Molecular marker related to hypoxia adaptability of Tibetan chicken and application of molecular marker

Abstract

The invention relates to a molecular marker related to hypoxia adaptability of Tibetan chicken and application of the molecular marker. By analyzing the genetic structure and gene exchange of the Tibetan chicken under different altitude gradients and combining heart transcriptome data, the PARK7 gene shows a stable expression level in the Tibetan chicken and is not influenced by the plateau environment. Therefore, the breeding method for improving the survival rate and adaptability of the Tibetan chicken in the plateau low-oxygen environment is screened out. The method not only enriches the understanding of the hypoxia adaptation mechanism of the Tibetan chicken, but also provides technical support for breed conservation and breeding of the Tibetan chicken.

Description

Technical Field

[0001] The invention belongs to the field of animal genetic breeding, and specifically relates to a molecular marker related to the hypoxia adaptability of Tibetan chickens and its application. The method improves the adaptability of Tibetan chickens in plateau hypoxia environments by molecular genetics. Background Art

[0002] In high-altitude environments, animals face the challenge of hypoxia and need to adapt to the plateau environment through changes in tissues and organs, especially those related to oxygen exchange, transportation and metabolism. Tibetan chickens are small chickens unique to the Qinghai-Tibet Plateau. They have lived in high-altitude, low-oxygen environments for a long time, forming unique physiological and genetic characteristics. The low-oxygen environment in plateau areas poses a severe challenge to the survival and reproduction of poultry. Traditional breeding methods mainly rely on phenotypic selection, but this method has some shortcomings. Phenotypic selection usually only focuses on external characteristics and ignores diversity at the genetic level, which may lead to the loss of genetic diversity and reduce the resistance of chickens to environmental changes and diseases. In addition, phenotypic selection is less efficient, especially when complex traits such as disease resistance or adaptability are involved, and it takes a long time to observe and evaluate individual performance. In addition, phenotypic characteristics are easily affected by environmental factors, which may lead to inaccurate selection results. For example, in high-altitude areas, low-oxygen environments may affect the growth and reproductive performance of chickens, and these changes are not necessarily reflected at the genetic level. Traditional methods also have difficulty identifying recessive traits, and some important traits may be recessive and difficult to directly observe through phenotypes. Finally, in special environments (such as high-altitude hypoxia), traditional methods may not be able to quickly and effectively breed more adaptable varieties. Molecular genetics have not been fully utilized to improve the hypoxia adaptability of Tibetan chickens. It is difficult to effectively improve the hypoxia adaptability of Tibetan chickens.

[0003] Therefore, the development of a molecular marker related to the hypoxia adaptability of Tibetan chickens and its application can not only improve the survival rate and production performance of Tibetan chickens in the plateau hypoxic environment, but also provide a reference for the breeding of other plateau poultry. Summary of the invention

[0004] In order to solve the adaptability challenges and survival problems faced by Tibetan chickens in the plateau hypoxic environment, the present invention explores a breeding method based on the PARK7 gene to improve the adaptability of Tibetan chickens in the hypoxic environment.

[0005] To achieve the above objectives, this application adopts the following technical solutions: In a first aspect, the present application provides a nucleic acid molecular marker for the breeding of Tibetan chickens adapted to hypoxia, the nucleotide sequence of the nucleic acid molecular marker being as 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 at nucleotide position 264837 of chromosome 21 of the Gallusgallus domesticuss reference genome GRCg6a version; its position in the sequence SEQ ID NO: 1 is the 224th at the 5' end.

[0006] The second aspect of the present application provides a primer pair for detecting the above-mentioned nucleic acid molecule marker, the primer pair is used to detect the nucleic acid molecule marker, 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.

[0007] The third aspect of the present application provides a kit for detecting the above-mentioned nucleic acid molecule marker, the kit comprising the above-mentioned primer pair.

