CN112226519B - Sinocyclocheilus sinensis paternity test kit based on microsatellite marker and method thereof - Google Patents
Sinocyclocheilus sinensis paternity test kit based on microsatellite marker and method thereof Download PDFInfo
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Abstract
The invention discloses a sinobarbus sinobrei paternity test kit based on a microsatellite marker and a method thereof. Extracting genome DNA of an individual sample of the Sinocyclocheilus sinensis; and (3) PCR amplification: marking a fluorescent substance on the 5' end of a forward primer of each pair of primers of 15 pairs of microsatellite primers in a primer group for paternity test of sinobarbus sinensis, performing gradient PCR amplification on a DNA genome obtained by using each marked fluorescent primer pair in the primer group, performing capillary electrophoresis on an amplification product on a sequencer, reading the size of an allele of each individual, and obtaining genotyping data; paternity test and analysis of sinobarbus sinobrei family: and (3) carrying out parent and offspring data analysis based on the genotype data, and determining the paternity between the offspring and the parent according to the correlation between the offspring genotype and the parent genotype. The method is simple and rapid, can be popularized and applied in the aspects of germplasm identification and family genetic management of the sinocyclocheilus sinensis, and provides a new technical means for evaluating the proliferation and releasing effects.
Description
Technical Field
The invention relates to the technical field of fish breeding and molecular marking, and relates to a technology for paternity test of sinobarbus sinensis based on microsatellite markers, in particular to a sinobarbus sinensis paternity test kit and a microsatellite PCR (polymerase chain reaction) test method thereof.
Background
Sinocyclocheilus sinensis (Spinibarbus sinonsis) belongs to Cypriniformes, Cyprinidae and Sinocyclocheilus, is commonly called as 'green wave', is one of important economic fishes in an upstream trunk-tributary water system of Yangtze river, and has higher proportion of fishery harvests in a Yangtze river basin and a downstream river basin. The Sinocyclocheilus sinensis has large size, is lively, is fond of a torrent river beach with clear water quality, has quick growth and omnivorous property, and is deeply loved by people. However, in recent years, due to the influence of over-fishing, serious water pollution, habitat destruction caused by hydraulic engineering construction and operation and other factors, the wild resource of the sinocyclocheilus sinensis is increasingly exhausted, and the research on the conservation, recovery and proliferation of seed resources is very important.
The method for protecting the sinobarbus sinensis resource mainly adopts an artificial propagation mode, namely, the sinobarbus sinensis parents are bred, and after the sinobarbus sinensis parents are bred and grown out of seedlings in a large scale, the sinobarbus sinensis parents are artificially released to a river to supplement wild species of the sinobarbus sinensis, so that the wild resource amount is rapidly increased. Because the Sinocyclocheilus sinensis has large individuals, fast growth and wide sources of feeding baits, the Sinocyclocheilus sinensis is suitable for intensive culture in ponds, lakes, reservoirs, fish breeding stations and the like. For years, researches on nutrient characteristics, artificial culture, propagation technology and the like of the sinocyclocheilus sinensis boon are mature, large-scale adult fish production can be basically realized, and a foundation is laid for large-scale proliferation and releasing of seedlings. However, on the one hand, the continuous captive breeding of the sinobarbus sinobrevichensis breeding population is easy to cause inbreeding of parents, thereby causing the genetic diversity level of the population to be reduced; on the other hand, the proliferation and release performed by the multi-province and multi-institution for many years are from different families, how to quickly and effectively distinguish different sinobarbus families and sources and efficiently evaluate the proliferation and release effects of the sinobarbus, and the method becomes one of the main obstacles and important topics for the sinobarbus population resource recovery.
