CN109182546B - SSR fluorescence labeling primer for paternity test of pangolin scales and application thereof - Google Patents

SSR fluorescence labeling primer for paternity test of pangolin scales and application thereof Download PDF

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CN109182546B
CN109182546B CN201811220793.2A CN201811220793A CN109182546B CN 109182546 B CN109182546 B CN 109182546B CN 201811220793 A CN201811220793 A CN 201811220793A CN 109182546 B CN109182546 B CN 109182546B
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陈金平
李慧明
李林妙
晋学君
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Abstract

The invention discloses an SSR fluorescence labeling primer for parent-child identification of pangolin and application thereof. The invention successfully screens and synthesizes 15 pairs of fluorescent labeled satellite primers which can be applied to paternity test of pangolin scales, can also be prepared into a kit for paternity test of pangolin scales, or can be used for germplasm identification and family management of pangolin scales, thereby reasonably guiding artificial breeding work of pangolin scales and providing a powerful tool for protecting the genetic diversity, population genetic organization and other researches of the pangolin scales. The invention also provides a method for identifying the paternity of pangolins, which only needs to synthesize one M13 fluorescence labeling primer for PCR expansion by adding the M13 joint, thereby saving the cost and time for synthesizing a large amount of fluorescence primers.

Description

SSR fluorescence labeling primer for paternity test of pangolin scales and application thereof
Technical Field
The invention relates to the technical field of paternity test of animals, in particular to SSR fluorescent labeled primers for paternity test of pangolin scales and application thereof.
Background
Pangolin scales are mysterious mammals with atypical morphological characteristics belonging to the Mammalia (Mammalia) order Lepidoptera (Pholidota) family Pangolidea (Manideae) genus Dioscorea (Manis). For a long time, the medical requirement of people for squama manitis penetration leads to the increasing illegal trade of pangolins, and the large-scale destruction of the habitat of pangolins causes the wild population quantity of pangolins to be sharply reduced, and currently, 8 varieties (Gaubert and antonnes, 2005; Zhang et al, 2015) are listed in the International trade protection list of endangered wild animals (Convention on International trade in ended individuals of world and flora I, CITES I).
At present, the fragmentation and population isolation of habitat make pangolins in small population easy to have close-relative breeding, so that the problems of germplasm degradation, growth performance and reproductive capacity reduction and the like are very prominent. Furthermore, the reproduction rate of pangolins is low, the individual number in small populations is difficult to recover after being suddenly reduced, and the small populations isolated from each other are extremely easy to be extinct in local areas. Although in situ protection is an important component of the protection work of endangered species, artificial breeding and ex situ protection have become important measures for protecting endangered species in order to enlarge the size of the emergent population and increase genetic diversity. Under the condition of artificial feeding, the pangolin can ensure to obtain comprehensive and sufficient food and nutrition, obtain good health guarantee and avoid the harm of natural enemies. Therefore, it is important to establish a stable artificial breeding population, avoid inbreeding depression and genetic drift, increase effective population size, and perform breeding and optimal pairing with the purpose of long-term gene exchange with the individual with the smallest genetic relationship. Therefore, the method not only can meet the social requirements of scientific research and public education at home and abroad, but also can create certain conditions for captive breeding individual release to supplement wild population.
Early paternity testing used blood type or enzyme type as genetic markers. At present, the DNA molecular marker replaces the traditional genetic marker and becomes an important genetic marker used in paternity test research. The microsatellite marker has rich polymorphism, relatively conservative single copy sequences are presented at two ends of the sequences, the number of alleles is highly variable, the heterozygosity is high, the genetic stability is good, and the microsatellite marker is in co-dominant inheritance, so that the microsatellite marker is widely applied to construction of a gene map, determination of genetic relationship and identification of individual or group genetic relationship.
Microsatellite, also known as Simple Sequences (SSR), refers to a class of repetitive Sequences having repeat units of 2-6 bases. The SSR molecular marker has the advantages of high polymorphism, co-dominant inheritance, neutral selection, easy operation and the like, has reliable obtained results, simple method, time and labor conservation, can be quickly developed into a genetic marker with great value, and can be widely applied to the research fields of paternity test between individuals, population genetic analysis, diagnosis of genetic diseases, gene mapping, genetic maps and the like.
