1) Disinfect the seed surface with 75% alcohol for 25-40 seconds, disinfect with 0.1% mercuric chloride for 12-17 minutes, then rinse with sterile water, dry the seeds with sterile paper, inoculate the seeds on the first culture medium, and incubate at 23-25℃, with daytime light for 12-16 hours and light intensity of 2000-3000 LX, and nighttime dark culture for 8-12 hours.
[0017] (2) When the seeds grow to 1-2 true leaves in the first culture medium, cut the plant from the hypocotyl and transfer it to the second culture medium to continue growing.
[0018] (3) After the plant grows to the point where lateral buds differentiate, the lateral buds and the plant are transferred to the third culture medium for rooting culture. After 14 days of rooting culture, the plant grows strong roots.
[0019] (4) Harden the plants at room temperature for 3-7 days, rinse the culture medium from the plant roots with tap water, and soak them in soaking buffer for 15-30 minutes before transplanting them into the greenhouse. The greenhouse temperature should be 16-25℃ and the relative humidity should be 60-80% to ensure a transplant survival rate of over 95%.
[0020] Furthermore, the first culture medium described above consists of the following components in the following proportions: 1L of MS medium, 0.5-1.5mg of 6-benzyladenine, 30-70mg of colchicine, 20-30g of sucrose, and 8-10g of agar, with a pH of 5.8-6.
[0021] The second culture medium consists of the following components in the following proportions: 1L MS medium, 0.5-1mg 6-benzyladenine, 20-40mg colchicine, 20-30g sucrose, and 8-10g agar, with a pH of 5.8-6.
[0022] The third culture medium consists of the following components in the following proportions: 1L MS medium, 0.03-0.5mg 6-benzyladenine, 5-20mg colchicine, 20-30g sucrose, and 8-10g agar, with a pH of 5.8-6.
[0023] The above soaking buffer consists of the following components in the following proportions: 1L water, 0.6-1.2g of Eboscin or Chloroform, and 0.5-1mg of α-naphthaleneacetic acid.
[0024] Furthermore, flow cytometry was used to determine the ploidy of their offspring.
[0025] Furthermore, the aforementioned special molecular markers are Bn09-1, Bn09-2, and Bn09-3.
[0026] Furthermore, the forward primer sequence of Bn09-1 is shown in SEQ ID NO.1, and the reverse primer sequence of Bn09-1 is shown in SEQ ID NO.2.
[0027] Furthermore, the forward primer sequence of Bn09-2 is shown in SEQ ID NO.3, and the reverse primer sequence of Bn09-2 is shown in SEQ ID NO.4.
[0028] Furthermore, the forward primer sequence of Bn09-3 is shown in SEQ ID NO.5, and the reverse primer sequence of Bn09-3 is shown in SEQ ID NO.6.
[0029] The above-mentioned method for breeding Brassica napus-radish cytoplasmic restorer lines that do not rely on restorer sources was used to select Brassica napus-radish cytoplasmic restorer lines.
[0030] The above-mentioned Brassica napus-radish cytoplasmic restorer line was used in the breeding of new radish cytoplasmic three-line hybrid varieties with strong advantages.
[0031] In summary, the present invention has the following beneficial effects:
[0032] 1. This method does not rely on radish cytoplasmic sterility restoration sources.
[0033] 2. Restorer lines bred using this method are genetically stable.
[0034] 3. This invention has a short improvement cycle, high efficiency, and low cost. Attached Figure Description
[0035] Figure 1 Flowchart for the breeding of Brassica napus radish cytoplasmic restorer lines that do not depend on restorer sources;
[0036] Figure 2 Phenotypic observation diagrams of the induced maternal parent and induced offspring plants;
[0037] Figure 3 Anther sections of 4508A at different stages of development are shown in the images.
[0038] Figure 4 Anther sections of 4211A at different stages of development are shown in the images.
[0039] Figure 5 Images of anther sections at different stages of 4211C.
