CN117089633A - Molecular marker combination for analyzing goat hair performance and application - Google Patents
Molecular marker combination for analyzing goat hair performance and application Download PDFInfo
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Abstract
The application discloses a molecular marker combination for analyzing goat performance and application thereof, relates to the field of biotechnology, and provides 948 molecular markers capable of analyzing goat performance, wherein physical position information of the 948 molecular markers is determined based on comparison of genome sequences of goat reference genome ARS1, and a molecular probe combination, a gene chip and a kit prepared from 948 SNP molecular markers can be used for carrying out genetic evaluation on goat individuals, and individual selection is carried out on the goat performance which is difficult to measure in early stage, so that generation interval is shortened, breeding process is accelerated, and a large amount of breeding cost is saved.
Description
Technical Field
The application relates to the technical field of biology, in particular to the technical field of biological detection, and more particularly relates to a goat hair performance SNP locus combination and application thereof.
Background
During the natural environment selection or artificial breeding process, the species can gradually form unique appearance morphology and body type characteristics. According to statistics, 69 goat breeds exist in China at present, and 58 goat breeds exist in China. Under the environment of complex environment and accurate feeding, the rapid and efficient selection of goats with stable wool production performance has important significance, and aiming at goats with various varieties and changeable characters at present, how to rapidly screen the goats, and whether goats are wool goats or not is a problem to be solved urgently, therefore, the design of the SNP chip which is suitable for Chinese goats and can rapidly and effectively detect wool characters has very important significance.
Disclosure of Invention
In order to meet the requirements of the current goat variety research and agricultural production in China on the performance detection of goat, the application provides a molecular probe combination, a gene chip, a kit and application for analyzing the performance of goat, and the site information provided by the application can be used for rapidly and accurately realizing the performance evaluation, variety screening, variety identification, variety tracing and goat breeding of goat, is favorable for germplasm resource protection and germplasm resource improvement, and has the advantages of short time consumption, low cost and wide market benefit.
In order to achieve the technical purpose of the application, the application provides the following technical scheme:
in a first aspect, the present application provides an application of 948 SNP site combinations in analysis of goat hair performance, wherein the physical positions of the 948 SNP site combinations are shown in table 1:
TABLE 1 position information for 948 position combinations
Its physical location was determined based on goat reference genome ARS1 genome sequence alignment.
A second aspect provides a method of analyzing goat performance by comparing 948 SNP locus genotypes of genomic DNA of a goat to be tested with the 948 SNP locus genotypes of genomic DNA of a control goat;
wherein the 948 SNP loci are 948 SNP loci described in Table 1.
In a third aspect, a molecular probe set for analyzing goat hair performance is provided, wherein the molecular probe set detects SNP site combinations as shown in Table 1 in a sample to be tested, and the physical position information of the site combinations in Table 1 is determined based on goat reference genome ARS1 genome sequence alignment.
The fourth aspect is a gene chip for analyzing goat hair performance, the gene chip being loaded with the molecular probe set according to the third aspect.
The fifth aspect is a kit for analyzing goat hair performance, comprising the molecular probe set according to the third aspect or the gene chip according to the fourth aspect.
The sixth aspect is a method for analyzing goat hair performance, which uses the molecular probe combination of the third aspect or the gene chip of the fourth aspect or the kit of the fifth aspect to detect a sample to be detected.
The molecular probe set according to the seventh aspect, the third aspect or the gene chip according to the fourth aspect or the kit according to the fifth aspect has any of the following uses:
(1) Application in performance evaluation of goat hair;
(2) Application in goat variety screening;
(3) Application in goat variety identification;
(4) Application in the tracing of goat breeds;
(5) Application in goat breeding;
(6) Application in germplasm resource protection;
(7) Application in germplasm resource improvement;
(8) The application in goat pedigree reconstruction.
Advantageous effects
1. The SNP locus combination for analyzing goat performance, which is composed of 948 SNP loci only, is provided based on the research of genetic resources of a plurality of goats at home and abroad, has good domestic and foreign universality, can rapidly evaluate the goat performance which cannot be shown at early stage from the gene level, acquires more accurate breeding evaluation information, controls the breeding process, can screen, identify and trace the goat variety by utilizing the locus combination, and provides technical support for germplasm resource protection and germplasm resource improvement.
2. The probe combination, the gene chip and the kit for analyzing the goat hair performance provided by the application have the characteristics of small flux, low cost and easier analysis, and have wide universality and wide market prospect.
