Method for cultivating salt and alkali tolerant hybrid barley seeds based on three-line method

Abstract

The application discloses a method for cultivating saline-alkali tolerant hybrid barley seeds based on barley three-line method, takes a barley male sterile line parent 270A as a sterile source, takes a known saline-alkali tolerant variety Yanshima No. 11 as a recurrent parent and a maintainer line, introduces a major saline-alkali QTL site carried by Yanshima No. 11 into the sterile line background through continuous multiple generations of backcrossing, simultaneously fixes the agronomic traits, and finally breeds a stable saline-alkali tolerant male sterile line Yanshima No. 16; then takes Yanshima No. 11 as the maintainer line, plants parent and offspring in a 2:1 ratio in a saline-alkali stress field, implements artificial assisted pollination and dynamic hybrid removal during the heading stage to the flowering stage, and harvests seeds formed by sterile line plants, so that saline-alkali tolerant hybrid barley F1 generation seeds are obtained. The application realizes precise coupling of the saline-alkali tolerance of barley and the three-line matching system for the first time, overcomes the bottleneck of long breeding period and difficult utilization of hybrid vigor in traditional saline-alkali tolerance breeding, and the F1 seeds produced have a yield increase of 23.6% under saline-alkali soil compared with conventional varieties.

Description

Technical Field

[0001] This invention belongs to the field of crop hybrid breeding technology, specifically involving a method for introducing salt-alkali tolerant major gene loci into a barley male-sterile three-line system and achieving efficient and high-purity hybrid seed production under salt-alkali stress. It aims to solve the industrial bottleneck of low barley yield and lack of salt-alkali tolerant hybrid varieties in coastal saline-alkali land. Background Technology

[0002] my country has over 100 million mu (approximately 6.67 million hectares) of saline-alkali land along its coast and inland areas, representing a significant reserve of arable land. Barley, as a high-quality forage crop, has long relied on conventional hybridization and backcrossing for salt-alkali tolerance breeding, which suffers from long cycles (typically requiring 8-10 generations), severe trait linkage burden, and the inability to fix heterosis. Although there have been reports on using male-sterile lines (such as Qin 270A) for barley hybridization breeding (see CN 112369320 A), current technologies have not overcome the core obstacle of "the male-sterile line itself being intolerant to salt and alkali"—that is, the male-sterile line's fertility fluctuates and its flower development is abnormal under salt and alkali stress, leading to seed production failure. While Yanshimai 11 possesses excellent salt and alkali tolerance (with plant variety rights application number 20221005812), it is a conventional variety and cannot be directly used for three-line breeding. Therefore, how to accurately and stably introduce salt-alkali tolerance traits into the male-sterile line background and establish seed production technology procedures adapted to saline-alkali environments remains a long-standing technical challenge in this field. Summary of the Invention

[0003] This invention provides a method for breeding hybrid barley seeds that can be stably inherited, have high seed purity, and still ensure the expression of male sterility traits and effective pollen dispersal under salt and alkali stress, thereby achieving the synergistic utilization of salt and alkali tolerance traits and heterosis.

[0004] The core innovation of this invention lies in the pioneering dual-track technical approach of "salt-alkali tolerant QTL directional backcross breeding + dynamic seed production management under salt-alkali stress".

[0005] First Track: Creation of Salt-Tolerant Sterile Lines Unlike CN 112369320 A, which only focuses on backcrossing for morphological and yield traits, this invention introduces salt-alkali stress selection pressure from the BC4F1 generation and combines it with the molecular marker Barley-SaltQTL2H for early, non-destructive, and high-throughput selection. This compresses the traditional 6–8 year breeding cycle to 5 years (2019–2025) and avoids the negative linkage between salt-alkali tolerance genes and sterility genes. The sterile cytoplasm (CMS-T) of parent 270A successfully recombines with the nuclear genome of Yanshimai 11, forming a "nucleocytoplasmic interaction stable" salt-alkali tolerant sterile line.

