An evaluation method for identifying fast-growing traits of nereis divergens and application thereof

Abstract

The present application belongs to the technical field of aquatic animal breeding, germplasm creation and trait identification, and particularly relates to a method for evaluating fast-growing traits of Eudratylus mongololicus and application thereof. Specifically, the body wall tissue of Eudratylus mongololicus to be tested is collected; part of the body wall tissue is used for tissue section, staining and digital image quantitative analysis to obtain body wall histological quantitative parameters; part of the body wall tissue is used for extracting total RNA, and the expression levels of muscle-related marker genes and collagen-related marker genes are detected; the body wall histological quantitative parameters and the expression levels of the marker genes are jointly analyzed, and compared with reference individuals, reference groups or the same batch of individuals to be tested to evaluate whether the individuals or groups to be tested have fast-growing traits or fast-growing development advantages. The present application can effectively reduce the interference of factors such as coelomic fluid, feeding state and intestinal contents on the evaluation results of traditional body weight and body length, and improve the objectivity, accuracy and stability of the determination of fast-growing traits of Eudratylus mongololicus.

Description

Technical Field

[0001] This invention belongs to the technical field of aquatic animal breeding, germplasm creation and trait identification, specifically involving an evaluation method for identifying the fast-growing trait of *Urechis unicinctus* (an evaluation method for identifying the fast-growing trait of *Urechis unicinctus* based on a combination of body wall histological parameters and marker gene expression) and its application. Background Technology

[0002] Uni-ringed thorn beetle ( Urechis unicinctus The *Ulva unicinctus* is an intertidal benthic aquatic animal of significant economic value along the Yellow and Bohai Sea coasts of my country, possessing value for food, medicine, and industrial development. During artificial breeding and cultivation, a significant asynchronous growth phenomenon is commonly observed among seedlings from the same batch, with substantial differences in growth rates between individuals. Accurately identifying individuals or groups with rapid growth potential, and accordingly conducting superior germplasm screening and family breeding, has become one of the key issues in *Ulva unicinctus* breeding.

[0003] Currently, the evaluation of growth traits in *Urtica dioica* mainly relies on macroscopic phenotypic indicators such as body weight and body length. However, due to the unique coelomic sac-like structure of *Urtica dioica*, its body weight is easily affected by factors such as coelomic fluid content, feeding status, and intestinal contents, and the measured results cannot consistently reflect its true tissue growth level. Relying solely on macroscopic phenotypic criteria for rapid growth screening is prone to misjudgment, thus affecting the early identification and breeding efficiency of superior individuals.

[0004] With the application of molecular biology techniques in aquaculture breeding, researchers have begun to explore using growth-related genes as molecular markers to assist in the screening of superior individuals. Such methods can reveal growth-related molecular changes to some extent, but relying solely on gene expression or molecular markers still has limitations, because differences at the molecular level do not necessarily correspond entirely to terminal tissue structure and actual growth phenotype. On the other hand, relying solely on histological observation lacks molecular-level support, making cross-validation from the perspective of growth regulation mechanisms difficult.

[0005] The body wall tissue of *Urtica diochotomata* is a major component of its body. Body wall thickness, muscle fiber development, and the deposition level of extracellular matrix such as collagen can directly reflect its tissue growth capacity and body mass accumulation potential. Therefore, if quantitative parameters of body wall histology can be combined with the expression levels of growth-related marker genes to establish a joint auxiliary assessment method that considers both "molecular expression potential" and "tissue construction level," it is expected to improve the objectivity, accuracy, and stability of identifying the rapid-growth trait of *Urtica diochotomata*. Currently, there is a lack of auxiliary assessment methods that combine specific body wall sampling areas, marker gene expression, and quantitative histological analysis to jointly determine the rapid-growth trait of *Urtica diochotomata*.

[0006] Zhao JC, He ZH, Chen XY, Huang YY, Xie JJ, Qin X, Ni ZT&Sun CB (2021) Growth trait gene analysis of kuruma shrimp ( Marsupenaeus japonicus ) bytranscriptome study. Comp. Biochem. Physiol. D-Genomics Proteomics 40:100874. Saavedra C, Milan M, Leite RB, Cordero D, Patarnello T, Cancela ML&Bargelloni L (2017) A microarray study of carpet-shell clam ( Ruditapes decussatus ) shows common and organ-specific growth-related gene expressiondifferences in gills and digestive gland. Frontiers in Physiology 8:943. Summary of the Invention

