A method for screening and directional breeding improvement of potato stress-resistant high-quality germplasm resources

Abstract

The application provides a potato stress-resistant high-quality germplasm resource screening and directional breeding improvement method, and belongs to the technical field of crop germplasm resource screening and breeding.The application builds a compound stress garden by standardizing virus-free seedling of potato germplasm, and simultaneously applies four gradient stresses of drought, saline-alkali, night low temperature and continuous cropping of root knot nematode;combined with progressive agronomic phenotype observation of potato seedling stage and tuber bulking stage, inferior single plants are eliminated in advance;nondestructive batch quality detection of tubers is carried out by using near-infrared spectroscopy technology, and the four-dimensional weighted comprehensive scoring system is relied on to complete germplasm grading and screening;finally, the excellent germplasm obtained by screening is divided into groups for seed saving, and through cross-group directional crossing and continuous breeding under stress, a stress-resistant high-quality potato improved line is bred.The new line has multiple stress resistance and excellent commodity and processing quality, and is suitable for large-scale potato germplasm screening and directional breeding popularization and application.

Description

Technical Field

[0001] This invention relates to the field of crop germplasm resource screening and breeding technology, and in particular to a method for screening and targeted breeding improvement of stress-resistant and high-quality potato germplasm resources. Background Technology

[0002] Potatoes are the world's fourth largest food crop, widely cultivated in my country, and play a vital role in ensuring food security. However, potato production often faces multiple abiotic stresses, including drought, salinity, low-temperature freezing damage, and root-knot nematode diseases. These stresses often occur simultaneously and interact, severely impacting potato yield and quality. Therefore, screening potato germplasm resources with multiple stress resistances and excellent processing qualities, and conducting targeted breeding improvements, is a crucial issue that urgently needs to be addressed in the field of potato breeding.

[0003] In existing technologies, potato multiple stress resistance screening and quality testing face a dilemma: tuber quality testing under combined stress requires destructive sampling, resulting in the inability to retain superior germplasm for seed production; while non-destructive testing lacks precision and cannot support breeding decisions. Simultaneously, germplasm obtained through single stress screening degrades under combined field stress, while simply applying multiple stresses leads to excessive screening pressure, complete germplasm death, or excessively low seed setting rates, making it impossible to obtain effective breeding materials.

[0004] In summary, the existing technology lacks an efficient screening and breeding method for potato germplasm resources that can simultaneously simulate multiple abiotic stresses, requires no molecular biology operations, non-destructively test quality, and organically combine screening with targeted improvement. Summary of the Invention

[0005] The purpose of this invention is to provide a method for screening and targeted breeding improvement of potato germplasm resources with stress resistance, so as to achieve efficient and non-destructive screening of potato germplasm and targeted breeding of new potato lines with multiple stress resistance, high quality and high yield.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for screening and targeted breeding improvement of stress-resistant and high-quality potato germplasm resources, comprising the following steps: 1) Virus-free potato germplasm was detoxified, tissue culture propagation was carried out, and seedlings were hardened off in a greenhouse. Strong seedlings were selected as planting materials. 2) Construct a tiered, progressively challenging stress nursery and plant the plants; 3) Conduct agronomic phenotypic observations during the seedling stage and tuber enlargement stage, and eliminate unqualified individual plants according to the elimination criteria; 4) Conduct appearance evaluation and quality testing on the harvested tubers; 5) The germplasm is scored based on four dimensions: physiological phenotype of resistance, commercial appearance of tubers, internal processing quality and yield under stress. The germplasm is graded according to the comprehensive score and the core superior germplasm is selected. 6) The selected core superior germplasm was divided into different parent groups according to the scoring results of each dimension. Cross-group directional hybridization strategy was adopted for hybridization, and the hybrid offspring were placed in a medium-stressed compound aquarium for continuous pressure selection until the traits were stable.

[0007] Preferably, in step 1), the virus removal is carried out using the shoot tip peeling culture method; MS basal medium is used for tissue culture propagation; the tissue culture temperature is 25±1℃, the light intensity is 2000~3000 lx, and the photoperiod is 14~16 h light / 8~10 h dark; the hardening substrate is peat moss:vermiculite:perlite = 2~3:1~2:1~2 by mass, and 1 / 2 Hoagland nutrient solution is applied, with a hardening period of 26~30 days; the standard for planting material is a plant height of 5~8 cm and ≥5 leaves.

