Method for analyzing skeletal muscle fiber heterogeneity of white feather broiler chicken based on space transcriptome technology

Abstract

The invention discloses a space transcriptome technology-based white feather broiler skeletal muscle fiber heterogeneity analysis method. The method comprises the following steps: preparing a white feather broiler skeletal muscle tissue embedding sample; the skeletal muscle tissue embedded sample obtained in the step 1 is sliced, and part of the sliced sample is dyed and then photographed; carrying out space transcriptome library construction on partial slice samples, and finally sequencing and carrying out data analysis. A space transcriptome technology is utilized to construct a space transcriptome map of four skeletal muscles of the white feather broiler chicken, the heterogeneity of the skeletal muscle muscle fibers of the white feather broiler chicken is revealed, and the space distribution of I-type and II-type muscle fibers of four skeletal muscle tissues of lumbar muscle, semimembrane muscle, soleus muscle and gastrocnemius muscle is verified; the functional difference between the I-type muscle fiber gene and the II-type muscle fiber gene is proved, and a new detailed skeletal muscle muscle fiber heterogeneity data support is provided for chicken breed improvement.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of poultry breeding, and particularly relates to a method for analyzing skeletal muscle fiber heterogeneity of white-feathered broiler chickens based on spatial transcriptome technology. BACKGROUND

[0002] Chicken is the most widely consumed meat product in the world and is a source of high-quality protein. Skeletal muscle accounts for the majority of meat and is one of the most important economic traits of agricultural animals, closely related to meat production and quality. Animal skeletal muscle is composed of different types of muscle fibers, and muscle fibers, i.e., muscle cells, are the basic building blocks of muscle tissue, and their types are an important factor in determining meat quality. The density, number, diameter, cross-sectional area (CSA), and proportion of different types of muscle fibers are closely related to meat quality. Currently, muscle fibers are characterized as types I, IIa, IIb, and IIx according to the presence of myosin heavy chain (MyHC) subtypes, and the expression levels of muscle fiber type marker genes are detected based on conventional transcriptome sequencing technology, but the spatial distribution information of muscle cells and the related information of the microenvironment in which the cells are located are lacking. Therefore, it is particularly important to explore new skeletal muscle typing techniques for studying the types of skeletal muscle fibers and analyzing the gene expression profiles of different muscle fibers. SUMMARY

[0003] The present application aims to explore new research techniques for typing skeletal muscle fibers of white-feathered broiler chickens, and further provides a method for analyzing chicken skeletal muscle fiber heterogeneity based on spatial transcriptome technology.

[0004] To achieve the above-mentioned purpose, the technical scheme of the present application is as follows: In a first aspect, the present application provides a method for analyzing skeletal muscle fiber heterogeneity of white-feathered broiler chickens based on spatial transcriptome technology, comprising the following steps: Step 1, preparing a white-feathered broiler chicken skeletal muscle tissue embedding sample; Step 2, slicing the skeletal muscle tissue embedding sample obtained in step 1, and performing staining treatment on part of the sliced sample, and then taking a photograph; Step 3, constructing a spatial transcriptome library for the remaining skeletal muscle slice sample of step 2, which comprises: Step 3-1, performing total RNA integrity evaluation on the skeletal muscle slice sample in step 2, and selecting a skeletal muscle slice sample with a RIN value > 7; Step 3-2, performing permeation on the skeletal muscle slice sample obtained in step 1 to capture the RNA therein; Step 3-3, performing reverse transcription on the captured RNA to obtain cDNA, and then constructing a spatial transcriptome sequencing library and performing sequencing; Step 4, analyzing the sequencing data.

[0005] Preferably, in the step 1, the preparation of the white-feathered broiler skeletal muscle tissue embedding sample comprises the following steps: After removing the feathers and fascia on the skeletal muscle, the sample is trimmed to a size of no more than 6.5 mm x 6.5 mm, and the trimmed sample is frozen in liquid nitrogen; then, according to the muscle fiber direction, the cross-section is exposed, and the OCT cryo-embedding agent is used for embedding, so as to prepare the sample required for the spatial transcriptome of the white-feathered broiler skeletal muscle tissue, and store it at -80℃.

