CN117126948B - Molecular marker combination for analyzing goat ear characters and application thereof - Google Patents
Molecular marker combination for analyzing goat ear characters and application thereof Download PDFInfo
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Abstract
The invention discloses a molecular marker combination for analyzing goat ear traits and application thereof, relates to the technical field of biology, and provides 1173 molecular markers capable of analyzing goat ear traits, wherein physical position information of the 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 1173 SNP molecular markers can be used for carrying out genetic evaluation on goat individuals, judging and selecting early-stage difficult-to-measure goat ear traits, shortening generation intervals and accelerating breeding processes, so that a large amount of breeding cost is saved.
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 goat ear size 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. According to the natural geographical region division of breeding, goats in China can be divided into three regions: comprises the north of a yellow river and a Wei river, the Qinghai-Tibet plateau and the north area thereof, a region: the goat is mainly in the form of wool and milk, the wool length is dense, the wool is more and thicker, and the region is favorable for developing the wool goat and the milk goat; two areas: the method comprises the steps of a region between a yellow river, a Wei river and a Yangtze river, wherein the climate of the region is neither high cold nor dry nor too high temperature and high humidity, and goats in the region have the characteristics of a first region and a second region at the same time, are mainly purified and rejuvenated, and have great potential for developing goat production for cultivating specialized goats; three regions: the sheep in the three areas mainly comprise meat and fur or skin, meat and milk in the regions of Yangtze river, the fur is short and thin, the rainwater in the south is sufficient, the climate is mild, the growth period of forage grass is long, the forage grass resources and agricultural and sideline products are rich, the development of varieties with meat, fur or skin, meat and milk is facilitated, and the local goats have the characteristics of high fertility and rapid growth of lambs. Goats can be generally classified into the following six classes according to production uses: firstly, a meat goat: the meat is delicious and has small smell of mutton, and the meat is taken as a main feeding target; secondly, goat for wool: the fineness and the count of the wool are different with the high wool yield as the main feeding target; thirdly, goat for down production: the fluff of Mao Naceng is the main product; fourthly, a leather goat: the main product is sheepskin; fifthly, a goat for milk: the main feeding goal is high lactation; and six, the goat is a common local goat. However, there were few studies on goat ears.
Ear type is an important trait of current livestock breeds, and ear size is one of its important phenotypes. The goat ears are not only one of important goat traits, but also popular food materials with high nutritional value and medicinal value and good taste; in addition, the ear character of the goat has important significance in the source tracing of the goat variety and the protection, development and utilization of germplasm resources.
Aiming at the current goats with various varieties and changeable characters, how to rapidly screen the goats and distinguish the types of the ears of the goats becomes an 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 characters of the ears 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 detection of the goat ear traits, the invention provides a molecular probe combination, a gene chip, a kit and application for analyzing the goat ear traits, and the site information provided by the invention can be used for rapidly and accurately realizing the deposition evaluation of the goat ear, variety screening, variety identification, variety tracing and goat breeding, 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 invention, the invention provides the following technical scheme:
in a first aspect, the present invention provides an application of 1173 SNP site combinations in analyzing goat ear sizes, wherein physical positions of the 1173 SNP site combinations are shown in table 1:
table 1 1173 location information for a combination of sites
Its physical location was determined based on goat reference genome ARS1 genome sequence alignment.
A second aspect provides a method of analyzing the ear size of a goat, comparing 1173 SNP site genotypes of genomic DNA of a goat to be tested with the 1173 SNP site genotypes of genomic DNA of a control goat;
wherein the 1173 SNP sites are 1173 SNP sites as set forth in Table 1.
In a third aspect, a molecular probe set for analyzing the ear size of a goat 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 a reference genome of the goat.
The fourth aspect is a gene chip for analyzing goat ear size, the gene chip being loaded with the molecular probe combination according to the third aspect.
The fifth aspect is a kit for analyzing goat ear size, which has 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 size of goat ears, 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 goat ear size evaluation;
(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 provided by the invention has good domestic and foreign universality, can rapidly evaluate the early-period non-obvious goat ear size characters from the gene level, acquires more accurate breeding evaluation information, controls the breeding process, can screen, identify and trace the source of goat varieties 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 ear size of the goat 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 Pak-angora, kamori, daer, matebele Vs Toggenburg set;
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 size of the goat ear is divided according to the size of the ear area, the ear area of the conventional area is a big ear, the ear length is generally 25 to 35 cm, and the flat width of the ear is 12 to 15 cm; the malformation of the mutated small ear is the small ear.
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 obtaining ear trait 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 related to the ear size 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
Grouping according to the remarkable differences of goat varieties in ear traits worldwide, summarizing all varieties of individuals with big ears and small ears, and performing selective elimination analysis according to the two groups of big ears and small ears. The selected Pak-angora (Pakistan angora), kamori, daer (Jianzhou Dart), matebile (Martebeli) and Toggenburg (ipenburg goat) small-ear sheep were grouped for elimination analysis.
