Method for identifying Langya chicken and Non-Langya chicken by using SNP (Single Nucleotide Polymorphism) molecular marker

Abstract

The invention relates to a method for identifying Langya chicken and Non-Langya chicken by using SNP molecular markers, and belongs to the technical fields of animal husbandry and molecular biology. The SNP molecular marker for Langya chicken identification comprises 12 loci. The invention further provides a primer combination which comprises SNP loci for amplifying Langya chicken identification. According to the method, the 12 specific SNP sites of the Langya chicken are utilized, the site information is obtained through PCR amplification and Sanger sequencing, prediction is conducted through machine learning, the Langya chicken variety identification model is established based on the 12 SNP site combination, and the method has the advantages of being low in cost, simple in sequencing method, high in speed and high in accuracy.

Description

Technical Field

[0001] The present invention relates to a method for identifying Langya chickens and non-Langya chickens using SNP molecular markers, belonging to the technical fields of animal husbandry and molecular biology. Background Art

[0002] Agricultural germplasm resources are the material basis for the original innovation of agricultural science and technology and the development of modern seed industry. Identifying and evaluating high-quality and efficient germplasm resources provides a reliable basis for scientific protection and utilization, and is an important guarantee for the high-quality development of modern agriculture in the future.

[0003] Langya chickens are local characteristic chicken germplasm resources in Shandong Province, with advantages such as small body size, good meat flavor, high egg production, strong disease resistance, good adaptability, and high feed conversion rate. The hens of Langya chickens have sparrow feathers all over their bodies, and their feather colors are yellowish-brown or flax-yellow. They are small and solid in body, with small eyes and short legs, showing the characteristics of egg-laying chickens. Their eggs are known as "Langya eggs", which are famous for being "red-skinned, large-sized, and of good quality". The egg yolk content is high, accounting for 33.3% of the egg weight, with a strong color and fragrant smell, making them excellent dishes. The roosters have bright and harmonious feather colors all over their bodies, with high and thick legs, and they hold their heads high and stare, being famous for the "fiery red roosters". They are important local chicken resources in Shandong Province. By developing the Langya chicken variety identification technology, it lays a foundation for the identification, conservation, and genetic breeding of Langya chickens.

[0004] Gene detection technology has been widely used in the identification of animal and plant species because it can almost identify all biological materials. Gene detection technology for identifying animal and plant species is the fastest-developing and most accurate technical means in species identification. The traditional method of identifying chicken breeds based on morphological markers is greatly affected by environmental factors, and the results obtained through morphological identification are prone to errors. Applying molecular markers, a molecular biology means, can accurately identify chicken breeds based on specific variations between different chicken breeds. Summary of the Invention

[0005] In view of the above problems, the present invention provides a method for identifying Langya chicken breeds using SNP molecular markers. In order to achieve the above invention purpose, the technical solutions adopted by the present invention are as follows: Whole-genome resequencing is performed on 9 chicken breeds, 12 SNP sites unique to Langya chickens are screened out, and a Langya chicken breed identification model is established based on the combination of 12 SNP sites. The technical solutions of the present invention are as follows: An SNP molecular marker for identifying Langya chickens, and the specific SNP sites of the SNP molecular marker are shown in Table 1: Table 1 Langya chicken breed-specific SNP sites

[0006] Furthermore, PCR primers for amplifying the SNP molecular marker for identifying Langya chickens: 1_187885629F is as shown in SEQ ID NO.1; 1_187885629R is as shown in SEQ ID NO.2; 2_50761092F is as shown in SEQ ID NO.3; 2_50761092R is as shown in SEQ ID NO.4; 4_36833907F is as shown in SEQ ID NO.5; 4_36833907R is as shown in SEQ ID NO.6; 4_36866207F is as shown in SEQ ID NO.7; 4_36866207R is as shown in SEQ ID NO.8; 9_7892234F is as shown in SEQ ID NO.9; 9_7892234R is as shown in SEQ ID NO.10; 11_18456698F is as shown in SEQ ID NO.11; 11_18456698R is as shown in SEQ ID NO.12; 14_13682661F is as shown in SEQ ID NO.13; 14_13682661R is as shown in SEQ ID NO.14; 14_13707470F is as shown in SEQ ID NO.15; 14_13707470R is as shown in SEQ ID NO.16; 14_13755909F is as shown in SEQ ID NO.17; 14_13755909R is as shown in SEQ ID NO.18; 14_13763133F is as shown in SEQ ID NO.19; 14_13763133R is as shown in SEQ ID NO.20; 14_13771563F is as shown in SEQ ID NO.21; 14_13771563R is as shown in SEQ ID NO.21; 18_413573F is as shown in SEQ ID NO.23; 18_413573R is as shown in SEQ ID NO.24.