At present, a microsatellite molecular marker is not reported to be applied to parent-child identification and genetic management of pangolins, the invention aims to establish a gill identification technology of the malayan pangolins by utilizing a microsatellite fluorescent marker, provides scientific basis for establishing a family pedigree of a pangolin farm in order to solve 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 pangolins breeding process, and reasonably guides the breeding and conservation work of artificial pangolins.
Disclosure of Invention
The invention aims to provide an SSR fluorescent labeled primer for paternity test of pangolin, and the primer can be used for paternity test of pangolin.
The invention also aims to provide application of the SSR fluorescent labeled primers for paternity test of pangolin scales, which comprises the step of performing paternity test on pangolin scales by using the primers or preparing a kit for paternity test of pangolin scales by using the primers.
The invention is realized by the following technical scheme:
obtaining a transcriptome of pangolin according to a multi-tissue mixed sample of the pangolin, and designing a primer according to a microsatellite locus provided in the transcriptome data; secondly, performing PCR amplification by using DNA (deoxyribonucleic acid) of a pangolin saliva sample as a template and a microsatellite marker primer, and primarily screening polymorphism of a PCR product to obtain a microsatellite marker primer with high polymorphism; carrying out FAM fluorescent modification on a microsatellite marker primer with high polymorphism, carrying out PCR analysis by taking pangolin saliva sample DNA as a template, and carrying out genotype judgment on a PCR product; and (4) identifying the paternity relationship by jointly using an exclusion method and a likelihood method according to the genotype analysis result.
Specifically, the SSR fluorescence labeling primer for parent-child identification of pangolin comprises the following components:
M13MJ-F2F:TTTCATACCGGGAAGTCCAC；R:ATGGTCCTAACACCACGGAG；
M13MJ-F3F:CACCTGCATGTACCCCTTTT；R:CCCCCTCAAAATACCACCTT；
M13MJ-F4F:GAGAGAAAGGGGAAAATCGG；R:TGATAGGATGTGAGGAGGGG；
M13MJ-F5F:TGGGGTCTGCTGTTTTCATT；R:CTCCCTCTGTAGGTTGCCCT；
M13MJ-F7F:AGAAGTGATTTGCACCCCTG；R:CAGTGGCCAGAATGGAGATT；
M13MJ-F8F:GTCATGCATGGAAGACAGGA；R:GTCTGGCTGAAATGGAAAGG；
M13MJ-F9F:CCCTATGAGGTGGGCACTAA；R:AACTCCATCAAAGGTGTGGC；
M13MJ-F10F:CCCAGATCCAAAATGAATGG；R:TGCTGATGTTCACTCTTGCC；
M13MJ-F11F:ATCCACCTAGGAACCTCAGC；R:GACTCTTCGGGATTTCACACA；
M13MJ-F12F:ATCCACCTAGGAACCTCAGC；R:GACTCTTCGGGATTTCACACA；
M13MJ-F16F:TTCCCTTCATCTGTTTTGCC；R:CAGCACTGCCAAGGTCTGTA；
M13MJ-F17F:GTAATGGGGTATGTGGTGGG；R:TCCCTGTTCAAACGGAATTT；
M13MJ-F20F:CAGTGCTCATCACATAGCAGG；R:CATGCCTAGTGTTTCACGTTG
M13MJ-F24F:TTCAGCCAGGGTCTCTCAGT；R:TGGGGTTTTTCCTCAATCTG；
M13MJ-F25F:CCAGAGAAAGGTAGGAGCCA；R:TCCAGAAAACAGACCCAAGG。
further, M13 adaptor is connected to the positive 5 'end of the primer pair, a positive primer of M13 adaptor added to the 5' end is synthesized, and the sequence of the M13 adaptor is CACGACGTTGTAAAACGAC; and connecting a fluorescent group to the forward 5' end of the universal primer M13 to synthesize a fluorescent group modified M13 forward primer, wherein the forward primer sequence of the universal primer M13 is CACGACGTTGTAAAACGAC. The fluorescent group is FAM.
The application of the SSR fluorescence labeling primer for paternity test of pangolin scales comprises the steps of utilizing the primer to perform paternity test of pangolin scales, or utilizing the primer to prepare a kit for paternity test of pangolin scales, or utilizing the primer to perform germplasm identification and family management.
