CN106755516B - Identify the reagent set and method of Beijing duck parent child relationship - Google Patents
Identify the reagent set and method of Beijing duck parent child relationship Download PDFInfo
- Publication number
- CN106755516B CN106755516B CN201710072905.3A CN201710072905A CN106755516B CN 106755516 B CN106755516 B CN 106755516B CN 201710072905 A CN201710072905 A CN 201710072905A CN 106755516 B CN106755516 B CN 106755516B
- Authority
- CN
- China
- Prior art keywords
- single stranded
- stranded dna
- beijing duck
- sequence
- parent
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses the reagent set and method of identification Beijing duck parent child relationship.Identification disclosed by the invention or the reagent set of auxiliary identification Beijing duck parent child relationship, are reagent set 1, reagent set 2 and reagent set 3；Reagent set 1 is made up of IAS05 P, IAS07 P and IAS11 P；Reagent set 2 is made up of reagent set 1 and reagent A；Reagent A is made up of IAS06 P and IAS08 P；Reagent set 3 is made up of reagent set 2 and reagent B；Reagent B is made up of IAS12 P, IAS13 P, IAS04 P, IAS03 P, IAS01 P and IAS14 P.It is demonstrated experimentally that the accumulation probability of exclusion of the reagent set identification Beijing duck of the present invention, up to more than 0.99, chaotic Beijing duck breeding pedigree occurs for the identification of complete, accurate pedigree and individual identification available for Beijing duck, recoverable.
Description
Technical field
The present invention relates in biological technical field, the reagent set and method of Beijing duck parent child relationship are identified.
Background technology
China is to carry out one of country that wild duck domestication is raised, duck and its processed side product duck neck, duck earliest in the world
Intestines, duck pawl etc. have very extensive mass foundation in China.Beijing duck is ground by Chinese Academy of Agricultural Sciences's Beijing animal and veterinary
The kind that Jiu Suo aquatic birds research department forms after many decades seed selection.
Accurate pedigree information is the basis that poultry genetic breeding can smoothly be carried out, and is the basic guarantor for obtaining genetic progress
Barrier.The pedigree information of mistake can influence the accuracy of genetic evaluation, reduce genetic progress, brought to breeding station and manufacturing enterprise huge
Big economic loss.In actual breeding work, collected from hatching egg, hatching, the raising of kind duck, the links such as assemble to family and all need
Manually to be operated and be recorded, cause pedigree inaccurate unavoidable because of mistake.Therefore, it is necessary to be reflected by parent-offspring
Surely the mistake of pedigree record is corrected, to obtain genetic progress as much as possible.
In recent years, Protocols in Molecular Biology is quickly grown, and AFLP, RAPD, SSR equimolecular mark are widely used in heredity
Diversity evaluation and paternity test.It is at most microsatellite marker to be applied at present in livestock and poultry parenthood determination field, and FAO is
Recommend the microsatellite marker system suitable for the main Application of Animal Genetic diversity such as pig, chicken, ox, sheep and paternity test.But use
In the microsatellite marker system still vacancy of duck paternity test.At present, NCBI websites only report hundreds of duck microsatellite markers,
And reason mostly cannot be used for paternity test to these marks because its stability is poor, polymorphism is low etc., and there is the 3-4 of application prospect
Base repeat high polymorphism microsatellite sequence then and its lack.Therefore, it is necessary to be built by excavating and screening microsatellite marker
The vertical paternity test system for being suitable for Beijing duck.
The content of the invention
The technical problems to be solved by the invention are how to identify the parent child relationship of Beijing duck.
In order to solve the above technical problems, present invention firstly provides identification or auxiliary to identify the complete of Beijing duck parent child relationship
Reagent.
