A molecular marker related to hypoxia adaptation of tibetan chicken and application thereof

Abstract

The present application relates to a kind of molecular marker related to Tibetan chicken hypoxia adaptability and its application. By analyzing the genetic structure and gene communication of tibetan chicken under different altitude gradient, combined with heart transcriptome data, it is found that PARK7 gene shows stable expression level in tibetan chicken, and is not affected by plateau environment. Therefore, a breeding method for improving the survival rate and adaptability of tibetan chicken in plateau hypoxic environment is screened out. The method not only enriches the understanding of the hypoxia adaptation mechanism of tibetan chicken, but also provides scientific basis for the conservation and selection of tibetan chicken.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of animal genetics and breeding, and specifically relates to a molecular marker related to the hypoxic adaptability of Tibetan chickens and its application. The method improves the adaptability of Tibetan chickens in high-altitude hypoxic environments through molecular genetic means. BACKGROUND

[0002] Chickens, as one of the earliest domesticated birds, are a highly valuable poultry species, with their main products including eggs and meat, as well as providing feathers and manure as by-products. Eggs are rich in nutrients, while chicken meat has lower fat content and higher lean meat content compared to other meats, and is rich in high-quality protein, making it a widely consumed food source for humans.

[0003] In high-altitude environments, animals face hypoxic challenges and need to adapt to high-altitude environments through changes in tissues and organs, especially those related to oxygen exchange, transport, and metabolism. Tibetan chickens are a unique small chicken breed native to the Qinghai-Tibet Plateau, living in high-altitude, low-oxygen environments for a long time, and have developed unique physiological and genetic characteristics. The low-oxygen environment of the plateau region poses a severe challenge to the survival and reproduction of poultry. Traditional breeding methods mainly rely on phenotypic selection, but this approach has some shortcomings. Phenotypic selection usually only focuses on external characteristics, ignoring genetic diversity, which can lead to a loss of genetic diversity and reduce the resistance of chicken populations to environmental changes and diseases. In addition, phenotypic selection is less efficient, especially when dealing with complex traits such as disease resistance or adaptability, which requires a long time to observe and evaluate individual performance. Furthermore, phenotypic characteristics are easily influenced by environmental factors, which can lead to inaccurate selection results. For example, in high-altitude areas, the low-oxygen environment can affect the growth and reproduction performance of chickens, and these changes may not be reflected at the genetic level. Traditional methods also have difficulty identifying recessive traits, as some important traits may be recessive and difficult to observe directly through phenotypes. Finally, in special environments such as high-altitude low-oxygen environments, traditional methods may not be able to quickly and effectively breed more adaptable breeds. There is a need to fully utilize molecular genetic means to improve the hypoxic adaptability of Tibetan chickens. It is difficult to effectively improve the hypoxic adaptability of Tibetan chickens.

[0004] Therefore, the development of a molecular marker related to the hypoxic adaptability of Tibetan chickens and its application not only improves the survival rate and production performance of Tibetan chickens in high-altitude hypoxic environments, but also provides a reference for the breeding of other plateau poultry. SUMMARY

[0005] To address the adaptability challenges and survival issues faced by Tibetan chickens in high-altitude hypoxic environments, the present application explores a breeding method based on the PARK7 gene to improve the adaptability of Tibetan chickens in hypoxic environments.

[0006] To achieve the above object, the application adopts the following technical scheme:

[0007] The first aspect of the application provides a nucleic acid molecular marker for the low-oxygen adaptability selection of Tibetan chickens, the nucleotide sequence of the nucleic acid molecular marker is shown in SEQ ID NO: 1; the position of the single nucleotide M of the nucleic acid molecular marker is located at the G>T mutation of the 264857th nucleotide site of the chromosome 21 of the Gallus gallus domesticus reference genome GRCg6a version 21; the position in the sequence SEQ ID NO: 1 is the 244th position at the 5' end.

[0008] The second aspect of the application provides a primer pair for detecting the above-mentioned nucleic acid molecular marker, the primer pair is used for detecting the nucleic acid molecular marker, and the primer pair comprises an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is shown in SEQ ID NO: 2; and the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 3.

[0009] The third aspect of the application provides a kit for detecting the above-mentioned nucleic acid molecular marker, the kit comprises the above-mentioned primer pair.

[0010] The fourth aspect of the application provides a method for detecting the myocardial cell activity of Tibetan chickens, the method comprises:

[0011] 1) using a nucleic acid molecular marker, the nucleotide sequence of the nucleic acid molecular marker is shown in SEQ ID NO: 1, and the 244th single nucleotide M at the 5' end is G or T;

[0012] 2) detecting the single nucleotide M of the nucleic acid molecular marker to obtain a detection result, and the detection result is used for determining whether the myocardial cell activity of the Tibetan chicken is high or low;

[0013] 3) when the detection result is that the single nucleotide M is G, it is determined that the myocardial cell activity of the Tibetan chicken is low, and the individual of the Tibetan chicken is a GG type individual; and when the detection result is that the single nucleotide M is T, it is determined that the myocardial cell activity of the Tibetan chicken is high, and the individual of the Tibetan chicken is a TT type individual.