[0008] Preferably, the stratified composite stress field in step 2) includes mild composite stress cells, moderate composite stress cells, and severe composite stress cells; The stress conditions of the mild combined stress plot were: soil water holding capacity 45%~50%, soil salinity 1.0‰~1.5‰, nighttime temperature controlled at 10~12℃, and root-knot nematodes 400~600 per 100g dry soil. The stress conditions of the moderate combined stress plot were: soil water holding capacity of 30%~35%, soil salinity of 2.0‰~3.0‰, nighttime temperature control of 6~8℃, and root-knot nematode stress of 1000~1400 nematodes / 100g dry soil. The stress conditions of the severe combined stress plot were as follows: soil moisture content of 18% to 23%, soil salinity of 3.5‰ to 4.5‰, nighttime temperature of 2 to 4℃, and root-knot nematode stress of 1800 to 2200 nematodes per 100g of dry soil.

[0009] Preferably, the agronomic phenotypic observation during the seedling stage in step 3) is as follows: 20-30 days after transplanting, observe the recovery ability from wilting under strong light and the proportion of yellow leaves. The elimination criteria are: wilting under strong light cannot be recovered within 2 hours or the proportion of yellow leaves exceeds 30%. The agronomic phenotypic observation during the tuber enlargement period is as follows: 60-80 days after transplanting, the disease incidence and tuber formation characteristics of the plants are observed. The elimination criteria are: leaf lesion coverage rate exceeds 35% or the number of tubers per plant is less than 3 or the proportion of deformed tubers is higher than 40%.

[0010] Preferably, the quality detection in step 4) is to detect the content of tuber dry matter, starch, and reducing sugar.

[0011] Preferably, the scoring criteria in step 5) are: 40 points for stress resistance physiological phenotype, 20 points for tuber appearance marketability, 25 points for internal processing quality, and 15 points for stress yield; the grading criteria are: a comprehensive score ≥75 points is core superior germplasm, 60~74 points is candidate germplasm, and a score <60 points is eliminated.

[0012] Preferably, the stress resistance physiological phenotype is scored based on seedling wilting recovery ability, yellow leaf ratio, lesion coverage rate during the tuber enlargement stage, and plant growth vigor; the tuber appearance marketability is scored based on the tuber shape regularity, skin smoothness, tuber size uniformity, and the ratio of green-skinned and rotten tubers after harvest; the internal processing quality is scored based on dry matter, starch, and reducing sugar content; and the stress yield is calculated by proportionally adjusting the relative yield under each stress level based on the yield of the blank control.

[0013] Preferably, the different parent groups in step 6) are stress-resistant parent groups, tuber appearance parent groups, high starch parent groups, and stress-yield parent groups; the directional hybridization strategy is: the stress-resistant parent group is hybridized with the tuber appearance parent group, the high starch parent group is hybridized with the stress-yield parent group, and then the two types of hybrid combinations are hybridized a second time.

[0014] Preferably, the stress-resistant parental group consists of germplasm with a stress resistance physiological phenotype score ≥36; the tuber appearance parental group consists of germplasm with a tuber appearance commercial quality score ≥18; the high-starch parental group consists of germplasm with a high-starch processing quality score ≥22.5; and the high-yield parental group under adverse conditions consists of germplasm with an adverse yield score ≥13.5.

[0015] This invention also provides the application of the method in potato breeding.

[0016] Beneficial effects

[0017] 1. This invention applies four abiotic stressors—drought, salinity, low nighttime temperature, and root-knot nematodes—to the same screening system simultaneously, and sets three progressive levels: mild, moderate, and severe. This can realistically simulate the complex environment of multiple stressors superimposed in field production, and the performance of the selected germplasm under combined stress conditions is more valuable for reference.

[0018] 2. This invention detects the quality of micro-tubers during the tuber enlargement stage and mature commercial potatoes. The entire process uses near-infrared spectroscopy scanning, without crushing or using chemical reagents. The tubers after detection can be completely recovered and reused for seed production, thus achieving efficient preservation and recycling of germplasm resources.

[0019] 3. This invention allows for progressive observation and early elimination of inferior individual plants during the three growth stages: seedling stage, fruit enlargement stage, and maturity stage. This avoids the waste of ineffective germplasm on later testing resources and significantly improves screening efficiency.

