A molecular marker for purification and breeding of Loba chicken and its application

Abstract

The present invention belongs to the technical field of biomolecules, and specifically relates to a molecular marker for purification and breeding of Loba chicken and its application. By collecting chicken breeds of different geographical origins and genetic backgrounds for population genomics research, the characteristic genetic background of pure line of Loba chicken is identified by using population genetic structure analysis, and characteristic SNP markers are mined therefrom, and two loci of Chr2:46800892 (GRCg7b) and Chr5:58219282 (GRCg7b) are screened out for accurate identification of pure line individuals and non-pure line individuals in the Loba chicken population. The operation is simple, the reproducibility is good, and the accuracy is high, which effectively overcomes the problems of high cost and long time period when molecular purification and breeding of Loba chicken is carried out based on whole genome resequencing or gene chip.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomolecules, and in particular relates to a molecular marker for the purification and breeding of Loba chicken and its application. Background Art

[0002] The Luoba chicken is another representative local chicken breed of the Qinghai-Tibet Plateau, besides the Tibetan chicken. It is light and small, with a long, low body, well-proportioned and compact, with a high head and low tail, forming a boat-shaped pattern. Its pectoral muscles are well-developed and protrude forward, making it a lively, nervous, and aggressive breed. Its wing and tail feathers are well-developed, making it an adept soarer. The male's sickle feathers can reach up to 40-60 cm in length. Its head is delicate, with a single red crest, often with a bean-shaped crest. The male's single crest is large and upright, with four to six teeth. Hens have a smaller crest with crest feathers. Their wattles are red, and their beaks are mostly black, with a few being flesh-colored or yellow. Their iridescence is often orange, with a yellowish-chest coloration to a lesser extent. Their earlobes are mostly white, with a few being red and white. Their shanks are mostly black, with a few flesh-colored, and a few have shank feathers, resulting in a complex plumage pattern.

[0003] Currently, genetic evaluation of germplasm based on genome-wide SNP molecular markers, including population genetic diversity analysis, systematic relationship analysis, population genetic structure analysis, inbreeding coefficient assessment, and gene flow analysis, is very mature. However, obtaining all SNP molecular markers within the entire genome region is expensive and time-consuming, which seriously limits its large-scale application in germplasm genetic evaluation. At the same time, there are currently no simplified molecular markers for germplasm purification within the Loba chicken population. Therefore, discovering characteristic molecular markers of pure-line Loba chicken populations and conducting molecular marker-assisted breeding for Loba chickens are of great significance for the efficient purification of Loba chickens.

[0004] In 2021, the Zhuhai Modern Agriculture Development Center, the Institute of Animal Science of the Guangdong Academy of Agricultural Sciences, and the Tibet Yuelian Milin Tibetan Chicken Development Co., Ltd. collected individuals that met the characteristics of the Luoba chicken population from individual households in Milin County, Nyingchi. The Institute of Animal Science of the Guangdong Academy of Agricultural Sciences conducted off-site conservation of Luoba chickens (the conservation site is located at No. 1, Dafeng 1st Street, Tianhe District, Guangzhou), and simultaneously conducted genetic evaluation and performance testing of the Luoba chicken germplasm. Considering that some individuals in the Luoba chicken conservation population have genes introgressed from other local chicken breeds or commercial chicken breeds into the conservation population, the purification and breeding of Luoba chickens is imperative. Summary of the Invention

[0005] In view of the above problems, the purpose of the present invention is to provide a molecular marker for the purification and breeding of Loba chicken and its application.

[0006] The technical contents of the present invention are as follows:

[0007] The present invention provides an application of a SNP molecular marker in the purification and breeding of Luoba chicken, wherein the SNP molecular marker is two polymorphic sites, Chr2:46800892 and Chr5:58219282, on the ENSEMBL GRCg7b genome (https: / / asia.ensembl.org / Gallus_gallus / Info / Index?db=core;r=4:75408776-75408786);

[0008] The SNP molecular marker is used to identify pure line individuals and non-pure line individuals of Luoba chicken;

[0009] The polymorphic site of Chr2:46800892 is C / A;

[0010] The polymorphic site of Chr5:58219282 is A / G;

[0011] The physical location of the site is GRCg7b (GCA_016699485.1) version.

[0012] When the genotype of Chr2:46800892 is CC and the genotype of Chr5:58219282 is AA, it is identified as a pure line Luoba chicken individual;

[0013] When the genotype of Chr2:46800892 is AA and the genotype of Chr5:58219282 is GG, it is identified as a non-pure line Luoba chicken individual;

[0014] When the genotype of Chr2:46800892 is AC, and / or the genotype of Chr5:58219282 is AG, and one or both of them are heterozygous, the individual is identified as a non-pure line Luoba chicken.