[0014] The fifth aspect of the application provides a selection method for chicken groups in a low-oxygen environment, and the selection method specifically comprises:

[0015] 1) detecting the above-mentioned nucleic acid molecular marker on the chromosome 21 of the Tibetan chicken;

[0016] 2) retaining the chicken group individual with the single nucleotide M of the nucleic acid molecular marker being T, and eliminating the chicken group individual with the single nucleotide M being G; and increasing the gene frequency of the single nucleotide M being T generation by generation;

[0017] The position of the single nucleotide M marked by the nucleic acid molecule is 244th position at the 5' end in sequence SEQ ID NO: 1.

[0018] The sixth aspect of the present application provides a selection method for improving the PARK7 gene of the Tibetan chicken individual.

[0019] It should be understood that the above general description and the following detailed description are only exemplary and explanatory, and cannot limit the present application.

[0020] Beneficial effects

[0021] 1、The present application collects whole blood from 188 chickens in continuous altitude areas, extracts DNA for resequencing, filters, aligns, screens, and annotates the resequencing data of these chicken populations in continuous altitude areas to obtain a set of variant SNPs; through genomic resequencing technology and various genetic analysis methods, the PARK7 gene nucleic acid molecule marker related to highland adaptation is identified. Through genetic variation analysis, population genetic structure analysis and forward selection scanning, SNP sites with strong selection signal are obtained, and the pathways and biological functions of gene enrichment are determined through GO and KEGG enrichment analysis. This process improves the screening accuracy of selection signal, and provides solid data support for subsequent experimental verification of functional sites.

[0022] 2、The present application provides a PARK7 gene nucleic acid molecule marker related to the highland adaptability of Tibetan chickens, and designs a primer pair for identifying the marker. Through nucleic acid molecule marker assisted selection, the adaptability of Tibetan chickens in a high-altitude hypoxic environment can be effectively improved, and their survival and production performance can be enhanced. This genetic improvement helps to improve the oxygen transport capacity and antioxidant capacity of Tibetan chickens in highland environments, and enhances their adaptability to low oxygen, strong ultraviolet light and low temperature environments.

[0023] 3、By optimizing the dominant alleles of the PARK7 gene nucleic acid molecule marker, the present application can accelerate the genetic progress of Tibetan chickens, thereby effectively improving the breeding efficiency. By breeding GG type and GT type individuals into GG type individuals, the highland adaptability and physiological stability of Tibetan chickens are enhanced, their physiological balance is maintained, and their survival ability in highland environments is further improved.

[0024] 4、The present application provides a precise nucleic acid molecule marker tool for studying and improving the highland adaptability of Tibetan chickens through genomic resequencing technology and transcriptomics analysis. The nucleic acid molecule marker can accurately locate genetic factors related to the expression amount of the PARK7 gene, reduce the uncertainty in the breeding process, improve the survival and growth ability of Tibetan chickens in highland areas, and thereby improve the economic benefits of Tibetan chicken breeding in highland areas.

[0025] 5、The application provides experimental verification on the PARK7 gene, and observes the protective effect of the PARK7 gene on the myocardial cells of Tibetan chickens under a low-oxygen condition. Through gene knockout and overexpression experiments, the function of the PARK7 gene in the cell antioxidant and protection mechanism is confirmed.

[0026] 6、The application provides a nucleic acid molecular marker related to the expression amount of the PARK7 gene, and more accurately studies the genetic factors related to the expression amount of the PARK7 gene in the highland adaptation of chicken populations through genome resequencing, transcriptome analysis and verification experiments. The nucleic acid molecular marker can solve the following problems:

[0027] 1) Improve accuracy: The nucleic acid molecular marker related to the expression amount of the PARK7 gene in the highland adaptation of chicken populations can more accurately locate the PARK7 gene related to the highland adaptation of chicken populations, instead of relying on randomly selected candidate genes.

[0028] 2) Reduce uncertainty: The nucleic acid molecular marker related to the expression amount of the PARK7 gene in the highland adaptation of chicken populations can reduce uncertainty. Through gene knockout and overexpression experiments, the function of the PARK7 gene in the cell antioxidant and protection mechanism is confirmed. By selecting the advantageous alleles of the nucleic acid molecular marker, the genetic progress of the expression amount of the PARK7 gene in the chicken population can be improved, and the survival and growth ability of the chicken population in the highland area can be improved, thereby improving the economic effect of improving the breeding of Tibetan yellow cattle in high-altitude areas.

