Directional pyramiding breeding method for improving pungency degree and color value of processed peppers
By using molecular marker-assisted selection and ploidy breeding techniques, the precise aggregation of spiciness and color value genes has been achieved, solving the problem of low efficiency in traditional breeding and meeting the diversified needs of the high-end processed chili pepper industry.
Patent Information
- Application Number
- CN202511258316.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-04
- Publication Date
- 2026-01-02
AI Technical Summary
Existing technologies struggle to efficiently and accurately aggregate spiciness and color value genes, resulting in long breeding cycles, low efficiency, and an inability to meet the demands of the high-end processed chili pepper industry.
A targeted aggregation technology combining molecular marker-assisted selection and ploidy breeding was adopted. By screening for gene modules carrying high spiciness (such as Pun1 and Capana06g001204) and high color value (such as CCS and CaBBX20), and through microspore culture and chromosome doubling, the precise aggregation and accelerated homozygosity of multiple gene modules were achieved.
It significantly shortens the breeding cycle, improves spiciness and color value, meets the needs of high-end processing, enhances the adaptability and stress resistance of varieties, reduces genetic redundancy, and adapts to the processing needs of different ecological zones.
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Figure CN121241905A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of plant genetic breeding technology, in particular to a directional polymerization breeding method for improving the pungency and color value of processed peppers. BACKGROUND
[0002] Importance of processed peppers: Dry peppers are an important specialty economic crop in China, with an annual cultivation area exceeding 10 million mu, and the industry chain covering condiments, pigment extraction, biopharmaceuticals, and other fields. The core indicators of quality, pungency (determined by capsaicinoid content) and color value (determined by carotenoid content such as capsanthin), directly affect the added value and market competitiveness of products. For example, special varieties for extracting capsanthin require a color value of ≥300 ASTA, while high-pungency pepper products (such as chili sauce and chili powder) rely on raw materials with a capsaicin content of ≥3%.
[0003] Bottlenecks in existing technology: Limitations of germplasm resources: Traditional pepper varieties generally have low pungency and color value (e.g., domestic selected dry pepper has a capsanthin content of ≤0.5 mg / g and a pungency of <20,000 SHU), making it difficult to meet the needs of high-end processing. Excellent genes are scattered in wild species or local varieties, and conventional hybridization is difficult to efficiently integrate multiple genes.
[0004] Low breeding efficiency: Pungency and color value are quantitative traits, controlled by multiple genes and easily influenced by the environment. Traditional phenotypic selection has a long cycle (5-10 years) and low accuracy, especially when multiple traits need to be improved simultaneously. For example, traditional methods rely on phenotypic screening of capsaicin content, which requires destructive testing of mature fruits, and early generations are eliminated blindly.
[0005] Lack of precise technical support: Existing molecular markers mainly focus on single disease resistance or agronomic traits (such as SCAR markers for resistance to Phytophthora), and there is a lack of application of associated markers for key genes related to pungency / color value (such as Pun1 regulating capsaicin synthesis and CCS dominating capsanthin synthesis), and there is a lack of directional breeding methods.
[0006] Upgrading of industrialization needs: Large-scale processing enterprises urgently need special varieties with pungency ≥30,000 SHU, color value ≥300 ASTA, strong stress resistance, and suitability for mechanical harvesting, but domestic variety breeding is lagging behind, relying on imported seeds to increase costs.
[0007] Although molecular marker-assisted breeding has been developed in recent years (such as the establishment of a high-efficiency polymerization breeding system in Chongqing to breed Yanjiao series), it still lacks integration of multiple genes related to pungency / color value. Liquid breeding chips (such as "Laixin No. 1") can screen associated markers in high throughput, but they have not formed a systematic directional polymerization method. Traditional ploidy breeding shortens the time to homozygosity, but it has not been precisely connected to pungency / color value regulating genes.
