CN117701732A - Application of SNP locus combination in identification of Xuyi mountain buffalo - Google Patents
Application of SNP locus combination in identification of Xuyi mountain buffalo Download PDFInfo
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- 230000002068 genetic effect Effects 0.000 claims abstract description 39
- 230000004069 differentiation Effects 0.000 claims abstract description 8
- 239000000523 sample Substances 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 5
- 239000003147 molecular marker Substances 0.000 claims description 5
- 239000002773 nucleotide Substances 0.000 claims description 5
- 125000003729 nucleotide group Chemical group 0.000 claims description 5
- 238000002864 sequence alignment Methods 0.000 claims description 2
- 238000004458 analytical method Methods 0.000 description 10
- 238000009826 distribution Methods 0.000 description 10
- 238000001514 detection method Methods 0.000 description 6
- 238000012163 sequencing technique Methods 0.000 description 6
- 238000001914 filtration Methods 0.000 description 5
- 239000012634 fragment Substances 0.000 description 5
- 239000008280 blood Substances 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- 230000035772 mutation Effects 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 238000007400 DNA extraction Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000003908 quality control method Methods 0.000 description 3
- 210000000349 chromosome Anatomy 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 235000013372 meat Nutrition 0.000 description 2
- 238000005096 rolling process Methods 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 108700028369 Alleles Proteins 0.000 description 1
- 238000007399 DNA isolation Methods 0.000 description 1
- 241000357437 Mola Species 0.000 description 1
- 238000009412 basement excavation Methods 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000007621 cluster analysis Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q2600/156—Polymorphic or mutational markers
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Abstract
The invention discloses an application of SNP locus combinations in identifying Xuyi mountain buffalo, wherein the SNP locus combinations consist of 1086 SNP loci and are used for constructing a Xuyi mountain buffalo DNA fingerprint, identifying the authenticity of Xuyi mountain buffalo varieties, analyzing the genetic information, genetic similarity and genetic differentiation degree of Xuyi mountain buffalo.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to an application of SNP locus combination in identification of Xuyi mountain buffalo.
Background
Xuyi mountain buffalo is a local variety of buffalo with both meat and service functions, and is also one of the well-known breed buffalo in China. Xuyi mountain area buffalo is native to Xuyi county, huai 'an city, jiangsu province, and surrounding Huai' an, yangzhou, nanjing and other hilly mountain areas have scattered distribution, and Nantong, taizhou, changzhou and Wuxi have scattered distribution. The number of the buffalo stock in the Xuyi mountain area is about 9000 heads in Jiangsu province in 2006, and 5400 heads in the central production area. But the current service use is gradually reduced, the beef production machine is mainly used for producing meat, and the improvement of the Mola buffalo is more in recent years. The number of the buffalo stock in the Xuyi mountain area gradually decreases in the last 20 years, and the buffalo stock is in an endangered state. To understand the genetic background, genetic structure and genetic diversity of Xuyi mountain buffalo and individual identification. This is difficult to perform only from the aspect of appearance, etc., and therefore, it is necessary to develop a more accurate identification method.
DNA fingerprinting refers to the collective term that a DNA sample is treated with a specific molecular marker technique to reveal a specific DNA fragment. With the progress and development of biotechnology, the DNA fingerprint technology is widely applied to variety resource diversity and variety identification research. The SNP molecular marker for constructing the DNA fingerprint has the advantages of high polymorphism detection rate, wide distribution in genome, stable and reliable result and the like.
At present, the application of the DNA fingerprint technology on buffalo varieties is not much, and the construction of the Xuyi buffalo DNA fingerprint by using SNP molecular markers is not reported. The construction of the DNA fingerprint of the Xuyi mountain area buffalo based on the SNP markers can provide accurate and efficient basis for distinguishing and identifying the Xuyi mountain area buffalo varieties.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, provides application of SNP locus combination in identifying Xuyi mountain buffalo, and solves the problems of distinguishing identification, DNA fingerprint construction, genetic similarity and genetic differentiation analysis of Xuyi mountain buffalo individuals in the background art.
The technical scheme adopted for solving the technical problems is as follows: the application of the SNP locus combination in the identification of Xuyi mountain buffalo is provided, and the SNP locus combination is used for constructing a Xuyi mountain buffalo DNA fingerprint; identifying the authenticity of the variety of the buffalo in Xuyi mountain areas; analyzing the genetic information, genetic similarity and genetic differentiation degree of the Xuyi mountain buffalo;
the SNP locus combination consists of 1086 SNP loci, and the physical positions of the 1086 SNP loci are determined based on the genome sequence alignment of the Xuyi buffalo.
Preferably, the probe comprises a molecular marker probe for detecting 1086 SNP loci, and the nucleotide sequence of the probe is SEQ ID NO. 1-SEQ ID NO. 1086.