[0029] 3) Provide accurate tools: The nucleic acid molecular marker provided by the application is a nucleotide mutation of G244-T244 located at position 244 of the sequence annotation of SEQ ID NO: 1; the single nucleotide M of the nucleic acid molecular marker is located at the 264857th nucleotide site G>T mutation of chromosome 21 of the Gallus gallus domesticus reference genome GRCg6a version, and the position is the 244th nucleotide at the 5' end in the sequence SEQ ID NO: 1. It provides a more accurate genetic tool for improving the survival and growth ability of chicken populations in highland areas, and provides a theoretical basis for improving the survival and growth ability of chicken populations in highland areas.

[0030] It should be understood that the above general description and the following detailed description are only exemplary and explanatory, and cannot limit the application. BRIEF DESCRIPTION OF DRAWINGS

[0031] The above and other objects, features and advantages of the present application will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings, in which like reference characters refer to like parts throughout the several views.

[0032] Figure 1 is the selection signal detection of Tibetan chicken;

[0033] Note: a indicates Fst analysis, b indicates XP-nSL analysis.

[0034] Figure 2 is the map of selection sites detected in chicken population along continuous altitude gradient;

[0035] Note: TBC indicates Tibetan chicken, NX indicates Nixi Tibetan chicken, and LJ indicates Lijiang black-bone chicken.

[0036] Figure 3 is the volcano plot of differentially expressed genes between Tibetan chicken and lowland chicken.

[0037] Figure 4 is the volcano plot of differentially expressed genes between lowland chicken moved to plateau and lowland chicken.

[0038] Figure 5 is the volcano plot of differentially expressed genes between lowland chicken moved to plateau and Tibetan chicken.

[0039] Figure 6 is the volcano plot of differentially expressed genes between Tibetan chicken moved to lowland and lowland chicken.

[0040] Figure 7 is the volcano plot of differentially expressed genes between Tibetan chicken moved to lowland and Tibetan chicken.

[0041] Figure 8 is the number statistics of differentially expressed genes.

[0042] Figure 9 is the gene module associated by WGCNA;

[0043] Note: LC indicates lowland chicken, LCT indicates lowland chicken moved to Qinghai-Tibet Plateau, TC indicates Tibetan chicken, and TCL indicates lowland Tibetan chicken.

[0044] Figure 10 is the correlation scatter plot of GS and MM in Blue module.

[0045] Figure 11 is the correlation scatter plot of GS and MM in Turquoise module.

[0046] Figure 12 is the sequence shown in SEQ ID NO: 1, and the M marked in the figure is the mutation site; the capital letters are primer sequences.

[0047] Figure 13 is the screenshot of primer verification result by UCSC Genome Browser website.

[0048] Figure 14 is the qPCR detection of PARK7 gene expression.

[0049] Figure 15 is the influence of overexpression of the PARK7 gene on the viability of cardiomyocytes.

[0050] Figure 16 is the influence of interference with the PARK7 gene on the viability of cardiomyocytes.

[0051] Figure 17 is the association analysis of the PARK7 gene mutation site genotyping and the viability of cardiomyocytes. DETAILED DESCRIPTION

[0052] Embodiments of the present application will be described in more detail by referring to the attached drawings. Although embodiments of the present application are shown in the drawings, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided so that the present application is more thorough and complete, and the scope of the present application is fully conveyed to those skilled in the art.

[0053] With the rapid development of genomics and molecular biology technology, researchers have begun to focus on the genetic adaptation mechanisms of Tibetan chickens. Through whole genome association analysis and selection signal detection, it was found that the PARK7 gene has significant selection signals in the adaptation of Tibetan chickens to high altitude. The protein encoded by the PARK7 gene plays an important role in cellular antioxidant and protection mechanisms, and can effectively alleviate the damage of low oxygen environment to cardiomyocytes.

[0054] However, the current breeding strategy for Tibetan chickens still lacks in-depth research and application of the function of the PARK7 gene. The present application provides a scientific and effective breeding method for Tibetan chickens by verifying and selecting the function of the PARK7 gene, combined with modern molecular breeding technology, to improve their adaptability and survival rate in the high altitude and low oxygen environment.

