A reagent for detecting the genotype of SNP loci associated with the egg-laying traits of Leizhou black ducks

Abstract

This application is a divisional application of the invention patent application with the application date of December 22, 2020, application number 2020115303638, and invention title of "A reagent for detecting the genotype of SNP loci related to the egg-laying traits of Leizhou black ducks". The present invention discloses a reagent for detecting the genotype of SNP loci related to the egg-laying traits of Leizhou black ducks. The present invention has discovered 7 SNP loci in the HAL gene of Leizhou ducks, and their genotypes are all significantly correlated with the egg-laying traits. In particular, individuals with the genotype of AA at SNP locus 3, the genotype of CC at SNP locus 4, the genotype of TT at SNP locus 5, and the genotype of AA at SNP locus 6 can be used as the optimal choice for screening individuals with low age at first laying, low body weight at first laying, and high egg production in the Leizhou black duck population. This provides a theoretical basis for molecular marker-assisted breeding of Leizhou black ducks and the establishment of a superior high-yield population.

Description

[0001] This application is a divisional application of the invention patent application with the application date of December 22, 2020, application number 2020115303638, and invention name of "A reagent for detecting the genotype of SNP loci related to the egg-laying traits of Leizhou black ducks". Technical Field

[0002] The present invention relates to the field of molecular breeding technology. Specifically, it relates to a reagent for detecting the genotype of SNP loci related to the egg-laying traits of Leizhou black ducks. Background Art

[0003] China is a major country in poultry farming. The annual output of poultry products ranks among the top in the world. It is currently statistically obtained that the egg production and poultry meat production in 2018 ranked first and second in the world respectively. Moreover, China is rich in local poultry breeds, but the reproductive capacity of local breeds is relatively low. The increase in farmers' input and the decrease in output have caused the problem of the loss of local breed resources. There are various local poultry breeds in the western region of Guangdong Province. Among them, the Leizhou black duck is a local breed mainly distributed in the Leizhou Peninsula. It belongs to the black-feathered duck for both egg and meat production, with a small body size, delicious meat, high egg production rate, resistance to roughage, strong disease resistance and strong environmental adaptability, and is widely liked by local people. However, there is currently little research on it. Some studies have used the method of fitting the egg production curve to obtain that the peak egg production period of Leizhou black ducks is from 27 weeks to 41 weeks of age, and the peak egg production rate is above 90%; the egg weight of Leizhou black ducks is higher than that of other local breeds, such as Jinyun Ma ducks, Huainan Ma ducks, Liancheng white ducks, Youxian Ma ducks, etc.; the meat and egg use values of Leizhou black ducks are rich, containing a variety of trace elements, especially the Fe element required by the human body, and the nutritional content is high.

[0004] Chinese Patent 201810410892.0, a molecular marker related to the egg-laying performance of laying ducks and its application in breeding, discloses a molecular marker that is significantly related to the age at first lay, egg production at 34 weeks of age, and egg production at 72 weeks of age in ducks. However, since egg-laying traits are quantitative traits, quantitative traits are usually controlled by multiple genes. The discovery of more molecular markers affecting related traits can more accurately judge egg-laying traits.

[0005] Histidine ammonia-lyase (HAL), also known as histidase or histidine deaminase, is a member of the carbon-nitrogen lyase family, catalyzing the first reaction in the catabolism of histidine, that is, catalyzing the deamination of histidine to generate urocanic acid. Previous studies have shown that mutations in the HAL gene are related to the risk of histidinemia, non-melanoma skin cancer, and the incidence of coronary heart disease. The role of the HAL gene in egg-laying performance has not been reported. Summary of the Invention

[0006] The object of the present invention is to overcome the above-mentioned deficiencies of the prior art and provide a reagent for detecting the genotypes of SNP loci related to the egg-laying traits of Leizhou black ducks.

[0007] The first object of the present invention is to provide a reagent for detecting the genotypes of SNP loci related to the egg-laying traits of Leizhou black ducks.

[0008] The second object of the present invention is to provide the application of the reagent in the preparation of a kit for evaluating the egg-laying performance of Leizhou black ducks.

[0009] The third object of the present invention is to provide the application of the reagent in evaluating the egg-laying performance of Leizhou black ducks.

[0010] The fourth object of the present invention is to provide a kit for evaluating the egg-laying performance of Leizhou black ducks.

[0011] The fifth object of the present invention is to provide the application of one or both of the reagent and / or the kit in evaluating the egg-laying performance of Leizhou black ducks.