Microsatellites (microsatellites) are a class of simple repetitive DNA sequences with high variability that are widely present in eukaryotic genomes. The gene type detection method has the characteristics of separation according to a Mendelian mode, rich polymorphic information content, co-dominant inheritance and the like, can be used for simply, conveniently and quickly detecting the gene type, and is a widely applied genetic molecular marker. Meanwhile, the microsatellite marker is simple to use, stable in result and low in cost, so that the microsatellite marker has more applications in paternity and child identification. At present, no research report of applying microsatellite markers to paternity test of sinobarbus sinensis is found. The invention aims to establish a paternity test technology of the Sinocyclocheilus sinensis based on microsatellite markers, and provides a technical approach for the family management, population genetic management and proliferation and releasing effect evaluation of the Sinocyclocheilus sinensis.
Disclosure of Invention
The invention relates to a sinobarbus sinobrei paternity test kit based on a microsatellite marker and a microsatellite PCR identification method thereof.
The technical scheme adopted by the invention is as follows:
the method provides primer groups for parent-child identification of the sinocyclocheilus sinensis based on microsatellite markers, and the primer groups are respectively as follows: ZHDCB24, ZHDCB27, ZHDCB28, ZHDCB29, ZHDCB33, ZHDCB35, ZHDCB41, ZHDCB43, ZHDCB50, ZHDCB57, ZHDCB58, ZHDCB66, ZHDCB79, ZHDCB80 and ZHDCB88, wherein the sequences of the primers are shown in the following table:
according to the above scheme, the 5' end of the forward primer of each pair of primers is labeled with a fluorescent substance. The fluorescent substance may be selected from FAM fluorescent substance.
Provides a sinobarbus sinensis paternity test kit based on microsatellite markers, comprising a Taq enzyme PCR pre-mixed solution and the primer group for the sinobarbus sinensis paternity test, wherein the main components of the Taq enzyme PCR pre-mixed solution are 0.1U/mul Taq DNA polymerase, 2X PCR reaction buffer solution and 3mM MgCl2And 0.4mM dNTPs; the concentration of the sinobarbus sinensis genome DNA is 30-50 ng/uL; paternity test of sinobarbus sinensisThe primers used (15 primers) were: ZHDCB24, ZHDCB27, ZHDCB28, ZHDCB29, ZHDCB33, ZHDCB35, ZHDCB41, ZHDCB43, ZHDCB50, ZHDCB57, ZHDCB58, ZHDCB66, ZHDCB79, ZHDCB80, ZHDCB 88.
According to the scheme, the sinobarbus sinensis paternity test kit further comprises double distilled water.
A microsatellite PCR identification method is provided, which comprises the following steps:
(1) extracting genome DNA of individual sample of Sinocyclocheilus sinensis: obtaining the genome DNA of each individual by using a high-salt extraction method for the sinobarbus sinobrei parent fin and offspring fry individual, and storing for later use;
(2) and (3) PCR amplification: marking FAM fluorescent substances on 5' ends of forward primers of each pair of primers in 15 pairs of microsatellite primers ZHDCB24, ZHDCB27, ZHDCB28, ZHDCB29, ZHDCB33, ZHDCB35, ZHDCB41, ZHDCB43, ZHDCB50, ZHDCB57, ZHDCB58, ZHDCB66, ZHDCB79, ZHDCB80 and ZHDCB88 in a primer group for paternity test of the sinobarbus sinensis, performing gradient PCR amplification on the DNA genome obtained in the step (1) by using each marked fluorescent primer in the primer group, performing capillary electrophoresis on an amplification product on a sequencer, reading the size of an allele of each individual and obtaining genotyping data;
(3) paternity test and analysis of sinobarbus sinobrei family: and (4) converting the genotype data acquired in the step (3) into a format which can be read by biological software, analyzing parent and offspring data, and determining the paternity between the offspring and the parent according to the correlation between the offspring genotype and the parent genotype.
According to the scheme, the PCR amplification procedure comprises the following steps: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 62 ℃ → 52 ℃ gradient temperature (touch down) for 30s, extension at 72 ℃ for 30s, and performing 10 cycles; denaturation at 95 ℃ for 30s, annealing at 52 ℃ for 30s, and extension at 72 ℃ for 30s, for 22 cycles; finally, the extension is carried out for 20min at 72 ℃.