[0006] Preferably, in the step 2, the slicing of the skeletal muscle tissue embedding sample obtained in the step 1 refers to the slicing of the skeletal muscle tissue embedding sample at -12±1℃, and the thickness of the slice is 9-10 μm.

[0007] Preferably, in the step 2, the staining treatment comprises HE staining, SDH staining, and ATPase staining.

[0008] Preferably, in the step 3-2, the permeabilization is to treat the skeletal muscle slice sample with a permeabilization agent for 15-30 min, preferably 20-22 min, to destroy the cell membrane and release the RNA; then, the permeabilized mRNA is combined with the barcode capture probe.

[0009] Preferably, in the step 3-3, the sequencing depth is that each tissue covers nearly 5000 spots on the capture region.

[0010] Preferably, in the step 4, the data analysis comprises: Step 4-1, after obtaining the original sequencing data, first align the fiducial points with the image, then use 10x SpaceRanger to split the barcode, UMI and cDNA insert of Reads, and obtain the quantitative analysis result of the chicken skeletal muscle sample spatial transcriptome data; preferably, the total data information at least includes: total spot number, total reads number, average gene number of each spot; Step 4-2, according to the quantitative analysis result obtained in the step 4-1, quality control is performed on the spatial data set; Step 4-3, dimensionality reduction clustering analysis is performed on the quality-controlled data; Step 4-4, difference gene analysis and function enrichment analysis are performed on the data after dimensionality reduction clustering analysis, and integration analysis of skeletal muscles in different parts is also performed.

[0011] Further preferably, in the step 4-2, the parameters of quality control include: 1) the number of unique features detected for each cell; 2) the proportion of mitochondrial genes detected for each cell; more preferably, the spatial data set is quality controlled by using Rstudio (R 4.2.2) software.

[0012] Further preferably, in the step 4-4, the different parts of skeletal muscle are quadratus lumborum, semimembranosus, soleus and gastrocnemius, and the integrated analysis includes: using the merge function to integrate and analyze the data of the four parts of quadratus lumborum, semimembranosus, soleus and gastrocnemius, using the SCTransform function to standardize it, and finally using the FindAllMarkers function to find the differential genes of the four parts.

[0013] Preferably, in the step 4, the data analysis further includes: skeletal muscle fiber marker gene analysis and muscle fiber type proportion calculation analysis.

[0014] In a second aspect, the present application provides a spatial transcriptome of chicken skeletal muscle obtained by the method of the first aspect.

[0015] Compared with the prior art, the present application has the following beneficial effects: The present application uses spatial transcriptome technology to construct the spatial transcriptome of four kinds of skeletal muscle of white-feathered broilers, reveals the skeletal muscle fiber heterogeneity of white-feathered broilers, verifies the spatial distribution of type I and type II muscle fibers of quadratus lumborum, semimembranosus, soleus and gastrocnemius, and confirms the functional differences between type I and type II muscle fiber genes, thereby providing new detailed skeletal muscle fiber heterogeneity data support for chicken breed improvement. BRIEF DESCRIPTION OF DRAWINGS

[0016] The drawings described herein are used to provide further understanding of the present application, and form a part of the present application. The illustrative embodiments of the present application and their descriptions serve to explain the present application, and do not constitute an improper limitation on the present application. In the drawings: Figure 1 Figure 1 shows the Loupe Browser 5 alignment results in Example 1 of the present application, wherein A is quadratus lumborum; B is semimembranosus; C is soleus; and D is gastrocnemius.

[0017] Figure 2Figure 1 is a comparison chart of four skeletal muscle samples before and after quality control in Example 1 of the present application, wherein A1 and A2 are gene data of quadratus lumborum muscle samples before and after quality control, respectively; B1 and B2 are gene data of semimembranosus muscle samples before and after quality control, respectively; C1 and C2 are gene data of soleus muscle samples before and after quality control, respectively; D1 and D2 are gene data of gastrocnemius muscle samples before and after quality control, respectively; nCount_Spatial: UMI count; nFeature_Spatial: number of detected genes; percent.mt: proportion of detected mitochondrial genes; percent.rb: proportion of detected ribosomal genes.