[0012] In order to achieve the above object, the present invention is realized through the following scheme:

[0013] The present invention discovers 7 SNP loci in introns 1 and 2 of the HAL gene of Leizhou ducks, namely SNP locus 1, SNP locus 2, SNP locus 3, SNP locus 4, SNP locus 5, SNP locus 6 and / or SNP locus 7:

[0014] Among them, SNP locus 1 is located at the 288th base of the first intron where C mutates to T (I1-288C>T). The body weight at the onset of lay of samples with the CC genotype is significantly higher than that of samples with the TT genotype, while the egg production number at 300 days of samples with the CC genotype is significantly lower than that of samples with the TT genotype;

[0015] SNP locus 2 is located at the 373rd base of the first intron where T mutates to G (I1-373T>G). The egg production number at 300 days of samples with the TT genotype is significantly higher than that of samples with the GG genotype;

[0016] SNP locus 3 is located at the 438th base of the first intron where A mutates to G (I1-438A>G). The age at first lay of samples with the AA genotype is extremely significantly lower than that of samples with the AG genotype;

[0017] SNP locus 4 is located at the 16th base of the second intron where C mutates to A (I2-16C>A). The age at first lay of samples with the AA genotype is significantly lower than that of samples with the AC genotype;

[0018] The SNP locus 5 is located at the 123rd base of the second intron, where A mutates to T (I2-123A>T). The age at first laying of the samples with the TT genotype is significantly lower than that of the samples with the AT genotype, and the number of eggs laid at 300 days of the samples with the TT genotype is significantly higher than that of the samples with the AT genotype;

[0019] The SNP locus 6 is located at the 141st base of the second intron, where G mutates to A (I2-141G>A). The age at first laying of the samples with the AA genotype is significantly lower than that of the samples with the GG genotype. The body weight at first laying of the samples with the AG genotype is extremely significantly higher than that of the GG genotype samples, and the number of eggs laid at 300 days of the samples with the AG genotype is significantly lower than that of the samples with the GG genotype;

[0020] The SNP locus 7 is located at the 168th base of the second intron, where A mutates to G (I2-168A>G). The age at first laying of the samples with the GG genotype is extremely significantly lower than that of the AA type. The body weight at first laying of the samples with the AG genotype is significantly higher than that of the GG type, and the number of eggs laid at 300 days of the samples with the AG genotype is significantly lower than that of the GG type.

[0021] Further findings show that for individuals with the genotype of AG at SNP locus 3, CA at SNP locus 4, TA at SNP locus 5, and AG at SNP locus 6, their age at first laying is significantly higher than that of individuals with the genotype of AA at SNP locus 3, CC at SNP locus 4, TT at SNP locus 5, and AA at SNP locus 6, and also higher than that of individuals with the genotype of AA at SNP locus 3, CC at SNP locus 4, TA at SNP locus 5, and AG at SNP locus 6;

[0022] The body weight at first laying of individuals with the genotype of AG at SNP locus 3, CA at SNP locus 4, TA at SNP locus 5, and AG at SNP locus 6 and those with the genotype of AA at SNP locus 3, CC at SNP locus 4, TT at SNP locus 5, and AG at SNP locus 6 is significantly higher than that of individuals with the genotype of AA at SNP locus 3, CC at SNP locus 4, TT at SNP locus 5, and AA at SNP locus 6;

[0023] The number of eggs laid at 300 days of individuals with the genotype of AA at SNP locus 3, CC at SNP locus 4, TT at SNP locus 5, and AA at SNP locus 6 is significantly higher than that of individuals with the genotype of AA at SNP locus 3, CC at SNP locus 4, TA at SNP locus 5, and AG at SNP locus 6.

[0024] Therefore, individuals with the genotype AA at SNP locus 3, genotype CC at SNP locus 4, genotype TT at SNP locus 5, and genotype AA at SNP locus 6 can be the optimal choice for screening individuals with low age at first laying, low body weight at first laying, and high egg production in the Leizhou black duck population.

[0025] Therefore, the present invention claims to protect a reagent for detecting the genotypes of SNP loci related to the egg-laying traits of Leizhou black ducks, and the reagent is used for detecting the genotypes of SNP locus 1, SNP locus 2, SNP locus 3, SNP locus 4, SNP locus 5, SNP locus 6, and / or SNP locus 7;

[0026] The SNP locus 1 is located at the 288th base of the first intron of the HAL gene. The body weight at first laying of samples with the CC genotype is significantly higher than that of samples with the TT genotype, while the number of eggs produced at 300 days of samples with the CC genotype is significantly lower than that of samples with the TT genotype;

[0027] The SNP locus 2 is located at the 438th base of the first intron of the HAL gene. The number of eggs produced at 300 days of samples with the TT genotype is significantly higher than that of samples with the GG genotype;

[0028] The SNP locus 3 is located at the 438th base of the first intron of the HAL gene. The age at first laying of samples with the AA genotype is extremely significantly lower than that of samples with the AG genotype;

[0029] The SNP locus 4 is located at the 16th base of the second intron of the HAL gene. The age at first laying of samples with the AA genotype is significantly lower than that of samples with the AC genotype;

[0030] The SNP locus 5 is located at the 123rd base of the second intron of the HAL gene. The age at first laying of samples with the TT genotype is significantly lower than that of samples with the AT genotype, and the number of eggs produced at 300 days of samples with the TT genotype is significantly higher than that of samples with the AT genotype;