[0040] Figure 6 Flow cytometry ploidy map of 4508A cells;
[0041] Figure 7 Flow cytometry ploidy plot of cells 4211;
[0042] Figure 8 ploidy diagram of 178 flow cytometry cells of the hybrid offspring of 4508A and 4211;
[0043] Figure 9 The chromosome diagram for 4211;
[0044] Figure 10 Image showing the BSA identification results;
[0045] Figure 11 Electrophoresis results for the specific molecular markers Bn09-1, Bn09-2, and Bn09-3;
[0046] Figure 12 Electrophoresis results of molecular marker identification for Bn09-1, Bn09-2, and Bn09-3 in the F2 generation of the cross between 4508A and 4211C. Detailed Implementation
[0047] The present invention will be further described below with reference to embodiments and accompanying drawings, but this does not constitute a limitation thereof. The flowchart for the breeding of a Brassica napus-radish cytoplasmic restorer line independent of restorer sources provided by the present invention is shown below. Figure 1 As shown.
[0048] Example 1: Breeding an early generation stable line with parthenogenesis genetic characteristics
[0049] 1. The F1 generation seeds obtained by crossing Brassica napus F009 with Brassica rapa Yaan yellow rape YH were disinfected with 75 wt% alcohol for 30 seconds, then with 0.1 wt% mercuric chloride for 15 minutes. The mercuric chloride on the seed surface was then rinsed off with sterile water, and the seeds were dried with sterile paper. The seeds were then inoculated on the first culture medium and cultured under the following conditions: temperature 25℃, daytime light for 12 hours, light intensity 2500 LX, and nighttime dark culture for 12 hours.
[0050] 2) When the seeds have grown to 1-2 true leaves in the first culture medium, cut the plant from the hypocotyl and transfer it to the second culture medium to continue growing;
[0051] 3) After the plant has grown to the point where lateral buds differentiate, the lateral buds and the plant are transferred to the third culture medium for rooting culture. After two weeks of rooting culture, the plant grows strong roots.
[0052] 4) Harden the plants at room temperature for 5 days, rinse the culture medium from the plant roots with tap water, soak them in soaking buffer for 20 minutes, and then transplant them into the greenhouse. The greenhouse temperature is 20℃ and the relative humidity is 70% to ensure that the transplant survival rate is over 95%.
[0053] The first culture medium described above consists of the following components in the following proportions: 1 L of MS medium, 1.0 mg of 6-benzyladenine, 40 mg of colchicine, 20 g of sucrose, and 8 g of agar, with a pH of 5.8.
[0054] The second culture medium consists of the following components in the following proportions: 1L MS medium, 0.5mg 6-benzyladenine, 20mg colchicine, 30g sucrose, and 8g agar, with a pH of 5.8.
[0055] The third culture medium consists of the following components in the following proportions: 1L MS medium, 0.5mg 6-benzyladenine, 10mg colchicine, 30g sucrose, and 10g agar, with a pH of 6.
[0056] The above soaking buffer comprises the following components: 1 L of water, 1.0 g of Ebo or Chloro, and 1 mg of α-naphthaleneacetic acid.
[0057] 2. The F1 generation plants were self-pollinated or forced bud removal self-pollinated to obtain the F2 generation. The F2 generation was observed in the field and the fertility of each individual plant was identified. Fertile offspring were selected for self-pollination to obtain the F3 generation. The homozygosity of the F3 generation was identified by morphology, cytology and molecular markers. The results showed that the molecular marker patterns of each individual plant were consistent and that each plant was a hybrid offspring of the two parents. This was a homozygous early generation stable line P3-2.
[0058] 3. The obtained early-generation stable line P3-2 was subjected to reciprocal crosses with 10 conventional homozygous stable lines of rapeseed. The genetic characteristics of the early-generation stable lines in the F1 and F2 generations of the reciprocal crosses were identified. The genetic characteristics of the early-generation stable lines were parthenogenetic. When the identification results showed that the F1 generation was segregated and the F2 generation had stable lines, the corresponding early-generation stable line was an early-generation stable line with parthenogenetic inheritance.