[0015] The present invention also provides a method for purifying and breeding Loba chicken, comprising the following steps:

[0016] 1) Extracting genomic DNA from the germplasm resources of the Luoba chicken population to be identified;

[0017] 2) Perform PCR amplification to determine the genotypes of the two sites Chr2:46800892 and Chr5:58219282 on the extracted genomic DNA;

[0018] The obtained genotypes were used to identify the chicken population according to the above identification method;

[0019] When the genotype of Chr2:46800892 is CC and the genotype of Chr5:58219282 is AA, it is identified as a pure line Luoba chicken individual;

[0020] When the genotype of Chr2:46800892 is AA and the genotype of Chr5:58219282 is GG, it is identified as a non-pure line Luoba chicken individual;

[0021] When the genotype of the Chr2:46800892 locus is AC, and / or the genotype of the Chr5:58219282 locus is AG, and one or both of them are heterozygous, the individual is identified as a non-pure line Luoba chicken.

[0022] The beneficial effects of the present invention are as follows:

[0023] The molecular markers for the purification and breeding of Loba chickens of the present invention are prepared by collecting chicken breeds of different geographical origins and genetic backgrounds for population genomics research, using population genetic structure analysis to identify the characteristic genetic background of the pure line of Loba chickens, mining characteristic SNP markers therefrom, and screening out two sites, Chr2:46800892 (GRCg7b) and Chr5:58219282 (GRCg7b), for accurately identifying pure line individuals and non-pure line individuals in a Loba chicken population. The molecular markers of the present invention are simple to operate, have good reproducibility, and high accuracy, and effectively overcome the problems of high cost and long time period in the molecular purification and breeding of Loba chickens based on whole genome resequencing or gene chips. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is an individual characteristic diagram of an adult Luoba chicken;

[0025] Figure 2 This is the Admixture genetic mixture analysis result diagram of Luoba chicken and other chicken breeds / groups in the optimal genetic grouping (K=K);

[0026] Figure 3 This is the genotype sequencing peak diagram of the identification sites of pure and non-pure individuals in the Luoba chicken population. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below through specific implementation cases and accompanying drawings. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention. After reading the present invention, various equivalent modifications of the present invention by those skilled in the art are all within the scope of the claims attached to this application.

[0028] Unless otherwise specified, all raw materials and reagents in the present invention are raw materials and reagents from conventional markets.

[0029] Example

[0030] Screening of characteristic SNP molecular markers for pure line individuals in a Luoba chicken population

[0031] 1. Blood Sample Collection

[0032] A total of 125 chickens of different geographical origins and genetic backgrounds were selected as experimental subjects, including 10 Luoba chickens, 86 other local chickens (10 Huaixiang chicken A line, 10 Huaixiang chicken B line, 9 Huaixiang chicken C line, 6 Huiyang bearded chickens, 6 Hetian chickens, 6 white-eared yellow chickens, 6 Xianju chickens, 6 Liyang chickens, 6 Yunyang large chickens, 6 Jining 100-day chickens, 6 Lindian chickens, 6 Turpan fighting chickens and 3 Laotian fighting chickens), 24 commercial broilers and laying hens (6 recessive white-feathered Rock chickens, 6 Ross 308 broilers, 6 Luodao Red chickens and 6 White Leghorn chickens) and 5 red jungle fowl individuals;

[0033] The appearance characteristics of adult Luoba chicken are as follows Figure 1 As shown, Figure 2 This is a genetic admixture analysis of the above chicken breeds / populations at the optimal genetic grouping, K = 5. HB represents the Huiyang Bearded Chicken, BEH represents the White-Eared Yellow Chicken, BR represents the Jining Hundred-Day Chicken, Laos represents the Laotian Fighting Chicken, LD represents the Lindian Chicken, LH represents the White Leghorn Chicken, LY represents the Liyang Chicken, RIR represents the Luodao Red Chicken, RJF represents the Red Jungle Fowl, RS represents the Ross 308 Broiler, TLF represents the Turpan Fighting Fowl, WRR represents the Recessive White-Feathered Rock Chicken, XJ represents the Xianju Chicken, YY represents the Yunyang Large Chicken, and HT represents the Hetian Chicken. HX_A, HX_B, and HX_C represent three different conservation lines of Huaixiang Chicken, and LB represents the Luoba Chicken. It can be observed that individuals LB05, LB07, LB08, LB09, and LB10 contain genetic components from both commercial and other local breeds.

