CN106834517B - SSR (simple sequence repeat) labeled primer group for parent-child identification of parva and application of SSR labeled primer group - Google Patents
SSR (simple sequence repeat) labeled primer group for parent-child identification of parva and application of SSR labeled primer group Download PDFInfo
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- CN106834517B CN106834517B CN201710179132.9A CN201710179132A CN106834517B CN 106834517 B CN106834517 B CN 106834517B CN 201710179132 A CN201710179132 A CN 201710179132A CN 106834517 B CN106834517 B CN 106834517B
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- 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
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Abstract
The invention discloses an SSR labeled primer group for parent-child identification of seal and application thereof, wherein 15 pairs of fluorescence labeled microsatellite primer groups are successfully screened and synthesized, and can be used for establishing a seal parent-child identification technology and disclosing a kit for parent-child identification. The identification method provided by the invention has the characteristics of accuracy, high efficiency, reliability and the like, can guide the artificial breeding and the field release work of the seal, and is popularized and applied to seal family management and germplasm identification.
Description
Technical Field
The invention belongs to the technical field of paternity test of marine mammals, and particularly relates to an SSR primer group for paternity test of seal and application thereof.
Background
The zebra sea leopard (Phoca largha) belongs to Carnivora (Carnivora) Canine suborder (Caniformia) and Phoenidae (Phocidae) and the zebra sea leopard (Phoca), is a secondary protective animal in China, is mainly distributed in the northern and western regions of the North Pacific ocean and the coasts and islands thereof, is mainly distributed in the Bohai sea and the yellow sea in China and is occasionally seen in the south sea. The parva is the only finfoot animal which can be bred in the sea area of China, and the ice-forming region of the Bay of the Liaodong of the Bohai sea is the soutest one of 8 breeding regions of the parva. The wild population of the zebra sea leopards is threatened, artificial propagation becomes an important means for protecting the species, taking the great liangya tourism stock control limited company (hereinafter referred to as the great liangya) as an example, the 44 zebra sea leopards are successfully bred since the manual reproduction of the zebra sea leopards is carried out, 8-10 individuals are born under artificial conditions every year, the F1 generation individuals account for 60 percent of the whole breeding population up to now, and many F1 generations reach sexual maturity. Over time, numerous generations of F1 will inevitably participate in reproduction, and unfortunately the parental relationship of these F1 generations is not clear, and similar situations exist in other zebra aquaria.
Paternity testing, also known as paternity testing, microsatellite paternity testing, a paternity testing technique that has prevailed in recent years, has found wide application in human forensic identification, identification of genetic lineages of important economic animals and endangered animals. However, this technique is currently found in only a few whale animals such as dolphin's nose (tursio spp), Yangtze river finless porpoise (Neophorcaena orientalis) among marine mammals. In recent years, the excavation work of genome and transcriptome sequences of the species of the family of the seal, such as the plaque seal, and the like, makes a breakthrough, thereby providing a chance for the research, development and application of the microsatellite paternity test technology of the species.
At present, there is no report of applying microsatellite molecular markers to paternity testing and genetic management of the seal. The invention aims to establish the plaque seal paternity test technology by utilizing the microsatellite primer group and adopting the fluorescence labeling capillary electrophoresis technology, and provides a basis for the construction of the plaque seal propagation population genetic pedigree, the exchange and the communication of the plaque seals among all aquariums so as to reasonably guide the artificial propagation of the plaque seals.
Disclosure of Invention
The invention aims to provide an SSR labeled primer group for the paternity test of the seal, the primer can be used for the paternity test of the seal, and the SSR labeled primer group comprises 15 pairs of primers, and the nucleotide sequence of the SSR labeled primer group is shown in SEQ ID NO. 1-SEQ ID NO. 30.
The invention also aims to provide application of the SSR fluorescence labeling primer group for paternity test of the seal. The method comprises the steps of preparing a kit for paternity test of the seal by using the primer group, or performing paternity test on the seal by using the primer group, or performing family management by using the primer group.
The kit comprises all/partial primer groups with nucleotide sequences shown as SEQ ID NO. 1-SEQ ID NO.30, 10 × PCR buffer solution and MgCl2dNTPs, Ex Taq DNA polymerase and double distilled water.
