CN111662989B - SSR fluorescence labeling primer for paternity test of deer on slope and identification method - Google Patents

SSR fluorescence labeling primer for paternity test of deer on slope and identification method Download PDF

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CN111662989B
CN111662989B CN202010548407.3A CN202010548407A CN111662989B CN 111662989 B CN111662989 B CN 111662989B CN 202010548407 A CN202010548407 A CN 202010548407A CN 111662989 B CN111662989 B CN 111662989B
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primer
deer
hainan
paleo
dna
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CN111662989A (en
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李慧明
陈兴永
陈金平
苏力
胡倩雯
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Institute of Zoology of Guangdong Academy of Sciences
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/156Polymorphic or mutational markers

Abstract

The invention discloses SSR fluorescence labeling primers and an identification method for paternity test of deer on a sloping cam. The SSR fluorescence labeling primer for parent-child identification of the Hainan paleo-deer can be used for parent-child identification and family management of the Hainan paleo-deer, so that artificial breeding work of the Hainan paleo-deer is reasonably guided, and a powerful tool is provided for researches on protection of genetic diversity, population genetic organization and the like of the Hainan paleo-deer. In addition, the invention also adopts a method of adding M13 joints, and only one M13 fluorescence labeled primer needs to be synthesized for PCR extension, thereby saving the cost and time for synthesizing a large amount of fluorescence primers.

Description

SSR fluorescence labeling primer for paternity test of deer on slope and identification method
The technical field is as follows:
the invention belongs to the field of paternity test of animals, and particularly relates to SSR fluorescence labeling primers and a method for paternity test of deer.
Background art:
china is the country with the most deer species in the world, the variety diversity of the deer animals is an important biological resource in China, and the number of 9 deer species is 16. Among them, Hainan deer (Cervus eldi hainanus) is a unique species in China and is only distributed in Hainan island. Since the 70 s of the 20 th century, the wild population of Hainan deer is rapidly reduced due to serious illegal killing and large-scale destruction of habitat, and only a few individuals (only about 26 individuals) are remained in the most serious period and are mainly distributed in natural protection areas of Hainan field countries and the like. At present, Hainan paleo-deer is listed in International trade Convention on endangered wild animal and plant species (Convention on International trade in endangered species of wild fauna and flora, CITES), is listed as endangered species by the world alliance for natural protection (IUCN), is listed as endangered-grade protected animals in Chinese endangered animal red book, and belongs to the first-grade important protected animals in China.
Currently, artificial breeding and in-situ protection have become important measures for protecting endangered species in order to enlarge the size of the current flora, increase genetic diversity. Although the population quantity of the Hainan paleo deer is recovered by long-term in-situ protection measures, the newly-built population and the source population are genetically differentiated due to the bottleneck effect and the founder effect, and the genetic diversity of the newly-built population is lower than that of the source population. And the small population of the sika deer is easy to have close reproduction, so that the problems of the reduction of the growth performance and the reproductive capacity and the like are very prominent. Therefore, the establishment of a stable artificial breeding population, the avoidance of population decline caused by inbreeding, the increase of effective population and the breeding and optimization pairing work aiming at adopting the individual with the minimum genetic relationship to carry out long-term gene communication are very important. At present, breeding research aiming at Hainan paleo deer is carried out more on original habitats in Hainan area, but research reports about ex-situ protection and breeding are still less.
Microsatellite DNA (Microsate), also known as Simple Sequences (SSR), is a common DNA molecular marker, has the advantages of rich polymorphism, high heterozygosity, good co-dominant inheritance and genetic stability and the like, and has reliable obtained results, Simple method, time and labor saving, thus being widely applied to the research fields of constructing genetic maps, detecting genetic variation, identifying the paternity relationship among individuals, analyzing population genetics and the like.
At present, the report of applying microsatellite molecular markers to parent-child identification and genetic management of Hainan paleo deer does not exist.
The invention content is as follows:
the invention aims to solve the problems, provides an SSR fluorescence labeling primer and a paternity test method applied to paternity test of Hainan deer, is used for solving the problems of difficult individual identification, unclear pedigree and the like caused by mating of small populations and mixed culture of different parents in the breeding and pairing process of Hainan deer, and provides a scientific basis for establishing the pedigree of the family in the Hainan deer farm so as to reasonably guide the breeding and conservation work of artificial populations of the Hainan deer.