[0020] 4. This invention comprehensively evaluates four dimensions: stress resistance physiological phenotype, tuber appearance and marketability, internal processing quality, and stress yield. The weighting is scientific and reasonable, which can fully reflect the comprehensive value of germplasm, screen out core superior germplasm, and further divide the screened core superior germplasm into four parent groups according to the dominant stress resistance traits. Cross-group directional hybridization and continuous selection under pressure in a composite stress nursery are adopted to achieve rapid aggregation and stable inheritance of superior genes, shortening the breeding cycle. Detailed Implementation

[0021] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0022] Example 1

[0023] 1. Standardized and unified pretreatment of germplasm

[0024] Two hundred potato germplasm samples were all detoxified and virus-free plantlets were obtained through shoot tip separation culture. These plantlets were then uniformly propagated in a tissue culture laboratory using MS basal medium (3% sucrose, 0.7% agar, pH 5.8–6.0). The culture temperature was 25±1℃, the light intensity was 2000–3000 lx, and the photoperiod was 16 h light / 8 h dark. After propagation, the plantlets were transferred to a greenhouse for hardening off. The hardening substrate was peat moss:vermiculite:perlite = 2:1:1 (mass ratio), and the plants were uniformly irrigated with 1 / 2 Hoagland nutrient solution. After 30 days of cultivation, healthy, disease-free seedlings with uniform height (5–8 cm), ≥5 leaves, and well-developed root systems were selected for transplanting, while weak, deformed, and unevenly growing seedlings were removed.

[0025] 2. Construct a three-tiered, progressively layered composite stress nursery and plant in designated areas.

[0026] A stress nursery was established on a flat, well-drained plot with consistent soil type. The total area of ​​the stress nursery was approximately 2000 m², divided into four areas: a mild combined stress plot (400 m²), a moderate combined stress plot (400 m²), a severe combined stress plot (400 m²), and a no-stress control plot (400 m²). A 2-m wide buffer zone was set up between each plot to prevent interference between stress factors.

[0027] A randomized block design was used in each plot, with three biological replicates for each germplasm sample in each plot. Five plants were planted in each replicate, with a plant spacing of 30 cm × 60 cm. Base fertilizer (2000 kg / mu of organic fertilizer and 50 kg / mu of compound fertilizer) was applied uniformly before transplanting. Except for stress treatment, other agronomic management measures were kept consistent across plots.

[0028] The standardized parameters for the four stresses are as follows: Drought stress: Soil moisture content was controlled at 45%–50% in mild cases, 30%–35% in moderate cases, 18%–23% in severe cases, and 70%–75% in the control group. Soil moisture was monitored in real time using soil moisture sensors, and water was precisely controlled using a drip irrigation system.

[0029] Salt-alkali stress: Soil salinity was adjusted to 1.2‰ for mild cases, 2.5‰ for moderate cases, and 4.0‰ for severe cases, while the blank control was <0.3‰. Soil salinity was adjusted by applying a mixed salt solution of NaCl and Na2SO4 (molar ratio 1:1).

[0030] Low temperature stress: Nighttime temperature control measures were implemented from the seedling stage to the fruit enlargement stage. The nighttime temperature in the mild stress plots was controlled at 10-12℃, in the moderate stress at 6-8℃, and in the severe stress at 2-4℃. The nighttime temperature in the control group was ≥15℃. A combination of movable insulation film and a ventilation system was used to regulate the nighttime temperature.

[0031] Root-knot nematode stress: Inoculate with a suspension of southern root-knot nematode (Meloidogyne incognita) eggs at planting. For mild stress, inoculate 500 nematodes / 100 g dry soil; for moderate stress, 1200 nematodes / 100 g dry soil; and for severe stress, 2000 nematodes / 100 g dry soil. A clean substrate without nematodes is used as a blank control. Apply the nematode egg suspension evenly into the planting hole during inoculation.

[0032] 3. Progressive agronomic phenotypic observation at different growth stages

[0033] (1) Seedling observation (20-30 days after transplanting): Investigate the recovery ability of each seedling under strong light wilting (the degree of recovery 2 hours after wilting under strong light at noon, with light intensity ≥800 µmol·m -2 ·s -1 ), proportion of yellow leaves.

