11K SNP (Single Nucleotide Polymorphism) liquid chip for Sichuan local chicken and application of 11K SNP liquid chip

Abstract

The invention provides a Sichuan local chicken 11K SNP liquid phase chip and application thereof, the liquid phase chip is prepared from a Sichuan local chicken 11K SNP molecular marker combination, the Sichuan local chicken 11K SNP molecular marker combination is composed of 11747 SNP molecular markers, and chromosome positions and numbers of the 11747 SNP molecular markers on a chicken reference genome GRCg7b are shown in a specification table 1. The chip is high in detection flux, high in target site detection rate (99.91%) and accurate and reliable in typing result (the repetition consistency rate is 99.82%), and is a molecular marker chip with low density, high accuracy and low cost; the method can be used for chicken germplasm resource identification and improvement, genetic diversity evaluation, population structure analysis, genetic relationship identification, transmission map construction and QTL positioning, whole genome association analysis, molecular marker-assisted selection, whole genome selective breeding and the like.

Description

Technical Field

[0001] The invention relates to the technical field of gene chips, and in particular to a Sichuan local chicken 11K SNP liquid phase chip and application thereof. Background Art

[0002] According to the statistical results of the third national livestock and poultry genetic resources census, there are 14 local chicken breed resources in Sichuan. These genetic resources and varieties are the stamina and driving force for the development of the poultry industry in our province. However, due to the large-scale introduction of supporting systems by foreign breeding companies in recent years, especially the unplanned and large-scale hybridization of introduced varieties (lines) with local varieties, Sichuan's chicken breed resources have been severely damaged, the breeding scale has been greatly reduced, many varieties are heading towards extinction, and many high-quality and characteristic traits have also disappeared. At present, the situation of Sichuan chicken breed resources is both good and bad. The good news is that there are sporadic reports of newly discovered "breeds", and the worry is that a large number of varieties have or are heading towards extinction, and many high-quality and characteristic traits have also disappeared. Even because the resource situation is unclear, the newly discovered "breeds" may not necessarily be breeds in the true sense. Therefore, there is an urgent need for accurate identification of these varieties, so as to accurately understand the current status of chicken breed resources in Sichuan Province and lay a solid foundation for the protection, development and utilization of local breed resources. The essence of variety resource identification is the difference in markers between varieties. At present, the main markers are morphological markers, cytological markers, biochemical markers and molecular markers. The first three markers have disadvantages such as low density, low repeatability and low versatility. Morphological markers such as body shape and appearance are important manifestations of breed differences and are also one of the important characteristics of germplasm resources. However, the classification and identification of breeds based on body shape, feather skin color, etc. requires the identifier to have rich experience, and it is highly subjective and has a high misjudgment rate, which makes it difficult to promote and apply. Compared with morphological markers, molecular markers can directly reflect the differences in breeds at the DNA level, are stable and reliable, and have good polymorphism. However, for molecular markers that are sparsely distributed, the information provided for the genome is very limited.

[0003] In summary, it can be found that for molecular markers with sparse or single distribution, the information provided for the genome is very limited, so the number and range of molecular markers used for variety identification and evaluation or high-quality broiler breeding selection need to be expanded. Whole genome selection does not rely on phenotypic information, has higher selection accuracy and shortens the breeding cycle, but due to its high cost, it is only used in several poultry breeding companies such as Hubbard and Avigen; the detection cost also limits the application of chips such as Illumina 60K, Affymetrix 600K, and "Beijing Core No. 1" 55K. Therefore, screening a reasonable number of SNPs subsets with accurate variety classification from the whole genome or developing low-density dedicated breeding chips is an effective way to reduce the cost of accurate identification and breeding of poultry and other small animal varieties. With the continuous optimization of chip design, genotype filling and genomic selection technology, low-cost dedicated chips for important economic traits and specific target traits preferred by the market have better application prospects and will be the future development direction. Summary of the invention

[0004] In view of this, the present invention provides a Sichuan local chicken 11K SNP liquid phase chip and its application, aimed at the severe situation faced by Sichuan local chicken breed resources. The chip can be used for Sichuan local chicken germplasm resource identification, genetic diversity assessment, population structure analysis, kinship identification, breeding material genetic background analysis, etc.

[0005] In order to achieve the above technical objectives, the technical solutions adopted in this application are as follows:

[0006] In a first aspect, the present invention provides a Sichuan local chicken 11K SNP molecular marker combination, which consists of 11747 SNP molecular markers, and the chromosome positions and numbers of the 11747 SNP molecular markers on the chicken reference genome GRCg7b are shown in Table 1 of the specification.

