CN107177698B - Primer, kit and method for paternity test of deer animals - Google Patents
Primer, kit and method for paternity test of deer animals Download PDFInfo
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- CN107177698B CN107177698B CN201710627976.5A CN201710627976A CN107177698B CN 107177698 B CN107177698 B CN 107177698B CN 201710627976 A CN201710627976 A CN 201710627976A CN 107177698 B CN107177698 B CN 107177698B
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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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- 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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- C—CHEMISTRY; METALLURGY
- 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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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- C—CHEMISTRY; METALLURGY
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention provides a primer, a kit and a method for paternity test of a deer animal; the primer is selected from at least one primer pair of: 1-20 of SEQ ID NO. The primers have 10 pairs in total, the amplified band is clear, the polymorphism is high, the accumulated non-paternal exclusion rate is as high as 99.99 percent, and the method can be applied to the paternity test practice of deer.
Description
Technical Field
The invention relates to the field of molecular genotyping, in particular to a primer, a kit and a method for paternity test of a cervidae animal.
Background
Microsatellites are short tandem repeats (2-6bp) in the genome that exhibit different alleles due to different repeat numbers. There are many incomparable advantages to other identification methods using microsatellite markers for paternity testing. Among them, the most significant advantage is his co-dominant genetic characteristic, which allows the distinction of homozygous and heterozygous genotypes, greatly increasing the accuracy of paternity testing. In addition, microsatellite markers have high polymorphisms, i.e., multiple alleles at the same locus, and since it is unlikely that non-paternity individuals will share the same allele, paternity can be identified for a large number of individuals using a small number of high polymorphism microsatellite markers. Finally, because microsatellite analysis uses the Polymerase Chain Reaction (PCR), only small amounts of DNA are required for identification, and highly degraded DNA can be used, such as DNA extracted from feces, hair, feathers, and skin.
Sika deer (Cervus nippon) is mainly distributed in east asia, ranging from siberia to korea, eastern and vietnam of china; there are also distributions in western pacific islands such as japan. Chinese sika deer is mainly distributed in jilin province, while japanese sika deer is mainly distributed in hokkaido. Sika in the 19 th century was once hunted to almost the dead species, legislative protection began in the 20 th century, and the population recovered rapidly from the 1950 s to the 1980 s. Sika deer was also introduced in australia, europe and the united states. Originally intended as park ornamentation animals, many now become wild. At present, the subfamily of sika deer distributed in China and Japan is already listed as the most dangerous or endangered grade by IUCN red name, and the sika deer in China is also the first-grade national protection animal.
At present, microsatellite molecular marker technology is most applied in paternity test research of other domestic animals of human, but primers of the microsatellite molecular marker used for paternity test of the species are different due to difference of genomes of the species. At present, no accurate deer paternity test method exists in China, because the relationship of cattle is very similar to that of deer, some zoos adopt cattle microsatellite markers to be used on deer, but the invalid gene sites are many, and the identification efficiency is not high. In previous researches, paternity test is carried out on 27 zoo raglus by using microsatellite loci of 7 cattle, and the 7 microsatellites have a non-paternity exclusion rate of 83.6 percent and show lower polymorphism. Researches prove that only 56% of the bovine microsatellite primers can be applied to sheep, wherein only 42% of the microsatellite loci show polymorphism on the sheep, and the identification effect is poor.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention provides a set of microsatellite primers for paternity test of deer, the set of primers has 10 pairs in total, the cumulative non-paternity exclusion rate is as high as 99.99 percent, and the microsatellite primers can be applied to paternity test practice of deer.
The invention relates to a primer for paternity test of deer animals, which is selected from at least one primer pair of the following:
1 and 2, 3 and 4, 5 and 6, 7 and 8, 9 and 10, 11 and 12, 13 and 14, 15 and 16, 17 and 18, and 19 and 20.
