CN107177698B - Primer, kit and method for paternity test of deer animals - Google Patents

Abstract

The invention provides a primer, a kit and a method for paternity identification of deer animals; the primer is selected from at least one primer pair of the following: SEQ ID NO: 1-20. This set of primers has a total of 10 pairs, and the amplified bands are clear and highly polymorphic, and the cumulative non-father exclusion rate is as high as 99.99%, which can be applied to the practice of deer paternity testing.

Description

Primers, kits and methods for paternity testing in deer

technical field

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

Background technique

Microsatellites are short tandem repeats (2-6 bp) in the genome that exhibit different alleles due to different repeat numbers. Compared with other identification methods, the use of microsatellite markers for paternity testing has many incomparable advantages. Among them, the most significant 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-parental individuals are unlikely to share the same allele, a small number of high polymorphisms are used. Morphological microsatellite markers can identify the parent-child relationship of a large number of individuals. Finally, because microsatellite analysis uses the polymerase chain reaction (PCR), only a small amount of DNA is required 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 South Korea, eastern China and Vietnam; it is also distributed in Japan and other western Pacific islands. China's sika deer are mainly distributed in Jilin Province, while Japan's sika deer are mainly distributed in Hokkaido. In the 19th century, the sika deer were hunted to the point of extinction. In the middle of the 20th century, legislative protection began, 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 animals for park decoration, many are now wild. At present, the sika deer subspecies distributed in China and Japan have been listed as critically endangered or endangered by the IUCN Red List, and the sika deer in my country is also a national first-class protected animal.

At present, the microsatellite molecular marker technology is the most widely used in the paternity testing research of human and other domestic animals, but the primers used for the microsatellite molecular marker for paternity testing are also different due to the difference of their genomes between species. At present, there is no accurate paternity identification method for deer in China. Because the kinship of cattle is very similar to that of deer, some zoos use bovine microsatellite markers for deer, but there are many invalid gene loci, and the identification efficiency is not high. A previous study used 7 bovine microsatellite loci to perform paternity testing on 27 dolphin deer in zoos. The non-paternal 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, of which only 42% of microsatellite loci show polymorphism in sheep, and the identification effect is poor.

In view of this, the present invention is proposed.

SUMMARY OF THE INVENTION

The invention provides a set of microsatellite primers for paternity identification of deer animals, the primers have 10 pairs in total, and the cumulative non-paternal exclusion rate is as high as 99.99%, which can be applied to the practice of deer paternity identification.

The present invention relates to a kind of primer for paternity test of deer, 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 NOs: 15 and 16, SEQ ID NOs: 17 and 18, and SEQ ID NOs: 19 and 20.

Preferably, the primers provided by the present invention for paternity testing of deer 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, and are the original data obtained by simplifying genome sequencing. Afterwards, high polymorphism microsatellite markers are obtained through strict screening. After repeated application in practice, 10 stable A highly accurate and polymorphic microsatellite marker.

Preferably, in the above-mentioned primers for paternity testing of deer, the 5' end of the upstream primer of each pair of primers is labeled with a fluorescent dye.

Preferably, the above-mentioned primer for paternity test of deer, the fluorescent dye is selected from 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 deer paternity testing, which includes the primers used for deer paternity testing as described above;

The kit preferably further includes one or more of PCR reaction buffer, DNA polymerase, dNTPs, and water.

Preferably, the above-mentioned cervidae paternity test kit, the DNA polymerase is selected from Taq, Bst, Vent, Phi29, Pfu, Tru, Tth, Tl1, Tac, Tne, Tma, Tih, Tf1, Pwo, Any 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 hot-start Taq DNA polymerase.

Preferably, in the above-mentioned cervidae paternity test kit, the water is selected from double distilled water or deionized water.

The present invention also relates to a deer parentage identification method, comprising:

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

The size of the amplified product was detected, and paternity inference was performed using software.

Preferably, in the above-mentioned cervidae paternity test method, when the primer pairs for the cervidae paternity test are used for amplification, 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 above-mentioned method for determining the paternity of the deer family, the deer family sample to be detected includes the blood, saliva, semen, bones or hair of the deer family animal to be detected.

Preferably, in the above-mentioned method for determining the paternity of the deer family, the method for extracting the DNA of the deer family sample includes extraction by saturated phenol-chloroform method, resin extraction method or magnetic bead extraction method.

Preferably, the above-mentioned cervidae paternity test method, wherein when extracting the DNA of the cervidae sample to be detected and amplifying it using the above-mentioned cervidae paternity test primers, the primers used are at least 2 pairs above;

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

Preferably, the above-mentioned method for paternity testing of cervidae uses all 10 pairs of primers to perform paternity testing;

After the amplification is completed, when detecting the size of the amplification products, 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 NOs: 9 and 10, SEQ ID NOs: 11 and 12;

The fourth group: SEQ ID NOs: 13 and 14, SEQ ID NOs: 15 and 16;

Fifth group: SEQ ID NO: 17 and 18;

The sixth group: SEQ ID NOs: 19 and 20;

Preferably, the primer pairs in the same group are marked with different colors of fluorescent dyes;

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

Preferably, in the above primers for cervidae paternity test, cervidae paternity test kit and cervidae paternity test method, the cervaceae animals include cervaceae, muntjac, roe, and American deer subfamily;

More preferably, the deer family includes animals of the genus Ceratus, Deer, Deer and Elk;

More preferably, the deer animals include red deer, white-lipped deer, eld deer, alder deer, Philippine black muntjac, siberian deer, hyena, marsh deer, sambar deer and sika deer.

