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 a scientific basis 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] As one of the earliest domesticated birds, chicken is a poultry with extremely high economic value. Its main products include eggs and meat, as well as by-products such as feathers and feces. Eggs are rich in nutrients, and chicken has a lower fat content, more lean meat, and is rich in high-quality protein compared to other meats. Therefore, it is widely used as one of the important food sources for humans.

[0003] 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 are also difficult to identify 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.

[0004] 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

[0005] 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.

[0006] To achieve the above objectives, this application adopts the following technical solutions:

[0007] In a first aspect, the present application provides a nucleic acid molecular marker for the breeding of Tibetan chickens adaptable 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 264857 of chromosome 21 of the Gallusgallus domesticuss reference genome GRCg6a version; its position in the sequence SEQ ID NO: 1 is the 244th at the 5' end.

[0008] 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.

[0009] 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.

[0010] The fourth aspect of the present application provides a method for detecting the viability of Tibetan chicken cardiomyocytes, the method comprising:

[0011] 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 244th single nucleotide M at the 5' end is G or T;

[0012] 2) detecting the single nucleotide M of the nucleic acid molecule marker to obtain a test result, which is used to determine whether the activity of the Tibetan chicken cardiomyocytes is high or low;

[0013] 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 low, 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 high, and the Tibetan chicken individual is a TT type individual.

[0014] The fifth aspect of the present application provides a breeding method for chickens in a hypoxic environment, and the breeding method specifically comprises:

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

[0016] 2) retaining chicken individuals whose single nucleotide M of the nucleic acid molecule marker is T, and eliminating chicken individuals whose single nucleotide M is G, so as to increase the gene frequency of the single nucleotide M being T generation by generation;

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

[0018] In a sixth aspect, the present application provides a breeding method as described above for improving the PARK7 gene in Tibetan chicken individuals.

[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.

[0020] Beneficial Effects

[0021] 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.

[0022] 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.

[0023] 3. By selecting the dominant allele of the PARK7 gene nucleic acid molecule marker, the present invention 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 plateau adaptability and physiological stability of Tibetan chickens are enhanced, their physiological balance is maintained, and their survival ability in the plateau environment is further improved.

[0024] 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.

[0025] 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.

[0026] 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:

[0027] 1) Improve accuracy: Provide a nucleic acid molecule marker related to the expression level of PARK7 gene related to plateau adaptation of chickens, which can more accurately locate the PARK7 gene related to plateau adaptation of chickens, rather than relying solely on randomly selected candidate genes.

[0028] 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.

[0029] 3) Providing accurate tools: The nucleic acid molecular marker provided by the present application is a nucleotide mutation of G244-T244 located at position 244 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 264857 of the reference genome GRCg6a version 21 of Gallusgallus domesticuss; its position in the sequence SEQ ID NO:1 is the 244th 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.

[0030] 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

[0031] 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.

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

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

[0034] Figure 2 It is a map of the selection sites for flock detection in a continuous altitude gradient;

[0035] Note: TBC stands for Tibetan chicken, NX stands for Nepali Tibetan chicken, and LJ stands for Lijiang black-bone chicken.

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

[0037] Figure 4 This is the volcano map of differentially expressed genes between lowland chickens and highland chickens.

[0038] Figure 5 This is a volcano map of differentially expressed genes between lowland chickens and plateau chickens and Tibetan chickens.

[0039] Figure 6 This is the volcano map of differentially expressed genes between Tibetan lowland chicken and lowland chicken.

[0040] Figure 7 This is the volcano map of differentially expressed genes between lowland Tibetan chicken and Tibetan chicken.

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

[0042] Fig. 9 It is the WGCNA associated gene module;

[0043] Note: LC means lowland chicken, LCT means lowland chicken of the Qinghai-Tibet Plateau, TC means Tibetan chicken, and TCL means lowland Tibetan chicken.

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

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

[0046] 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.

[0047] Fig.13 This is a screenshot of the primer verification results on the UCSC Genome Browser website.

[0048] Fig.14 qPCR was used to detect PARK7 gene expression.

[0049] Fig.15 The effect of overexpression of PARK7 gene on cardiomyocyte viability.

[0050] Fig.16 It is the effect of interfering with the PARK7 gene on cardiomyocyte viability.