Drawings
FIG. 1 is a Manhattan diagram of a goat breeder for nap and a goat breeder for non-nap for elimination analysis;
fig. 2 is a graph of the results of the present application for performing a significance test on the determination results of population threshold analysis.
Detailed Description
The application will be further elucidated with reference to the following detailed description of specific embodiments, which are merely illustrative and are not to be construed as limiting the application. Unless otherwise indicated, the techniques used in the examples are conventional and well known to those skilled in the art, and may be carried out with reference to the "bioinformatics and functional genomics" original third edition or related books, and the bioinformatics software and products used are also commercially available. The various processes and methods not described in detail are conventional methods known in the art, the source of the materials used, the trade names and those necessary to list the constituents are all indicated at the first occurrence, and the same reagents used thereafter, unless otherwise indicated, are all the same as the first indicated.
In addition, it should be noted that the site combination and application provided by the application are accomplished by the inventor through hard creative work and optimization work.
The features and advantages described in the previous site combination section herein are equally applicable to the combination of molecular probes formed based on site combination, gene chips, kits and uses thereof, and are not described in detail herein.
The goat wool performance referred to in the present application means goat wool and is classified according to the presence or absence of wool.
The SNP referred to in the present application means a single nucleotide polymorphism (Single Nucleotide Polymorphism), mainly means a DNA sequence polymorphism caused by a variation of a single nucleotide including a variation caused by a single base transition, a transversion, an insertion or a deletion at the genome level.
It should be noted that the molecular marker of the present application is any heritable and detectable DNA sequence or protein, including, but not limited to, molecular markers based on molecular hybridization, such as RFLP, minisatelliteDNA; molecular markers based on PCR technology, such as RAPD, STS, SSR and SCAR; DNA markers based on restriction and PCR techniques; molecular markers based on DNA chip technology, such as SNPs; analytical marking techniques developed based on EST databases, and the like. The molecular marker provided by the application can be used for genome mapping, gene localization research, gene cloning based on a map, species genetic relationship, system classification and the like.
The probe referred to in the present application is a nucleic acid sequence (DNA or RNA) with a known sequence complementary to the target gene, such as Taqman-MGB probe.
It should be noted that the kit of the present application is any one of the kits conventionally used in the art, which contains reagents used for detection or experiment, and is convenient for operators to get rid of the heavy reagent preparation and optimization process. In one embodiment of the application, the kit comprises a primer for amplifying the site information provided by the application, a molecular marker or a probe or a gene chip for detecting the site information provided by the application, an enzyme and a buffer used for amplification, or a fluorescent marker used for detection.
Example 1 acquisition of wool Performance SNP site combinations
1. Selection of goat individuals
In order to realize more comprehensive coverage of domestic and foreign goat breeds, 361 individuals of 68 goat breeds in the global scope are re-sequenced, and the specific breeds are as follows:
the preparation method comprises the following steps of, menabe coat Mei Nabei Goat.
It should be noted that, some kinds of goats have no formal Chinese translations, and English names are still used at present.
2. Acquisition of Total SNP set of goat Whole Gene
The sample carrying genetic information from the individual goat in step 1 is collected using methods conventional in the art, including but not limited to blood, cells, tissue, skin, hair, excrement, etc. Genetic information (such as DNA) in the sample is extracted for high-depth sequencing, and comparison is carried out on a reference genome (obtained from NCBI) of a goat published in 2016 by using two modes of SAMtools and GATK, and a common result obtained by the comparison of the two modes forms a SNP set which contains 17,668,737 single nucleotide variations at the whole genome level. 82 goats related to villus were selected from these breeds.
The genetic information (genetic information) referred to herein refers to information that an organism is to replicate the same thing as itself, is transferred from a parent to a progeny, or is transferred from a cell to a cell each time each cell divides.
It should be noted that, the high-depth sequencing by extracting genetic information (e.g., DNA) in the sample may be performed by a biological company, such as hua da gene company, illuminea company, etc., and the high-depth sequencing method is a conventional method in the art or a method of the biological company, and in one embodiment of the present application, the high-depth sequencing is performed by using an average sequencing depth of about 25.7×, and using a re-sequencing analysis procedure.
3. Screening of candidate genes and located functional regions
Grouping according to the remarkable differences of goat varieties in wool traits worldwide, summarizing all varieties of individuals with and without wool, and performing selective elimination analysis by dividing the wool-use goat varieties Pak-angora goat (Pakistan Angola goat), liaoning cashmere goat (Liaoning down goat), cashmere goat (down goat), white chanthangi pashimina goat (Baiqiang pond down goat) and the non-appearance goat Toggenburg goat (ipenser goat) into two groups.
