Goose molecular identity card construction method based on SNP (Single Nucleotide Polymorphism) molecular marker

Abstract

The invention provides a Taizhou goose germplasm analysis and identification method based on whole genome re-sequencing. The Taizhou goose germplasm analysis and identification method is used for accurately identifying and identifying different varieties of geese. Rapid identification and identity traceability of local goose germplasm are realized by constructing a local purebred goose germplasm reference library and combining with a lineage analysis and molecular identity card technology.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular markers, in particular to a goose molecular identity card construction method based on SNP molecular markers. BACKGROUND

[0002] Taizhou goose is a local excellent livestock and poultry resource in Taizhou City with both meat and egg functions. The body weight of Taizhou goose can reach 4.0 kg at 10 weeks of age, and the meat quality is excellent. The annual egg production of a female goose at first laying can reach 60-65 eggs, which is higher than that of most other large and medium-sized goose breeds at home and abroad. At present, morphological, biochemical marker and microsatellite marker methods are mainly used for goose breed identification and individual identification. However, these methods have certain limitations, such as morphological markers being easily affected by the environment, and the genetic background of biochemical markers and microsatellite markers being unclear. With the development of molecular biology technology, individual identification technology based on DNA molecular markers has been gradually applied in the field of animal identification. SNP, as a kind of efficient DNA molecular marker, has broad application prospects.

[0003] SNP (Single Nucleotide Polymorphism) is a common molecular marker widely used in genomics and genetics research. SNP molecular markers have the advantages of high density, high repeatability, high genetic stability and easy detection, and therefore have wide application value in animal breed identification, individual identification, genetic disease diagnosis and other aspects. Compared with other identification methods, SNP molecular markers have higher polymorphism and stronger anti-interference ability, and therefore are more suitable for goose breed identification and individual identification. By using SNP molecular markers, a molecular identity card of goose can be constructed to realize individual identification and breed identification of goose. This method has the advantages of simplicity, rapidness, accuracy and economy, and can be applied not only to individual identification and breed identification in livestock production, but also to ecological protection, wildlife protection and endangered species protection.

[0004] In summary, the present application provides a goose molecular identity card construction method based on SNP molecular markers, which has wide application prospects and market prospects. SUMMARY

[0005] The present application aims to provide a Taizhou goose molecular identity card identification method based on whole genome resequencing, which can accurately identify and identify different breeds of geese.

[0006] The present application provides a Taizhou goose molecular identity identification method based on whole genome resequencing, which comprises the following steps: Step 1: Sample collection and preparation (1) Collect 30-50 blood samples of each of 10 goose breeds including Taizhou goose, half male and half female; (2) Number the samples and record the phenotypic data and other related data; Step 2: Whole genome resequencing and data processing (1) Extract the DNA of blood samples of each sample, construct a sequencing library, and perform second-generation high-throughput genome resequencing; (2) Mark and sample quality control the data obtained by sequencing, and retain sites with higher quality; (3) Count the remaining samples and SNP sites after quality control; Step 3: Construction of purebred local goose germplasm reference library (1) Select 80% of the samples from the data after quality control for the construction of the purebred goose germplasm reference library; (2) The remaining 20% of the samples are used as test samples, and two purebred samples selected from the screening are used as positive control samples for each breed; Step 4: Pedigree analysis and population differentiation index analysis (1) Using the constructed purebred goose germplasm reference library, the pedigree of the test samples is analyzed; (2) PCA analysis is performed to observe the hierarchical situation of each population; (3) Calculate the FST value and select different numbers of site sets; Step 5: Taizhou goose molecular identity identification site screening and testing (1) Based on the reference library samples and the remaining sites after quality control, the population differentiation index analysis between 10 breeds is performed; (2) The SNP site set with the highest FST value is selected to determine the selection of 10K sites as the Taizhou goose molecular identity marker; (3) Use online tools to interconnect two-dimensional codes for quick inquiry; Preferably, the 10 goose breeds in step 1 include Taizhou goose, Taihu goose, Wanxi white goose, Guangfeng white-feather goose, Huayan goose, Magan goose, Sichuan white goose, Xupu goose, Youjiang goose and Zhedong white goose.

[0007] Preferably, in step 2, the nucleic acid extraction kit is used to extract the DNA of the blood samples of each sample through centrifugation, precipitation, washing, drying and dissolution. The extracted blood sample DNA is subjected to end repair, adapter ligation, PCR amplification and other steps to construct a sequencing library that can be used for second-generation high-throughput genome resequencing. Sequencing is performed through a high-throughput sequencing platform to obtain genomic resequencing data for each sample. Quality control is performed to remove low-quality sites. The data after quality control processing is subjected to sample and SNP site statistics.

[0008] Preferably, in step 3, 80% of the samples are screened from the samples subjected to the forensic treatment for constructing the reference library, and the remaining 20% of the samples are used as test samples, and 2 screened purebred samples of each breed are selected as positive control samples. This construction method can determine the germplasm characteristics and genetic background of purebred geese.