The SSR fluorescence labeling primer group applied to parent-child identification of Hainan paleo deer of the invention is that more than 4 primer pairs are selected optionally
CES11:
F: 5'-TGGAAGAAACCAAAAGAAGAACA-3', the nucleotide sequence is shown in SEQ ID NO.1, R: 5'-GTCGGACACGACTGAAGCTACT-3', the nucleotide sequence of which is shown in SEQ ID NO. 2;
CES19:
f: 5'-ACTTGAGATTTAGACAAGCCTGG-3', the nucleotide sequence is shown in SEQ ID NO.3, R: 5'-TTGATTGTGCCAACTTTAGATGA-3', the nucleotide sequence of which is shown in SEQ ID NO. 4;
CES30:
f: 5'-TCTGTTTCTGTTTTGCAAGTTCA-3', the nucleotide sequence is shown in SEQ ID NO.5, R: 5'-ACTTTGGAGAACTACACAGCCC-3', the nucleotide sequence of which is shown in SEQ ID NO. 6;
CES31:
f: 5'-TGAGAGTTAGGAAGAGAGTGAGCA-3', the nucleotide sequence is shown in SEQ ID NO.7, R: 5'-TAACACTTCCACACACAGGTGAC-3', the nucleotide sequence of which is shown in SEQ ID NO. 8;
CES32:
f: 5'-GCACAGAGTTGAACATGACTGAA-3', the nucleotide sequence is shown in SEQ ID NO.9, R: 5'-AAATAGGCGAAGGACCTAAACAG-3', the nucleotide sequence of which is shown in SEQ ID NO. 10;
CES33:
f: 5'-CTCCAGTATTCTTGCCTGGATAA-3', the nucleotide sequence is shown in SEQ ID NO.11, R: 5'-AATTTTTAGTAAATTGGCCTGACG-3', the nucleotide sequence of which is shown in SEQ ID NO. 12;
CES38:
f: 5'-GCTAGGCAGATTACAAATGAAGC-3', the nucleotide sequence is shown in SEQ ID NO.13, R: 5'-AAAGGAAATACTTTCTGGTGAGTGA-3', the nucleotide sequence of which is shown in SEQ ID NO. 14;
CES39:
f: 5'-AGCCTGGTGTGCTATAATCCATA-3', the nucleotide sequence is shown in SEQ ID NO.15, R: 5'-TAAAGGAATTTCATCAGCCAAAA-3', the nucleotide sequence of which is shown in SEQ ID NO. 16;
CES40
f: 5'-AGGGAAGAGAGCATCACATCAT-3', the nucleotide sequence is shown in SEQ ID NO.17, R: 5'-TCCAACTAAGGTAGTGCTGGGTA-3', the nucleotide sequence of which is shown in SEQ ID NO. 18;
CES41:
f: 5'-GTCACAAAGAGTCAAACACGACA-3', the nucleotide sequence is shown in SEQ ID NO.19, R: 5'-CCTGCATTTTCAGAGCAAAGTAT-3', the nucleotide sequence of which is shown in SEQ ID NO. 20;
CES42:
f: 5'-CAGTGTTCTTGCCTGGAGAAT-3', the nucleotide sequence is shown in SEQ ID NO.21, R: 5'-TCCTGCTTGTACATCTGGAAGTT-3', the nucleotide sequence of which is shown in SEQ ID NO. 22;
CES43:
f: 5'-CACAGAGTCGGACAAAACTGAA-3', the nucleotide sequence is shown in SEQ ID NO.23, R: 5'-CTACTCTCCCTCAAAAGTCAGCA-3', the nucleotide sequence of which is shown in SEQ ID NO. 24;
CES44:
f: 5'-GGACACGACTGAAGCGACTTA-3', the nucleotide sequence is shown in SEQ ID NO.25, R: 5'-TGGTTAAGAGCACGGACTTTCTA-3', the nucleotide sequence of which is shown in SEQ ID NO. 26;
CES45:
f: 5'-GGATCTCCAGTATTCTTGCCTG-3', the nucleotide sequence is shown in SEQ ID NO.27, R: 5'-CTGTAGGTAGGTGACCACACCAT-3', the nucleotide sequence of which is shown in SEQ ID NO. 28;
CES46:
f: 5'-CTTTTTCTTTTGCCAAATTGATG-3', the nucleotide sequence is shown in SEQ ID NO.29, R: 5'-CAGACACACCTGAACGACTAACA-3', the nucleotide sequence of which is shown in SEQ ID NO. 30;
CES47:
f: 5'-AACCCACTGAAATGTTGTGAAGT-3', the nucleotide sequence is shown in SEQ ID NO.31, R:
5'-ATCACACCTTCGTGATTATCTGG-3', the nucleotide sequence of which is shown in SEQ ID NO. 32;
CES48:
f: 5'-GTAAACTCTGGGAATTGGTGATG-3', the nucleotide sequence is shown in SEQ ID NO.33, R: 5'-TACCTCTAGCTCCATCTGGGAAT-3', the nucleotide sequence is shown in SEQ ID NO. 34;
CES49:
f: 5'-GGACCCCAGATTTGCTCTTATTA-3', the nucleotide sequence is shown in SEQ ID NO.35, R: 5'-ATACAGGGAGAGAGAGGGAGAGA-3', the nucleotide sequence of which is shown in SEQ ID NO. 36;
CES50:
f: 5'-GCGACTGAACATTAACACATCAT-3', the nucleotide sequence is shown in SEQ ID NO.37, R: 5'-AGAATTTTGAACAGGGTAGAGGG-3', the nucleotide sequence is shown in SEQ ID NO. 38.
The second purpose of the invention is to provide a method for identifying the paternity of the Hainan paleo-deer, which uses the SSR fluorescence labeling primer group for identifying the paternity of the Hainan paleo-deer as a primer and uses the genome DNA of the Hainan paleo-deer as a template to carry out fluorescence PCR reaction to obtain a PCR product, and then carries out genotyping to judge whether the samples are in paternity relationship.
Preferably, the genomic DNA of the Hainan paleo deer is the genomic DNA of the feces of the Hainan paleo deer.
The reaction system of the fluorescence PCR reaction is as follows: the system was 10. mu.l, comprising 1. mu.l of 100 ng/. mu.l DNA template, 2 XPCR Mix 5. mu.l, ddH2O 1.6.6. mu.l, 0.4. mu.l of forward primer with M13 linker at the 5' end, 1. mu.l of reverse primer, 1. mu.l of FAM-modified M13 forward primer, and the amplification reaction procedure used touchdown PCR: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 15 s; the annealing temperature is 30s from 62 to 52 ℃, the annealing temperature is reduced by 2 ℃ every 2 cycles, and the annealing temperature is extended for 2min at 72 ℃; denaturation at 95 ℃ for 15 s; the annealing temperature is 30s at 52 ℃; extending for 2min at 72 ℃; 30 cycles; extension at 72 ℃ by 10 mi. The forward primer of which the 5 'end is added with the M13 joint is formed by adding the M13 joint to the 5' end of the forward primer, the FAM modified M13 forward primer is formed by modifying and adding a FAM group to the M13 universal primer, and the sequences of the M13 joint and the M13 universal primer are as follows: 5'-CACGACGTTGTAAAACGAC-3' are provided.
The third purpose of the invention is to provide a kit for identifying the parent-child of the Hainan paleo deer, which comprises the fluorescent SSR labeled primers and the fluorescent PCR reaction reagent for identifying the parent-child of the Hainan paleo deer.
The SSR fluorescence labeling primer for parent-child identification of the Hainan paleo-deer can be used for parent-child identification and family management of the Hainan paleo-deer, so that artificial breeding work of the Hainan paleo-deer is reasonably guided, and a powerful tool is provided for researches on protection of genetic diversity, population genetic organization and the like of the Hainan paleo-deer. In addition, the invention also adopts a method of adding M13 joints, and only one M13 fluorescence labeled primer needs to be synthesized for PCR extension, thereby saving the cost and time for synthesizing a large amount of fluorescence primers.
Drawings
FIG. 1 is a design roadmap for the present invention;
FIG. 2 shows the principle of fluorescent PCR with the addition of M13 linker.