[0031] The SNP locus 6 is located at the 141st base of the second intron of the HAL gene. The age at first laying of samples with the AA genotype is significantly lower than that of samples with the GG genotype. The body weight at first laying of samples with the AG genotype is extremely significantly higher than that of samples with the GG genotype, and the number of eggs produced at 300 days of samples with the AG genotype is significantly lower than that of samples with the GG genotype;

[0032] The SNP locus 7 is located at the 168th base of the second intron of the HAL gene. The age at first laying of samples with the GG genotype is extremely significantly lower than that of samples with the AA genotype. The body weight at first laying of samples with the AG genotype is significantly higher than that of samples with the GG genotype, and the number of eggs produced at 300 days of samples with the AG genotype is significantly lower than that of samples with the GG genotype.

[0033] Preferably, SNP locus 3, SNP locus 4, SNP locus 5 and SNP locus 6 are detected.

[0034] Individuals with the genotype AG at SNP locus 3, the genotype CA at SNP locus 4, the genotype TA at SNP locus 5, and the genotype AG at SNP locus 6 have a significantly higher age at first egg than individuals with the genotype AA at SNP locus 3, the genotype CC at SNP locus 4, the genotype TT at SNP locus 5, and the genotype AA at SNP locus 6, and individuals with the genotype AA at SNP locus 3, the genotype CC at SNP locus 4, the genotype TA at SNP locus 5, and the genotype AG at SNP locus 6;

[0035] Individuals with the genotype AG at SNP locus 3, the genotype CA at SNP locus 4, the genotype TA at SNP locus 5, and the genotype AG at SNP locus 6 and individuals with the genotype AA at SNP locus 3, the genotype CC at SNP locus 4, the genotype TT at SNP locus 5, and the genotype AG at SNP locus 6 have a significantly higher body weight at first egg than individuals with the genotype AA at SNP locus 3, the genotype CC at SNP locus 4, the genotype TT at SNP locus 5, and the genotype AA at SNP locus 6;

[0036] Individuals with the genotype AA at SNP locus 3, the genotype CC at SNP locus 4, the genotype TT at SNP locus 5, and the genotype AA at SNP locus 6 have a significantly higher number of eggs laid at 300 days than individuals with the genotype AA at SNP locus 3, the genotype CC at SNP locus 4, the genotype TA at SNP locus 5, and the genotype AG at SNP locus 6.

[0037] More preferably, the reagent is a primer.

[0038] Even more preferably, the nucleotide sequences of the primers are as shown in SEQ ID NO.1 - 2.

[0039] SEQ ID NO.1: 5’-GAATGGCACCTGAGTTAGCA-3’;

[0040] SEQ ID NO.2: 5’-CCTGTTCTGGTTCTCGGTATTTGCT-3’.

[0041] The present invention also claims the use of any of the above-mentioned reagents in the preparation of a kit for evaluating the egg-laying performance of Leizhou black ducks.

[0042] The present invention also claims the use of any of the above-mentioned reagents in evaluating the egg-laying performance of Leizhou black ducks.

[0043] Furthermore, the present invention also claims to protect a method for evaluating the egg-laying performance of Leizhou black ducks, including the said reagent.

[0044] Preferably, the said reagent is a primer with a nucleotide sequence as shown in SEQ ID NO.1-2.

[0045] Most preferably, a kit for evaluating the egg-laying performance of Leizhou black ducks contains a primer with a nucleotide sequence as shown in SEQ ID NO.1-2 and 2×Green Taq Mix;

[0046] The usage method is as follows:

[0047] 1. Extract the sample DNA;

[0048] 2. Perform PCR amplification

[0049] The PCR reaction system is 20 μL: 10 μL of 2×Green Taq Mix, 0.4 μL each of the upstream / downstream primers (with a nucleotide sequence as shown in SEQ INNO.1-2), 1 μL of template, and 8.2 μL of ddH2O;

[0050] The PCR reaction procedure: Heat denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 1 min, 35 cycles, extension at 72°C for 5 min, and store at 4°C.

[0051] 3. Sequence the PCR product

[0052] Perform electrophoresis, recovery, and send to a sequencing company for sequencing using 1% agarose gel;

[0053] Result interpretation:

[0054] SNP site 1 is located at the 288th base of the first intron of the HAL gene, that is, at the 250th base at the 5' end of the amplification product, with a C / T single nucleotide polymorphism;

[0055] SNP site 2 is located at the 438th base of the first intron of the HAL gene, that is, at the 335th base at the 5' end of the amplification product, with a T / G single nucleotide polymorphism;

[0056] SNP site 3 is located at the 438th base of the first intron of the HAL gene, that is, at the 410th base at the 5' end of the amplification product, with an A / G single nucleotide polymorphism;

[0057] SNP site 4 is located at the 16th base of the second intron of the HAL gene, that is, at the 489th base at the 5' end of the amplification product, with a C / A single nucleotide polymorphism;

[0058] SNP locus 5 is located at the 123rd base of the second intron of the HAL gene, that is, at the 596th base at the 5' end of the amplification product, and there is an A / T single nucleotide polymorphism;

[0059] SNP locus 6 is located at the 141st base of the second intron of the HAL gene, that is, at the 614th base at the 5' end of the amplification product, and there is a G / A single nucleotide polymorphism;

[0060] SNP locus 7 is located at the 168th base of the second intron of the HAL gene, that is, at the 641st base at the 5' end of the amplification product, and there is an A / G single nucleotide polymorphism.