A method for paternity test of pangolin scales comprises the following steps:
A. synthesizing the 15 primer pairs (M13MJ-F2, M13MJ-F3, M13MJ-F4, M13MJ-F5, M13MJ-F7, M13MJ-F8, M13MJ-F9, M13MJ-F10, M13MJ-F11, M13MJ-F12, M13MJ-F16, M13MJ-F17, M13MJ-F20, M13MJ-F24 and M13MJ-F25) as forward primers by adding M13 linker to the forward 5' end, wherein the M13 linker sequence is CACGACGTTGTAAAACGAC; synthesizing a forward primer with a fluorophore added to the forward 5' end of the universal primer M13; the sequence of the forward primer of the universal primer M13 is CACGACGTTGTAAAACGAC.
B. Extracting pangolin DNA as a template, performing PCR amplification by using the 15 primers and a forward primer with an M13 joint added at the forward 5 'end, a reverse primer and a forward primer with a fluorophore added at the forward 5' end of the universal primer M13, performing genotype judgment on a PCR product, calculating an allelic factor by using Cervus software, observing heterozygosity and expected heterozygosity, performing Hardy-Weinberg equilibrium test, detecting polymorphic information content, individual exclusion rate and accumulated exclusion rate, and performing paternity test by jointly using an exclusion method and a likelihood method according to a genotype analysis result.
In the above method for paternity test of squama Manis, touchdown PCR is used as PCR amplification program, and the specific conditions are as follows: 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 ℃ for 10 min.
Compared with the prior art, the invention has the advantages that:
(1) the SSR fluorescence labeling primer provided by the invention can be used for paternity test and family management of the malayan pangolin, so that artificial breeding work of the malayan pangolin is reasonably guided, and a powerful tool is provided for researches such as protection of genetic diversity, population genetic organization and the like of the pangolin;
(2) the method of adding M13 joint is adopted, and only one M13 fluorescence labeling primer needs to be synthesized for PCR extension, so that the cost and time for synthesizing a large amount of fluorescence primers are saved.
Drawings
FIG. 1 is a schematic diagram of a fluorescent PCR reaction with M13 linker added.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1 screening of microsatellite markers from manis pentadactyla
Dissecting and taking dead samples of scaly malay (dead) tongue, stomach, large intestine, pancreas, liver, saliva and the like, mixing and grinding, extracting total RNA for transcriptome sequencing, splicing, assembling and searching microsatellite loci. 18693 SSR sites were detected in total, in which 12120 mononucleotide repeats, 3202 dinucleotide repeats, 1594 trinucleotide repeats, 259 tetranucleotide repeats, 52 pentanucleotide repeats and 23 hexanucleotide repeats were detected. 56079 pairs of primers were designed using Primer 5 based on the SSR sites obtained.
Extracting 35 (15 female and 20 male) malayan pangolin saliva sample DNA by using an animal Tissue DNA minikit (Hipure Tissue DNA Mini Kit, magenta), and detecting the concentration and quality of the DNA by using a NanoDropDN-1000 ultraviolet spectrophotometer; the obtained DNA was diluted with double distilled water to 100 ng/. mu.l of the working solution, and stored at-20 ℃ for further use.
Using the Malaysia mala saliva DNA as a template, 40 pairs of primers were randomly selected and added with M13 sequence (CACGACGTTGTAAAACGAC) at the forward 5 'end of the obtained microsatellite primers for synthesis, and the PCR amplification system was 10. mu.l, comprising 1. mu.l of 100 ng/. mu.l DNA template, 5. mu.l of 2 XPCR Mix, 2. mu.l of ddH2O 2. mu.l, 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. The PCR products were detected by polyacrylamide gel electrophoresis and 15 pairs of primers with polymorphic sites were screened out by preliminary analysis (Table 1).
TABLE 1 microsatellite primer sequence information of pangolin malayan selected in the present invention having polymorphism
Figure BDA0001834714810000061
Figure BDA0001834714810000071
Figure BDA0001834714810000081
Example 2 polymorphic microsatellite locus PCR amplification and genotyping
(1) Microsatellite fluorescent labeling primer PCR reaction
Adding a FAM fluorescent group to the forward 5' end of the universal primer M13 for modification (Boutin-Ganache et al, 2001) to obtain a FAM modified M13 forward primer, wherein the sequence of the M13 forward primer is CACGACGTTGTAAAAC GAC; preliminary analysis of example 1 screened 15 pairs of primers with polymorphic sites for forward 5 'addition of M13 sequence (CACGACGTTGTAAAACGAC) to synthesize a forward primer with 5' addition of M13 linker.