The invention scans the functional area related to the ear size 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 ear size by taking intersection of the calculation results of the two methods, screens out the functional area related to the ear size, screens out genes in the area by referring to the Manhattan diagram which is generated by different groups of ear size characters and is made by pi value and is shown in figure 1, and finally determines 7 candidate genes SIX2, CXCL14, POU4F3, TCOF1, HOXA1, MSRB3 and GATA6 which are related to the ear characters and have very definite functions. And then determining the functional region corresponding to the candidate gene by perl script.
4. Ear-sized SNP site combination acquisition
Searching SNP loci corresponding to the functional regions of the candidate genes determined in the step 3 in the total SNP set by utilizing bedtools to obtain ear trait locus combinations which are related to 7 ear traits in total and only comprise 1173 SNP loci, wherein the functional genes are related to SIX2, CXCL14, POU4F3, TCOF1, HOXA1, MSRB3 and GATA6.
Example 2 ear trait SNP locus combination for preparation of primer combination and Probe combination
The technical personnel designs the primer according to the sequence information of each site in the ear character 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 with the ear size can be obtained without 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 ear size for preparation of Gene chip
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 can prepare an SNP gene chip for detecting the ear size of a goat in any one of the ways, and can also entrust the preparation of biological companies, but all SNP gene chips prepared based on the combination of SNP loci for ear size analysis provided by the application belong to the protection scope of the invention.
Example 4 analysis kit for goat ear traits
The SNP detection kit for ear size analysis 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.
Different SNP kits for detecting the ear traits of the goat are configured by a person skilled in the art according to different using modes, but the SNP detection kits for detecting the ear traits of the goat based on the ear trait SNP locus combination configuration provided by the application belong to the protection scope of the invention.
Example 5 detection of goat ear Properties
Based on the SNP locus combination for analyzing the ear size of the goat provided in the embodiment 1 of the application, the ear size of the goat with known ear size is detected, and according to the detection result, the accuracy of detection is judged by combining with the known ear size, 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 ear phenotype.
The population threshold value is obtained by analyzing populations with different ear sizes, and the method is the same as the above.
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 large-ear goat population and the small-ear 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 extremely significant, and the accuracy and the effectiveness of the judging result by adopting the method are realized.
Industrial application
The SNP locus combination which is only composed of 1173 SNP loci and can be used for analyzing the ear size of a goat and can be prepared by a person skilled in the art, a gene chip and a kit can analyze the ear size of a goat individual from genome level, genetic information evaluation, variety screening and variety identification can be realized, the breeding process can be controlled, 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 (6)
- Application of 1.1173 SNP locus combinations in analysis of goat ear sizes, physical positions of 1173 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. A method for analyzing the ear size of a goat, wherein 1173 SNP locus genotypes of genomic DNA of the goat to be detected are compared with 1173 SNP locus genotypes of genomic DNA of a control goat;wherein the 1173 SNP sites are 1173 SNP sites of claim 1.
- 3. Analyzing a goat ear size molecular probe set which detects the SNP site set as set forth in claim 1 in a test sample, the physical location information of the site set in claim 1 being determined based on goat reference genome ARS1 genome sequence alignment.
- 4. A gene chip for analyzing the size of goat ears, which is loaded with the molecular probe combination of claim 3.
- 5. A kit for analyzing the size of goat ears, which has the molecular probe set according to claim 3 or the gene chip according to claim 4.
- 6. A method for analyzing the size of goat ears, which comprises detecting a sample to be detected using the molecular probe set of claim 3 or the gene chip of claim 4 or the kit of claim 5.
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| KR102243308B1 (en) * | 2019-11-08 | 2021-04-22 | 대한민국 | Novel SNP marker for identification of black goat and uses thereof |
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| CN113293220A (en) * | 2021-07-23 | 2021-08-24 | 中国农业大学 | Gene chip for analyzing ear size of sheep, molecular probe combination, kit and application |
| CN113396866A (en) * | 2021-05-28 | 2021-09-17 | 河南省畜牧总站 | Method for breeding new variety of meat goats |
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| KR102243308B1 (en) * | 2019-11-08 | 2021-04-22 | 대한민국 | Novel SNP marker for identification of black goat and uses thereof |
| CN113396866A (en) * | 2021-05-28 | 2021-09-17 | 河南省畜牧总站 | Method for breeding new variety of meat goats |
| CN113265475A (en) * | 2021-07-21 | 2021-08-17 | 中国农业大学 | Gene chip for analyzing sheep fat tail, molecular probe combination, kit and application |
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