The invention also discloses a method for identifying the paternity of the seals, which comprises the following operation steps of extracting parent and filial generation sample DNA to be identified, marking the forward primer of the primer group with fluorescent dyes of FAM, HEX, TAM or ROX, amplifying the extracted sample DNA by using all/part of the primer group marked with the fluorescent dyes, performing capillary electrophoresis, genotyping according to the electrophoresis peak chart result, performing genotyping by using GeneMapper software, outputting the analysis result in an Excel table, using Cervus software to count allele factors (Na), average heterozygosity (He), information content (PIC), accumulated first exclusion rate (CE-1P), accumulated second primer exclusion rate (CE-2P) and biparental accumulated exclusion rate (CE-PP), calculating the value of L OD according to the Parentiage module and the Delta value standard, displaying the result as "+" (namely under the confidence level of 80%) as the possible primer group, displaying the result as the confidence level of the "+" (namely the confidence level of the primer group "), displaying the result as the confidence level of 95% of the parent and the SSR, and providing the effective molecular marker for identifying the parent and the parent molecular propagation guidance of the present invention.
Drawings
FIG. 15 of the present invention is a partial sequence chart of the primer set in paternity test, showing the correctness of designed primers, which is:
FIG. 1 is a sequencing diagram of primer P L MS10, which comprises a female parent 59368(275bp,278bp), a progeny 87492(278bp) and a male parent 59347(275bp,278bp) from left to right.
FIG. 2 is a sequencing diagram of primer P L MS12, which comprises female parent 59368(280bp), offspring 87492(277bp,280bp) and male parent 59347(277bp) from left to right.
FIG. 3 is a sequencing diagram of the primer P L MS17, which comprises a female parent 59368(261bp), a progeny 87492(258bp,261bp) and a male parent 59347(258bp,261bp) from left to right.
FIG. 4 is a sequencing diagram of primer P L MS24, which comprises a female parent 59368(223bp), a progeny 87492(223bp) and a male parent 59347(223bp) from left to right.
FIG. 5 is a sequencing diagram of primer P L MS28, which comprises female parent 59368(263bp), offspring 87492(263bp) and male parent 59347(263bp) from left to right.
FIG. 6 is a sequencing diagram of primer P L MS44, which is a female parent 59368(226bp), a child 87492(226bp) and a male parent 59347(226bp) from left to right.
FIG. 7 is a sequencing diagram of primer P L MS45, which is a female parent 59368(189bp), a child 87492(189bp) and a male parent 59347(189bp) from left to right.
FIG. 8 is a sequencing diagram of primer P L MS68, which comprises female parent 59368(250bp,258bp), offspring 87492(250bp,258bp) and male parent 59347(247bp,258bp) from left to right.
FIG. 9 is a sequencing diagram of primer P L MS55, which comprises female parent 59368(211bp,223bp), offspring 87492(211bp) and male parent 59347(211bp) from left to right.
FIG. 10 is a sequencing diagram of primer P L MS58, which includes female parent 59368(231bp), offspring 87492(231bp), and male parent 59347(231bp) from left to right.
FIG. 11 is a sequencing diagram of primer P L MS71, which comprises female parent 59368(248bp), offspring 87492(248bp,295bp) and male parent 59347(248bp,295bp) from left to right.
FIG. 12 is a sequencing diagram of primer P L MS76, which comprises female parent 59368(264bp,280bp), offspring 87492(264bp,280bp) and male parent 59347(280bp) from left to right.
FIG. 13 is a sequencing diagram of primer P L MS79, which comprises female parent 59368(314bp,318bp), offspring 87492(314bp,316bp) and male parent 59347(316bp) from left to right.
FIG. 14 is a sequencing diagram of primer P L MS83, which includes female parent 59368(217bp), offspring 87492(217bp), and male parent 59347(217bp) from left to right.
FIG. 15 is a sequencing diagram of primer P L MS90, which includes female parent 59368(287bp), child 87492(287bp), and male parent 59347(287bp) from left to right.
Detailed Description
The technical scheme of the invention is a conventional scheme in the field if not specifically stated.
Collecting samples: the applicant of the invention carries out blood sample collection on 53-head seal housed in great lian san xia in 2014 to 2016, wherein the maternal-child relationship of 10 seal young animals to be identified is recorded to be known, and the candidate male parent to be identified is 17 heads. During the examination of the seal, about 5ml of blood is collected from the tail vein by a veterinarian or a trainer by using a disposable syringe, is filled in a medical blood conventional tube (EDTA-K2 anticoagulation), is stored at 4 ℃, and is stored at-20 ℃ for a long time after being taken back to a laboratory. The DNA extraction adopts a blood genome DNA purification kit of Tiangen company, and the specific steps refer to an operation manual.