The application scans the functional area related to the wool by calculating the nucleotide base polymorphism and locus allele frequency difference between pi value and fst value and scans the functional area related to the wool by taking intersection of the calculation results of the two methods, screens out the functional area related to the existence of the wool, generates Manhattan diagram as shown in figure 1 according to the different groups of the existence of the wool by pi value, screens the genes in the area by referring to the published gene research results, and finally determines 4 candidate genes with quite definite functions and related to wool performanceBLZF1,TUBB2A,DLGAP5,CMTM4. And then determining the functional region corresponding to the candidate gene by perl script.
4. Acquisition of SNP site combinations for analysis of villus
Searching SNP loci corresponding to the functional regions of the candidate genes determined in the step 3 in the total SNP set by using bedtools to obtain the SNP loci corresponding to the functional regionsBLZF1,TUBB2A,DLGAP5,CMTM4A total of 4 wool performance-related functional genes are associated and comprise only a wool performance site combination of 948 snp sites.
EXAMPLE 2 preparation of primer set and probe set Using wool Performance SNP site set
The technical personnel designs the primer according to the sequence information of each site in the SNP site combination with wool performance, and carries out secondary structure evaluation and Tm value evaluation on the designed primer, thereby finally obtaining the primer which has good specificity and high sensitivity and can realize the detection purpose under the same reaction condition.
The secondary structure and Tm value can be evaluated by any means commonly used in the art, for example, by using DNA shaping Form to evaluate the secondary structure, see (http:// unfold. Rna. Albany. Edu// q=mfold/DNA-shaping-Form), and then using software RaW-Probe to evaluate the Tm value.
The method is a conventional method, and the site information in the SNP site combination of the villus can be obtained without creative labor, so that the primer obtained by the SNP site combination provided by the application also belongs to the protection scope of the application.
Likewise, probes prepared by using the SNP site combinations provided by the application, such as tanqman probes, also belong to the scope of the application.
Example 3 SNP site combination for analysis of fluff for preparation of Gene chip
The SNP gene chip of the application is obtained by immobilizing the primer or probe obtained in example 2 on a polymer substrate such as nylon membrane, nitrocellulose membrane, plastic, silica gel wafer, micro magnetic beads or the like by a conventional method, or by immobilizing the probe on a glass plate, or by directly synthesizing the primer or probe obtained in example 2 on a hard surface such as glass, and the method of using the SNP gene chip of the application is the same as the conventional method.
It should be noted that, a person skilled in the art may prepare the SNP gene chip for detecting the performance of goat hair in any mode, and may also entrust the preparation of the bio-company, but all SNP gene chips prepared based on the SNP locus combination for goat hair performance analysis provided by the application belong to the protection scope of the application.
Example 4 analysis kit for goat wool Performance
The SNP detection kit for mountain analysis wool performance provided by the application comprises a primer or a probe or a gene chip obtained based on the SNP locus combination obtained in example 1. Depending on the type of use, corresponding detection reagents are also included, such as buffers, ligases, aceQUniversal u+ Probe Master Mix V2, taqman probes, etc., conventionally used for fluorescent quantitative PCR reactions when Taqman probes are obtained based on the SNP site combinations obtained in example 1.
The SNP kits for detecting the wool performance are configured differently according to the use modes by the technicians in the field, but the SNP locus combination configuration for wool performance SNP kits for detecting the wool performance provided by the application belong to the protection scope of the application.
Example 5 detection of goat wool Performance
The SNP locus combination for analyzing the performance of goat wool provided by the embodiment 1 of the application is used for detecting the existence of known villus, and the accuracy of detection is judged according to the detection result by combining the existence of the known villus, specifically:
collecting peripheral blood of goats by adopting a conventional method, and extracting whole genome DNA (deoxyribonucleic acid) in the peripheral blood to obtain a whole genome DNA sample;
the gene chip is designed according to the site information in the SNP site combination provided by the application by adopting a conventional method, a whole genome DNA sample of the goat is detected, a typing result of each site (namely, whether each site is homozygote, heterozygote, mutant homozygote or base deletion result) in the goat is obtained, the frequency value of the typing result of each site is calculated, the frequency value is compared with a population threshold value, and the comparison result shows that the gene detection result is consistent with the phenotype of the corresponding goat villus part.