[0034] 1 mL of sterile blood was collected from the wing vein of each individual, mixed with sodium citrate anticoagulant, and stored at -80°C until use. Sample information is shown in Table 1. Genomic DNA was extracted from the samples using the phenol-chloroform method.

[0035] Table 1 Experimental sample information

[0036]

[0037]

[0038] 2. Whole-genome resequencing and SNP variation detection

[0039] Whole-genome sequencing of the 125 chickens was performed using a high-throughput, next-generation sequencing platform. Whole-genome resequencing of 125 chickens from 19 populations was performed using the Illumina High-seq 2000 or 4000 platform (Illumina Inc, USA) to generate raw sequencing reads. Sequences containing adapters, N content ratios greater than 10, and low-quality base content (Q ≤ 5) greater than 50% were further removed from the raw sequencing reads to generate clean reads for subsequent genome alignment and genetic variation detection (Table 2).

[0040] Table 2 Statistics of whole-genome resequencing data of 125 chickens

[0041]

[0042]

[0043]

[0044] Clean reads from each individual were aligned to the chicken reference genome (Ensembl GRCg7b) using BWA version 0.7.15 to generate a SAM file for each individual. Furthermore, the SAM file for each individual was filtered and sorted using Picard version 2.9.0 to generate an indexed BAM file for each individual, and repetitive reads were also marked. For SAMtools calling, the SAMtools summary package with default parameters was used to call raw SNPs and raw indels. Quality control of the resulting population SNPs was performed using VCFtools version 0.1.14.

[0045] 3. Data quality control

[0046] The quality control standards are:

[0047] 1) max-missing 0.1;

[0048] 2) --maf 0.05;

[0049] 3)--minQ 20;

[0050] 4) --min-meanDP 5;

[0051] 5) max-meanDP 1000;

[0052] 6)--minGQ 5.

[0053] 4. Population genetic structure-Admixture analysis

[0054] Admixture version 1.3.0 software was used to perform genetic admixture analysis on 19 groups of 125 chickens. The cross-validation errors (CV) corresponding to the K value were calculated. The K value with the smallest CV value was the optimal grouping number. The results showed that the CV value was the smallest when K = 5.

[0055] Admixture analysis showed that when the optimal genetic grouping K=5, some individuals of Loba chicken (LB05, LB07, LB08, LB09, LB10) were significantly affected by the introgression of commercial chicken genes, while the genetic components of Loba chicken LB01, LB02, LB03, LB04, and LB05 were relatively simple.

[0056] 5. Specific site screening

[0057] Selection signal analysis was performed on the autosomal SNPs after quality control. The genetic differentiation coefficient (Fst) of each SNP locus was calculated using VCFtools version 0.1.14. With Fst = 0 as the standard, SNP marker loci that were completely differentiated between the two populations were mined. The results are shown in Table 3:

[0058] Table 3 SNP sites with complete genetic differentiation between pure-line and non-pure-line Luoba chicken populations

[0059] physical location genetic differentiation coefficient Purebred Luoba chicken Non-pure Luoba chicken 2:46800892 1 CC AA 5:58219282 1 AA GG

[0060] As can be seen above, a total of two specific SNPs were identified, namely Chr2:46800892 and Chr5:58219282. The genotypes of the pure-line Loba chicken individuals at these two loci were CC and AA, respectively, while those of the non-pure-line Loba chicken individuals were AA and GG. In the actual identification process, the genotypes of the two loci may be heterozygous, namely AC and AG. If one or both of them are heterozygous, the individual is considered to be a non-pure-line Loba chicken.

[0061] Example 2

[0062] Molecular marker combinations and their applications in Loba chicken

[0063] 1. Blood Sample Collection

[0064] Blood was randomly collected from 10 Luoba chickens at the original breeding farm of the Institute of Animal Science, Guangdong Academy of Agricultural Sciences, added with sodium citrate anticoagulant, mixed, and stored at -80°C for later use.

[0065] Genomic DNA was extracted from the samples using the phenol-chloroform method.

[0066] 2. SNP marker detection of breed-specific genes in Luoba chicken

[0067] Primer design: The SNP sites of the Luoba chicken breed characteristic genes screened were located on the chromosomes in the reference genome to obtain a sequence containing these SNPs sites.