[0051] Fig.17 It is an analysis of the association between genotyping of PARK7 gene mutation sites and cardiomyocyte viability. DETAILED DESCRIPTION

[0052] 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.

[0053] 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.

[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 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.

[0055] The above-mentioned invention object of the present application is specifically achieved as follows:

[0056] Example 1 Screening process of PKPA7 gene

[0057] Experimental animals

[0058] The experimental animal samples for the genome of this study 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 2500m; Nisi chicken (NX), Linzhi Tibetan chicken (LZ), Gongbujiangda Tibetan chicken (GB) and Shannan Tibetan chicken (SN) at an altitude of more than 3000m; 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 for details).

[0059] Table 1 Resequencing sample information

[0060]

[0061] 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.

[0062] Table 2 Transcriptome sample information

[0063]

[0064] Genetic screening

[0065] In order to study the plateau adaptation selection signal of Tibetan chicken, this study took the 500m low-altitude chicken flock as the reference group and used 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. 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 1a). These selected genes and their biological pathways are closely related to plateau hypoxia adaptation and can be further studied in the future.

[0066] In order to further screen candidate genes, in addition to the Fst method based on differentiation selection signal, this study also used the XP-nSL method based on linkage disequilibrium detection selection site, and the threshold was 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 a G>T mutation at nucleotide position 264857 on 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)

[0067] In order to detect whether the selection signal of plateau chickens is affected by the altitude environment, this study used four chickens with altitude gradient as detection analysis, and used the XtXst statistical method in BayPass to detect the sites selected by the environment, and obtained 249613 variant sites, including the G>T mutation at nucleotide site 264857 of chromosome 21 of the reference genome GRCg6a version of Gallus gallus domesticuss. This site was annotated by the reference genome and was related to the RKPA7 gene of myocardial contraction. In addition, the selected genes of the Nisi chicken and Tibetan chicken genomes were intersected, and a total of 716 candidate genes related to the plateau environment were obtained, including not only the DYNC2H1, ARHGAP42, HBAD, MMRN1, HRG, GABBR2, PXDN, and ZMIZ1 genes that were strongly selected in the Fst test, but also newly discovered genes such as GJA5, ASIC2, CACNA2D1, and RKPA7 genes that were not detected by the previous selection signal. In addition, this study also found that there were significantly differentiated missense mutation sites in the selected genes MMRN1, HBAD and HRG. The gene frequency changed according to the altitude trend, that is, the mutation gene frequency was highest in the high-altitude group, the mutation frequency decreased in the middle-altitude group, and the mutation frequency was lowest in the low-altitude group (see Figure 2 ).

[0068] In order to explore the specific expression genes of Tibetan chicken heart under plateau environment, this study compared and analyzed the differential gene levels between native plateau Tibetan chicken and native lowland chicken. A total of 744 differentially expressed genes were detected, of which 268 were up-regulated genes and 476 were down-regulated genes ( Figure 3 ).

[0069] According to the results of sample expression correlation analysis, this study combined the Tibetan chickens and lowland chickens raised in the original place as the reference group not affected by environmental changes, and compared the differential genes with the two chicken groups affected by the foreign environment. A total of 3350 differentially expressed genes were obtained by comparing the differential genes between the lowland chickens migrating to the plateau group (LCT) and the lowland native chickens, of which 1252 were upregulated genes and 2098 were downregulated genes ( Figure 4 ); A total of 3758 differentially expressed genes were obtained by comparative analysis of differentially expressed genes with those of the plateau Tibetan chicken population, including 1348 up-regulated genes and 2410 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 ); 973 differentially expressed genes were found in comparison with Plateau Tibetan Chicken, of which 635 were up-regulated and 338 were down-regulated ( 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.

[0070] 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 ).

[0071] In this study, the selection of PARK7 gene as the key research object was based on comprehensive considerations from many aspects. The Fst sliding window method and XP-nSL method were used to detect the plateau adaptation selection signal, and multiple genes were identified to be related to hypoxia response, oxygen transport, vasoconstriction, heart rate regulation and coagulation regulation, indicating that plateau adaptation 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 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 protection 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.