[0059] 2. The F1 generation plants were self-pollinated or forced bud removal self-pollinated to obtain the F2 generation. The F2 generation was observed in the field and the fertility of each individual plant was identified. Fertile offspring were selected for self-pollination to obtain the F3 generation. The homozygosity of the F3 generation was identified by morphology, cytology and molecular markers. The results showed that the molecular marker patterns of each individual plant were consistent and that each plant was a hybrid offspring of the two parents. These plants were homozygous early generation stable lines.
[0060] 3. The obtained early-generation stable lines were crossed with 10 conventional homozygous stable lines of rapeseed in reciprocal crosses. The genetic characteristics of the early-generation stable lines in the F1 and F2 generations of the reciprocal crosses were identified. The genetic characteristics of the early-generation stable lines were parthenogenetic. When the identification results showed that the F1 generation was segregated and the F2 generation had stable lines, the corresponding early-generation stable lines were early-generation stable lines with parthenogenetic inheritance.
[0061] Example 2: Breeding polyploid rapeseed carrying dominant genetic traits, possessing parthenogenesis characteristics, and stable ploidy inheritance.
[0062] 1. The early stable line P3-2 with parthenogenesis genetic characteristics obtained in Example 1 was crossed with rapeseed with purple leaf dominant trait to obtain F1 generation seeds. The F1 generation seeds were then subjected to chromosome doubling using the chromosome doubling method used in Example 1.
[0063] 2. Chromosome ploidy was determined in the F1 generation plants with dominant traits after doubling. Plants with dominant traits were selected, while abnormally doubled plants, aneuploid plants, and doubled plants without dominant traits were discarded. Polyploid plants with dominant traits are hexaploid or octoploid rapeseed plants with stable ploidy inheritance, good fruit setting, parthenogenesis inheritance characteristics, and dominant traits.
[0064] Example 3: Identification and Induction Ability Determination of Rapeseed Double Haploid Inducible Lines
[0065] 1. The dominant gene in the polyploid plant with stable ploidy inheritance, parthenogenesis inheritance characteristics, and dominant trait obtained in Example 2 can remove the hybrid plants produced in the test cross offspring of rapeseed. If a plant with a dominant trait or an aneuploid plant appears in the test cross offspring, it indicates that the plant is produced by hybridization of a polyploid plant and the maternal parent, and the plant should be removed.
[0066] 2. The above-mentioned single plants were testcrossed with rapeseed. The offspring showed rapeseed that was completely sterile, had normal ploidy, and did not carry the dominant trait. This indicates that the paternal gene corresponding to the testcross offspring did not enter the testcross offspring. The dominant polyploid plants are rapeseed double haploid induction lines, denoted as rapeseed double haploid induction lines Y3380\Y3560; the above-mentioned normal ploidy is diploid or tetraploid.
[0067] Example 4: Ploidy determination of induced line offspring
[0068] 1. Test cross or backcross multiple generations of superior and genetically stable Brassica napus F009 with Brassica napus-radish cytoplasmic male sterile line (OguarCMS) 4508A to form radish cytoplasmic hybrid F1 generation or stable new radish cytoplasmic male sterile line.
[0069] 2. Use the rapeseed double haploid inducible line without the radish cytoplasmic restorer gene obtained in Example 2 to pollinate the radish cytoplasmic hybrid F1 generation or a stable new radish cytoplasmic male sterile line.
[0070] Flow cytometry was used to determine the ploidy of the offspring. A tetraploid Brassica napus before induction was used as a control for ploidy determination. If the fluorescence intensity of the first peak in flow cytometry was the same as that of the control, the plant was identified as tetraploid. If the fluorescence intensity of the first peak was 1.5 times that of the control, the plant was identified as hexaploid. If three or more peaks appeared, the plant was identified as hybrid. Hexaploids and hybrids were discarded, and only fertile tetraploid plants were selected.
[0071] Example 5: Screening of stable Brassica napus-radish cytoplasmic restorer lines
[0072] The tetraploid progeny of fertile single plants selected in Example 4 were bagged and self-crossed, and the pollen of the tetraploid fertile single plants was testcrossed with the genetically stable radish cytoplasmic male sterile line. The tetraploid fertile single plants were then tested for three specific molecular markers: Bn09-1, Bn09-2, and Bn09-3.