Identification provided by the present invention or the reagent set of auxiliary identification Beijing duck parent child relationship, are reagent set 3, complete
Reagent 2 or reagent set 1；
The reagent set 1 is made up of the primer pair that title is respectively IAS05-P, IAS07-P and IAS11-P；
The single stranded DNA that the IAS05-P is respectively IAS05-F and IAS05-R by title forms；
The IAS05-F is following a1) to a4) any of single stranded DNA：
A1) the single stranded DNA in sequence table shown in sequence 1；
A2) in a1) 5 ' ends and/or 3 ' ends add the single stranded DNAs that one or several nucleotides obtain；
A3) and a1) or a2) single stranded DNA of the single stranded DNA with more than 85% homogeneity that limits；
A4) under strict conditions with a1) or a2) limit single stranded DNA hybridization single stranded DNA；
The IAS05-R is following b1) to b4) any of single stranded DNA：
B1) the single stranded DNA in sequence table shown in sequence 2；
B2) in b1) 5 ' ends and/or 3 ' ends add the single stranded DNAs that one or several nucleotides obtain；
B3) and b1) or b2) single stranded DNA of the single stranded DNA with more than 85% homogeneity that limits；
B4) under strict conditions with b1) or b2) limit single stranded DNA hybridization single stranded DNA；
The single stranded DNA that the IAS07-P is respectively IAS07-F and IAS07-R by title forms；
The IAS07-F is following c1) to c4) any of single stranded DNA：
C1) the single stranded DNA in sequence table shown in sequence 3；
C2) in c1) 5 ' ends and/or 3 ' ends add the single stranded DNAs that one or several nucleotides obtain；
C3) and c1) or c2) single stranded DNA of the single stranded DNA with more than 85% homogeneity that limits；
C4) under strict conditions with c1) or c2) limit single stranded DNA hybridization single stranded DNA；
The IAS07-R is following d1) to d4) any of single stranded DNA：
D1) the single stranded DNA in sequence table shown in sequence 4；
D2) in d1) 5 ' ends and/or 3 ' ends add the single stranded DNAs that one or several nucleotides obtain；
D3) and d1) or d2) single stranded DNA of the single stranded DNA with more than 85% homogeneity that limits；
D4) under strict conditions with d1) or d2) limit single stranded DNA hybridization single stranded DNA；
The single stranded DNA that the IAS11-P is respectively IAS11-F and IAS11-R by title forms；
The IAS11-F is following e1) to e4) any of single stranded DNA：
E1) the single stranded DNA in sequence table shown in sequence 5；
E2) in e1) 5 ' ends and/or 3 ' ends add the single stranded DNAs that one or several nucleotides obtain；
E3) and e1) or e2) single stranded DNA of the single stranded DNA with more than 85% homogeneity that limits；
E4) under strict conditions with e1) or e2) limit single stranded DNA hybridization single stranded DNA；
The IAS11-R is following f1) to f4) any of single stranded DNA：
F1) the single stranded DNA in sequence table shown in sequence 6；
F2) in f1) 5 ' ends and/or 3 ' ends add the single stranded DNAs that one or several nucleotides obtain；
F3) and f1) or f2) single stranded DNA of the single stranded DNA with more than 85% homogeneity that limits；
F4) under strict conditions with f1) or f2) limit single stranded DNA hybridization single stranded DNA；
The reagent set 2 is made up of the reagent set 1 and reagent A；
The reagent A is entitled IAS06-P primer pair and/or entitled IAS08-P primer pair；
The single stranded DNA that the IAS06-P is respectively IAS06-F and IAS06-R by title forms；
The IAS06-F is following g1) to g4) any of single stranded DNA：
G1) the single stranded DNA in sequence table shown in sequence 7；
G2) in g1) 5 ' ends and/or 3 ' ends add the single stranded DNAs that one or several nucleotides obtain；
G3) and g1) or g2) single stranded DNA of the single stranded DNA with more than 85% homogeneity that limits；
G4) under strict conditions with g1) or g2) limit single stranded DNA hybridization single stranded DNA；
The IAS06-R is following h1) to h4) any of single stranded DNA：
H1) the single stranded DNA in sequence table shown in sequence 8；
H2) in h1) 5 ' ends and/or 3 ' ends add the single stranded DNAs that one or several nucleotides obtain；
H3) and h1) or h2) single stranded DNA of the single stranded DNA with more than 85% homogeneity that limits；
H4) under strict conditions with h1) or h2) limit single stranded DNA hybridization single stranded DNA；
The single stranded DNA that the IAS08-P is respectively IAS08-F and IAS08-R by title forms；
The IAS08-F is following i1) to i4) any of single stranded DNA：
I1) the single stranded DNA in sequence table shown in sequence 9；