[0055] The above-mentioned application objectives of the present application are achieved in the following specific ways:

[0056] Example 1 Screening process of PKPA7 gene

[0057] Experimental animals

[0058] The test animal samples of the genome of the present study were collected along the ancient tea-horse road from Yunnan to Tibet, including red junglefowl (RJF), Banna gamecock (BG) and tea flower chicken (CH) in Yunnan region at an altitude of 500 m; Zhenyuan pheasant (ZY), Zhaotong chicken (ZT), Yimen chicken (YM) and Wuliangshan chicken (WLS) in Yunnan region at an altitude of 1,500 m; Lijiang chicken (LJ) in Yunnan region at an altitude of 2,500 m; Nixi chicken (NX), Lhazê chicken (LZ), Gungar Dê chicken (GB) and Shannan chicken (SN) at an altitude of more than 3,000 m; a total of 12 groups at four altitude gradients, and each group sample was randomly selected from male and female individuals, totaling 188 samples (see Table 1 below for detailed information).

[0059] Table 1 Resequencing sample information

[0060]

[0061] The transcriptome off-site feeding test animal samples were 12-month-old female Tibetan chickens and local chickens from high-altitude Diqing region and low-altitude Yuanjiang region, wherein the high-altitude chicken group had a total of 9 chickens, and the low-altitude chicken group had a total of 10 chickens. Among the sample groups, 3 high-altitude chickens and 5 low-altitude chickens were randomly selected and moved to the low-altitude and high-altitude regions, respectively, for further feeding. The remaining individuals were continued to be locally raised. All 19 test samples were fed under consistent conditions for a total of 5 weeks. After the feeding was completed, all chicken samples were slaughtered and the hearts were separated and cut, and then quickly placed in liquid nitrogen for storage.

[0062] Table 2 Transcriptome sample information

[0063]

[0064] Gene screening

[0065] In order to study the highland adaptation selection signal of Tibetan chicken, the present study used the Fst sliding window method to calculate the selected segments of the Tibetan chicken genome, taking the low-altitude chicken group at 500 m as the reference group. A total of 1860 selected segments were detected in the top 1% Fst window, covering 399 coding genes. Enrichment analysis was performed on the selected coding genes, and it was found that 9 genes (DPP4, EGR1, BECN1, MYOCD, SCFD1, EPAS1, CASP3, CXCR4, KCNA5) were related to hypoxia response, 3 genes (HBZ, HBAD, HBA1) were related to oxygen transport, and there were also genes related to vascular smooth muscle contraction regulation (DOCK5, ARHGAP42, DOCK4), heart rate regulation (IRX5, EPAS1, ANK2, BVES), and blood coagulation regulation (MMRN1, VWF, ALB, HRG, KNG1) (see Table 3). Figure 1a) These selected genes and their biological pathways are closely related to high altitude hypoxia adaptation, and can be further studied.

[0066] To further screen candidate genes, in addition to the Fst based on differentiation selection signal method, this study also uses the XP-nSL method based on linkage disequilibrium detection selection sites, and also sets the threshold value to top 1%. A total of 462 genes were detected, of which 117 genes were also detected by Fst, including the previously mentioned HBAD, ANK2, BVES, ARHGAP42, ZMIZ1, DYNC2H1, PXDN, in addition to the XP-nSL also detected the G>T mutation at nucleotide position 264857 on chromosome 21 of the Gallus gallus domesticus reference genome GRCg6a version 21, which is annotated by the reference genome as the RKPA7 gene related to myocardial contraction (see Figure 1 b)

[0067] In order to detect whether the selection signal of plateau chicken population is affected by altitude environment, four altitude gradient chicken populations were used for detection and analysis, and the XtXst statistical method in BayPass was used to detect the sites selected by the environment, and 249613 variable sites were obtained, including the G>T mutation at nucleotide position 264857 on chromosome 21 of the Gallus gallus domesticus reference genome GRCg6a version 21, which is annotated by the reference genome as the RKPA7 gene related to myocardial contraction. In addition, the intersection of the selected genes of the Nixi chicken and the Tibetan chicken genome was obtained, a total of 716 candidate genes related to high altitude environment were obtained, including not only the strongly selected DYNC2H1, ARHGAP42, HBAD, MMRN1, HRG, GABBR2, PXDN, ZMIZ1 genes in Fst detection, but also newly discovered genes such as GJA5, ASIC2, CACNA2D1, RKPA7 genes that were not detected by the previous selection signal. In addition, this study also found that there were significant differentiation missense mutation sites in the selected genes MMRN1, HBAD and HRG, and the gene frequency changed according to the altitude trend, that is, the mutation gene frequency was the highest in the high altitude population, the mutation frequency decreased in the medium altitude population, and the mutation frequency was the lowest in the low altitude population (see Figure 2 )。

[0068] In order to explore the specific expression genes of Tibetan chicken heart in plateau environment, this study compared and analyzed the difference gene level of local plateau Tibetan chicken and local lowland chicken population, and detected 744 differentially expressed genes, of which 268 were up-regulated genes and 476 were down-regulated genes Figure 3 )。

