Three kinds of wild excellent liquorice germplasm H2. W, SS3. H and J2. W

Abstract

The invention discloses three kinds of wild excellent liquorice germplasm H2. W, SS3. H and J2. W. Belongs to the technical field of traditional Chinese medicine germplasm resource evaluation and improved variety breeding. The three kinds of high-quality licorice germplasm are obtained based on correlation analysis and screening of phenotypic characters of fruit pods and seeds and the glycyrrhizic acid content, and have specific phenotypic characteristics and high glycyrrhizic acid content. By means of the method, high-quality germplasm capable of being used for glycyrrhiza uralensis improved variety breeding and standardized production is provided, and germplasm guarantee is provided for standardized production and quality improvement of glycyrrhiza uralensis.

Description

Technical Field

[0001] It belongs to the technical field of germplasm resource evaluation and breeding of superior varieties of Chinese medicinal materials. Background Technology

[0002] With the sharp decline in wild licorice reserves, cultivated licorice has taken over the market, but its inconsistent quality is becoming increasingly prominent. Achieving high-quality and standardized licorice cultivation hinges on overcoming the following industry bottlenecks: Quality Dilemma: Quality Fluctuations Due to Genetic Diversity Interspecific hybridization is common among plants of the genus Glycyrrhiza, resulting in a complex genetic background. This directly leads to severe phenotypic segregation in cultivated offspring, with varying levels of core quality indicators (such as glycyrrhizic acid content) and difficulty in ensuring quality stability.

[0003] Germplasm Dilemma: Severe Shortage of High-Quality Germplasm Resources Among existing licorice germplasm resources, there is a lack of known high-quality germplasm with consistently high glycyrrhizic acid content that has undergone systematic identification. Growers cannot obtain germplasm with clear quality assurance, resulting in inconsistent quality of cultivated licorice and making it difficult to meet the requirements of industrialization for uniform quality.

[0004] Identification Dilemmas: Limitations of Traditional Screening Techniques Currently, germplasm identification and screening mainly rely on complex techniques such as visual judgment of plant morphology or molecular markers. The former is highly subjective and requires a high level of expertise; the latter is costly and complex to operate. Neither of these methods can meet the practical needs of production for rapid and accurate identification of large quantities of seedlings, becoming a major bottleneck for industrialization.

[0005] Screening Dilemma: The Gap in Early Quality Prediction Systems In breeding and production practices, there is still a lack of reliable technologies that can effectively predict the future quality of medicinal materials at the seed stage. Breeders have to conduct quality evaluations only after a long cultivation period throughout the entire growth cycle, resulting in long breeding cycles, high costs, and low efficiency.

[0006] In summary, overcoming the limitations of traditional technologies, providing specific licorice germplasm with stable, high-quality traits, and establishing rapid and accurate identification standards are of great significance for improving the quality of licorice medicinal materials from the source and promoting industrial upgrading. The three high-glycyrrhizic acid content licorice germplasms H2.W, SS3.H, and J2.W provided by this invention are direct solutions to the aforementioned industrial bottlenecks. Summary of the Invention

[0007] (a) Purpose To address the industry bottleneck of lacking stable, high-glycyrrhizic acid content licorice germplasm resources, this invention provides three high-quality licorice germplasms—H2.W, SS3.H, and J2.W—identified through correlation analysis based on systematic screening of pod and seed phenotypic traits. These germplasms possess clear, identifiable phenotypic characteristics and stable glycyrrhizic acid content, and can be directly used for improved seed breeding and standardized production.

[0008] (II) Technical Solution To achieve the above objectives, this invention obtains three high-quality licorice germplasms based on the following systematic screening process: Basic principles of systematic screening of pod and seed phenotypes By collecting germplasm from multiple geographical regions, we systematically measured the quantitative traits (length, width, and thickness of the ear) and qualitative traits (swelling degree, glandular hairs, constriction, etc.) of pods, as well as the quantitative traits (weight per 100 seeds, length, width, and thickness) and qualitative traits (color, shape, halo, and surface ornamentation) of seeds, and constructed a phenotypic database.