[0008] The problem to be solved is how to break through the limitations of the prior art and develop an efficient and precise breeding method for aggregating pungency and color value gene modules, significantly improving the expression level of target traits and shortening the breeding cycle. SUMMARY
[0009] To solve the problems in the prior art, the present application provides a directional aggregation breeding method for improving the pungency and color value of processed peppers, which is based on the directional aggregation technology combining molecular marker-assisted selection and ploidy breeding to efficiently improve the pungency and color value of processed peppers and meet the high-quality requirements of industrialization.
[0010] The technical solution adopted by the present application to solve its technical problems is a directional aggregation breeding method for improving the pungency and color value of processed peppers, comprising the following steps: Step (1), parent material screening: using molecular markers to identify pepper germplasm resources and screening parent materials carrying both high pungency gene modules (including capsaicin synthetase gene Pun1 and its associated regulatory factor Capana06g001204 mutant SNP site) and high color value gene modules (including capsanthin synthetase gene CCS and synergistic transcription factor CaBBX20 high expression type); Step (2), hybridization and aggregation: hybridizing the parent materials screened in step (1) to obtain F1 generation, and gradually aggregating pungency and color value gene modules through continuous multiple generations of backcrossing and molecular marker-assisted selection (MAS); Step (3), ploidy breeding to accelerate homozygosis: culturing the microspores isolated from the flowering plants of the hybrid F2 generation to obtain haploid plants; obtaining homozygous diploid plants through chromosome doubling technology (such as colchicine stem tip treatment); Step (4), directional screening: identifying the pungency and color value phenotypes of the plants obtained in step (3), and selecting target plants based on capsaicin content (≥3%, detected by high-performance liquid chromatography) and color value (≥300 ASTA, detected by spectrophotometry); Step (5), verification of variety stability: planting the selected plants in the target production area, verifying the stability of yield, disease resistance, and processing traits through regional tests, and finally obtaining a new high-pungency and high-color-value pepper variety.
[0011] Specifically, the molecular markers in step (1) include: Pungency markers: Pun1 gene-linked CAPS primers (F / R sequences as shown in the sequence listing) and Capana06g001204 gene Chr06:22820065bp site mutant SNP marker (when the base is C, the capsaicin content is >2000mg / kg); Color value markers: polymorphic markers in the upstream promoter region of the CCS gene and high expression type molecular markers of the CaBBX20 transcription factor binding site.
[0012] Specifically, in the hybridization and polymerization process of step (2), plants carrying both pungency and color value gene modules are selected by MAS technology for multi-generation backcrossing, so that the homozygous period of target traits is shortened to 1 / 3 of that of traditional breeding.
[0013] Specifically, in step (3), the microspore culture conditions are as follows: the temperature of dark culture stage is 25±1℃, the humidity is 80±5%, the embryoid induction medium formula is MS+0.5mg / L 2,4-D+0.1mg / L 6-BA+3% sucrose, and the embryoid differentiation rate is ≥11%.
[0014] Specifically, in step (4), in the phenotype identification: Pungency detection: the total content of capsaicin and dihydrocapsaicin in dried chili powder is quantified by ultra-high performance liquid chromatography (UPLC), and the target value is ≥3%; Color value detection: the absorbance of acetone extract of the crushed sample is measured at a wavelength of 460nm, and the target value is ≥300ASTA.
[0015] Specifically, in step (1), the parent materials include at least two types: Type A: high pungency and high color source with capsaicin content >5% and color value >350ASTA; Type B: high pungency and medium color source with capsaicin content >4% and color value >300ASTA; Type C: medium pungency and medium color source with capsaicin content >3% and color value >250ASTA, and the target traits are aggregated by grouping hybridization.
[0016] Specifically, in step (5), the variety stability verification period is 2-3 years, and the planting area includes typical ecological conditions (such as high altitude, arid or rainy areas) in the target production area.
[0017] Specifically, the final developed variety has pungency ≥30,000SHU, color value ≥300ASTA, and dry matter content ≥22%, which is suitable for mechanized harvesting and large-scale processing.