[0007] Furthermore, the Sichuan local chicken breeds are selected from the following 14 types: Liangshan Rock Eagle Chicken, Jinyang Silky Chicken, Qiangshan Cloud Chicken, Tibetan Chicken, Pingwu Red Chicken, Pengxian Yellow Chicken, Miyi Chicken, Luning Chicken, Emei Black Chicken, Guangyuan Gray Chicken, Jiuyuan Black Chicken, Caoke Chicken, Heishui Phoenix Tail Chicken, and Sichuan Mountain Black-bone Chicken.

[0008] In a second aspect, the present invention provides the use of the Sichuan local chicken 11K SNP molecular marker combination and / or a probe for detecting the Sichuan local chicken 11K SNP molecular marker combination described in the first aspect in preparing a Sichuan local chicken 11K SNP molecular marker product.

[0009] Preferably, the Sichuan local chicken 11K SNP molecular marker product is a liquid phase chip or a kit.

[0010] The present invention also provides a Sichuan local chicken 11K SNP liquid phase chip or a Sichuan local chicken 11K SNP kit, comprising the Sichuan local chicken 11K SNP molecular marker combination described in the first aspect and / or a probe for detecting the Sichuan local chicken 11K SNP molecular marker combination.

[0011] The present invention also provides the use of the Sichuan local chicken 11K SNP liquid phase chip or the Sichuan local chicken 11K SNP kit in the identification and improvement of Sichuan local chicken germplasm resources.

[0012] The present invention also provides the use of the Sichuan local chicken 11K SNP liquid phase chip or the Sichuan local chicken 11K SNP kit in the genetic diversity assessment and / or genetic map construction and QTL positioning of Sichuan local chicken.

[0013] The present invention also provides the use of the Sichuan local chicken 11K SNP liquid phase chip or the Sichuan local chicken 11K SNP kit in the Sichuan local chicken whole genome association analysis.

[0014] The present invention also provides the use of the Sichuan local chicken 11K SNP liquid phase chip or the Sichuan local chicken 11K SNP kit in the molecular marker-assisted selection of Sichuan local chicken.

[0015] The present invention also provides the use of the Sichuan local chicken 11K SNP liquid phase chip or the Sichuan local chicken 11K SNP kit in the whole genome selection breeding of Sichuan local chicken.

[0016] Compared with the prior art, the present invention has the following excellent effects:

[0017] The present invention is based on the resequencing data of the rich diversity of Sichuan local chicken materials, and selects 11,747 molecular markers with strong representativeness, high polymorphism, good versatility and uniform distribution on chromosomes to form a liquid phase chip; the chip has high detection throughput, high target site detection rate (99.91%), and accurate and reliable typing results (repeat consistency rate is 99.82%); it is a low-density, high-accuracy, low-cost molecular marker chip; it can be used for chicken germplasm resource identification and improvement, genetic diversity assessment, population structure analysis, kinship identification, genetic map construction and QTL positioning, whole genome association analysis, molecular marker-assisted selection, whole genome selection breeding, etc.

[0018] Compared with other chips of the same type, this chip has rich functional loci, including 1,670 loci significantly associated with chicken growth traits, body weight, residual feed intake, muscle development, breast muscle weight, etc., and 1,900 QTL loci related to 169 traits such as chicken body weight, egg production, ketone bodies, muscle development, feed intake, age at first laying, feed conversion rate, breast muscle weight, average daily weight gain, femur length, comb morphology, reproduction, and disease. It can be used for molecular marker-assisted selection, important trait gene mining and identification, and functional analysis.

[0019] The chip contains 1,590 breed-specific loci of 14 local chicken breeds and can be used for identification of Sichuan local chicken germplasm resources, genetic diversity assessment, population structure analysis, kinship identification, and genetic background analysis of breeding materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0021] Figure 1 This is the distribution of Sichuan local chicken 11K chip loci on chromosomes.

[0022] Figure 2 This is the flow chart for the preparation of Sichuan local chicken 11K liquid phase chip.

[0023] Figure 3 It is the principle and process of cGPS liquid chip technology.

[0024] Figure 4 It is the genotype detection rate of Sichuan local chicken 11K chip locus in the sample.

[0025] Figure 5 It is the genotype consistency rate of repeated samples of Sichuan local chicken 11K chip.

[0026] Figure 6 This is the phylogenetic tree of 78 local chicken samples.

[0027] Figure 7 This is the PCA plot of 78 local chicken samples.