Preferably, the primer for parentage determination of the deer animal provided by the invention is selected from any 2 pairs, 3 pairs, 4 pairs, 5 pairs, 6 pairs, 7 pairs, 8 pairs, 9 pairs or all of the following primer pairs.
The microsatellite markers used by the invention are all from deer, are original data obtained by simplifying genome sequencing, are strictly screened to obtain high-polymorphism microsatellite markers, and are repeatedly applied in practice, so that 10 stable microsatellite markers with high accuracy and high polymorphism are obtained by screening.
Preferably, the primers for paternity test of cervidae animals as described above, the 5' end of the upstream primer of each pair of primers is labeled with a fluorescent dye.
Preferably, the primer for paternity test of cervidae animals as described above, wherein the fluorescent dye is selected from one or more selected from FAM, FITC, HEX, VIC, JOE, TAMRA, TET, ROX, Cy3, Cy5, TEXAS-Red, PET, NED, Alexa Fluor, DyLight and FTM.
The invention also relates to a kit for paternity test of deer animals, which comprises the primer for paternity test of deer animals;
the kit preferably further comprises one or more of a PCR reaction buffer, a DNA polymerase, dNTPs and water.
Preferably, the kit for paternity test of deer animal as described above, wherein the DNA polymerase is selected from any one of Taq, Bst, Vent, Phi29, Pfu, Tru, Tth, Tl1, Tac, Tne, Tma, Tih, Tf1, Pwo, Kod, Sac, Sso, Poc, Pab, Mth, Pho, ES4DNA polymerase and Klenow fragment;
in some embodiments, the DNA polymerase is Taq DNA polymerase;
more preferably, hot start Taq DNA polymerase.
Preferably, the kit for paternity test of cervidae animals as described above, wherein the water is selected from double distilled water or deionized water.
The invention also relates to a method for paternity test of the deer animal, which comprises the following steps:
extracting DNA of a sample of a cervidae animal to be detected and amplifying the DNA by using the primer for paternity test of the cervidae animal;
the size of the amplified product was detected and paternity inference was performed using the software.
Preferably, in the method for identifying the paternity of the deer animal, when the primer pair for identifying the paternity of the deer animal is used for amplification, the annealing temperature of each primer pair is 58-62 ℃; more preferably from 59 ℃ to 61 ℃ and most preferably 60 ℃.
Preferably, in the method for paternity test of a cervidae animal as described above, the sample of the cervidae animal to be detected includes blood, saliva, semen, bone or hair of the cervidae animal to be detected.
Preferably, the method for paternity test of deer animal as described above, wherein the method for extracting DNA from deer sample comprises saturated phenol-chloroform extraction, resin extraction or magnetic bead extraction.
Preferably, the method for paternity test of cervidae animals as described above, wherein when DNA of a sample of cervidae animals to be detected is extracted and amplified using the primer for paternity test of cervidae animals as described above, at least 2 pairs of primers are used;
more preferably, the primers used are 3, 4, 5, 6, 7, 8, 9 or 10 pairs.
Preferably, the method for paternity testing of cervidae animals as described above, wherein paternity testing is performed using all 10 pairs of primer pairs;
after amplification is completed and the sizes of the amplification products are detected, the amplification products are grouped according to the following primer pairs:
a first group: 1 and 2, 3 and 4;
second group: 5 and 6 SEQ ID NOS, 7 and 8 SEQ ID NOS;
third group: 9 and 10 SEQ ID NOS, 11 and 12 SEQ ID NOS;
and a fourth group: 13 and 14 SEQ ID NOS, 15 and 16 SEQ ID NOS;
and a fifth group: 17 and 18;
a sixth group: 19 and 20 in SEQ ID NO;
preferably, the colors of the fluorescent dye labels carried by the primer pairs in the same group are different;
amplification is carried out by the grouping method as described above, and the amplification products are grouped according to their sizes, whereby the amplification can be carried out more economically and simply.
