SSR fluorescence labeled primer for paternity test of pangolin and application thereof

Abstract

Disclosed are an SSR fluorescent labeled primer for paternity test of pangolin and application thereof. 15 pairs of fluorescent labeled satellite primers are successfully screened and synthesized, theprimers can be used in pangolin paternity test, be used for making pangolin paternity testing kit, and be used for pangolin germplasm test and pedigree management, which can guide pangolin breeding and provide a powerful tool for protecting pangolin genetic diversity and population genetic institutions. The invention also provides a method for paternity testing of pangolin. In the method, an M13linker is added, and only one M13 fluorescent label primer is synthesized to carry out PCR expansion, thereby saving the cost and time of synthesizing a large number of fluorescent primers.

Description

technical field

[0001] The invention relates to the field of animal paternity identification technology, in particular to SSR fluorescently labeled primers for pangolin paternity identification and its application. Background technique

[0002] The pangolin is a mysterious mammal with atypical morphological characteristics, which belongs to the genus Manis in the family Manidae of the class Mammalia and the order Pholidota. For a long time, people’s demand for medicinal pangolin scales has led to the increasing illegal trade of pangolins, coupled with the large-scale destruction of their habitats, resulting in a sharp decline in the wild population of pangolins. Currently, there are 8 species (Gaubert and Antunes, 2005 ; Zhang et al., 2015) are included in the international trade protection list of endangered wild animals (Convention on international trade in endangered species of wild fauna and flora I, CITES I).

[0003] At present, habitat fragmentation and population is...

Examples

Embodiment 1

[0038] Example 1 Screening of Malayan pangolin microsatellite markers

[0039] Dissected Malayan pangolin (dead) tongue, stomach, large intestine, pancreas, liver, saliva and other samples were mixed and ground, and total RNA was extracted for transcriptome sequencing, splicing, assembly and searching for microsatellite sites. A total of 18,693 SSR sites were detected, including 12,120 mononucleotide repeats, 3,202 dinucleotide repeats, 1,594 trinucleotide repeats, 259 tetranucleotide repeats, and pentanucleotide repeats. There are 52 repeat sequences and 23 hexanucleotide repeat sequences. According to the obtained SSR loci, 56079 pairs of primers were designed using Primer 5.

[0040] DNA was extracted from the saliva samples of 35 Malayan pangolins (15 females and 20 males) using the Animal Tissue DNA Mini Kit (Hipure Tissue DNA Mini Kit, Magen), and the DNA concentration and quality were detected by a NanoDrop DN-1000 UV spectrophotometer; Dilute the obtained DNA with do...

Embodiment 2

[0046] Example 2 PCR Amplification and Genotyping of Polymorphic Microsatellite Sites

[0047] (1) Microsatellite fluorescently labeled primer PCR reaction

[0048] Add 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. The sequence of the M13 forward primer is CACGACGTTGTAAAAC GAC; for Example 1 Preliminary analysis screened out 15 pairs of primers with polymorphic sites, and added M13 sequence (CACGACGTTGTAAAACGAC) to the forward 5' end of the primers to synthesize forward primers with M13 linker at the 5' end.

[0049] The saliva sample DNA of 35 Malayan pangolins extracted in Example 1 was used as a template for fluorescent PCR reaction. The amplification system was 10 μl, including 1 μl of 100ng / μl DNA template, 5 μl of 2×PCR Mix, and 1.6 μl of ddH2O, which had polymorphism 0.4 μl of forward primer with M13 linker added to the forward 5’ end of the 15 pairs ...