[0009] Phenotypic clustering and subtype classification Based on the above traits, Q-type cluster analysis was performed to establish a phenotypic classification system for pods and seeds, dividing the germplasm into different phenotypic subtypes, providing a classification basis for quality association screening.

[0010] Identification and Confirmation of High-Quality Germplasm Through standardized cultivation and glycyrrhizic acid content determination, combined with phenotypic and content correlation analysis, the following three high-quality germplasm varieties with high glycyrrhizic acid content and stable phenotypic characteristics were screened out: Germplasm H2.W It belongs to the Ural licorice family and originates from Hami, Xinjiang. The seed phenotype is II-1, with a 100-seed weight of approximately 1.073g. The seeds are green, round-kidney shaped, with an expansive triangular halo and intermediate surface ornamentation; the glycyrrhizic acid content is not less than 0.94%.

[0011] Germplasm SS3.H It belongs to the hybrid group of inflated and smooth licorice, originating from Shanshan, Xinjiang. The seed phenotype is subtype IV-1, with a 100-seed weight of about 0.610g. The seeds are yellow, round, with a circular halo and striped surface ornamentation; the glycyrrhizic acid content is not less than 0.507%.

[0012] Germplasm J2.W It belongs to the Ural licorice family and originates from Jiuquan, Gansu. The seed phenotype is subtype II-1, with a 100-seed weight of approximately 1.025g. The seeds are green, round, with an expansive triangular halo and intermediate surface ornamentation; the glycyrrhizic acid content is not less than 0.45%.

[0013] (III) Beneficial Effects Compared with the prior art, the present invention has the following advantages: High-quality and reliable germplasm: All three germplasms were selected based on the "phenotype-component" correlation analysis. In the homogeneous garden experiment, it was found that the glycyrrhizic acid content was significantly higher than that of ordinary germplasm, and the quality was stable.

[0014] Strong early identification: Target germplasm can be quickly identified in the early stages of planting based on the phenotype of pods and seeds, without the need for complex equipment or destructive testing, greatly improving the efficiency of improved seed application.

[0015] The screening system is scientific and complete: The multi-trait clustering and association analysis method of pods and seeds on which this invention is based provides a reliable phenotypic basis for germplasm screening, which has both theoretical value and practical feasibility.

[0016] Low barriers to promotion: The germplasm and phenotypic identification standards provided are simple and intuitive, making them easy for seed management, planting bases and medicinal herb farmers to use directly, which helps to promote the quality improvement of the licorice industry from the source. Attached Figure Description

[0017] Figure 1 Q-clustering diagram of ear of fruit based on 9 morphological traits. Figure 2 Scatter plot of 100-seed weight (A), length (B), width (C), and thickness (D) of 52 licorice seeds (G: Glycyrrhiza glabra; H: Hybrid group; M: Glycyrrhiza nematodes; W: Glycyrrhiza uracilii; Z: Glycyrrhiza inflata). Figure 3 Four types of licorice seed color classification. Figure 4 Illustration of the kidney-shaped and round shape of licorice seeds. Figure 5 Different types of halo patterns on medicinal licorice seeds. Figure 6 Types of surface ornamentation on the seed coat of medicinal licorice from different germplasms. Figure 7 Q-clustering of 52 licorice seed samples from different sources based on 8 morphological indicators. Figure 8 Results of glycyrrhizic acid content determination in 28 different licorice seedlings. Figure 9 Identification spectrum of H2.W. Figure 10 : Identification spectrum of SS3.H. Figure 11 : Identification spectrum of J2.W; Detailed Implementation

[0018] Systematic identification and screening of high-quality licorice germplasm resources 1. Survey of Experimental Materials and Resources A systematic survey of wild licorice resources was conducted in 13 major distribution areas in Xinjiang Uygur Autonomous Region (Alar, Korla, Hami, Yanqi, Shanshan, Turpan, Bachu, Shihezi) and Gansu Province (Jiuquan, Guazhou, Jinta, Zhangye, Jiayuguan). From each population, 20-30 healthy plants were randomly selected, and their typical fruit spikes were collected, resulting in a total of 542 specimens.

