CN115896303B - SNP primer set for panda sex identification and application thereof - Google Patents
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Abstract
The invention discloses an SNP primer set for panda sex identification and application thereof, wherein the SNP primer set comprises 5 primer sets, and the nucleotide sequence of the SNP primer set is SEQ ID NO.1-15. The SNP primer set has high stability and rich polymorphic information content, and can be used for identifying the sex of pandas.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a SNP primer set for panda sex identification and application thereof.
Background
The appearance characteristics of the female and male pandas are not obvious, the appearance difference of the genitals is also not obvious, and the hairiness is thick. In a containment environment, it is often necessary to accurately identify gender after anesthesia. The sex of the pandas in the wild is more difficult to judge, so that the sex of the pandas is often required to be identified by means of DNA molecular markers.
In the past two decades, DNA molecular markers used for panda identification have been mainly microsatellite (STR) markers, the STR markers consist of motifs that are repeated in tandem, PCR amplification is prone to the generation of a slide chain phenomenon, and STRs are mostly multicopy sequences, a large number of similar or identical sequences exist in the genome, and multiple amplification and interference typing are prone to occur. A Single Nucleotide Polymorphism (SNP) is one of the DNA sequence polymorphisms and is considered to be a third generation genetic marker following STR. SNPs are widely existing in mammalian genome, and one SNP exists approximately every 1000bp, so that the SNP has rich polymorphism and strong superiority in forensic identification. With the advent of second generation sequencing technology, a large number of SNP sites were identified by large-scale genome re-sequencing. In the early stage, 61 SNP loci suitable for panda individual identification have been developed, and panda individuals can be identified economically, stably and rapidly through nucleic acid mass spectrometry.
In the breeding program of pandas, sex identification of pandas is a very important task. In the conventional method for identifying sex of pandas by molecular genetic analysis, the fifth exon fragment of the AMEL gene on sex chromosome is amplified by PCR, and the polymorphism of the amplified fragment (the length of the amplified fragment AMEL. X is 237bp, and the length of the amplified fragment AMEL. Y is 174 bp) is identified to judge the sex. However, DNA degradation is very serious in samples collected in the field, DNA amounts are also very rare, and it is often difficult to amplify a target fragment. Therefore, we developed a technique for identifying the sex of pandas based on SNP marker loci, replacing the conventional identification technique based on DNA length polymorphism. The technique contemplates shortening the PCR amplified fragments to below 120bp for better detection of highly degraded DNA samples, while using a high sensitivity nucleic acid mass spectrometer, as few as 5ng of DNA samples can be detected. It is therefore necessary to provide a SNP primer set for panda sex determination.
Disclosure of Invention
In view of the above, the invention provides a SNP primer set for panda sex determination and application thereof, which can amplify 5 nucleotide sequences with polymorphism and good repeatability, and the SNP primer set detects 'point' mutation, aiming at the problem that the marking typing result of the AMEL amplified fragment length polymorphism in a severely degraded DNA sample is unstable; the SNP primer set has high stability and rich polymorphic information content, and can rapidly identify the sex of pandas.
In order to solve the technical bottleneck that the genome DNA which is severely degraded is difficult to amplify longer DNA fragments, the invention discloses a SNP primer set for panda sex identification, which comprises a mh0XGP-001 primer set, a mh0XGP-002 primer set, a mh0XGP-001# (1) primer set, a mh0XGP-001# (2-3) primer set and a mh0XGP-002# (1-2) primer set, wherein the nucleotide sequence of the SNP primer set is SEQ ID NO.1-15.
As still further aspects of the invention: wherein, the SNP primer set is applied to panda sex identification.
Of course, it is not necessary for any of the products embodying the invention to achieve all of the technical effects described above at the same time.
Description of the drawings:
FIG. 1 is a pattern of peaks in a chromatogram of 5 SNP sites for sex determination of pandas.
Detailed Description
The following will describe embodiments of the present invention in detail by referring to examples, so that the implementation process of how to apply the technical means to solve the technical problems and achieve the technical effects of the present invention can be fully understood and implemented.
Example 1 preparation of SNP primer set for panda sex determination
(1) Extracting DNA of 83 pandas, wherein the number of female is 49, and the number of male is 34;
collecting panda blood, tissue and other samples, extracting DNA strictly according to QIAGEN DNA hr extraction kit, adding appropriate amount of TE buffer solution to dissolve precipitate, and storing at 4deg.C or-20deg.C for use.