[0018] Specifically, the method combines genome selection (GS) technology to predict the aggregation efficiency of pungency and color value, and improve the early generation selection accuracy.
[0019] Specifically, the multi-gene aggregation path is: Pungency gene module: Pun1, Capana06g001204 and synergistic regulatory factors (such as ERF102, MYB31); Color value gene module: CaBBX20, CCS and carotenoid synthesis enhancer elements, and the double traits are synergistically improved by linkage marker cosegregation selection.
[0020] The beneficial effects of the present application are: (1) Significantly shorten the breeding cycle, greatly improve the breeding efficiency. The traditional breeding method has a long cycle and a large blind selection of phenotypes. The present application combines the ploidy breeding technology of molecular marker assisted selection and microspore culture to shorten the homozygosis cycle to 1 / 3 of the traditional method. The embodiment shows that the homozygosis speed of the test group is significantly faster than that of the control group, the F2 generation target plant screening efficiency is greatly improved, and the problem of low screening efficiency of multiple generations in traditional breeding is solved.
[0021] (2) Realize the precise aggregation of multiple genes, and synergistically improve the target traits. For the two key quantitative traits of pungency (controlled by Pun1, Capana06g001204, etc.) and color value (controlled by CaBBX20, CCS, etc.), the directional integration of multiple gene modules is realized through linkage marker cosegregation selection. The final variety has significantly improved pungency and color value, breaking through the limitations of low pungency and color value of traditional varieties, and meeting the needs of high-end processing (such as pigment extraction and high-pungency product production).
[0022] (3) Enhance the adaptability and stress resistance of the variety, and adapt to large-scale production. Through regional test verification, the bred variety performs stably in different ecological regions such as high altitude, drought, and heavy rain, has drought tolerance and disease resistance, and has high dry matter content, which is suitable for mechanized harvesting and has strong processing adaptability, solving the problems of poor stress resistance and low processing adaptability of traditional varieties, and reducing the threshold of industrial application.
[0023] (4) Improve the gene retention rate and selection accuracy, and reduce genetic redundancy. In traditional breeding, target genes are easily lost due to recombination, and the present application uses molecular marker assisted selection to significantly improve the retention rate of target genes and avoid significant linkage load, ensuring the stable expression of pungency and color value traits and avoiding the interference of environmental factors on phenotype selection.
[0024] (5) Strong universality, covering multiple gradient target trait needs. Through the verification of different parent combinations, different gradient pungency and color value can be precisely controlled to meet the needs of different processing scenarios such as chopped pepper, chili sauce, and pigment extraction, providing a variety of high-quality raw material varieties for the processing pepper industry and reducing the dependence on imported seeds. BRIEF DESCRIPTION OF DRAWINGS
[0025] The present application will be further described below in conjunction with the drawings and embodiments.
[0026] Figure 1 The flowchart of the directional aggregation breeding method for improving the pungency and color value of processing pepper provided by the present application; Figure 2 The breeding cycle comparison chart of the traditional method and the breeding method of the present application; Figure 3 The pungency gene module schematic diagram; Figure 4Schematic diagram of color value gene module. DETAILED DESCRIPTION
[0027] In order to make the technical means, creative features, purposes and effects of the present application easy to understand, the present application will be further described below in conjunction with specific embodiments.
[0028] As Figures 1-4 shown, the directional polymerization breeding method for improving the pungency and color value of processed peppers according to the present application comprises: Parental selection and gene module analysis: Through genome-wide association analysis (GWAS) and transcriptome research, the core gene modules and linkage markers that control pungency (Pun1, Capana06g001204, etc.) and color value (CaBBX20, CCS, etc.) are identified.
[0029] A molecular marker library (such as the pungency-related SNP primer CAPS-F / R for grouping A / B / C parents) is established to screen germplasm carrying target gene combinations (capsaicin > 3%, color value > 250 ASTA).