[0019] All collected materials were identified by Professor Lu Jiahui of Shihezi University, covering *Glycyrrhizauralensis* Fisch., *Glycyrrhiza inflata* Bat., *Glycyrrhiza glabra* L., *Glycyrrhiza glabra* var. *glandulosa* XY Li, and their natural hybrids. Ultimately, 52 representative germplasm accessions with significant differentiation in spikelet and seed morphology were selected as experimental materials for in-depth research.

[0020] 2. Systematic determination and classification of ear phenotypic traits Measurement method: Ten mature ears of fruit were randomly selected from each sample, and their length, width, and thickness were accurately measured using digital vernier calipers (accuracy 0.01 mm). At the same time, six key quality traits were observed and recorded in detail: pod swelling degree, presence of glandular hairs, degree of constriction between seeds, dorsoventral curvature, ear density, and overall ear morphology.

[0021] Classification results: Q-type cluster analysis was performed based on 9 phenotypic traits (3 quantitative traits and 6 qualitative traits). The results are as follows: Figure 1 As shown, the 52 ear morphologies were clearly divided into 4 major categories and 26 subtypes. Specifically, *Glycyrrhiza uralensis* clustered into 5 subtypes (ZⅠ-ZⅤ), *Glycyrrhiza ursaria* clustered into 9 subtypes (WI-WⅨ), *Glycyrrhiza glabra* and *Glycyrrhiza nectarine* each had 2 subtypes (GI / GⅡ, MI / MIⅡ), and hybrids clustered into 8 subtypes (HI-HⅧ). This systematic ear phenotypic classification provides important macroscopic morphological background for subsequent seed phenotypic analysis.

[0022] 3. Multi-level systematic determination of seed phenotypic traits Precise measurement of quantitative traits: Size measurement: 100 plump seeds were randomly selected from each germplasm sample and mixed. The "W" shaped sampling method was used, taking 10 seeds each time. The length, width and thickness of the seeds were measured using a digital vernier caliper. The measurement was repeated 3 times and the average value was taken.

[0023] Weight determination: Using an electronic analytical balance (accuracy 0.0001g), weigh 100, 50 and 30 seeds in sequence, convert the weight to the weight of 100 seeds and calculate the average value.

[0024] Measurement results ( Figure 2 The results showed that *Glycyrrhiza uralensis* seeds were generally the largest, with a 100-seed weight reaching 1.557g; *Glycyrrhiza inflata* seeds were the second largest; while *Glycyrrhiza glabra* and *Glycyrrhiza nigra* seeds were relatively small. This result provides an important basis for the preliminary screening of high-quality germplasm.

[0025] Systematic observation and standardized coding of quality traits: Color and Shape: Observe and photograph the seeds under natural light, and classify the seed colors into 4 categories: yellow (0), green (1), dark green (2), and dark brown (3). Figure 3 Seed shapes were divided into two categories: round-kidney-shaped (0) and round (1) ( Figure 4 ).

[0026] Halo morphology: Under a stereomicroscope (OLYMPUS SZX7), the halo shapes were divided into three categories: circular (0), overflowing triangular (1), and triangular (2). Figure 5 ).

[0027] Surface ornamentation: Mature and plump seeds were vacuum sputtered with gold and then observed using a Hitachi SU8010 field emission scanning electron microscope. The surface ornamentation of the seed coat was divided into three categories: striped (0), intermediate (1), and flaky (2). Figure 6 ).

[0028] Anatomical structure: Representative seeds were selected, fixed with FAA, embedded in paraffin, sectioned, stained with safranin-fast green, and observed under an optical microscope (OLYMPUS BX51). The length of palisade cells and the length and width of supporting cells were measured using Digimizer software, and the length-to-width ratio of supporting cells was calculated. Their morphology was classified into dumbbell type (length-to-width ratio <1.5) and long dumbbell type (length-to-width ratio ≥1.5).

