Fine seed breeding method of fokienia hodginsii

Abstract

The invention belongs to the field of forest tree genetic breeding, and discloses an improved variety breeding method of Fujian cypress, which comprises the following steps: (1) performing high performance liquid chromatography on a Fujian cypress test sample solution to obtain an HPLC (High Performance Liquid Chromatography) fingerprint spectrum; and (2) based on the HPLC fingerprint spectrum and the control fingerprint spectrum in the step (1), combined with principal component analysis and clustering analysis, carrying out improved variety breeding of the fokienia hodginsii. According to the method, the HPLC fingerprint spectrum and chemical mode recognition are tried to be combined for fine seed breeding of the Fujian cypress for the first time, the HPLC fingerprint spectrum can comprehensively reflect chemical characteristics of a sample, key information can be extracted from mass data in combination with principal component analysis and clustering analysis, comprehensive evaluation and scientific classification of germplasm quality are achieved, and the method has the advantages of being high in accuracy and high in accuracy. Potential high-quality germplasm is found, and blindness and hysteresis of traditional breeding depending on phenotype are overcome.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of forest tree genetics and breeding, and relates to a method for breeding a fine variety of Fokienia hodginsii. BACKGROUND

[0002] Fokienia hodginsii (Dunn) Henry et Thomas is a plant of Fokienia belonging to Cupressaceae, and is a national key second-class protected wild plant. The heartwood has the effects of promoting qi, relieving pain, reducing adverse and stopping vomiting in traditional Chinese medicine. Fokienia hodginsii

[0003] At present, researches on Fokienia hodginsii are mostly focused on chemical component separation and identification and essential oil analysis, and in the field of forest tree fine variety breeding, there is a lack of a fast and scientific quality evaluation and screening method. Traditional breeding methods are mostly based on external phenotypes such as tree height and diameter at breast height, and the period is long and the internal chemical components cannot be accurately reflected.

[0004] Chemical components are the key to determine the medicinal value and economic value of Fokienia hodginsii. Therefore, developing a method for directly, quickly and comprehensively evaluating the internal chemical components and guiding fine variety breeding has important significance for screening, protection and development and utilization of high-quality germplasm resources of Fokienia hodginsii. SUMMARY

[0005] The application provides a method for breeding a fine variety of Fokienia hodginsii, so as to solve or alleviate one or more technical problems.

[0006] The application provides a method for breeding a fine variety of Fokienia hodginsii, which comprises the following steps: (1) performing high performance liquid chromatography (HPLC) analysis on a test solution of Fokienia hodginsii to obtain an HPLC fingerprint; (2) breeding a fine variety of Fokienia hodginsii based on the HPLC fingerprint obtained in step (1) and a control fingerprint, combined with principal component analysis and cluster analysis.

[0007] Preferably, a configuration method of the test solution of Fokienia hodginsii in step (1) comprises the following steps: S1, mixing Fokienia hodginsii leaf powder in ethanol to form a mixed solution, and then refluxing to obtain a reflux solution; S2, supplementing ethanol to the reflux solution obtained in step S1 to obtain an extraction solution, and the mass of the extraction solution is the same as that of the mixed solution; S3, concentrating the extraction solution, dissolving and constant volume with ethanol, filtering, and obtaining the test solution of Fokienia hodginsii.

[0008] Preferably, the temperature of the refluxing in step S1 is 70-80℃, and the refluxing time is 50-70 min.

[0009] ​Preferably, based on the addition amount of 5 g of the Fujian cypress leaf powder, the addition amount of ethanol in step S1 is 70-80 mL.

[0010] Preferably, the chromatographic conditions for the high-performance liquid chromatography analysis in step (1) include: Liquid chromatography column: C-18 column; Flow rate: 0.7-0.9 mL / min; Column temperature: 25-35℃; Injection volume: 5-15 μL; Detector wavelength: 230-250 nm.