The specific implementation mode is as follows:
the following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1:
1. detection of SSR loci based on simplified genome (RAD) sequencing
The method comprises the steps of carrying out subcutaneous venous blood collection on an adult healthy Hainan deer by about 8ml, using sodium citrate as an anticoagulant, refrigerating in liquid nitrogen in a 10ml freezing tube, bringing the frozen tube back to a laboratory, and storing in a refrigerator of 80 ℃ below zero to extract DNA. Extracting the total DNA of the Hainan paleo deer blood sample, and accurately quantifying the DNA concentration. The whole library preparation is completed by the steps of enzyme digestion, random interruption, end repair, A tail addition, sequencing joint addition, purification, PCR amplification and the like. The constructed library was sequenced by Illumina (sequencer). Removing joints of the original sequence, performing quality filtering to obtain a high-quality sequence, assembling the high-quality sequence by using Trinity software, and performing SSR detection on the Unigene by using MISA. Primers were designed using Primer5 software.
2. Screening of polymorphic SSR sites based on PCR technology
DNA in the feces was extracted using a Hipure Tissue DNA Mini Kit (magenta) using Hainan deer feces DNA as a template. Among the microsatellite primers obtained, 50 pairs of primers were randomly selected and synthesized, and the PCR amplification system was 10. mu.l, including 1. mu.l of 100 ng/. mu.l DNA template, 5. mu.l of 2 XPCR Mix, 2. mu.l of ddH2O 2, 1. mu.l of forward primer with M13 linker at the 5' end, and 1. mu.l of reverse primer. The amplification reaction procedure used touchdown pcr (touchdown pcr): pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 15 s; the annealing temperature is 30s from 62 to 52 ℃, the annealing temperature is reduced by 2 ℃ every 2 cycles, and the extension time is 2min at 72 ℃; denaturation at 95 ℃ for 15 s; the annealing temperature is 30s at 52 ℃; extending for 2min at 72 ℃; 30 cycles; extension at 72 deg.C for 10min, and final storage at 4 deg.C. And (3) detecting the polymorphism of the PCR product by utilizing polyacrylamide gel electrophoresis, screening out 19 pairs of amplification primers of SSR sites with higher polymorphism, and obtaining the Hainan paleo deer microsatellite molecular marker. The main design route is shown in fig. 1. The forward primer with the 5 'end added with the M13 joint is formed by adding an M13 joint at the 5' end of the forward primer, and the sequence of the M13 joint is as follows:
5'-CACGACGTTGTAAAACGAC-3’。
3. screening of Hainan paleo-deer microsatellite marker
The simplified genome sequencing obtains Raw data 11.383Gb, the filtered Clean data 11.33G, the Raw data of each sample is between 11383.172M and 11383.172M, the sequencing quality is high (Q20> -96.99%, Q30> -91.69%), the GC content is between 41.74% and 41.74%, and the RAD-Tag capture rate is between 98.9% and 98.9%. The reads after deduplication are aligned into the assembly results. And (4) carrying out variation detection, wherein the detection result shows that most heterozygous SNPs and a small amount of homozygous SNPs are reliable.
The reads after deduplication are aligned into the assembly results. And (4) carrying out variation detection, wherein the detection result shows that most heterozygous SNPs and a small amount of homozygous SNPs are reliable. The sample assembly contigs were filtered to yield SSR fragments with 100bp (for later primer design) on each end. The number of fragments from which primers were generated was 19743 (Table 1). Of the 30 randomly selected primers, 19 of them were successfully amplified to obtain clear PCR products using Hainan deer DNA (Table 2).
TABLE 1 SSR test results statistics
Sample 865 Total ssr Primer ssr Primer/Total
865 20,505 19,743 96.28
(1) Sample: the name of the sample;
(2) totalssr: reserving 100bp at each end as the SSR number of the primer design;
(3) primerssr: (ii) has the number of primer fragments SSR;
(4) Primer/Total: fragment primer design rate.
TABLE 2 19 pairs of SSR loci and primers screened in this study
TABLE 3 genetic diversity of 19 microsatellite loci of Hainan deer
K: number of SSR locus genotypes;
na: number of SSR polymorphic sites;
hobs: observing the heterozygosity;
hexp: a desired heterozygosity;
PIC: polymorphic information content;
NE-1P is the elimination rate of the known duplicate information;
NE-2P is the exclusion rate of known parent information of one party;
NE-PP is the exclusion rate unknown by both pieces of information;
HW: harden equilibrium, NS stands for insignificant.