[0007] Furthermore, the present invention relates to the SNP molecular markers for identifying Langya chickens and their application in identifying Langya chickens.

[0008] The present invention also includes a method for identifying Langya chickens using SNP molecular markers, the steps of which are as follows: (1) Extract the genomic DNA of the sample and amplify it using the above PCR primers; The PCR reaction program is: 95°C for 10 min, 95°C for 30 s, 60°C for 30 s, 72°C for 50 s, for a total of 32 cycles; 72°C for 10 min; The PCR reaction system is 20 μl in total: 0.5 μl of template DNA, 0.5 μl of 10 pmol / μl upstream primer, 0.5 μl of 10 pmol / μl downstream primer, 10 μl of 2*Mastermix, 8.5 μl of ddH2O; (2) Perform allele sequencing on the amplification product using the direct sequencing method; (3) Obtain the gene table of the SNP corresponding sites of the sample to be tested; (4) Prepare the input file for analysis, convert the file into the plink format, and then convert the genotypes in the file into the 012 format using plink; (5) Input the data file into a randomly sampled training SVM (Support Vector Machine) model for prediction.

[0009] The present invention has the following advantages compared with the prior art: This patent overcomes the limitation of using experience to evaluate external characteristics to identify whether it is a Langya chicken. By using 12 specific SNP sites of Langya chickens, through PCR amplification and direct sequencing, site information is obtained, and machine learning is used for prediction, which has the advantages of low cost, simple sequencing method, fast speed, and high accuracy. Specific Embodiments

[0010] The following will further describe the present invention in combination with specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, the embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that without departing from the spirit and scope of the present invention, details and forms of the technical solutions of the present invention can be modified or replaced, but such modifications and replacements all fall within the protection scope of the present invention.

[0011] Example 1: Screening Breed-Specific SNPs 1. Experimental Materials Randomly select 20 Laiwu Black Chickens, 20 Langya Chickens, 20 Shouguang Chickens, 20 Wenshang Luhua Chickens (including 10 original Luhua Chickens and 10 directionally selected Luhua Chickens), 20 Luxi Gamecocks, 20 Jining 100-day Chickens, 20 Luxi Miniature Chickens, and 20 Recessive White Chickens from the Shandong Province Chicken Live Gene Bank and each Shandong Province local chicken purebred farm.

[0012] 2. Genome resequencing Collect blood from the wing veins of the experimental chickens. Total genomic DNA was extracted using the conventional phenol-chloroform method. The quality and quantity of the DNA were determined using a NanoDrop ND-2000 spectrophotometer and agarose gel electrophoresis. For each individual, a DNA library was constructed using the standard protocol of the Nextera DNA Library Preparation Kit (Illumina Inc., San Diego, CA, USA). All libraries were sequenced on the Illumina® HiSeq X 10 sequencing system with an average sequencing depth of 10X.

[0013] 3. Identification of Langya Chicken breed-specific SNPs Genomic information of 160 chickens was obtained through whole-genome resequencing. We used the genetic differentiation (Fst) between Langya Chickens and other chicken breeds (chicken breeds other than Langya Chickens) to identify the selected genomic regions in the Langya Chicken population. Considering the high genomic nucleotide diversity and heterozygosity of the chicken population, when calculating the Weir-Fst value for each window, we reduced the window size and step size to 40 kb and 10 kb, respectively. The top 1% of the windows were considered as the putative selected genomic regions.