[0018] Figure 3 Figure 2 is a UMAP plot of type I muscle fiber marker genes in Example 1 of the present application, wherein A-D correspond to quadratus lumborum muscle, semimembranosus muscle, soleus muscle, and gastrocnemius muscle, respectively.

[0019] Figure 4 Figure 3 is a UMAP plot and spatial location plot in Example 1 of the present application, wherein A-C correspond to quadratus lumborum muscle, soleus muscle, and gastrocnemius muscle, respectively.

[0020] Figure 5 Figure 4 is a UMAP plot of type I muscle fiber marker genes in Example 1 of the present application, wherein A-D correspond to quadratus lumborum muscle, semimembranosus muscle, soleus muscle, and gastrocnemius muscle, respectively.

[0021] Figure 6 Figure 5 is an ATPase staining and spatial location plot in Example 1 of the present application, wherein A-D correspond to quadratus lumborum muscle, semimembranosus muscle, soleus muscle, and gastrocnemius muscle, respectively.

[0022] Figure 7 Figure 6 is a functional enrichment analysis in Example 1 of the present application, wherein A1 corresponds to type I of quadratus lumborum muscle; A2 corresponds to type II of quadratus lumborum muscle; B1 corresponds to type I of soleus muscle; B2 corresponds to type II of soleus muscle; C1 corresponds to type I of gastrocnemius muscle; C2 corresponds to type II of gastrocnemius muscle. The p-value represents the probability of the result being annotated to other more random pathways, and the cumulative hypergeometric distribution test is used.

[0023] Figure 8 Figure 7 is a graph showing the expression of four sites integrated standardization clustering (A), MYH7B (B), MYH1A (C), and MYH1E (D) in Example 1 of the present application, wherein in the A graph, BMJ is semimembranosus muscle, BMYJ is soleus muscle, FCJ is gastrocnemius muscle, and YFJ is quadratus lumborum muscle.

[0024] Figure 9SDH, ATPase staining chart (10x) of skeletal muscle sample in embodiment 1 of the present application, wherein A, B, C, D are the SDH and ATPase staining results of the quadratus lumborum muscle, the semimembranosus muscle, the soleus muscle and the gastrocnemius muscle in turn. The first one of each part is the SDH staining result, and the second to fourth ones are the ATPase staining results, and the three sections of ATPase staining are taken from different areas of the same slide. DETAILED DESCRIPTION

[0025] The test animals in the experimental part of the embodiment of the present application come from the Yu Tai Poultry Farm of Zongda Food Suining Co., Ltd., white-feathered broilers, breed: Aberega (AA+), female, 61 weeks old. The white-feathered broilers are evaluated for quality according to "NY / T 631-2002 Chicken Quality Grading", and a total of 4 muscle part tissue samples are taken, which are the quadratus lumborum muscle, the semimembranosus muscle, the soleus muscle and the gastrocnemius muscle. The present experiment is carried out in accordance with the provisions of the Animal Ethics Committee of Sichuan Agricultural University.

[0026] Further, the method for preparing frozen sections of skeletal muscle samples is as follows: the white-feathered broiler is placed on the operating table, and the quadratus lumborum muscle, the semimembranosus muscle, the soleus muscle and the gastrocnemius muscle tissue samples are collected. The collected skeletal muscles are trimmed to a size of not more than 6.5 mm x 6.5 mm, and then gently placed in liquid nitrogen for quick freezing. The frozen samples are embedded with OCT cryopreservation embedding agent according to the muscle fiber exposure cross section, and then moved to a -80℃ ultra-low temperature refrigerator for standby use. Before sectioning, the sample is taken out, balanced at -20℃ for about 30 min, and then fixed on the sample holder of the microtome. The section is upward, the section thickness parameter is adjusted, and then sectioning is carried out. After obtaining the complete section, the front of the glass slide is downward, and the tissue section is slowly adsorbed to prepare for the following staining.