[0008] A fourth aspect of the present application provides a breeding method for Tibetan chickens in a hypoxic environment, the breeding method specifically comprising: 1) by detecting the nucleic acid molecular markers on chromosome 21 of the Tibetan chicken; 2) Keep the chickens whose single nucleotide M is G in the nucleic acid molecule marker and eliminate the chickens whose single nucleotide M is T, so as 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 molecule marker is the 224th position at the 5' end in the sequence SEQ ID NO: 1.

[0009] The fifth aspect of the present application provides a breeding method as mentioned above to improve the PARK7 gene in Tibetan chicken individuals.

[0010] It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present application.

[0011] Beneficial Effects 1. This application collects whole blood from 188 chickens in continuous altitude areas, extracts DNA for resequencing, filters, aligns, screens, and annotates the resequencing data of chicken flocks in these continuous altitude areas to obtain a set of variant SNPs; through genome resequencing technology and a variety of genetic analysis methods, the PARK7 gene nucleic acid molecular markers related to plateau adaptation are identified. Through genetic variation analysis, population genetic structure analysis, and positive selection scanning, SNP sites with strong selection signals are obtained, and GO and KEGG enrichment analysis are used to determine the pathways and biological functions of gene enrichment. This process improves the screening accuracy of selection signals and provides solid data support for subsequent experimental verification of functional sites.

[0012] 2. The present invention provides a PARK7 gene nucleic acid molecule marker related to the plateau 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 plateau hypoxia environments 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 plateau environments, and enhance their adaptability to environments such as hypoxia, strong ultraviolet rays and low temperatures.

[0013] 3. By selecting the dominant allele of the PARK7 gene nucleic acid molecule marker, the present invention can improve the genetic progress of Tibetan chickens, thereby effectively improving the breeding efficiency. By breeding GT-type and TT-type individuals into GG-type individuals, the plateau adaptability and physiological stability of Tibetan chickens can be enhanced, their physiological balance can be maintained, and their ability to survive in the plateau environment can be further improved.

[0014] 4. The present invention provides an accurate nucleic acid molecular marker tool for studying and improving the plateau adaptability of Tibetan chickens through genome resequencing technology and transcriptomic analysis. The nucleic acid molecular marker can accurately locate the genetic factors related to the expression of the PARK7 gene, reduce the uncertainty in the breeding process, improve the survival and growth ability of Tibetan chickens in plateau areas, and thus improve the economic benefits of Tibetan chicken farming in plateau areas.

[0015] 5. The present invention provides experimental verification of the PARK7 gene, observing its protective effect on Tibetan chicken cardiomyocytes under hypoxic conditions. Through gene knockout and overexpression experiments, the function of the PARK7 gene in cell anti-oxidation and protection mechanisms is confirmed.

[0016] 6. This application provides a nucleic acid molecular marker related to the expression of PARK7 gene, and uses genome resequencing, transcriptome analysis and verification experiments to more accurately study the genetic factors of PARK7 gene expression related to plateau adaptation of chickens. This nucleic acid molecular marker can solve the following problems: 1) Improved accuracy: A nucleic acid molecular marker related to the expression level of PARK7 gene related to plateau adaptation of chickens is provided, which can more accurately locate the PARK7 gene related to plateau adaptation of chickens, rather than relying solely on randomly selected candidate genes.

[0017] 2) Reduce uncertainty: A nucleic acid molecular marker related to the expression of PARK7 gene related to the plateau adaptation of chickens is provided to reduce uncertainty. Through gene knockout and overexpression experiments, the function of PARK7 gene in cellular antioxidant and protection mechanism is confirmed. By optimizing the dominant allele of the nucleic acid molecular marker, the genetic progress of PARK7 gene expression in chickens can be improved, and the survival and growth ability of chickens in plateau areas can be improved, thereby improving the economic effect of improved Tibetan cattle breeding in high-altitude areas.

[0018] 3) Providing accurate tools: The nucleic acid molecular marker provided by the present application is a nucleotide mutation of G224-T224 located at position 224 of the sequence annotation of SEQ ID NO: 1; the position of the single nucleotide M of the nucleic acid molecular marker is located at the nucleotide position G>T mutation of chromosome 264837 of the reference genome GRCg6a version 21 of Gallus gallus domesticuss; its position is in the sequence SEQ ID NO: 1, which is the 224th nucleotide at the 5' end. It will provide more accurate genetic tools for improving the survival and growth ability of chickens in plateau areas, and provide a theoretical basis for improving the survival and growth ability of chickens in plateau areas.

