CN113278715A - Gene chip for analyzing sheep papilla number, molecular probe combination, kit and application - Google Patents
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Abstract
The invention discloses a gene chip, a molecular probe combination, a kit and application for analyzing the number of sheep nipples, and relates to the technical field of biology.
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 gene chip, a molecular probe combination, a kit and application for analyzing the number of sheep papilla.
Background
Sheep is one of the most important economic animals in the world, and can be used as food for human beings and also used as a source of dairy products. The sheep breed resources are rich in China, and the breed with the property of multiple papillae also generally exists, and researches show that the property of the number of papillae is obviously related to the breeding of female animals, the growth of offspring, susceptibility to mastitis and other properties, but the source of the breed is still controversial, and more researchers research the property of the papillae of the sheep by utilizing gene analysis in order to better analyze and research the property of the papillae of the sheep and protect the breed of the sheep and research the source of the breed.
At present, the molecular marker technology is more and more emphasized due to its advantages of high accuracy, strong operability, etc., and among them, the molecular marker technology based on Single Nucleotide Polymorphisms (SNPs) is more and more widely applied. SNP is taken as a genetic molecular marker in biological genomes, and plays more and more important roles in the aspects of animal and plant genetic evolution analysis, important economic shape screening, molecular breeding and the like. The SNP chip based on the SNP is a convenient and efficient tool for modern genetic breeding, and is easy to realize high-throughput and automatic detection of the SNP, can detect the change of each base pair on genomic DNA, including insertion, deletion, inversion, conversion and the like, becomes an ideal SNP detection technology, and is increasingly applied to the field of sheep breeding.
Although commercial Sheep SNP chips (Illumina provine SNP50 Beadchip (50K), Illumina sheet HD Genotyping Beadchip (680K) and Illumina provine LD (5K)) cover more than 54K SNP sites of Sheep whole genome, the chips are used for genetic breeding, whole genome association analysis, quantitative trait locus positioning, gene optimization, comparative genomics and other researches. However, the existing sheep SNP chip is mainly based on western sheep data, lacks of SNP data of combination of Chinese sheep varieties and foreign sheep varieties, and still has the problems of insufficient site uniformity, insufficient embodiment of functional sites and regions, extremely low frequency sites of over 10 percent of sites in Chinese sheep populations and the like, so that the design of the SNP chip which is suitable for Chinese sheep populations and can rapidly and effectively detect sheep wool traits is of great significance.
Disclosure of Invention
The invention provides a molecular probe combination, a gene chip, a kit and application for analyzing the number of sheep papilla for meeting the requirements of sheep papilla number detection in the current sheep variety research and agricultural production in China.
To achieve the technical object of the present invention, the present invention provides:
the application of 1782 site combination in analyzing the number of sheep papilla is shown in the table 1:
TABLE 11782 physical location of site combinations
Wherein the physical position information is determined based on sheep v4.0 genome sequence alignment.
The genotype of 1782 SNP sites of the genomic DNA of the sheep to be detected is compared with the genotype of 1782 SNP sites of the genomic DNA of the control sheep; the 1782 SNP sites are the 1782 SNP sites described in Table 1.
Thirdly, analyzing the molecular probe combination of the number of the sheep papilla, detecting the SNP locus combination shown in the table 1 in a sample to be detected by the molecular probe combination, and determining the physical position information of the locus combination in the table 1 based on the sheep v4.0 genome sequence alignment.
Fourthly, the gene chip for analyzing the number of the sheep papilla is loaded with the molecular probe combination.
The kit for analyzing the number of the sheep papilla comprises the molecular probe combination or the gene chip.
Sixthly, a method for analyzing the number of sheep papilla, and a method for detecting a sample to be detected by applying the molecular probe combination or the gene chip or the kit.
The molecular probe combination or the gene chip or the kit has the following applications: (1) the application in evaluation of sheep papilla number; (2) the application in screening sheep varieties; (3) the application in sheep variety identification; (4) the application in tracing sheep varieties; (5) the application in sheep breeding; (6) the application in germplasm resource protection; (7) the application in germplasm resources improvement; (8) application in sheep system reconstruction.
Has the advantages that:
1. the SNP locus combination for analyzing the number of sheep nipples is provided based on the research on genetic resources of a plurality of sheep at home and abroad, the SNP locus combination only consists of 1782 SNP loci, has good universality at home and abroad, can quickly evaluate the number of sheep nipples on a gene level, knows the nipple number characters at the early stage of the sheep, realizes the identification and traceability of sheep varieties, and provides technical support for sheep genealogical reconstruction, germplasm resource protection and germplasm resource improvement.
2. The probe combination, the gene chip and the kit formed by the SNP locus combination for analyzing the number of the sheep papilla provided by the invention have the characteristics of small flux, low cost, easier analysis, wide universality and wide market prospect.