According to the scheme, the PCR reaction system is as follows: 5.0uL Taq enzyme PCR preliminary mixture (major components of 0.1U/. mu.l Taq DNA polymerase, 2 XPCR reaction buffer, 3mM MgCl)2And 0.4mM dNTPs), 1.0uL template genomic DNA (in concentration)Degree 30-50ng/uL), upstream and downstream amplification primers 0.4uL each (10 uM concentration), add 3.2uL ddH2O to total volume 10 uL.
Based on 15 carefully screened polymorphic microsatellite loci, the invention develops fluorescence labeling microsatellite primers (primers with stable amplification, clear result, strong specificity and high heterozygosity are screened out from a laboratory self-screening and separating sinobarbus sinensis microsatellite sequence) after obtaining clear and stable effects, and screens out 15 pairs: ZHDCB24, ZHDCB27, ZHDCB28, ZHDCB29, ZHDCB33, ZHDCB35, ZHDCB41, ZHDCB43, ZHDCB50, ZHDCB57, ZHDCB58, ZHDCB66, ZHDCB79, ZHDCB80 and ZHDCB88) to obtain microsatellite typing information of parents and filial generations, obtain genotype data of parents and filial generations in a family, finally carry out paternity analysis, analyze the correlation between parents and filial generations, thereby determining the paternity relationship between filial generations and parents.
Compared with the prior art, the invention has the following advantages:
(1) according to the invention, the fluorescence-labeled microsatellite loci are combined with the capillary electrophoresis technology, and paternity analysis is carried out on the sinocyclocheilus grahami family according to the microsatellite typing of high-throughput sequencing;
(2) the 15 polymorphic microsatellite loci screened by the invention are mainly loci with three-base or four-base repeating units, the amplification effect is stable and clear, and objective basis is provided for later data normalization.
(3) The establishment of the invention provides a new technical means for the germplasm identification, the family genetic management and the proliferation and releasing effect evaluation of the sinocyclocheilus sinensis.
Drawings
FIG. 1 is a sequencing diagram of a primer ZHDCB027 (sequentially comprising a parent F1 (genotype 203/207), a parent M1 (genotype 199/203) and a first filial generation Z1-1 (genotype 199/207)), wherein two alleles of the first filial generation are respectively from a male parent and a female parent and accord with Mendel's law of segregation
FIG. 2 is a sequencing diagram of primer ZHDCB043 (sequentially including parent F1 (genotype 228/232), parent M1 (genotype 228/232) and first filial generation Z1-1 (genotype 228/228)), wherein two alleles of the first filial generation are respectively from male parent and female parent and accord with Mendel's law of segregation
FIG. 3 is a sequencing diagram of primer ZHDCB080 (sequentially including parent F1 (genotype 274/274), parent M1 (genotype 274/278) and first filial generation Z1-1 (genotype 274/278)), wherein two alleles of the first filial generation are respectively from male parent and female parent and accord with Mendel's law of segregation
Detailed Description
Example (b):
the present invention will be further described with reference to examples.
A sinocyclocheilus sinensis parent-child identification method based on microsatellite markers comprises the following steps:
(1) extracting genome DNA of individual sample of Sinocyclocheilus sinensis: and (3) taking out the samples of the 6-tailed parent and 96-tailed offspring fries of the family obtained by artificial pairing, and extracting genome DNA according to a high-salt method. The method comprises the following specific steps: shearing about 0.5g of fin-shaped tissue (all the offspring fry are used as the fries), washing with double distilled water to preserve alcohol attached to the sample, sufficiently shearing, and putting into a 1.5mL centrifuge tube; adding 500 mu L of cell lysate and 6 mu L of proteinase K into a centrifuge tube, digesting in water bath at 55 ℃ overnight, and shaking for several times in the first 30 minutes; adding 500 μ L sodium chloride (4.5mol/L) and 300 μ L chloroform, mixing at medium speed in shaker for 20min, and centrifuging at 13000rpm and 10 deg.C for 10 min; transferring the supernatant to a new tube (about 850. mu.L), adding 595. mu.L of anhydrous isopropanol, mixing uniformly at medium speed in a shaker for 20 minutes, centrifuging at 13000rpm at 10 ℃ for 10 minutes, and pouring off the supernatant; adding 500 μ L of 75% ethanol, digesting in 55 deg.C water bath for 5min, centrifuging at 13000rpm at 10 deg.C for 20min, and removing supernatant; and (3) placing the centrifuge tube in a super-clean workbench, drying for 1 hour, adding 50-100 mu L of TE 8.0, dissolving DNA overnight at 4 ℃, and placing the centrifuge tube in a refrigerator at-20 ℃ for later use.