[0011] Preferably, in step (1), the high performance liquid chromatography uses acetonitrile as mobile phase A and an aqueous solution containing 0.6-1.0% acetic acid and 0.05-0.15% diethylamine as mobile phase B for gradient elution.

[0012] Preferably, the gradient elution procedure is as follows: From 0 to 15 min, the volume fraction of mobile phase A is 38-42%; In 16-20 minutes, the volume fraction of mobile phase A increased from 38-42% to 48-52%. 20-45 min, the volume fraction of mobile phase A is 48-52%; Over 45-48 minutes, the volume fraction of mobile phase A increased from 48-52% to 73-77%. After 48-70 min, the volume fraction of mobile phase A is 73-77%.

[0013] Preferably, the comparative fingerprint spectrum in step (2) includes characteristic peaks of paclitaxel flavonoids, ginkgo biloba flavonoids and trans-cyperic acid.

[0014] Preferably, the principal component analysis in step (2) includes: Principal components were selected from the common peak areas in the HPLC fingerprint as the original variables. The principal components were then linearly weighted based on their overall scores and corresponding variance contribution rates to perform principal component analysis.

[0015] Preferably, the cluster analysis in step (2) includes: Cluster analysis was performed using the peak area of ​​common peaks in HPLC fingerprints as the original variable and the squared Euclidean distance as the interval.

[0016] Preferably, the breeding of superior varieties of Fujian cypress in step (2) includes: Based on the results of cluster analysis and principal component analysis, different germplasms of Fujian cypress were classified, and germplasms with significantly higher chemical content than other groups were selected as candidates for breeding superior varieties.

[0017] Technical features and beneficial effects of the present invention: This invention is the first attempt to combine HPLC fingerprinting with chemical pattern recognition for the breeding of Fujian Bai Liang variety. HPLC fingerprinting can comprehensively reflect the chemical characteristics of the sample. Combined with principal component analysis and cluster analysis, it can extract key information from massive amounts of data, realize the comprehensive evaluation and scientific classification of germplasm quality, discover potential high-quality germplasm, and overcome the blindness and lag of traditional breeding that relies solely on phenotypic selection. Attached Figure Description

[0018] Figure 1 This is a superimposed HPLC fingerprint of 20 batches of different germplasms of Fujian cypress leaves in Example 3; Figure 2 The HPLC comparison chromatograms of Fujian cypress sample (A) and mixed reference standard (B) in Example 3 are shown. Figure 3 This is a dendrogram of cluster analysis of 20 batches of Fujian cypress leaf samples in Example 4. Detailed Implementation

[0019] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. In the drawings, for clarity, the dimensions of layers, regions, elements, and their relative dimensions may be exaggerated. Throughout, the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. It should be noted that, unless otherwise specified, the embodiments and features in the embodiments of the present invention can be combined with each other.

[0020] It should be understood that when an element or layer is referred to as "on," "adjacent to," "connected to," or "coupled to" other elements or layers, it may be directly on, adjacent to, connected to, or coupled to other elements or layers, or there may be intervening elements or layers. Conversely, when an element is referred to as "directly on," "directly adjacent to," "directly connected to," or "directly coupled to" other elements or layers, there are no intervening elements or layers. It should be understood that although the terms first, second, third, etc., may be used to describe various elements, components, areas, layers, and / or portions, these elements, components, areas, layers, and / or portions should not be limited by these terms. These terms are only used to distinguish one element, component, area, layer, or portion from another element, component, area, layer, or portion. Therefore, without departing from the teachings of this invention, the first element, component, area, layer, or portion discussed below may be referred to as a second element, component, area, layer, or portion. And when a second element, component, area, layer, or portion is discussed, it does not imply that the first element, component, area, layer, or portion necessarily exists in this invention.