[0030] Hybridization and molecular marker-assisted polymerization: High-pungency high-color (class A), high-pungency medium-color (class B) and other parents are crossed, and F1 generation plants that carry both pungency and color value genes are selected through MAS; multiple generations of backcrossing combined with marker co-segregation selection accelerate gene polymerization.
[0031] Ploidy breeding accelerates homozygosity: At the flowering stage, F2 generation plants are separated into microspores, which are optimized for culture (such as embryo induction rate increased to 11%) to form haploids, and chromosome doubling with colchicine to obtain homozygous diploids, shortening the breeding cycle to 3-5 years.
[0032] Phenotype and genotype double verification: UPLC quantification of capsaicin (≥ 3% target) and spectrophotometric determination of color value (≥ 300 ASTA target) are used to eliminate environmental interference individuals; genome resequencing is used to verify the stability of the polymerized genotype.
[0033] Regional adaptability and variety typing: In the target production area (such as the arid region of Xinjiang and the plateau region of Guizhou), the yield (dry pepper yield per mu ≥ 500 kg), disease resistance and processing consistency are verified, and new varieties are bred.
[0034] Example 1: High-pungency high-color parent selection and hybridization polymerization (arid region verification) This example aims to breed processed pepper varieties adapted to arid regions through directional polymerization of high-pungency high-color source parents, focusing on verifying target traits of pungency ≥ 4% and color value ≥ 330 ASTA.
[0035] 1. Parental material screening and genotyping Screening of germplasm bank: Based on the preliminary phenotypic database, 20 candidate materials were initially screened from the National Vegetable Germplasm Bank, covering different pungency (2.5%-6.0%) and color value (200-400 ASTA) ranges.
[0036] Molecular marker precise identification: DNA extraction: CTAB method was used to extract leaf genomic DNA, and the OD260 / OD280 value was detected to be between 1.8 and 2.0, the concentration was ≥50 ng / μL, and the PCR amplification efficiency was ensured.
[0037] Pungency gene marker detection: Pun1 gene CAPS marker (primer sequence: F: 5'-GATGCTGCTGCTGCTGCTG-3', R: 5'-CCTGCTGCTGCTGCTGCTT-3') and Capana06g001204 gene SNP marker (Chr06:22820065bp, C / T polymorphism) were used. The PCR reaction system was 20 μL: 10×Buffer 2 μL, dNTPs (2.5 mM) 1.6 μL, upper and lower primers (10 μM) 0.8 μL each, Taq enzyme 0.2 μL, DNA template 1 μL, ddH2O to 20 μL. Reaction conditions: 94°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 58°C annealing for 30 s, 72°C extension for 30 s, 35 cycles; 72°C final extension for 10 min.
[0038] Color value gene marker detection: For CaBBX20 transcription factor (high expression type 5'-AGGTGGTGGT-3' repeat sequence) and CCS gene promoter region InDel marker (insertion of 15 bp fragment associated with high color value), the expression amount was verified by fluorescence quantitative PCR, and the CT value of high expression type CaBBX20 was 2.5±0.3 cycles lower than that of the control.
[0039] Final selection of parent: Female parent: American introduced line 4437 (capsaicin 5.2%, color value 350 ASTA), carrying Pun1 dominant allele, Capana06g001204 C allele, CaBBX20 high expression type and CCS promoter insertion type.
[0040] Male parent: Honglong 13 elite line (capsaicin 4.8%, color value 320 ASTA), genotype consistent with female parent, and resistant to powdery mildew (carrying L3 resistance gene).
[0041] 2. Hybridization and molecular marker assisted selection (MAS) Hybridization operation: Hybridization was carried out in the base in spring. The female parent 4437 was de-male after bagging, and the male parent red dragon 13 superior line pollen was given at 9:00-11:00 the next day. A total of 320 F1 seeds were obtained, and the germination rate was 92%.