[0019] It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The above and other objects, features and advantages of the present application will become more apparent by describing in more detail the exemplary embodiments of the present application in conjunction with the accompanying drawings, wherein the same reference numerals generally represent the same components in the exemplary embodiments of the present application.

[0021] Figure 1 It is the selection signal detection of Tibetan chicken; a represents Fst analysis, and b represents XP-nSL analysis.

[0022] Figure 2 This is a map of the selection sites for detecting chickens at a continuous altitude gradient; TBC represents Tibetan chicken, NX represents Nishi Tibetan chicken, and LJ represents Lijiang black-bone chicken.

[0023] Figure 3 This is the volcano map of differentially expressed genes between Tibetan chicken and lowland chicken.

[0024] Figure 4 This is the volcano map of differentially expressed genes between lowland chickens and highland chickens.

[0025] Figure 5 This is a volcano map of differentially expressed genes between lowland chickens and plateau chickens and Tibetan chickens.

[0026] Figure 6This is the volcano map of differentially expressed genes between Tibetan lowland chicken and lowland chicken.

[0027] Figure 7 This is the volcano map of differentially expressed genes between lowland Tibetan chicken and Tibetan chicken.

[0028] Figure 8 is the statistical number of differentially expressed genes.

[0029] Fig. 9 It is the WGCNA association gene module; LC represents lowland chicken, LCT represents lowland chicken in the Qinghai-Tibet Plateau, TC represents Tibetan chicken, and TCL represents lowland Tibetan chicken.

[0030] Fig.10 It is the correlation scatter plot of GS and MM in the Blue module.

[0031] Fig.11 It is the correlation scatter plot of GS and MM in Turquoise module.

[0032] Fig.12 It is the sequence shown in SEQ ID NO: 1, the M marked in the figure is the mutation site; the uppercase is the primer sequence.

[0033] Fig.13 This is a screenshot of the primer verification results on the UCSC Genome Browser website.

[0034] Fig.14 qPCR was used to detect PARK7 gene expression.

[0035] Fig.15 The effect of overexpression of PARK7 gene on cardiomyocyte viability.

[0036] Fig.16 It is to interfere with the effect of PARK7 gene on cardiomyocyte vitality. DETAILED DESCRIPTION

[0037] The embodiments of the present application will be described in more detail below with reference to the accompanying drawings. Although the embodiments of the present application are shown in the accompanying drawings, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to make the present application more thorough and complete, and to fully convey the scope of the present application to those skilled in the art.

[0038] With the rapid development of genomics and molecular biology technologies, researchers have begun to focus on the genetic adaptation mechanism of Tibetan chickens. Through genome-wide association analysis and selection signal detection, it was found that the PARK7 gene has a significant selection signal in the plateau adaptability of Tibetan chickens. The protein encoded by the PARK7 gene plays an important role in the cell's antioxidant and protective mechanisms, and can effectively alleviate the damage of hypoxic environments to myocardial cells.

[0039] However, the current breeding strategy for Tibetan chickens still lacks in-depth research and application of the function of the PARK7 gene. The present invention provides a scientific and effective Tibetan chicken breeding method by verifying and selecting the function of the PARK7 gene and combining modern molecular breeding technology to improve its adaptability and survival rate in the plateau hypoxic environment.