[0021] In this invention, unless otherwise explicitly specified and limited, the terms "installation," "connection," "linking," and "fixing," etc., should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components, unless otherwise explicitly limited. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0022] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such terms can be used interchangeably where appropriate so that embodiments of the invention described herein can be implemented, for example, in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0023] In this invention, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the distribution of selectable values ​​within a numerical interval is considered continuous, and includes the two endpoints of the numerical interval (i.e., the minimum and maximum values), as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints, which is equivalent to directly listing every integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical ranges disclosed in this invention should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, percentage, ratio, etc. The term "numerical interval" can be broadly included to include percentage intervals, ratio intervals, proportion intervals, etc.

[0024] In this specific implementation, the instruments and reagents used are all obtained through purchase, including: Waters W2695-QDA high-performance liquid chromatograph (Waters Corporation, USA); PX124ZH electronic analytical balance (Ohaus Instruments Changzhou Co., Ltd.); Millipore ultrapure water system (Millipore Corporation, USA); KQ50ODE CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); HH-1 constant temperature water bath (Changzhou Guohua Electric Co., Ltd.). Methanol, acetonitrile (chromatographic grade, Merck GmbH, Germany); ethanol (analytical grade, Sinopharm Chemical Reagent Co., Ltd.)

[0025] It should be noted that: Twenty batches of different germplasm of *Cupressus fragrans* were transplanted from their place of origin to Banlin State-owned Forest Farm in Anxi County, Quanzhou, Fujian Province. They were identified as *Cupressus fragrans* by Researcher Zou Shuangquan of the College of Forestry, Fujian Agriculture and Forestry University. Fokienia hodginsii (Dunn) Henry et Thomas).

[0026] The standards for cyperine biflavonoids, ginkgo biflavonoids, and trans-cyperic acid were prepared in the laboratory, and the 1H and 13C data obtained by nuclear magnetic resonance (NMR) detection were completely consistent with those in the literature.

[0027] This invention provides a technical solution for the breeding of superior varieties of Fujian cypress, as detailed below.

[0028] Exemplary embodiments of the present invention will now be described in more detail with reference to the accompanying drawings. It should be understood that these exemplary embodiments may be implemented in many different forms and should not be construed as being limited to the embodiments set forth herein.

[0029] The method for breeding superior varieties of Fujian cypress provided by this invention includes: (1) The Fujian Bai sample solution was analyzed by high performance liquid chromatography to obtain the HPLC fingerprint spectrum; (2) Based on the HPLC fingerprint spectrum and the control fingerprint spectrum of step (1), combined with principal component analysis and cluster analysis, the breeding of superior varieties of Fujian cypress is carried out.

[0030] This invention is the first attempt to combine HPLC fingerprinting with chemical pattern recognition for the breeding of Fujian Bai Liang variety. HPLC fingerprinting can comprehensively reflect the chemical characteristics of the sample. Combined with principal component analysis and cluster analysis, it can extract key information from massive amounts of data, realize the comprehensive evaluation and scientific classification of germplasm quality, discover potential high-quality germplasm, and overcome the blindness and lag of traditional breeding that relies solely on phenotypic selection.

[0031] In some embodiments, the preparation method of the Fujian cypress test solution in step (1) includes: S1. Mix Fujian cypress leaf powder in ethanol to form a mixture, then reflux to obtain reflux liquid; S2. Add ethanol to the reflux liquid obtained in step S1 to obtain an extract, the mass of which is the same as the mass of the mixture. S3. Concentrate the extract, then dissolve it in ethanol and bring the volume to a fixed level. Filter to obtain the Fujian Bai sample solution.

[0032] In this invention, the synergistic effect of "heated reflux extraction" and "weight compensation" ensures that the target chemical components (especially biflavonoids and trans-piperidine) are extracted efficiently, stably, and quantitatively. The key step of "weight compensation" effectively compensates for the evaporation loss of solvent during reflux, ensuring the consistency of pretreatment between different batches of samples from the source, and laying a solid foundation for the subsequent construction of highly reproducible fingerprint spectra.

