CN107177698A - Primer, kit and method for animal in deer family paternity test - Google Patents

Abstract

The invention provides a kind of primer, kit and method for animal in deer family paternity test；The primer is selected from least one following primer pair：SEQ ID NO:1‑20.This set primer has 10 pairs, and the band amplified is clear, polymorphism is high, and its accumulative parentage exclusion probability is up to 99.99%, in the paternity test practice that can be applied to deer.

Description

Primers, kits and methods for paternity testing of cervidae animals

technical field

The invention relates to the field of molecular genotyping, in particular to a primer, a kit and a method for paternity identification of deer animals.

Background technique

Microsatellite is a short tandem repeat sequence (2-6bp) in the genome, which shows different alleles due to different repeat numbers. Compared with other identification methods, paternity testing with microsatellite markers has many incomparable advantages. Among them, the most notable advantage is his co-dominant genetic characteristics, which allows homozygous and heterozygous genotypes to be distinguished, greatly increasing the accuracy of paternity testing. In addition, microsatellite markers have high polymorphism, that is to say, there are multiple alleles at the same locus. Since non-parent-child individuals are unlikely to share the same allele, a small number of high polymorphisms is used. Morphological microsatellite markers can identify the parent-child relationship of a large number of individuals. Finally, because microsatellite analysis uses polymerase chain reaction (PCR), only small amounts of DNA are needed for identification, and highly degraded DNA can also be used, such as DNA extracted from feces, hair, feathers, and skin.

The sika deer (Cervus nippon) is mainly distributed in East Asia, ranging from Siberia to Korea, eastern China and Vietnam; it is also distributed in Japan and other western Pacific islands. Chinese sika deer are mainly distributed in Jilin Province, while Japanese sika deer are mainly distributed in Hokkaido. In the 19th century, the sika deer was hunted to almost extinction. Legislation and protection began in the middle of the 20th century, and the population recovered rapidly from the 1950s to the 1980s. Sika deer have also been introduced to Australia, Europe and the United States. Originally intended as park animals, many are now wild. At present, the subspecies of sika deer distributed in China and Japan have been listed as critically endangered or endangered by the IUCN Red List. my country's sika deer is also a first-class national protected animal.

At present, microsatellite molecular marker technology is most widely used in the paternity test of human and other domestic animals, but because the genomes of species are different, the primers for the microsatellite molecular markers used in the paternity test are also different. At present, there is no accurate paternity test method for deer in China. Because the genetic relationship between cattle and deer is very similar, some zoos use microsatellite markers of cattle for deer, but there are many invalid gene sites, and the identification efficiency is not high. A previous study used 7 bovine microsatellite loci to test the paternity of 27 hog deer in zoos. The non-parent exclusion rate of these 7 microsatellites was 83.6%, showing low polymorphism. Studies have shown that only 56% of bovine microsatellite primers can be applied to sheep, and only 42% of the microsatellite sites are polymorphic in sheep, and the identification effect is poor.

In view of this, the present invention is proposed.

Contents of the invention

The invention provides a set of microsatellite primers for paternity identification of deer animals. There are 10 pairs of primers in this set, and the accumulative non-father exclusion rate is as high as 99.99%, which can be applied in the practice of paternity identification of deer.

The invention relates to a primer for paternity identification of deer animals, at least one primer pair selected from the following:

SEQ ID NO: 1 and 2, SEQ ID NO: 3 and 4, SEQ ID NO: 5 and 6, SEQ ID NO: 7 and 8, SEQ ID NO: 9 and 10, SEQ ID NO: 11 and 12, SEQ ID NO : 13 and 14, SEQ ID NO: 15 and 16, SEQ ID NO: 17 and 18 and SEQ ID NO: 19 and 20.

Preferably, the primers provided by the present invention for parentage identification of cervidae animals are selected from any 2, 3, 4, 5, 6, 7, 8, 9 or all of the following primer pairs.

The microsatellite markers used in the present invention are all derived from deer. They are raw data obtained by simplified genome sequencing, and then undergo strict screening to obtain highly polymorphic microsatellite markers. After repeated application in practice, 10 stable microsatellite markers obtained by screening Microsatellite markers with high accuracy and high polymorphism.

Preferably, the 5' end of the upstream primer of each pair of primers is labeled with a fluorescent dye.

Preferably, the above-mentioned primers for paternity identification of cervidae animals, the fluorescent dye is selected from the group consisting of FAM, FITC, HEX, VIC, JOE, TAMRA, TET, ROX, Cy3, Cy5, TEXAS-Red, PET One or more of , NED, Alexa Fluor, DyLight and FTM.

The present invention also relates to a kit for paternity testing of cervids, which includes the above-mentioned primers for paternity testing of cervids;

Preferably, the kit further includes one or more of PCR reaction buffer, DNA polymerase, dNTP and water.