[0034] (2) Tuber enlargement period observation (60-80 days after transplanting): Investigate plant disease incidence (leaf lesion coverage rate) and tuber formation characteristics. Representative plants are randomly selected from each plot, and miniature tubers are carefully dug out. After non-destructive quality scanning using a near-infrared spectrometer, the tubers are backfilled in situ. Elimination criteria: fewer than 3 tubers per plant, malformed tubers exceeding 40%, and leaf lesion coverage exceeding 35%.

[0035] (3) Harvest at maturity (100-120 days after transplanting): Harvest the plants after the above-ground parts have naturally withered and turned yellow. Record the yield per plant, the number of rotten tubers, and the number of green-skinned tubers. Also, evaluate the appearance of the tubers (oblong, round, oval, etc.).

[0036] 4. Near-infrared non-destructive physical quality batch testing of tubers

[0037] A portable near-infrared spectrometer (wavelength range 900–2500 nm) was used for non-destructive quality testing of mature commercial potatoes. Before testing, a spectral calibration model for the dry matter, starch, and reducing sugar content of potato tubers was established. Two hundred representative potato tuber samples were collected, and near-infrared spectral data were acquired. Quality parameters were determined using conventional chemical methods (dry matter determination by drying method, starch determination by acid hydrolysis method, and reducing sugar determination by DNS method). A calibration model between the spectra and quality parameters was established using partial least squares (PLS) method, and the coefficient of determination R0 was used for cross-validation. 2 ≥0.90.

[0038] During testing, the surface of the intact tubers is wiped clean and placed directly on the sample stage of a near-infrared spectrometer for scanning. Each tuber is scanned three times, and the average spectrum is obtained. After being input into the calibration model, the system automatically outputs quality parameters such as dry matter content (%), starch content (%), and reducing sugar content (%). The entire testing process does not involve crushing or using any chemical reagents. The tested tubers are collected intact, marked, and then used for seed preservation.

[0039] 5. Four-dimensional weighted comprehensive scoring system for hierarchical screening

[0040] 5.1 Physiological phenotype of stress resistance (40 points): The scores are based on the comprehensive indicators of seedling wilting recovery ability, yellow leaf ratio, lesion coverage rate during the swelling stage and plant growth vigor during the mature stage. The specific scores are shown in Table 1 below.

[0041] Table 1. Physiological phenotype scoring criteria for stress resistance

[0042] Among them, the proportion of yellow leaves in the seedling stage is estimated by visual inspection. The percentage of leaves with obvious chlorophyll loss, yellowing or withered yellowing is estimated out of the total number of leaves.

[0043] Disease coverage during tuber enlargement: During the tuber enlargement period, three functional leaves from the upper, middle, and lower parts of the plant were randomly selected, and the average percentage of disease area (including fungal diseases, bacterial diseases, and physiological necrotic spots) to the total leaf area was estimated.

[0044] Plant growth vigor: Measure plant height (from ground to growing point) and stem diameter (diameter at the base of the main stem) at maturity, and calculate relative growth = (plant height / stem diameter increase under stress ÷ plant height / stem diameter increase in blank control) × 100%.

[0045] 5.2 Appearance and marketability of tubers (20 points): Scored based on indicators such as tuber shape regularity, skin smoothness, tuber size uniformity, and the proportion of green-skinned and rotten tubers. Specific scoring standards are shown in Table 2.

[0046] Table 2. Standards for Commercial Appearance Scoring of Tubers

[0047] Among them, tuber shape regularity: observe all commercial tubers of a single plant (single tuber weight ≥50 g), and count the percentage of tubers with consistent shape and no deformities (no dumbbell shape, pear shape, cracks, tumor-like protrusions, etc.).

[0048] Sweet potato skin smoothness: Visually inspect the roughness of the sweet potato skin surface, including the size of the lenticels, the presence of corky patches, and the presence of cracks.

[0049] Tuber size uniformity: coefficient of variation of single tuber weight of all marketable tubers from a single plant (CV = standard deviation / mean × 100%).

[0050] Green-skinned potatoes + rotten potatoes ratio: the percentage of the total weight of green-skinned potatoes and rotten potatoes in a single plant.

[0051] 5.3 High Starch Processing Quality (25 points): Based on the comprehensive score obtained from near-infrared spectroscopy, the dry matter, starch, and reducing sugar content are measured. Higher dry matter and starch content result in higher scores, while lower reducing sugar content results in higher scores. Specific scoring criteria are shown in Table 3.