[0009] Preferably, in step 4, the pedigree analysis of the test samples is performed by using the constructed purebred goose germplasm reference library, PCA analysis is performed, high-dimensional data is converted into low-dimensional data by dimensionality reduction processing of the genotype data, the hierarchical situation of each population is observed, and different numbers of locus sets are screened according to the FST value. The pedigree analysis and population differentiation analysis can be effectively performed, and the genetic relationship and differentiation degree between different breeds or populations can be understood.

[0010] Preferably, in step 5, the population differentiation index between 10 breeds is calculated by statistically analyzing the reference library samples and the remaining loci after quality control. By screening the SNP locus set with the highest FST value, the most representative locus can be selected as the Taizhou goose molecular ID marker. Selecting 10K loci can ensure the accuracy and reliability of the identification result. By using the online tool interconnection two-dimensional code, the identification result can be quickly queried and verified, and the work efficiency and convenience are improved.

[0011] Compared with the prior art, the goose molecular ID construction method based on SNP molecular markers provided by the present application has the following beneficial effects: (1) By using whole genome resequencing technology, more comprehensive and accurate genetic information of local goose germplasm resources is provided.

[0012] (2) The local purebred goose germplasm reference library is constructed, which provides a reliable basis for the identification and identity tracing of goose breeds.

[0013] (3) Combined with pedigree analysis and molecular ID technology, rapid and accurate Taizhou goose germplasm identification is realized. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 It is the technical route for constructing the molecular ID of Taizhou geese in Example 1; Figure 2 It is the PCA analysis result of the local goose germplasm reference library samples in Example 1; Figure 3 It is the result analysis diagram of the test samples in Example 1; Figure 4 It is the chromosome distribution density diagram of 10K SNP loci in Example 1; Figure 5 It is the pedigree analysis result diagram of 10K-SNP identification locus set in Example 1; Figure 6 The Taizhou goose molecular identity card identification two-dimensional code of Example 1. DETAILED DESCRIPTION

[0015] The preferred embodiments of the present application will be described in detail below with reference to the drawings, wherein the drawings constitute a part of the present application and are used to explain the principles of the embodiments of the present application, but are not used to limit the scope of the present application.

[0016] A Taizhou goose molecular identity card identification method based on whole genome resequencing, comprising the following steps: Step 1: sample collection and preparation Collect 30-50 blood samples of Taizhou geese, Taihu geese, Wanxi white geese, Guangfeng white-feather geese, Huanyan geese, Ma'gan geese, Sichuan white geese, Xupu geese, Youjiang geese and Zhexiang white geese, half male and half female, number the samples and record the phenotypes and other related data.

[0017] Step 2: whole genome resequencing and data processing Using a nucleic acid extraction kit, the DNA of each sample is extracted through centrifugation, precipitation, washing, drying and dissolution. The extracted blood sample DNA is subjected to end repair, adapter ligation and PCR amplification to construct a sequencing library that can be used for second-generation high-throughput genome resequencing. Sequencing is performed on a high-throughput sequencing platform to obtain the genome resequencing data of each sample. Quality control is performed to remove low-quality sites. The data after quality control processing is subjected to sample and SNP site statistics.

[0018] Step 3: construction of pure-bred local goose germplasm reference library 80% of the samples after quality control are selected to construct the reference library, and the remaining 20% of the samples are used as test samples. Two pure-bred samples of each breed are selected as positive control samples.

[0019] Step 4: pedigree analysis and population differentiation index analysis The constructed pure-bred goose germplasm reference library is used to analyze the pedigree of the test samples, and PCA analysis is performed. The genotype data is reduced to process the high-dimensional data into low-dimensional data to observe the hierarchical situation of each population, and the FST value is calculated and a different number of site sets are selected.

[0020] Step 5: Taizhou goose molecular identity card identification site screening and testing The population differentiation index between the 10 varieties is calculated by statistical analysis of the reference library samples and the remaining sites after quality control. By screening the SNP site set with the highest FST value, the most representative site can be selected as the Taizhou goose molecular identity marker. Selecting 10K sites can ensure the accuracy and reliability of the identification results. By using the online tool interconnection two-dimensional code, the identified results can be quickly queried and verified, improving work efficiency and convenience.

[0021] Example 1 A Taizhou goose molecular identity recognition method based on whole genome resequencing 1. Through sample collection and preparation, data processing of samples by whole genome resequencing, construction of pure breed local goose germplasm reference library, pedigree analysis and population differentiation index analysis, Taizhou goose molecular identity identification site screening and testing. The construction technology route of Taizhou goose molecular identity is as shown in Figure 1 2. Collect 30-50 blood samples of Taizhou goose, Taihu goose, Wanxi white goose, Guangfeng white ling goose, Huayan goose, Magan goose, Sichuan white goose, Xupu goose, Youjiang goose and Zhedong white goose, half male and half female. Number each sample and record the phenotype and other related data. Use nucleic acid extraction kit to extract DNA from each sample by centrifugation, precipitation, washing, drying and dissolution. The extracted blood sample DNA is subjected to end repair, adapter ligation, PCR amplification and other steps to construct a sequencing library that can be used for second-generation high-throughput genome resequencing. Sequencing is performed on a high-throughput sequencing platform to obtain genomic resequencing data for each sample. Quality control is performed to remove low-quality sites. After quality control, the data is statistically analyzed for samples and SNP sites (Table 1).

