CN117089634B - Molecular marker combination for analyzing goat milk performance and application - Google Patents
Molecular marker combination for analyzing goat milk performance and application Download PDFInfo
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Abstract
The invention discloses a molecular marker combination for analyzing the performance of goat milk and application thereof, relates to the technical field of biology, and provides 1699 molecular markers capable of analyzing the performance characteristics of goat milk, wherein the physical position information of the molecular markers is determined based on the comparison of genome sequences of goat reference genome ARS1, and a molecular probe combination, a gene chip and a kit prepared by using 1699 SNP molecular markers can be used for carrying out genetic evaluation on goat individuals, so that the early difficult-to-measure goat milk performance is selected individually, the generation interval is shortened, and the breeding process is accelerated, thereby saving a large amount of breeding cost.
Description
Technical Field
The invention relates to the technical field of biology, in particular to the technical field of biological detection, and more particularly relates to a goat milk performance SNP locus combination and application thereof.
Background
Goats are one of the earliest animals domesticated in the world, so that not only are the goats provided with low cholesterol, high protein, rich nutrition and delicious meat resources for human society, but also goat milk is easier to absorb by human bodies compared with milk because of the fact that the goats contain more short chain fatty acids, and also contains rich nutrient substances, so that the goats are favored by consumers more and more because of the unique advantages of the goats, but also the goats have genetic polymorphism (sheep for milk or sheep for non-milk, and genetic variation exists) in milk performance. Aiming at the current goats with various varieties and changeable characters, how to rapidly screen the goats and distinguish the goat milk type becomes the urgent problem to be solved, therefore, the design of the SNP chip which is applicable to the Chinese goats and can rapidly and effectively detect the milk performance 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 milk, the invention provides a molecular probe combination, a gene chip, a kit and application for analyzing the performance of goat milk, and the site information provided by the invention can be used for rapidly and accurately realizing the performance deposition evaluation, variety screening, variety identification, variety tracing and goat breeding of goat milk, is beneficial to 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 invention, the invention provides the following technical scheme:
in a first aspect, the present invention provides an application of 1699 SNP site combinations in analysis of goat milk performance, wherein physical positions of the 1699 SNP site combinations are shown in table 1:
TABLE 1 position information for 1699 combinations of sites
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Its physical location was determined based on goat reference genome ARS1 genome sequence alignment.
The second aspect provides a method for analyzing goat milk performance, which compares 1699 SNP locus genotypes of genomic DNA of a goat to be tested with the 1699 SNP locus genotypes of genomic DNA of a control goat;
wherein the 1699 SNP sites are 1699 SNP sites described in table 1.
In a third aspect, a molecular probe set for analyzing the performance of goat milk 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 the alignment of ARS1 genome sequences of goat reference genome.
The fourth aspect is a gene chip for analyzing goat milk performance, the gene chip being loaded with the molecular probe combination according to the third aspect.
The fifth aspect is a kit for analyzing the performance of goat milk, 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 the performance of goat milk, wherein the molecular probe set according to the third aspect or the gene chip according to the fourth aspect or the kit according to the fifth aspect is used for detecting 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 milk;
(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.
The beneficial effects are that:
1. the SNP locus combination for analyzing goat milk performance, which is composed of 1699 SNP loci only, is provided based on the research on genetic resources of a plurality of goats at home and abroad, has good domestic and foreign universality, can rapidly evaluate the goat milk performance which cannot be displayed 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 milk performance provided by the invention 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 group of dairy and non-dairy goats;
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 invention 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 invention. 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 invention 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 milk production referred to in the present invention is divided into a milk goat and a non-milk goat.
The SNP referred to in the present invention 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 invention 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 invention 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 invention 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 invention 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 invention, the kit comprises a primer for amplifying the site information provided by the invention, a molecular marker or a probe or a gene chip for detecting the site information provided by the invention, an enzyme and a buffer used for amplification, or a fluorescent marker used for detection.
EXAMPLE 1 acquisition of milk 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 each breed is:
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. From these breeds 36 goat individuals with and without milk production were selected.
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 invention, 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
All individuals of the milk goats and the non-milk goats were grouped according to significant differences in milk performance throughout the world, and selective elimination analysis was performed by dividing the milk and non-milk goats into two groups, namely, milk goats varieties Kamor, dera Din Pannah goat (delatin pana goats), maurego (mox goats), galla goats, sofia goats, togengurg goats, saanen goats, provence goats, fosses goats, pointe goats, savieie goats, alpine goats, applicator goats (albopine goats Peng Ceer goats), and non-milk goats black bengal goat (bangla goats), grisons striped goat (gray), and love goats (Luo Fu) as stripes.
The invention scans the functional area related to milk property by calculating the nucleotide base polymorphism and locus allele frequency difference between pi value and fst value of two groups of goats, and the calculated results of the two methods are used for taking intersection, screening out the functional area related to milk property, and the functional areas are produced by different groups of milk property according to pi valueAs shown in the Manhattan diagram of FIG. 1, genes in a region are screened by referring to the published gene research results, and finally 5 candidate genes which are relevant to milk performance and have well-defined functions are determinedSYT16、PI16、MAPK13、LHFP、YPEL2. And then determining the functional region corresponding to the candidate gene by perl script.
