阅读说明：本技术 一种利用y染色体单核苷酸遗传标记鉴定瘤牛公牛来源的方法 (Method for identifying source of bull ruminants by utilizing Y chromosome mononucleotide genetic markers ) 是由 陈宁博 雷初朝 许星龙 夏小婷 党瑞华 黄永震 蓝贤勇 陈宏� 于 2021-10-29 设计创作，主要内容包括：本发明公开了一种利用Y染色体单核苷酸遗传标记鉴定瘤牛公牛来源的方法。本发明利用基因组重测序结果,对主要瘤牛群体的Y染色体DNA序列进行扫描,发现了东亚瘤牛、非洲瘤牛等瘤牛群体特有的Y染色体SNP位点,根据这些SNP位点可以在DNA水平上准确鉴别瘤牛公牛所属群体,能有效用于不同瘤牛群体的地方品种的引种及分子标记辅助育种,为快速建立遗传资源优良的瘤牛种群奠定基础。(The invention discloses a method for identifying a source of a bull tumor by utilizing a Y chromosome mononucleotide genetic marker. The invention utilizes the genome re-sequencing result to scan the Y chromosome DNA sequence of the main tumor cattle group, finds the specific Y chromosome SNP sites of the tumor cattle groups such as east Asia tumor cattle, Africa tumor cattle and the like, can accurately identify the tumor cattle bull group on the DNA level according to the SNP sites, can be effectively used for the introduction of local varieties of different tumor cattle groups and the molecular marker assisted breeding, and lays a foundation for quickly establishing the tumor cattle group with excellent genetic resources.)

1. A method for identifying the source of a tumor bull by utilizing a Y chromosome mononucleotide genetic marker is characterized by comprising the following steps: the method comprises the following steps:

extracting the genome DNA of the bull stud to be identified, detecting the polymorphism of the single nucleotide site corresponding to the specific SNP associated with the group identification of the genome DNA in the Y chromosome reference genome of the bull, and if the mutant allele is detected, determining the group to which the bull stud to be identified belongs according to the single nucleotide site of the detected mutant allele; the group of bulls comprises eastern Asia tumor cattle, south Asia tumor cattle and Africa tumor cattle; the specific SNP of the east Asian tumor cattle is selected from one or more SNPs positioned at 3839042, 3861238, 42456991, 42685548, 42882874 and 43143941 th positions of a cattle Y chromosome reference genome, and the specific SNP is used for identifying that the mutant allele of the east Asian tumor cattle corresponds to G, T, T, T, T, C.

2. The method for identifying the source of bulls using the Y chromosome mononucleotide genetic marker according to claim 1, wherein the method comprises the following steps: the peculiar SNPs of african-born cattle are selected from one or more of the SNPs located at positions 43180361, 42393839, 3467637 of the reference genome of the Y chromosome of cattle, and the mutant allele used for identifying african-born cattle corresponds to C, C, C.

3. The method for identifying the source of bulls using the Y chromosome mononucleotide genetic marker according to claim 1, wherein the method comprises the following steps: the specific SNP shared by the southern Asian tumor cattle and the African tumor cattle is selected from one or more SNPs positioned at 42282365 and 43282424 of a cattle Y chromosome reference genome, and the mutant allele used for identifying the southern Asian tumor cattle and the African tumor cattle is A, A correspondingly.

4. The method for identifying the source of bulls using the Y chromosome mononucleotide genetic marker according to claim 1, 2 or 3, wherein the method comprises the following steps: the genotype of the individual of the bull with the tumor at a special SNP locus is in a homozygous state.

5. The method for identifying the source of bulls using the Y chromosome mononucleotide genetic marker according to claim 1, 2 or 3, wherein the method comprises the following steps: the bovine Y chromosome reference genome is selected from Btauu 5.0.1.

6. The method for identifying the source of bulls using the Y chromosome mononucleotide genetic marker according to claim 1, 2 or 3, wherein the method comprises the following steps: and traversing and detecting the specific SNP by adopting a sequencing-based genotyping method or a probe chip-based genotyping method.

7. The use of the method for identifying the source of bulls seye using Y chromosome mononucleotide genetic markers according to any one of claims 1 to 3 in the introduction or molecular marker assisted breeding of bulls seye.