[0022] Table 1 Number of SNP site sets for molecular identification and FST value distribution statistics 3. From the samples after quality control, 80% of the samples are selected to construct the reference library, and the remaining 20% of the samples are used as test samples. Two purebred samples selected from each variety are selected as positive control samples. As shown in Figure 2 and Figure 3 The constructed purebred goose germplasm reference library is used for pedigree analysis of the test samples, PCA analysis is performed to observe the stratification of each population, and FST value is calculated and different numbers of site sets are screened.

[0023] 4. The population differentiation index between the 10 varieties is calculated by statistical analysis of the reference library samples and the remaining sites after quality control. As shown in Figure 4 and Figure 5 ​As shown by screening the SNP site set with the highest FST value, the most representative site can be selected as the Taizhou goose molecular identity card marker. Figure 4 The chromosome distribution density map shows that 10K sites can ensure the accuracy and reliability of the identification result. By using an online tool such as Figure 6 As shown, the identified result can be quickly inquired and verified.

[0024] The above merely describes preferred specific embodiments of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be covered within the protection scope of the present application.

Claims

1. A method for molecular identification of Taizhou goose based on whole genome resequencing, comprising the following steps: Step 1: sample collection and preparation (1) Collect blood samples of 30-50 geese for each of 10 goose breeds including Taizhou goose, half male and half female; (2) Number each sample and record related data such as phenotype; Step 2: whole genome resequencing and data processing (1) Extract DNA from each sample, construct sequencing library, and perform second-generation high-throughput genome resequencing; (2) Label and sample quality control the data obtained by sequencing, and retain sites with higher quality; (3) Count the remaining samples and SNP sites after quality control; Step 3: construction of purebred local goose germplasm reference library (1) Select 80% of the samples after quality control for the construction of purebred goose germplasm reference library; (2) Use the remaining 20% of the samples as test samples, and select two purebred samples screened from each breed as positive control samples; Step 4: pedigree analysis and population differentiation index analysis (1) Use the constructed purebred goose germplasm reference library to perform pedigree analysis on the test samples; (2) Perform PCA analysis to observe the hierarchical situation of each population; (3) Calculate FST value and select different numbers of site sets; Step 5: screening and testing of Taizhou goose molecular identification sites (1) Based on the reference library samples and the remaining sites after quality control, perform population differentiation index analysis between the 10 breeds; (2) Select the SNP site set with the highest FST value to determine 10K sites as Taizhou goose molecular identification markers; (3) Use online tools to interconnect two-dimensional codes for quick inquiry.

2. The method according to claim 1, wherein The 10 goose breeds in step 1 include Taizhou goose, Taihu goose, Wanxi white goose, Guangfeng white-feathered goose, Huoyan goose, Magan goose, Sichuan white goose, Xupu goose, Youjiang goose, and Zhedong white goose.

3. The method according to claim 1, wherein In step 2, the nucleic acid extraction kit is used to extract the blood sample DNA of each sample through centrifugation, precipitation, washing, drying, and dissolution steps. The extracted blood sample DNA is subjected to end repair, adapter ligation, and PCR amplification steps to construct a sequencing library that can be used for second-generation high-throughput genome resequencing. Sequencing is performed on a high-throughput sequencing platform to obtain genomic resequencing data for each sample. Quality control is performed to remove low-quality sites. The data after quality control processing is subjected to sample and SNP site statistics.

4. The method according to claim 1, wherein In step 3, 80% of the samples after quality control are selected for the construction of the reference library, and the remaining 20% of the samples are used as test samples. Two purebred samples screened from each breed are selected as positive control samples. This construction method can determine the germplasm characteristics and genetic background of purebred geese.

5. The method according to claim 1, wherein In step 4, the constructed purebred goose germplasm reference library is used to perform pedigree analysis on the test samples, PCA analysis is performed, and the high-dimensional data is converted to low-dimensional data by dimensionality reduction processing to observe the hierarchical situation of each population. FST value is calculated and different numbers of site sets are selected. This can effectively perform pedigree analysis and population differentiation analysis to understand the genetic relationship and differentiation degree between different breeds or populations.

6. The method according to claim 1, wherein The population differentiation index between 10 breeds is calculated by statistical analysis on the reference library samples and the remaining sites after quality control in step 5; by screening the SNP site set with the highest FST value, the most representative site can be selected as the Taizhou goose molecular identity card mark, and the selection of 10K sites can ensure the accuracy and reliability of the identification result; by using the online tool interconnection two-dimensional code, the identified result can be quickly inquired and verified, and the work efficiency and convenience are improved.

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