Fluorescence PCR was Performed using 35 DNA samples of the squama Manis obtained in example 1 as templates, and the amplification system was 10. mu.l including 100 ng/. mu.l DNA template 1. mu.l, 2 XPCR Mix 5. mu.l, ddH2O 1.6.6. mu.l, forward primer 0.4. mu.l of M13 linker added to the forward 5' end of 15 pairs of Primers with polymorphic sites, reverse primer 1. mu.l, FAM modified M13 forward primer 1. mu.l (see reaction scheme 1, references: Boutin-Ganache et al, 2001. M13-labelled Primers improving the reactivity and use of Microatellite analysis Performed with Two Different reagent Primers-reagents, Biotechniques,31(1): 25-28). 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 ℃.
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, processing, and then putting into an ABI3730XL high-throughput DNA sequencer for capillary electrophoresis and STR analysis to determine the allele sizes of the microsatellite markers of the pangolin on different individuals.
(2) Genetic diversity analysis
The allele fraction (k) was calculated using Cervus software, observed heterozygosity (Ho) versus expected heterozygosity (He), Hardy Winberg Equilibrium (HWE) test, Polymorphic Information Content (PIC) and exclusion rates of candidate parents were used to characterize the Malaysia malayi associated microsatellite DNA polymorphisms.
The results of diversity analysis of 15 microsatellite genetic markers in 35 DNA samples of pangolin malayan are shown in Table 2, and the nucleotide sequences of 15 microsatellite loci are respectively shown in SEQ ID NO.1-SEQ ID NO. 15. Wherein the nucleotide sequence of the microsatellite locus amplified by the primer M13MJ-F2 is shown as SEQ ID NO. 1; the nucleotide sequence of the microsatellite locus amplified by the primer M13MJ-F3 is shown as SEQ ID NO. 2; wherein the nucleotide sequence of the microsatellite locus amplified by the primer M13MJ-F4 is shown as SEQ ID NO. 3; wherein the nucleotide sequence of the microsatellite locus amplified by the primer M13MJ-F5 is shown as SEQ ID NO. 4; wherein the nucleotide sequence of the microsatellite locus amplified by the primer M13MJ-F7 is shown as SEQ ID NO. 5; wherein the nucleotide sequence of the microsatellite locus amplified by the primer M13MJ-F8 is shown as SEQ ID NO. 6; wherein the nucleotide sequence of the microsatellite locus amplified by the primer M13MJ-F9 is shown as SEQ ID NO. 7; wherein the nucleotide sequence of the microsatellite locus amplified by the primer M13MJ-F10 is shown as SEQ ID NO. 8; wherein the nucleotide sequence of the microsatellite locus amplified by the primer M13MJ-F11 is shown as SEQ ID NO. 9; wherein the nucleotide sequence of the microsatellite locus amplified by the primer M13MJ-F12 is shown as SEQ ID NO. 10; wherein the nucleotide sequence of the microsatellite locus amplified by the primer M13MJ-F16 is shown as SEQ ID NO. 11; wherein the nucleotide sequence of the microsatellite locus amplified by the primer M13MJ-F17 is shown as SEQ ID NO. 12; wherein the nucleotide sequence of the microsatellite locus amplified by the primer M13MJ-F20 is shown as SEQ ID NO. 13; wherein the nucleotide sequence of the microsatellite locus amplified by the primer M13MJ-F24 is shown as SEQ ID NO. 14; wherein the nucleotide sequence of the microsatellite locus amplified by the primer M13MJ-F25 is shown as SEQ ID NO. 15.
As can be seen from Table 2, 15 microsatellite loci detect 4-11 alleles, and the average number of alleles is 8; observing a heterozygosity between 0.206 and 0.857, and expecting a heterozygosity between 0.454 and 0.869; the Polymorphic Information Content (PIC) value is between 0.413 and 0.84. Thus, the primer of the microsatellite locus screened by the invention is proved to have higher genetic polymorphism. When the parent genetic information is unknown, the individual exclusion rate (NE-PP) of the excluded offspring and the assumed parent in a paternity relationship is 0.117-0.599; when the genetic information of the parent is known, the individual exclusion rate (NE-2P) of the excluded filial generation and the assumed parent is in a paternity relationship is 0.317-0.752; when the genetic information of both parents is known, the individual exclusion rate (NE-1P) of the excluded offspring and the unrelated assumed parent pair to be in-child relationship is 0.53-0.896. The cumulative exclusion probabilities NE-1P, NE-2P, NE-PP are 0.9999, 0.995, respectively.