[0027] In the description of the embodiment of the present application, HE staining, SDH staining and ATPase staining are all conventional operations in the art, which will not be described in detail here.

[0028] Further, in the description of the present application, it needs to be explained that the specific conditions not mentioned in the embodiment are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments not mentioned by the manufacturer are all conventional products that can be purchased on the market.

[0029] The technical solutions in the embodiments of the present application will be described clearly and completely in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0030] Embodiment 1

[0031] The present embodiment develops an analysis method for skeletal muscle fiber heterogeneity of white-feathered broilers, as follows: (1) Quality inspection of white-feathered broilers According to NY / T 631-2002 Chicken Quality Grading, the quality of the test animals, white-feathered broilers, was evaluated. The test animals were rated as level 1, meeting the requirements of the test animals.

[0032] (2) Skeletal muscle section staining The H&E staining of the frozen section samples of the quadratus lumborum muscle, semimembranosus muscle, soleus muscle, and gastrocnemius muscle showed that the boundaries between muscle fibers were clear, the ice crystals generated during sampling were few, and the tissues were relatively complete.

[0033] (3) Spatial transcriptome data analysis In the present test, the Visium spatial transcriptome technology of the 10x Genomics company was used to study the heterogeneity of different types of muscle fibers. The specific process was as follows: the fresh frozen tissue embedded with OCT was sectioned and placed on the capture area of the spatial transcriptome technology slide. One slide contained four capture areas, each containing 5000 capture points, the diameter of each capture point was 55 μm, the distance between the centers of two adjacent capture points was 110 μm, each capture point contained a unique spatial position barcode and contained millions of oligos containing PolyT. The tissue was permeabilized with a permeabilizing agent such as Triton X-100, and the released mRNA PolyA binding probe oligo (dT) was reverse transcribed into cDNA. Then, the library required for sequencing was prepared by eluting from the slide, ready for the next sequencing. After that, the sequencing results were analyzed and visualized by using the easy-to-use data analysis and visualization software Loupe Browser, Space Ranger, Seurat, and Rstudio. The specific analysis process was as follows: (3-1) Loupe Browser 5 analysis After obtaining the raw sequencing data, the Space Ranger software was used for preliminary processing: first, using LoupeBrowser 5, align the slide spots with the image to generate a json file containing coordinate information, that is, the data information is matched with the position information in the tissue section, as shown in Figure 1 .

[0034] (3-2) Space Ranger quantitative analysis The "spaceranger mkfastq" pipeline in Space ranger (version 1.3.1; 10X Genomics) was used to align the FASTQ files to the reference genome sequence and gene annotation file (GTF) of chicken from (http: / / asia.ensembl.org). Then the microscope slide images and FASTQ files were scanned using the Space ranger count pipeline, and alignment, organization detection, benchmark detection, barcode counting, UMI counting were performed to generate the gene expression matrix.

[0035] The final four tissue sample cases are as follows: a, the soleus muscle sample covers 2190 spots in the capture area, with an average of 159285 reads per spot, the Q30 value of sample UMI sequence is 94.7%, the reads uniquely aligned to the genome accounts for 87.3%, and the sequencing saturation depth is 94.2%. b, the cecum muscle sample covers 3624 spots in the capture area, with an average of 95467 reads per spot, the Q30 value of sample UMI sequence is 94.6%, the reads uniquely aligned to the genome accounts for 84.8%, and the sequencing saturation depth is 94.2%. c, the psoas muscle sample covers 3167 spots in the capture area, with an average of 122342 reads per spot, the Q30 value of sample UMI sequence is 94.3%, the reads uniquely aligned to the genome accounts for 86.9%, and the sequencing saturation depth is 95.3%. d, the semimembranosus muscle sample covers 1702 spots in the capture area, with an average of 226831 reads per spot, the Q30 value of sample UMI sequence is 94.5%, the reads uniquely aligned to the genome accounts for 83.1%, and the sequencing saturation depth is 98% (Table 1). Overall, the quality is qualified, the sequencing depth is high, and the coverage of the reference genome of chicken is good, which can be used for subsequent data analysis.