I2) in i1) 5 ' ends and/or 3 ' ends add the single stranded DNAs that one or several nucleotides obtain；
I3) and i1) or i2) single stranded DNA of the single stranded DNA with more than 85% homogeneity that limits；
I4) under strict conditions with i1) or i2) limit single stranded DNA hybridization single stranded DNA；
The IAS08-R is following j1) to j4) any of single stranded DNA：
J1) the single stranded DNA in sequence table shown in sequence 10；
J2) in j1) 5 ' ends and/or 3 ' ends add the single stranded DNAs that one or several nucleotides obtain；
J3) and j1) or j2) single stranded DNA of the single stranded DNA with more than 85% homogeneity that limits；
J4) under strict conditions with j1) or j2) limit single stranded DNA hybridization single stranded DNA；
The reagent set 3 is made up of the reagent set 2 and reagent B；
The reagent B is that title is respectively IAS12-P, IAS13-P, IAS04-P, IAS03-P, IAS01-P and IAS14-P
Primer pair in six kinds, it is wantonly five kinds, wantonly four kinds, wantonly three kinds, any two or it is any；
The single stranded DNA that the IAS12-P is respectively IAS12-F and IAS12-R by title forms；
The IAS12-F is following k1) to k4) any of single stranded DNA：
K1) the single stranded DNA in sequence table shown in sequence 11；
K2) in k1) 5 ' ends and/or 3 ' ends add the single stranded DNAs that one or several nucleotides obtain；
K3) and k1) or k2) single stranded DNA of the single stranded DNA with more than 85% homogeneity that limits；
K4) under strict conditions with k1) or k2) limit single stranded DNA hybridization single stranded DNA；
The IAS12-R is following l1) to l4) any of single stranded DNA：
L1) the single stranded DNA in sequence table shown in sequence 12；
L2) in l1) 5 ' ends and/or 3 ' ends add the single stranded DNAs that one or several nucleotides obtain；
L3) and l1) or l2) single stranded DNA of the single stranded DNA with more than 85% homogeneity that limits；
L4) under strict conditions with l1) or l2) limit single stranded DNA hybridization single stranded DNA；
The single stranded DNA that the IAS13-P is respectively IAS13-F and IAS13-R by title forms；
The IAS13-F is following m1) to m4) any of single stranded DNA：
M1) the single stranded DNA in sequence table shown in sequence 13；
M2) in m1) 5 ' ends and/or 3 ' ends add the single stranded DNAs that one or several nucleotides obtain；
M3) and m1) or m2) single stranded DNA of the single stranded DNA with more than 85% homogeneity that limits；
M4) under strict conditions with m1) or m2) limit single stranded DNA hybridization single stranded DNA；
The IAS13-R is following n1) to n4) any of single stranded DNA：
N1) the single stranded DNA in sequence table shown in sequence 14；
N2) in n1) 5 ' ends and/or 3 ' ends add the single stranded DNAs that one or several nucleotides obtain；
N3) and n1) or n2) single stranded DNA of the single stranded DNA with more than 85% homogeneity that limits；
N4) under strict conditions with n1) or n2) limit single stranded DNA hybridization single stranded DNA；
The single stranded DNA that the IAS04-P is respectively IAS04-F and IAS04-R by title forms；
The IAS04-F is following o1) to o4) any of single stranded DNA：
O1) the single stranded DNA in sequence table shown in sequence 15；
O2) in o1) 5 ' ends and/or 3 ' ends add the single stranded DNAs that one or several nucleotides obtain；
O3) and o1) or o2) single stranded DNA of the single stranded DNA with more than 85% homogeneity that limits；
O4) under strict conditions with o1) or o2) limit single stranded DNA hybridization single stranded DNA；
The IAS04-R is following p1) to p4) any of single stranded DNA：
P1) the single stranded DNA in sequence table shown in sequence 16；
P2) in p1) 5 ' ends and/or 3 ' ends add the single stranded DNAs that one or several nucleotides obtain；
P3) and p1) or p2) single stranded DNA of the single stranded DNA with more than 85% homogeneity that limits；
P4) under strict conditions with p1) or p2) limit single stranded DNA hybridization single stranded DNA；
The single stranded DNA that the IAS03-P is respectively IAS03-F and IAS03-R by title forms；
The IAS03-F is following q1) to q4) any of single stranded DNA：
Q1) the single stranded DNA in sequence table shown in sequence 17；
Q2) in q1) 5 ' ends and/or 3 ' ends add the single stranded DNAs that one or several nucleotides obtain；
Q3) and q1) or q2) single stranded DNA of the single stranded DNA with more than 85% homogeneity that limits；
Q4) under strict conditions with q1) or q2) limit single stranded DNA hybridization single stranded DNA；
The IAS03-R is following r1) to r4) any of single stranded DNA：