[0007] The purpose of this invention is to address the shortcomings of existing methods for evaluating the rapid growth trait of *Ulva monocyclicis*, which mainly rely on macroscopic phenotype, susceptibility to body fluid, feeding status, and intestinal contents, and lack a combined molecular and histological quantitative standard. This invention provides an assessment method for identifying the rapid growth trait of *Ulva monocyclicis* (an assessment method for identifying the rapid growth trait of *Ulva monocyclicis* based on a combination of body wall histological parameters and marker gene expression) and its application.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: An evaluation method for identifying the fast-growing trait of *Urtica dioica*. (1) Collect the body wall tissue of the *Ulva unicornuate* to be tested; (2) Part of the body wall tissue was used for tissue sectioning, staining and digital image quantitative analysis to obtain quantitative parameters of body wall histology; (3) A portion of the body wall tissue was used to extract total RNA, and the expression levels of muscle-related marker genes and collagen-related marker genes were detected; (4) The quantitative parameters of body wall histology are analyzed together with the expression level of the marker gene, and compared with the reference individual, reference group or the same batch of test individuals to assess whether the test individual or group has the rapid growth trait or rapid growth development advantage.

[0009] The quantitative parameters of the body wall histology include morphological parameters and extracellular matrix parameters; wherein, the morphological parameters include one or more of the following: average body wall thickness, mid-longitudinal muscle fiber density, and long axis of muscle fibers; the extracellular matrix parameters include relative collagen content or collagen volume fraction.

[0010] The average thickness of the body wall is the average value obtained by measuring the thickness of the entire body wall layer in a direction perpendicular to the body wall surface under low magnification. The relative collagen content or collagen volume fraction is calculated as the percentage of the collagen region area to the effective tissue area after separating the collagen fiber staining signal using the color deconvolution function. The muscle fiber density of the mid-longitudinal muscle layer is the number of muscle fibers per unit area counted under high magnification. The long diameter of the muscle fiber is the average value obtained by measuring the diameter of the long axis of the muscle fiber in the mid-longitudinal muscle layer under high magnification.

[0011] In step (1), the body wall tissue is collected from the middle 1 / 3 region of the trunk of the single-ringed cricket, which is equidistant from the front and rear ends.

[0012] The staining in step (2) is Masson trichrome staining, which is used to differentiate muscle fibers and collagen fibers in the body wall tissue and to perform digital image quantitative analysis of subsequent body wall morphological parameters and extracellular matrix parameters.

[0013] In step (2), digital image analysis software is used to perform quantitative analysis on the stained tissue sections, and the collagen fiber content and muscle fiber parameters are analyzed using the color deconvolution function; preferably, the digital image analysis software is FijiImageJ.

[0014] The muscle-related marker gene is the myosin heavy chain gene. MHC The collagen-related marker gene is the collagen gene. COL12A1 .

[0015] The COL12A1 and MHC The detection primer sequences are as follows: COL12A1 Upstream primer: 5′-TGACGAACTCCATCAGCGAAGAAG-3′ COL12A1 Downstream primer: 5′-AGGCGACTGACCAGCGTAGC-3′; MHC Upstream primer: 5′-CCACCCACCGCCTACAAATCC-3′ MHCDownstream primer: 5′-AAGTCTTGACGAGTAGTCGCCATC-3′.

[0016] When the individual or group to be tested COL12A1 and MHC When the expression level is higher than that of the reference group or known slow-growing individuals, and at least two of the following are higher than those of the reference group or known slow-growing individuals: average body wall thickness, relative collagen content, mid-longitudinal muscle fiber density, and muscle fiber long diameter, they are judged to have fast-growing traits or fast-growing development advantages. Alternatively, when making relative comparisons among individuals or groups in the same batch, individuals or groups with higher overall performance in quantitative parameters of body wall histology and expression levels of marker genes are identified as candidate individuals or groups with a relative advantage in rapid growth and development.

[0017] The comparisons include comparisons with reference individuals, reference groups, known dominant individuals, or known slow-growing individuals, as well as relative comparisons among multiple individuals or groups to be tested in the same batch, at the same age, and under the same breeding conditions. When making relative comparisons within the same batch, individuals or groups with higher overall performance in body wall histological parameters and marker gene expression levels can be identified as candidate individuals or candidate groups with relative fast growth and development advantages.

[0018] An application of the evaluation method for identifying the fast-growing trait of *Urechis unicinctus*, wherein the evaluation method is used for screening fast-growing individuals of *Urechis unicinctus*, breeding fast-growing families, grading seedling growth potential, or evaluating germplasm resources.