[0040] The above-mentioned invention object of the present application is specifically achieved as follows: Example 1 Screening process of PKPA7 gene Experimental animals The experimental animal samples of the genome of the present invention were collected along the Yunnan-Tibet Tea-Horse Road, including red jungle fowl (RJF), Banna fighting chicken (BG) and Camellia chicken (CH) in Yunnan at an altitude of 500m; Zhenyuan chicken (ZY), Zhaotong chicken (ZT), Yimen chicken (YM) and Wuliangshan black-bone chicken (WLS) in Yunnan at an altitude of 1,500m; Lijiang black-bone chicken (LJ) in Yunnan at an altitude of 2,500m; Nisi chicken (NX), Linzhi Tibetan chicken (LZ), Gongbujiangda Tibetan chicken (GB) and Shannan Tibetan chicken (SN) at an altitude of more than 3,000m; a total of 12 populations in four altitude gradients, and male and female individuals were randomly selected from each population sample, totaling 188 samples (see Table 1 below for details).

[0041] The animal samples of transcriptome ex situ feeding experiments were 12-month-old female Tibetan chickens and native chickens of similar weight, selected from the high-altitude Diqing area and the low-altitude Yuanjiang area, including 9 high-altitude chickens and 10 low-altitude chickens. Three high-altitude chickens and five low-altitude chickens were randomly selected from the sample population and moved to low-altitude and high-altitude areas for continued feeding, and the remaining individuals continued to be raised locally. The feeding conditions of all 19 test samples were kept consistent and were raised for a total of 5 weeks. After the feeding, all chicken samples were slaughtered and the hearts were cut and separated, and quickly placed in liquid nitrogen for preservation.

[0042] Table 1 Resequencing sample information Table 2 Transcriptome sample information Genetic screening In order to study the selection signals of plateau adaptation of Tibetan chickens, the present invention uses the 500m low-altitude chicken flock as the reference group, and uses the Fst sliding window method to calculate the selected segments of the Tibetan chicken genome. A total of 1860 selected segments were detected in the Top1%Fst window, covering 399 coding genes. The selected coding genes were enriched and analyzed, and it was found that 9 genes (DPP4, EGR1, BECN1, MYOCD, SCFD1, EPAS1, CASP3, CXCR4, KCNA5) were related to hypoxic 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 coagulation regulation (MMRN1, VWF, ALB, HRG, KNG1) (see Figure 1 These selected genes and their biological pathways are closely related to plateau hypoxia adaptation and can be further studied in the future.

[0043] In order to further screen candidate genes, in addition to the Fst method based on differentiation selection signal, the present invention also uses the XP-nSL method based on linkage disequilibrium detection selection site, and the threshold is also set to top1%. As a result, a total of 462 genes were detected, of which 117 genes were also detected by Fst, including the aforementioned HBAD, ANK2, BVES, ARHGAP42, ZMIZ1, DYNC2H1, PXDN, and XP-nSL also detected the G>T mutation at the nucleotide position 264837 of chromosome 21 of the reference genome GRCg6a version of Gallus gallus domesticuss, which was annotated by the reference genome and was related to the RKPA7 gene related to myocardial contraction (see Figure 1 b) In order to detect whether the selection signal of plateau chickens is affected by the altitude environment, the present invention uses four altitude gradient chickens as detection analysis, uses the XtXst statistical method in BayPass to detect the sites selected by the environment, and obtains 249613 variable sites, including the G>T mutation at the nucleotide site 264837 of the 21st chromosome of the reference genome GRCg6a version of Gallus gallus domesticuss, and the RKPA7 gene related to myocardial contraction is annotated by the reference genome. In addition, the selected genes of the Nisi chicken and the Tibetan chicken genomes are intersected, and a total of 716 candidate genes related to the plateau environment are obtained, including not only the DYNC2H1, ARHGAP42, HBAD, MMRN1, HRG, GABBR2, PXDN, and ZMIZ1 genes that are strongly selected in the Fst detection, but also newly discovered genes such as GJA5, ASIC2, CACNA2D1, and RKPA7 genes that have not been detected by the previous selection signal. In addition, the present invention also found that there were significantly differentiated 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 group, the mutation frequency decreased in the middle-altitude group, and the mutation frequency was the lowest in the low-altitude group (see Figure 2 ).

[0044] In order to explore the specific expression genes of Tibetan chicken hearts under plateau environment, the present invention compared and analyzed the differential gene levels of native plateau Tibetan chickens and native lowland chickens, and detected 744 differentially expressed genes, of which 268 were up-regulated genes and 476 were down-regulated genes ( Figure 3 ).