[0028] Figure 8 This is a population structure diagram of 78 local chicken samples. DETAILED DESCRIPTION

[0029] In the description of the present invention, it should be noted that, if the specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0030] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments, which are explanations of the present invention rather than limitations. Therefore, the following embodiments are only examples and cannot be used to limit the scope of protection of the present invention. In addition, it should be noted that, unless otherwise specified, the technical terms or scientific terms used in this application should be the usual meanings understood by those skilled in the art to which the present invention belongs.

[0031] Example 1

[0032] The design and preparation method of Sichuan local chicken 11K liquid phase chip is as follows:

[0033] 1.1 Obtaining the 11K molecular marker combination of Sichuan local chicken

[0034] (1) Genome-wide background sites

[0035] The selection of loci in the present invention is based on whole genome resequencing of 14 Sichuan local chicken breeds (Liangshan Rock Eagle Chicken, Jinyang Silky Chicken, Qiangshan Cloud Chicken, Tibetan Chicken, Pingwu Red Chicken, Pengxian Yellow Chicken, Miyi Chicken, Luning Chicken, Emei Black Chicken, Guangyuan Gray Chicken, Jiuyuan Black Chicken, Caoke Chicken, Heishui Fengwei Chicken, Sichuan Mountain Black-bone Chicken), and resequencing data of 121 diverse materials were obtained. The resequencing data analysis process is as follows:

[0036] Sentieon was used to align reads to the chicken reference genome GRCg7b (GCF_016699485.2), position sorting and marking duplicate reads. The variant sites were detected for each sample to obtain the variant information of each sample. The gVCF of all samples was jointly analyzed to obtain the variant results of each individual in the population. To ensure the accuracy of SNP, the SNP sites obtained after the joint analysis were initially hard filtered (SNP hard filtering standard: "QD<2.0||FS>60.0||MQ<40.0||SOR>3.0||MQRankSum<-12.5||ReadPosRankSum<-8.0").

[0037] The MAF value, detection rate, heterozygosity rate and sequencing depth of the loci were calculated and counted from the vcf file containing the SNP variation information of all samples, and polymorphic SNP loci with minimum allele frequency MAF ≥ 0.2, deletion rate < 0.1, heterozygosity rate ≤ 0.4, and sequencing depth ≥ 8X were selected as initial candidate loci. All the above candidate loci were used for probe design. The probe design method is to design probes within 100 bp upstream and downstream of each locus. The probe length is generally 100 bp and the GC content is between 20 and 80%. Probes that cannot be uniquely aligned on the genome and contain repetitive sequences in the flanking sequence are removed. According to the principles of uniform distribution of loci on chromosomes and preferred gene region loci, SNP loci uniformly distributed on chromosomes are selected as whole genome background loci, of which gene region loci account for 69.5%.

[0038] (2) Variety-specific loci

[0039] VCFtools-0.1.13 was used to calculate the Fst values ​​between the SNP loci of 14 Sichuan local chicken breeds and other broiler chicken breeds, and the loci with Fst>0.5 were selected. At the same time, according to the principle of uniform density distribution, 1,590 SNP loci with population quality indicators that met the minimum allele frequency MAF≥0.05, missing rate <0.1, and sequencing depth ≥8X were selected to distinguish the 14 chicken breeds.

[0040] (3) Significantly associated loci and QTL loci

[0041] The reported loci significantly associated with chicken growth traits, body weight, residual feed intake, muscle development, breast muscle weight, etc. were collected and screened, and the population indicators of these loci were extracted from the resequencing data. 1,670 high-quality significant trait association loci were retained, including 106 growth trait association loci, 740 body weight association loci, 624 residual feed intake association loci, 162 muscle development association loci, and 38 breast muscle weight association loci. The reported QTL loci associated with 169 traits such as chicken body weight, egg production, ketone bodies, muscle development, feed intake, age at first laying, feed conversion rate, breast muscle weight, average daily weight gain, femur length, comb morphology, reproduction, and disease were collected and screened, and the population indicators of these loci were extracted from the resequencing data. 1,900 high-quality QTL loci were retained.

[0042] 1.2 Preparation of chicken 11K SNP molecular marker combination

[0043] All marker sites obtained in Example 1.1 were integrated, and according to the quality indicators of these sites, 11,747 molecular markers with strong representativeness, high polymorphism, good versatility, and uniform distribution on chromosomes were finally screened, including 1,590 14 Sichuan local chicken breed-specific sites, 1,670 trait-significantly associated sites, and 1,900 QTL sites, which together constituted the Sichuan local chicken 11K liquid phase chip. The chromosome positions and numbers of the 11,747 SNP molecular markers on the chicken reference genome GRCg7b are shown in Table 1 of the specification.