8. The application of the special SNP of the bull in the introduction of the bull, the molecular marker assisted breeding or the manufacture of SNP chips is characterized in that: among the characteristic SNPs of bulls, the characteristic SNP for identifying eastern asian tumor cattle is selected from one or more of SNPs located at positions 3839042, 3861238, 42456991, 42685548, 42882874, 43143941 of a reference genome of a cattle Y chromosome; the unique SNPs for identifying african knot cattle are selected from one or more of the SNPs located at positions 43180361, 42393839, 3467637 of the reference genome of the bovine Y chromosome, and the unique SNPs for identifying south asian knot cattle and african knot cattle are selected from one or more of the SNPs located at positions 42282365, 43282424 of the reference genome of the bovine Y chromosome.

9. The application of the special SNP of the bull as the paternal origin marker of the bull is characterized in that: among the characteristic SNPs of bulls, the characteristic SNP for identifying eastern asian tumor cattle is selected from one or more of SNPs located at positions 3839042, 3861238, 42456991, 42685548, 42882874, 43143941 of a reference genome of a cattle Y chromosome; the unique SNPs for identifying african knot cattle are selected from one or more of the SNPs located at positions 43180361, 42393839, 3467637 of the reference genome of the bovine Y chromosome, and the unique SNPs for identifying south asian knot cattle and african knot cattle are selected from one or more of the SNPs located at positions 42282365, 43282424 of the reference genome of the bovine Y chromosome.

10. An SNP chip, characterized in that: the typing sites of the chip comprise SNP sites positioned at 3839042, 3861238, 42456991, 42685548, 42882874, 43143941, 43180361, 42393839, 3467637, 42282365 and 43282424 th sites of a bovine Y chromosome reference genome.

Technical Field

The invention belongs to the technical field of molecular marker-assisted identification of introduction and breeding, and relates to identification of a group of Bull oxus by using a Bos indicus Y chromosome single nucleotide genetic marker (SNP).

Background

Single Nucleotide Polymorphism (SNP) is the phenomenon in which single nucleotides differ in allelic sequences of genomic DNA of different individuals of the same species. Single-stranded DNA of the same length will form different conformations due to different sequences or single base differences. The human genome project has identified a number of SNPs, which are the most widespread type of variation in the genome of organisms due to insertions, deletions, transitions and transversions of individual nucleotides.

The Male specific region (MSY) of the mammalian Y chromosome does not recombine with the X chromosome during meiosis, and is therefore typically inherited paternally and is present only in Male individuals. The Y-SNPs markers are widely used for genetic diversity and origin research of the world cattle population, and the tumor cattle have been reported to have 2Y chromosome haplotypes Y3A and Y3B at present, but the tumor cattle of the two haplotypes are mainly discovered to be Chinese tumor cattle and American tumor cattle, and the American tumor cattle cannot represent south Asia tumor cattle and Africa tumor cattle; and can not be used for distinguishing the groups of east Asia tumor cattle, south Asia tumor cattle and Africa tumor cattle due to the limited number of SNPs.

Disclosure of Invention

Aiming at the problems that the number of SNP sites for identifying the cattle (Bos indicus) group is limited, the cattle can be only divided into two paternal origins of Y3A and Y3B, and the groups (east Asia cattle, south Asia cattle and Africa cattle) can not be finely distinguished, the invention provides a method for identifying the source of the bull cattle by using a Y chromosome mononucleotide genetic marker.

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

the invention searches Y chromosome genetic markers for distinguishing east Asia tumor cattle, south Asia tumor cattle and Africa tumor cattle by performing re-sequencing on the east Asia tumor cattle, south Asia tumor cattle and Africa tumor cattle. According to the sequencing result of the individual Y-stain weight of different groups of bulls, combining certain experimental and analytical means, the site information (SNP site must be male specific) of the specific SNP of the corresponding group of bulls in the reference genome sequence (Btau5.0.1) of the bovine Y chromosome is determined, wherein:

the specific SNP of the east Asia oncous cattle is positioned at 3839042, 3861238, 42456991, 42685548, 42882874 and 43143941 th sites of a cattle Y chromosome reference genome, and the corresponding allele for identifying the east Asia oncous cattle in the sites (without heterozygous state genotypes) is a mutant base G, T, T, T, T, C;

the peculiar SNP of the African tumor cattle is positioned at 43180361 th, 42393839 th and 3467637 th sites of a cattle Y chromosome reference genome, and the corresponding allele for identifying the African tumor cattle in the sites (without heterozygous state genotypes) is a mutant base C, C, C;

the common unique SNP of the southern Asia and African tumor cattle is positioned at 42282365 and 43282424 th sites of a cattle Y chromosome reference genome, and the corresponding allele used for identifying the southern Asia and African tumor cattle in the loci (without heterozygous state genotypes) is a mutant base A, A.