TABLE 2 polymorphic characteristics of the 15 microsatellite loci of Pangolin margarita
Locus k N HObs HExp PIC NE-1P NE-2P NE-PP HW
M13MJ-F2 6 35 0.657 0.706 0.644 0.721 0.553 0.373 NS
M13MJ-F3 6 33 0.606 0.74 0.682 0.686 0.513 0.333 ND
M13MJ-F4 5 34 0.382 0.65 0.6 0.764 0.588 0.397 NS
M13MJ-F5 8 32 0.469 0.692 0.645 0.714 0.534 0.334 NS
M13MJ-F7 11 35 0.743 0.853 0.821 0.485 0.317 0.145 ND
M13MJ-F8 11 32 0.469 0.869 0.84 0.446 0.285 0.117 ND
M13MJ-F9 4 34 0.206 0.454 0.413 0.896 0.752 0.599 ND
M13MJ-F10 9 35 0.857 0.814 0.777 0.556 0.38 0.195 ND
M13MJ-F11 9 35 0.6 0.822 0.785 0.546 0.37 0.189 ND
M13MJ-F12 7 35 0.429 0.78 0.737 0.616 0.435 0.246 ND
M13MJ-F16 7 34 0.382 0.673 0.611 0.751 0.584 0.403 NS
M13MJ-F17 11 35 0.657 0.82 0.789 0.53 0.355 0.166 ND
M13MJ-F20 9 32 0.625 0.825 0.789 0.537 0.362 0.179 ND
M13MJ-F24 6 32 0.656 0.709 0.654 0.713 0.538 0.352 NS
M13MJ-F25 8 34 0.735 0.743 0.692 0.669 0.491 0.302 NS
Note: n is the number of detected sample individuals; k is the allelic factor; hobs is used for observing heterozygosity; HExp is the desired heterozygosity; HW is Hardy Winberg equilibrium test; PIC is polymorphic information content.
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 117 alleles altogether, the average allele is 8, the average observed heterozygosity is 0.565, the average expected heterozygosity is 0.743, the smaller difference between the two indicates that the 15 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 15 microsatellite markers are all greater than 0.99. In conclusion, the invention develops 15 polymorphic microsatellite loci of the malayan pangolin, a primer sequence for amplifying the 15 microsatellite loci and an amplification method, can be applied to the genetic diversity research of different geographical populations of the malayan pangolin, has good repeatability and is a reliable and effective molecular marker.
Example 3 application of microsatellite markers to paternity test of Pangolin malayan
And (3) carrying out paternity test on 35 manis pentadactyla by applying the 15 microsatellite markers and using an exclusion method and a likelihood method, and comparing the result of the paternity test with the stock farm pedigree record to verify the accuracy of the method. The LOD value calculated using CERVUS 3.0 software was 5.75 at 95% confidence and 2 at 80% confidence. When the confidence coefficient is 95%, the numbers 246 and C46 are identified, and A26 and BB are in parent-child relationship; a25 was identified as having a parent-child relationship to BB. The results of the in-person identification were compared to pedigree records, and the accuracy was found to be 100%.