[0044] Table 1 Information of 11747 SNP molecular markers

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[0264] The average spacing of Sichuan local chicken 11K liquid phase chip sites on chromosomes is 16.9Kb, and the 11K sites are evenly distributed on each chromosome. The density distribution diagram is shown in Figure 1 The 11K chip locus of Sichuan local chicken has a high polymorphism, and the average minor allele frequency (MAF value) is 0.34.

[0265] The 11,747 selected molecular markers were developed into Sichuan local chicken 11K liquid phase chip using the liquid phase probe precise positioning sequencing typing technology (Genotyping by Pinpoint Sequencing of liguid captured target, cGPS) independently developed by Huazhi. The preparation flow chart of Sichuan local chicken 11K liquid phase chip is as follows: Figure 2 As shown. cGPS liquid phase chip technology is based on an optimized thermodynamic stability algorithm model to design specific probes for target interval sequences, and uses synthetic specific probes to perform liquid phase hybridization capture and enrichment of multiple different target sequences located at different genomic locations, and then constructs libraries and performs second-generation sequencing on the captured and enriched target intervals to obtain the genotypes of the SNP / InDel sites in the target region.

[0266] Example 2: Genotyping process of Sichuan local chicken 11K liquid phase chip

[0267] This example provides a method for using the liquid phase chip in Example 1 to perform genotyping on chicken samples. The steps are as follows:

[0268] (1) Extraction and quality control of genomic DNA

[0269] The sample DNA was extracted by magnetic bead method and the quality of DNA samples was tested. The quality test included measuring DNA concentration with Qubit fluorescence quantification instrument and testing DNA integrity with 1% agarose gel electrophoresis. Samples that passed the quality test were used for library preparation.

[0270] (2) cGPS library construction and quality control

[0271] a. Use fragmentase to digest the DNA sample, repair the end of the digestion, add A base to the 3' end, and use agarose gel electrophoresis to detect the fragment size. b. Use T4 ligase to connect the sequencing adapter and DNA fragment, and use magnetic beads to purify the connection product. The concentration of the purified product is detected by Qubit fluorescence quantification instrument, and the fragment size is detected by agarose gel electrophoresis. c. Amplify and enrich the purified connection product, and use magnetic beads to screen the fragments of the amplified product. The concentration of the product after fragment screening is detected by Qubit fluorescence quantification instrument, and the fragment size is detected by agarose gel electrophoresis. d. Take 200ng of the completed library, add probes and hybridization reagents, and incubate at 50℃ for 16-24 hours to complete the hybridization reaction. Use magnetic beads to capture the target segment, use cleaning solution to clean the captured product, remove non-specific binding fragments, and then perform another round of PCR amplification. Use Qubit fluorescence quantification instrument to detect the library concentration, and agarose gel electrophoresis to detect the fragment size. After the concentration and fragment size are qualified, the construction of cGPS sequencing library is completed. The prepared library was sequenced using a BGI sequencer with a high-throughput sequencing strategy of PE150.

[0272] (3) Data analysis

[0273] The raw data after high-throughput sequencing was processed by quality control filtering, and the adapter fragments and low-quality reads were removed using FASTP software to obtain high-quality Clean Reads. The obtained Cleanreads were compared with the reference genome using BWA software, and the positions were sorted to obtain the bam file after the sample sorting. The sequencing results were analyzed for variant sites using GATK software to obtain the genotyping results of the target sites, as shown in Table 1. The principle and process of cGPS liquid phase chip technology are shown in Figure 3 .

[0274] Example 3: Evaluation of the Genotyping Effect of Sichuan Local Chicken 11K Liquid Phase Chip

[0275] To verify the genotyping effect of Sichuan local chicken 11K liquid phase chip, the liquid phase chip designed in Example 1 was used to perform genotyping tests on 104 chicken materials, including 4 technical replicates. The specific method of use is shown in Example 2.

[0276] After sequencing and data analysis, the site detection rate of 104 chicken materials was between 99.82% and 99.98%, with an average detection rate of 99.91%. Figure 4 ; The genotype consistency of technical replicate samples was between 99.71% and 99.89%, with an average consistency of 99.82%. Figure 5 .

[0277] Among the 104 chicken materials, 20 were resequencing samples. The cGPS liquid phase chip results of the 20 resequencing samples were compared with the resequencing results. The genotype consistency rate was between 95.07% and 98.11%, and the average consistency rate was 96.50%.