[0073] If fertile plants appear in the testcross progeny and the molecular markers can amplify specific bands, retain these induced fertile plants that can produce fertile plants in the testcross and whose molecular markers can detect specific bands; discard fertile plants whose testcross progeny are all sterile and whose molecular markers cannot detect specific bands.
[0074] The retained tetraploid fertile single plants are continuously self-pollinated for 2-3 generations until no more sterile plants separate from the self-pollinated offspring. Then, the fertile plants are testcrossed with the radish cytoplasmic male sterile line. If all the testcross offspring are fertile, the corresponding tetraploid fertile line is a stable Brassica napus-radish cytoplasmic restorer line.
[0075] Phenotypic identification in Experiment 1
[0076] During the flowering period, field observations revealed that inducing the Brassica napus radish-sterile line 4508A using the double haploid inducible lines Y3380 / Y3560 produced fertile plants 4211C and sterile plants 4211A. The phenotypes of 4211C and 4211A are as follows: Figure 2 As shown in the figure. Among them, A and B are plants of 4508A and 4211, respectively; C and D are the floral organs of 4508A and 4211, respectively; E and F are the leaves of 4508A and 4211, respectively; G and H are the young plants of 4508A and 4211, respectively.
[0077] Depend on Figure 2 It can be seen that, apart from the difference in flower development and flower size between the induced fertile offspring 4211C and the parent plant 4508A, there are no significant differences between the two in terms of plant morphology, leaf size, and margin teeth. Both the male and female pistils develop normally. However, 4211C shows that it bolts earlier than 4508A. The stamens of 4508A are completely without pollen, while the anthers of 4211C are full and have pollen.
[0078] Experimental Example 2: Identification of Anther Sections
[0079] To determine the sterility and fertility of plants and whether there is a specific abortion stage in anther development, semi-thin cross-sections of anthers were prepared from flower buds of the sterile line 4508A and induced progeny plants at different developmental stages. The results are as follows: Figure 3-5 As shown. Among them, Figure 3 In the figure, Figure A shows an anther section of 4508A with a diameter of less than 2 mm; Figures B and E show anther sections of 4508A with a diameter of 2-6 mm respectively; and Figure F shows anther sections of 4508A with a diameter of more than 6 mm. Figure 4 In the figure, Figure A shows an anther section of 4211A with a diameter of less than 2 mm; Figures B and E show anther sections of 4211A with a diameter of 2-6 mm respectively; and Figure F shows anther sections of 4211A with a diameter of more than 6 mm. Figure 5 In the figure, Figure A shows anther sections of 4211C with a diameter of less than 2 mm; Figures B and E show anther sections of 4211C with a diameter of 2 mm to 6 mm; and Figure F shows anther sections of 4211C with a diameter of more than 6 mm.
[0080] Depend on Figure 3It is known that the different cell layers of rapeseed anthers from the inside out are: tapetum, medial layer, inner layer, and anther cell wall. In 2mm flower buds, microsporogenesis and tapetum development are similar, representing the meiotic and tetrad stages. Furthermore, the sterile anthers were observed to contain a proliferating tapetum, which partially blocked the anther cell and eventually filled most of it. Anther abnormalities were first observed in subsequent stages, accompanied by tapetum shrinkage and microspore abortion. In subsequent stages, abnormal cell division and vacuolation of the tapetum cells in 4508A were very obvious. During the gradual development of the anther, the pollen sacs slowly grew from a shriveled, butterfly-shaped state to a full state, and the tapetum slowly grew from containing a large amount of media to the media gradually disappearing, consistent with the phenotype of mature anthers being full and transparent. However, throughout the entire process, no mature pollen grains were found in the anther cells of the four pollen sacs and stamens, consistent with the results of pollen viability identification, indicating the presence of many residues within the anther cells. Each anther of 4508A has four pollen sacs, which are irregularly butterfly-shaped. Each pollen sac is in a different state of abortion, and the abortion period of 4508A may be in the later stage between the tetrad and the pollen grain.