Preferably, in the cervid paternity test kit as described above, the DNA polymerase is selected from Taq, Bst, Vent, Phi29, Pfu, Tru, Tth, Tl1, Tac, Tne, Tma, Tih, Tf1, Pwo, Any one of Kod, Sac, Sso, Poc, Pab, Mth, Pho, ES4 DNA polymerase and Klenow fragment;

In some embodiments, the DNA polymerase is Taq DNA polymerase;

More preferred is a hot-start Taq DNA polymerase.

Preferably, in the cervid paternity testing kit as described above, the water is selected from double distilled water or deionized water.

The present invention also relates to a method for paternity testing of deer animals, comprising:

Extract the DNA of the cervid sample to be detected and amplify it using the primers for paternity testing of the cervid as described above;

Detect the size of the amplified product, and use software to infer parentage.

Preferably, in the method for paternity testing of cervidae described above, when amplification is performed with the primer pairs for paternity testing of cervidae, the annealing temperature of each primer pair is 58° C. to 62° C.; more preferably 59° C. ~61°C, most preferably 60°C.

Preferably, in the method for paternity testing of the cervid animal described above, the sample of the cervid animal to be detected includes the blood, saliva, semen, bone or hair of the cervid animal to be detected.

Preferably, in the method for paternity testing of cervidae described above, the DNA extraction method of the cervidae sample includes saturated phenol-chloroform method, resin extraction method or magnetic bead extraction method.

Preferably, the method for paternity testing of cervid animals as described above, wherein when the DNA of the cervid family animal sample to be detected is extracted and amplified using the primers for the parent-child testing of cervid family animals as described above, at least 2 pairs of primers are used above;

More preferably, 3, 4, 5, 6, 7, 8, 9 or 10 pairs of primers are used.

Preferably, in the method for paternity identification of cervid animals as described above, all 10 pairs of primers are used for paternity identification;

After the amplification is completed, when the size of the amplification product is detected, the amplification products of the following primer pairs are grouped:

The first group: SEQ ID NO:1 and 2, SEQ ID NO:3 and 4;

The second group: SEQ ID NO:5 and 6, SEQ ID NO:7 and 8;

The third group: SEQ ID NO:9 and 10, SEQ ID NO:11 and 12;

The fourth group: SEQ ID NO:13 and 14, SEQ ID NO:15 and 16;

The fifth group: SEQ ID NO: 17 and 18;

The sixth group: SEQ ID NO: 19 and 20;

Preferably, the fluorescent dye labels carried by the primer pairs in the same group have different colors;

Using the above-mentioned grouping method for amplification and grouping according to the size of the amplified products can make the amplification more economical and convenient.

Preferably, in the above-mentioned primers for paternity testing of cervidae, kits for paternity testing of cervidae and methods for paternity testing of cervidae, the cervidae include cervidae, muntinae, swertinae and American deer Subfamily;

More preferably, the animals of the genus Cervidae include animals of the genus Deer, Cervus, Fallow and Elk;

More preferably, the deer family animals include red deer, white-lipped deer, eld's deer, Ashi's deer, Philippine black muntjac, Su's deer, hyena, marsh deer, sambar deer and sika deer.

Compared with prior art, the beneficial effect of the present invention is:

The primer pair provided by the invention can amplify microsatellite DNA bands with clear bands and high polymorphism in samples of cervidae animals, and the cumulative non-parent exclusion rate is as high as 99.99%. The identification method is simple and the identification results are reliable .

detailed description

Embodiments of the present invention will be described in detail below in conjunction with examples, but those skilled in the art will understand that the following examples are only for illustrating the present invention, and should not be considered as limiting the scope of the present invention. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased from the market.

Example

1. Primer Synthesis

Synthesized 10 pairs of microsatellite primers for paternity identification of deer, with different fluorescent markers (FAM, HEX, TAMRA) at the 5' ends of the primers. The specific information is as follows:

Table 1 10 pairs of microsatellite primers and their groupings for paternity testing of deer

2. Genome extraction of the sika deer to be tested

Take 300ul of sika deer blood, use the Biotake blood extraction kit to extract the sika deer genome, use 1% agarose gel electrophoresis to detect the integrity of the DNA, measure the DNA concentration and purity with a microplate reader, and store it at -20°C for later use.

3. Using the sika deer genome to be detected as a template, the above 10 microsatellite loci were amplified by PCR.

The PCR amplification system and conditions are shown in Table 2 and Table 3.

Table 2 PCR reaction system

Table 3 PCR reaction program

4. Capillary electrophoresis was used to detect the size of PCR products.

The size of the PCR product was detected using an ABI3730 genetic analyzer, and then genotyped using GeneScan and GenoTyper (ABI) software.