Preferably, the genome sequence version number of the Xuyi mountain buffalo is NDDB_SH_1.
Compared with the background technology, the technical proposal has the following advantages:
according to the invention, the genomic sequences of the Xuyi mountain buffalos are compared and determined 1086 SNP loci are used as combinations, so that the DNA fingerprint of the Xuyi mountain buffalos can be constructed, the authenticity of the Xuyi mountain buffalo varieties can be accurately and efficiently identified, and the genetic information, the genetic similarity and the genetic differentiation degree of the Xuyi mountain buffalos can be analyzed.
Drawings
FIG. 1 is a distribution of SNP sites selected in example 1 on a chromosome;
FIG. 2 is a DNA fingerprint of a 25-head Xuyi buffalo in example 2;
fig. 3 is an NJ cluster map in example 3.
Detailed Description
The technical scheme of the invention is further described below with reference to the accompanying drawings.
According to the invention, through collecting blood samples of the Xuyi with different geographical distributions, a Xuyi mountain area buffalo DNA database based on the re-sequenced Xuyi is constructed, the sites of the high polymorphism of the Xuyi mountain area buffalo are screened and filtered in the database, the genetic similarity among the samples is calculated based on the sites of the high polymorphism, and a phylogenetic tree is made. The method comprises the following specific steps: 1. collecting a Jiangsu Xuyi mountain area buffalo blood sample; 2. respectively extracting DNA of the collected Xuyi mountain buffalo, and carrying out mass analysis on the extracted DNA; 3. resequencing the fragments with qualified quality control; 4. detecting the quality distribution, the base distribution and the pollution of the re-sequencing data; 5. comparing reads of each sample with a reference genome by adopting software, setting reasonable screening and filtering parameters, and constructing a specific site (SNP site) database of the Xuyi mountain buffalo; 6. and (5) performing mutation detection and statistics on SNP loci. 7. Screening finger print loci; 8. constructing a Xuyi mountain area buffalo DNA fingerprint or SNP molecular identity card.
According to the invention, large-scale high-depth sequencing excavation is carried out on a Xuyi mountain area buffalo population, a DNA fingerprint of the Xuyi mountain area buffalo is established, the DNA fingerprint comprises 1086 SNP loci, and the physical positions of the 1086 SNP loci are determined based on genome sequence comparison of the Xuyi mountain area buffalo. The genome sequence version number of the Xuyi mountain area buffalo is NDDB_SH_1, and the 1086 SNP locus combinations and specific nucleotides thereof are shown in table 1:
table 1 1086 SNP site combinations and specific nucleotide tables thereof
The invention also comprises a molecular marker probe for detecting the 1086 SNP loci, and the nucleotide sequence of the probe is SEQ ID NO. 1-SEQ ID NO. 1086.
Example 1
The embodiment is the establishment of the DNA fingerprint of the buffalo in the Xuyi mountain area, and the specific establishment flow is as follows:
1. and collecting blood of 25 Xuyi mountain buffalos in the region, and performing mutation detection and statistics on SNP loci.
2. The collected blood was extracted and quality-examined using a DNA extraction kit.
DNA extraction was performed using a DNA extraction kit (FastPure Cell/Tissue DNA Isolation Mini Kit, DC 102-01) from Norvezan, OD value and concentration were measured according to the instructions, and the samples were used after electrophoresis to detect quality. Wherein OD260/OD280 should be between 1.8-2.0; when the sample amount is 2. Mu.l, the sample concentration is more than or equal to 50 ng/. Mu.l. And the DNA quality detection results are all qualified.
3. And (5) resequencing the fragments after quality control is qualified.
Sequencing by using an MGISEQ T7 sequencer, randomly breaking the DNA sample which is qualified in detection, and screening the DNA fragments which meet the requirements and are suitable for the size. The purified DNA fragments were ligated to sequencing adaptors, rolling circle amplified to prepare DNA, and then sequenced on an arrayed chip.
4. Detecting and analyzing the resequencing data, detecting the quality distribution, detecting the base distribution and monitoring the pollution. And the data quality is ensured, and the original data is filtered and evaluated in quality before information analysis.
5. Nddb_sh_1 was selected as the reference genome. And (3) comparing the detected clear Reads with a reference genome by adopting a BWA-MEM algorithm, sequencing the positions by using samtools software, repeating the Reads by using picard software marks, and finally counting various comparison indexes. The comparison rate and coverage index can reflect the quality of samples, library construction, sequencing, reference sequences and the like.