[0061] For the said SNP locus 1, the body weight at the onset of lay of the samples with the CC genotype is significantly higher than that of the samples with the TT genotype, while the egg production number at 300 d of the samples with the CC genotype is significantly lower than that of the samples with the TT genotype;

[0062] For the said SNP locus 2, the egg production number at 300 d of the samples with the TT genotype is significantly higher than that of the samples with the GG genotype;

[0063] For the said SNP locus 3, the age at first lay of the samples with the AA genotype is extremely significantly lower than that of the samples with the AG genotype;

[0064] For the said SNP locus 4, the age at first lay of the samples with the AA genotype is significantly lower than that of the samples with the AC genotype;

[0065] For the said SNP locus 5, the age at first lay of the samples with the TT genotype is significantly lower than that of the samples with the AT genotype, while the egg production number at 300 d of the samples with the TT genotype is significantly higher than that of the samples with the AT genotype;

[0066] For the said SNP locus 6, the age at first lay of the samples with the AA genotype is significantly lower than that of the samples with the GG genotype, the body weight at the onset of lay of the samples with the AG genotype is extremely significantly higher than that of the samples with the GG genotype, and the egg production number at 300 d of the samples with the AG genotype is significantly lower than that of the samples with the GG genotype;

[0067] For the said SNP locus 7, the age at first lay of the samples with the GG genotype is extremely significantly lower than that of the AA genotype, the body weight at the onset of lay of the samples with the AG genotype is significantly higher than that of the GG genotype, and the egg production number at 300 d of the samples with the AG genotype is significantly lower than that of the GG genotype.

[0068] Individuals with the genotype AG at SNP locus 3, genotype CA at SNP locus 4, genotype TA at SNP locus 5, and genotype AG at SNP locus 6 have a significantly higher age at first egg than individuals with the genotype AA at SNP locus 3, genotype CC at SNP locus 4, genotype TT at SNP locus 5, and genotype AA at SNP locus 6, and individuals with the genotype AA at SNP locus 3, genotype CC at SNP locus 4, genotype TA at SNP locus 5, and genotype AG at SNP locus 6;

[0069] Individuals with the genotype AG at SNP locus 3, genotype CA at SNP locus 4, genotype TA at SNP locus 5, and genotype AG at SNP locus 6 and individuals with the genotype AA at SNP locus 3, genotype CC at SNP locus 4, genotype TT at SNP locus 5, and genotype AG at SNP locus 6 have a significantly higher body weight at first egg than individuals with the genotype AA at SNP locus 3, genotype CC at SNP locus 4, genotype TT at SNP locus 5, and genotype AA at SNP locus 6;

[0070] Individuals with the genotype AA at SNP locus 3, genotype CC at SNP locus 4, genotype TT at SNP locus 5, and genotype AA at SNP locus 6 have a significantly higher number of eggs laid at 300 days than individuals with the genotype AA at SNP locus 3, genotype CC at SNP locus 4, genotype TA at SNP locus 5, and genotype AG at SNP locus 6.

[0071] The application of the reagent and / or one or both of the evaluations of the egg-laying performance of Leizhou black ducks in the evaluation of the egg-laying performance of Leizhou black ducks also belongs to the protection scope of the present invention.

[0072] Compared with the prior art, the present invention has the following beneficial effects:

[0073] The present invention discovers 7 SNP loci of the HAL gene of Leizhou ducks, and their genotypes are significantly correlated with egg-laying traits. In particular, individuals with the genotype AA at SNP locus 3, genotype CC at SNP locus 4, genotype TT at SNP locus 5, and genotype AA at SNP locus 6 can be used as the optimal choice for screening individuals with a low age at first egg, a low body weight at first egg, and a high egg production in the Leizhou black duck population. This provides a theoretical basis for molecular marker-assisted breeding of Leizhou black ducks and the establishment of a superior high-yield population. Description of the Drawings

[0074] Figure 1 It is the electrophoresis diagram of the amplification product of the HAL gene of Leizhou ducks; lanes 1-4 are the PCR amplification products of different individuals respectively.

[0075] Figure 2 Sequencing maps of three genotypes at SNP locus 1 of the HAL gene; Figure Ⅰ An individual with the genotype CT; Figure Ⅱ An individual with the genotype CC; Figure Ⅲ An individual with the genotype TT.