[0045] According to the results of sample expression correlation analysis, the present invention combined the Tibetan chickens and lowland chickens raised in situ as reference groups not affected by environmental changes, and compared the differentially expressed genes with the two chicken groups affected by the foreign environment. A total of 3350 differentially expressed genes were obtained by comparing the differentially expressed genes between the lowland chickens migrating to the plateau group (LCT) and the lowland native chickens, including 1252 upregulated genes and 2098 downregulated genes ( Figure 4 ); A total of 3758 differentially expressed genes were obtained by comparative analysis of differentially expressed genes with the plateau Tibetan chicken population, of which 1348 were up-regulated genes and 2410 were down-regulated genes ( Figure 5 ). A comparative analysis of differentially expressed genes between the Tibetan chicken lowland group (TCL) and the lowland native chicken group was performed, and 986 differentially expressed genes were screened out, of which 514 were up-regulated genes and 472 were down-regulated genes ( Figure 6 ); Compared with Plateau Tibetan Chicken, 973 differentially expressed genes were found, of which 635 were up-regulated genes and 338 were down-regulated genes ( Figure 7). Through the number of differentially expressed genes, it can be found that the gene expression of the heart under ex situ rearing changes greatly, and the number of differentially expressed genes under high and low altitude rearing is less, which is consistent with the results of the previous sample correlation analysis.

[0046] The Venn diagram showed that when LCT and reference chicken groups were compared, there were 2466 differentially expressed genes, while TCL comparison analysis detected 409 differentially expressed genes. The obvious difference in the number of differentially expressed genes also reflects the relatively low altitude environment. The high altitude environment has a greater regulatory level on the expression of cardiac genes in lowland chickens ( Figure 8 ).

[0047] In the present invention, the selection of PARK7 gene as the key research object is based on comprehensive considerations from many aspects. The plateau adaptation selection signal was detected by the Fst sliding window method and the XP-nSL method, and multiple genes were identified to be related to hypoxia response, oxygen transport, vasoconstriction, heart rate regulation and coagulation regulation, indicating that plateau adaptability and hypoxia response are the core adaptation mechanisms of Tibetan chickens in the plateau environment. A large number of sites selected by the environment were detected using the XtXst statistical method of BayPass. Comprehensive comparative analysis showed that the expression level of the PARK7 gene decreased after low-altitude chickens migrated to the plateau, while no obvious changes were found in local plateau Tibetan chickens, suggesting that PARK7 may play an important role in hypoxia adaptation. In addition, the PARK7 gene plays an important role in cellular antioxidant and protective mechanisms, and is closely related to the biological processes of mitochondrial autophagy and oxidative stress, which are important physiological processes for cells to adapt to the plateau environment under hypoxic conditions. Finally, combined with the differential expression data, the PARK7 gene may play an important role in the hypoxia adaptation mechanism of Tibetan chickens. Therefore, the PARK7 gene was selected for subsequent functional identification research, aiming to reveal its specific contribution to plateau adaptability and provide new directions for breeding and genetic improvement. The PARK7 gene is considered to be an important candidate gene for studying the plateau adaptability of Tibetan chickens. Its in-depth study will help understand the physiological effects of hypoxia on Tibetan chickens and their adaptation mechanisms.

[0048] Example 2 Weighted gene co-expression network analysis and enrichment analysis According to the breed of chicken samples and the environmental variables of the altitude at which the chickens grow, the chickens are divided into four groups, namely, the lowland chicken group (LC), the Tibetan chicken group (TC), the lowland chicken migrating to the plateau group (LCT) and the Tibetan chicken migrating to the lowland group (TCL). The FPKM (Fragments Per Kilobase Million) matrix information is obtained from all sample GTF files calculated by the StringTie software through the getFPKM.py script, which is used to count the expression level of each gene and the weighted gene co-expression network analysis (WGCNA).