(2) Screening for polymorphic microsatellite loci: according to the sino-barbus-cubeba microsatellite sequence of laboratory self-screening separation, design and synthesize series upper and lower stream primer to respectively 8 sino-barbus-cubeba individuals (the genomic DNA who draws in step (1)) carry out PCR amplification, select that the amplification is stable, the result is clear, the specificity is strong, primer that the heterozygosity is high. The PCR reaction system is as follows: 5.0uL 2 XPower Taq PCR Pre-PCRThe mixture (major components of 0.1U/. mu.l Taq DNA polymerase, 2 XPCR reaction buffer, 3mM MgCl)20.4mM dNTPs), 1.0uL template genomic DNA (concentration 30-50ng/uL), 0.4uL each of the upstream and downstream amplification primers (concentration 10uM), and 3.2uL ddH2O to a total volume of 10 uL. The PCR amplification procedure was: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 62 ℃ → 52 ℃ gradient temperature (touch down) for 30s, extension at 72 ℃ for 30s, and performing 10 cycles; denaturation at 95 ℃ for 30s, annealing at 52 ℃ for 30s, and extension at 72 ℃ for 30s, for 22 cycles; finally, the extension is carried out for another 20min at 72 ℃. After the obtained PCR product is quantitatively diluted, 1ul of the PCR diluted product is taken, 7ul of formamide (containing 4 per mill of internal fluorescence standard LIZ500) is added for denaturation, and then capillary fluorescence electrophoresis detection and genotyping are carried out on an ABI 3730xl DNA sequencer. The invention screens out 15 pairs of available sinobarbus sinensis microsatellite primers: ZHDCB24, ZHDCB27, ZHDCB28, ZHDCB29, ZHDCB33, ZHDCB35, ZHDCB41, ZHDCB43, ZHDCB50, ZHDCB57, ZHDCB58, ZHDCB66, ZHDCB79, ZHDCB80, ZHDCB 88. The primer sequence, the repeating unit and annealing denaturation information of the sinobarbus sinensis microsatellite are shown in table 1;
table 115 microsatellite locus information table
The invention specifically verifies each primer, and the result shows that each primer accords with Mendelian separation law, wherein: the specific analysis is carried out by taking primers ZHDCB027, ZHDCB043 and ZHDCB080 as examples as follows: FIG. 1 is a sequencing diagram of a primer ZHDCB027 (sequentially including a parent F1 (genotype 203/207), a parent M1 (genotype 199/203) and a first filial generation Z1-1 (genotype 199/207)), wherein two alleles of the first filial generation are respectively from a male parent and a female parent and accord with Mendel's law of segregation; FIG. 2 is a sequencing diagram of a primer ZHDCB043 (sequentially including a parent F1 (genotype is 228/232), a parent M1 (genotype is 228/232) and a first filial generation Z1-1 (genotype is 228/228)), wherein two alleles of the first filial generation are respectively from a male parent and a female parent and accord with Mendel's law of segregation; FIG. 3 is a sequencing diagram of primer ZHDCB080 (sequentially including a parent F1 (genotype 274/274), a parent M1 (genotype 274/278) and a first filial generation Z1-1 (genotype 274/278)), wherein two alleles of the first filial generation are respectively from a male parent and a female parent and accord with Mendelian segregation law.