Genome resequencing was performed on 83 pandas using the Illumina HiSeq 2000 platform. The raw data filtering criteria for the project are as follows: (1) filtering the joint to remove pollution; (2) In order to obtain higher quality data, if a read has more than half of its base number of low quality (Q.ltoreq.5 (E)) than the entire read, the reads are removed; (3) removing the tag sequence for sample identification. After data filtering, clear data of each individual is obtained; SNP locus primer set for panda sex identification can amplify SNP with higher polymorphism and stable typing result, and the nucleotide sequence is shown in the following table 1:
TABLE 1 SNP primer set for panda sex determination
The SNP primer set designs primers aiming at SNP loci of stable inheritance in homologous fragments of X and Y of pandas, 10bp tails, namely ' ACGTTGGATG ', are added to 5' of the 5 SNP amplification primers so that the molecular mass of the PCR primers exceeds the detection range of the nucleic acid mass spectrum, the detection of extension products is not influenced, the pandas SNP loci with polymorphism and good repeatability consistency are obtained, a corresponding genotyping technology is established, and sex identification of pandas can be realized.
Example 2 detection of SNPs by individual resequencing methods
(1) And (3) comparing the clean data of each sample to the panda genome by using BWA comparison software to obtain an initial comparison result file in the BAM format. Duplicate reads (where duplicate refers to identical reads resulting from PCR amplification) were removed using a Picard tool and ordered by alignment position;
(2) The SNP and Indel were simultaneously detected using the replotype teller tool of GATK software (alignment file used for detection. BAM has passed correction of the base matrix values and correction of the incorrect alignment positions by Indel);
(3) The SNPs and indels in the output VCF result file labeled "PASS" are the trusted set of variations that passed the filtering conditions.
Example 3 SNP screening for panda gender identification:
(1) Extracting all mutation sets located within the interval 116,833,061-116,838,887 of panda X chromosome;
(2) Dividing the variation sets into two groups according to the sex of pandas, wherein one group is all male pandas, and the other group is all female pandas;
(3) Screening SNP loci with the same genotypes of all pandas in each group;
(4) Reserving SNP loci which are heterozygous genotypes in the male variation set and homozygous genotypes in the female variation set;
according to the above requirements, 5 SNP markers from the genome were selected for panda sex determination.
EXAMPLE 4 panda sexing based on Sanger sequencing
Primer design: the NCBI/BLAST online tool is used for designing primers, wherein the genome sequence of panda crystal is used as a background, the amplified fragment is below 120bp, and the TM value is controlled between 45 ℃ and 65 ℃. A total of 2 sets of specific primers were obtained (Table 2).
TABLE 2 primer set for identifying sex of pandas by Sanger method
Table 3 details the amplification length, tm values, SNP relative positions and SNP site peak patterns.
TABLE 3 SNP site information for identifying sex of pandas based on Sanger method
The Sanger typing method comprises the following specific steps:
step one, randomly selecting fecal DNA samples of 1 panda individual as templates, wherein a reaction system is as follows: in a 50uL system, 2uL of DNA template, 4uL of 10mmol/L forward and reverse primers each, 25uL of 2 XTiangen PCR premix, and ddH were added 2 O was added to the total volume of the reaction system of 50uL. The reaction conditions are as follows: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30sec, extension at 58℃for 15sec, extension at 72℃for 15sec for a total of 35 cycles; the end extension is carried out for 5min at 72 ℃,preserving at 4 ℃;
preparing agarose gel with concentration of 2% (w/v), taking 5uL of sample, adding the sample into a sample application hole, simultaneously taking 5uL of DNA Marker, adding the DNA Marker into an adjacent hole, and switching on a power supply to carry out electrophoresis under the conditions of 80V and 40 min;
thirdly, after electrophoresis, placing the gel block in a gel imager for photographing, and observing the size of a target amplified fragment in a gel chart;
step four, taking 45uL amplification products, and sequencing according to Sanger standard sequencing steps;
step five, opening a chromatogram in the sequencing result, namely an ab1 format file, and observing the peak type condition of the corresponding SNP loci in the chromatogram, wherein in fig. 1, the first column is a reverse primer sequencing result, the rest is a forward primer sequencing result, the peak types of the positions corresponding to the male pandas are all double peaks, however, the peak types of the female pandas on 5 SNP loci are all single peaks.
EXAMPLE 5 panda sexing based on nucleic acid Mass Spectrometry detection method
Primer design: the primers were designed again with the analysis Designer 4.0 software of Agena company for the 5 SNP sites selected above. Wherein the length of the amplified fragment is controlled between 80 and 120 bp; avoiding primer mismatch, hairpin structure and dimer structure; the TM value of the extension primer is controlled between 45 ℃ and 100 ℃. The 3' -end of the extension primer is located 1bp upstream or downstream of the SNP site, and the effective primer length is 17-20bp, i.e., in Table 4, and all primers are purified by PAGE.
TABLE 4 SNP primer set for panda sex determination
The primers designed at the 5 sites are tested and verified by selecting tissue DNA of 5 panda individuals, fecal DNA of 12 containment pandas and fecal DNA of 4 field pandas.