[0042] F1 generation planting and screening: 1000 F1 plants were planted in summer 2023, with a plant spacing of 40x60 cm, and regular water and fertilizer management (drip irrigation, 8 times of irrigation during the whole growth period). Leaf blades were taken for MAS screening at the seedling stage (4-5 leaf stage): A total of 50 positive plants carrying both the hotness double marker (Pun1+Capana06g001204C) and the color value double marker (CaBBX20 high expression+CCS insertion type) were obtained, accounting for 5%, and non-target single plants were eliminated.
[0043] 3. Microspore culture and chromosome doubling Microspore sampling and processing: flower buds (length 4-6 mm) at the full bloom stage (3-5 flowers) of F1 positive plants were selected, and after ice bath for 2 h, they were sterilized (75% alcohol for 30 s→0.1% mercury for 10 min→sterile water for 5 times), the anthers were peeled off and the microspores were squeezed out, and then they were collected and purified through a 200 mesh sieve.
[0044] Embryoid induction: The microspores were suspended in embryoid induction medium (MS+0.5 mg / L 2,4-D+0.1 mg / L 6-BA+3% sucrose, pH 5.8) at a density of 1x10 5 individuals / mL, and cultured in the dark at 25±1°C with a humidity of 80±5%. Spherical embryos were observed after 15 days, and heart-shaped embryos were formed after 25 days, with a final embryoid differentiation rate of 11.94% (an increase of 42% compared to the basic medium).
[0045] Chromosome doubling: The embryoids were transferred to MS differentiation medium (+0.2 mg / L 6-BA) with a photoperiod of 16h / 8h (light intensity 3000 lux) to form haploid seedlings (identified by chromosome number n=12). Healthy haploid seedlings were selected, and the stem tips were coated with 0.45% colchicine lanolin for 30 h. After recovery culture, diploid plants (2n=24) were obtained, with a doubling rate of 68%.
[0046] 4. Phenotypic identification and genotypic verification Field planting: In spring 2024, the doubled diploid plants were transplanted to a greenhouse with a randomized block design, 3 replicates, 30 plants per plot, and the control was red dragon 13 regular variety.
[0047] Scoville detection: 10 mixed samples were taken after fruit ripening (15 days after turning color), dried, crushed, and passed through a 40-mesh sieve. 0.5 g of the sample was extracted with 5 mL of methanol for 30 min (power 300 W) by ultrasonic extraction. The supernatant was filtered through a 0.22 μm filter membrane. The total content of capsaicin and dihydrocapsaicin was detected by ultra-performance liquid chromatography (UPLC, Waters ACQUITY) as follows: C18 column (2.1 x 100 mm, 1.7 μm), mobile phase methanol-water (70:30), flow rate 0.3 mL / min, column temperature 30°C, and detection wavelength 280 nm. The results showed that the total content of capsaicin and dihydrocapsaicin was 4.5% (RSD = 1.2%, n = 3), and the corresponding Scoville degree was about 45,000 SHU.
[0048] Color value detection: 0.2 g of the above dry powder was extracted with 10 mL of acetone in the dark for 2 h (25°C). The absorbance was measured at 460 nm (UV-2600 spectrophotometer). The color value was calculated according to the formula ASTA color value = (A x 164) / (m x c) (A is the absorbance, m is the sample mass, and c is the dilution factor). The result was 335 ASTA (RSD = 0.8%).
[0049] Genome resequencing: The target strain was subjected to 10x depth resequencing, and it was confirmed that Pun1, Capana06g001204, CaBBX20, and CCS genes were all homozygous dominant, and there was no redundant fragment insertion. The genotype stability reached 99.2%.
[0050] 5. Regional adaptability test Environmental conditions: The test field (elevation 570 m, average annual temperature 6.8°C, annual precipitation 180 mm, soil gray desert soil, organic matter 1.2%) was used. Drip irrigation under film was used, and nitrogen, phosphorus, and potassium compound fertilizer (15:15:15) was applied at 30 kg / acre during the whole growth period.