(3) Carrying out PCR amplification by using a fluorescence-labeled microsatellite primer: labeling FAM fluorescent substance on the 5' end of the forward primer of each pair of primers in the 15 pairs of microsatellite primers in the step (2), and performing gradient PCR amplification on the DNA genome obtained in the step (1) by using the labeled fluorescent primers, wherein the PCR reaction system is as follows: 5.0uL of 2 XPower Taq PCR preliminary mixture (major components of 0.1U/. mu.l Taq DNA polymerase, 2 XPCR reaction buffer, 3mM MgCl2And 0.4mM dNTPs), 1.0uL template genomic DNA (concentration 30-50ng/uL), 0.4uL each of the upstream and downstream amplification primers (concentration 10uM), and 3.1uL ddH2O to a total volume of 10 uL. The PCR amplification procedure was: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 62 ℃ → 52 ℃ gradient temperature (touch down) for 30s, extension at 72 ℃ for 30s, and performing 10 cycles; denaturation at 95 ℃ for 30s, annealing at 52 ℃ for 30s, and extension at 72 ℃ for 30s, for 22 cycles; finally, the extension is carried out for another 20min at 72 ℃. After the obtained PCR product is quantitatively diluted, 1ul of the PCR diluted product is taken, 7ul of formamide (containing 4 per mill of fluorescence internal standard LIZ500) is added for denaturation, capillary fluorescence electrophoresis detection is carried out on an ABI 3730xl DNA sequencer, the size of the allele of each individual is read by software, and the original data of genotyping is obtained. Based on the typing results obtained by the sequencer, the size of the individual alleles was read using the software GeneMarker v 2.2.0(Holland and Parson, 2011) and manually corrected for the repeat unit composition of the microsatellites.
(4) Paternity test and analysis of sinobarbus sinobrei family: and (3) performing allele frequency calculation, simulation analysis and paternity analysis on the artificially corrected genotype data obtained in the step (3) on parent and offspring data by using software Cervus 3.0.7(Kalinowski et al, 2010), analyzing the correlation of the parent and the offspring data by using the genotype Likelihood (LOD) value of the offspring to be detected and the parent, and determining the relationship between the offspring to be detected and the parent at a 95% confidence level. The results show that the cumulative probability of exclusion of non-paternity (NE-1P) for 15 sites is 0.9682947 for the single parent when the parents are unknown; for another parent when the uniparental genotype is known, the cumulative probability of exclusion of non-paternity (NE-2P) for 15 sites is 0.99839823; for the parental combination when parents were unknown, the cumulative probability of exclusion of non-paternity (NE-PP) for 15 loci was 0.99997443. The genetic diversity and exclusion probability information of 15 microsatellite loci of Sinocyclocheilus sinensis are detailed in table 2. In order to ensure the accuracy of the identification result, the relationship can be confirmed only if the LOD value is greater than 0 and is consistent with the family record data. The result shows that 92 progeny individuals in 96 progeny confirm the paternity, and the identification accuracy is 95.83%. The results show that the paternity test kit and the paternity test method for the sinocyclocheilus sinensis based on the microsatellite markers can efficiently and quickly meet the requirements of paternity test analysis, germplasm management and proliferation and releasing effect evaluation of the sinocyclocheilus sinensis family.