Preferably, the southern Asian tumor bovine can also be identified by adopting a haplotype consisting of alleles (reference bases G, T and A) of the sites of the three characteristic SNPs at positions 3467637, 43180361 and 42393839 and alleles (mutant bases A, A) of the sites of more than one characteristic SNP at positions 42282365 and 43282424.

The invention has the beneficial effects that:

according to the invention, through the discovered specific SNP sites on the Y chromosomes of the bull of different groups, the bull of different group sources can be effectively distinguished by utilizing the SNP sites, and the method can be used for identifying the breeding bull group, introducing and molecular marker-assisted breeding, and lays a foundation for rapidly establishing the bull group with excellent genetic resources.

Detailed Description

The present invention will be described in further detail with reference to examples.

The invention provides a method for identifying a group of Bull bull by using a Bomb bull Y chromosome mononucleotide genetic marker (SNP marker)

Determining cattle Y chromosome genetic variation information for accurately distinguishing source information of a tumor cattle bull population by using a genome re-sequencing technology and a bioinformatics analysis method

(1) Re-sequencing bull genome (cow genome sequence as control)

191 eastern, southern and African bull bulls were re-sequenced and 85 other bull bulls genome re-sequencing results were downloaded from the NCBI (containing Simmental, Jersey, Angus, Gelbvieh, Hereford, Holstein, Kazakh, Mongolian, Chaidamu, Yakutian, Yanbian, Hanwood, Tibetan, Muturu, N' Dama, Abergelle, Arado, Arsi, Bale, Afar, Bagaria, Begaiit, Ethipia, Semien, Choke, Erob, Fogera, Goffa, Horro, Mursi, Raya, Ogaden, Borana, Kenana, Sri Lanka, Cholini, Paragnia, Burbank, Banhai, Guilian, Banhai, Guihua, Banhai, Guihua, Banhuana, Guihuana, Guihuan, Lanhuan, Shisanshan, Lanhua, Lanhuan, and Lanhuan, and Lanhuan, and Lanhuan, and.

(2) Genome alignment

The resequencing results for each bull were aligned to the bovine reference genome Btau5.0.1(NCBI version number GCF _000003205.7) using BWA software.

(3) The SNP sites were found by Samtools software and GATK software and filtered on the Y chromosome as follows: SNPs must be present only in individuals from bulls, and not in all individuals from cows (i.e., SNPs present only in the male-specific region of the Y chromosome); the SNP loci are homozygous in the individual bull, and are ensured to be positioned in the single copy area of the Y chromosome.

(4) Deletion sites were filled in using Beagle software.

(5) Screening of individual groups for unique single nucleotide mutation sites (i.e., unique SNPs)

Specific SNP sites of three groups of east Asia tumor cattle, south Asia tumor cattle and Africa tumor cattle are found through screening, see table 1, the frequency of part of specific SNPs in the corresponding group is 100%, and the frequency of other groups is basically 0; some characteristic SNPs have a frequency of less than 100% in the corresponding population, but not more than 5% in other populations.

TABLE 1 statistics of SNPs loci and their mutant allele frequencies for different bull populations

According to Table 1, African nodules have 3 unique SNP sites (i.e., SNP1, SNP2, SNP3), and the frequencies in African nodules are 89% (31/35), 89% (31/35), and 86% (30/35), respectively. The frequencies of other cattle breeds are respectively 5% (11/241), 5% (11/241) and 0(0/241), the genotypes of the African tumor cattle at the three sites are SNP1-CC, SNP2-CC and SNP3-CC, and the African tumor cattle can be identified by using the genotype of any one SNP site.

According to table 1, eastern asian nodules of cattle have 6 unique SNP sites (i.e. SNP4, SNP5, SNP6, SNP7, SNP8, SNP9), the frequencies in eastern asian nodules of cattle are 84% (73/87), 84% (73/87), 84% (73/87), 84% (73/87), 84% (73/87), 84% (73/87), and the frequencies in other cattle are all 0. The genotypes corresponding to the six sites of the east Asia tumor cattle are SNP4-GG, SNP5-TT, SNP6-TT, SNP7-TT, SNP8-TT and SNP9-CC respectively, and the genotype of any one SNP site can be used for identifying the east Asia tumor cattle.