[0049] The present invention uses the WGCNA package in the R software to perform weighted gene co-expression network analysis. First, a gene similarity matrix is ​​obtained by matrix operation, and then the similarity matrix is ​​converted into an adjacency matrix. Based on the principle of approximate scale-free topology, the pickSoftThreshold function is used to perform network topology analysis (RsquaredCut = 0.85), and an appropriate soft threshold is calculated to construct a weighted gene network for the adjacency matrix. When detecting co-expressed genes, the correlation between genes is calculated using topological overlap to form a topological overlap matrix; hierarchical clustering is then performed to calculate the eigenvector of each module, wherein the first principal component in each module is referred to as ME. According to the standard of the dynamic shear tree, the minimum number of module genes is set to 30 (minModuleSize = 30), the medium sensitivity (deepSplit = 3) cluster splicing, and the threshold parameter for merging modules is set to 0.25 (mergeCutHeight = 0.25). Through cluster analysis of modules, modules with close distances are merged into one module, and different colors represent different modules. In each module, the quantitative index MM (module membership) is used to represent the correlation between the module characteristic genes and the expression values ​​of each gene, and GS (gene significance) represents the correlation between genes and traits and their biological significance. The present invention uses |GS|≥0.7 and |MM|≥0.8 to screen the target module genes, and identifies the core genes with high weight and high module identity in the target module through GS and MM values.

[0050] At the same time, in order to better explore the physiological functions of modules related to plateau hypoxia, the present invention combined the differential expression results with the co-expression network analysis results and used DAVID (version 2022q2) for enrichment analysis.

[0051] According to the correlation comparison analysis, the present invention focuses on the modules with the greatest correlation with the LCT group, including the largest positively correlated blue module and the largest negatively correlated turquoise module ( Fig. 9 The blue module contains 1343 known genes, and the turquoise module contains 4175 known genes. According to the GS and MM standards, the core genes related to the modules and LCT traits were further screened, and a total of 106 core genes were obtained for the blue module ( Fig.10 ), a total of 1448 core related genes were obtained in the turquoise module ( Fig.11 ).

[0052] The present invention performed intersection detection on the 2466 differentially expressed genes related to hypoxia obtained in the previous analysis, and a total of 102 differentially expressed genes were obtained in the blue module, while a total of 850 differentially expressed genes were obtained in the turquoise module. The results of pathway enrichment analysis showed that the biological pathways enriched by the differentially expressed genes in the blue module included pathways such as negative regulation of erythrocyte differentiation (KLF13, MAFB), cellular nitrogen compound metabolism (CPS1, PM20D1), and Rho protein signal transduction (ROCK2, C15ORF62, EPS8L3). In contrast, biological pathways enriched with key differentially regulated genes in the turquoise module may be more 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), etc.

[0053] By detecting the selection signal of Tibetan chicken plateau adaptation, and also detecting the genes that are not selected in lowland chickens, combined with the transcriptome differentially expressed genes and weighted gene co-expression network analysis, not only the plateau response genes of lowland chickens are explored, but also the plateau adaptation genes of Tibetan chickens. In addition to the basic transcriptome research, the enrichment analysis results combined with the selection signal found that the significantly enriched pathways are more detailed, and some important biological process pathways similar to the transcriptome analysis were also found, such as regulating the action potential of myocardial cells, regulating apoptosis, and redox reactions, etc., reflecting that the genes involved in these pathways are the core genes of Tibetan chicken plateau adaptation. Therefore, based on the effects of mitochondrial autophagy and oxidative stress on cells, the present invention finally identified the PARK7 gene in the selected sites as a subsequent research object. The transcriptome data found that compared with other treatment groups, the expression of the PARK7 gene decreased in the LCT group, while in the TC group, that is, the expression of the local plateau Tibetan chicken did not change significantly, so it is speculated that the PARK7 gene will play a role in hypoxia adaptation, and later it is also a gene for functional identification.