TABLE 2 Sinocyclocheilus sinensis 15 microsatellite locus genetic diversity and non-paternity exclusion probability information
Note: NS indicates no significant deviation (P > 0.05).; indicates significant deviation (P < 0.05)
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Nucleotide and amino acid sequence listing of the specification
<110> institute of Water engineering ecology of national academy of sciences in Water conservancy department
<120> Sinocyclocheilus sinobrevicilii paternity test kit based on microsatellite markers and method thereof
<160> 30
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ZHDCB24 F
<400> 1
CTTGTCTCCT CGTTCCCTTG
<210> 2
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ZHDCB24 R
<400> 2
TTGATGCTTA TCAATGGCTC C
<210> 3
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ZHDCB27 F
<400> 3
CAGTGTTCTA TCTCATGGAC GG
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ZHDCB27 R
<400> 4
TTCATGTTTG GGGGAGAGAG
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ZHDCB28 F
<400> 5
CCAGCTAAAG CCAAATGAGG
<210> 6
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ZHDCB28 R
<400> 6
GCTGCATCCT GATCTGCAC
<210> 7
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ZHDCB29 F
<400> 7
ATTCTATGTC GACCCGAACG
<210> 8
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ZHDCB29 R
<400> 8
ACATCATCAG ACTAGCCCCG
<210> 9
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ZHDCB33 F
<400> 9
TCGAAAAATC GTGTCGTCAA
<210> 10
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ZHDCB33 R
<400> 10
TCGGCCTGGA TTTTGAATTA
<210> 11
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ZHDCB35 F
<400> 11
GCAGGATTAG TTTCGCTGTT G
<210> 12
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ZHDCB35 R
<400> 12
AACCGTAATC CAATAAGCTT GG
<210> 13
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ZHDCB41 F
<400> 13
TGGGCTGATC TCAGTCTCTG
<210> 14
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ZHDCB41 R
<400> 14
TGGACAAAAA CAAGGAGCTG
<210> 15
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ZHDCB43 F
<400> 15
CCCGGAGCTA ATTGTGGTAA
<210> 16
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ZHDCB43 R
<400> 16
AACACTCGAA GGTGATTGGG
<210> 17
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ZHDCB50 F
<400> 17
CTGCTTTGGC TCTTCATTCC
<210> 18
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ZHDCB50 R
<400> 18
CGTGCACTTC CAACATCATC
<210> 19
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ZHDCB57 F
<400> 19
TGCATGCAAT CATTCAGTAT CTC
<210> 20
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ZHDCB57 R
<400> 20
TCTAGCAATC CAAAATCACC A
<210> 21
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ZHDCB58 F
<400> 21
CTCATTCCTT GATGTCCCGT
<210> 22
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ZHDCB58 R
<400> 22
GGTTAACCCA TTTAACCCAG C
<210> 23
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ZHDCB66 F
<400> 23
TTCAGACAAG AATCAGACCA ACA
<210> 24
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ZHDCB66 R
<400> 24
GCAAACCTTT TGGGAAGGAT
<210> 25
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ZHDCB79 F
<400> 25
TAGTCACTGC AGCGTCCTG
<210> 26
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ZHDCB79 R
<400> 26
TTGAGCAGAA ATGTTGAGCG
<210> 27
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ZHDCB80 F
<400> 27
CCATATGTGA ATTATGTGGA TGTG
<210> 28
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ZHDCB80 R
<400> 28
GTGACCGAGT GCAGAAGTGA
<210> 29
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ZHDCB88 F
<400> 29
TGTTCCTCAC TGTTTGGCAG
<210> 30
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ZHDCB88 R
<400> 30
TCACTAACCA GGCATGCAAA
Claims (9)
1. A primer group for paternity test of sinobarbus sinensis based on microsatellite markers is characterized in that: the primer group comprises ZHDCB24, ZHDCB27, ZHDCB28, ZHDCB29, ZHDCB33, ZHDCB35, ZHDCB41, ZHDCB43, ZHDCB50, ZHDCB57, ZHDCB58, ZHDCB66, ZHDCB79, ZHDCB80 and ZHDCB88, and the sequence of each primer is as follows:
。
2. The primer set for parent-child identification of sinobarbus sinensis according to claim 1, wherein: the 5' -end of the forward primer of each pair is labeled with a fluorescent substance.