5. Genotyping results were analyzed using Cervus3.0 software for polymorphism and parent-child relationship.

(2) Technical effect

We used 16 sika deer individuals from 4 families to verify the paternity testing effect of the 10 microsatellites of the present invention. In the results, all offspring are matched with their biological parents (offspring of F1 are Z2, Z3, Z4; offspring of F5 are Z6, Z7, Z8; offspring of F9 are Z10, Z11, Z12; offspring of F13 Progeny are Z14, Z15, Z16). If the LOD is a positive number, it can be sure that it is a successful match, and the larger the value is, the more credible it is. If it is a negative number, it means that the match is not credible. For example, in Table 4, the LOD value of the unrelated match between F1 and F5 is negative, and the LOD of other correct matches are all positive. number:

Table 4 Individual verification results of paternity testing

Table 5 Non-parent exclusion rate and other parameter values of 10 microsatellite values

Locus: the name of the site; K: the number of alleles; N: the number of genotypes at the site; Hobs: observed heterozygosity; PIC: polymorphic information content; PE1: single site non-parent exclusion when there is only one candidate parent PE2:: Knowing one parent, the non-parent exclusion rate of a single site when another parent is inferred; PE3: the non-parent exclusion rate of a single site when a pair of parents is inferred; I: a single site between two unrelated individuals Point individual recognition rate; SI: single-site individual recognition rate between two related individuals; CPE1: cumulative non-parent exclusion rate when there is only one candidate parent; CPE2: cumulative non-parent exclusion rate when one parent is known and another parent is inferred Non-parent exclusion rate; CPE3: cumulative non-parent exclusion rate when a pair of parents is speculated; CI: cumulative individual identification rate between two unrelated individuals; CSI: cumulative individual identification rate between two related individuals.

Its cumulative non-parent exclusion rate is:

95.45% where neither parent is known

99.66% where one parent is known

99.99% when both parents are known

It should be noted that at last: above each embodiment is only in order to illustrate technical scheme of the present invention, and is not intended to limit; Although the present invention has been described in detail with reference to foregoing each embodiment, those of ordinary skill in the art should understand that: It is still possible to modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the various embodiments of the present invention. range.

SEQUENCE LISTING

<110> Institute of Specialty Products, Chinese Academy of Agricultural Sciences

<120> Primers, kits and methods for paternity testing of cervidae

<130>PA17029713SC

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Claims (10)

1. Primers for paternity testing of deer animals, at least one primer pair selected from the following: SEQ ID NO: 1 and 2, SEQ ID NO: 3 and 4, SEQ ID NO: 5 and 6, SEQ ID NO: 7 and 8, SEQ ID NO: 9 and 10, SEQ ID NO: 11 and 12, SEQ ID NOs: 13 and 14, SEQ ID NOs: 15 and 16, SEQ ID NOs: 17 and 18, and SEQ ID NOs: 19 and 20. 2. The primer for paternity testing of cervidae according to 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 identification of cervidae according to claim 2, wherein the fluorescent dye is selected from the group consisting of FAM, FITC, HEX, VIC, JOE, TAMRA, TET, ROX, Cy3, Cy5 One or more of , TEXAS-Red, PET, NED, AlexaFluor, DyLight and FTM. 4. A kit for paternity testing of cervidae, comprising the primers for paternity testing of cervidae described in any one of claims 1 to 3; Preferably, the kit further includes one or more of PCR reaction buffer, DNA polymerase, dNTP and water. 5. cervid parentage test kit according to claim 4, is characterized in that, described DNA polymerase is selected from Taq, Bst, Vent, Phi29, Pfu, Tru, Tth, Tl1, Tac, Tne, Tma, Any one of Tih, Tf1, Pwo, Kod, Sac, Sso, Poc, Pab, Mth, Pho, ES4 DNA polymerase, and Klenow fragment. 6. A method for paternity testing of deer animals, comprising: extracting the DNA of the cervidae sample to be detected and amplifying it using the primers for paternity testing of the cervidae described in any one of claims 1 to 3; Detect the size of the amplified product, and use software to infer parentage. 7. The method for paternity testing of cervidae according to claim 6, characterized in that, when amplifying with the primer pairs for paternity testing of cervidae, the annealing temperature of each primer pair is 58° C. to 62° C. . 8 . The method for paternity testing of cervids according to claim 6 , wherein the samples of the deer to be detected comprise blood, saliva, semen, bones or hair of the deer to be detected. 9 . 9. the deer parent-child identification method according to claim 6, is characterized in that, use all 10 pairs of primers to carry out parent-child identification; After the amplification is completed, when the size of the amplification product is detected, the amplification products of the following primer pairs are grouped: The first group: SEQ ID NO:1 and 2, SEQ ID NO:3 and 4; The second group: SEQ ID NO:5 and 6, SEQ ID NO:7 and 8; The third group: SEQ ID NO:9 and 10, SEQ ID NO:11 and 12; The fourth group: SEQ ID NO:13 and 14, SEQ ID NO:15 and 16; The fifth group: SEQ ID NO: 17 and 18; The sixth group: SEQ ID NO: 19 and 20; Preferably, the fluorescent dye labels carried by the primer pairs in the same group are of different colors. 10. The method according to any one of claims 6-9, characterized in that, the cervidae include deer subfamily, muntinae, deer subfamily and American deer subfamily; More preferably, the animals of the genus Cervidae include animals of the genus Deer, Cervus, Fallow and Elk; More preferably, the deer family animals include red deer, white-lipped deer, eld's deer, Ashi's deer, Philippine black muntjac, Su's deer, hyena, marsh deer, sambar deer and sika deer.