[0052] Table 3. Quality Scoring Standards for High Starch Processing

[0053] 5.4 Stress Yield (15 points): Based on the yield of the blank control, the relative yield at each stress level is proportionally converted into a score. The specific scoring criteria are shown in Table 4.

[0054] Table 4. Adversity Yield Scoring Standards

[0055] After the comprehensive score was calculated, the germplasm was graded according to the following criteria: a comprehensive score ≥75 was considered core superior germplasm, 60-74 was considered candidate germplasm, and <60 was directly eliminated. From 200 germplasm samples, 23 were selected as core superior germplasm, 67 as candidate germplasm, and 110 were eliminated. The scores of the core superior germplasm are shown in Table 5 below.

[0056] Table 5. Scores of Core Superior Germplasm

[0057] 6. Improve the quality of superior germplasm by isolating and reproducing it through targeted hybridization breeding.

[0058] The 23 core superior germplasm accessions were divided into four parental groups based on their scores (if a parental group meets multiple requirements, it can be classified into any one of the parental groups): Resistant parental group (7 copies): Select germplasm with a score ≥36 based on the physiological phenotype score of stress resistance; Tuber appearance parental group (5 copies): Select germplasm with a score ≥18 based on the commercial value of tuber appearance; High-starch parental group (6 copies): Select germplasm with a score ≥22.5 based on the high-starch processing quality score; High-yielding parental line under adverse conditions (5 copies): Select germplasm with a score ≥13.5 based on the yield under adverse conditions score.

[0059] Each parental group was isolated and preserved separately, and homozygosity was maintained by net-room isolation or bagging self-pollination.

[0060] A cross-group directional hybridization strategy was adopted: germplasm from the stress-resistant parent group and the tuber appearance parent group were hybridized in pairs to obtain combination 1; germplasm from the high-starch parent group and the high-yield parent group under stress were hybridized in pairs to obtain combination 2; then, germplasm from the above two combinations were hybridized in pairs again to achieve the aggregation of four stress resistance and excellent processing traits. The offspring of the second hybridization were placed in a moderately stressed compound stress nursery (i.e., a moderately stressed compound stress plot) for continuous pressure selection. The top 10% of individual plants with the comprehensive score were selected for seed in each generation, and this selection was continued for more than two generations until the traits were stable. A new potato improvement line with completely stable comprehensive traits and excellent performance was obtained.

[0061] Example 2

[0062] The potato population with completely stable and excellent comprehensive traits selected in Example 1 underwent two consecutive years of fixed-point observation, indoor quality testing, and multi-point regional trials in a moderately stressed compound stress nursery and a non-stressed field. A systematic evaluation was conducted from the dimensions of plant agronomic traits, multiple stress resistance, yield traits, tuber marketability traits, and processing quality traits. The specific performance is as follows: 1. Main agronomic traits of the plant The plants grow vigorously and have a compact shape, with a seedling uniformity of ≥98%. During the seedling stage, the root system is well-developed with a large number of fibrous roots and strong root vitality. The roots can extend normally even in soils with low water holding capacity and saline-alkali environments. The leaves are dark green throughout the entire growth period, with very little natural yellowing or premature aging.

[0063] 2. Comprehensive resistance to four types of adversity

[0064] It exhibits excellent overall resistance under moderate quadruple combined stress conditions, and is also tolerant to drought, salinity, low nighttime temperatures, and continuous cropping stress caused by root-knot nematodes. Drought resistance: When the soil moisture content is maintained at 30%~35%, the plants do not wilt significantly. After 15 days of continuous drought, they wilt briefly under strong light, but can fully recover within 2 hours of shading. Salt and alkali resistance: In an environment with a soil salinity of 2.5‰, plant height, stem diameter, and leaf quantity are not significantly inhibited, and the incidence of salt damage symptoms is <5%; Low temperature resistance: During the seedling stage to the tuber enlargement stage, under the continuous low temperature of 6~8℃ at night, the plant growth rhythm is normal and there are no frozen or stunted seedlings. Root-knot nematode resistance: The root-knot nematode incidence index is only 7.6, which is much lower than that of conventional varieties. The number of root knots is very small, and the varieties are highly tolerant to continuous cropping obstacles.