[0081] Depend on Figure 4 It is known that each anther of 4211A has four pollen sacs, arranged in a butterfly shape. Similar to 4508A, a proliferating tapetum was observed in the sterile anthers of this 2mm flower bud, with only a few tissue cells observed in the inner tapetum, possibly remnants of some pretaenial cells. Subsequently, the tapetum also began to shrink abnormally, accompanied by microspore abortion. In contrast to 4508A, as the anther chamber of 4211A gradually matures, the tapetum remnant matrix gradually decreases, and the tapetum gradually shrinks. However, the pollen sacs also gradually shrink from their initial plump state to a shriveled state, and each pollen sac is in a different state of abortion. The abortion stage of 4211A may also be in the early stage between the tetrad and the pollen grain.
[0082] Depend on Figure 5It is evident that in 2mm flower buds, microsporogenesis and tapetal development are similar, representing the meiotic and tetrad stages. The 4211C pollen mother cell undergoes normal meiosis, forming normal tetrads and pollen grains; all developmental processes are normal. The meiotic phase of pollen mother cells is observed in the anthers of 2mm flower buds. Microsporid tetrads mostly appear in 2-3mm flower buds, occasionally in specimens smaller than 2mm. This then progresses to the next developmental stage, continuing until mid-microsporogenesis, characterized by small cytoplasmic vacuoles and a central nucleus. Fertile microsporids undergo pollen mitosis, forming pollen grains with both vegetative and reproductive cells. Finally, as the ovary wall gradually disintegrates, the pollen grains mature and are released from the ovary chamber. Tapetal cells gradually disintegrate with the formation of tetrad cells, providing ample nutrition for microsporogenesis and pollen grain formation until the tapetum completely disappears during pollen formation.
[0083] Experimental Example 3: Identification of Plant Plumpness and Chromosome Number
[0084] To better determine the relationship between the ploidy of the induced progeny, the ploidy of the induced line, and the ploidy of the maternal parent 4508A, flow cytometry was used to determine the ploidy of different plants from the sterile maternal parent 4508A, the induced progeny 4211, and their F1 hybrid 178. The conventional tetraploid rapeseed variety Zhongshuang (ZS11) was used as a control. The results are as follows: Figure 6-8 As shown in Table 1, the peak values and ploidy of the induced maternal parent, induced offspring, and their F1 hybrids are represented by flow cytometry data.
[0085] Table 1. Peak values and ploidy tables of induced maternal parents, induced offspring, and induced F1 generation flow cytometry cells.
[0086] Material peak Plant ploidy Material peak Plant ploidy Material peak Plant ploidy 4508A-1 493,182.10 Tetraploid 4211-1 504,960.19 Tetraploid 178-1 541,941.81 Tetraploid 4508A-2 499,763.38 Tetraploid 4211-2 470,924.31 Tetraploid 178-2 533,351.01 Tetraploid 4508A-3 496,333.67 Tetraploid 4211-3 526,423.50 Tetraploid 178-3 474,846.66 Tetraploid 4508A-4 476,639.84 Tetraploid 4211-4 484,176.08 Tetraploid 178-4 502,470.85 Tetraploid 4508A-5 494,143.85 Tetraploid 4211-5 523,022.60 Tetraploid 178-5 513,437.29 Tetraploid 4508A-6 506,747.64 Tetraploid 4211-6 483,556.09 Tetraploid 178-6 502,829.80 Tetraploid 4508A-7 474,492.24 Tetraploid 4211-7 527,705.57 Tetraploid 178-7 522,605.93 Tetraploid 4508A-8 453,988.11 Tetraploid 4211-8 494,914.50 Tetraploid 178-8 505,619.32 Tetraploid 4508A-9 461,213.16 Tetraploid 4211-9 494,832.05 Tetraploid 178-9 502,994.31 Tetraploid 4508A-10 439,380.52 Tetraploid 4211-10 497,956.00 Tetraploid 178-10 528,531.48 Tetraploid 4508A-11 477,780.88 Tetraploid 4211-11 519,318.16 Tetraploid 178-11 498,480.70 Tetraploid 4508A-12 471,759.82 Tetraploid 4211-12 468,391.00 Tetraploid 178-12 511,720.75 Tetraploid 4508A-13 468,842.90 Tetraploid 4211-13 475,299.76 Tetraploid 178-13 506,961.61 Tetraploid 4508A-14 449,507.74 Tetraploid 4211-14 451,191.62 Tetraploid 178-14 512,357.78 Tetraploid 4508A-15 482,869.26 Tetraploid 4211-15 466,502.74 Tetraploid 178-15 518,013.20 Tetraploid China Double 11-1 506,498.80 Tetraploid China Double 11-2 517,940.89 Tetraploid China Double 11-3 535,824.11 Tetraploid