Preferably, in the primer for deer animal paternity test, the deer animal paternity test kit and the deer animal paternity test method described above, the deer animals include cervidae, chamois, swidae and american cervidae;
more preferably, said cervidae animals include malus, cervi, fallow deer and elk animals;
more preferably, the Cervidae animals include Cervus Elaphus L, Cervus elaphus L, Ardisia, Philippine suede, Soy deer, bristles deer, marsh deer, water deer and sika deer.
Compared with the prior art, the invention has the beneficial effects that:
the primer provided by the invention can be used for amplifying microsatellite DNA bands with clear bands and high polymorphism in a sample of a deer animal, the accumulated non-parentage exclusion rate is up to 99.99%, the identification method is simple and convenient, and the identification result is reliable.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Examples
1. Primer synthesis
10 pairs of microsatellite primers for paternity test of deer are synthesized, and different fluorescent labels (FAM, HEX and TAMRA) are carried at the 5' ends of the primers. The specific information is as follows:
TABLE 110 pairs of microsatellite primers and their grouping for paternity test of deer
2. Extracting genome of sika deer to be detected
Taking 300ul of sika deer blood, extracting sika deer genome by using a Biotake blood extraction kit, detecting the integrity of DNA by using 1% agarose gel electrophoresis, measuring the concentration and purity of the DNA by using an enzyme-labeling instrument, and storing at-20 ℃ for later use.
3. And (3) amplifying the 10 microsatellite loci by using a PCR method by using a sika deer genome to be detected as a template.
The PCR amplification systems and conditions are shown in tables 2 and 3.
TABLE 2 PCR reaction System
TABLE 3 PCR reaction procedure
4. The size of the PCR product is detected by a capillary electrophoresis method.
The PCR products were size-checked using an ABI3730 genetic Analyzer and then genotyped using GeneScan and GenoTyper (ABI) software.
5. Genotyping results polymorphism and paternity relationships were analyzed using Cervus3.0 software.
(2) Technical effects
We verified the paternity test effect of 10 microsatellites of the invention with 16 sika individuals from 4 families. In the results, all the progeny matched their parents (progeny of F1 was Z2, Z3, Z4; progeny of F5 was Z6, Z7, Z8; progeny of F9 was Z10, Z11, Z12; progeny of F13 was Z14, Z15, Z16). LOD is positive and can be confident as a successful match, and the larger the value is, the more credible the LOD is, and if negative the LOD is, the matching is not credible, for example, in Table 4, the LOD value of F1 and F5 which are not related is negative, and the LOD of other correct matches is positive:
TABLE 4 paternity test individual verification results
Non-parentage exclusion rates and other parameter values for table 510 microsatellite values
Locus: the name of the site; k is the allelic base factor; n is the genotype number of the locus; hobs, observing heterozygosity; PIC is polymorphic information content; PE1 Single site non-paternal exclusion ratio with only one candidate parent; PE2: knowing one parent and inferring the non-paternal exclusion rate of a single site in the other parent; PE3, estimating the non-father exclusion rate of a single locus when a pair of parents exist; i, individual recognition rate of single site between two unrelated individuals; SI, individual recognition rate of single site between two individuals with genetic relationship; CPE1 cumulative non-parent exclusion rate for only one candidate parent; CPE 2: knowing the cumulative non-paternal exclusion rate for one parent when inferring the other parent; CPE3, the cumulative non-father exclusion rate when guessing a pair of parents; CI is the accumulated individual recognition rate between two unrelated individuals; CSI is the accumulated individual recognition rate between two individuals with relationship.
The cumulative non-paternal exclusion rate is:
95.45% under the condition that both parents are unknown
99.66% in the case of a parent known to one parent
99.99% under the condition of both parents knowing
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
SEQUENCE LISTING
<110> institute of specialty products of Chinese academy of agricultural sciences
<120> primer, kit and method for paternity test of Cervidae animal
<130>PA17029713SC
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Claims (8)
1. The primers for paternity test of sika deer are all the following primer pairs:
1 and 2, 3 and 4, 5 and 6, 7 and 8, 9 and 10, 11 and 12, 13 and 14, 15 and 16, 17 and 18, and 19 and 20.