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

[0073] In this study, chickens were divided into four groups according to the breed of the sample and the environmental variables of the altitude at which the chickens grew, namely, Low chicken (LC), Tibetan chicken (TC), Low chicken in Tibet (LCT) and Tibetan chicken in lowland (TCL). The FPKM (Fragments Per Kilobase Million) matrix information was obtained from all sample GTF files calculated by StringTie software using the getFPKM.py script, which was used to count the expression level of each gene and perform weighted gene co-expression network analysis (WGCNA).

[0074] In this study, the WGCNA package in R software was used to perform weighted gene co-expression network analysis. First, the gene similarity matrix was obtained through 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), and the appropriate soft threshold was calculated to construct a weighted gene network using the adjacency matrix. When detecting co-expressed genes, the topological overlap was used to calculate the association between genes 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 was called ME. According to the standard of dynamic shear tree, the minimum number of module genes was set to 30 (minModuleSize=30), the medium sensitivity (deepSplit=3) cluster splicing, 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) 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. This study used |GS| ≥ 0.7 and |MM| ≥ 0.8 to screen the target module genes, and identified the core genes with high weight and high module identity in the target module through GS and MM values.

[0075] At the same time, in order to better explore the physiological functions of modules related to plateau 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 greatest correlation with the LCT group, including the blue module with the greatest positive correlation and the turquoise module with the greatest negative correlation ( Fig. 9 ). The blue module contains 1343 known genes, and the turquoise module contains 4175 known genes. According to the GS and MM set standards, the core genes related to the module and LCT traits were further screened, and a total of 106 core genes were obtained for the blue module ( Fig.10 ,) turquoise module obtained a total of 1448 core related genes ( Fig.11 ).

[0077] This study performed an intersection test on the 2466 differentially expressed genes related to hypoxia obtained in the previous analysis. A total of 102 differentially expressed genes were obtained in the blue module, and 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.

[0078] By detecting the selection signal of Tibetan chicken plateau adaptation, and also detecting the genes that are not selected in lowland chickens, combined with 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, 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, this study 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. Therefore, it is speculated that the PARK7 gene will play a role in hypoxia adaptation, and it will also be used as a gene for functional identification in the later stage.

[0079] Example 3 Identification of hypoxia adaptation gene function

[0080] Experimental Materials

[0081] The materials for quantitative verification and analysis of gene expression were the chicken heart RNA samples with different treatments that were successfully extracted and tested in Chapter 3 of this study. In addition, this study collected Tibetan chicken and Camellia chicken breeding eggs from the experimental chicken farm of China Agricultural University, with 30 eggs in each group. After the eggshells were cleaned and disinfected, they were placed in an automatic incubator for incubation. The incubator temperature was set at 37.8℃, 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 of the PARK7 gene in Tibetan chicken, which is a 979 bp nucleotide sequence in chromosome 21. The nucleotide sequence of the nucleic acid molecule marker is shown in SEQ ID NO: 1 (see Fig.13 ), qPCR primers were designed using Primer Premier 5.0 software, and the upstream and downstream primer sequences for sequence amplification were:

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

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

[0086] 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.

[0087] 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 ).

[0088] 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 this study, 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, indicating the importance of the PARK7 gene in the plateau adaptation of chickens ( Fig.14 ).

[0089] Example 4 Cell validation experiment

[0090] Genotyping verification of PARK7 gene mutation sites

[0091] In order to verify the G>T mutation at nucleotide site 264857 on chromosome 21 of the Gallus gallus domesticuss reference genome GRCg6a version, which is annotated by the reference genome with the RARK7 gene related to myocardial contraction to affect the activity of chicken embryo cardiomyocytes, further enhance the Tibetan chicken's ability to adapt to hypoxic environments.

[0092] PARK7 gene overexpression analysis

[0093] In this study, PARK7 gene was overexpressed in Camellia chicken (CH) and Tibetan chicken (TBC) embryonic cardiomyocytes under normoxic and hypoxic conditions, respectively, and normoxic Camellia chicken and Tibetan chicken embryonic cardiomyocytes were used as controls. CCK8 detection found that the relative cell viability of Camellia chicken cardiomyocytes after hypoxic culture 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 embryonic cardiomyocytes are highly sensitive to hypoxic stress. After overexpressing the PARK7 gene, the viability of Camellia chicken embryonic cardiomyocytes was restored under hypoxic conditions, and was even slightly higher than that of the normoxic culture group, indicating that the PARK7 gene can offset the adverse reactions in the hypoxic environment and protect the growth of chicken embryonic cardiomyocytes under hypoxic conditions ( Fig.15 In 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 ).