4. Polymorphic microsatellite locus PCR amplification and genotyping
A FAM fluorescent group is added to the forward 5 'end of the universal primer M13 for modification (Boutin-Ganache et al, 2001), 13 Hainan deer saliva sample DNAs are used as templates for carrying out fluorescent PCR reaction, and the amplification system is 10 ul and comprises 1 ul of 100 ng/ul DNA template, 5 ul of 2 XPCR Mix, 1.6 ul of ddH2O 1.6, 0.4 ul of forward primer with M13 joint at the 5' end, 1 ul of reverse primer and 1 ul of FAM modified M13 forward primer (the reaction principle is shown in figure 2). The amplification reaction procedure used touchdown pcr (touchdown pcr): pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 15 s; the annealing temperature is 30s from 62 to 52 ℃, the annealing temperature is reduced by 2 ℃ every 2 cycles, and the extension time is 2min at 72 ℃; denaturation at 95 ℃ for 15 s; the annealing temperature is 30s at 52 ℃; extending for 2min at 72 ℃; 30 cycles; extension is carried out for 10min at 72 ℃, and finally, the product is stored in the dark at 4 ℃. The forward primer of which the 5 'end is added with the M13 joint is formed by adding the M13 joint to the 5' end of the forward primer, the FAM modified M13 forward primer is formed by modifying and adding a FAM group to the M13 universal primer, and the sequences of the M13 joint and the M13 universal primer are as follows: 5'-CACGACGTTGTAAAACGAC-3' are provided.
And (3) identifying the concentration of the PCR product amplified by the fluorescent primer by using 1% agarose gel electrophoresis, diluting to the concentration suitable for capillary electrophoresis detection, adding a GS500LIZ internal standard used for capillary electrophoresis analysis, and after treatment, putting the PCR product into an ABI3730XL high-throughput DNA sequencer to perform capillary electrophoresis and STR analysis so as to determine the sizes of alleles of microsatellite markers of Hainan deer on different individuals.
The characteristic of DNA polymorphism of related microsatellites of the Hainan deer is described by calculating an allelic factor (k) by using Cervus software, observing heterozygosity (Ho) and expected heterozygosity (He), testing Hardy-Weinberg equilibrium (HWE), Polymorphic Information Content (PIC) and exclusion rate of candidate parents.
The results of the diversity analysis of 19 microsatellite genetic markers in 13 Hainan deer samples show that (Table 3): the SSR sites of 13 Hainan paleo-deer show certain genetic variability, and 19 sites are polymorphic, wherein 12 sites at least contain 4 alleles. The allelic base factor (Na) of each site is 2-8, the observed heterozygosity (Ho) of the Hainan paleo-deer is 0.077-1, the expected heterozygosity (He) is 0.077-0.841, and the Polymorphic Information Content (PIC) is 0.071-0.78. The average values for Na, Ho, He and PIC were 3.81, 0.628, 0.475 and 0.414, respectively.
The exclusion probability (NE-PP) of each locus is 0.193 to 0.933 when parent information is unknown, 0.377 to 0.964(NE-2P) when single parent information is known, and 0.552 to 0.997(NE-1P) when parent information is known. When 4 SSR loci are adopted, the joint exclusion probability values of NE1-P, NE-2P and NE-PP are both more than 99%. Furthermore, when 3 SSR sites were used, both NE-1P and NE-2P had values higher than 99.99% (Table 4).
TABLE 4 cumulative exclusion rates of 19 primers in this study
It is generally accepted that microsatellite markers with an allelic factor of at least 4 can be better used for genetic analysis and paternity testing of species. The invention detects 78 alleles altogether, the average allele is 4, the average observed heterozygosity is 0.628, the average expected heterozygosity is 0.525, the smaller difference between the two indicates that the 19 microsatellite markers have better gene heterozygosity, and can reflect the genetic characteristics of the population more accurately. Only when the cumulative exclusion rate is greater than 0.8, the paternity identified by the microsatellite markers has certain reliability, and in the invention, the cumulative exclusion rates of 19 microsatellite markers are all greater than 0.99. In conclusion, the invention develops 19 polymorphic microsatellite loci of Hainan deer, a primer sequence for amplifying the 19 microsatellite loci and an amplification method, can be applied to the genetic diversity research of different geographic populations of Hainan deer, has good repeatability and is a reliable and effective molecular marker.