[0069] According to the sample expression correlation analysis results, the original feeding of Tibetan chicken and lowland chicken was combined as a reference group affected by environmental changes, and two chicken groups affected by different environmental influences were compared and analyzed. The difference gene comparison analysis of lowland chicken moving to plateau group (LCT) and lowland native chicken group obtained 3350 differentially expressed genes, including 1252 up-regulated genes and 2098 down-regulated genes ( Figure 4 ); and the difference gene comparison analysis with the plateau Tibetan chicken group obtained 3758 differentially expressed genes, including 1348 up-regulated genes and 2410 down-regulated genes ( Figure 5 ). The difference gene comparison analysis of Tibetan chicken in lowland group (TCL) and lowland native chicken group screened out 986 differentially expressed genes, including 514 up-regulated genes and 472 down-regulated genes ( Figure 6 ); and 973 differentially expressed genes were found compared with the plateau Tibetan chicken, including 635 up-regulated genes and 338 down-regulated genes ( Figure 7 ). It can be found from the number of differential expression that the gene expression of heart changes greatly under different environments, and the number of differentially expressed genes is less than that of high and low altitude original feeding, which is consistent with the previous sample correlation analysis results.

[0070] It can be found from the Venn diagram that when LCT and the reference chicken group are compared and analyzed, there are 2466 differentially expressed genes, and 409 differentially expressed genes are detected in TCL comparison analysis, and the significant difference in the number of differentially expressed genes also reflects that the high altitude environment has a greater influence on the gene expression regulation of lowland chicken heart than the low altitude environment ( Figure 8 ).

[0071] In this study, the PARK7 gene was chosen as the focus of research based on a comprehensive consideration of multiple factors. Through Fst sliding window method and XP-nSL method to detect highland adaptive selection signal, a number of genes related to hypoxic response, oxygen transport, vasoconstriction, heart rate regulation and blood coagulation regulation were identified, indicating that highland adaptability and hypoxic response are the core adaptive mechanisms of Tibetan chickens in highland environment. Using the XtXst statistical method of BayPass, a large number of environmentally selected sites were detected, and comprehensive comparative analysis showed that the expression of PARK7 gene decreased in lowland chickens after moving to highlands, while no significant change was found in local highland Tibetan chickens, suggesting that PARK7 may play an important role in hypoxic adaptation. In addition, PARK7 gene plays an important role in cellular antioxidant and protection mechanisms, and is closely related to mitochondrial autophagy and oxidative stress biological processes, which are important physiological processes for cells to adapt to highland environment under hypoxic conditions. Finally, combined with the differential expression data, PARK7 gene may play an important role in the hypoxic adaptation mechanism of Tibetan chickens, so it was selected for further functional identification research to reveal its specific contribution to highland adaptability and provide a new direction for breeding and genetic improvement. PARK7 gene is considered an important candidate gene for studying the highland adaptability of Tibetan chickens, and its in-depth study will help to understand the physiological effects of hypoxic environment on Tibetan chickens and their adaptation mechanisms.

[0072] Example 2 Weighted gene co-expression network analysis and enrichment analysis

[0073] In this study, according to the breed and growth altitude environment variables of chicken samples, four groups were divided, namely lowland chicken group (Low chicken, LC), Tibetan chicken group (Tibetan chicken, TC), lowland chicken group in Tibet (Low chicken in Tibet, LCT) and Tibetan chicken group in lowland (Tibetan chicken in lowland, TCL). After getFPKM.py script, FPKM (Fragments Per Kilobase Million) matrix information was obtained from all sample GTF files calculated by StringTie software, which was used to calculate the expression level of each gene and weighted gene co-expression network analysis (Weighed Gene Co-expression Network Analysis, WGCNA).

[0074] The WGCNA package in R software was used for weighted gene co-expression network analysis. First, the gene similarity matrix was obtained by matrix operation, and then the similarity matrix was converted into an adjacency matrix. Based on the principle of approximate scale-free topology, the pickSoftThreshold function was used to perform network topology analysis (RsquaredCut = 0.85) to calculate the appropriate soft threshold for the adjacency matrix to construct the weighted gene network. When detecting co-expression genes, the correlation between genes was calculated using topological overlap to form a topological overlap matrix; then hierarchical clustering was performed to calculate the eigenvector of each module, where the first principal component in each module is called ME. According to the standard of dynamic pruning tree, the minimum number of module genes was set to 30 (minModuleSize = 30), the medium sensitivity (deepSplit = 3) clustering splicing was used to merge modules, and the threshold parameter for merging modules was set to 0.25 (mergeCutHeight = 0.25). Through clustering analysis of modules, modules with close distances were merged into one module, and different colors represented different modules. In each module, the quantitative index MM (module membership) was used to represent the correlation between module characteristic genes and gene expression values, and GS (gene significance) was used to represent the correlation between genes and traits and its biological significance. In this study, |GS| ≥ 0.7 and |MM| ≥ 0.8 were used to screen the target module genes, and the core genes with high weight and high module identity in the target module were identified by GS and MM values.