[0006] Second track: Salt-alkali adapted seed production technology Breaking away from the conventional understanding that seed production must be carried out under non-stress conditions, this method utilizes Ca²⁺ to regulate anther dehiscence, artificial pollination to enhance pollination, and dynamic impurity removal under a salt-alkali gradient (eliminating false fertile plants caused by salt stress). This transforms the seed production process itself into a "field validation ground" for salt-alkali tolerance traits. The 2:1 parent-to-parent ratio, optimized through three years of saline-alkali land trials, ensures sufficient pollen while avoiding excessive competition from the male parent that could negatively impact the female parent's yield.

[0007] Due to the adoption of the above technical solution, the advantages and positive effects of this invention are as follows: 1. Improved breeding efficiency: MAS shortens the breeding cycle by 40%, and the accuracy rate of introducing salt and alkali tolerance traits is >99.5%; 2. Enhanced seed production reliability: Seed production purity in saline-alkali land is ≥99.8%, which is 1.6 percentage points higher than that in non-saline-alkali land (purity 98.2%). F1 generation seeds were grown in a typical coastal saline-alkali land (pH 8.9, EC 6.5) in Yancheng, Jiangsu Province. e When planted at 8.5 dS / m, the average yield reached 412.3 kg per mu, which is 23.6% higher than the self-crossed variety Yanshimai 11 and 31.2% higher than the local main variety Yangnongpi 5. Attached Figure Description

[0008] Figure 1 This invention provides a technical roadmap for the three-line breeding and seed production of salt-tolerant barley; Figure 2 The PCR detection results of the Barley-SaltQTL2H site provided by the present invention in Yanshimai 16; Figure 3 Microscopic images comparing anther development of Yanshimai 16 and Qin 270A under salt-alkali stress, provided by this invention; Figure 4 The diagram illustrates the implementation steps and contents of this invention. Detailed Implementation

[0009] Example 1: Creation of Yanshimai 16 (2019–2025) Spring 2019: Qin 270A (♀) × Yanshi Wheat 11 (♂) → F0; Autumn sowing at Yancheng Teachers College Experimental Station (non-saline-alkali soil) → 19N18; 2020–2024: Every spring for BC n The F1 population underwent anther microscopy (sterility rate ≥99.5% was the selection criterion), and selected individual plants were backcrossed after autumn sowing; the BC6F1 (2024) population size was 3200 plants; In the spring of 2025: the BC6F1 population was subjected to hydroponic salt stress (0.5% NaCl + 0.1% NaHCO3, 14 days), and individual plants with a survival rate of less than 85% were eliminated (2173 plants remained). DNA was extracted from the surviving plants, and PCR was performed using SaltQTL2H primers to screen for individual plants with a homozygous band of 186 bp (892 plants were obtained). The 892 plants were then identified in a field plot in saline-alkali soil (pH 9.0), and finally 12 individual plants with the best comprehensive traits were selected. After autumn propagation in 2025, they were named "Yanshimai 16".

[0010] Example 2: Hybrid Seed Production in Saline-Alkali Land (Autumn 2025) Location: Saline-alkali experimental station of Binhuai Farm, Binhai County, Yancheng (pH 8.8, EC) e =8.2 dS / m); Planting: Sow on October 28th, with the female parent Yanshimai 16 in 2 rows (density 150,000 plants / 667m²) and the male parent Yanshimai 11 in 1 row (density 120,000 plants / 667m²), with a row spacing of 25 cm. Pollination: Spray with 0.1% CaCl2 on December 15 (when 50% of the female parent has emerged); brush with pollen four times a day from December 20 to 25 (peak flowering period); Weed removal: Weed removal was carried out twice in the field on December 10 (early heading stage) and December 18 (before flowering), removing a total of 37 abnormal plants; Harvest: On April 20, 2026 (late waxy maturity stage), the female parent was harvested alone, yielding 216.5 kg / 667m² of F1 seeds. The heterozygosity rate was 99.4% according to SSR testing, and the salt and alkali tolerance uniformity was 100% verified in field planting.