[0033] In some embodiments, the reflux temperature in step S1 is 70-80°C (e.g., 70°C, 72°C, 75°C, 78°C, 80°C, etc.), and the reflux time is 50-70 min (e.g., 50 min, 52 min, 55 min, 57 min, 60 min, 62 min, 65 min, 67 min, 70 min, etc.).

[0034] Preferably, based on the addition amount of 5 g of Fujian cypress leaf powder, the addition amount of ethanol in step S1 is 70-80 mL (e.g., 70 mL, 72 mL, 75 mL, 78 mL, 80 mL, etc.).

[0035] Preferably, the chromatographic conditions for the high-performance liquid chromatography analysis in step (1) include: Liquid chromatography column: C-18 column; Flow rate: 0.7-0.9 mL / min (e.g., 0.7 mL / min, 0.75 mL / min, 0.8 mL / min, 0.85 mL / min, 0.9 mL / min, etc.); Column temperature: 25-35℃ (e.g., 25℃, 28℃, 30℃, 32℃, 35℃, etc.); Injection volume: 5-15 μL (e.g., 5 μL, 8 μL, 10 μL, 12 μL, 15 μL, etc.); Detector wavelength: 230-250 nm (e.g., 230 nm, 235 nm, 240 nm, 245 nm, 250 nm, etc.).

[0036] Preferably, in step (1), the high performance liquid chromatography uses acetonitrile as mobile phase A and an aqueous solution containing 0.6-1.0% (e.g., 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, etc.) acetic acid and 0.05-0.15% (e.g., 0.05%, 0.08%, 0.10%, 0.12%, 0.15%, etc.) diethylamine as mobile phase B for gradient elution.

[0037] In this invention, the components of Fujian cypress are relatively complex, making the separation of effective components difficult. By setting these conditions, it is possible to achieve better chromatographic separation, peak shape, and detection sensitivity for its complex chemical components (especially structurally similar biflavonoids) within a reasonable time.

[0038] In this invention, an acetonitrile-amine aqueous solution is used as the mobile phase. The addition of acetic acid can inhibit the ionization of acidic components (such as trans-piperic acid), improve peak shape, and prevent tailing. Diethylamine, as a modifier, can interact with residual silanol groups on the chromatographic column and basic groups in the sample, further improving the symmetry and separation efficiency of the chromatographic peaks.

[0039] Preferably, the gradient elution procedure is as follows: For 0-15 min, the volume fraction of mobile phase A is 38-42% (e.g., 38%, 39%, 40%, 41%, 42%, etc.). Over 16-20 minutes, the volume fraction of mobile phase A increases from 38-42% (e.g., 38%, 39%, 40%, 41%, 42%, etc.) to 48-52% (e.g., 48%, 49%, 50%, 51%, 52%, etc.). 20-45 min, the volume fraction of mobile phase A is 48-52% (e.g., 48%, 49%, 50%, 51%, 52%, etc.). Over 45-48 minutes, the volume fraction of mobile phase A increases from 48-52% (e.g., 48%, 49%, 50%, 51%, 52%, etc.) to 73-77% (e.g., 73%, 74%, 75%, 76%, 77%, etc.). For 48-70 min, the volume fraction of mobile phase A is 73-77% (e.g., 73%, 74%, 75%, 76%, 77%, etc.).

[0040] In this invention, the HPLC fingerprint spectrum includes 11 common peaks through gradient elution; Using the trans-cyperric acid chromatographic peak as reference peak S, the relative retention times of other common peaks include: Peak 1 has a relative retention time of 0.2-0.3 min; Peak 2 has a relative retention time of 0.25–0.35 min; Peak 3 has a relative retention time of 0.30–0.40 min; Peak 4 has a relative retention time of 0.40–0.50 min; Peak 5 has a relative retention time of 0.50–0.60 min; Peak 6 has a relative retention time of 0.60–0.70 min; Peak 7 has a relative retention time of 0.70–0.80 min; Peak 8 has a relative retention time of 0.80–0.90 min; Peak 9 has a relative retention time of 0.90–1.00 min; Peak 10 has a relative retention time of 1.00–1.10 min; The relative retention time for reference peak S is 1.00 min.