[0076] Figure 3 Sequencing maps of three genotypes at SNP locus 2 of the HAL gene; Figure Ⅰ An individual with the genotype TG; Figure Ⅱ An individual with the genotype GG; Figure Ⅲ An individual with the genotype TT.

[0077] Figure 4 Sequencing maps of three genotypes at SNP locus 3 of the HAL gene; Figure Ⅰ An individual with the genotype AG; Figure Ⅱ An individual with the genotype AA; Figure Ⅲ An individual with the genotype GG.

[0078] Figure 5 Sequencing maps of three genotypes at SNP locus 4 of the HAL gene; Figure Ⅰ An individual with the genotype CA; Figure Ⅱ An individual with the genotype CC; Figure Ⅲ An individual with the genotype AA

[0079] Figure 6 Sequencing maps of three genotypes at SNP locus 5 of the HAL gene; Figure Ⅰ An individual with the genotype AT; Figure Ⅱ An individual with the genotype TT; Figure Ⅲ An individual with the genotype AA.

[0080] Figure 7 Sequencing maps of three genotypes at SNP locus 6 of the HAL gene; Figure Ⅰ An individual with the genotype GA; Figure Ⅱ An individual with the genotype AA; Figure Ⅲ An individual with the genotype GG.

[0081] Figure 8 Sequencing maps of three genotypes at SNP locus 7 of the HAL gene; Figure Ⅰ An individual with the genotype AG; Figure Ⅱ An individual with the genotype AA; Figure Ⅲ An individual with the genotype GG.

[0082] Figure 9 Linkage reaction of SNPs loci in the dairy cattle population of the HAL gene. Detailed implementation mode

[0083] The present invention will be further elaborated in detail below in conjunction with the accompanying drawings of the specification and specific embodiments. The embodiments are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used are all reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0084] Main reagents: The whole-genome blood DNA extraction kit, purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; agarose, purchased from Boge Lin Biological Co., Ltd.

[0085] Main instruments: The PCR instrument was purchased from Hangzhou Langji Scientific Instruments Co., Ltd.; the DYY-6C electrophoresis instrument was purchased from Beijing Liuyi Biotechnology Co., Ltd.; the gel imaging system; the Tianmei electronic analytical balance FA1204B was purchased from Shanghai Jingke.

[0086] Example 1 Construction of DNA pooling and PCR amplification

[0087] 1. Experimental method

[0088] (1) Test animals

[0089] In this experiment, the F4 generation of the Leizhou black duck foundation group from a certain farm in Zhanjiang City was selected. 93 Leizhou black ducks at 300 days old were collected with 2 mL of jugular vein blood, collected with a heparin anticoagulation tube, and stored at -20 °C for later use to extract blood genomic DNA.

[0090] (2) Extraction of blood genomic DNA

[0091] The blood DNA was extracted using the blood genomic DNA extraction kit from Tiangen. The steps were referred to the kit instructions, and the DNA concentration and purity were detected using a concentration analyzer, and stored at -20 °C for later use.

[0092] (3) Construction of DNA pooling

[0093] Randomly select 30 - 40 DNA samples for equal mixing to prepare a DNA pooling sample.

[0094] (4) Primer design

[0095] According to the HAL genomic sequence provided by the Ensemble database (number: (ENSAPLG00000003775.2)), the primers for the HAL gene were designed using Primer 5.0 software, and the primers were sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis:

[0096] Forward primer: 5’-GAATGGCACCTGAGTTAGCA-3’ (SEQ IN NO.1);

[0097] Reverse primer: 5’-CCTGTTCTGGTTCTCGGTATTTGCT-3’ (SEQ IN NO.2).

[0098] (5) PCR amplification and verification of the target gene

[0099] PCR reaction system of 20 μL: 10 μL of 2×Green Taq Mix, 0.4 μL each of the forward / reverse primers (nucleotide sequences as shown in SEQ IN NO.1 - 2), 1 μL of template, 8.2 μL of ddH2O;

[0100] PCR reaction procedure: Heat denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 1 min, 35 cycles, extension at 72°C for 5 min, store at 4°C.

[0101] After electrophoresis verification with 1% agarose gel, it was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing.

[0102] 2. Experimental results

[0103] The PCR amplification product of the HAL gene of Leizhou black ducks was detected by 1% gel electrophoresis, and the results are as Figure 1 shown. The PCR product has good amplification specificity, no non-specific bands, and clear bands, and can be directly used for sequencing and identification.