[0054] Example 3 Identification of hypoxia adaptation gene function Experimental Materials The gene expression quantitative verification analysis material is the chicken heart RNA samples with different treatments that were successfully extracted and tested in Chapter 3 of the present invention. In addition, the present invention collects Tibetan chicken and Camellia chicken breeding eggs from the experimental chicken farm of China Agricultural University, 30 eggs in each group, and the eggshells are cleaned and disinfected and placed in an automatic incubator for incubation. The incubator temperature is set to 37.8°C, the humidity is 60%, and the eggs are turned every 2 hours.

[0055] qPCR primer design and synthesis According to the mRNA sequence of chicken PARK7 published on the NCBI website, the sequence contains a nucleic acid molecular marker for regulating the expression of the PARK7 gene in Tibetan chicken, which is a 939 bp nucleotide sequence in chromosome 21. The nucleotide sequence of the nucleic acid molecular marker is shown in SEQ ID NO: 1 (see Fig.12 , the uppercase in the figure is the primer information, the total length is 979bp), qPCR primers were designed using Primer Premier5.0 software, and the upstream and downstream primer sequences for sequence amplification are: Upstream primer (SEQ ID NO. 2): 5′-AGCAAAGTCACAACGCATCC-3′; Downstream primer (SEQ ID NO. 3): 5′-GCTTCAACAATGGCCAACCC-3′; PCR amplification: 1uL DNA template, 3.4uL double distilled water, 5uL 2×Tag PCR StanMixwith Loading Dye, 0.3ul each of primers P001 and P002 were added to the 10uL reaction system. The PCR reaction conditions were: 94℃ pre-denaturation for 2min, 94℃ denaturation for 30s, 55℃ annealing for 20s, 72℃ extension for 30s, 35 cycles, and finally 72℃ extension for 10min.

[0056] Primer verification: In-Silico PCR was performed on the UCSC Genome Browser website (see 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 domesticuss reference genome GRCg6a version as template. The corresponding PCR product fragment was amplified as shown in the figure below. The obtained PCR product fragment was 979 bp (see Fig.13 ).

[0057] The qPCR results showed that the relative expression of PARK7 gene in the LCT group was the lowest, significantly lower than that in the TC, LC and TCL groups, while there was no significant difference in the relative expression between the TC, LC and TCL groups. This result is consistent with the transcriptome sequencing results of the present invention, that is, in the LCT group, the expression of PARK7 was significantly downregulated, and the PARK7 gene was one of the genes with the highest correlation module in the WGCNA analysis, which shows the importance of the PARK7 gene in the plateau adaptation of chickens ( Fig.14 ).

[0058] Example 4 Confirmatory Experiment PARK7 gene overexpression analysis The present invention overexpresses the PARK7 gene in Camellia chicken (CH) and Tibetan chicken (TBC) chicken embryo cardiomyocytes under normoxic and hypoxic conditions, respectively, and uses Camellia chicken and Tibetan chicken cardiomyocyte embryo cell groups cultured in normoxic conditions as controls. CCK8 detection found that after Camellia chicken cardiomyocytes were treated with hypoxia, their relative cell viability was significantly lower than that of the normoxic culture treatment group, indicating that Camellia chicken cardiomyocytes showed slow cell growth or apoptosis under hypoxic stress, reflecting that Camellia chicken embryo cardiomyocytes have a high sensitivity to hypoxic stress. After overexpressing the PARK7 gene, the viability of Camellia chicken embryo cardiomyocytes was restored under hypoxic conditions, and was even slightly higher than that of the normoxic culture group, indicating that the PCRK7 gene can offset the adverse reactions in the hypoxic environment and protect the growth of chicken embryo cardiomyocytes under hypoxic conditions ( Fig.15In addition to Camellia chicken, the chicken embryo cardiomyocytes of Tibetan chicken also had similar test results. The difference was that the cardiomyocytes of Tibetan chicken did not show a significant downward trend in cell viability under hypoxic conditions, indicating that the cardiomyocytes of Tibetan chicken had strong adaptability to hypoxic conditions. After overexpressing the PARK7 gene under hypoxia, the detection found that the cardiomyocytes of Tibetan chicken also increased significantly, indicating that the PARK7 gene may not only protect the cardiomyocytes viability under hypoxic conditions, but also promote cell growth ( Fig.16 ).