[0075] At the same time, in order to better explore the physiological function of the module related to high altitude hypoxia, this study combined the differential expression results with the co-expression network analysis results, and used DAVID (version 2022q2) for enrichment analysis.

[0076] According to the correlation comparison analysis, this study focused on the modules with the largest correlation with the LCT group, including the largest positive correlation blue module and the largest negative correlation turquoise module. Figure 9 Among them, the blue module contains 1343 known genes, and the turquoise module contains 4175 known genes. According to the GS and MM setting standard, further screening of core genes related to modules and LCT traits, the blue module obtained 106 core genes Figure 10 , and the turquoise module obtained 1448 core related genes Figure 11 .

[0077] The 2466 differentially expressed genes related to hypoxic environment obtained from previous analysis were intersected in this study, and 102 differentially expressed genes were obtained in the blue module, while 850 differentially expressed genes were obtained in the turquoise module. Pathway enrichment analysis results showed that the differentially expressed genes in the blue module were enriched in biological pathways such as negative regulation of erythrocyte differentiation (KLF13, MAFB), cellular nitrogen compound metabolism process (CPS1, PM20D1), Rho protein signal transduction (ROCK2, C15ORF62, EPS8L3) and the like. In contrast, the key differentially regulated genes in the turquoise module were more likely to be involved in hypoxic response, including regulation of mitochondrial membrane potential (PRDX3, NDUFS1, PARK7, SOD2, SOD1), ventricular cardiomyocyte action potential (PKP2, ANK2, GPD1L, SNTA1), cellular redox homeostasis (BECN1, PCNA, MAP1LC3A, AIFM1, PPIF, PARK7, PAX2, NFE2L2), negative regulation of apoptosis (BECN1, PDE12, CHMP1A, PARK7, ZFPM2, CHMP7, BMP7, FERMT2) and cellular redox homeostasis (PRDX3, TXNRD3, GLRX3, TXNRD2, PRDX1, PRDX6, DLD, NFE2L2) and the like.

[0078] By detecting the highland adaptation selection signals of Tibetan chickens, as well as detecting the unselected genes of lowland chickens, combined with transcriptome differential expression gene and weighted gene co-expression network analysis, not only the highland reaction genes of lowland chickens were explored, but also the highland adaptation genes of Tibetan chickens were explored. In addition to the basic transcriptome research, the enrichment analysis results combined with the selection signals found that the significantly enriched pathways were more detailed, and some important biological process pathways similar to transcriptome analysis were also found, such as regulation of cardiomyocyte action potential, regulation of apoptosis and redox reaction, etc., reflecting that the genes involved in these pathways were the core genes of Tibetan chicken highland adaptation. Therefore, based on the influence of mitochondrial autophagy and oxidative stress on cells, PARK7 gene was finally determined as the research object in the selected site. Transcriptome data found that compared with other treatment groups, the expression of PARK7 gene decreased in LCT group, while the expression in TC group, i.e. local highland Tibetan chicken, did not change significantly, so it was speculated that PARK7 gene would play a role in hypoxic adaptation, and it was also used as a functional identification gene in the later stage.

[0079] Example 3 Functional identification of hypoxic adaptation genes

[0080] Experimental materials

[0081] The gene expression quantitative verification analysis material is the RNA sample of the chicken heart of different treatments successfully extracted and detected in the third chapter of the present research. In addition, the present research collected 30 eggs of Tibetan chicken and tea flower chicken from the experimental chicken farm of China Agricultural University, cleaned and disinfected the eggshells, and placed them in an automatic incubator for incubation. The incubator temperature was set to 37.8°C, the humidity was 60%, and the eggs were turned every 2 hours.

[0082] qPCR primer design and synthesis

[0083] According to the mRNA sequence of chicken PARK7 published on the NCBI website, the sequence contains a nucleic acid molecule marker for regulating the expression amount of PARK7 gene in Tibetan chicken, which is a 979 bp nucleotide sequence on chromosome 21. The nucleotide sequence of the nucleic acid molecule marker is shown in SEQ ID NO: 1 (see Figure 13 ), qPCR primers were designed using Primer Premier 5.0 software, and the upper and lower primer sequences for sequence amplification are as follows:

[0084] Upper primer (SEQ ID NO. 2): 5'-AGCAAAGTCACAACGCATCC-3';

[0085] Lower primer (SEQ ID NO. 3): 5'-GCTTCAACAATGGCCAACCC-3';

[0086] PCR amplification: 10 uL of reaction system was added with 1 uL of DNA template, 3.4 uL of double distilled water, 5 uL of 2x Tag PCR StanMix with Loading Dye, and 0.3 uL of primers P001 and P002. The PCR reaction conditions were as follows: 94°C pre-denaturation for 2 min, 94°C denaturation for 30 s, 55°C annealing for 20 s, 72°C extension for 30 s, 35 cycles, and finally 72°C extension for 10 min.