[0104] Seven SNP sites were found in introns 1 and 2 of the HAL gene. Among them, SNP site 1, SNP site 2, and SNP site 3 are all located in intron 1 of the HAL gene, and SNP site 4, SNP site 5, SNP site 6, and SNP site 7 are all located in intron 2 of the HAL gene. The sequencing results of each genotype of the seven SNPs are as Figures 2 to 8 shown,

[0105] Specifically, SNP site 1 is located at the 250th base at the 5' end of the amplification product, with a C / T single nucleotide polymorphism;

[0106] SNP site 2 is located at the 335th base at the 5' end of the amplification product, with a T / G single nucleotide polymorphism;

[0107] SNP site 3 is located at the 410th base at the 5' end of the amplification product, with an A / G single nucleotide polymorphism;

[0108] SNP site 4 is located at the 489th base at the 5' end of the amplification product, with a C / A single nucleotide polymorphism;

[0109] The SNP locus 5 is located at the 596th base at the 5' end of the amplification product, with an A / T single nucleotide polymorphism;

[0110] The SNP locus 6 is located at the 614th base at the 5' end of the amplification product, with a G / A single nucleotide polymorphism;

[0111] The SNP locus 7 is located at the 641st base at the 5' end of the amplification product, with an A / G single nucleotide polymorphism.

[0112] Analysis of population genetic characteristics of 7 SNP loci of HAL gene in Example 2

[0113] 1. Experimental method

[0114] Use SeqMan software for sequence alignment, count the SNP loci and genotypes of each individual, and calculate the gene frequencies, genotype frequencies, genetic heterozygosity (H), effective number of alleles (Ne), polymorphism information content (PIC) and chi-square test value χ 2 。

[0115] 2. Experimental results

[0116] The results are shown in Table 1. The dominant genotypes at SNP locus 1 to SNP locus 7 are CT, TG, AA, CC, AT, GA and AG respectively, and the dominant alleles are C, T, A, C, T, A and G respectively, and their frequencies are all greater than 0.50; the gene heterozygosity (H) and polymorphism information content (PIC) of the 7 SNPs are both in the range of 0.25 - 0.5, indicating that the polymorphism of this population belongs to moderate polymorphism. The chi-square test shows that the 7 SNPs are all in Hardy-Weinberg equilibrium in this Leizhou black duck population (P>0.05).

[0117] Table 1 Population genetic characteristics of 7 SNPs of HAL gene

[0118]

[0119] Analysis of the correlation between SNPs of HAL gene and egg production performance of Leizhou black duck in Example 3

[0120] 1. Experimental method

[0121] Use the multiple comparison analysis in the one-way ANOVA of SPSS 22.0 software to conduct a significant difference test on the association between the egg production traits, genotypes and haplotypes of Leizhou black duck, and the results are expressed as "mean ± standard error". When P<0.05, it indicates a significant difference, and when P<0.01, it indicates a highly significant difference.

[0122] 2. Experimental results

[0123] The results are shown in Table 2. Therefore, it can be seen that the body weight at the onset of lay of individuals with the CC genotype at SNP locus 1 is significantly higher than that of individuals with the TT genotype, while the egg production number at 300 days of individuals with the CC genotype is significantly lower than that of individuals with the TT genotype (P<0.05); the egg production number at 300 days of individuals with the TT genotype at SNP locus 2 is significantly higher than that of individuals with the GG genotype (P<0.05); the age at first lay of individuals with the AA genotype at SNP locus 3 is extremely significantly lower than that of individuals with the AG genotype (P<0.01); the age at first lay of individuals with the AA genotype at SNP locus 4 is significantly lower than that of individuals with the AC genotype (P<0.01); similarly, the age at first lay of individuals with the TT genotype at SNP locus 5 is significantly lower than that of individuals with the AT genotype, while the egg production number at 300 days of individuals with the TT genotype is significantly higher than that of individuals with the AT genotype (P<0.05); the age at first lay of individuals with the AA genotype at SNP locus 6 is significantly lower than that of individuals with the GG genotype (P<0.05), the body weight at the onset of lay of individuals with the AG genotype is extremely significantly higher than that of individuals with the GG genotype (P<0.01), and the egg production number at 300 days of individuals with the AG genotype is significantly lower than that of individuals with the GG genotype (P<0.05); similarly, the age at first lay of individuals with the GG genotype at SNP locus 7 is extremely significantly lower than that of individuals with the AA genotype (P<0.01), the body weight at the onset of lay of individuals with the AG genotype is significantly higher than that of individuals with the GG genotype (P<0.05), and the egg production number at 300 days of individuals with the AG genotype is significantly lower than that of the GG genotype (P<0.05). All 7 SNPs have no significant effect on the egg weight at the onset of lay (P>0.05).

[0124] Table 2 Correlation analysis of 7 SNPs of HAL gene with egg production performance of Leizhou ducks

[0125]

[0126]

[0127] From the above results, it can be seen that except for SNP locus 1 and SNP locus 2, the remaining SNP loci are all significantly correlated with the age at first lay of Leizhou black ducks. It is speculated that the remaining 5 SNP loci can be used as molecular marker loci for the age at first lay trait of Leizhou black ducks; SNP locus 1, SNP locus 6 and SNP locus 7, these 3 loci are all significantly correlated with the body weight at the onset of lay of Leizhou black ducks, indicating that these 3 SNPs may play an important guiding role in improving the body weight at the onset of lay; except for SNP locus 3 and SNP locus 4, the remaining loci are all significantly associated with the egg production at 300 days of Leizhou black ducks, indicating that the remaining loci can be used as important markers for improving the egg production of Leizhou black ducks.