[0065] 3. Yield characteristics

[0066] Production data has remained stable for two consecutive years, demonstrating both high and stable yields. In the moderate compound stress test area: the average yield was 2862 kg / mu, which was 22.7% higher than the local main control variety; the number of tubers per plant was 5-8, the tuber weight per plant was uniform, and there was no serious problem of large and small tuber differentiation. In normal field without stress: the average yield was 3315 kg / mu, an increase of 18.3% compared to the control variety; The annual output fluctuation range is less than 4.2%, and it is less affected by climate and adverse conditions, with strong stable production capacity.

[0067] 4. Appearance characteristics of tuber products

[0068] The tubers are regular in shape, mainly oval, with smooth skin and shallow eyes, meeting the requirements of the commercial potato market: the rate of deformed tubers is 5.2%, the rate of rotten tubers is 2.1%, and the rate of green-skinned tubers is 1.3%, all three indicators are better than the mainstream local varieties; the rate of large and medium-sized tubers reaches 89.4%, the commercial potato grade is high, and the economic benefits are obvious.

[0069] 5. Internal processing quality characteristics

[0070] The use of near-infrared non-destructive testing combined with national standard chemical verification testing ensures stable and compliant quality indicators, catering to both fresh food and processed food needs. Dry matter content: average 22.3%; Starch content: Average 17.6%, which is medium to high starch content and suitable for starch processing, French fries, and potato chip processing. Reducing sugar content: average 0.12%, far below the critical standard of 0.3% for processed potatoes, making it less prone to browning during high-temperature processing and resulting in excellent processed appearance; The potato flesh has a delicate texture and a soft, satisfying mouthfeel, making it excellent for fresh consumption and achieving a balance between processing and fresh eating properties.

[0071] Comparative Example 1

[0072] 200 potato germplasm samples from the same batch as in Example 1 were selected and set up as a single stress independent screening group. Only four types of single stresses were applied: drought, salinity, low nighttime temperature, and root-knot nematodes. The other procedures for seedling raising, transplanting, observation, quality testing, and four-dimensional scoring were completely the same as those for the compound stress group in Example 1, except for the stress treatment conditions. The intensity of the single stress was consistent with the single parameter corresponding to the moderate compound stress in Example 1.

[0073] 1. Single Adversity Grouping Settings

[0074] A total of 4 single-stress plots and a control group were set up, with each plot having an area of ​​400 m². 2 The isolation buffer zone, planting density, number of replicates, and water and fertilizer management are completely consistent with those in Example 1. ①Single drought group: soil moisture content 30%~35%, salinity <0.3‰, nighttime temperature ≥15℃, no insects; ②Single saline-alkali group: soil salinity 2.0‰~3.0‰, soil water holding capacity 70%~75%, nighttime temperature ≥15℃, no insects; ③ Single nighttime low temperature group: nighttime temperature 6~8℃, soil moisture holding capacity 70%~75%, salt content <0.3‰, no insects; ④ Single root-knot nematode group: 1000~1400 nematodes / 100g dry soil, soil moisture content 70%~75%, salt content <0.3‰, nighttime temperature ≥15℃; ⑤ Blank control group: Soil water holding capacity 70%~75%, salt content <0.3‰, nighttime temperature ≥15℃, no insects.

[0075] 2. The progressive agronomic phenotypic observation of growth stages, batch testing of tuber near-infrared non-destructive physical quality, and grading and screening using a four-dimensional weighted comprehensive scoring system were exactly the same as in Example 1. The final screening results were as follows: Single drought group: 62 eliminated germplasm samples, 95 candidate germplasm samples, and 43 core superior germplasm samples; Single saline-alkali group: 68 eliminated germplasm samples, 92 candidate germplasm samples, and 40 core superior germplasm samples; Single nighttime low temperature group: 59 eliminated germplasm samples, 98 candidate germplasm samples, and 43 core superior germplasm samples; Single root-knot nematode group: 65 eliminated germplasm samples, 94 candidate germplasm samples, and 41 core superior germplasm samples. A total of 167 core superior germplasm samples were selected.

[0076] 3. Improve the quality of superior germplasm by isolating and reproducing it through targeted hybridization breeding.