Drawings
FIG. 1 is a Manhattan chart of the hole sheep Vs Dorper sheep (WDS verses DPS) group;
FIG. 2 is a Manhattan plot for the hole sheep Vs savock sheep (WDS veruss SFK) group;
fig. 3 is a result graph of the significance test performed on the determination result of the population threshold analysis according to the present application.
Detailed Description
The invention is further illustrated by reference to the following detailed description of specific embodiments, which are intended to be illustrative only and not to be construed as limiting the invention. Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art, and can be performed with reference to the third edition of the original book "bioinformatics and functional genomics" or related books, and bioinformatics software and products used therein are commercially available. Various procedures and methods not described in detail are conventional methods well known in the art, and the sources of materials used, trade names, and components thereof, if necessary, are indicated at the time of first appearance, and the same reagents used thereafter, if not specifically indicated, are the same as those indicated at the time of first appearance.
In addition, it should be noted that the site combination and application provided by the present invention are completed by the inventors of the present application through hard creative work and optimization work.
The features and advantages described in the site combination section above are also applicable to the molecular probe combination, gene chip, kit and application thereof formed based on the site combination, and are not described herein again.
The number of sheep papillae referred to in the present invention means the number of sheep papillae, and in one embodiment of the present invention, the number of small papillae means only two papillae, and the number of large papillae means 3 or more papillae.
The SNP refers to a Single Nucleotide Polymorphism (Single Nucleotide Polymorphism) and mainly refers to a DNA sequence Polymorphism caused by a variation of a Single Nucleotide on a genome level, wherein the variation of the Single Nucleotide includes a variation caused by a transition, a transversion, an insertion or a deletion of a Single base.
It should be noted that the molecular markers referred to in the present invention are all heritable and detectable DNA sequences or proteins, 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 scarr; DNA labeling based on restriction and PCR techniques; molecular markers based on DNA chip technology, such as SNP; analytical labeling techniques based on the development of EST databases, and the like. The molecular marker provided by the invention can be used for genome mapping, gene positioning research, map-based gene cloning, species genetic relationship, systematic classification and the like.
The probe of the present invention is a nucleic acid sequence (DNA or RNA) having a detection label and a known sequence, which is 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 cassettes conventionally used in the art, which contains reagents for detection or experiment, and is convenient for operators to be able to get rid of the heavy reagent preparation and optimization process. In one embodiment of the present invention, the primer for amplifying the site information provided by the present invention, the molecular marker or probe or gene chip for detecting the site information provided by the present invention, the enzyme and buffer solution for amplification, or the fluorescent marker for detection are also included.
Example 1 acquisition of Nipple trait SNP site combinations
1. Selection of sheep individuals
In order to achieve a more comprehensive coverage of the domestic and foreign Sheep breeds, the applicant carried out the acquisition of genetic information on 248 Sheep individuals throughout asia, europe, africa and the middle east, including 16 wild Sheep asian moloren breeds, 172 local breeds and 60 breeds, specifically related to the small tailed Sheep, Sishui fur Sheep, big tail han Sheep, hole Sheep, hu Sheep of china jiang, ningxia tan Sheep of china, alexan Sheep of china new jiang, bashme Sheep, dupont Sheep, merino Sheep (fine wool Sheep), merino Sheep (superfine wool Sheep), saffron, zelerian black Sheep, surf Sheep, wagggir Sheep, finland Sheep (filsheep) of finland, weisang Sheep island Sheep (oesnant), teddy Sheep (sheed), sheds (shedullander), soyoho tiger knone, gold island (solhelvet), gotten-down Sheep (gold Sheep), and heaven Sheep (drayagarland), and Sheep (dracawarrio Sheep), and Sheep) Afaham Sheep (Afar Sheep), Nigerl's Muboro Sheep (MbioSheep), Nigeria's Yanka Sheep (Yankasa Sheep), African shorn West African Sheep (West African Dwarf Sheep), Wuda Sheep (Uda Sheep), African Bruna's Jialonka Sheep (Djallonk Sheep), Morse Sheep (Mossi Sheep), Sahler Sheep (Sahalian Sheep), West non-Kernel Sheep (Cameroon Sheep), Irelak Arwashi Sheep (Awassia Sheep), Hadamni Sheep (Hamdani Sheep), Asahi Kanzi Sheep (Makh Sheep), Ashira Sheep Shez-Shez (Shirafay), ganzel Sheep (Ghezel Sheep), Afrika Sheep (Afshii Sheep), Sharl Sheep (Shal Sheep), marqui Sheep (Makui Sheep), mohni Sheep (Moghani Sheep), Karakul Sheep of Pakistan (Karakl Sheep), Asian molar Flon of Iranshenschel.