[0019] Compared with the prior art, the present invention has the following beneficial effects: This invention combines the expression information of growth-related marker genes with quantitative parameters of body wall histology to assist in the evaluation of the rapid growth and developmental advantages of *Ulva uniflora*, providing technical support for the screening of superior individuals, breeding of fast-growing families, and evaluation of germplasm resources of *Ulva uniflora*. 1. Improve the objectivity and accuracy of the assessment: This invention achieves an objective quantitative assessment of the rapid growth trait of *Urtica monocyclicis* through the joint analysis of body wall histological parameters and marker gene expression, which can more realistically reflect its body wall tissue development level and growth potential.

[0020] 2. Effectively reduce interference factors in traditional phenotypic evaluation: This invention can effectively reduce the interference of factors such as body wall thickness, relative collagen content and muscle fiber development status on traditional weight and body length evaluation results by quantitatively analyzing body wall thickness, relative collagen content and muscle fiber development status.

[0021] 3. Achieving cross-validation at the molecular and tissue levels: This invention will... COL12A1 and MHCBy combining marker gene expression information with quantitative parameters of body wall histology, the rapid growth trait can be jointly verified at both the molecular and tissue levels, which improves the stability and reliability of the evaluation results.

[0022] 4. Applicable to breeding screening and germplasm evaluation: The auxiliary evaluation method established in this invention can be used for screening fast-growing individuals of *Ulva uniflora*, breeding fast-growing families, grading seedling growth potential, and evaluating germplasm resources, and has good application prospects. Attached Figure Description

[0023] Figure 1 This is a diagram showing the epimorphic differences of individuals from the same batch of *Ulva unicornu* in this embodiment of the invention, where (1) represents the candidate inferior group individuals and (2) represents the candidate superior group individuals; this epimorphic grouping is only used for pre-screening of candidate samples, and the final grouping is based on body wall histological parameters and marker gene expression results.

[0024] Figure 2 These are Masson's trichrome stained micrographs of the body wall of *Ulva uniflora* in the candidate dominant and candidate inferior groups in this embodiment of the invention. Figure 3 This is a quantitative statistical chart showing the average thickness of the body wall, relative collagen content, long diameter of muscle fibers, and density of *Ulva uniflora* in the candidate dominant group (FG) and candidate inferior group (SG) in this embodiment of the invention.

[0025] Figure 4 These are marker genes in the body wall tissues of *Ulva unicornu* from the candidate dominant group (FG) and candidate inferior group (SG) in this embodiment of the invention. COL12A1 and MHC A comparison chart of relative expression levels.

[0026] Figure 3 and Figure 4 All data in this paper are expressed as mean ± standard deviation. Indicates a significant difference ( P <0.05), This indicates that the difference is highly significant ( P <0.01). Detailed Implementation

[0027] To more clearly illustrate the purpose, technical solution, and beneficial effects of the present invention, the present invention will be further described below in conjunction with specific embodiments, but the scope of protection of the present invention is not limited to the following embodiments.

[0028] This invention provides an auxiliary assessment method for identifying the rapid growth trait of *Urtica unicinctus*. The method uses individuals from the same batch, age, and farming conditions as research subjects. Candidate samples are pre-grouped based on differences in individual morphology, body wall fullness, and overall developmental status. Subsequently, histological parameters such as average body wall thickness, relative collagen content, mid-longitudinal muscle fiber density, and muscle fiber long diameter are obtained through body wall tissue sections, Masson's trichrome staining, and digital image quantitative analysis. Based on these parameters, fast-growing and slow-growing groups are identified. Further detection of collagen-related marker genes is then performed. COL12A1 Muscle-related marker genes MHC The expression level was used to verify the histological grouping results at the molecular level, thereby enabling an auxiliary assessment of the rapid growth trait or rapid growth advantage of *Ulva uniflora*.

[0029] Example 1: An auxiliary assessment method for identifying the rapid growth trait of *Urtica monocyclicis* based on a combination of body wall histological parameters and marker gene expression. 1. Sample selection, apparent pregrouping, and tissue sampling To ensure comparability between different samples, this embodiment selected individuals of *Urtica monoringensis* from the same breeding batch, of the same age, and under the same breeding conditions as the test subjects. Figure 1 As shown, there were significant differences in apparent body shape among individuals of the same batch of *Urtica unicinctus*. Based on individual appearance, body wall fullness, and overall developmental status, the individuals were initially divided into candidate inferior and candidate superior groups based on visual differences. Figure 1 The individuals shown in (1) are relatively small and have relatively weak body wall development, and are preliminarily classified as candidate disadvantage group (SG). Figure 1 The individuals shown in (2) are relatively large and have fuller body walls, and are initially classified as candidate dominant group (FG).