[0128] Example 4 Correlation analysis of 7 SNP loci with egg production performance of Leizhou ducks

[0129] 1. Experimental method

[0130] Perform linkage analysis on 7 SNP loci using HaploView 4.2 software.

[0131] 2. Experimental results

[0132] The results are shown in Figure 9 and Table 3. It was found that there was strong linkage inheritance among 4 loci, namely SNP locus 3, SNP locus 4, SNP locus 5, and SNP locus 6. These 4 loci constituted a domain, with a total of 10 different haplotypes, among which only 7 haplotypes had biological statistical significance (the number of individuals was greater than 3), namely H1H1 (AA - CC - TT - AA), H1H2 (AG - CA - TA - AG), H1H3 (AA - CC - TA - AG), H1H4 (AA - CC - TT - AG), H2H2 (GG - AA - AA - GG), H2H3 (AG - CA - AA - GG), and H3H4 (AA - CC - TA - GG).

[0133] Use SPSS 22.0 for one - way ANOVA to analyze the correlation between 7 haplotypes and the egg - laying performance of Leizhou black ducks. The results are shown in Table 4. It can be seen from this that the age at first egg of the H1H2 haplotype was significantly higher than that of the H1H1 and H1H3 haplotypes (P < 0.05); the body weight at first egg of the H1H2 and H1H4 haplotypes was significantly higher than that of the H1H1 haplotype (P < 0.05); while the number of eggs laid at 300 days of the H1H1 haplotype was significantly higher than that of the H1H3 haplotype (P < 0.05). There were no significant differences between the remaining haplotypes and the egg - laying performance of Leizhou black ducks (P > 0.05).

[0134] Table 3 Haplotype frequencies of the HAL gene

[0135]

[0136] Table 4 Correlation analysis between the HAL gene haplotypes and the egg - laying performance of Leizhou ducks

[0137]

[0138] Note: Among the same indicators in the same column, those marked with completely different letters indicate significant differences (P < 0.05); those without marked letters indicate no significant differences (P > 0.05).

[0139] Example 5 A kit for evaluating the egg - laying performance of Leizhou black ducks

[0140] I. Composition

[0141] Primers with nucleotide sequences as shown in SEQ ID NO.1 - 2 and 2×Green Taq Mix.

[0142] II. Usage method

[0143] 1. Extract the sample DNA;

[0144] 2. PCR amplification

[0145] The PCR reaction system is 20 μL: 10 μL of 2×Green Taq Mix, 0.4 μL each of the upstream / downstream primers (nucleotide sequences are shown in SEQ INNO.1 - 2), 1 μL of template, and 8.2 μL of ddH2O;

[0146] The PCR reaction program: Heat denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 1 min, 35 cycles, extension at 72°C for 5 min, and store at 4°C.

[0147] 3. Sequencing of the PCR product

[0148] Perform electrophoresis, recovery, and send for sequencing by a sequencing company using 1% agarose gel.

[0149] III. Result interpretation

[0150] SNP site 1 is located at the 288th base of the first intron of the HAL gene, that is, at the 250th base at the 5' end of the amplification product, with a C / T single nucleotide polymorphism;

[0151] SNP site 2 is located at the 438th base of the first intron of the HAL gene, that is, at the 335th base at the 5' end of the amplification product, with a T / G single nucleotide polymorphism;

[0152] SNP site 3 is located at the 438th base of the first intron of the HAL gene, that is, at the 410th base at the 5' end of the amplification product, with an A / G single nucleotide polymorphism;

[0153] SNP site 4 is located at the 16th base of the second intron of the HAL gene, that is, at the 489th base at the 5' end of the amplification product, with a C / A single nucleotide polymorphism;

[0154] SNP site 5 is located at the 123rd base of the second intron of the HAL gene, that is, at the 596th base at the 5' end of the amplification product, with an A / T single nucleotide polymorphism;

[0155] SNP site 6 is located at the 141st base of the second intron of the HAL gene, that is, at the 614th base at the 5' end of the amplification product, with a G / A single nucleotide polymorphism;

[0156] SNP site 7 is located at the 168th base of the second intron of the HAL gene, that is, at the 641st base at the 5' end of the amplification product, with an A / G single nucleotide polymorphism.