Example 1
The application of primer group screening of microsatellite marker for parent-child leopard identification and fluorescence marker primer group in parent-child leopard identification comprises the following steps:
(I) screening of primer set
Since the published genomic information of the seal is very little, the inventors perform SSR site search on Unigenes sequences by using MISA software through only transcriptome data, the search criteria are that the minimum repetition times of dinucleotides, trinucleotides, tetranucleotides, pentanucleotides and hexanucleotides are respectively 14, 9, 6, 4 and 4, and the sequence containing EST-SSR is used for designing a pair of microsatellite primers 195 by using Primer Premier 5.0 software, and the design principle of the primers is as follows: length of the primer: 18-21bp, primer sequence GC content: 33-72%, the Tm value of the primer pair is between 53-61.5 ℃ (the Tm value difference of the upstream primer and the downstream primer is within 5 ℃), the size of the amplified fragment is between 185-315bp, and the occurrence of secondary structures of primers such as hairpin structures, dimers, mismatching, primer dimers and the like is avoided. The primers were synthesized by Biotechnology engineering (Shanghai) Inc. Firstly, randomly selecting 20-head plaque seal DNA to carry out primer amplification, carrying out primary screening by using 8% polyacrylamide gel electrophoresis, and preliminarily obtaining a primer group with stable amplification and polymorphism after removing primers which have no amplification product, no polymorphism, weak bands and incapability of repeated amplification according to an electrophoresis result; and (3) carrying out PCR amplification on random 2-head seal individuals by using the primer subjected to primary screening, and sending the PCR product to the company Limited in the biological engineering (Shanghai) for clone sequencing.
Simultaneously, the upstream primers of the primer group obtained by the preliminary screening are randomly marked by different fluorescent dyes FAM, HEX, TAM and ROX, and the primers are synthesized by the company of biological engineering (Shanghai).
(II) primer set screening results
The PCR reaction system for the primary screening of the primers was 25. mu.l, in which 50ng of template DNA was mixed with 0.2mM dNTP, 200nM each for the upstream and downstream primers, 1 × PCR buffer, 1.5mM MgCl2Ex Taq (Takara corporation) 1U. Amplification was performed under the following PCR reaction conditions: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing for 30s (table 3), extension at 72 ℃ for 30s, for 35 cycles; finally, extension is carried out for 10min at 72 ℃. Screening by 8% polyacrylamide gel electrophoresisSelecting and separating, and obtaining clear, accurate and consistent allele information from 25 sites through screening and comparison repetition.
The PCR reaction system and reaction conditions of the fluorescent primers are shown in the following tables 1 and 2, and 15 pairs of microsatellite markers (shown in SEQ ID NO. 1-SEQ ID NO. 30) which have high polymorphism and can be stably amplified are finally obtained by combining the detection of capillary electrophoresis and the clone sequencing result.
TABLE 1 fluorescent primer reaction System
| Stencil (10 ng/. mu.l) | 1μl |
| Primer F (10 mmol/. mu.l) | 0.5μl |
| Primer R (10 mmol/. mu.l) | 0.5μl |
| dNTP 10mM | 0.5μl |
| 10×PCR Buffer | 2.5μl |
| 25mM MgCl2(μl) | 2.0μl |
| Ex Taq enzyme (5U/. mu.l) | 0.2 |
| Water (ul) | 17.8 |
TABLE 2 fluorescent primer reaction conditions
Example 2
Microsatellite genotype analysis
The extracted DNA of the large Lissajous Asian seal group (53 individuals) is amplified by using 15 pairs of fluorescence labeling primer groups obtained by screening, a PCR product is detected by 1% agarose gel and then is detected by a 3730X L sequencer (ABI company, USA) through capillary electrophoresis, geneMapper software is used for genotyping, and the analysis result is output in an Excel table.
Characterization of the microsatellite gene bases
The capillary electrophoresis peak patterns of the 15 pairs of fluorescence-labeled primers of 53 individuals were genotyped using GeneMapper software, and the typing results were output in an Excel table and introduced into Cervus3.0 software, and the results are shown in Table 3. The number of alleles of each microsatellite locus is 2-8, the average allele factor is 4.1, the average expected value (He) of the gene heterozygosity is 0.3669, and the average value of the Polymorphic Information Content (PIC) is 0.3206.
TABLE 3 sequence, annealing temperature, Gene frequency distribution, and paternity test exclusion ratio of each microsatellite
Example 3
(I) paternity determination
And (3) counting the allelic gene factors (Na), the average expected value heterozygosity (He) and the Polymorphic Information Content (PIC) of each microsatellite locus by using Cervus3.0 software. And calculating parameters such as exclusion rate of each locus on the basis of the above parameters.