[0030] It should be noted that the above epigenetic grouping is only a preliminary screening criterion for candidate samples and is not the final criterion for determining the rapid growth trait of *Ulva uniflora*. The final grouping will be based on subsequent body wall histological parameters and marker gene expression results.

[0031] Subsequently, body wall tissue was harvested from the middle third of the trunk at equidistant points from both the anterior and posterior ends of individuals in the candidate inferior and candidate superior groups. After sampling, a portion of the tissue was immediately fixed in 4% paraformaldehyde solution for 24 h for subsequent tissue sectioning, Masson's trichrome staining, and quantitative analysis of histological parameters; another portion of the body wall tissue was used to extract total RNA and detect marker gene expression.

[0032] 2. Tissue section preparation and Masson's trichrome staining Body wall tissue samples fixed in 4% paraformaldehyde for 24 h were subjected to routine dehydration, clearing, and paraffin embedding to prepare continuous transverse sections with a thickness of 5 μm. After dewaxing and hydration, the sections were stained with Masson's trichrome staining reagent. After staining, collagen fibers in the body wall tissue appeared blue, and muscle fibers appeared red, thus clearly distinguishing the muscle components and extracellular matrix components in the body wall tissue. After staining, the sections were further dehydrated with graded ethanol, cleared with xylene, and mounted with neutral resin.

[0033] like Figure 2 As shown, the cross-sections of the body walls of *Ulva unicornu* from the candidate dominant group and the candidate inferior group exhibited significant differences after Masson's trichrome staining. Individuals in the candidate dominant group had thicker body walls overall, denser muscle fiber arrangement in the mid-longitudinal muscle layer, and richer collagen fiber distribution; individuals in the candidate inferior group had relatively thinner body walls, looser muscle fiber arrangement, and relatively lower collagen fiber deposition.

[0034] 3. Microscopic image acquisition and quantitative analysis of histological parameters The stained tissue sections were observed and photographed using a biological microscope equipped with a digital imaging system, and the morphological parameters of the body wall and the extracellular matrix were quantitatively analyzed using digital image analysis software. In this embodiment, Fiji ImageJ software was used for image processing and analysis.

[0035] Under low magnification, regions with intact cross-sections of the body wall, clear structures, and no obvious wrinkles were selected. The full-thickness of the body wall was measured along a direction perpendicular to the body wall surface, and the average of multiple measurements was taken as the average body wall thickness of the sample. The results showed that the average body wall thickness of the candidate dominant group was 736.44±6.40 μm, which was significantly higher than that of the candidate inferior group (329.54±28.16 μm).

[0036] Masson staining images were acquired at the same magnification. Color deconvolution was used in image analysis software to extract staining signal images representing collagen fibers. Collagen regions were identified through threshold segmentation, and the percentage of collagen region area to effective tissue area was calculated as the relative collagen content or collagen volume fraction. The results showed that the relative collagen content of the candidate dominant group was 81.01±3.85%, significantly higher than the 67.78±2.12% of the candidate inferior group.

[0037] Focusing on the longitudinal muscle layer of the body wall under high magnification, regions of interest with uniform area were selected. The long axis diameter of the muscle fibers was measured, and the number of muscle fibers per unit area was counted, serving as the long axis diameter and density indicators of the muscle fibers, respectively. The results showed that the long axis diameter of the muscle fibers in the candidate dominant group was 5.13±0.36 μm, and the muscle fiber density was 12875±359 fibers / mm². 2The longest diameter of the muscle fibers in the candidate inferior group was 4.40±0.09 μm, and the muscle fiber density was 8900±1174 fibers / mm². 2 .

[0038] like Figure 3 As shown, the candidate dominant group had higher histological parameters than the candidate inferior group in terms of average body wall thickness, relative collagen content, myofiber long diameter, and myofiber density. These results indicate that the candidate dominant group has a higher level of body wall tissue development, richer collagen matrix deposition, and stronger myofiber development characteristics.