[0041] In some embodiments, the comparative fingerprint spectrum in step (2) includes characteristic peaks of paclitaxel flavonoids, ginkgo biloba flavonoids and trans-cyperic acid.

[0042] This invention discovers that the cypress biflavonoids and ginkgo biflavonoids in Fujian cypress possess various biological activities such as antitumor, antiimmune, antibacterial, and antioxidant effects, while trans-piperidine has a significant antibacterial effect against plant pathogenic fungi. By screening the content of cypress biflavonoids, ginkgo biflavonoids, and trans-piperidine in Fujian cypress, it is easier to screen Fujian cypress with antitumor and antibacterial properties.

[0043] In some embodiments, the principal component analysis in step (2) includes: Principal components were selected from the common peak areas in the HPLC fingerprint as the original variables. The principal components were then linearly weighted based on their overall scores and corresponding variance contribution rates to perform principal component analysis.

[0044] In this invention, principal component analysis can remove a large amount of chromatographic data and effectively extract useful information.

[0045] In some embodiments, the clustering analysis in step (2) includes: Cluster analysis was performed using the peak area of ​​common peaks in HPLC fingerprints as the original variable and the squared Euclidean distance as the interval.

[0046] In this invention, automatic grouping based on the similarity of chemical components greatly reduces the risk of misselection.

[0047] In some embodiments, the breeding of superior varieties of Fujian cypress in step (2) includes: Based on the results of cluster analysis and principal component analysis, different germplasms of Fujian cypress were classified, and germplasms with significantly higher chemical content than other groups were selected as candidates for breeding superior varieties.

[0048]

Example 1

[0049] Table 1 Sample Information

[0050]

Example 2

[0051]

Example 3

[0052] (2) Precision test: Take the mixed reference solution and determine it six times consecutively under the above chromatographic conditions. Use trans-cadal acid (peak 11) as the reference peak and calculate the relative retention time and relative peak area of ​​each common peak. The results show that the relative standard deviation (RSD) of the relative retention time of each common peak is 0.53-0.68%, and the RSD of the relative peak area is 1.0-2.0%, indicating that the instrument used has good precision.

[0053] (3) Repeatability test: Six portions of Fujian cypress leaf powder (S12) were prepared according to the method in Example 1, and the chromatographic conditions described above were used for determination. Using trans-cyperic acid (peak 11) as the reference peak, the relative retention time and relative peak area of ​​each common peak were calculated. The results showed that the RSD of the relative retention time of each common peak was 0.17-0.67%, and the RSD of the relative peak area was 0.38-2.96%, indicating that the method has good repeatability.

[0054] (4) Stability test: Six portions of Fujian cypress leaf powder (S3) were prepared according to the method in Example 1. The samples were injected at 0, 2, 4, 8, 16, and 24 hours under the chromatographic conditions described above. Using trans-cyperic acid (peak 11) as a reference peak, the relative retention time and relative peak area of ​​each common peak were calculated. The results showed that the RSD of the relative retention time of each common peak was 0.42-0.92%, and the RSD of the relative peak area was 1.77-2.39%, indicating that the test solution had good stability within 24 hours.

[0055] (5) Fingerprint mapping and similarity evaluation: Accurately pipette 10 μL each of the test solution and the mixed reference solution from Examples 1 and 2, inject them into the high-performance liquid chromatograph, and determine the chromatograms within 70 min under the chromatographic conditions described above.

[0056] Results: HPLC chromatograms of 20 batches of *Phellodendron chinense* samples were imported into the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)". The median method was used, with a time window width of 0.1 min. Peak matching was performed through multi-point calibration to generate fingerprint chromatograms for different *Phellodendron chinense* germplasms. (See attached data). Figure 1 The top-down graph follows the direction from S1 to S20.