3. The utility model provides a sinobarbus sinensis parent-child appraisal kit based on microsatellite marker which characterized in that: comprises thatTaqThe primer set for parent-child identification of Sinocyclocheilus sinobreviciparous according to claim 1, wherein the primer set is prepared from a mixture of enzymatic PCR primersTaqThe major component in the enzyme PCR preliminary mixture was 0.1U/. mu.lTaqDNA polymerase, 2 XPCR reaction buffer, 3mM MgCl2And 0.4mM dNTPs; the primer sets for paternity test of sinobarbus sinensis are respectively as follows: ZHDCB24, ZHDCB27, ZHDCB28, ZHDCB29, ZHDCB33, ZHDCB35, ZHDCB41, ZHDCB43, ZHDCB50, ZHDCB57, ZHDCB58, ZHDCB66, ZHDCB79, ZHDCB80, ZHDCB 88.
4. The sinobarbus sinobrevicilus paternity test kit according to claim 3, characterized in that: also comprises genome DNA of the Sinocyclocheilus sinensis and double distilled water.
5. A microsatellite PCR identification method is characterized in that: the method comprises the following steps:
(1) extracting genome DNA of an individual sample of the Sinocyclocheilus sinensis;
(2) and (3) PCR amplification: labeling fluorescent substances on the 5' end of each pair of forward primers in 15 pairs of microsatellite primers ZHDCB24, ZHDCB27, ZHDCB28, ZHDCB29, ZHDCB33, ZHDCB35, ZHDCB41, ZHDCB43, ZHDCB50, ZHDCB57, ZHDCB58, ZHDCB66, ZHDCB79, ZHDCB80 and ZHDCB88 in the primer group for paternity test of the sinobarbus sinensis as claimed in claim 1, performing gradient PCR amplification on the DNA genome obtained in the step (1) by using each labeled fluorescent primer in the primer group, performing capillary electrophoresis on the amplification product on a sequencer, reading the size of the allele of each individual, and obtaining genotyping data;
(3) paternity test and analysis of sinobarbus sinobrei family: and (3) analyzing parent and offspring data based on the genotype data obtained in the step (2), and determining the paternity between the offspring and the parent according to the correlation between the offspring genotype and the parent genotype.
6. The method of claim 5, wherein: the PCR amplification procedure is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, gradient temperature annealing at 62 ℃ → 52 ℃ for 30s, and extension at 72 ℃ for 30s, for 10 cycles; denaturation at 95 ℃ for 30s, annealing at 52 ℃ for 30s, and extension at 72 ℃ for 30s, for 22 cycles; finally, the extension is carried out for another 20min at 72 ℃.
7. The method of claim 5, wherein: the PCR reaction system is as follows: 5.0 uLTaq enzyme PCR preliminary mixed solution, 1.0uL template genomic DNA, upstream and downstream amplification primers 0.4uL each, 10uM concentration, and 3.2uL ddH2O to the total volume of 10 uL;
the main components in the Taq enzyme PCR pre-mixed solution are 0.1U/. mu.l Taq DNA polymerase, 2X PCR reaction buffer solution and 3mM MgCl2And 0.4mM dNTPs.
8. The method of claim 7, wherein: the concentration of the sinobarbus sinensis genome DNA is 30-50ng/uL, and the upstream and downstream amplification primer concentration is 10 uM.
9. The method of claim 5, wherein: the step (1) is as follows: and (3) obtaining the genome DNA of each individual by using a high-salt extraction method for the sinobarbus sinobrei parent fin line and the offspring fry individual, and storing for later use.
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| CN106282365A (en) * | 2016-09-09 | 2017-01-04 | 广州大学 | A kind of test kit quickly light agnail different groups classified |
| CN106399530A (en) * | 2016-10-17 | 2017-02-15 | 华南师范大学 | Spinibarbus dneticulatus microsatellite family identification method |
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| CN106282365A (en) * | 2016-09-09 | 2017-01-04 | 广州大学 | A kind of test kit quickly light agnail different groups classified |
| CN106399530A (en) * | 2016-10-17 | 2017-02-15 | 华南师范大学 | Spinibarbus dneticulatus microsatellite family identification method |
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