[0036] Table 1 Space transcriptome sequencing data of skeletal muscle samples Sample_ID Psoas Semimembranosus Soleus Gastrocnemius Number of Spots Under Tissue 3167 1702 2190 3624 Number of Reads 387456139 386065994 348834835 345973323 Mean Reads per Spot 122341 226830 159285 95467 Mean Reads Under Tissue per Spot 97827 173192 116414 77258 Fraction of Spots Under Tissue 0.63 0.34 0.44 0.73 Median Genes per Spot 554 620.5 971 732 Median MMI Counts per Spot 2824 2146 4695 2618 Valid Barcodes 0.973 0.974 0.972 0.974 Valid MMIs 0.999 0.999 0.999 0.999 Sequencing Saturation 0.953 0.980 0.942 0.942 Q30 Bases in Barcode 0.954 0.955 0.951 0.951 Q30 Bases in RNA Read 0.915 0.907 0.919 0.921 Q30 Bases in MMI 0.943 0.945 0.947 0.946 Reads Mapped to Genome 0.927 0.910 0.923 0.916 Reads Mapped Confidently to Genome 0.869 0.831 0.873 0.848 Fraction Reads in Spots Under Tissue 0.821 0.789 0.755 0.833 Total Genes 13222 12605 13698 14193 (3-3) Sequencing data quality control Based on the quantification results of Space Ranger, the spatial dataset was pre-processed using Seurat 4.1.1 in Rstudio (R 4.2.2). The main parameters for quality control were two: a, the number of unique features detected by each cell (unique features represent the number of genes detected by a cell), in this case, genes expressed in less than 10 spots were considered to be low-quality expressed genes and were filtered out; b, the proportion of mitochondrial genes detected by each cell, theoretically, the mitochondrial genome accounts for a very small part compared to the nuclear genome. Therefore, cells with too high mitochondrial gene expression ratio will be filtered out. The mitochondrial gene expression ratio is based on the percentage of 13 mitochondrial genes (ND6, ND5, ND4L, ND4, ND3, MT-ND2, ND1, CYTB, COX2, COX3, ATP8, ATP6, MT-CO1) in the total gene expression number in each spot. The spots and expressed genes selected by the above steps were used for subsequent analysis (gene data comparison before and after quality control of four skeletal muscle samples, as shown in Figure 2 ). After quality control, 3119 spots and 8874 genes were retained in the lumbar quadratus muscle sample, 1699 spots and 7935 genes were retained in the semimembranosus muscle sample, 2187 spots and 9501 genes were retained in the soleus muscle sample, and 3573 spots and 9976 genes were retained in the gastrocnemius muscle sample for downstream clustering analysis and cell type identification (Table 2).

[0037] Table 2. Sample quality control information Sample Name Pre-QC _spot_count Post-QC _spot_count Pre-QC _gene_count Post-QC _gene_count Psoas 3167 3119 13222 8874 Semimembranosus 1702 1699 12605 7935 Soleus 2190 2187 13698 9501 Gastrocnemius 3624 3573 14193 9976 (3-4) Dimensionality reduction and clustering analysis The results of dimensionality reduction and clustering analysis using Seurat are shown in Figure 2 . Principal component analysis was performed on the sequencing data, and then clustering was performed. Then, the defined slow muscle genes TNNT1 and MYH7 were used as marker genes of type I muscle fibers, and the lumbar quadratus muscle, semimembranosus muscle, soleus muscle, and gastrocnemius muscle in the four skeletal muscle samples were clustered and visualized by UMAP (Fig. Figure 3 A~D). It was found that the UMAP plots of TNNT1 and MYH7 were clustered in similar positions, and compared with the third UMAP plot which had distinguished type I and type II, the UMAP plot with MYH7 as the marker gene was closer to the typing result. At the same time, it was found that the slow muscle genes TNNT1 and MYH7 were not expressed in the selected semimembranosus muscle. And the clustering results were displayed on the whole expression profile with MYH7 as the marker gene, showing the spatial position map (Fig. Figure 4 A~C).

