CN113278714A

CN113278714A - Gene chip for analyzing whether sheep has horns or not, molecular probe combination, kit and application

Info

Publication number: CN113278714A
Application number: CN202110834345.7A
Authority: CN
Inventors: 李孟华; 李心; 罗凌云; 杨继; 吕锋骅
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2021-07-23
Filing date: 2021-07-23
Publication date: 2021-08-20
Anticipated expiration: 2041-07-23
Also published as: CN113278714B

Abstract

The invention discloses a gene chip, a molecular probe combination, a kit and application for analyzing whether sheep are horned, and relates to the technical field of biology.

Description

Gene chip for analyzing whether sheep has horns or not, molecular probe combination, kit and application

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 for analyzing whether sheep has horns, a molecular probe combination, a kit and application.

Background

Sheep are one of the most important economic animals in the world. Among the various sheep breeds, some sheep have no horn and some sheep have horns, but with the development of livestock production and the change of market demand, some sheep breeds begin to disappear or are in a dangerous state, and because the high-horn sheep have certain aggressiveness, the facilities are often damaged, and the sheep are mutually blown, so that farmers are not willing to raise the high-horn ewes, the study on whether the sheep horns are horns is urgent, however, the phenotype of the sheep horns is difficult to find in the early stage and can only be determined until the sheep horns grow to half to 1 year. Indirect determination of the polyhedrosis phenotype is therefore an area of interest to researchers in favor of DNA-based diagnostics. 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 gene chip based on SNP is a convenient and efficient tool for modern genetic breeding, and because the gene chip can easily realize high-throughput and automatic detection of SNP, can detect the change of each base pair on genomic DNA, including insertion, deletion, inversion, conversion and the like, the gene chip 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 the Chinese sheep populations and can rapidly and effectively detect whether the sheep has horns has very important significance.

Disclosure of Invention

In order to meet the requirements of current sheep variety research and agricultural production on the detection of whether sheep is horned or not in China, the invention provides a 514 SNP locus combination for analyzing whether sheep is horned or not, a molecular probe combination, a gene chip, a kit and application prepared based on the locus combination.

In order to achieve the technical purpose of the invention, the invention provides the following technical scheme:

1. the application of 514 SNP loci in analyzing whether sheep have horns or not, wherein the physical positions of the 514 SNP loci are determined based on sheep v4.0 genome sequence alignment, and the 514 SNP loci are shown in Table 1.

TABLE 1514 SNP site information

。

2. A method for analyzing whether sheep have horns or not, comprising the step of comparing the 514 SNP site genotypes of the genomic DNA of a test sheep variety with the 514 SNP site genotypes of the genomic DNA of a control sheep variety; the 514 SNP loci are 514 loci described in Table 1.

3. And (3) analyzing whether the sheep have horned molecular probe combinations, and detecting 514 SNP sites in the genome of the sheep to be detected, wherein the SNP sites are described in Table 1.

4. And (3) analyzing whether the sheep has horns or not by using a gene chip, wherein the gene chip is loaded with the molecular probe combination of 3.

5. A kit for analyzing whether sheep have horns, which comprises the molecular probe set of 3 or the gene chip of 4.

6. And (3) a method for analyzing whether the sheep has horns or not, and detecting the genome of the sheep to be detected by using the molecular probe combination described in the step 3 or the gene chip described in the step 4 or the kit described in the step 5.

7. The molecular probe combination or the gene chip or the kit has the following applications: a. use in assessing whether a sheep has horns; b. the application in screening sheep varieties; c. the application in sheep variety identification; d. the application in tracing sheep varieties; e. the application in sheep breeding; f. the application in germplasm resource protection; g. the application in germplasm resources improvement; use in sheep pedigree reconstitution.

Has the advantages that:

1. the SNP locus combination which is only composed of 514 SNP loci and is used for judging whether the sheep has horns or not is provided based on the research on genetic resources of a plurality of sheep at home and abroad, the SNP locus combination provided by the invention has good universality at home and abroad, can quickly judge whether the sheep has horns or not from the gene level so as to obtain more accurate breeding evaluation information, selects the shape which is difficult to measure at the early stage, shortens the generation interval, accelerates the breeding process and saves the breeding cost; in addition, whether the angular SNP locus combination exists or not can be used for identifying and tracing sheep varieties, and technical support is provided for germplasm resource protection and germplasm resource improvement.

2. The probe combination, the gene chip and the kit formed based on the SNP locus combination provided by the invention are used for detecting a sheep sample, and have the characteristics of small flux, low cost, easier analysis, wide universality and wide market prospect.

Drawings

FIG. 1 is a Manhattan chart of a group of Hu sheep Vs small tailed Han sheep (HUS verses SXW);

fig. 2 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 apply equally to the molecular probe combinations, gene chips, kits and uses thereof formed based on site combination and will not be described in detail here.

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 SNP site combination for horny trait

1. Selection of sheep individuals

In order to achieve a more comprehensive coverage of the Sheep species at home and abroad, the present invention provides a genetic information acquisition of 248 Sheep individuals throughout asia, europe, africa and the middle east, including 16 wild Sheep asian moloren species, 172 local species and 60 breeds, specifically related to caucasian, Sishui fur, Dacaudal han, pothole Sheep, Husu Sheep, Nick Sheep in China, Ariety Sheep in Xinjiang, Pasteur Sheep, Dorper Sheep, merino Sheep (fine wool Sheep), merino Sheep (superfine wool), saffron, Lei black Sheep, Dolang Sheep, Waggir Sheep, Finland Sheep (Fisheep) in Finland, Weisang Sheep (Ouessant) in Netherlands, Shetland Land Sheep (Shetland), Sorano knoont, Gothene island (Gothene), Gothene and Lantern Sheep (Dragne and Lancey Sheep), and Sheep (Dragne-and Lancey Sheep), and Sheep (Dreche) in Sheep), and Sheep (Tanay Sheep) in China 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 genetic information of sheep with horns or not

Screening whether horned sheep varieties exist or not, grouping corresponding genetic information samples according to the obvious difference of different sheep varieties on whether horned characters exist, and in one embodiment of the invention, dividing genetic information of Hu sheep Vs small tailed Han sheep (HUS overturs SXW) into a group.