[0057] In the fingerprint chromatogram of *Platycladus orientalis*, peak 11 has the highest peak area and best separation; therefore, peak 11 was selected as the reference peak. In addition, 11 common peaks were identified in the *Platycladus orientalis* fingerprint chromatogram. Comparison with the reference standard showed that... Figure 2 As shown, curve A represents the Fujian cypress sample, curve B represents the mixed control, peak 4 is identified as paclitaxel flavonoids, peak 10 as ginkgo biloba flavonoids, and peak 11 as trans-cypressic acid.

[0058] The similarity between the fingerprint patterns of different germplasms of *Platycladus orientalis* and the control fingerprint pattern was evaluated, and the results are shown in Table 2. Table 2. Similarity of Fujian cypress pedigrees from different germplasms

[0059] As shown in Table 2, the similarity calculation results of the 20 batches of samples and the control spectrum R are all greater than 0.900, indicating that the chemical composition of different germplasms of Fujian cypress is relatively consistent, but the content of each component is different.

[0060]

Example 4

[0061] Using eigenvalues ​​> 0.9 as the criterion, four principal components were extracted, with a cumulative variance contribution rate of 83.674%. The test results are shown in Table 3. Table 3. Results of principal component eigenvalues ​​and variance contribution rates

[0062] As shown in Table 3, germplasm S6 (number 473) has a significantly higher overall score (10.4621) than other germplasms, indicating that its overall chemical composition is the best. The overall scores and rankings are shown in Table 3.

[0063] The factor loading matrix reflects the correlation coefficients between the 11 common peaks (i.e., the original variables) and the 4 principal components. The factor loading matrix is ​​shown in Table 4. Table 4 Initial Factor Loading Matrix

[0064] Table 4 shows the contributions of the 11 common peaks to the four principal components. The absolute values ​​of the coefficients of each factor in the principal component expression reflect the strength of the contribution of that factor to its principal component. The peaks with larger loadings corresponding to the first principal component are peaks 8, 5, 11 (trans-cyperric acid), 9, 4 (catecholone), and 1; the peaks with larger loadings corresponding to the second principal component are peaks 3 and 10 (ginkgolone); the peak with a larger loading corresponding to the third principal component is peak 6; and the peak with a larger loading corresponding to the fourth principal component is peak 2.

[0065] The scores of the four principal components and their corresponding variance contribution rates were used as weights for linear weighting to comprehensively score the 20 batches of samples. The eigenvectors of the 11 factors were calculated from the loading vectors and eigenvalues ​​of the 11 common peak factors. The neutralization scores were calculated by constructing linear equations for the four principal components using the eigenvectors as functions. The results are shown in Table 5. S6 (germ 473) had a particularly high comprehensive score, which was found to be outstanding in the direction of principal component 1.

[0066] Table 5. Principal component factor scores and rankings of Fujian Baiji.

[0067] (2) Cluster analysis (CA): The common peak area data of fingerprint spectra of 20 batches of different germplasms of *Platycladus orientalis* were also imported into SPSS 27.0. Hierarchical clustering was performed using the intergroup linkage method with squared Euclidean distance as the metric. When the inter-cluster distance was 10-25, the 20 batches of samples were clearly divided into two classes: S6 was in one class alone, and the remaining 19 germplasms were in another class.

[0068] This result is completely consistent with the PCA analysis showing that S6's overall score was far ahead. See the clustering dendrogram below. Figure 3 .

[0069]

Example 5

[0070] The linear regression equation for cyperus tinctoria flavonoids is y = 3E + 0.7x - 613782(R) 2=0.9995), showing a good linear relationship in the range of 0.071-0.36 mg / mL; The linear regression equation for ginkgo biloba flavonoids is y = 3E + 0.7x - 204774 (R 2 =0.9994), showing a good linear relationship in the range of 0.048-0.24 mg / mL; The linear regression equation for trans-cypermethrin is y = 6E + 0.6x - 2E + 0.6(R) 2 =1.000), showing a good linear relationship in the range of 0.5-2 mg / mL.