According to table 1, south asian and african born cows (common to south asian born cows and african born cows) have 2 unique SNP sites (i.e., SNP10, SNP11), with frequencies of 93% (80/86), 93% (80/86) in south asian and african born cows, respectively, and frequencies of 9.1% (8/87) and 9.1% (8/87) in east asian born cows, respectively, and 0 in normal cows. The southern Asia and Africa tumor cows can be identified by utilizing the combined genotypes of 1, 2 or 3 sites in 3 special SNP sites (SNP1, SNP2 and SNP3) of the Africa tumor cows and 1 or 2 sites in SNP10 and SNP11, wherein the tumor cows with the genotypes of the 5 sites of SNP1-GG, SNP2-TT, SNP3-AA, SNP10-AA and SNP11-AA can be identified as the southern Asia tumor cows.

(II) identifying the group of the bull belonging to the tumor

According to the results, African tumor bulls from different groups can be distinguished by utilizing the site 1-3 of SNP listed in Table 1, east Asian tumor bulls from different groups can be distinguished by utilizing the site 4-9 of SNP, and African tumor bulls and south Asian tumor bulls can be distinguished by utilizing the combined genotypes of SNP1-3 and SNP 10-11.

When identifying the group to which the individual bull belongs, extracting the genomic DNA of the bull to be identified, detecting the polymorphism of the genomic DNA at the single nucleotide site corresponding to the specific SNP in the reference genome of the Y chromosome of the bull, and determining the group to which the bull to be identified belongs according to the detection result and the specific SNP site of the bull of the corresponding group, wherein the specific flow is illustrated as follows.

Example 1 method for identifying the group of Bull with the use of Y chromosome Single nucleotide genetic marker (SNP marker) (DNA sequencing method)

Taking eastern Asia tumor cattle as an example, one or more of SNP4, SNP5, SNP6, SNP7, SNP8 and SNP9 are selected by utilizing a cattle Y chromosome reference genome sequence, and the two sides of each of the two parts are extended by a certain length, so that PCR amplification primers are designed by taking the two sides as templates. And (3) performing PCR amplification (the cow should have no amplification band, and the bull has a bright amplification band) by using the bull genome DNA to be detected as a template and the cow genome DNA as a recessive control, and sequencing the amplification product. And comparing the sequencing result with a reference genome sequence by using DNASTAR software, and determining whether the selected SNP site is a reference base or a mutant base, wherein if the SNP site is the mutant base, the bull is an east Asia oncous cow, and otherwise, the bull is not the east Asia oncous cow.

The experimental result shows that the accuracy or consistency of the identification by the method reaches 97%.

Example 2 method for identifying the group of Bull with the use of Y chromosome Single nucleotide genetic marker (SNP marker) (SNP chip method)

(1) A section of 50bp single-stranded probe primer is designed according to each SNP site in the table 1.

(2) These primers are immobilized on the chip material in a certain order.

(3) And carrying out whole genome amplification on the genomic DNA of the bull to be detected.

(4) And (3) fragmenting the amplified product by using random endonuclease enzyme.

(5) Hybridizing the DNA fragment with the chip to ensure that the enzyme digestion product of the genome DNA is specifically combined with the probe.

(6) Single base extension by the addition of two fluorescently labeled dideoxynucleotide triphosphates (ddATP, ddGTP) only extended with probes that bind complementary to genomic DNA.

(7) And scanning the chip after cleaning.

(8) Detecting the fluorescence intensity and wavelength of each site, and judging the SNP typing result.

The experimental result shows that the accuracy or consistency of the identification by the method reaches 95%, and by the method, samples of a plurality of bulls to be detected can be detected at one time, and respective groups (belonging to east Asia tumor bulls, Africa tumor bulls or south Asia tumor bulls) of different bulls can be identified.

In a word, the invention provides a method for identifying bulls of different rumble cattle groups on the DNA level, which is suitable for quickly identifying bulls of east Asia rumble cattle, Africa rumble cattle and south Asia rumble cattle, can be effectively used for introducing and molecular marker-assisted breeding of bulls of various local varieties belonging to the groups, can also be used for paternal origin markers of the bulls groups, and lays a foundation for quickly establishing the rumble cattle groups with excellent genetic resources.