3.2 processing of genetic information on whether there is a horned sheep

And (2) scanning out a functional region related to whether the sheep has horns or not by sweeping the multi-locus allele frequency difference between each sheep group through XP-CLR (a Manhattan graph obtained by scanning is shown in figure 1, and meanwhile, excavating the functional region related to whether the sheep has horns or not in the sheep variety in each group by utilizing a pi ratio (namely a pi value), and then taking the intersection of the two results to screen out the functional region related to whether the sheep has horns or not.

Screening genes in the region by referring to published gene research results, finally determining 3 candidate genes ADAR, RXFP2 and NPR3 which are related to whether the genes have corners and have quite definite functions, and further determining the functional regions corresponding to the candidate genes by perl scripts.

4. Acquisition of whether there is a combination of horned SNP sites

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 functional genes related to whether 3 genes are related to angles or not, wherein the functional genes are related to the ADAR, the RXFP2 and the NPR3, and only 514 SNP loci are included.

Example 2 use of SNP site combinations judged to have horns for preparing primer combinations and Probe combinations

The skilled in the art designs a primer according to the sequence information of each site in the 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, such as DNA folding form evaluation of the secondary structureStructures, see in particular (

) The Tm value was then evaluated by using the software RaW-Probe.

The methods are all conventional methods, and can be obtained according to the site information in the angular SNP site combination provided by the application without creative labor, so that the primer obtained according to the angular SNP site combination provided by the application also belongs to the protection scope of the invention.

Similarly, it is within the scope of the present invention to use the combination of angular SNP sites provided by the present invention to prepare probes, such as tanqman probes.

Example 3 SNP site combination for preparing 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, the skilled person can prepare the SNP gene chip for detecting whether sheep has horns in any way, and also can prepare the SNP gene chip by entrusting a biological company, but the SNP gene chip prepared based on the combination of whether there are horns and SNP sites provided in this application is within the protection scope of the present invention.

Example 4 kit for analyzing whether sheep are horned or not

The kit for analyzing whether or not there is a corner provided by the present invention includes primers or probes or gene chips obtained based on the combination of SNP sites 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 skilled in the art configures different SNP kits according to different using modes, but the kit for analyzing whether sheep have horns or not configured based on the combination of the SNP sites provided by the invention is in the protection scope of the invention.

EXAMPLE 5 detection of whether sheep are horned

Whether the purchased lambs have horns or not is detected based on the combination of the horns or not and the SNP loci of the sheep provided by the embodiment 1 of the application, and meanwhile, whether the sheep have horns or not is observed, so that the accuracy of the invention is verified, and the method specifically comprises the following steps:

collecting peripheral blood of lambs by a conventional method, wherein the peripheral blood is an in vitro biological sample taken from the lambs, extracting whole genome DNA in the sample by a conventional DNA extraction kit to obtain a whole genome DNA sample, measuring the concentration of the DNA sample by a Thermo Scientific NanoDrop 2000 ultraviolet spectrophotometer, and diluting to 20 ng/mul for later use;

the primer is designed by using the site information in the SNP site combination by a conventional method, the whole genome DNA of the lamb 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 in the detection sample is consistent with the observation result. It should be noted that the population threshold value in the present application is obtained by analyzing the angular population and the polled population, and the method is the same as the above method.

According to the method, the significance test (independent sample ManWhitney U test) is carried out on the judgment results of the analysis of the angular population and the non-angular population, the results are shown in figure 2, 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 the method have accuracy and effectiveness.

Industrial applications

The technical personnel in the field can analyze and judge whether sheep have horns or not on the genome level based on the SNP site combination of whether sheep have horns or not and the SNP probe combination and the SNP chip for detecting whether sheep have horns or not, which are provided by the application and only consist of 514 SNP sites, on the sheep horns or not, and carry out genetic evaluation or variety screening or variety identification, thereby not only shortening the generation interval, accelerating the breeding process and saving the breeding cost, but also being applied to 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

The application of 1.514 SNP loci in analyzing whether sheep have horns or not, wherein the physical positions of the 514 SNP loci are determined based on sheep v4.0 genome sequence alignment, and the 514 SNP loci are shown in Table 1.
2. A method for analyzing whether sheep have horns or not, comprising the step of comparing the 514 SNP site genotypes of the genomic DNA of a test sheep variety with the 514 SNP site genotypes of the genomic DNA of a control sheep variety; the 514 SNP sites are the 514 sites of claim 1.
3. Analyzing whether sheep have horns or not by using a molecular probe combination, wherein the molecular probe combination detects 514 SNP sites as set forth in claim 1 in the genome of a sheep to be detected.
4. A gene chip for analyzing whether sheep has horns, wherein said gene chip is loaded with the combination of molecular probes according to claim 3.
5. A kit for analyzing whether sheep have horns or not, comprising the molecular probe set according to claim 3 or the gene chip according to claim 4.
6. A method for analyzing whether sheep have horns, which is characterized in that the genome of a test sheep is detected by using the molecular probe combination of claim 3, the gene chip of claim 4 or the kit of claim 5.
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) use in assessing whether a sheep has horns;

(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.