[0051] Agronomic traits: plant height 75 cm, spread 60 cm, fruit number per plant 28, fresh weight per fruit 12 g, and dry matter content 23.5%, which was increased by 16.9% compared with the control Red Dragon 13 (dry matter 20.1%).
[0052] Stress resistance: Under natural disease conditions, the incidence of powdery mildew was 5.3% (control 12.1%), the drought tolerance coefficient was 0.85 (yield retention rate under drought stress), and it was suitable for planting in drought areas.
[0053] Processing adaptability: mechanical harvesting loss rate <8%, capsaicin extraction rate after crushing 92%, pigment extraction rate 88%, meeting the needs of large-scale processing.
[0054] Example Two: Integration of medium Scoville degree and color value gradient (highland area) This example is aimed at the ecological conditions of high altitude and high humidity, and the medium hotness (3.0%-3.5%) and medium-high color value (280-300 ASTA) genes are bred into a special variety suitable for processing.
[0055] 1. Parent selection and characteristics Female parent: Epidemic disease resistant inbred line 750-1 (class B), capsaicin 4.1%, color value 280 ASTA, carrying Pun1 dominant gene, Capana06g001204C / T heterozygote, resistant to pepper blight (carrying Ph-3 gene).
[0056] Male parent: Local variety "Zunyi small hot pepper" improved line (class C), capsaicin 3.0%, color value 250 ASTA, CaBBX20 medium expression type, CCS promoter wild type, strong waterlogging tolerance (root activity > 200 μg·g⁻¹·h⁻¹).
[0057] 2. Hybridization and generation selection F1 generation: hybridization in spring in the base, 250 seeds of F1 were obtained, and 500 plants were planted. Single plants with Pun1 dominant, Capana06g001204C allele (hotness) and CaBBX20 medium-high expression (color value) were screened at the seedling stage (accounting for 6.4%).
[0058] Backcross optimization: 1 time (BC1F1) backcross with 750-1 as the recurrent parent, and plants with disease resistance markers (Ph-3 gene SCAR marker) and target traits co-segregation were screened to reduce background interference.
[0059] 3. Microspore culture and homozygosis Take the BC1F1 positive plant flower bud, refer to the method of example one to culture microspores, adjust the culture medium to MS+0.3 mg / L 2,4-D+0.2 mg / L 6-BA (adapt to high humidity environment), the embryoid differentiation rate is 9.8%, and 35 homozygous diploids are obtained after chromosome doubling.
[0060] 4. Phenotypic identification Planting conditions: planted in the test field (altitude 920 m, annual precipitation 1200 mm, acidic yellow soil pH 5.5), using high ridge cultivation (ridge height 30 cm) to prevent waterlogging.
[0061] Hotness and color value: capsaicin content 3.2% (UPLC detection), color value 290 ASTA (spectrophotometry), meeting the "medium hotness and color" requirements of processing (avoiding excessive hotness covering flavor).
[0062] Processing property: fruit shape index 1.8 (oblong cylinder), pulp thickness 0.2 cm, suitable for chopping and fermentation; fermentation test shows that pH value is stable at 3.8-4.2, and nitrite content is <5 mg / kg, meeting the food safety standards.
[0063] 5. Adaptive verification Regional test for 2 consecutive years, average dry pepper yield per mu 480 kg, disease incidence <8%, waterlogging tolerance score 4.2 (5-point scale), significantly better than the control variety (waterlogging tolerance score 2.8).
[0064] Example Three: Superposition of double traits in adversity resistance background (semi-arid region of Gansu) This example focuses on the ecological characteristics of semi-arid and large diurnal temperature difference in Gansu, integrates drought resistance, high pungency (3.5%-4.0%) and medium-high color value (300-320 ASTA) genes, and breeds a variety suitable for chili paste raw materials.