Compared with the prior art, the beneficial effects of the present invention are:

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

Detailed ways

The embodiments of the present invention will be described in detail below with reference to the examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. If the specific conditions are not indicated in the examples, it is carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used without the manufacturer's indication are conventional products that can be purchased from the market.

Example

1. Primer Synthesis

Ten pairs of microsatellite primers for deer paternity testing were synthesized, with different fluorescent labels (FAM, HEX, TAMRA) at the 5' end of the primers. The specific information is as follows:

Table 1 Ten pairs of microsatellite primers used for deer paternity test and their groups

2. Genome extraction of sika deer to be tested

Take 300 ul of sika deer blood, extract the genome of sika deer with Biotake blood extraction kit, detect the integrity of DNA by 1% agarose gel electrophoresis, measure DNA concentration and purity with a microplate reader, and store 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 method.

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. The size of PCR product was detected by capillary electrophoresis.

PCR products were sized using ABI3730 Genetic Analyzer, and then genotyped using GeneScan and GenoTyper (ABI) software.

5. Genotyping results Using Cervus3.0 software to analyze the polymorphism and parent-child relationship.

(2) Technical effect

We verified the paternity testing effect of the 10 microsatellites of the present invention with 16 sika deer individuals from 4 families. In the results, all the offspring matched their biological parents (Z2, Z3, Z4 for F1; Z6, Z7, Z8 for F5; Z10, Z11, Z12 for F9; Z10, Z11, Z12 for F13 The offspring are Z14, Z15, Z16). If the LOD is a positive number, it can be considered as a successful match, and the larger the value, the more credible it is. If it is a negative number, the match is unreliable. For example, in Table 4, the matching LOD value of F1 and F5 with no relationship is a negative number, and the other correct matching LODs are positive. number:

Table 4 Individual verification results of paternity test

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

Locus: name of locus; K: number of alleles; N: number of genotypes at the locus; Hobs: observed heterozygosity; PIC: polymorphic information content; PE1: single locus non-paternal exclusion when there is only one candidate parent rate; PE2: the non-father exclusion rate of a single locus when one parent is known and the other parent is inferred; PE3: the non-father exclusion rate of a single locus when a pair of parents is inferred; I: a single locus between two unrelated individuals Point individual identification rate; SI: single locus individual identification rate between two related individuals; CPE1: cumulative non-father exclusion rate when there is only one candidate parent; CPE2: cumulative when one parent is known and the other parent is inferred Non-paternal exclusion rate; CPE3: Cumulative non-paternal exclusion rate when inferring a pair of parents; 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% when both parents are unknown

99.66% where one parent is known

99.99% where both parents are known

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand: It is still possible to modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements to 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 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 in deer

<130> PA17029713SC

<160> 20

<170> PatentIn version 3.3

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Claims (8)

1. A primer for paternity test of sika deer, the primers are all of the following primer pairs: 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 NOs: 15 and 16, SEQ ID NOs: 17 and 18, and SEQ ID NOs: 19 and 20. 2. The primer for paternity testing of sika deer 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 test of sika deer according to claim 2, is characterized in that, described fluorescent dye is selected from FAM, FITC, HEX, VIC, JOE, TAMRA, TET, ROX, Cy3, Cy5, TEXAS-Red , PET, NED, Alexa Fluor, DyLight and a variety of FTM. 4. a sika deer paternity test kit, which comprises the primer for the sika deer paternity test described in any one of claims 1 to 3; The kit also includes PCR reaction buffer, DNA polymerase, dNTPs, and water. 5. A method for identifying paternity of sika deer, comprising: Extract the DNA of the sika deer sample to be detected and amplify it using the primer of the sika deer paternity test described in any one of claims 1 to 3; The size of the amplified product was detected, and paternity inference was performed using software. 6 . The method for paternity testing of sika deer according to claim 5 , wherein when the primers for the paternity testing of sika deer are used for amplification, the annealing temperature of each primer pair is 58° C. to 62° C. 7 . 7 . The paternity identification method of sika deer according to claim 5 , wherein the sika deer sample to be detected comprises blood, saliva, semen, bones or hair of the sika deer to be detected. 8 . 8. The method for paternity test of sika deer according to claim 5, characterized in that, using the primer for paternity test of sika deer to carry out paternity test; After the amplification is completed, when detecting the size of the amplification products, the amplification products of the following primer pairs are grouped: Group 1: 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 NOs: 13 and 14, SEQ ID NOs: 15 and 16; Fifth group: SEQ ID NOs: 17 and 18; The sixth group: SEQ ID NOs: 19 and 20; The primer pairs in the same group are marked with different colors of fluorescent dyes.