R1) the single stranded DNA in sequence table shown in sequence 18；
R2) in r1) 5 ' ends and/or 3 ' ends add the single stranded DNAs that one or several nucleotides obtain；
R3) and r1) or r2) single stranded DNA of the single stranded DNA with more than 85% homogeneity that limits；
R4) under strict conditions with r1) or r2) limit single stranded DNA hybridization single stranded DNA；
The single stranded DNA that the IAS01-P is respectively IAS01-F and IAS01-R by title forms；
The IAS01-F is following s1) to s4) any of single stranded DNA：
S1) the single stranded DNA in sequence table shown in sequence 19；
S2) in s1) 5 ' ends and/or 3 ' ends add the single stranded DNAs that one or several nucleotides obtain；
S3) and s1) or s2) single stranded DNA of the single stranded DNA with more than 85% homogeneity that limits；
S4) under strict conditions with s1) or s2) limit single stranded DNA hybridization single stranded DNA；
The IAS01-R is following t1) to t4) any of single stranded DNA：
T1) the single stranded DNA in sequence table shown in sequence 20；
T2) in t1) 5 ' ends and/or 3 ' ends add the single stranded DNAs that one or several nucleotides obtain；
T3) and t1) or t2) single stranded DNA of the single stranded DNA with more than 85% homogeneity that limits；
T4) under strict conditions with t1) or t2) limit single stranded DNA hybridization single stranded DNA；
The single stranded DNA that the IAS14-P is respectively IAS14-F and IAS14-R by title forms；
The IAS14-F is following u1) to u4) any of single stranded DNA：
U1) the single stranded DNA in sequence table shown in sequence 21；
U2) in u1) 5 ' ends and/or 3 ' ends add the single stranded DNAs that one or several nucleotides obtain；
U3) and u1) or u2) single stranded DNA of the single stranded DNA with more than 85% homogeneity that limits；
U4) under strict conditions with u1) or u2) limit single stranded DNA hybridization single stranded DNA；
The IAS14-R is following v1) to v4) any of single stranded DNA：
V1) the single stranded DNA in sequence table shown in sequence 22；
V2) in v1) 5 ' ends and/or 3 ' ends add the single stranded DNAs that one or several nucleotides obtain；
V3) and v1) or v2) single stranded DNA of the single stranded DNA with more than 85% homogeneity that limits；
V4) under strict conditions with v1) or v2) limit single stranded DNA hybridization single stranded DNA.
One or several nucleosides acid-specifics of addition can add one to five nucleotides.
Term " homogeneity " used herein refers to the sequence similarity with native sequence nucleic acid." homogeneity " includes and this hair
Any sequence has 75% or higher, or 85% or higher, or 90% or higher, or 95% or higher together in bright sequence 1-22
The nucleotide sequence of one property.Homogeneity can with the naked eye or computer software is evaluated.Using computer software, two or more
Homogeneity between individual sequence can use percentage (%) to represent, it can be used for evaluating the homogeneity between correlated series.
The stringent condition is in 2 × SSC, 0.1%SDS solution, hybridizes at 68 DEG C and washes film 2 times, every time
5min, and in 0.5 × SSC, 0.1%SDS solution, hybridize at 68 DEG C and wash film 2 times, each 15min；Or, 0.1 ×
SSPE (or 0.1 × SSC), 0.1%SDS solution in, hybridize under the conditions of 65 DEG C and wash film.
Above-mentioned more than 85% homogeneity, can be 85%, 90% or more than 95% homogeneity.
The primer pair of the reagent set of the identification or auxiliary identification Beijing duck parent child relationship can be marked by fluorescent material.
The IAS01-P, the IAS07-P, the IAS11-P and the IAS04-P can be marked by HEX.The IAS03-P, institute
Stating IAS06-P, the IAS08-P and the IAS05-P can be marked by FAM.The IAS13-P, the IAS12-P and described
IAS14-P can be marked by TAMRA.
Each primer pair of the reagent set can be used alone, and can also use together, no matter be used alone or one
Rise and use, the proportioning of each primer pair can determine according to specific needs.Two single stranded DNAs in each primer pair of reagent set
Mol ratio can be 1:1.Two single stranded DNAs of each primer pair or each primer pair can independent packaging.
In order to solve the above technical problems, present invention also offers identification or the system of auxiliary identification Beijing duck parent child relationship.
Identification provided by the present invention or the system of auxiliary identification Beijing duck parent child relationship, including the identification or auxiliary mirror
Determine the reagent set of Beijing duck parent child relationship.