[0071] (2) Precision test: Take an appropriate amount of the mixed reference solution prepared in Example 2, and determine it six times consecutively under the above chromatographic conditions, recording the peak area of ​​each component.

[0072] The results showed that the RSD values ​​of the peak areas of Taxodium biflavonoids, Ginkgo biflavonoids, and trans-cyperic acid were 1.33%, 1.44%, and 1.20%, respectively, indicating that the instrument used had good precision.

[0073] (3) Repeatability test Take 6 portions of Fujian cypress leaf powder (S12), prepare samples according to the method in Example 1, and determine them under the above chromatographic conditions, recording the peak areas of each component.

[0074] The results showed that the RSD values ​​of the peak areas of Taxodium biflavonoids, Ginkgo biflavonoids, and trans-cyperic acid were 1.82%, 1.93%, and 1.90%, respectively, indicating that the method had good reproducibility.

[0075] (4) Stability Take 6 portions of Fujian cypress leaf powder (S3) and prepare samples according to the method in Example 1. Inject the samples at 0, 2, 4, 8, 16 and 24 h according to the above chromatographic conditions, and record the peak area of ​​each component.

[0076] The results showed that the RSD values ​​of the peak areas of each of the following compounds were 0.53%, 2.00%, and 1.04%, respectively, indicating that the test solution had good stability within 24 hours.

[0077] (5) Recovery rate: Take 5g of Fujian cypress leaf powder (S2), and accurately add it to a mixed reference solution with a known content equivalent to the analyte in the sample. Prepare the sample according to the method in Example 1. Prepare the sample in parallel 6 times. Perform the determination under the above chromatographic conditions and record the peak areas of cypress biflavonoids, ginkgo biflavonoids, and trans-cypressic acid. Calculate the average recovery rates as 92.6%, 100.7%, and 102.0%, and the RSD values ​​as 2.95%, 3.49%, and 3.74%, respectively.

[0078] (6) Determination of sample content Twenty batches of Fujian cypress leaf powder were taken and samples were prepared according to the method in Example 1. The samples were determined under the above chromatographic conditions, and the content of each component was calculated. The results are shown in Table 6.

[0079] Table 6. Results of content determination of different germplasm of Fujian cypress

[0080] (7) Combining the results of PCA and CA analysis: Based on the combined results of PCA and CA analyses, germplasm S6 (number 473) can be definitively identified as a superior germplasm. Its HPLC fingerprint exhibits distinct characteristics, and the content determination results of the three main active ingredients (taxanthin, ginkgo biloba flavonoids, and trans-pinenic acid) (see Table 4) also confirm that its trans-pinenic acid content is as high as 2.52%, significantly higher than other germplasms. Therefore, germplasm S6 can be used as a core material for the breeding and promotion of superior Fujian cypress varieties.

[0081] It should be noted that the terms "center," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," "axial," "radial," and "circumferential," etc., are used only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the present invention. The directional terms "inner" and "outer" refer to the inside or outside relative to the outline of the component itself. For example, if a device in the drawings is inverted, a device described as "above" or "on top of other devices or structures" will subsequently be positioned as "below" or "under" other devices or structures. Thus, the exemplary term "above" can include both "above" and "below." The device may also be positioned in other different ways (rotated 90 degrees or in other orientations), and the spatial relative descriptions used herein are interpreted accordingly.

[0082] It should also be noted that the terms "one embodiment," "another embodiment," and "embodiment" used in this invention refer to specific features, structures, or characteristics described in connection with that embodiment, which are included in at least one embodiment of the general description of this invention. The appearance of the same expression in multiple places in the specification does not necessarily refer to the same embodiment. Furthermore, when a specific feature, structure, or characteristic is described in connection with any embodiment, the intention is to suggest that implementing such a feature, structure, or characteristic in conjunction with other embodiments also falls within the scope of this invention.