The population threshold value of the present application is obtained by analyzing whether different populations exist in the fluff, and the method is the same as above.
According to the application, the significance test (independent sample Manwhitney U test) is carried out on the judging result of the analysis of the large-villus goat population and the small-villus goat population, the result is shown in figure 2, according to the result in the figure, P is less than 0.01, the difference is very significant, and the result judged by the method has accuracy and effectiveness.
Industrial application
Based on the SNP locus combination which is only composed of 948 SNP loci and is used for analyzing goat villus, the SNP probe combination, the gene chip and the kit for analyzing goat villus can be prepared by the person skilled in the art, the villus of a goat individual can be analyzed on the genome level, genetic information evaluation, variety screening and variety identification can be realized, the breeding process can be controlled, and the method can be applied to goat variety tracing, goat pedigree reconstruction, germplasm resource protection and germplasm resource improvement.
The above description is only for the purpose of aiding in understanding the preferred embodiments of the present application and is not intended to limit the present application, and various changes and modifications may be made to the present application by those skilled in the art without departing from the spirit of the present application, and it should also be considered that the present application shall fall within the scope of the present application.
Claims (7)
- Use of 1.948 SNP locus combinations for analysis of goat hair performance, the physical positions of the 948 SNP locus combinations are shown in the following table:；its physical location is determined based on genomic sequence alignment of goat reference genome ARS 1.
- 2. The method for analyzing goat performance comprises the steps of comparing 948 SNP locus genotypes of genome DNA of goats to be detected with 948 SNP locus genotypes of genome DNA of control goats;wherein the 948 SNP sites are 948 SNP sites according to claim 1.
- 3. And analyzing the molecular probe combination for goat hair performance, wherein the molecular probe combination detects SNP locus combinations shown in table 1 in a sample to be detected, and the physical position information of the locus combinations in table 1 is determined based on the goat reference genome ARS1 genome sequence alignment.
- 4. A gene chip for analyzing goat hair performance, the gene chip being loaded with the molecular probe set according to claim 3.
- 5. A kit for analyzing properties of goat hair, which comprises the molecular probe set according to claim 3 or the gene chip according to claim 4.
- 6. A method for analyzing the performance of goat hair, which comprises detecting a sample to be detected by using the molecular probe set according to claim 3 or the gene chip according to claim 4 or the kit according to claim 5.
- 7. The molecular probe combination of claim 3 or the gene chip of claim 4 or the kit of claim 5 has the following uses:(1) Application in performance evaluation of goat hair;(2) Application in goat variety screening;(3) Application in goat variety identification;(4) Application in the tracing of goat breeds;(5) Application in goat breeding;(6) Application in germplasm resource protection;(7) Application in germplasm resource improvement;(8) The application in goat pedigree reconstruction.
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| CN113278712A (en) * | 2021-07-23 | 2021-08-20 | 中国农业大学 | Gene chip, molecular probe combination, kit and application for analyzing sheep hair color |
| CN114959059A (en) * | 2022-05-30 | 2022-08-30 | 中国农业科学院兰州畜牧与兽药研究所 | SNP locus combination related to diameter variation coefficient of fine wool sheep wool fiber and application thereof |
| WO2023001212A1 (en) * | 2021-07-21 | 2023-01-26 | 中国农业大学 | Gene chip, molecular probe combination and kit for analyzing sheep milk production performance, and use |
| CN115838810A (en) * | 2022-10-19 | 2023-03-24 | 沈阳农业大学 | Application of molecular marker primer of cashmere fineness gene COL6A5 of cashmere goats |
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| WO2023001212A1 (en) * | 2021-07-21 | 2023-01-26 | 中国农业大学 | Gene chip, molecular probe combination and kit for analyzing sheep milk production performance, and use |
| CN113278716A (en) * | 2021-07-23 | 2021-08-20 | 中国农业大学 | Gene chip for analyzing characters for sheep wool, molecular probe combination, kit and application |
| CN113278712A (en) * | 2021-07-23 | 2021-08-20 | 中国农业大学 | Gene chip, molecular probe combination, kit and application for analyzing sheep hair color |
| CN114959059A (en) * | 2022-05-30 | 2022-08-30 | 中国农业科学院兰州畜牧与兽药研究所 | SNP locus combination related to diameter variation coefficient of fine wool sheep wool fiber and application thereof |
| CN115838810A (en) * | 2022-10-19 | 2023-03-24 | 沈阳农业大学 | Application of molecular marker primer of cashmere fineness gene COL6A5 of cashmere goats |
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