The system can specifically be identified the reagent set and A1 and/or A2 of Beijing duck parent child relationship by the identification or auxiliary
Composition；The A1 is into the reagent and/or instrument needed for performing PCR amplification；The A2 is progress microsatellite marker Genotyping institute
The instrument and/or software and/or module needed.It is described enter performing PCR amplification needed for reagent concretely Beijing flower bud wound biotechnology
2 × Taq PCR MasterMix of Co., Ltd.The instrument carried out needed for microsatellite marker Genotyping can be ABI
PRISM3730XL genetic analyzers.The software carried out needed for microsatellite marker Genotyping can be GeneMapper V3.2
Software.
In order to solve the above technical problems, present invention also offers complete point that Beijing duck parent child relationship is identified in identification or auxiliary
Son mark.
It is provided by the present invention identification or auxiliary identification Beijing duck parent child relationship complete molecular labeling, be complete mark 3,
Complete mark 2 or complete mark 1；
The complete mark 1 is made up of the molecular labeling that title is respectively IAS05, IAS07 and IAS11；
The IAS05, the IAS07 and the IAS11 are respectively using Beijing Duck genome DNA as described in template utilization
IAS05-P, the IAS07-P and the IAS11-P enter the DNA molecular that performing PCR expands to obtain；
The complete mark 2 is made up of the complete mark 1 and complete mark A；
The complete mark A is entitled IAS06 molecular labeling and/or entitled IAS08 molecular labeling；
The IAS06 and IAS08 is respectively to utilize the IAS06-P and institute by template of Beijing Duck genome DNA
State IAS08-P and enter the DNA molecular that performing PCR expands to obtain；
The complete mark 3 is made up of the complete mark 2 and complete mark B；
The complete mark B is the molecule mark that title is respectively IAS12, IAS13, IAS04, IAS03, IAS01 and IAS14
Six kinds in note, it is wantonly five kinds, wantonly four kinds, wantonly three kinds, any two or it is any；
The IAS12, the IAS13, the IAS04, the IAS03, the IAS01 and the IAS14 be respectively with
Beijing duck genomic DNA, which is template, utilizes the IAS12-P, IAS13-P, the IAS04-P, the IAS03-P, described
The IAS01-P and IAS14-P enters the DNA molecular that performing PCR expands to obtain.
It is the IAS05, the IAS07, the IAS11, the IAS06, the IAS08, the IAS12, described
IAS13, the IAS04, the IAS03, the IAS01 and the IAS14 are microsatellite marker.
In order to solve the above technical problems, present invention also offers identification or the method for auxiliary identification Beijing duck parent child relationship.
Identification provided by the present invention or the method for auxiliary identification Beijing duck parent child relationship, including：Using it is described identification or
The reagent set of auxiliary identification Beijing duck parent child relationship detects the molecule of filial generation Beijing duck to be measured and parent's Beijing duck to be measured respectively
Mark, whether filial generation Beijing duck to be measured there is parent-offspring to close with parent's Beijing duck to be measured according to the molecular markers for identification
System；
The molecular labeling is the identification or the complete molecular labeling of auxiliary identification Beijing duck parent child relationship.
In actual applications, determine the molecular labeling be specially which mark when, can treated according to the molecular labeling
The accumulation probability of exclusion surveyed in Beijing duck colony determines, as long as the accumulation probability of exclusion of the molecular labeling meets to be more than or equal to
0.99 paternity test that can be used between further specific Beijing duck individual to be measured.
When identifying Beijing duck parent child relationship to be measured, (1) complete mark 1 as described in molecular labeling used is, and accumulate row
When being more than or equal to 0.99 except probability, then need to meet that molecular labeling used meets Mendelian inheritance and just can determine that described treat simultaneously
Surveying Beijing duck has parent child relationship, i.e.,：(1a) accumulation probability of exclusion is more than or equal to 0.99 and molecular labeling used meets Meng
During Dare heredity, the Beijing duck to be measured with or candidate with parent child relationship；(1b) accumulation probability of exclusion is more than or equal to 0.99
And molecular labeling used at least one when being unsatisfactory for Mendelian inheritance, the Beijing duck to be measured does not have or candidate does not have
Parent child relationship.
(2) if molecular labeling used is the complete mark 2, and when accumulating probability of exclusion and being more than or equal to 0.99, then need together
When meet that molecular labeling used complies fully with Mendelian inheritance and just can determine that the Beijing duck to be measured has parent child relationship, i.e.,：
(2a) accumulation probability of exclusion is more than or equal to 0.99 and molecular labeling used when meeting Mendelian inheritance, the Beijing duck to be measured
With or candidate with parent child relationship；(2b) accumulation probability of exclusion is more than or equal to 0.99 and molecular labeling used at least 1
When not meeting Mendelian inheritance, the Beijing duck to be measured does not have or candidate does not have parent child relationship.