[0068] Chicken genomic DNA was used as a template and PCR amplification was performed using primers designed from the NCBI website (see Table 4).

[0069] Table 4 Primer pair information for identifying Luoba chicken breeds

[0070]

[0071] 3. PCR amplification and detection:

[0072] The PCR reagents, methods, and reaction procedures were all purchased from Acryl Biotech Co., Ltd. (Changsha, China). The reaction system and reaction conditions are shown in Tables 5 and 6, respectively. After the PCR amplification reaction procedure was completed, the samples were stored at 4°C until use and sent for testing.

[0073] Table 5 PCR reaction system

[0074] Component name Final reaction concentration Addition amount 2X Pro Taq Master Mix(dye plus) 1X 25 μL Template ≤500ng - Primer F (10 μM) 0.2μM 1 μL Primer R (10μM) 0.2μM 1 μL RNase-free water - Add to 50 μL

[0075] Note: When using the 2X Pro Taq Master Mix (dye plus) for the first time, centrifuge it before use to avoid enzyme loss.

[0076] Generally, the recommended template addition amount is no more than 500ng; the template amount can be adjusted according to actual needs; the final concentration of primers is usually 0.2μM, which can be adjusted within the range of 0.2-1.0μM according to experimental results;

[0077] The above reaction system needs to be prepared on ice, and finally the prepared reaction solution is placed in a PCR instrument for reaction.

[0078] Table 6 PCR reaction conditions

[0079]

[0080] The results of Luoba chicken breed identification are shown in Table 7.

[0081] Table 7 Genotype identification of Luoba chicken breeds

[0082]

[0083]

[0084] like Figure 3Shown are the genotype sequencing peaks of the identification sites of the pure and non-pure individuals in the Luoba chicken population. According to the method of the present invention, when the genotypes of the two sites on Chr2:46800892 and Chr5:58219282 of the Luoba chicken individual are CC and AA respectively, it is judged to be a pure Luoba chicken individual. From Table 7, it can be judged that the individual with sample number LB7 is a pure Luoba chicken individual.

Claims

1. An application of SNP molecular markers in the purification and breeding of Loba chicken, characterized in that: The SNP molecular markers are two polymorphic sites, Chr2:46800892 and Chr5:58219282, on the ENSEMBL GRCg7b, version GCA_016699485.1 genome; The polymorphic site of Chr2:46800892 is C / A; The polymorphic site of Chr5:58219282 is A / G.

2. The use of the SNP molecular marker according to claim 1 in the purification and breeding of Loba chicken, characterized in that: The SNP molecular marker is used to identify pure line individuals and non-pure line individuals of Loba chicken.

3. The use of the SNP molecular marker according to claim 1 in the purification and breeding of Loba chicken, characterized in that: When the genotype of Chr2:46800892 is CC and the genotype of Chr5:58219282 is AA, it is identified as a pure line Loba chicken individual.

4. The use of the SNP molecular marker according to claim 1 in the purification and breeding of Loba chicken, characterized in that: When the genotype of Chr2:46800892 is AA and the genotype of Chr5:58219282 is GG, it is identified as a non-pure line Loba chicken individual.

5. The use of the SNP molecular marker according to claim 1 in the purification and breeding of Loba chicken, characterized in that: When the genotype of Chr2:46800892 is AC, and / or the genotype of Chr5:58219282 is AG, and one or both are heterozygous, the individual is identified as a non-pure line Loba chicken.

6. A method for purifying and breeding Loba chicken, characterized in that: The steps include: 1) Extract genomic DNA from the chickens to be identified; 2) Perform PCR amplification to determine the genotypes of the two loci Chr2:46800892 and Chr5:58219282 on the extracted ENSEMBL GRCg7b, version GCA_016699485.1 genomic DNA; 3) Use the obtained genotypes to identify the chicken population; When the genotype of Chr2:46800892 is CC and the genotype of Chr5:58219282 is AA, it is identified as a pure line Loba chicken individual; When the genotype of Chr2:46800892 is AA and the genotype of Chr5:58219282 is GG, it is identified as a non-pure line Loba chicken individual; When the genotype of Chr2:46800892 is AC, and / or the genotype of Chr5:58219282 is AG, and one or both are heterozygous, the individual is identified as a non-pure line Loba chicken.

7. The method for purifying and breeding Loba chicken according to claim 6, characterized in that: Step 2) In the PCR amplification, the upstream and downstream primer sequences used for genotyping the Chr2:46800892 site are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively; The upstream and downstream primer sequences used for genotyping the Chr5:58219282 site are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

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