4. Acquisition of SNP site combinations for milk production
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 regionsSYT16、PI16、MAPK13、LHFP、YPEL2A total of 5 milk trait related functional genes and a milk performance site combination comprising only 1699 snp sites.
EXAMPLE 2 use of milk Performance SNP site combination for preparation of primer set and Probe set
The technical personnel in the art designs the primer according to the sequence information of each site in the milk performance SNP site combination provided by the invention, and carries out secondary structure evaluation and Tm value evaluation on the designed primer, so that the primer with good specificity and high sensitivity can be finally obtained, and the detection purpose can be realized 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 for milk production provided by the application can be obtained without the need of creative labor, so that the primer obtained by the SNP site combination provided by the invention also belongs to the protection scope of the invention.
Likewise, probes prepared by using the SNP site combinations provided by the invention, such as tanqman probes, also belong to the scope of the invention.
Example 3 SNP site combination for analysis of milk Properties for Gene chip preparation
The SNP gene chip of the invention 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 immobilizing the probe on a glass plate, or 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 invention is the same as that of the conventional method.
It should be noted that, a person skilled in the art may prepare an SNP gene chip for detecting the performance of goat milk in any one of the ways, and may also entrust the biological company to prepare the SNP gene chip based on the SNP locus combination for milk performance analysis provided in the application all belong to the protection scope of the invention.
Example 4 analysis kit for goat milk Performance
The SNP detection kit for milk performance analysis provided by the application comprises a primer or a probe or a gene chip obtained based on the SNP locus combination obtained in the 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.
Different SNP kits for goat milk performance detection are configured by a person skilled in the art according to different use modes, but the goat milk performance SNP detection kits configured based on the milk performance SNP locus combination provided by the application belong to the protection scope of the invention.
Example 5 detection of goat milk Performance
The detection of milk performance is performed on known goats for milk and non-milk based on the SNP locus combination for analyzing goat milk performance provided in the embodiment 1 of the application, and according to the detection result, the accuracy of the detection is judged by combining the known goat phenotypes for milk and non-milk, 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 invention by adopting a conventional method, a whole genome DNA sample of the goat is detected, a typing result of each site in the goat (namely, whether each site is homozygote, heterozygote, mutant homozygote or base deletion result) 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 corresponding goat milk performance phenotype.
The population threshold value is obtained by analyzing different populations of milk goats and non-milk goats, and the method is the same.
The method has the advantages that the significance test (independent sample Manwhitney U test) is carried out on the judging result of the analysis of the non-dairy 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 accuracy and the effectiveness of the judging result are realized by adopting the method.
Industrial application
The SNP locus combination which is only composed of 1699 SNP loci and can be used for analyzing the performance of goat milk and can be prepared by a person skilled in the art based on the SNP locus combination which is provided by the application and is used for analyzing the performance of goat milk, a gene chip and a kit, the performance of goat individual milk can be analyzed from genome level, genetic information evaluation, variety screening, variety identification and breeding process control can be realized, and the SNP locus combination 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 invention and is not intended to limit the present invention, and various changes and modifications may be made to the present invention by those skilled in the art without departing from the spirit of the present invention, and it should also be considered that the present invention shall fall within the scope of the present invention.
Claims (7)
- Application of 1.1699 SNP locus combinations in analysis of goat milk performance, physical positions of the 1699 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. Comparing 1699 SNP locus genotypes of genome DNA of the goat to be detected with the 1699 SNP locus genotypes of genome DNA of the control goat by a method for analyzing the goat milk performance;wherein the 1699 SNP sites are 1699 SNP sites described in claim 1.
- 3. A molecular probe combination for analyzing the performance of goat milk, which detects the SNP site combination as set forth in claim 1 in a sample to be tested.
- 4. A gene chip for analyzing goat milk performance, the gene chip being loaded with the molecular probe combination of claim 3.
- 5. A kit for analyzing the performance of goat milk, which comprises the molecular probe set according to claim 3 or the gene chip according to claim 4.
- 6. A method for analyzing goat milk performance, which uses the molecular probe combination of claim 3 or the gene chip of claim 4 or the kit of claim 5 to detect a sample to be detected.
- 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 milk;(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) The application in goat pedigree reconstruction.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106434955A (en) * | 2016-11-04 | 2017-02-22 | 中国农业科学院北京畜牧兽医研究所 | SNP (single nucleotide polymorphism) molecular marker related with Chinese goat milk production traits and application of SNP molecular marker |
| CN113151507A (en) * | 2021-05-21 | 2021-07-23 | 内蒙古自治区农牧业科学院 | SNP molecular marker related to milk production traits of milk goats, liquid phase chip prepared from SNP molecular marker and application of SNP molecular marker |
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| CN106434955A (en) * | 2016-11-04 | 2017-02-22 | 中国农业科学院北京畜牧兽医研究所 | SNP (single nucleotide polymorphism) molecular marker related with Chinese goat milk production traits and application of SNP molecular marker |
| CN113151507A (en) * | 2021-05-21 | 2021-07-23 | 内蒙古自治区农牧业科学院 | SNP molecular marker related to milk production traits of milk goats, liquid phase chip prepared from SNP molecular marker and application of SNP molecular marker |
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| Comparative genomic analysis uncovers candidate genes related with milk production and adaptive traits in goat breeds;Zeinab Amiri Ghanatsaman等;《Scientific Reports》;第13卷(第8722期);第1-10页 * |
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