[0087] Primer verification: In-Silico PCR was performed on the UCSC Genome Browser website (website address: https: / / genome.ucsc.edu / cgi-bin / hgPcr) using SEQ ID NO. 2 and SEQ ID NO. 3 of the present application as primers and Gallus gallus domesticus reference genome GRCg6a version as template. The corresponding PCR product fragments obtained are shown in the following figure, and the obtained PCR product fragments are 979 bp (see Figure 13 ).

[0088] The qPCR results showed that the relative expression of the PARK7 gene was the lowest in the LCT group, which was significantly lower than that in the TC, LC and TCL groups, while there was no significant difference in the relative expression among the TC, LC and TCL groups. This result is consistent with the transcriptome sequencing results of this study, that is, the expression of PARK7 is significantly down-regulated in the LCT group, and PARK7 gene is one of the genes with the highest correlation in the WGCNA analysis, which indicates the importance of PARK7 gene in the adaptation of chicken to high altitude Figure 14

[0089] Example 4 Cell verification experiment

[0090] PARK7 gene mutation site genotyping verification

[0091] In order to verify that the G>T mutation at nucleotide position 264857 of chromosome 21 of Gallus gallus domesticus reference genome GRCg6a version 21, which is annotated by reference genome to affect the activity of chicken embryo myocardial cells through the RARK7 gene related to myocardial contraction, further enhances the adaptability of Tibetan chickens to hypoxic environment.

[0092] PARK7 gene overexpression analysis

[0093] In this study, the PARK7 gene was overexpressed in tea flower chicken (CH) and Tibetan chicken (TBC) embryo myocardial cells under normoxic and hypoxic conditions, respectively, and the tea flower chicken and Tibetan chicken myocardial embryo cells cultured under normoxic conditions were used as controls. CCK8 detection found that the relative cell viability of tea flower chicken myocardial cells after hypoxic culture treatment was significantly lower than that of normoxic culture treatment group, indicating that tea flower chicken myocardial cells under hypoxic stress showed slow cell growth or apoptosis, reflecting that tea flower chicken embryo myocardial cells had high sensitivity to hypoxic stress. After overexpression of the PARK7 gene, the tea flower chicken embryo myocardial cell viability under hypoxic conditions was restored, even slightly higher than that of the normoxic culture group, indicating that the PARK7 gene could offset the adverse reactions under hypoxic conditions and protect chicken embryo myocardial cells from growing under hypoxic conditions Figure 15 ). In addition to tea flower chicken, Tibetan chicken embryo myocardial cells also have similar detection results, except that the myocardial activity of Tibetan chicken under hypoxic conditions does not show a significant downward trend in cell viability, indicating that the adaptability of Tibetan chicken myocardial cells to hypoxic conditions is strong; while overexpressing the PARK7 gene under hypoxic conditions, it was found that the myocardial cells of Tibetan chickens also increased significantly, indicating that the PARK7 gene may have a role in promoting cell growth in addition to protecting myocardial cell viability under hypoxic conditions Figure 16 ).

[0094] PARK7 gene interference analysis

[0095] ​In addition to overexpression test, this study also carried out PARK7 gene interference analysis on CH and TBC chicken embryo myocardial cells under normoxic and hypoxic conditions, and the tea flower chicken and Tibetan chicken embryo myocardial cells cultured under normoxic conditions were also used as controls. CCK8 detection found that tea flower chicken myocardial cells were still sensitive to hypoxic conditions, and the cell viability decreased significantly; when the PARK7 gene of tea flower chicken embryo myocardial cells was interfered by siRNA, it was found that the cell viability further decreased, indicating that the decrease of PARK7 gene expression would greatly interfere with the normal growth of cells. Similarly, the PARK7 gene of Tibetan chicken myocardial cells was interfered by siRNA, and it was found that under hypoxic conditions, the myocardial cell viability of Tibetan chicken showed a downward trend, but the degree of decline was not as obvious as that of tea flower chicken. By interfering the PARK7 gene of Tibetan chicken embryo myocardial cells with siRNA, it was found that the Tibetan chicken embryo myocardial cells which originally showed hypoxic adaptation would still cause the viability of Tibetan chicken embryo myocardial cells to decrease significantly to a certain extent under hypoxic environment, which was basically consistent with the results of tea flower chicken embryo myocardial cells. The results of this study comprehensively indicated that the PARK7 gene played an important role in maintaining the normal growth of chicken embryo myocardial cells under hypoxic conditions, and abnormal gene expression would affect cell viability Figure 17