2. The primers for paternity testing of sika deer as claimed in claim 1, wherein the 5' end of the upstream primer of each pair of primers is labeled with a fluorescent dye.
3. The primer for paternity testing of sika deer according to claim 2, wherein said fluorescent dye is selected from a plurality of FAM, FITC, HEX, VIC, JOE, TAMRA, TET, ROX, Cy3, Cy5, TEXAS-Red, PET, NED, Alexa Fluor, DyLight and FTM.
4. A sika deer paternity test kit, comprising the primer for sika deer paternity test of any one of claims 1-3;
the kit also comprises PCR reaction buffer solution, DNA polymerase, dNTP and water.
5. A method for identifying parent-child sika deer comprises the following steps:
extracting DNA of a sika deer sample to be detected and amplifying the DNA by using the sika deer paternity test primer as claimed in any one of claims 1-3;
the size of the amplified product was detected and paternity inference was performed using the software.
6. The method for paternity test of sika deer as claimed in claim 5, wherein the annealing temperature of each primer pair is 58-62 ℃ when the primers for paternity test of sika deer are used for amplification.
7. The method according to claim 5, wherein the sika deer sample to be detected comprises blood, saliva, semen, bone or hair of the sika deer to be detected.
8. The method of claim 5, wherein the paternity test is carried out using primers for paternity test of Cervus nippon;
after amplification is completed and the sizes of the amplification products are detected, the amplification products are grouped according to the following primer pairs:
a first group: 1 and 2, 3 and 4;
second group: 5 and 6 SEQ ID NOS, 7 and 8 SEQ ID NOS;
third group: 9 and 10 SEQ ID NOS, 11 and 12 SEQ ID NOS;
and a fourth group: 13 and 14 SEQ ID NOS, 15 and 16 SEQ ID NOS;
and a fifth group: 17 and 18;
a sixth group: 19 and 20 in SEQ ID NO;
the fluorescent dye labels carried by the primer pairs in the same group have different colors.
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| CN111662989B (en) * | 2020-06-16 | 2022-05-10 | 广东省科学院动物研究所 | SSR fluorescence labeling primer for paternity test of deer on slope and identification method |
| CN112458180A (en) * | 2020-11-02 | 2021-03-09 | 北京麋鹿生态实验中心 | SNP marker combination for geographic traceability identification of Beijing and elk in Jiangsu and identification method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1932041A (en) * | 2005-07-06 | 2007-03-21 | 韩国韩医学研究院 | The primers specific to cervus elaphus, c. nippon, c. canadensis and rangifer tarandus gene and the method to identify cervi parvum cornu species |
| CN102465181A (en) * | 2010-11-19 | 2012-05-23 | 同济大学 | Paternity test method for goats and microsatellite primer and kit thereof |
| CN107164468A (en) * | 2017-05-16 | 2017-09-15 | 中国农业科学院特产研究所 | Polymorphic micro-satellite DNA molecular marker for deer and application thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1932041A (en) * | 2005-07-06 | 2007-03-21 | 韩国韩医学研究院 | The primers specific to cervus elaphus, c. nippon, c. canadensis and rangifer tarandus gene and the method to identify cervi parvum cornu species |
| CN102465181A (en) * | 2010-11-19 | 2012-05-23 | 同济大学 | Paternity test method for goats and microsatellite primer and kit thereof |
| CN107164468A (en) * | 2017-05-16 | 2017-09-15 | 中国农业科学院特产研究所 | Polymorphic micro-satellite DNA molecular marker for deer and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| Pedigree Analysis of the Sika Deer (Cervus nippon) using Microsatellite Markers;Ayumi Okada等;《Zoological Science》;20001231;第17卷(第3期);335-340 * |
| 梅花鹿3个种群遗传多样性的微卫星标记分析;孙海涛等;《动物学杂志》;20091231;第44卷(第3期);30-35 * |
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