[0038] Next, the fast muscle fiber genes TNNT3 and MYH1A were used as marker genes for type II muscle fibers. The clustering and UMAP method visualization of the four skeletal muscle samples, the quadratus lumborum, the semimembranosus, the soleus, and the gastrocnemius, were performed (Figs. 3A-3D). Figure 5 The clustering positions of the UMAP plots of TNNT3 and MYH1A were found to be almost identical to the clustering positions of the UMAP plots of type I and type II in the third panel.

[0039] The ATPase staining method was used to distinguish type I and type II muscle fibers. The sections of the spatial transcriptome samples were subjected to ATPase staining, as shown in Figs. 3A-3D. The ATPase staining was type I, which corresponded to the distribution of type I muscle fibers in the spatial plot. The semimembranosus muscle, which had no expression of MYH7 in the above dimensionality reduction and clustering steps, had a staining result consistent with that. Figure 6

[0040] (3-5) Differential gene and functional enrichment analysis The Top10 differential genes highly expressed in type I and type II muscle fibers were counted after dimensionality reduction of the differential genes of the quadratus lumborum, the soleus, and the gastrocnemius (Table 3). As shown in Table 3, the Top10 differential genes highly expressed in type I muscle fibers were relatively fixed, including but not limited to (TNNT1, TNNC1, MYH7B, TNNI1, MYL10, TPM3), etc. The Top10 differential genes highly expressed in type II muscle fibers were diverse and not fixed.

[0041] Table 3 Top 10 differential genes of type I and type II Sample Name Type I Differential Genes Type II Differential Genes Psoas ENSGALG00000050515, ENSGALG00000049450, TNNI1, TNNC1, CD74, MYH7B, MYL10, TNNI1, MYL3, APOA1 ENSGALG00000036956, ENSGALG00000043598, TNN2, COII, TNNI2, COX3, HBAD, MT - CO1, TNNI3, GAPDH Soleus ACTA1, MYL10, MYL3, MYH7B, TPM3, ATP2A2, MYBPC1, TNNC1, TNNI1 ENSGALG00000050515, ENSGALG00000049450, ENSGALG00000036956, APOA1, ENSGALG00000043598, COX3, COII, RPS28, ATP6, GAPDH Gastrocnemius TNNC1, TNNI1, MYH7B, MYL3, MYL10, TNNI1, TPM3, ATP2A2, FHLI, FABP3 ENSGALG00000050515, ENSGALG00000049450, GAPDH, ENSGALG00000036956, APOA1, ACTA1, FTH1, ENSGALG00000043598, MYH1F, MYL1 ​The differentially expressed genes of the three skeletal muscle samples, including psoas major muscle, soleus muscle and gastrocnemius muscle, were converted into human genes by 1:1 homologous transformation, and then the functional enrichment analysis was performed using Metascape online tool (http: / / metascape.org). Among them, 224 high-expression differential genes (179 human homologous genes) were screened out in type I psoas major muscle, which were mainly functionally enriched in oxidoreductase activity (GO:0016491), cytoplasmic ribosome (GO:0022626) and peptide metabolic process (GO:0006518); 226 high-expression differential genes (183 human homologous genes) were selected in type II, which were mainly functionally enriched in mitochondrial protein-containing complex (GO:0098798), structural component of ribosome (GO:0003735), oxidoreductase activity acting on NAD(P)H (GO:0016651), peptide metabolic process (GO:0006518), ATP metabolic process (GO:0046034), aerobic respiration (GO:0009060) and cell macromolecule biosynthesis process (GO:0034645). 400 high-expression differential genes (348 human homologous genes) were screened out in type I soleus muscle, which were mainly functionally enriched in mitochondrial matrix (GO:0005959), peptide metabolic process (GO:0006518), oxidoreductase activity (GO:0016491), mitochondrial protein-containing complex (GO:0098798) and actin binding (GO:0003779); 206 high-expression differential genes (160 human homologous genes) were selected in type II, which were mainly functionally enriched in cell macromolecule biosynthesis process (GO:0034645), peptide metabolic process (GO:0006518), polypeptide biosynthesis process (GO:0043043), mitochondrial inner membrane protein complex (GO:0098800) and cytoplasmic ribosome (GO:0022626). There were 212 differential genes in type I gastrocnemius muscle fibers, which were mainly functionally enriched in mitochondrial matrix (GO:0005759), oxidoreductase activity (GO:0016491), mitochondrial protein-containing complex (GO:0098798) and aerobic respiration (GO:0009060). 259 high-expression differential genes (212 human homologous genes) were screened out in type I gastrocnemius muscle, and 243 high-expression differential genes (55 human homologous genes) were selected in type II, which were mainly functionally enriched in mitochondrial protein-containing complex (GO:0098798), oxidoreductase activity (GO:0016491), cell macromolecule biosynthesis process (GO:0034645), aerobic respiration (GO:0009060) and ATP metabolic process (GO:0046034) Figure 7 ).