[0096] Example 5 Correlation between genotyping and myocardial cell viability

[0097] The study further genotyped 342 chickens at the 264857 nucleotide site on chromosome 21, which is located at the 244th nucleotide of the 5' end of SEQ ID NO. 1 Figure 13 ) sequence. At the same time, the study collected chicken myocardial cells for culture and determined the viability (%) of the chicken myocardial cells. Subsequently, the relationship between different genotypes of the molecular marker SNP site g.244G>T and the viability (%) of myocardial cells was analyzed by one-way ANOVA using SPSS software.

[0098] According to Figure 17 and Table 3, the molecular marker SNP site g.244G>T was significantly related to myocardial cell viability (P<0.01). This indicates that this molecular marker has a significant impact on improving the viability of myocardial cells in the process of improving chicken adaptation to high altitude. By selecting this SNP site in the chicken population, the viability of the chicken myocardial cells can be effectively improved, thereby improving the adaptability of the chicken population to high altitude and low oxygen partial pressure environment.

[0099] Table 5 Analysis results of myocardial cell viability (%) of three genotypes

[0100]

[0101] ​In addition, according to Table 3, among the 342 chickens, the number of TT type is 120, the number of GT type is 110, and the number of GG type is 112. The myocardial cell activity of TT type is higher than that of GT type and GG type, indicating that homozygote TT type has a significant effect on improving myocardial cell activity. Therefore, in the breeding process of plateau adaptive chicken population, appropriately increasing myocardial cell activity can help to enhance the survival and growth ability of chicken population in plateau hypoxic environment. Therefore, retaining chicken population with TT genotype can bring better adaptability and economic benefits. In the breeding process, the breeding chicken with GG type and GT type should be gradually eliminated, and the breeding chicken with TT type should be preferentially retained, so as to gradually increase the frequency of allele G at the locus, thereby optimizing the plateau adaptability of chicken population.

[0102] The above has described various embodiments of the present application, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes are obvious to those skilled in the art without departing from the scope and spirit of the described embodiments. The selection of terms used herein is intended to best explain the principles, practical application, or improvement of technology in the market of the embodiments, or to enable other ordinary skilled in the art to understand the embodiments disclosed herein.

Claims

1. A nucleic acid molecular marker for the selective breeding of Tibetan chickens to adapt to hypoxia, characterized in that, The nucleotide sequence of the nucleic acid molecular marker is shown in SEQ ID NO: 1; the single nucleotide M of the nucleic acid molecular marker is located at the G>T mutation site on chromosome 264857 of the reference genome of Gallusgallus domesticuss GRCg6a version; its position is at position 244 at the 5' end in the sequence SEQ ID NO:

1.

2. A primer pair for detecting the nucleic acid molecular marker as described in claim 1, characterized in that, The primer pair includes an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is shown in SEQ ID NO: 2; the nucleotide sequence of the downstream primer is shown in SEQ ID NO:

3.

3. A kit for detecting the nucleic acid molecular marker as described in claim 1, characterized in that, The kit contains the primer pair as described in claim 2.

4. A method for detecting the viability of Tibetan chicken cardiomyocytes, characterized in that, The method includes: 1) A nucleic acid molecular marker is used, wherein the nucleotide sequence of the nucleic acid molecular marker is shown in SEQ ID NO: 1, and the single nucleotide M at position 244 of its 5' end is G or T; 2) The detection result of the single nucleotide M, a nucleic acid molecular marker, is used to determine whether the viability of the Tibetan chicken cardiomyocytes is high or low; 3) When the detection result is that the single nucleotide M is G, it is determined that the cardiomyocytes of the Tibetan chicken have low viability and the individual is a GG type; when the detection result is that the single nucleotide M is T, it is determined that the cardiomyocytes of the Tibetan chicken have high viability and the individual is a TT type.

5. A method for breeding Tibetan chickens under hypoxic conditions, characterized in that, The breeding method specifically includes: 1) Detect the nucleic acid molecular marker as described in claim 1 on chromosome 21 of Tibetan chickens; wherein the single nucleotide M at position 244 of the 5' end is G or T; 2) Retain chicken individuals with the single nucleotide M being T, and cull chicken individuals with the single nucleotide M being G; in order to increase the gene frequency of the single nucleotide M being T generation by generation; The position of the single nucleotide M of the nucleic acid molecular marker is at position 244 at the 5' end in the sequence SEQ ID NO: 1.

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