[0278] The above data show that when the Sichuan local chicken 11K SNP liquid phase chip is used for genotyping of the test materials, the target site detection rate is high, the stability is good, and the typing results are accurate and reliable.

[0279] Example 4: Application of Sichuan local chicken 11K molecular marker combination in genetic diversity assessment processes such as chicken material kinship identification and population structure analysis

[0280] The Sichuan local chicken 11K liquid phase chip prepared in Example 1 and the genotyping process of Example 2 were used to identify the genotypes of 78 local chicken materials (27 Tibetan chickens, 10 Pengxian yellow chickens, 9 Caoke chickens, 9 Guangyuan gray chickens, 9 Pingwu red chickens, 8 Liangshan rock eagle chickens, and 6 Jiuyuan black chickens). The genetic distance matrix of the 78 local chicken materials was calculated using Plink software and cluster analysis was performed. The results showed that the 78 local chicken materials were mainly divided into 7 subgroups, which was consistent with their actual grouping, such as Figure 6 shown.

[0281] Plink software was used to analyze the PCA components of 78 local chicken materials and construct a PCA scatter plot. Each site in the scatter plot represents a sample. The farther the distance between two samples in the plot, the greater the difference in the genetic background of the two samples. Individuals with similar genetic backgrounds will be clustered into one category in the plot. The results showed that the genetic backgrounds of Tibetan chickens, Pengxian yellow chickens, and Liangshan rock eagle chickens were quite different, while the genetic backgrounds of grass chickens, Guangyuan gray chickens, Pingwu red chickens, and Jiuyuan black chickens were relatively small. Figure 7 Admixture was used to analyze the population structure of local chicken materials. The analysis results were consistent with the PCA results. There were subpopulation hybridizations in Tibetan chickens and Liangshan rock eagle chickens. Figure 8 shown.

[0282] This indicates that the 11K molecular marker combination of Sichuan local chicken is highly representative and can be used to determine the kinship, evolutionary relationship, and composition structure of different materials.

[0283] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A Sichuan local chicken 11K SNP molecular marker combination, characterized in that: The Sichuan local chicken 11K SNP molecular marker combination consists of 11,747 SNP molecular markers, and the chromosome positions and numbers of the 11,747 SNP molecular markers on the chicken reference genome GRCg7b are shown in Table 1 of the specification.

2. The Sichuan local chicken 11K SNP molecular marker combination according to claim 1, characterized in that: The Sichuan local chicken breeds are selected from the following 14 types: Liangshan Rock Eagle Chicken, Jinyang Silky Chicken, Qiangshan Cloud Chicken, Tibetan Chicken, Pingwu Red Chicken, Pengxian Yellow Chicken, Miyi Chicken, Luning Chicken, Emei Black Chicken, Guangyuan Gray Chicken, Jiuyuan Black Chicken, Caoke Chicken, Heishui Phoenix Tail Chicken, and Sichuan Mountain Black-bone Chicken.

3. Use of the Sichuan local chicken 11K SNP molecular marker combination according to claim 1 or 2 and / or a probe for detecting the Sichuan local chicken 11K SNP molecular marker combination according to claim 1 or 2 in preparing a Sichuan local chicken 11K SNP molecular marker product.

4. A Sichuan local chicken 11K SNP liquid phase chip or a Sichuan local chicken 11K SNP kit, characterized in that: The invention comprises the Sichuan local chicken 11K SNP molecular marker combination as claimed in claim 1 or 2 and / or a probe for detecting the Sichuan local chicken 11K SNP molecular marker combination as claimed in claim 1 or 2.

5. Use of the Sichuan local chicken 11K SNP liquid phase chip or the Sichuan local chicken 11K SNP kit according to claim 4 in the identification and improvement of Sichuan local chicken germplasm resources.

6. Use of the Sichuan local chicken 11K SNP liquid phase chip or the Sichuan local chicken 11K SNP kit according to claim 4 in the genetic diversity assessment and / or genetic map construction and QTL positioning of Sichuan local chicken.

7. Use of the Sichuan local chicken 11K SNP liquid phase chip or the Sichuan local chicken 11K SNP kit according to claim 4 in Sichuan local chicken genome-wide association analysis.

8. Use of the Sichuan local chicken 11K SNP liquid phase chip or the Sichuan local chicken 11K SNP kit according to claim 4 in molecular marker-assisted selection of Sichuan local chicken.

9. Use of the Sichuan local chicken 11K SNP liquid phase chip or the Sichuan local chicken 11K SNP kit according to claim 4 in Sichuan local chicken whole genome selection breeding.

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