[0059] PARK7 gene interference analysis In addition to the overexpression test, the present invention also performs PARK7 gene interference analysis on CH and TBC chicken embryo cardiomyocytes under normoxic and hypoxic conditions, respectively, and also uses Camellia chicken and Tibetan chicken embryo cardiomyocytes cultured in normoxic conditions as controls. CCK8 detection found that Camellia chicken cardiomyocytes are still sensitive to hypoxic conditions, and cell viability is significantly reduced; when the PARK7 gene of Camellia chicken embryo cardiomyocytes is interfered with by siRNA, it is found that cell viability is further reduced, indicating that the reduction in PARK7 gene expression will greatly interfere with the normal growth of cells. Similarly, the PARK7 gene of Tibetan chicken cardiomyocytes is interfered with by siRNA, and it is found that the cardiomyocyte viability of Tibetan chickens shows a downward trend under hypoxic conditions, but the degree of decline is not as obvious as that of Camellia chickens. By performing siRNA interference on the PARK7 gene of Tibetan chicken embryo cardiomyocytes, it is found that the Tibetan chicken embryo cardiomyocytes that originally showed hypoxia adaptation, when the PARK7 gene expression is disturbed, the hypoxic environment can still cause the viability of Tibetan chicken embryo cardiomyocytes to be significantly reduced to a certain extent, which is basically consistent with the results of Camellia chicken embryo cardiomyocytes. The results of the present invention comprehensively illustrate that the PARK7 gene has an important function in maintaining normal growth of chicken embryo cardiomyocytes under hypoxic conditions, and abnormal gene expression will affect cell viability.

[0060] The embodiments of the present application have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those of ordinary skill 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 of the embodiments, practical applications, or improvements to the technology in the market, or to enable other persons of ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. A nucleic acid molecular marker for breeding Tibetan chickens with hypoxia adaptability, characterized in that: The nucleotide sequence of the nucleic acid molecule marker is shown in SEQ ID NO: 1; the position of the single nucleotide M of the nucleic acid molecule marker is located at the G>T mutation at nucleotide position 264837 of chromosome 21 of the Gallusgallus domesticuss reference genome GRCg6a version; its position in the sequence SEQ ID NO: 1 is the 224th at the 5' end.

2. A primer pair for detecting the nucleic acid molecule marker according to 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 molecule marker according to claim 1, characterized in that: The kit comprises the primer pair according to claim 2.

4. A method for detecting the activity of Tibetan chicken cardiomyocytes, characterized in that: The method comprises: 1) using a nucleic acid molecule marker, wherein the nucleotide sequence of the nucleic acid molecule marker is as shown in SEQ ID NO: 1, and the 224th single nucleotide M at the 5' end is G or T; 2) Detecting the single nucleotide M of the nucleic acid molecule marker to obtain a detection result, which is used to determine whether the activity of the Tibetan chicken cardiomyocytes is high or low; 3) When the detection result shows that the single nucleotide M is G, it is determined that the cardiomyocyte activity of the Tibetan chicken is relatively high, and the Tibetan chicken individual is a GG type individual; when the detection result shows that the single nucleotide M is T, it is determined that the cardiomyocyte activity of the Tibetan chicken is relatively low, and the Tibetan chicken individual is a TT type individual.

5. A method for breeding Tibetan chickens in a hypoxic environment, characterized in that: The breeding method specifically comprises: 1) Detecting the nucleic acid molecular marker as claimed in claim 1 on chromosome 21 of Tibetan chicken; wherein the 224th single nucleotide M at the 5' end is G or T; 2) retaining the chickens whose single nucleotide M is G and eliminating the chickens whose single nucleotide M is T, so as to increase the gene frequency of the single nucleotide M being G generation by generation; The position of the single nucleotide M of the nucleic acid molecule marker is the 224th position at the 5' end in the sequence SEQ ID NO:

1.

6. Use of the breeding method according to claim 5 in increasing the expression level of the PARK7 gene in Tibetan chicken individuals.

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