5. Application example of using microsatellite markers to carry out paternity test on Hainan paleo-deer
And (3) carrying out paternity test on 13 Hainan deer by applying the 19 microsatellite marker primers in combination with an exclusion method and a likelihood method, specifically, sequencing the 13 Hainan deer by using 19 ssr fluorescent marker primers according to the polymorphic microsatellite locus PCR amplification and genotyping method in the step 4 to obtain matrix data of a genetic locus, analyzing and identifying the paternity by using CERVUS software according to the data of the matrix, and comparing the paternity test result with a feedlot pedigree record to verify the accuracy of the method. The LOD value calculated using CERVUS3.0 software was 5.75 at 95% confidence and 2 at 80% confidence. At a 95% confidence level, the paternal-child relationship was identified for each of numbers PL14, PL13, PL12, PL8PL6 and PL10, consistent with pedigree records for the guangzhou city zoo. In addition, the results of the study also supported PL11, PL7 and PL10 both in a parent-child relationship (table 5). These results indicate that selected SSRs have high parental analysis capabilities. The more sites we choose, the more accurate the paternity analysis.
TABLE 5 Main results of this paternity test
In summary, the genetic relationship detection and analysis method in the present study can provide an important reference for developing pedigree information verification and paternity identification. The microsatellite is used as a genetic marker, can objectively reflect the genetic diversity of the Hainan deer, and can be used for paternity test. Therefore, the microsatellite marker is combined with the traditional ecological research, the genetic diversity analysis and paternity test of the Hainan deer are carried out, the protection and reasonable development and utilization of rare or endangered species resources are promoted, and scientific guidance is provided for the management of related departments.
Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Sequence listing
<110> institute for biological resource application in Guangdong province
<120> SSR fluorescence labeling primer for paternity test of deer and identification method
<160> 38
<170> SIPOSequenceListing 1.0
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<400> 19
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<400> 20
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<400> 21
cagtgttctt gcctggagaa t 21
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<400> 22
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ctactctccc tcaaaagtca gca 23
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<212> DNA
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
tggttaagag cacggacttt cta 23
<210> 27
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
ggatctccag tattcttgcc tg 22
<210> 28
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
ctgtaggtag gtgaccacac cat 23
<210> 29
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
ctttttcttt tgccaaattg atg 23
<210> 30
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
cagacacacc tgaacgacta aca 23
<210> 31
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
aacccactga aatgttgtga agt 23
<210> 32
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
atcacacctt cgtgattatc tgg 23
<210> 33
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
gtaaactctg ggaattggtg atg 23
<210> 34
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
tacctctagc tccatctggg aat 23
<210> 35
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
ggaccccaga tttgctctta tta 23
<210> 36
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
atacagggag agagagggag aga 23
<210> 37
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
gcgactgaac attaacacat cat 23
<210> 38
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
agaattttga acagggtaga ggg 23

Claims (5)

1. A Hainan paleo-deer paternity test primer group for SSR fluorescent labeling is characterized in that 19 primer pairs are selected as follows:
CES11:
F:5'-TGGAAGAAACCAAAAGAAGAACA-3',R:5'-GTCGGACACGACTGAAGCTACT-3';
CES19:
F:5'-ACTTGAGATTTAGACAAGCCTGG-3',R:5'-TTGATTGTGCCAACTTTAGATGA-3';
CES30:
F:5'-TCTGTTTCTGTTTTGCAAGTTCA-3',R:5'-ACTTTGGAGAACTACACAGCCC-3';
CES31:
F:5'-TGAGAGTTAGGAAGAGAGTGAGCA-3',R:5'-TAACACTTCCACACACAGGTGAC-3';
CES32:
F:5'-GCACAGAGTTGAACATGACTGAA-3',R:5'-AAATAGGCGAAGGACCTAAACAG-3';
CES33:
F:5'-CTCCAGTATTCTTGCCTGGATAA-3',R:5'-AATTTTTAGTAAATTGGCCTGACG-3';
CES38:
F:5'-GCTAGGCAGATTACAAATGAAGC-3',R:5'-AAAGGAAATACTTTCTGGTGAGTGA-3';
CES39:
F:5'-AGCCTGGTGTGCTATAATCCATA-3',R:5'-TAAAGGAATTTCATCAGCCAAAA-3';
CES40
F:5'-AGGGAAGAGAGCATCACATCAT-3',R:5'-TCCAACTAAGGTAGTGCTGGGTA-3';
CES41:
F:5'-GTCACAAAGAGTCAAACACGACA-3',R:5'-CCTGCATTTTCAGAGCAAAGTAT-3';
CES42:
F:5'-CAGTGTTCTTGCCTGGAGAAT-3',R:5'-TCCTGCTTGTACATCTGGAAGTT-3';
CES43:
F:5'-CACAGAGTCGGACAAAACTGAA-3',R:5'-CTACTCTCCCTCAAAAGTCAGCA-3';
CES44:
F:5'-GGACACGACTGAAGCGACTTA-3',R:5'-TGGTTAAGAGCACGGACTTTCTA-3';
CES45:
F:5'-GGATCTCCAGTATTCTTGCCTG-3',R:5'-CTGTAGGTAGGTGACCACACCAT-3';
CES46:
F:5'-CTTTTTCTTTTGCCAAATTGATG-3',R:5'-CAGACACACCTGAACGACTAACA-3';
CES47:
F:5'-AACCCACTGAAATGTTGTGAAGT-3',R:5'-ATCACACCTTCGTGATTATCTGG-3';
CES48:
F:5'-GTAAACTCTGGGAATTGGTGATG-3',R:5'-TACCTCTAGCTCCATCTGGGAAT-3';
CES49:
F:5'-GGACCCCAGATTTGCTCTTATTA-3',R:5'-ATACAGGGAGAGAGAGGGAGAGA-3';
CES50:
F:5'-GCGACTGAACATTAACACATCAT-3',R:5'-AGAATTTTGAACAGGGTAGAGGG-3';
m13 adaptor is added to the 5' end of the forward primer in the above primer.
2. A method for paternity test of Hainan paleo-deer, characterized in that, the primer group of claim 1 is used as primer, the genome DNA of Hainan paleo-deer is used as template to respectively carry out fluorescence PCR reaction to obtain PCR products, and then genotyping, capillary electrophoresis and sequencing analysis are carried out to judge whether the samples are in paternity relationship.
3. The method of claim 2, wherein the genomic DNA of the Hainan deer is genomic DNA of the feces of Hainan deer.
4. The method for paternity test of Hainan poverty as claimed in claim 2, wherein the fluorescence PCR reaction comprises the following reaction steps: the system was 10. mu.l, including 1. mu.l of 100 ng/. mu.l DNA template, 2 XPCR Mix 5. mu.l, ddH2O1.6. mu.l, forward primer 0.4. mu.l with M13 linker at the 5' end, reverse primer 1. mu.l, FAM modified M13 forward primer 1. mu.l, amplification reaction program using touchdown PCR: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 15 s; the annealing temperature is 30s from 62 to 52 ℃, the annealing temperature is reduced by 2 ℃ every 2 cycles, and the extension time is 2min at 72 ℃; denaturation at 95 ℃ for 15 s; the annealing temperature is 30s at 52 ℃; extending for 2min at 72 ℃; 30 cycles; and (2) extending for 10min at 72 ℃, wherein the forward primer of which the 5 'end is added with the M13 joint is formed by adding the M13 joint to the 5' end of the forward primer, the FAM modified M13 forward primer is formed by modifying and adding a FAM group to the M13 universal primer, and the sequences of the M13 joint and the M13 universal primer are as follows: 5'-CACGACGTTGTAAAACGAC-3' are provided.
5. A kit for parent-offspring identification of Hainan paleo-deer, comprising the primer set of claim 1 and a fluorescent PCR reaction reagent, wherein the fluorescent PCR reaction reagent comprises a DNA template, 2 XPCR Mix and ddH2O, FAM modified M13 forward primer, wherein the 5 'end of the forward primer in the primer group is added with M13 joint, the 5' end of the forward primer added with M13 joint is added with M13 joint, the FAM modified M13 forward primer is modified on M13 universal primer and added with FAM group, and the M13 jointAnd the sequence of the M13 universal primer is: 5'-CACGACGTTGTAAAACGAC-3' are provided.
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CN107177698A (en) * 2017-07-28 2017-09-19 中国农业科学院特产研究所 Primer, kit and method for animal in deer family paternity test
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CN107177698A (en) * 2017-07-28 2017-09-19 中国农业科学院特产研究所 Primer, kit and method for animal in deer family paternity test
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