[0042] (3-6) MYH gene family expression integration analysis In the skeletal muscle of chicken, the expression of MYH7B, MYH1E and MYH1A in MYH gene family was also found to be high, in which MYH7B was highly expressed in type I muscle fiber, and MYH1E and MYH1A were highly expressed in type II muscle fiber. The clustering results after integration of the four parts are shown as follows Figure 8 The muscle fiber types of the four parts collected in the experiment were as follows: the soleus muscle was slow muscle, the psoas muscle and the gastrocnemius muscle were fast muscle and slow muscle, and the semimembranosus muscle was fast muscle. The clustering results after integration of the four parts were consistent with the results obtained in the previous experiment.

[0043] (4) Muscle fiber ratio calculation After SDH staining of the psoas muscle, the semimembranosus muscle, the gastrocnemius muscle and the soleus muscle of white-feathered broilers, the type I muscle fiber and the type II muscle fiber in the staining results were distinguished by different color blocks using Adobe Photoshop, and the proportion of type I muscle fiber was calculated using image j. The muscle fiber ratio was calculated as shown in Figure 9 and Table 4.

[0044] Table 4: Type I muscle fiber ratio Sample name ST type I ratio SDH type I ratio ATP type I ratio ST correlation analysis Psoas major 18.833 10.327 7.115 0.47 (with SDH) Semimembranosus 0 0 0 / Soleus 7.83 17.333 14.430 0.41 (with ATP) Gastrocnemius 9.22 16.202 8.131 / In summary, the spatial transcriptome technology is used to reveal the skeletal muscle fiber heterogeneity of white-feathered broilers, and the spatial distribution of type I and type II muscle fibers in four kinds of skeletal muscle tissues is verified. Through the expression amount of differential genes, in addition to the known marker genes, the slow muscle genes TNNT1 and MYH7, and the fast muscle genes TNNT3 and MYH1A are screened as marker genes for chicken skeletal muscle fiber typing to enhance the reliability of chicken skeletal muscle fiber typing. In addition, the clustering positions of MYH7B highly expressed in type I, and MYH1A and MYH1E highly expressed in type II are verified. In summary, the spatial transcriptome technology is used to construct the spatial transcriptional atlas of four kinds of skeletal muscle of white-feathered broilers, which provides new and detailed skeletal muscle fiber heterogeneity data support for chicken breed improvement.