The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should 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 pangolin scales and application thereof
<160> 15
<170> SIPOSequenceListing 1.0
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actgctggag aaaactgtga tgatgattgt atcttaagtg aatgatggcc ataatgggaa 180
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<213> Artificial sequence (M13MJF4)
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atatggcagt gaacaagaga gccaaaaatc cctctcctag gtaagcctgt gtgtgacagc 180
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tggggtctgc tgttttcatt atagaatact tttagttaag acaaggaaaa gtgaaaggaa 60
cagaagtctc tttttaatag tccatatgaa aatctcatat gcaattaatt aattaattaa 120
ttaattaatt aattaatatt tctctaaggt ctcctggctt ctgaaggttt atttagggca 180
acctacagag ggaggaagaa ataactgtga ctcatgttc 219
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agtgatttgc acccctgagg aaaggcctga cttctttttc ggtcctctcc tccttgttct 60
tatttctgtc tgtttgtttg ttcattttct cattcattca ttcattcatt cattcattca 120
ttcattcatt cattcattca ttcccatttg ctgcctcaag tgcacccact ttctcatggc 180
tgcacagggt ttaagaggtg actttaatct ccattctggc cactgggtcc atcatccca 239
<210> 6
<211> 250
<212> DNA
<213> Artificial sequence (M13MJF8)
<400> 6
gtcatgcatg gaagacagga ctaaggcaaa tgctattatg attagagcac tccaggtcat 60
gacactcact ggatattgtt ttttgtttgt ttgtttgttt gtttgtttgt ttgttttgtt 120
tcaaagctat tacaactaag ggatgatgtg ctcctctttt tccttaccag taaacatttt 180
agtggtagtg gtatattaaa tagcaaaaag ttagtggcca tccctttcca tttcagccag 240
actaacccat 250
<210> 7
<211> 300
<212> DNA
<213> Artificial sequence (M13MJF9)
<400> 7
ccctatgagg tgggcactaa tgttatttcc atgtcacaga tgaggaaaca gaggaccagc 60
aaggtacagt cagttgccca ggccacacag ccaggatttc agccctaaag tctgattcca 120
gagtttgagt tctccattga gccctaccgt cgtttggatg aatgaatgaa tgaatgaatg 180
aatgaatgag tgaatgaagc ccccggggag gggacagcag ggcccccact cctgcttaca 240
gcctttccca ccagctgcca cacctttgat ggagttttcc cagaggggcc tgtcttggga 300
<210> 8
<211> 210
<212> DNA
<213> Artificial sequence (M13MJF10)
<400> 8
cccagatcca aaatgaatgg agccggaaat tacctcatca gttggtatta atcagccaag 60
caattgacag gctgattgac aggctgggag cccctgaggc tgttgacaaa ggaacatgtt 120
gcttctggaa tgaatgaatg aatgaatgaa tgaatgaatg gggcttgatg tttgatactg 180
tggaagtaag gcaagagtga acatcagcaa 210
<210> 9
<211> 260
<212> DNA
<213> Artificial sequence (M13MJF11)
<400> 9
atccacctag gaacctcagc taacagcaac tttctaggtc tctgagttga ttgatttaac 60
atgttaaata gatatagata gatagatgga tagataatag atagatgata gatgatagat 120
acatagatga tagatgatag atgatagata gatagataga tagatagata gatagataga 180
tagatagata gaccaactga tcttgaaatg tgtgcatagt ttgtaaactg tgtgaaatcc 240
cgaagagtct ttgaagagaa 260
<210> 10
<211> 249
<212> DNA
<213> Artificial sequence (M13MJF12)
<400> 10
atccacctag gaacctcagc taacagcaac tttctaggtc tctgagttga ttgatttaac 60
atgttaaata gatatagata gatagatgga tagataatag atagatgata gatgatagat 120
acatagatga tagatgatag atagatagat agatagatag atagatagat agatagatag 180
atagaccaac tgatcttgaa atgtgtgcat agtttgtaaa ctgtgtgaaa tcccgaagag 240
tctttgaag 249
<210> 11
<211> 249
<212> DNA
<213> Artificial sequence (M13MJF16)
<400> 11
ttcccttcat ctgttttgcc cattctccta acccacccag tttgttctct acatttatga 60
gcatttttct ggttggttgg ttggttggtt ggttggttgg ttggttggtt gttggttgtc 120
ttgtttaaaa aaaagaaggg taaaaccata tctttggaaa tcaaataaca ctgaaaaaaa 180
tgctgatctg aagatgatgc acttacacca tctagaaaat tgtacagacc ttggcagtgc 240
tgccctatc 249
<210> 12
<211> 219
<212> DNA
<213> Artificial sequence (M13MJF17)
<400> 12
ctgtaatggg gtatgtggtg gggacttgat aatgggggga gtcagtaacc ataatgttgc 60
tcatgtgatt gtatattaat ggtaccataa taaataaata gatagataga tagatagata 120
gatagataga tagatagata gatagataat ctgcagcgtt atccatataa gtagtttgct 180
gagagagtat aattgaaatt ccgtttgaac agggaagat 219
<210> 13
<211> 199
<212> DNA