[0029] 4. Seed phenotypic cluster analysis and classification system establishment The numerical data of the eight core phenotypic traits (100-grain weight, length, width, thickness, color, shape, halo, and surface ornamentation) obtained above were standardized, and Q-type cluster analysis (Euclidean distance, sum of squared deviations) was performed using OriginPro 2022 software. The clustering results are as follows: Figure 7 As shown, the 52 licorice seed phenotypes were clearly divided into 6 major categories (I-VI) and 12 stable subtypes (I-1, I-2, II-1, II-2, II-3, II-4, III, IV-1, IV-2, IV-3, V, VI). This comprehensive classification system laid a solid foundation for subsequent quality association analysis and selection of high-quality germplasm.

[0030] 5. Standardized cultivation and precise quality evaluation Seedling cultivation and sample preparation: Twenty-eight representative germplasm accessions were selected from the 12 established seed phenotypic subtypes for standardized cultivation. Seeds were treated with 85% concentrated sulfuric acid for 1 hour to break dormancy before being sown in a sterilized substrate (native soil: vermiculite = 2:1). They were cultivated for 6 months in a temperature- and light-controlled artificial climate chamber (temperature: 25±5℃; photoperiod: 12h / 12h light / dark).

[0031] Glycyrrhizic acid content determination: An Agilent 1260 Infinity II high-performance liquid chromatography (HPLC) system was used. Chromatographic conditions: Agilent ZORBAX SB-C18 column (4.6 mm × 250 mm, 5 μm); mobile phase: methanol:water:36% glacial acetic acid = 71:28:1 (v / v / v); detection wavelength: 254 nm. The established standard curve regression equation was Y = 29.58462X - 9.89376 (R² = 0.99988), showing good linearity in the range of 3.125–50 μg / mL.

[0032] 6. Association Analysis and Identification of High-Quality Germplasm This study found that three samples had glycyrrhizic acid content higher than the three pure Ural licorice varieties, representing potentially high-quality licorice germplasm. Notably, phenotypic segregation was observed in the hybrid groups, with a wide range of glycyrrhizic acid content variation. Through systematic screening, we identified three special germplasm varieties whose glycyrrhizic acid content was even higher than that of the high-content control pure Ural licorice varieties: High-quality germplasm H2.W ( Figure 9 ): Source: Hami, Xinjiang; originally identified as Ural licorice. Seed phenotype: Belongs to subtype II-1 Phenotypic characteristics: 100-seed weight 1.073g; seed length 3.267mm, width 2.613mm, thickness 1.793mm; seed color green, round-kidney shaped, with an outward-spreading triangular halo, and intermediate surface ornamentation. Anatomical features: palisade cells are 72 μm long, and supporting cells are dumbbell-shaped (length-to-width ratio 1.33). Quality performance: The glycyrrhizic acid content is as high as 0.94%, the highest among all tested germplasms. High-quality germplasm SS3.H ( Figure 10 ): Source: Shanshan, Xinjiang; originally identified as a hybrid group. Seed phenotype: Belongs to subtype IV-1 Phenotypic characteristics: 100-seed weight 0.610g; seed length 2.633mm, width 2.303mm, thickness 1.773mm; seed color yellow, round, with a circular halo, and striped surface decoration. Anatomical features: Palisade cells are 90.4 μm long, and Supporting cells are significantly long dumbbell-shaped (length-to-width ratio 3.04). Quality characteristics: The glycyrrhizic acid content is 0.507%, which is particularly high among hybrid groups. High-quality germplasm J2.W ( Figure 11 ): Source: Jiuquan, Gansu; originally identified as Ural licorice. Seed phenotype: Belongs to subtype II-1 Phenotypic characteristics: 100-seed weight 1.025g; seed length 2.927mm, width 2.547mm, thickness 1.863mm; seed color green, round, with an outward-spreading triangular halo and intermediate surface ornamentation. Anatomical features: Palisade cells are 66 μm long, and Supporting cells are dumbbell-shaped (length-to-width ratio 0.85). Quality performance: The glycyrrhizic acid content is 0.45%, indicating excellent and stable quality. in conclusion This invention establishes a scientific evaluation system for licorice germplasm resources through a complete technical path: "resource survey → systematic phenotypic evaluation → standardized cultivation → precise quality testing → mathematical correlation analysis." Based on this, three high-quality licorice germplasms with high glycyrrhizic acid content—H2.W, SS3.H, and J2.W—were successfully identified. These germplasms possess stable and identifiable phenotypic characteristics and excellent quality indicators, providing valuable core germplasm materials for the breeding of superior licorice varieties and the construction of standardized production bases. This offers an effective solution to the problem of unstable quality in licorice medicinal materials from its source.