[0087] From Table 1 and Figure 6-8 It can be seen that the peak value of the flow cytometer of Zhongshuang 11 is about 450,000 to 550,000, and the peak value of the flow cytometer of the induced maternal parent and the offspring is about 450,000 to 550,000. This indicates that they are all tetraploid plants, not induced octoploids of the paternal parent. Therefore, it reveals the genetic relationship between the induced offspring and the parents, indicating that the paternal parent only plays an induction role, rather than a hybridization role.
[0088] Brassica napus is an allotetraploid with 38 chromosomes (2n = 4x = 38), consisting of A genome chromosomes (x = 10) and C genome chromosomes (x = 9). Crossing 4x ploidy with 8x ploidy may produce 4x, 4x+1, 6x, 6x+1, and 8x ploidy offspring. After testcrossing an 8x ploidy inducing line (Y3560 / Y3380) with a 4x ploidy sterile line (4508A), only 4x ploid offspring identical to the sterile maternal line appeared in the offspring. The chromosome results for 4211 are as follows... Figure 9 As shown.
[0089] Depend on Figure 9 It can be seen that 4211 has 38 chromosomes, which is consistent with the number of chromosomes in tetraploid Brassica napus.
[0090] Experiment 4: Initial gene localization results and molecular marker verification
[0091] The induced radish cytoplasmic recovery gene was initially mapped using extreme pool BSA and resequencing. The results are as follows: Figure 10 As shown in the diagram, genotyping analysis was performed using 4508A as the parent. Blue indicates heterozygous genotypes, white indicates homozygous genotypes identical to parent 4508A, and red indicates homozygous genotypes different from parent 4508A. Through genotyping analysis, segments meeting the following two conditions can be identified as candidate gene segments: first, segments where the genotype differs between the two extreme pools; second, within each pool, the genotype of the differing segment is identical to or heterozygous with the parental genotype that exhibits the same phenotype.
[0092] Depend on Figure 10 It is known that samples 4508A and 4211C are the parents. Mixed pool sample C1-20 has the same phenotype as parent 4211C and is fertile. The F1 generation is also fertile, so the gene loci associated with the fertile phenotype should be heterozygous. Mixed pool sample A1-11 has the same phenotype as parent 4508A and is sterile, so the phenotype-related loci should be homozygous. Based on the genotype data, the gene segments associated with the phenotype are located as follows: A09 chromosome 10.99-17.20 Mb, C03 chromosome 5.07-5.37 Mb, and C09 chromosome 21.12-36.60 Mb.
[0093] The testcross results of 4508A and 4211C show that the restorer gene was generated after induction, and the primers used for the restorer gene were effective. After initial mapping, analysis of the mapping region revealed that the molecular markers of the three restorer lines, Bn09-1, Bn09-2, and Bn09-3, fell precisely within this region. The molecular marker primers Bn09-1 (chrC09: 19590617-19590251; chrA0912956843-) 12956567), Bn09-2 (chrC09: 19086365-19086611), and Bn09-3 (chrC09: 19788489-19788380; chrA09: 13137389-13137280) can be used to identify the restoration gene. The molecular marker results for the F2 generation are shown in Table 2. The electrophoresis results of the special molecular markers Bn09-1, Bn09-2, and Bn09-3 are shown below. Figure 11 As shown, the electrophoresis results of molecular marker identification for Bn09-1, Bn09-2, and Bn09-3 in the F2 generation of the 4508A / 4211C hybridization are as follows: Figure 12 As shown.