<213> Artificial sequence (M13MJF20)
<400> 13
cagtgctcat cacatagcag gtactcagaa agtaaaatta gttcccttct tttagcctta 60
cttgatcata accattggag atagatagat agatagatag atagatagat agatagatag 120
atagatagat agatagatag atagcagtaa gagatacaac gtgaaacact aggcatgtaa 180
attctcaaag aaaacaatt 199
<210> 14
<211> 199
<212> DNA
<213> Artificial sequence (M13MJF24)
<400> 14
ttcagccagg gtctctcagt gactgctgag tagaacactc tgaaggtcca tgtcagtccc 60
atgaaataag aatgagaaag agactttgta tattaacctt ctgatacttt ggtgctcttt 120
tttgttttgt tttgttttgt tttgttttgt tttgttatag caacataaca gattgaggaa 180
aaaccccaaa tactcatgc 199
<210> 15
<211> 141
<212> DNA
<213> Artificial sequence (M13MJF25)
<400> 15
ccagagaaag gtaggagcca aaagctgggg gaaacaaaac aaaacaaaac aaaacaaaac 60
aaaacaaaac aaaacaacac tttgcagcct ctaattctgg gagtaaaacc ttgggtctgt 120
tttctggagt gaggagggaa g 141

Claims (5)

1. The SSR fluorescence labeling primer for parent-child identification of pangolin scales is characterized by comprising the following components in parts by weight:
M13MJ-F2 F: TTTCATACCGGGAAGTCCAC; R: ATGGTCCTAACACCACGGAG；
M13MJ-F3 F: CACCTGCATGTACCCCTTTT; R: CCCCCTCAAAATACCACCTT；
M13MJ-F4 F: GAGAGAAAGGGGAAAATCGG; R: TGATAGGATGTGAGGAGGGG；
M13MJ-F5 F: TGGGGTCTGCTGTTTTCATT; R: CTCCCTCTGTAGGTTGCCCT；
M13MJ-F7 F: AGAAGTGATTTGCACCCCTG; R: CAGTGGCCAGAATGGAGATT；
M13MJ-F8 F: GTCATGCATGGAAGACAGGA; R: GTCTGGCTGAAATGGAAAGG；
M13MJ-F9 F: CCCTATGAGGTGGGCACTAA; R: AACTCCATCAAAGGTGTGGC；
M13MJ-F10 F: CCCAGATCCAAAATGAATGG; R: TGCTGATGTTCACTCTTGCC；
M13MJ-F11 F: ATCCACCTAGGAACCTCAGC; R: GACTCTTCGGGATTTCACACA；
M13MJ-F12 F: ATCCACCTAGGAACCTCAGC; R: GACTCTTCGGGATTTCACACA；
M13MJ-F16 F: TTCCCTTCATCTGTTTTGCC; R: CAGCACTGCCAAGGTCTGTA；
M13MJ-F17 F: GTAATGGGGTATGTGGTGGG; R: TCCCTGTTCAAACGGAATTT；
M13MJ-F20 F: CAGTGCTCATCACATAGCAGG; R: CATGCCTAGTGTTTCACGTTG
M13MJ-F24 F: TTCAGCCAGGGTCTCTCAGT; R: TGGGGTTTTTCCTCAATCTG；
M13MJ-F25 F: CCAGAGAAAGGTAGGAGCCA; R: TCCAGAAAACAGACCCAAGG。
2. use of the primer of claim 1 for paternity testing of pangolin scales.
3. The use of the primer of claim 1 in the preparation of a kit for paternity testing of pangolin scales.
4. A method for paternity test of pangolin scales is characterized by comprising the following steps:
A. synthesizing a forward primer of the primer pair of claim 1 with a M13 linker at the forward 5' end, wherein the M13 linker sequence is CACGACGTTGTAAAACGAC; synthesizing a forward primer with a fluorophore added to the forward 5' end of the universal primer M13; the forward primer sequence of the universal primer M13 is CACGACGTTGTAAAACGAC;
B. extracting pangolin DNA as a template, performing PCR amplification by using the primer in claim 1 and a forward primer with an M13 joint added at the forward 5 'end, a reverse primer and a forward primer with a fluorophore added at the forward 5' end of a universal primer M13, performing genotype judgment on a PCR product, calculating an allelic factor by using Cervus software, observing heterozygosity and expected heterozygosity, performing Hardy-Weinberg equilibrium test, detecting polymorphic information content, individual exclusion rate and accumulated exclusion rate, and performing paternity test by jointly using an exclusion method and a likelihood method according to the genotype analysis result.
5. The method for paternity testing of pangolin scales according to claim 4, wherein said PCR amplification procedure employs touchdown PCR, specifically provided that: 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 ℃ for 10 min.
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CN116200504B (en) * 2023-01-06 2024-10-22 南京森林警察学院 Development and application of STR molecular markers for individual identification of the endangered Malayan pangolin

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