[0033] A more detailed description of the accompanying drawings is provided below: Figure 2 Scatter plot of 100-seed weight (A), length (B), width (C), and thickness (D) of 52 licorice seeds (G: Glycyrrhiza glabra; H: Hybrid group; M: Glycyrrhiza nematodes; W: Glycyrrhiza uracilii; Z: Glycyrrhiza inflata) The 100-seed weight of *Glycyrrhiza inflata* ranges from 0.894 to 1.377 g, with seed length, width, and thickness ranging from 2.960 to 3.433 mm, 2.323 to 2.653 mm, and 1.730 to 2.097 mm, respectively. The 100-seed weight, seed length, width, and thickness of *Glycyrrhiza uralensis* range from 0.894 to 1.557 g, 2.850 to 3.447 mm, 2.547 to 3.105 mm, and 1.627 to 2.060 mm, respectively. The 100-seed weight, seed length, width, and thickness of *Glycyrrhiza glabra* A2.G and S2.G are 0.911 g and 0.717 g, 3.067 mm and 2.707 mm, 2.570 mm and 2.203 mm, and 1.613 mm and 1.577 mm, respectively. The 100-seed weight, seed length, width, and thickness of *Glycyrrhiza uralensis* A3.M and S3.M are 0.460g and 0.681g, respectively; 2.517mm and 2.763mm; 2.210mm and 2.257mm; and 1.190mm and 1.503mm, respectively. The 100-seed weight, seed length, width, and thickness of hybrid licorice range from 0.61-1.276g, 2.633-3.560mm, 2.303-2.790mm, and 1.505-2.037mm, respectively. The seeds of *Glycyrrhiza glabra* and *Glycyrrhiza uralensis* are generally small. The seeds of *Glycyrrhiza ursaria* and *Glycyrrhiza inflata* are generally large; however, the seed width of *Glycyrrhiza inflata* is slightly smaller than that of *Glycyrrhiza ursaria*.

[0034] Figure 3 Four types of licorice seed color classification The seeds of inflatable licorice are mostly yellow; hybrid licorice is found in all four color groups, but is most common in the yellow and dark brown groups, while Ural licorice is basically dark green. Seed color shows significant differences among different medicinal licorice varieties.

[0035] Figure 4 Illustration of the kidney-shaped and round shapes of licorice seeds Seed shapes are mainly divided into two categories: reniform (a) and round (b) Figure 3 Except for H4.Z, the seeds of *Glycyrrhiza inflata* are all round-kidney shaped; the seeds of *Glycyrrhiza glabra* and *Glycyrrhiza nephrolepis* are all round; and the seeds of *Glycyrrhiza ursaria* are mainly round.

[0036] Figure 5 Different germplasm types of medicinal licorice seeds with halo patterns Seed halos can be divided into three main categories: circular (a), triangular (b), and expansive triangular (c). The halos of *Glycyrrhiza glabra* and *Glycyrrhiza nigra* are both expansive triangular, while the halos of *Glycyrrhiza inflata* are mainly triangular, and the halos of *Glycyrrhiza uralsii* are mainly circular, with some triangular and expansive triangular halos.

[0037] Figure 6 Types of surface ornamentation on the seed coat of medicinal licorice from different germplasms In the striped type, except for JT1.W, the rest are *Glycyrrhiza inflata* and hybrid licorice varieties. In the intermediate type, except for S3.M, the rest are all *Glycyrrhiza ursica* types. The flaky type includes most of *Glycyrrhiza ursica* and some *Glycyrrhiza inflata*, while all *Glycyrrhiza glabra* samples are flaky. Based on the results, the flaky type of licorice surface ornamentation includes *Glycyrrhiza ursica*, *Glycyrrhiza ursica*, *Glycyrrhiza glabra*, and hybrid licorice.