[0014] For the SNPs in the selected genomic regions, we used an unbiased estimator of pairwise Fst-based simple summary statistics (di) to measure the locus-specific differences in allele frequencies for each breed. We calculated the statistics for the SNPs in each important window using the following formula:

[0015] Where and represent the expected value and standard deviation of Fst between breeds i and j calculated from 22,083 SNPs in 40 regions.

[0016] The top 1% of the outliers in the window were considered as the selected genomic SNPs. A total of 1142 breed-specific SNPs were screened through Di analysis. Then, LD pruning was performed on the 1142 SNPs with a window size of 25 SNPs and a step size of 5 SNPs, and the r 2 threshold was 0.2, resulting in 12 Langya Chicken breed-specific SNP markers as shown in Table 1: Table 1 Langya chicken breed-specific SNP loci

[0017] Example 2: Using molecular marker detection to distinguish Langya chicken from non-Langya chicken Test samples: 113 Langya chickens, 1060 background chickens (including 462 Shouguang chickens, 20 Laiwu black chickens, 10 Wenshang reed chickens, 10 new Wenshang reed chicken strains, 17 Jining 100-day chickens, 20 Luxi fighting chickens, 20 Luxi miniature chickens, 17 recessive white-feathered chickens, 341 Jingxing yellow chickens, 9 bearded chickens, 24 Daweishan miniature chickens, 21 Liao chickens, 21 Wuding chickens, 10 Tibetan chickens, 7 Zhuanghe large-bone chickens, 30 red jungle fowl, and 4 eight-clawed chickens). Blood was collected from the wing vein of the test chickens for genomic DNA extraction.

[0018] PCR Reaction and Sequencing Methods: PCR amplification was performed in a BioThermocycler using upstream and downstream primers. The PCR reaction program was: 95°C for 10 min, 95°C for 30 s, 60°C for 30 s, and 72°C for 50 s, for a total of 32 cycles; 72°C for 10 min. The PCR reaction system (20 μl) consisted of: 0.5 μl template DNA, 0.5 μl of 10 pmol / μl upstream primer, 0.5 μl of 10 pmol / μl downstream primer, 10 μl of 2* Master Mix, and 8.5 μl of ddH2O. Allelic sequencing of the amplified products was performed by direct sequencing. The primer information is as follows: 1_187885629F is shown in SEQ ID NO. 1; 1_187885629R is shown in SEQ ID NO. 2; 2_50761092F is shown in SEQ ID NO. 3; 2_50761092R is shown in SEQ ID NO. 4; 4_36833907F is shown in SEQ ID NO.5; 4_36833907R is shown in SEQ ID NO.6; 4_36866207F is shown in SEQ ID NO.7; 4_36866207R is shown in SEQ ID NO.8; 9_7892234F is shown in SEQ ID NO.9; 9_7892234R is shown in SEQ ID NO.10; 11_18456698F is shown in SEQ ID NO. 11; 11_18456698R is as shown in SEQ ID NO.12; 14_13682661F is as shown in SEQ ID NO.13; 14_13682661R is as shown in SEQ ID NO.14; 14_13707470F is as shown in SEQ ID NO.15; 14_13707470R is as shown in SEQ ID NO.16; 14_13755909F is as shown in SEQ ID NO.17; 14_13755909R is as shown in SEQ ID NO.18; 14_13763133F is as shown in SEQ ID NO.19; 14_13763133R is as shown in SEQ ID NO.20; 14_13771563F is as shown in SEQ ID NO.21; 14_13771563R is as shown in SEQ ID NO.22; 18_413573F is as shown in SEQ ID NO.23; 18_413573R is as shown in SEQ ID NO.24.

[0019] Prediction of Langya chickens and non - Langya chickens: Prepare the input file for analysis, convert the file to Plink format, and then use plink to convert the genotypes in the file to the 012 format.

[0020] Using 12 SNPs as prediction variables and Langya chickens or non - Langya chickens as predicted values, 70% of the individuals in the test samples are used as the training group for 20 - time random sampling to train the SVM (Support Vector Machine) model. The best value of the Cost hyperparameter is 1, and the best value of the Gamma parameter is 0.05. The results of the 20 - time sampling show that the prediction accuracy of 30% of the test population is 88.36%, which can effectively achieve the variety identification of Langya chickens.