(3) if molecular labeling used is the complete mark 3, and when accumulating probability of exclusion and being more than or equal to 0.99, then need together
When meet that molecular labeling used has and Mendelian inheritance is not met no more than 1 just can determine that the Beijing duck to be measured has parent-offspring
Relation, i.e.,：(3a) accumulation probability of exclusion is more than or equal to 0.99 and molecular labeling used has and do not meet Mendel less than or equal to 1
When hereditary, the Beijing duck to be measured with or candidate with parent child relationship；(3b) accumulation probability of exclusion is more than or equal to 0.99 and institute
Molecular labeling has when not meeting Mendelian inheritance more than or equal to 2, and the Beijing duck to be measured does not have or candidate does not have
Parent child relationship.
Above, the purpose of the number of molecular labeling used in increase is accumulation probability of exclusion is more than or equal to 0.99, tired
Determine whether the Beijing duck to be measured has parent child relationship according to (2a) and (2b) when product probability of exclusion is more than or equal to 0.99.
Above, in the number of molecular labeling used in further increase to further determine that whether the Beijing duck to be measured has
When having parent child relationship, the increased molecular labeling of institute can increase successively according to probability of exclusion descending order.
Increased molecular labeling can specifically increase successively according to the order of table 5, you can according to the IAS05 mark, institute
State IAS07 marks, IAS11 marks, IAS06 marks, IAS08 marks, IAS12 marks, the IAS13
The order that mark, IAS04 marks, IAS03 marks, IAS01 marks and the IAS14 are marked increases successively.
In one embodiment of the invention, Beijing duck to be measured is being detected with father and mother to be measured to the presence or absence of parent child relationship
When, the molecular labeling is the IAS05, the IAS07 and the IAS11 of the complete mark 1.
One of filial generation Beijing duck to be measured known to parent, identify whether parent's Beijing duck to be measured is the filial generation north to be measured
During another parent of capital duck, the molecular labeling is the IAS05 of the complete mark 2, the IAS07, the IAS11,
The IAS06 and IAS08.
A parent of filial generation Beijing duck to be measured it is unknown, identify whether parent's Beijing duck to be measured is the filial generation to be measured north
During another parent of capital duck, the molecular labeling is the IAS05 of the complete mark 3, the IAS07, the IAS11,
The IAS06, the IAS08, the IAS12, the IAS13, the IAS04, the IAS03, the IAS01 and described
IAS14。
In the above method, the filial generation Beijing duck to be measured and the molecular labeling of parent's Beijing duck to be measured of detecting can be detection institute
State the genotype or sequence of filial generation Beijing duck to be measured and molecular labeling described in parent's Beijing duck to be measured.
Whether the filial generation Beijing duck to be measured according to the molecular markers for identification has with parent's Beijing duck to be measured
There is the genotype that parent child relationship can be the molecular labeling according to the filial generation Beijing duck to be measured and parent's Beijing duck to be measured
Or whether filial generation Beijing duck to be measured described in Sequence Identification has parent child relationship with parent's Beijing duck to be measured.
Whether the filial generation Beijing duck to be measured according to the molecular markers for identification has with parent's Beijing duck to be measured
There is parent child relationship concretely according to the molecular labeling between the filial generation Beijing duck to be measured and parent's Beijing duck to be measured
Whether meet the accumulation probability of exclusion of mendelian inheritance and the molecular labeling, identify the filial generation Beijing duck to be measured with
Whether parent's Beijing duck to be measured has parent child relationship.
The IAS05, the IAS07 and the IAS11 and accumulation in the molecular labeling is the complete mark 1
When probability of exclusion is more than 0.99, at least one mark does not meet Mendelian inheritance in being marked such as this 3, then the parent to be measured
Be not or candidate be not the filial generation to be measured parent；As this 3 marks all meet Mendelian inheritance, then the parent to be measured is
Or the parent that candidate is the filial generation to be measured.
It is the IAS05, the IAS07 in the molecular labeling is the complete mark 2, the IAS11, described
The IAS06 and IAS08 and accumulation probability of exclusion be more than 0.99 when, as this 5 mark in there is more than or equal to 1 mark not meet
Mendelian inheritance, then the parent to be measured is not or candidate is not the parent of the filial generation to be measured；As 5 marks meet Meng De
You are hereditary, then the parent that the parent to be measured is or candidate is the filial generation to be measured.