[0094] Table 2. Statistical table of F2 generation molecular marker results.
[0095]
[0096]
[0097] Note: Those containing the radish cytoplasmic restorer line linkage marker are indicated by +, and those not containing the radish cytoplasmic restorer line linkage marker are indicated by -.
[0098] As shown in Table 2, fertile individuals all exhibited specific bands, while infertile individuals did not. The results were largely consistent with those of the molecular markers, indicating that the identification markers were reliable and completely consistent with the phenotypic expression. Furthermore, it also demonstrated the presence of the restorer gene within the localized region.
[0099] Depend on Figure 11-12 It can be seen that the fertility phenotype and the molecular marker identification results are 100% consistent.
[0100] Although specific embodiments of the present invention have been described in detail with reference to the accompanying drawings, this should not be construed as limiting the scope of protection of this patent. Various modifications and variations that can be made by those skilled in the art without inventive effort within the scope described in the claims are still within the scope of protection of this patent.
Claims
1. A method for breeding Brassica napus-radish cytoplasmic restorer lines that do not depend on restorer sources, characterized in that, Includes the following steps: S1. Test cross or back cross multiple generations of genetically stable Brassica napus and Brassica napus-radish cytoplasmic male sterile lines to obtain radish cytoplasmic hybrid F1 generation or stable new radish cytoplasmic male sterile lines; S2. Using a rapeseed double haploid inducing line lacking the radish cytoplasmic restorer gene, pollinate the F1 generation of the radish cytoplasmic hybrid or a stable new radish cytoplasmic male sterile line obtained in step S1. Determine the ploidy of the offspring, select tetraploid fertile plants for bagged self-pollination, and then test cross the tetraploid fertile plants with a genetically stable radish cytoplasmic male sterile line to obtain the test cross offspring. Perform specific molecular marker detection on the tetraploid fertile plants. The breeding method for the rapeseed double haploid inducing line lacking the radish cytoplasmic restorer gene includes the following steps: S2-1. The F1 generation seeds from the cross between two rapeseed parent materials are subjected to artificial chromosome doubling to obtain doubled F1 generation plants. Then, the F1 generation plants are self-pollinated to obtain F2 generation. The fertile offspring of the F2 generation plants are selected for self-pollination to obtain F3 generation. The homozygosity of the F3 generation plants is identified. The plants that show consistent molecular marker maps among the individual plants and are all hybrid offspring of the two parents are selected as homozygous early generation stable lines. S2-2. Perform reciprocal crosses between the early generation stable lines obtained in step S1 and 10 conventional homozygous stable lines of rapeseed. Select the early generation stable lines that show segregation in the F1 generation and stable lines in the F2 generation as the early generation stable lines of parthenogenesis. S2-3. The early-generation stable line with parthenogenesis inheritance obtained in step S2 is crossed with rapeseed with dominant traits to obtain F1 hybrid seeds. The F1 hybrid seeds are then subjected to artificial chromosome doubling to obtain F1 plants with dominant traits. Abnormally doubled, aneuploid, and doubling plants without dominant traits are then eliminated from the F1 plants. Hexaploid or octoploid rapeseed plants with stable ploidy inheritance, good fruit setting, parthenogenesis inheritance characteristics, and dominant traits are selected. The method of artificial chromosome doubling includes the following steps: (1) Disinfect the seed surface with 75% alcohol for 25-40 seconds, disinfect with 0.1% mercuric chloride for 12-17 minutes, then rinse with sterile water, dry the seeds with sterile paper, inoculate the seeds on the first culture medium, and incubate at 23-25℃, with daytime light for 12-16 hours and light intensity of 2000-3000 LX, and nighttime dark culture for 8-12 