In the present invention, there are two cases of the exclusion probability of paternal identification:
the first condition is as follows: all genotypes of suspect individuals have been listed, but the parent-child relationships are unclear. In this case, the exclusion probability E for each microsatellite locusxAn algorithm according to the following formula of Jamieson (1965, 1994), PiAnd PjGene frequency for each locus allele:
Ex＝∑Pi 2(1-Pi)2-∑(PiPj)[4-3(Pi+Pj)]
case two: in the case of mother-known and father-unknown, the exclusion probability Ex for father identification of each site is calculated according to the algorithm of the following formula of Gaber et al:
Ex＝∑Pi 2(1-Pi)2+∑2(PiPj)(1-Pi-Pj)2
the joint exclusion rate E of a plurality of sites is calculated according to the method of the following formula such as Jamieson, i is the number of adopted microsatellite sites:
E＝1-(1-Ex1)(1-Ex2)(1-Ex3)…(1-Exi)
in the invention, the parents are known and the parents are unknown, the parent-child relationship is judged by applying Cervus3.0 software, the L OD value (natural logarithm value of likelihood) is calculated according to the genotype of the suspicious father, the parent-child relationship is judged according to the L OD score, the delta value is calculated:
n≥2，Δ＝LOD1-LOD2
n＝1，Δ＝LOD1
n is 0 and the value of Δ is undefined.
In the formula, L OD1And L OD2Is the L OD value of the two plaques most likely to be the true father.
In order to ensure that the microsatellite primer set used by the invention can provide guidance for paternity test of other fish-pond seals in China, when the exclusion rate of each microsatellite locus is calculated, statistics is carried out according to the collected data of all the seal-pond seals.
(II) identification result:
obtaining the genotype of each sample according to the allele condition of each microsatellite locus of the collected seal, calculating the exclusion rate of all loci (Table 3), wherein the accumulated first exclusion rate (CE-1P) is 0.8214, the accumulated second exclusion rate (CE-2P) and the accumulated biparental exclusion rate (CE-PP) are 0.9713 and 0.9977 respectively, and the seal mothers to be identified are known, which indicates that the used microsatellite marker can be effectively used for paternal identification of the seal.
The experimental population in the scheme is stored spotted seal, the female-child relationship of 10 young seals to be identified is recorded to be known, 17 heads are provided for the candidate male parent to be identified, the identification result is known that 10 heads of seal to be identified find the most similar parent under the confidence level of 80 percent, wherein 9 heads of the seal to be identified have confidence degree of more than 95 percent, the detailed relationship of the parent relationship is shown in the table 4 according to the L OD value calculated by the Parentage module and the Delta value standard, the filial seals with the numbers of 59361 and 20946 have a common father: 59331 which is the father relationship, and the filial seals with the numbers of 59347 are provided with 4 brothers which are the father parents, 59365, 59351 which are the father siblings respectively.
TABLE 4 results of paternity testing
Note: in the table, "+" indicates that the parent-child relationship is extremely obvious, and the confidence coefficient exceeds 95%; "+" indicates that the parent-child relationship is significant with a confidence level of over 80%.
Claims (5)
1. The SSR primer group for paternity test of the parva comprises 15 pairs of primers, and the nucleotide sequence of the SSR primer group is shown as SEQ ID NO. 1-SEQ ID NO. 30.
2. Use of the primer set of claim 1 for paternity testing of seals.
3. The use of the primer set of claim 1 for the preparation of a seal paternity test kit.
4. The use according to claim 3, wherein the kit comprises the full/partial primer set according to claim 1, 10 × PCR buffer, MgCl2dNTPs, Ex Taq DNA polymerase and double distilled water.
5. The method for identifying the parent of the plaque seal is characterized by comprising the following operation steps of extracting parent and filial generation sample DNA to be identified, labeling fluorescent dyes of FAM, HEX, TAM or ROX on a forward primer of a primer set according to claim 1, performing PCR amplification on the extracted sample DNA by using all/part of the primer set after the fluorescent dyes are labeled, performing capillary electrophoresis, performing genotyping according to an electrophoresis peak map result, counting allele factors (Na), average expected value heterozygosity (He), information content (PIC), accumulated first exclusion rate (CE-1P), accumulated second exclusion rate (CE-2P) and biparental accumulated exclusion rate (CE-PP) by using Cervus software, and displaying the result as "+" as a possible parent at a confidence level of 80% according to a value of L OD calculated by a Parentiage module and a Delta value standard, and displaying the result as "+" as a most possible parent at a confidence level of 95%.
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| CN103866004A (en) * | 2014-01-24 | 2014-06-18 | 中国水产科学研究院 | Molecular marker method for identifying fugu rubripes parentage as well as microsatellite and kit used for molecular marker method |
| CN105177143A (en) * | 2015-09-16 | 2015-12-23 | 中国科学院南海海洋研究所 | Method for identifying hippocampus kelloggi family by virtue of microsatelite |
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