(1) PCR system: ddH of 0.8uL 2 O,0.5uL containing 20mmol/L MgCl 2 10 XPCR Buffer, 0.4uL concentration25mmol/L MgCl 2 dNTP Mix with concentration of 25mmol/L at 0.1uL, primer Mix with concentration of 0.5umol/L at 1uL, PCR Enzyme with concentration of 5U/uL at 0.2uL, DNA with concentration of 5ng/uL at 2uL, total volume of reaction solution of 5uL, vortex shaking and centrifuging after mixing; the PCR reaction procedure was: pre-denaturation at 95℃for 2min; denaturation at 95℃for 30sec, annealing at 56℃for 30sec, elongation at 72℃for 60sec for 45 cycles; terminal extension at 72℃for 5min. Finally, the mixture is preserved at the temperature of 4 ℃.
(2) And (3) performing alkaline phosphatase treatment on the PCR reaction product of the previous step to eliminate redundant dNTPs in a reaction system. The reaction system is as follows: 1.53uL ddH 2 O, SAP Buffer 0.17uL, SAP 0.30uL concentration of 1.7U/uL, and total reaction solution volume of 2uL; the digestion reaction is carried out at 37deg.C for 40min, inactivating at 85deg.C for 5min, and preserving at 4deg.C.
(3) Single base extension is carried out on the treated PCR reaction product, and the reaction system is as follows: ddH of 0.619uL 2 O, 0.2uL of iPLEX Buffer, 0.2uL of iPLEX Termination mix, 0.94uL of Extend Primer Mix and 0.041uL of iPLEX enzyme, and the total volume of the reaction solution is 2uL. The PCR reaction procedure was: pre-denaturation at 94 ℃ for 30sec; denaturation at 94℃for 5sec; annealing at 52 ℃ for 5sec, extending at 80 ℃ for 5sec, and carrying out 5 internal cycles; denaturation at 94℃for 5sec, annealing at 52℃for 5sec, elongation at 80℃for 5sec, total of 35 outer cycles; terminal extension at 72℃for 3min. Finally, the extension product was stored at 4 ℃.
(4) 6mg of resin was added to the extension product, and the extension product was subjected to rotary shaking using a CPM apparatus (Continues Passive Motion) to efficiently remove the salt ion and other components.
(5) And carrying out mass spectrometry on the extension product after resin purification to obtain a molecular weight mass spectrum peak diagram corresponding to the SNP locus. The extension products after resin purification were transferred to 384-well SpectroCHIP bioarray, the spotted SpectroCHIP chip was analyzed by using a MALDI-TOF mass spectrometer, and the detection results were processed by TYPER 4.0 software to obtain a mass spectrum peak map of the extension products corresponding to the SNP sites. Table 5 details the amplicon length, extension primer molecular weight, and female and male genotypes at the time of SNP analysis.
TABLE 5 SNP information for identification of panda gender based on nucleic acid mass spectrometry
(6) The accuracy of primer and sex genotype was determined from the typing map. Through the verification of nucleic acid mass spectrum, the screened 5 panda SNP loci have stable genotypes in female and male individuals and prominent sex differences, and can be completely used as SNP markers for panda sex identification.
The genotypes of the 5 SNP loci screened in individuals can accurately identify the sex of pandas by comparing all demonstration data. More importantly, the length polymorphism method of the DNA fragment with earlier length which needs to be amplified shortens 140bp, is better applied to the DNA sample of the wild panda feces which is seriously degraded, and simultaneously adopts the nucleic acid mass spectrometry to detect the SNP locus genotype, thereby improving the detection sensitivity and being capable of detecting DNA samples with the length as small as 5 ng.
The panda SNP primer pair of the invention performs sex identification on pandas of the panda breeding research base, has very accurate identification result, and can be further applied to field panda sex identification work.
While the foregoing description illustrates and describes several preferred embodiments of the invention, it is to be understood that the invention is not limited to the forms disclosed herein, but is not to be construed as limited to other embodiments, and is capable of use in various other combinations, modifications and environments and is capable of changes or modifications within the spirit of the invention described herein, either as a result of the foregoing teachings or as a result of the knowledge or skill of the relevant art. And that modifications and variations which do not depart from the spirit and scope of the invention are intended to be within the scope of the appended claims.
Claims (2)
1. SNP primer set for panda sex determination, characterized by comprising mh0XGP-001 primer set, mh0XGP-002 primer set, mh0XGP-001# (1) primer set, mh0XGP-001# (2-3) primer set and mh0XGP-002# (1-2) primer set, wherein the nucleotide sequence is SEQ ID NO.1-15.
2. The use of the SNP primer set according to claim 1 for panda sex determination.
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| CN113215267A (en) * | 2021-04-20 | 2021-08-06 | 成都大熊猫繁育研究基地 | SNP primer set for panda individual identification and paternity test and application |
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| CN113215267A (en) * | 2021-04-20 | 2021-08-06 | 成都大熊猫繁育研究基地 | SNP primer set for panda individual identification and paternity test and application |
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