[0077] The 167 single-adversity superior germplasm accessions were assigned to corresponding parental groups based on their four-dimensional scores: Superior germplasm groups were selected based on single drought conditions: 22 stress-resistant parental lines, 9 tuber appearance parental lines, 7 high-starch parental lines, and 5 high-yielding parental lines under stress conditions. Superior germplasm groups were selected from single salt-alkali environments: 19 stress-resistant parental lines, 8 parental lines with tuber appearance, 7 parental lines with high starch content, and 6 parental lines with high yield under stress conditions. Superior germplasm groups were selected by single low-temperature screening: 23 stress-resistant parental lines, 7 tuber appearance parental lines, 6 high-starch parental lines, and 7 high-yielding parental lines under stress conditions; Superior germplasm groups were selected from single root-knot nematodes: 21 stress-resistant parental lines, 8 tuber appearance parental lines, 6 high-starch parental lines, and 6 high-yielding parental lines under adverse conditions.

[0078] According to the cross-population directional hybridization strategy of Example 1, the germplasm selected by each single stress was hybridized. All hybrid offspring were uniformly planted in a medium-severity quadruple-combination stress nursery for continuous pressure selection. Only the top 10% of individual plants with the comprehensive score were retained for seed in each generation. This selection was carried out for two generations until the traits were stable, thus obtaining a population of improved offspring from single-stress germplasm.

[0079] 4. The progeny populations obtained from single-stress germplasm improvement were observed at fixed-point sites for two consecutive years in a moderately compound stress nursery.

[0080] 4.1 Multiple aspects of resilience: Improved strains from a single drought source: Under the combined conditions of saline-alkali soil, low temperature and nematode, the salt damage incidence rate was 42.6%, the root-knot nematode disease index was 41.2, and most individual plants produced fewer than 3 tubers. Improved strains from a single saline-alkali source: stunted seedling rate of 38.5% under continuous nighttime low temperature, and wilting under strong light during moderate drought could not recover after 2 hours; Improved strains from a single source of low temperature: irreversible wilting occurs after soil water shortage, nematode infestation causes root rot, and large-scale premature aging of plants; Improved strains derived from a single root-knot nematode: Under the dual stress of drought and salinity, leaf lesion coverage exceeded 40%, the proportion of deformed potatoes was >45%, and the marketability was severely reduced.

[0081] 4.2 Production Comparison

[0082] The average yield of the single-stress germplasm hybrid improved population under moderate compound stress was 1916 kg / mu; the average yield of the new strain improved by four-fold compound stress screening in this invention was 2862 kg / mu, an increase of 33.05%; the annual yield fluctuation of the single-stress improved population was 13.7%, which was much higher than the 4.2% of the strain bred under compound stress, indicating poor yield stability.

[0083] 4.3 Comparison of tuber commercial and processing quality

[0084] Appearance and marketability: 18.7% of potatoes were deformed, 11.5% were green-skinned or rotten, and only 70.2% were large or medium-sized. Processing quality indicators: average dry matter content 19.1%, average starch content 14.3%, average reducing sugar content 0.27%.

[0085] In summary, while single-stress screening can specifically enhance resistance to certain conditions, it is insufficient to address the complex and varied compound stresses encountered in the field. Its improvement effect rapidly diminishes under multiple stresses, revealing limitations due to a single genetic background and a lack of synergistic stress resistance mechanisms. In contrast, this invention, through quadruple-combination stress synergistic screening, achieves deep aggregation and synergistic expression of stress-resistance genes, significantly improving plant survival rates in extreme environments and ensuring stability in both yield and quality.

[0086] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for screening and targeted breeding improvement of stress-resistant and high-quality potato germplasm resources, characterized in that, Includes the following steps: 1) Detoxify potato germplasm, propagate it through tissue culture, harden it in a greenhouse, and select strong seedlings as planting materials; 2) Construct a tiered, progressively challenging stress nursery and plant the plants; 3) Conduct agronomic phenotypic observations during the seedling stage and tuber enlargement stage, and eliminate unqualified individual plants according to the elimination criteria; 4) Conduct appearance evaluation and quality testing on the harvested tubers; 5) The germplasm is scored based on four dimensions: physiological phenotype of resistance, commercial appearance of tubers, internal processing quality and yield under stress. The germplasm is graded according to the comprehensive score and the core superior germplasm is selected. 6) The selected core superior germplasm was divided into different parent groups according to the scoring results of each dimension. Cross-group directional hybridization strategy was adopted for hybridization, and the hybrid offspring were placed in a medium-stressed compound aquarium for continuous pressure selection until the traits were stable.