Embodiment 3

[0059] Example 3 Application of Microsatellite Markers in Malayan Pangolin Paternity Test

[0060] The above-mentioned 15 microsatellite markers were used in conjunction with the exclusion method and the likelihood method to identify the paternity of 35 Malayan pangolins, and compared the results of the personal identification with the pedigree records of the farm to verify the accuracy of the method. The LOD value calculated by using CERVUS 3.0 software is 5.75 at the 95% confidence level and 2 at the 80% confidence level. When the confidence level is 95%, it is identified that number 246 and C46, ​​A26 and BB are mother-child relationship; A25 and BB are identified as father-son relationship. Comparing the personal identification results with the pedigree records gave a 100% accuracy rate.

technical field

[0001] The invention relates to the field of animal paternity identification technology, in particular to SSR fluorescently labeled primers for pangolin paternity identification and its application. Background technique

[0002] The pangolin is a mysterious mammal with atypical morphological characteristics, which belongs to the genus Manis in the family Manidae of the class Mammalia and the order Pholidota. For a long time, people’s demand for medicinal pangolin scales has led to the increasing illegal trade of pangolins, coupled with the large-scale destruction of their habitats, resulting in a sharp decline in the wild population of pangolins. Currently, there are 8 species (Gaubert and Antunes, 2005 ; Zhang et al., 2015) are included in the international trade protection list of endangered wild animals (Convention on international trade in endangered species of wild fauna and flora I, CITES I).

[0003] At present, habitat fragmentation and population is...

Examples

Embodiment 1

[0038] Example 1 Screening of Malayan pangolin microsatellite markers

[0039] Dissected Malayan pangolin (dead) tongue, stomach, large intestine, pancreas, liver, saliva and other samples were mixed and ground, and total RNA was extracted for transcriptome sequencing, splicing, assembly and searching for microsatellite sites. A total of 18,693 SSR sites were detected, including 12,120 mononucleotide repeats, 3,202 dinucleotide repeats, 1,594 trinucleotide repeats, 259 tetranucleotide repeats, and pentanucleotide repeats. There are 52 repeat sequences and 23 hexanucleotide repeat sequences. According to the obtained SSR loci, 56079 pairs of primers were designed using Primer 5.

[0040] DNA was extracted from the saliva samples of 35 Malayan pangolins (15 females and 20 males) using the Animal Tissue DNA Mini Kit (Hipure Tissue DNA Mini Kit, Magen), and the DNA concentration and quality were detected by a NanoDrop DN-1000 UV spectrophotometer; Dilute the obtained DNA with do...

Embodiment 2

[0046] Example 2 PCR Amplification and Genotyping of Polymorphic Microsatellite Sites

[0047] (1) Microsatellite fluorescently labeled primer PCR reaction

[0048] Add 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. The sequence of the M13 forward primer is CACGACGTTGTAAAAC GAC; for Example 1 Preliminary analysis screened out 15 pairs of primers with polymorphic sites, and added M13 sequence (CACGACGTTGTAAAACGAC) to the forward 5' end of the primers to synthesize forward primers with M13 linker at the 5' end.

[0049] The saliva sample DNA of 35 Malayan pangolins extracted in Example 1 was used as a template for fluorescent PCR reaction. The amplification system was 10 μl, including 1 μl of 100ng / μl DNA template, 5 μl of 2×PCR Mix, and 1.6 μl of ddH2O, which had polymorphism 0.4 μl of forward primer with M13 linker added to the forward 5’ end of the 15 pairs ...

Embodiment 3

[0059] Example 3 Application of Microsatellite Markers in Malayan Pangolin Paternity Test

[0060] The above-mentioned 15 microsatellite markers were used in conjunction with the exclusion method and the likelihood method to identify the paternity of 35 Malayan pangolins, and compared the results of the personal identification with the pedigree records of the farm to verify the accuracy of the method. The LOD value calculated by using CERVUS 3.0 software is 5.75 at the 95% confidence level and 2 at the 80% confidence level. When the confidence level is 95%, it is identified that number 246 and C46, ​​A26 and BB are mother-child relationship; A25 and BB are identified as father-son relationship. Comparing the personal identification results with the pedigree records gave a 100% accuracy rate.

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