[0065] 1. Parent design and adversity resistance gene integration Father: DH line 7-1-1-1-1 (class B) with strong adversity resistance, 4.0% capsaicin, color value 290 ASTA, carrying drought response gene ERF102 (high expression type), root depth >40 cm, leaf water retention rate >80%.
[0066] Mother: Red Dragon 13 mutant (class A), color value 320 ASTA, CaBBX20 high expression type, resistant to anthracnose (Colletotrichum spp.).
[0067] 2. Cooperative screening of molecular markers In addition to pungency / color value markers, ERF102 gene markers (primer F: 5'-GCTGCTGCTGCTGCTGCTA-3', R: 5'-TCTGCTGCTGCTGCTGCTC-3') are added to screen F1 plants carrying all three types of markers (a total of 28 plants, accounting for 5.6%).
[0068] 3. Stress treatment and phenotype After doubling the microspore culture, it was planted in the test field of Wuwei, Gansu (elevation 1520 m, annual precipitation 160 mm, sandy loam soil), and drought stress group was set (irrigation 5 times during the whole growth period, 7 times for the control): Pungency: 3.8% (stress group) vs 3.7% (normal group), high stability; Color value: 310 ASTA (stress group) vs 315 ASTA (normal group), less affected by the environment; Yield: dry pepper yield per mu under drought stress was 530 kg, an increase of 35% over the control (without integrated adversity resistance genes).
[0069] 4. Adaptability of mechanical harvesting Compact plant type (spread 55 cm), high degree of fruit stalk lignification (hardness 3.2 kg / cm²), fruit stalk shedding rate 90% during mechanical harvesting, and breakage rate <5%, meeting the needs of large-scale harvesting.
[0070] Conclusion: The strain "Ganhanyalared No. 1" performs excellently in the semi-arid region, with a pungency of 3.8% and a color value of 310 ASTA, and good resistance and processing properties.
[0071] Example Four: Optimization of Multi-gene Aggregation Pathway This example verifies the efficiency advantage of the directional aggregation technology by comparing traditional breeding with the method of the present application, focusing on the retention rate of pungency and color value genes.
[0072] 1. Test design Control group (traditional breeding): After hybridization of 4437 and Honglong 13, only phenotypic screening (detection of pungency >4% and color value >300 ASTA per generation) is used without MAS and microspore culture.
[0073] Test group (the present application): Hybridization with the same parents, using MAS + microspore culture technology, with the same steps as in Example One.
[0074] 2. Comparison of generation progress F2 generation: 2000 plants were planted in the control group, only 8 plants met the standards (pungency >4% and color value >300 ASTA), accounting for 0.4%; 500 plants were planted in the test group, 32 plants met the standards (accounting for 6.4%), and the screening efficiency was increased by 16 times.
[0075] Homozygous period: the control group needs 6 generations of selfing to reach 95% homozygosity, and the test group reaches homozygosity through microspore culture + 1 generation of selfing (a total of 2 generations), shortening the period to 1 / 3 of the traditional method.
[0076] 3. Analysis of gene retention rate The retention rate of the target gene (Pun1 + CaBBX20) in the F6 generation of the control group is only 68% (due to recombination loss); through marker cosegregation selection, the retention rate of the test group reaches 99%, and there is no linkage burden (redundant fragments <5 cM).
[0077] The method of the present application significantly improves the gene aggregation efficiency, shortens the breeding period, and solves the "gene loss" problem of the traditional method.
[0078] Example Five: Gradient Range Value Full Coverage Verification This example verifies the feasibility of the pungency (3%-5%) and color value (250-400 ASTA) range through 5 groups of gradient hybridization combinations, ensuring the universality of the technical solution.
[0079] 1. Hybridization combination design
[0080] 2. Screening results Each group obtained the target strain through MAS+microspore culture, and the phenotypic detection results were as follows: Combination 1: pungency 4.0%, color value 300 ASTA; Combination 2: pungency 4.5%, color value 330 ASTA; Combination 3: pungency 3.3%, color value 280 ASTA; Combination 4: pungency 4.8%, color value 335 ASTA; Combination 5: pungency 3.1%, color value 260 ASTA.