2. Acquisition of Total SNP set of sheep Whole Gene
A sample of the sheep individual carrying genetic information in step 1, including but not limited to blood, cells, tissue, skin, hair, feces, etc., is collected using methods conventional in the art. Genetic information (such as DNA) in a sample is extracted for high-depth sequencing, the two modes of SAMtools and GATK are compared with a sheep 4.0 reference genome (obtained from NCBI) released in 2015, and a common result obtained by the two modes forms a SNP set, wherein 2836 ten thousand SNP sites are counted and used as a total SNP set of a sheep whole gene.
The genetic information (genetic information) referred to in the present invention means information that an organism replicates the same thing as itself, is transmitted from a parent to a daughter, or is transmitted from a cell to a cell every time each cell divides.
It should be noted that, the extraction of genetic information (e.g., DNA) from a sample for high-depth sequencing may be performed by a biological company, such as watsda gene, illumina, etc., the high-depth sequencing method is a conventional method in the art or a method of the biological company, in an embodiment of the present invention, the average sequencing depth is-25.7 ×, and the high-depth sequencing is performed by using a re-sequencing analysis process.
3. Screening of candidate genes and functional regions thereof
3.1 processing samples of sheep genetic information for different papillary counts
Screening sheep varieties with different papillary numbers, and grouping corresponding genetic information samples according to the obvious difference of the different sheep varieties in papillary characters.
3.2 processing of genetic information of sheep with different papillary counts after grouping
The functional regions related to the number of the nipples are scanned by sweeping the multi-locus allele frequency difference between each sheep group through XP-CLR (the scanned Manhattan graphs are shown in figures 1 and 2, wherein figure 1 is a depressed sheep Vs Dorper sheep (WDS versus DPS) group, figure 2 is a depressed sheep Vs savock sheep (WDS versus SFK) group, the functional regions related to the number of the nipples in the sheep variety in each group are excavated by pi ratio (namely pi value), and then the intersection of the two results is taken to screen the functional regions related to the number of the nipples.
Screening genes in the region by referring to published gene research results, finally determining 8 candidate genes NRIP1, DAB1, PCBP2, FGF7, SYNDIG1L, VRTN, GRB10 and CTBP1 which are related to the number of the papilla and have quite determined functions, and further determining the functional region corresponding to the candidate genes by perl scripts.
4. Acquisition of SNP site combinations for analysis of papillary number
And (3) 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 SNP locus combinations which are related to 8 papilla-related functional genes including NRIP1, DAB1, PCBP2, FGF7, SYNDIG1L, VRTN, GRB10 and CTBP1 and only contain the analyzed papilla number of 1782 SNP loci.
Example 2 primer combination and Probe combination
The skilled in the art designs a primer according to the sequence information of each site in the SNP site combination provided by the invention, and the designed primer is used for secondary structure evaluation and Tm value evaluation, so that the primer which has good specificity and high sensitivity and can realize the detection purpose under the same reaction condition is finally obtained.
Wherein, the secondary structure evaluation and Tm value evaluation can be performed by any one of the methods commonly used in the art, for example, DNA folding form is used to evaluate the secondary structure, see the description
The Tm value was then evaluated by using the software RaW-Probe.
The methods are all conventional methods, and can be obtained without creative labor according to the site information in the SNP site combination for analyzing the number of the papillae provided by the application, so that the primer obtained according to the SNP site combination for analyzing the number of the papillae provided by the application also belongs to the protection scope of the invention.
Similarly, the invention also provides a probe prepared by utilizing the SNP site combination for analyzing the number of papillae, such as a tanqman probe.
Example 3 SNP site combinations for analysis of papillary number used for preparation of Gene chip
The SNP gene chip of the present application is prepared by immobilizing the primer or probe obtained in example 2 on a polymer substrate, such as a nylon membrane, nitrocellulose membrane, plastic, silica gel wafer, micro magnetic bead, etc., 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, etc., using the same method as the conventional method.
It should be noted that, those skilled in the art can prepare the SNP gene chip for detecting the number of sheep papilla in any way, and also can prepare the SNP gene chip by entrusted to the biology company, but the SNP gene chip prepared based on the combination of the SNP sites of the number of papilla provided in the present application is within the protection scope of the present invention.
EXAMPLE 4 kit for analysis of sheep papilla count
The SNP detection kit for papillary number provided by the present application includes primers or probes or gene chips obtained based on the SNP site combinations obtained in example 1. According to the type of use, corresponding detection reagents are also included, for example, when the Taqman Probe is obtained based on the SNP site combination obtained in example 1, a buffer, a ligase, AceQUniversal U + Probe Master Mix V2, TaqMan Probe, etc. which are conventionally used in a fluorescent quantitative PCR reaction are also included.