[0157] For the SNP locus 1, the body weight at the onset of lay of samples with the CC genotype is significantly higher than that of samples with the TT genotype, while the number of eggs laid at 300 days of samples with the CC genotype is significantly lower than that of samples with the TT genotype;

[0158] For the SNP locus 2, the number of eggs laid at 300 days of samples with the TT genotype is significantly higher than that of samples with the GG genotype;

[0159] For the SNP locus 3, the age at the onset of lay of samples with the AA genotype is extremely significantly lower than that of samples with the AG genotype;

[0160] For the SNP locus 4, the age at the onset of lay of samples with the AA genotype is significantly lower than that of samples with the AC genotype;

[0161] For the SNP locus 5, the age at the onset of lay of samples with the TT genotype is significantly lower than that of samples with the AT genotype, and the number of eggs laid at 300 days of samples with the TT genotype is significantly higher than that of samples with the AT genotype;

[0162] For the SNP locus 6, the age at the onset of lay of samples with the AA genotype is significantly lower than that of samples with the GG genotype, the body weight at the onset of lay of samples with the AG genotype is extremely significantly higher than that of samples with the GG genotype, and the number of eggs laid at 300 days of samples with the AG genotype is significantly lower than that of samples with the GG genotype;

[0163] For the SNP locus 7, the age at the onset of lay of samples with the GG genotype is extremely significantly lower than that of the AA type, the body weight at the onset of lay of samples with the AG genotype is significantly higher than that of the GG type, and the number of eggs laid at 300 days of samples with the AG genotype is significantly lower than that of the GG type.

[0164] Individuals with the genotype of AG for SNP locus 3, CA for SNP locus 4, TA for SNP locus 5, and AG for SNP locus 6 have a significantly higher age at the onset of lay than individuals with the genotype of AA for SNP locus 3, CC for SNP locus 4, TT for SNP locus 5, and AA for SNP locus 6, and individuals with the genotype of AA for SNP locus 3, CC for SNP locus 4, TA for SNP locus 5, and AG for SNP locus 6;

[0165] Individuals with the genotype AG at SNP locus 3, genotype CA at SNP locus 4, genotype TA at SNP locus 5, and genotype AG at SNP locus 6, and individuals with the genotype AA at SNP locus 3, genotype CC at SNP locus 4, genotype TT at SNP locus 5, and genotype AG at SNP locus 6 have significantly higher body weights at first egg than individuals with the genotype AA at SNP locus 3, genotype CC at SNP locus 4, genotype TT at SNP locus 5, and genotype AA at SNP locus 6;

[0166] Individuals with the genotype AA at SNP locus 3, genotype CC at SNP locus 4, genotype TT at SNP locus 5, and genotype AA at SNP locus 6 have significantly higher egg production at 300 days than individuals with the genotype AA at SNP locus 3, genotype CC at SNP locus 4, genotype TA at SNP locus 5, and genotype AG at SNP locus 6.

[0167] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made on the basis of the above description and ideas. It is not necessary and impossible to enumerate all the implementation manners here. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. Use of a reagent for detecting genotypes of SNP loci correlated with egg-laying traits of Leizhou black ducks in evaluating the egg-laying performance of Leizhou black ducks, characterized in that, The reagent is used to detect SNP locus 3, SNP locus 4, SNP locus 5 and SNP locus 6; SNP locus 3 is located at the 438th base of the first intron of the HAL gene, SNP locus 4 is located at the 16th base of the second intron of the HAL gene, SNP locus 5 is located at the 123rd base of the second intron of the HAL gene, and SNP locus 6 is located at the 141st base of the second intron of the HAL gene; Individuals with the genotype of AG at SNP locus 3, CA at SNP locus 4, TA at SNP locus 5, and AG at SNP locus 6 have significantly higher age at first egg than individuals with the genotype of AA at SNP locus 3, CC at SNP locus 4, TT at SNP locus 5, and AA at SNP locus 6, and individuals with the genotype of AA at SNP locus 3, CC at SNP locus 4, TA at SNP locus 5, and AG at SNP locus 6; Individuals with the genotype of AG at SNP locus 3, CA at SNP locus 4, TA at SNP locus 5, and AG at SNP locus 6 and individuals with the genotype of AA at SNP locus 3, CC at SNP locus 4, TT at SNP locus 5, and AG at SNP locus 6 have significantly higher body weight at first egg than individuals with the genotype of AA at SNP locus 3, CC at SNP locus 4, TT at SNP locus 5, and AA at SNP locus 6; Individuals with the genotype of AA at SNP locus 3, CC at SNP locus 4, TT at SNP locus 5, and AA at SNP locus 6 have significantly higher egg production at 300 days than individuals with the genotype of AA at SNP locus 3, CC at SNP locus 4, TA at SNP locus 5, and AG at SNP locus 6, The Ensemble database number of the HAL gene is ENSAPLG00000003775.

2.

2. The use according to claim 1, characterized in that, The reagent is a primer, and the nucleotide sequence of the primer is shown in SEQ ID NO.1-2.

3. Use of a kit for evaluating the egg-laying performance of Leizhou black ducks in evaluating the egg-laying performance of Leizhou black ducks, characterized in that, The kit includes the reagent described in claim 1.

4. The use according to claim 3, characterized in that, The reagent is a primer with the nucleotide sequence shown in SEQ ID NO.1-2.

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