It is the IAS05, the IAS07 in the molecular labeling is the complete mark 3, the IAS11, described
IAS06, the IAS08, the IAS12, the IAS13, the IAS04, the IAS03, the IAS01 and the IAS14
And when accumulating probability of exclusion more than 0.99, there are more than or equal to 2 marks not meet Mendelian inheritance in being marked such as this 11, then institute
State parent to be measured be not or candidate be not the filial generation to be measured parent；As thering are less than 2 marks not meet Meng in 11 marks
Dare heredity, the then parent that the parent to be measured is or candidate is the filial generation to be measured.
In the above method, point for detecting filial generation Beijing duck to be measured and parent's Beijing duck to be measured respectively using reagent set
Son mark, can be respectively to the filial generation Beijing duck to be measured and the gene of parent's Beijing duck to be measured using the reagent set
Group DNA enters molecular labeling described in performing PCR augmentation detection.
In the above method, when carrying out the PCR amplifications, the IAS05-P, the IAS07-P, the IAS11-P, institute
State the IAS06-P, IAS08-P, the IAS12-P, the IAS13-P, the IAS04-P, the IAS03-P, described
IAS01-P and the IAS14-P annealing temperature can be 55-60 DEG C.
In the above method, concretely 60 DEG C of the annealing temperature of the IAS05-P.
Concretely 58.5 DEG C of the annealing temperature of the IAS07-P.
Concretely 57.5 DEG C of the annealing temperature of the IAS11-P.
Concretely 58 DEG C of the annealing temperature of the IAS06-P.
Concretely 58.5 DEG C of the annealing temperature of the IAS08-P.
Concretely 57.5 DEG C of the annealing temperature of the IAS12-P.
Concretely 58 DEG C of the annealing temperature of the IAS13-P.
Concretely 57.5 DEG C of the annealing temperature of the IAS04-P.
Concretely 57.5 DEG C of the annealing temperature of the IAS03-P.
Concretely 58.5 DEG C of the annealing temperature of the IAS01-P.
Concretely 58.5 DEG C of the annealing temperature of the IAS14-P.
In the reaction system that each primer pair in using the reagent set carries out the PCR amplifications, each single stranded DNA
Concentration can be 0.4mM.
In order to solve the above technical problems, present invention also offers following any applications：
The application of X1, the reagent set in identification or auxiliary identification Beijing duck parent child relationship product is prepared；
The application of X2, the reagent set in identifying or aiding in identification Beijing duck parent child relationship；
The application of X3, the reagent set in Beijing duck breeding；
The application of X4, the system in identification or auxiliary identification Beijing duck parent child relationship product is prepared；
The application of X5, the system in identifying or aiding in identification Beijing duck parent child relationship；
The application of X6, the system in Beijing duck breeding；
The application of X7, the complete molecular labeling in identifying or aiding in identification Beijing duck parent child relationship；
The application of X8, the complete molecular labeling in Beijing duck breeding；
The application of X9, methods described in Beijing duck breeding.
In the present invention, after the evaluation by probability of exclusion, the reagent set, the system, the complete molecule mark
Note and methods described are also used equally for the duck colony of other kinds.
In the present invention, the probability of exclusion (Probability of paternity exclusion, PE) is that evaluation is lost
An important indicator of mark practical value size in paternity test is passed, is calculated using the gene frequency of each mark
The allele of candidate parent mark is not from natural father or the probability of exclusion of natural mother.The accumulation probability of exclusion
(Combined exclusion probabilities) can be calculated in accordance with the following methods with the probability of exclusion：
(1) when another parental information is unknown, exclusion filial generation is with the probability of exclusion P that candidate parent is parent child relationship：
The accumulation probability of exclusion CPE (1) of k microsatellite marker calculation formula is：
CPE (1)=1- (1-P1) (1-P2) (1-P3) ... (1-Pk), Pk is the probability of exclusion of k-th of microsatellite marker；
(2) when known to another parental information, exclusion filial generation is with the probability of exclusion R that candidate parent is parent child relationship：
The accumulation probability of exclusion CPE (2) of k microsatellite marker calculation formula is：
CPE (2)=1- (1-R1) (1-R2) (1-R3) ... (1-Rk), Rk is the probability of exclusion of k-th of microsatellite marker；
(3) the candidate father and mother of exclusion filial generation nothing to do with are to the probability of exclusion Q for being parent child relationship：
The accumulation probability of exclusion CPE (3) of k microsatellite marker calculation formula is：
CPE (3)=1- (1-Q1) (1-Q2) (1-Q3) ... (1-Qk), Qk is the probability of exclusion of k-th of microsatellite marker.