The SNP kit for detecting the number of the sheep papilla is configured by a person skilled in the art according to different using modes, but the SNP kit for detecting the sheep papilla, which is configured based on the combination of the SNP sites of the number of the papilla provided by the application, belongs to the protection scope of the invention.
EXAMPLE 5 detection of sheep papilla count
The method for detecting the number of the sheep papilla comprises the following steps of carrying out gene detection on the sheep with known number of the papilla based on the SNP locus combination for analyzing the number of the papilla of the sheep provided by the embodiment 1 of the application, and judging the detection accuracy, specifically:
collecting peripheral blood of a sheep to be detected, and extracting whole genome DNA of the sheep;
a conventional method is adopted to design a gene chip according to site information in the SNP site combination, the whole genome DNA of a sheep to be detected is detected, the typing result of each site in each lamb is obtained (namely whether each site is a homozygote, a heterozygote, a mutant homozygote or a base deletion result), the frequency value of the typing result of each site is calculated and compared with a population threshold value, and the comparison result shows that the gene detection result is consistent with the sheep papillary number phenotype.
It should be noted that the population threshold value in the present application is obtained by analyzing the small nipple population and the large nipple population, and the method is the same as above.
The significance test (independent sample ManWhitney U test) is carried out on the judgment results of the analysis of the small nipple population and the multiple nipple population, the results are shown in figure 3, as can be seen from the results in the figure, P is less than 0.01, the difference is extremely significant, and the judgment results obtained by adopting the method have accuracy and effectiveness.
Industrial applications
The SNP molecular marker for analyzing the number of the nipples, the SNP probe combination for detecting the number of the sheep nipples and the SNP chip which can be prepared by the SNP locus combination for analyzing the number of the nipples only consisting of 1782 SNP loci provided by the application can be used for evaluating the number of the nipples of sheep individuals on the genome level, carrying out genetic evaluation or variety screening and variety identification, and can also be used for sheep variety traceability, sheep genealogy reconstruction, germplasm resource protection and germplasm resource improvement.
The above description is only a preferred example for helping understanding the present invention, and is not intended to limit the present invention, and those skilled in the art can make various changes and modifications to the present invention without departing from the spirit of the present invention, and those skilled in the art should make various changes and modifications to the present invention without departing from the spirit of the present invention.
Claims (7)
1.1782 site combination, the physical location of the 1782 site combination is shown in table 1, wherein the physical location information is determined based on sheep v4.0 genome sequence alignment.
2. The method for analyzing the number of sheep nipples comprises the steps of comparing the gene type of 1782 SNP loci of genomic DNA of a sheep to be detected with the gene type of 1782 SNP loci of the genomic DNA of a control sheep; the 1782 SNP sites are the 1782 SNP sites of claim 1.
3. Analyzing the molecular probe combination of the sheep papilla number, wherein the molecular probe combination detects SNP locus combinations shown in a table 1 in a sample to be detected, and the physical position information of the locus combinations in the table 1 is determined based on sheep v4.0 genome sequence alignment.
4. A gene chip for analyzing the number of sheep papilla, said gene chip being loaded with the combination of molecular probes according to claim 3.
5. A kit for analyzing the number of sheep papilla comprising the molecular probe set according to claim 3 or the gene chip according to claim 4.
6. A method for analyzing the number of sheep papilla, wherein the molecular probe combination of claim 3 or the gene chip of claim 4 or the kit of claim 5 is used for detecting 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 for use as any one of:
(1) the application in evaluation of sheep papilla number;
(2) the application in screening sheep varieties;
(3) the application in sheep variety identification;
(4) the application in tracing sheep varieties;
(5) the application in sheep breeding;
(6) the application in germplasm resource protection;
(7) the application in germplasm resources improvement;
(8) the application in sheep pedigree reconstruction.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112695107A (en) * | 2021-03-23 | 2021-04-23 | 中国农业大学 | Growth performance SNP locus combination of meat sheep and application thereof |
| CN112695108A (en) * | 2021-03-23 | 2021-04-23 | 中国农业大学 | Reproductive performance SNP (single nucleotide polymorphism) locus combination of meat sheep and application thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112695107A (en) * | 2021-03-23 | 2021-04-23 | 中国农业大学 | Growth performance SNP locus combination of meat sheep and application thereof |
| CN112695108A (en) * | 2021-03-23 | 2021-04-23 | 中国农业大学 | Reproductive performance SNP (single nucleotide polymorphism) locus combination of meat sheep and application thereof |
Non-Patent Citations (3)
| Title |
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| PAULINE等: "Heritability and genome-wide association mapping for supernumerary teats in French Alpine and Saanen dairy goats", 《J. DAIRY SCI.》 * |
| PENG等: "A genome-wide association study reveals candidate genes for the supernumerary nipple phenotype in sheep (Ovis aries)", 《ANIMAL GENETICS》 * |
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Application publication date: 20210820 |