2. The method according to claim 1, characterized in that, In step 1), virus removal was performed using shoot tip stripping culture; tissue culture propagation used MS basal medium; the tissue culture temperature was 25±1℃, the light intensity was 2000~3000 lx, and the photoperiod was 14~16 h light / 8~10 h dark; the hardening substrate was prepared by a mass ratio of peat moss:vermiculite:perlite = 2~3:1~2:1~2, and 1 / 2 Hoagland nutrient solution was applied; the hardening period was 26~30 days; the standard for planting material was a plant height of 5~8 cm and ≥5 leaves.

3. The method according to claim 1, characterized in that, The stratified composite stress field in step 2) contains mild composite stress cells, moderate composite stress cells, and severe composite stress cells; The stress conditions of the mild combined stress plot were: soil water holding capacity 45%~50%, soil salinity 1.0‰~1.5‰, nighttime temperature controlled at 10~12℃, and root-knot nematodes 400~600 per 100g dry soil. The stress conditions of the moderate combined stress plot were: soil water holding capacity of 30%~35%, soil salinity of 2.0‰~3.0‰, nighttime temperature control of 6~8℃, and root-knot nematode stress of 1000~1400 nematodes / 100g dry soil. The stress conditions of the severe combined stress plot were as follows: soil moisture content of 18% to 23%, soil salinity of 3.5‰ to 4.5‰, nighttime temperature of 2 to 4℃, and root-knot nematode stress of 1800 to 2200 nematodes per 100g of dry soil.

4. The method according to claim 1, characterized in that, The agronomic phenotypic observation during the seedling stage in step 3) is as follows: 20-30 days after transplanting, observe the recovery ability of wilting under strong light and the proportion of yellow leaves. The elimination criteria are: wilting under strong light cannot be recovered within 2 hours or the proportion of yellow leaves exceeds 30%. The agronomic phenotypic observation during the tuber enlargement period is as follows: 60-80 days after transplanting, the disease incidence and tuber formation characteristics of the plants are observed. The elimination criteria are: leaf lesion coverage rate exceeds 35% or the number of tubers per plant is less than 3 or the proportion of deformed tubers is higher than 40%.

5. The method according to claim 1, characterized in that, The quality testing mentioned in step 4) involves testing the content of dry matter, starch, and reducing sugar in the tubers.

6. The method according to claim 1, characterized in that, The scoring criteria in step 5) are: stress resistance physiological phenotype 40 points, tuber appearance marketability 20 points, internal processing quality 25 points, and stress yield 15 points; the grading criteria are: a comprehensive score ≥75 points is core superior germplasm, 60~74 points is candidate germplasm, and a score <60 points is eliminated.

7. The method according to claim 6, characterized in that, The stress resistance physiological phenotype was scored based on seedling wilting recovery ability, yellow leaf ratio, lesion coverage rate during the tuber enlargement stage, and plant growth vigor. The tuber appearance and marketability were scored based on the tuber shape regularity, skin smoothness, tuber size uniformity, and the ratio of green-skinned and rotten tubers after harvest. The internal processing quality was scored based on dry matter, starch, and reducing sugar content. The stress yield was calculated by proportionally adjusting the relative yield under each stress level, using the yield of the blank control as a baseline.

8. The method according to claim 6, characterized in that, The different parental groups mentioned in step 6) are stress-resistant parental groups, tuber appearance parental groups, high-starch parental groups, and stress-yield parental groups; the directional hybridization strategy is as follows: the stress-resistant parental groups are hybridized with the tuber appearance parental groups, the high-starch parental groups are hybridized with the stress-yield parental groups, and then the two types of hybrid combinations are hybridized a second time.

9. The method according to claim 8, characterized in that, The stress-resistant parental group consists of germplasm with a stress resistance physiological phenotype score ≥36; the tuber appearance parental group consists of germplasm with a tuber appearance commercial quality score ≥18; the high-starch parental group consists of germplasm with a high-starch processing quality score ≥22.5; and the high-yield parental group under adverse conditions consists of germplasm with an adverse yield score ≥13.

5.

10. The application of the method according to any one of claims 1 to 9 in potato breeding.

Images