[0045] Although the embodiments of the present application have been shown and described, it should be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present application, and the scope of the present application is defined by the appended claims and their equivalents. The above-described embodiments only express several implementation manners of the present application, which are described in detail, but should not be understood as limiting the scope of the patent of the present application. It should be noted that for those skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are all within the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. A method for analyzing the heterogeneity of skeletal muscle fibers in broiler chickens based on spatial transcriptomics technology, characterized in that, Includes the following steps: Step 1: Prepare embedded skeletal muscle tissue samples from broiler chickens; Step 2: Section the skeletal muscle tissue embedded sample obtained in Step 1, stain some of the sections, and then take pictures. Step 3 involves constructing a spatial transcriptome library from the remaining skeletal muscle slices from Step 2. This process includes: Step 3-1: Assess the total RNA integrity of the skeletal muscle slices from Step 2 and select skeletal muscle slices with RIN values ​​> 7. Step 3-2: Permeabilize the skeletal muscle slice sample obtained in Step 1 to capture the RNA within it; Step 3-3: The captured RNA is reverse transcribed to obtain cDNA, and then a spatial transcriptome sequencing library is constructed and sequenced. Step 4: Analyze the sequencing data.

2. The method according to claim 1, characterized in that, Step 1, which involves preparing embedded skeletal muscle tissue samples from broiler chickens, includes the following steps: After removing feathers and fascia from the skeletal muscle, the samples were trimmed to a size not exceeding 6.5 mm × 6.5 mm. The trimmed samples were then frozen in liquid nitrogen. The samples were then embedded in OCT cryoembryographing agent according to the direction of the muscle fibers in the exposed cross section. The samples required for the spatial transcriptome of broiler skeletal muscle tissue were prepared and stored at -80℃.

3. The method according to claim 1, characterized in that, In step 2, slicing the skeletal muscle tissue embedded sample obtained in step 1 means slicing the skeletal muscle tissue embedded sample under -12±1℃ conditions, with a slice thickness of 9~10 μm; and / or, staining treatment includes HE staining, SDH staining, and ATPase staining.

4. The method according to claim 1, characterized in that, In step 3-2, the permeabilization involves treating the skeletal muscle slice sample with a permeabilizing agent for 15-30 minutes, preferably 20-22 minutes, to disrupt the cell membrane and release RNA; then the permeabilized mRNA is combined with a barcode capture probe.

5. The method according to claim 1, characterized in that, In step 3-3, the sequencing depth is such that each tissue in the capture region covers approximately 5000 spots.

6. The method according to claim 1, characterized in that, In step 4, data analysis includes: Step 4-1: After obtaining the raw sequencing data, first align the reference points with the image, and then use a 10× Space Ranger to split the barcode, UMI, and cDNA insert fragments of the reads to obtain the quantitative analysis results of the spatial transcriptome data of the chicken skeletal muscle sample; preferably, the overall data information includes at least: the total number of spots, the total number of reads, and the average number of genes per spot; Step 4-2: Based on the quantitative analysis results obtained in Step 4-1, perform quality control on the spatial dataset; Step 4-3: Perform dimensionality reduction and cluster analysis on the quality-controlled data; Step 4-4 involves performing differential gene analysis and functional enrichment analysis on the data after dimensionality reduction and clustering analysis, as well as integrating analysis of skeletal muscle from different sites.

7. The method according to claim 6, characterized in that, In step 4-2, the quality control parameters include: 1) the number of unique genes detected in each cell; 2) the proportion of mitochondrial genes detected in each cell; preferably, Rstudio (R4.2.2) software is used to perform quality control on the spatial dataset.

8. The method according to claim 6, characterized in that, In step 4-4, the skeletal muscles in different locations are the quadratus lumborum, semimembranosus, soleus, and gastrocnemius. The integration analysis includes: using the merge function to integrate the data of the four locations of quadratus lumborum, semimembranosus, soleus, and gastrocnemius, using the SCTransform function to standardize them, and finally using the FindAllMarkers function to find the differentially expressed genes in the four locations.

9. The method according to any one of claims 6 to 8, characterized in that, In step 4, the data analysis also includes: skeletal muscle fiber type analysis and muscle fiber ratio calculation analysis.

10. The spatial transcription map of chicken skeletal muscle obtained by the method according to any one of claims 1 to 9.

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