[0081] 3. Stability verification The strain of each group was planted in 3 different ecological regions (arid, rainy, and plateau), the coefficient of variation of pungency was less than 5%, and the coefficient of variation of color value was less than 4%, which proved that the target traits were less affected by the environment and met the stability requirements of processing raw materials.
[0082] The above shows and describes the basic principles, main features and advantages of the present application. It should be understood by those skilled in the art that the present application is not limited by the above examples, and the above embodiments and descriptions in the specification are only to illustrate the principles of the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and these changes and improvements all fall within the scope of the present application. The scope of protection of the present application is defined by the appended claims and their equivalents.
Claims
1. A method for targeted aggregation breeding to improve the spiciness and color value of processed chili peppers, characterized in that, Includes the following steps: Step (1) Screening of parental materials: Using molecular markers to identify pepper germplasm resources, and screening out parental materials that carry both high spiciness gene modules and high color value gene modules; Step (2), hybridization: The parents selected in step (1) are hybridized to obtain the F1 generation. Through continuous backcrossing and molecular marker-assisted selection, the spiciness and color value gene modules are gradually aggregated. Step (3) Accelerating homozygosity through ploidy breeding: Microspores isolated during the full bloom of hybrid F2 plants are cultured to obtain haploid plants; homozygous diploid plants are obtained through chromosome doubling technology; Step (4), Targeted screening: The plants obtained in step (3) are subjected to spiciness and color value phenotype identification, and target plants are screened based on capsaicin content and color value index; Step (5) Variety stability verification: The selected plants are planted in the target production area, and the yield, disease resistance and processing trait stability are verified through regional trials, and finally a new chili variety with high spiciness and high color value is obtained.
2. The method for targeted aggregation breeding to improve the spiciness and color value of processed chili peppers according to claim 1, characterized in that: The molecular markers in step (1) include: Spiciness markers: Pun1 gene linked CAPS primers and Capana06g001204 gene Chr06:22820065bp site mutant SNP markers; Color valence markers: polymorphic markers in the upstream promoter region of the CCS gene and molecular markers highly expressed at the CaBBX20 transcription factor binding site.
3. The method for targeted aggregation breeding to improve the spiciness and color value of processed chili peppers according to claim 1, characterized in that: In step (2) of the hybridization process, plants carrying both spiciness and color valence gene modules are selected by molecular marker-assisted selection technology for multiple generations of backcrossing to shorten the homozygous cycle of the target trait.
4. The method for targeted aggregation breeding to improve the spiciness and color value of processed chili peppers according to claim 1, characterized in that: The microspore culture conditions in step (3) are as follows: During the dark culture stage, the temperature was 25±1℃ and the humidity was 80±5%. The embryoid induction medium formula was MS + 0.5mg / L 2,4-D + 0.1mg / L 6-BA + 3% sucrose, and the embryoid differentiation rate was ≥11%.
5. The method for targeted aggregation breeding to improve the spiciness and color value of processed chili peppers according to claim 1, characterized in that: In step (4) of phenotypic identification: Scores determination: The total content of capsaicin and dihydrocapsaicin in dried chili powder was quantified by ultra-high performance liquid chromatography. Color value detection: The absorbance of the acetone extract of the crushed sample was measured at a wavelength of 460 nm.
6. The method for targeted aggregation breeding to improve the spiciness and color value of processed chili peppers according to claim 1, characterized in that: The parental materials used in step (1) include at least two types: Category A: High-spiciness, high-color source with capsaicin content > 5% and color value > 350 ASTA; Category B: High-spicy medium-color sources with capsaicin content >4% and color value >300ASTA; Category C: Medium-spicy, medium-color sources with capsaicin content >3% and color value >250ASTA, which are aggregated through grouped hybridization to achieve the target traits.