hours. (2) When the seeds have grown to 1-2 true leaves in the first culture medium, cut the plant off from the hypocotyl and transfer it to the second culture medium to continue growing; (3) After the plant grows to the point where lateral buds differentiate, the lateral buds and the plant are transferred to the third culture medium for rooting culture. After 14 days of rooting culture, the plant grows strong roots. (4) Harden the plants at room temperature for 3-7 days, rinse the culture medium from the plant roots with tap water, and soak them in soaking buffer for 15-30 minutes before transplanting them into the greenhouse. The greenhouse temperature should be 16-25℃ and the relative humidity should be 60-80% to ensure a transplant survival rate of over 95%. In steps S2-4 and S2-3, the dominant trait in polyploid plants with stable ploidy inheritance, parthenogenesis, and dominant traits can remove hybrid plants produced in the testcross offspring. If a plant with a dominant trait or an aneuploid plant appears in the testcross offspring, it indicates that the plant is produced by crossing a polyploid plant with the maternal parent. Remove the plant. If the single-plant testcross offspring is completely sterile, has normal ploidy (i.e., diploid or tetraploid rapeseed), and does not carry a dominant trait, it indicates that the paternal gene corresponding to the testcross offspring has not entered the testcross offspring. The dominant polyploid plant is a rapeseed double haploid induction line. S3. Retain fertile single plants in the testcross progeny obtained in step S2 that can amplify specific bands of molecular markers, and continue self-pollination until homozygous fertile lines are selected. Then, test cross with the cytoplasmic male sterile line of Brassica napus and radish. The fertile lines in the testcross progeny that are all fertile are the stable cytoplasmic restorer lines of Brassica napus and radish.
2. The method for breeding a Brassica napus-radish cytoplasmic restorer line independent of restorer sources as described in claim 1, characterized in that, In step (2), the first culture medium is composed of the following components in the following proportions: 1L MS medium, 0.5-1.5mg of 6-benzyladenine, 30-70mg of colchicine, 20-30g of sucrose and 8-10g of agar, with a pH of 5.8-6; The second culture medium consists of the following components in the following proportions: 1L MS medium, 0.5-1mg 6-benzyladenine, 20-40mg colchicine, 20-30g sucrose and 8-10g agar, with a pH of 5.8-6.
3. The method for breeding a Brassica napus radish cytoplasmic restorer line independent of restorer sources as described in claim 1, characterized in that, In step (3), the third culture medium is composed of the following components in the following proportions: 1L MS medium, 0.03-0.5mg 6-benzyladenine, 5-20mg colchicine, 20-30g sucrose and 8-10g agar, with a pH of 5.8-6.
4. The method for breeding a Brassica napus-radish cytoplasmic restorer line independent of restorer sources as described in claim 1, characterized in that, In step (4), the soaking buffer consists of the following components in the following proportions: 1L of water, 0.6-1.2g of Eboss or Klebsiella pneumoniae and 0.5-1mg of α-naphthaleneacetic acid.
5. The method for breeding a Brassica napus-radish cytoplasmic restorer line independent of restorer sources as described in claim 1, characterized in that, In step S2, flow cytometry was used to determine the ploidy of the offspring.
6. The method for breeding a Brassica napus-radish cytoplasmic restorer line independent of restorer sources as described in claim 1, characterized in that, In step S2, the special molecular markers are Bn09-1, Bn09-2, and Bn09-3; The forward primer sequence of Bn09-1 is shown in SEQ ID NO.1, and the reverse primer sequence of Bn09-1 is shown in SEQ ID NO.2; The forward primer sequence of Bn09-2 is shown in SEQ ID NO.3, and the reverse primer sequence of Bn09-2 is shown in SEQ ID NO.4; The forward primer sequence of Bn09-3 is shown in SEQ ID NO.5, and the reverse primer sequence of Bn09-3 is shown in SEQ ID NO.
6.
7. The application of the Brassica napus-radish cytoplasmic restorer line as described in any one of claims 1-6 in the breeding of new cytoplasmic three-line hybrid radish varieties with strong dominance.