[0039] Based on the quantitative histological results, the candidate dominant group was identified as the rapid-growth group (FG), and the candidate inferior group was identified as the slow-growth group (SG). The histological parameters of each individual are shown in Table 1. Therefore, the FG and SG groups were not determined solely by visual observation, but rather based on quantitative histological parameters of the body wall.

[0040] Table 1: Comparison of morphological parameters of body wall tissues of *Ulva stolonifera* from candidate dominant group / FG and candidate inferior group / SG.

[0041] 4. Marker gene expression detection and molecular validation After confirming the FG and SG groups based on the above histological parameters, three individuals from each group were further examined for collagen-related marker genes in the body wall tissues of both groups. COL12A1 Muscle-related marker genes MHC The expression level was determined to validate the histological grouping results at the molecular level.

[0042] Total RNA was extracted from the body wall tissue, and cDNA was obtained by reverse transcription. qRT-PCR was then used for detection. COL12A1 and MHC The expression level. The primer sequences used are as follows: COL12A1 Upstream primer: 5′-TGACGAACTCCATCAGCGAAGAAG-3′; COL12A1 Downstream primer: 5′-AGGCGACTGACCAGCGTAGC-3′; MHC Upstream primer: 5′-CCACCCACCGCCTACAAATCC-3′; MHC Downstream primer: 5′-AAGTCTTGACGAGTAGTCGCCATC-3′.

[0043] In this embodiment, the total volume of the qRT-PCR reaction system was 10 μL, including: 5 μL of 2×SYBR Green qPCR premix, 1 μL of cDNA template, 0.2 μL of 10 μmol / L upstream primer, 0.2 μL of 10 μmol / L downstream primer, and 3.6 μL of ddH2O. The amplification program was: 95℃ pre-denaturation for 2 min; followed by 40 cycles, each cycle including 95℃ denaturation for 10 s and 60℃ annealing / extension for 30 s. The amplification results were used to obtain the concentrations of cDNA in each sample. COL12A1 and MHC The relative expression levels, results are shown in Figure 4 And Table 2.

[0044] Table 2: Comparison of marker gene expression levels of *Ulva unicircle* between candidate dominant group / FG and candidate inferior group / SG

[0045] Note: COL12A1 and MHC Relative expression levels of internal reference genes β-actin Calculated after correction; the FG and SG groups were determined based on quantitative parameters of body wall histology.

[0046] Compared to the SG group, the FG group showed a higher level of group mean. COL12A1 and MHC Level of expression, among which MHC The differences are even more pronounced. COL12A1 The relative expression level in the FG group was 1.84±0.37, and in the SG group it was 1.13±0.49. MHC The relative expression level was 3.71±0.36 in the FG group and 1.02±0.21 in the SG group. This result indicates that fast-growing individuals have a stronger molecular basis in collagen construction and muscle development.

[0047] 5. Joint Judgment and Result Analysis A comprehensive evaluation was conducted combining histological quantitative parameters and marker gene expression results. The candidate superior group (FG) was identified as the fast-growing group (FG) because it exhibited higher histological parameters than the candidate inferior group in terms of average body wall thickness, relative collagen content, mid-longitudinal muscle fiber density, and muscle fiber long diameter; the candidate inferior group was identified as the slow-growing group (SG). Further analysis... COL12A1 and MHC The results showed that, compared with the SG group, the FG group exhibited a higher level of group mean. COL12A1 and MHC Level of expression, among which MHC The differences were more pronounced, suggesting that the FG group had stronger growth potential at the molecular level related to muscle development.

[0048] In this embodiment, the detection data are expressed as mean ± standard deviation, and statistical methods were used to analyze the comparisons between groups. P <0.05 indicates a significant difference. P <0.01 indicates a highly significant difference.

[0049] When at least two of the following parameters of the tested individual or group are higher than those of the reference group or other tested individuals in the same batch: average body wall thickness, relative collagen content, mid-longitudinal muscle fiber density, and long axis of muscle fibers, and the tested individual or group is higher than those of the reference group or other tested individuals in the same batch, and COL12A1 and MHC When expression levels are high, it can be determined that the organism possesses a rapid growth trait or a rapid growth and development advantage. In other words, this invention does not rely on a single macroscopic phenotype or visual observation result for judgment, but rather uses a combined evaluation based on body wall histological parameters and marker gene expression results.

[0050] In summary, this embodiment establishes an auxiliary assessment method for *Urtica monocyclicis* that reduces errors in macroscopic weight and body shape judgment and more objectively reflects the growth potential of the body wall tissue through a technical route of "pre-screening based on epigenetic body shape differences—quantitative confirmation of histological parameters—verification of marker gene expression." This method can be used for screening fast-growing individuals, breeding fast-growing families, grading seedling growth potential, and evaluating germplasm resources.