[0094] PARK7 gene interference analysis

[0095] In addition to the overexpression test, this study also conducted PARK7 gene interference analysis on CH and TBC chicken embryo cardiomyocytes under normoxic and hypoxic conditions, and also used Camellia chicken and Tibetan chicken embryo cardiomyocytes cultured in normoxic conditions as controls. CCK8 detection found that Camellia chicken cardiomyocytes were still sensitive to hypoxic conditions, and cell viability decreased significantly; when the PARK7 gene of Camellia chicken embryo cardiomyocytes was interfered with by siRNA, it was found that cell viability was further reduced, indicating that the reduction of PARK7 gene expression would greatly interfere with the normal growth of cells. Similarly, siRNA interference was performed on the PARK7 gene of Tibetan chicken cardiomyocytes, and it was found that the cardiomyocyte viability of Tibetan chicken showed a downward trend under hypoxic conditions, but the degree of decline was not as obvious as that of Camellia chicken. By performing siRNA interference on the PARK7 gene of Tibetan chicken embryo cardiomyocytes, it was found that the Tibetan chicken embryo cardiomyocytes, which originally showed hypoxia adaptation, could still cause the viability of Tibetan chicken embryo cardiomyocytes to a certain extent to be significantly reduced under the condition of disturbed PARK7 gene expression, which was basically consistent with the results of Camellia chicken embryo cardiomyocytes. The results of this study comprehensively demonstrate that the PARK7 gene plays an important role in maintaining normal growth of chicken embryo cardiomyocytes under hypoxic conditions, and abnormal gene expression will affect cell viability ( Fig.17 ).

[0096] Example 5: Correlation between genotyping verification and cardiomyocyte viability

[0097] The study further conducted genotyping tests on 342 chickens at the 264857 nucleotide site on chromosome 21, which is located at SEQ ID NO.1 ( Fig.13 ) sequence at the 244th nucleotide at the 5' end. At the same time, the study collected chicken cardiomyocytes for culture and measured the viability (%) of the chicken cardiomyocytes. Subsequently, SPSS software was used to perform a one-way ANOVA on the relationship between different genotypes of the molecular marker SNP site g.244G>T and cardiomyocyte viability (%).

[0098] according to Fig.17 The results in Table 3 show that the molecular marker SNP site g.244G>T is significantly correlated with cardiomyocyte viability (P<0.01). This indicates that this molecular marker has a significant effect on the improvement of cardiomyocyte viability in the improved chicken flock during plateau adaptation. Through the auxiliary selection of this SNP site in the chicken flock, the viability of the cardiomyocytes of the chicken flock can be effectively improved, thereby improving the adaptability of the lowland chicken flock in the high-altitude low oxygen partial pressure environment.

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

[0100]

[0101] In addition, according to Table 3, among the 342 chickens, there are 120 TT-type chickens, 110 GT-type chickens, and 112 GG-type chickens. The cardiomyocyte activity of TT-type chickens is higher than that of GT-type and GG-type chickens, indicating that the homozygous TT-type has a significant effect on improving cardiomyocyte activity. Therefore, in the selection and breeding process of plateau-adaptive chickens, appropriately improving cardiomyocyte activity can help enhance the survival and growth ability of chickens in the plateau hypoxic environment. Therefore, chickens with the TT genotype can bring better adaptability and economic benefits. In the breeding process, GG-type and GT-type breeders should be gradually eliminated, and TT-type breeders should be retained first to increase the frequency of the allele G at this site generation by generation, thereby optimizing the plateau adaptability of the chickens.

[0102] 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 264857 of chromosome 21 of the Gallusgallus domesticuss reference genome GRCg6a version; its position in the sequence SEQ ID NO: 1 is the 244th 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 244th 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 test 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 low, 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 high, 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 244th single nucleotide M at the 5' end is G or T; 2) retaining the chickens whose single nucleotide M is T and eliminating the chickens whose single nucleotide M is G, 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 244th 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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