6. And (5) performing mutation detection and statistics on SNP loci. And detecting mutation sites of each sample by using GATK software to obtain gVCF files of each sample. In the subsequent joint-rolling step, gVCF of all samples is subjected to joint analysis to obtain a variation result of each individual in the population. In order to ensure SNP accuracy, quality control is required, and the SNP loci obtained after combination are subjected to preliminary filtration through the filtration parameters recommended by GATK: QD < 2.0|FS > 200.0|SOR > 10.0|MQRankSum < -12.5|ReadPosRankSum < -8.0, 92,085,106 SNPs were obtained.
7. Based on the SNP loci, screening fingerprint loci is carried out by the following steps:
(1) Filtration is performed by deletion rate (missing rate), temperature delta linkage disequilibrium (HWE), site heterozygosity rate (HET), and the like. The filtering conditions are as follows: 1) A two-position locus; 2) A transmission rate <1%; 3) HWE <1e-6; 4) HET <0.7, 6,704,862 SNPs were obtained.
(2) Further, a site with MAF >0.4 was selected to obtain 6,704,862 SNPs, i.e., a site with high polymorphism.
(3) And further dividing the genome into windows with the size of 2Mb by adopting a sliding window mode aiming at the obtained SNP loci, selecting 1 locus with the highest MAF value in each window, and finally obtaining 1086 SNP loci. FIG. 1 shows the distribution of SNP sites on chromosomes.
8. Constructing a Xuyi mountain area buffalo DNA fingerprint.
According to the reference genome: nddb_sh_1 evaluates 1086 sites obtained above, giving evaluation results and probe sequences. And constructing Xuyi mountain area buffalo DNA fingerprint according to the screened SNP loci, wherein the 25 Xuyi mountain area buffalo DNA fingerprint is shown in figure 2.
Example 2
The embodiment is the application of SNP marker combination in the analysis of the genetic similarity of the buffalo population in the Xuyi mountain area.
Using 1086 SNP markers obtained in example 1, 25 Xuyi buffalo were compared in pairs for genetic similarity. Genetic similarity is represented by the formula: gs=2n ij /(N i +N j ) Calculated, where N ij For the number of effective markers common to both individuals, ni is the number of effective markers for individual 1, N j Number of active markers for individual 2. The results are shown in Table 2, and the results have higher consistency with the prior pedigree records, which indicates that the results are also applicable to analysis application of genetic similarity.
TABLE 2 analysis of genetic similarity
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Note that: the upper triangle is the number of effective markers shared by two individuals, and the lower triangle is the genetic similarity of two individuals.
Example 3
The embodiment is the application of SNP locus combination in the genetic differentiation analysis of the Xuyi buffalo population.
Using the molecular markers obtained in example 1, the genetic distance between samples was calculated to discriminate the samples. Genetic distance (Genetic distance) is an indicator that measures the Genetic differentiation between species or between populations of the same species. For a gene, two populations have similar alleles and are similar in frequency, with a smaller genetic distance from each other, indicating that their common origin is relatively close. For 1086 sites, the Nei's standard distance (Nei's standard genetic distance) between samples was calculated using VCF2Dis software, and all samples could be separated based on genetic distance, the magnitude of the genetic distance matrix value indicated the distance of the genetic distance, the smaller the value, the closer the relationship between samples was, and the results are shown in Table 3. Further, according to the Nei's genetic distance, a Xuyi mountain buffalo population cluster analysis was performed, and an NJ cluster map was drawn (FIG. 3). A branching diagram or tree describing the evolutionary sequence among the populations, representing the evolutionary relationship among the populations. The relatedness of the populations can be inferred from their commonalities or differences in physical or genetic characteristics, etc. The results are consistent with the genealogy records and the genetic similarity results obtained in the example 2, which show that the method is also applicable to analysis application of population genetic differentiation.
TABLE 3 genetic distance analysis Table
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The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (3)
1. An application of SNP locus combination in identifying Xuyi mountain buffalo, which is characterized in that: the method is used for constructing a Xuyi mountain area buffalo DNA fingerprint; identifying the authenticity of the variety of the buffalo in Xuyi mountain areas; analyzing the genetic information, genetic similarity and genetic differentiation degree of the Xuyi mountain buffalo;
the SNP locus combination consists of 1086 SNP loci, and the physical positions of the 1086 SNP loci are determined based on the genome sequence alignment of the Xuyi buffalo.
2. The use of a SNP locus combination as set forth in claim 1 for identifying a Xuyi buffalo, characterized in that: comprises a molecular marker probe for detecting 1086 SNP loci, wherein the nucleotide sequence of the probe is SEQ ID NO. 1-SEQ ID NO. 1086.
3. The use of a SNP locus combination as set forth in claim 1 for identifying a Xuyi buffalo, characterized in that: the genome sequence version number of the Xuyi mountain area buffalo is NDDB_SH_1.
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