[0083] In the above embodiments, the descriptions of each embodiment have different focuses. For parts not described in detail in a certain embodiment, please refer to the relevant descriptions in other embodiments.

[0084] It should also be noted that the above are merely preferred embodiments of the present invention and do not limit the scope of patent protection of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention’s specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. A method for selecting a superior strain of Fokienia hodginsii, characterized by, The method comprises the following steps: (1) performing high performance liquid chromatography (HPLC) analysis on the test sample solution of Fokienia Hodginsii to obtain an HPLC fingerprint; (2) based on the HPLC fingerprint obtained in step (1) and a control fingerprint, and combined with principal component analysis (PCA) and cluster analysis, breeding fine varieties of Fokienia Hodginsii.

2. The method of claim 1, wherein the method is characterized by, The method for preparing the test sample solution of Fokienia Hodginsii in step (1) comprises the following steps: S1, mixing Fokienia Hodginsii leaf powder with ethanol to form a mixed solution, and then refluxing to obtain a refluxed solution; S2, supplementing the refluxed solution obtained in step S1 with ethanol to obtain an extraction solution, wherein the mass of the extraction solution is the same as that of the mixed solution; S3, concentrating the extraction solution, dissolving and constant volume with ethanol, and filtering to obtain the test sample solution of Fokienia Hodginsii.

3. The method of claim 2, wherein the selection of the superior variety is based on the evaluation of the yield of the plant. The temperature of the refluxing in step S1 is 70-80°C, and the refluxing time is 50-70 min; The amount of the ethanol added in step S1 is 70-80 mL, based on the amount of the Fokienia Hodginsii leaf powder added, which is 5 g.

4. The method of claim 1, wherein the method is characterized by, The chromatographic conditions for the HPLC analysis in step (1) comprise the following: Liquid chromatography column: C-18 column; Flow rate: 0.7-0.9 mL / min; Column temperature: 25-35°C; Injection volume: 5-15 μL; Detector wavelength: 230-250 nm.

5. The method of claim 1, wherein the method is performed on a plant of the genus Brassica. The HPLC analysis in step (1) is performed by gradient elution with acetonitrile as mobile phase A and an aqueous solution containing 0.6-1.0% acetic acid and 0.05-0.15% diethylamine as mobile phase B.

6. The method of claim 5, wherein the selection of the superior variety is based on the number of the plants having the desired trait. The gradient elution program is as follows: 0-15 min, the volume fraction of mobile phase A is 38-42%; 16-20 min, the volume fraction of mobile phase A is increased from 38-42% to 48-52%; 20-45 min, the volume fraction of mobile phase A is 48-52%; 45-48 min, the volume fraction of mobile phase A is increased from 48-52% to 73-77%; 48-70 min, the volume fraction of mobile phase A is 73-77%.

7. The method of claim 1, wherein the method is performed on a plant of the genus Brassica. The control fingerprint in step (2) comprises characteristic peaks of amentoflavone, bilobalide and trans-abelskaya acid.

8. The method of claim 1, wherein the method is used for selecting a superior variety of a plant. The principal component analysis in step (2) comprises the following steps: Taking the peak area of the common peaks in the HPLC fingerprint as the original variables, screening the principal components from the original variables, and performing linear weighting based on the comprehensive scores of the principal components and the corresponding variance contribution rates, thereby performing the principal component analysis.

9. The method of claim 1, wherein the method is used for selecting a superior variety of a plant. The cluster analysis in step (2) comprises the following steps: Taking the peak area of the common peaks in the HPLC fingerprint as the original variables, taking the squared Euclidean distance as the interval, and performing cluster analysis by the between-group linkage method.

10. The method of selective breeding of elite seeds according to claim 1, wherein, The breeding of fine varieties of Fokienia Hodginsii in step (2) comprises the following steps: According to the results of the cluster analysis and the principal component analysis, classifying the different germplasms of Fokienia Hodginsii, and screening germplasms with significantly higher content of chemical components than other groups as the objects for fine variety breeding.

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