Claims

1. An evaluation method for identifying the fast-growing trait of *Urtica monocyclicis*, characterized in that, (1) Collect the body wall tissue of the *Ulva unicornuate* to be tested; (2) Part of the body wall tissue was used for tissue sectioning, staining and digital image quantitative analysis to obtain quantitative parameters of body wall histology; (3) A portion of the body wall tissue was used to extract total RNA, and the expression levels of muscle-related marker genes and collagen-related marker genes were detected; (4) The quantitative parameters of body wall histology are analyzed together with the expression level of the marker gene, and compared with the reference individual, reference group or the same batch of test individuals to assess whether the test individual or group has the rapid growth trait or rapid growth development advantage.

2. The evaluation method for identifying the fast-growing trait of *Urtica monocyclic anguilla* according to claim 1, characterized in that: The quantitative parameters of the body wall histology include morphological parameters and extracellular matrix parameters; wherein, the morphological parameters include one or more of the following: average body wall thickness, mid-longitudinal muscle fiber density, and long axis of muscle fibers; the extracellular matrix parameters include relative collagen content or collagen volume fraction.

3. The evaluation method for identifying the fast-growing trait of *Urtica monocyclic anguilla* according to claim 1 or 2, characterized in that: In step (1), the body wall tissue is collected from the middle 1 / 3 region of the trunk of the single-ringed cricket, which is equidistant from the front and rear ends.

4. The evaluation method for identifying the fast-growing trait of *Urtica monocyclicis* according to claim 1, characterized in that: The staining in step (2) is Masson trichrome staining, which is used to differentiate muscle fibers and collagen fibers in the body wall tissue and to perform digital image quantitative analysis of subsequent body wall morphological parameters and extracellular matrix parameters.

5. The evaluation method for identifying the fast-growing trait of *Urtica monocyclic anguilla* according to claim 1 or 4, characterized in that: In step (2), digital image analysis software is used to perform quantitative analysis on the stained tissue sections, and the collagen fiber content and muscle fiber parameters are analyzed using the color deconvolution function.

6. The evaluation method for identifying the fast-growing trait of *Urtica monocyclicis* according to claim 1, characterized in that: The muscle-related marker gene is the myosin heavy chain gene. MHC The collagen-related marker gene is the collagen gene. COL12A1 .

7. The evaluation method for identifying the fast-growing trait of *Urtica monocyclic anguilla* according to claim 6, characterized in that: The COL12A1 and MHC The detection primer sequences are as follows: COL12A1 Upstream primer: 5′-TGACGAACTCCATCAGCGAAGAAG-3′ COL12A1 Downstream primer: 5′-AGGCGACTGACCAGCGTAGC-3′; MHC Upstream primer: 5′-CCACCCACCGCCTACAAATCC-3′ MHC Downstream primer: 5′-AAGTCTTGACGAGTAGTCGCCATC-3′.

8. The evaluation method for identifying the fast-growing trait of *Urtica monocyclic anguilla* according to claim 1, characterized in that: The comparisons include comparisons with reference individuals, reference groups, known dominant individuals, or known slow-growing individuals, or relative comparisons between multiple individuals or groups to be tested in the same batch, at the same age, and under the same breeding conditions.

9. The evaluation method for identifying the fast-growing trait of *Urtica monocyclic anguilla* according to claim 1 or 8, characterized in that: When the individual or group to be tested COL12A1 and / or MHC When the expression level is higher than that of the reference group or known slow-growing individuals, and at least two of the following are higher than those of the reference group or known slow-growing individuals: average body wall thickness, relative collagen content, mid-longitudinal muscle fiber density, and muscle fiber long diameter, the individual is judged to have a fast-growing trait or fast-growing development advantage. Alternatively, when making relative comparisons among individuals or groups in the same batch, individuals or groups with higher overall performance in quantitative parameters of body wall histology and expression levels of marker genes are identified as candidate individuals or groups with a relative advantage in rapid growth and development.

10. The application of the evaluation method for identifying the fast-growing trait of *Urtica monocyclic* as described in any one of claims 1 to 9, characterized in that: The evaluation method is used for screening fast-growing individuals of *Ulva uniflora*, breeding fast-growing families, grading seedling growth potential, or evaluating germplasm resources.

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