[0038] Figure 8 Identification spectrum of H2.W The fruit spike of H2.W is short and fist-shaped, and the pods are covered with glandular hairs in a zigzag pattern. The seed phenotype is II-1. The weight of 100 seeds is 1.073 g, the length is 3.267 mm, the width is 2.613 mm, the thickness is 1.793 mm, the color is green, the seed shape is round-kidney-shaped, the halo is triangular, the surface ornamentation is intermediate, the palisade cells are 72 μm long, and the palisade cells smaller than 75 μm are shorter, and the supporting cells are dumbbell-shaped.

[0039] Figure 9 Identification spectrum of SS3.H SS3.H: The pods are short, slightly swollen, and covered with glandular hairs. The seed phenotype is IV-1. The weight of 100 seeds is 0.610 g. The seeds are 2.633 mm long, 2.303 mm wide, and 1.773 mm thick. They are yellow in color, round in shape, with a round halo. The surface ornamentation is striped. The palisade cells are 90.4 μm long, and the supporting cells are long dumbbell-shaped.

[0040] Figure 10 The identification chart of J2.W shows that the spikelet morphology of J2.W is of the type of Ural licorice, with disordered and compact spikelets and slender S-shaped pods; the seed phenotype is the same as H2.W as II-1, with a 100-seed weight of 1.025 g, a length of 2.927 mm, a width of 2.547 mm, a thickness of 1.863 mm, and a green color. The seeds are round in shape, with a triangular halo, and the surface ornamentation is intermediate. The palisade cells are 66 μm long, and those less than 75 μm are shorter. The supporting cells are dumbbell-shaped.

[0041] Figure 7 The glycyrrhizic acid content among species was as follows: Ural licorice > glycyrrhiza glabra > hybrid licorice > inflated licorice > nectarine licorice.

Claims

1. Three high-quality licorice germplasms, characterized in that, The germplasm is any one or a combination of H2.W, SS3.H, or J2.W; The characteristics of the pods of each of the described germplasms are as follows: The germplasm H2.W has short, fist-shaped spikes and pods covered with glandular hairs and zig-shaped. The pods of the SS3.H germplasm are short, slightly swollen, and covered with glandular hairs; The ears of the J2.W germplasm are disordered and compact, and the pods are slender and S-shaped.

2. The high-quality licorice germplasm according to claim 1, characterized in that, The seed characteristics of each of the described germplasms are as follows: The seed weight of the germplasm H2.W is 1.073g per 100 seeds. The seeds are green, round and kidney-shaped, with an outward-spreading triangular halo and intermediate surface ornamentation. The seed weight of the germplasm SS3.H is 0.610g per 100 seeds. The seeds are yellow, round, with a circular halo and striped surface decoration. The seed weight of the J2.W germplasm is 1.025g per 100 seeds. The seeds are green, round, with an outward-spreading triangular halo and intermediate surface ornamentation.

3. The high-quality licorice germplasm according to claim 1, characterized in that, The seed coat micromorphology and cellular structure of each germplasm are as follows: The palisade cells of the H2.W germplasm are 72 μm in length, and the supporting cells are dumbbell-shaped. The palisade cells of the SS3.H germplasm are 90.4 μm in length, and the supporting cells are long dumbbell-shaped. The palisade cells of the J2.W germplasm are 66 μm in length, and the supporting cells are dumbbell-shaped.

4. The high-quality licorice germplasm according to claim 1, characterized in that, The glycyrrhizic acid content of each of the described germplasms is as follows: The glycyrrhizic acid content of the germplasm H2.W is not less than 0.94%; The glycyrrhizic acid content of the germplasm SS3.H is not less than 0.507%; The glycyrrhizic acid content of the germplasm J2.W is not less than 0.45%.

5. The high-quality licorice germplasm according to claim 1, characterized in that, The sources and locations of the germplasm described are as follows: The germplasm H2.W originated from Hami, Xinjiang, and was originally identified as Ural licorice. The germplasm SS3.H originated from Shanshan, Xinjiang, and was originally identified as a hybrid group; The germplasm J2.W originated from Jiuquan, Gansu, and was originally identified as Ural licorice.

Images