Claims

1. A SNP molecular marker for Langya chicken identification, characterized in that, The specific SNP loci of the Langya chicken are as follows: 1_187885629 is located at position 187885629 on chromosome 1, and its deoxynucleotide is T; 2_50761092 is located at position 50761092 on chromosome 2, and its deoxynucleotide is C; 4_36833907 is located at position 36833907 on chromosome 4, and its deoxynucleotide is A; 4_36866207 is located at position 436866207, and its deoxynucleotide is C; 9_7892234 is located at position 97892234, and its deoxynucleotide is A; 11_18456698 is located at position 18456698 on chromosome 11, and its deoxynucleotide is C; 14_13682661 is located at position 13682661 on chromosome 14, and its deoxynucleotide is A; 14_13707470 is located at position 13707470 on chromosome 14, and its deoxynucleotide is C; 14_13755909 is located at position 14052713 on chromosome 14, and its deoxynucleotide is G; 14_13763133 is located at position 14101173 on chromosome 14, and its deoxynucleotide is A; 14_13771563 is located at position 14116818 on chromosome 14, and its deoxynucleotide is T; 18_413573 is located at position 413573 on chromosome 18, and its deoxynucleotide is T.

2. A SNP primer combination for identifying Langya chicken breed, characterized in that, The primer combination includes a primer set for amplifying all SNP loci in the SNP locus combination described in claim 1; The sequences of the primers for each SNP locus are as follows: 1_187885629F is as shown in SEQ ID NO.1; 1_187885629R is as shown in SEQ ID NO.2; 2_50761092F is as shown in SEQ ID NO.3; 2_50761092R is as shown in SEQ ID NO.4; 4_36833907F is as shown in SEQ ID NO.5; 4_36833907R is as shown in SEQ ID NO.6; 4_36866207F is as shown in SEQ ID NO.7; 4_36866207R is as shown in SEQ ID NO.8; 9_7892234F is as shown in SEQ ID NO.9; 9_7892234R is as shown in SEQ ID NO.10; 11_18456698F is as shown in SEQ ID NO.11; 11_18456698R is as shown in SEQ ID NO.12; 14_13682661F is as shown in SEQ ID NO.13; 14_13682661R is as shown in SEQ ID NO.14; 14_13707470F is as shown in SEQ ID NO.15; 14_13707470R is as shown in SEQ ID NO.16; 14_13755909F is as shown in SEQ ID NO.17; 14_13755909R is as shown in SEQ ID NO.18; 14_13763133F is as shown in SEQ ID NO.19; 14_13763133R is as shown in SEQ ID NO.20; 14_13771563F is as shown in SEQ ID NO.21; 14_13771563R is as shown in SEQ ID NO.21; 18_413573F is as shown in SEQ ID NO.23; 18_413573R is as shown in SEQ ID NO.

24.

3. Use of the SNP molecular marker according to claim 1 in identifying the breed of Langya chickens.

4. A method for identifying Langya chickens using SNP molecular markers, characterized in that, The steps are as follows: (1) Extract the genomic DNA of the sample and amplify it using the PCR primers according to claim 2; The PCR reaction program is: 95°C for 10 min, 95°C for 30 s, 60°C for 30 s, 72°C for 50 s, for a total of 32 cycles; 72°C for 10 min; The PCR reaction system is calculated as 20 μl: template DNA 0.5 μl, 10 pmol / μl upstream primer 0.5 μl, 10 pmol / μl downstream primer 0.5 μl, 2*Mastermix 10 μl, ddH2O 8.5 μl; (2) Use the direct sequencing method to sequence the alleles of the amplification product; (3) Obtain the gene table of the SNP corresponding site of the sample to be tested; (4) Prepare the input file for analysis, convert the file to Plink format, and then convert the genotypes in the file to the 0 1 2 format using plink; (5) Input the data file into the randomly sampled training SVM (Support Vector Machine) model for prediction.

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