Claims

1. A method for breeding salt-tolerant hybrid barley seeds based on the three-line method, characterized in that, Includes the following steps: Step 1: Creation of salt-tolerant male sterile lines: Using the male-sterile barley line 270A as the female parent and the salt-tolerant barley variety Yanshimai 11 as the male parent, the first hybridization was conducted in the spring of 2019 to obtain F0 hybrid seeds. In the autumn of 2019, the F0 seeds were sown in a non-salt-alkali control field, designated 19N18. In the spring of 2020, anther microscopy was performed on the 19N18 population to screen for sterile individual plants, which were then backcrossed with Yanshimai 11 as the male parent to obtain BC1F1 seeds. Subsequently, BC1F1 seeds were sown annually in non-salt-alkali fields in the autumn. n In the spring, sterile individual plants were selected from F1 and backcrossed with Yanshimai 11 to the BC6F1 generation (2024). In 2025, the BC6F1 population was subjected to salt tolerance phenotype identification and molecular marker-assisted selection (MAS) to screen out individual plants that simultaneously meet the following criteria: ① anther abortion rate ≥99.5%; ② survival rate ≥85% under salt stress of pH 9.0 and total salt content 0.5%, plant height reduction ≤12%, and thousand-grain weight reduction ≤15%; ③ homozygous for Barley-SaltQTL2H locus. After self-pollination stabilization, these plants were named "Yanshimai 16". Step 2: Construction of the three-line seed production system: It was confirmed that Yanshimai 11 has complete retention ability for Yanshimai 16, that is, the sterility rate of the hybrid offspring of the two is 100%, and the agronomic traits are highly consistent with those of Yanshimai 11. Therefore, Yanshimai 11 is established as the dedicated maintainer line of Yanshimai 16. Step 3: Hybridization and seed production under salt and alkali stress: From October 25 to November 5, in saline-alkali land (soil pH 8.5–9.2, EC 50000), e Plant 6.0–9.0 dS / m) in a ratio of 2 rows of Yanshimai 16 (female parent) to 1 row of Yanshimai 11 (male parent); when the female parent has a heading rate of 50%, spray with 0.1% calcium chloride solution to induce anther dehiscence; from the beginning of flowering to the full bloom of the male parent, gently sweep the male parent's ear with a soft brush four times a day at 9:00, 11:00, 13:00 and 15:00, and simultaneously gently brush the stigma of the female parent to achieve artificial cross-pollination; at the beginning of heading and before flowering, remove all single plants with external anthers, abnormal glumes, or significantly deviated plant height to ensure the purity of the seed production field; Step 3, Seed Harvesting and Verification: Harvest the female parent at the end of the waxy ripening stage of the ear, thresh, winnow, and coat the seeds separately. The resulting seeds are the F1 generation seeds of salt-tolerant hybrid barley. Genotyping of the SSR marker Barley-SaltQTL2H showed that the heterozygosity at this locus in F1 seeds was ≥99.2%, and the field phenotype uniformity for salt tolerance reached 100%.

2. The method according to claim 1, characterized in that, The salt and alkali tolerance phenotype identification described in step (1) is as follows: BC6F1 single plants are transplanted into Hoagland nutrient solution containing 0.5% NaCl + 0.1% NaHCO3 and hydroponically cultured for 14 days. The root length inhibition rate, the decrease in chlorophyll fluorescence parameter Fv / Fm and the amount of proline accumulation are measured. The tolerant single plants are screened by comprehensive scoring.

3. The method according to claim 1, characterized in that, The primer pair used for molecular marker-assisted selection (MAS) in step (1) is: upstream primer SaltQTL2H-F, downstream primer SaltQTL2H-R; the PCR amplification product was subjected to 2.5% agarose gel electrophoresis, the target band size was 186bp, and the homozygous tolerance genotype was expressed as a single 186bp band.

4. The method according to claim 1, characterized in that, In step (3), the total salinity of the soil in the saline-alkali land seed production field needs to be controlled at 0.4%–0.6%, and the seed production field needs to be isolated with a spatial isolation distance of ≥500 meters from non-salt-alkali tolerant barley varieties.

5. The method according to claim 1, characterized in that, The botanical characteristics of Yanshimai 16 are as follows: semi-erect growth habit (code 2), flag leaf auricles with anthocyanin coloration (code 9), moderately waxy leaf sheath (code 5), fully emerged spike (code 3), erect spike (code 1), medium plant height (code 4), biangular spike (code 1), medium spike length (code 5), hulled caryopsis (code 2), yellow kernels (code 2), oblong (code 2), medium to high thousand-kernel weight (code 6), semi-wintering (code 2).

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