Wherein, n represents the number of alleles of each microsatellite marker, and pi represents the frequency of i-th of microsatellite marker.
It is demonstrated experimentally that the reagent set and complete molecule of identification provided by the invention or auxiliary identification Beijing duck parent child relationship
Mark can be used for identifying Beijing duck, and 11 mark polymorphisms in the complete molecular labeling are high, and not chain and fragment is big between mark
It is closely-spaced appropriate, it is easy to detect simultaneously.Using the present invention reagent set and complete molecular labeling detect Beijing duck to be measured with
Accumulation probability of exclusion when father and mother to be measured whether there is parent child relationship is up to 0.999999, in a parent of filial generation Beijing duck to be measured
Known to this, identify that accumulation probability of exclusion when whether parent's Beijing duck to be measured is another parent of the filial generation Beijing duck to be measured can
Up to 0.99974, a parent of filial generation Beijing duck to be measured it is unknown, identify whether parent's Beijing duck to be measured is the filial generation to be measured
Accumulation probability of exclusion during another parent of Beijing duck is up to 0.991419.The present invention also (is such as moved back by optimizing PCR amplification conditions
Fiery temperature) and PCR amplification system (such as primer concentration), establish the microsatellite based on above-mentioned reagent set Yu complete molecular labeling
Marker gene classifying method.The reagent set of the present invention can be used for complete, the accurate pedigree of Beijing duck with complete molecular labeling
Chaotic Beijing duck breeding pedigree occurs for identification and individual identification, recoverable, enters so as to improve breeding accuracy and accelerate breeding
Journey, has a good application prospect and economic benefit.
Brief description of the drawings
Fig. 1 is the genotyping of IAS07 marks and IAS08 marks.
Embodiment
The present invention is further described in detail with reference to embodiment, the embodiment provided is only for explaining
The bright present invention, the scope being not intended to be limiting of the invention.
Experimental method in following embodiments, it is conventional method unless otherwise specified.
Material used, reagent, instrument etc., unless otherwise specified, are commercially obtained in following embodiments.
The reagent set of embodiment 1, identification or auxiliary identification Beijing duck parent child relationship can be used to identify that Beijing duck parent-offspring reflects
It is fixed
First, identify or aid in the preparation of the reagent set of identification Beijing duck parent child relationship
The identification or the reagent set of auxiliary identification Beijing duck parent child relationship that the present embodiment provides, are reagent set 1, complete
Reagent 2 and reagent set 3；
Reagent set 1 is made up of the primer pair that title is respectively IAS05-P, IAS07-P and IAS11-P；IAS05-P is by name
Claim respectively IAS05-F and IAS05-R single stranded DNA composition；IAS07-P is by list that title is respectively IAS07-F and IAS07-R
Chain DNA forms；The single stranded DNA that IAS11-P is respectively IAS11-F and IAS11-R by title forms；
Reagent set 2 is made up of reagent set 1 and reagent A；Reagent A is respectively drawing for IAS06-P and IAS08-P by title
Thing is to composition；The single stranded DNA that IAS06-P is respectively IAS06-F and IAS06-R by title forms；IAS08-P is respectively by title
IAS08-F and IAS08-R single stranded DNA composition；
Reagent set 3 is made up of reagent set 2 and reagent B；Reagent B by title be respectively IAS12-P, IAS13-P,
IAS04-P, IAS03-P, IAS01-P and IAS14-P primer pair composition；IAS12-P by title be respectively IAS12-F and
IAS12-R single stranded DNA composition；The single stranded DNA that IAS13-P is respectively IAS13-F and IAS13-R by title forms；IAS04-P
It is respectively that IAS04-F and IAS04-R single stranded DNA forms by title；IAS03-P is respectively IAS03-F and IAS03-R by title
Single stranded DNA composition；The single stranded DNA that IAS01-P is respectively IAS01-F and IAS01-R by title forms；IAS14-P is by title
Respectively IAS14-F and IAS14-R single stranded DNA composition.
The PCR primer of each primer pair has different fragments length, in coloured differently body position, belong to 11 kinds it is different micro-
Satellite markers, be respectively designated as IAS05 mark, IAS07 mark, IAS11 mark, IAS06 mark, IAS08 mark,
IAS12 marks, IAS13 marks, IAS04 marks, IAS03 marks, IAS01 marks and IAS14 marks.Two lists in each primer pair
The mol ratio of chain DNA is 1:1, two equal independent packagings of single stranded DNA of each primer pair, each primer pair is by fluorescent material mark
Note, specifying information are as shown in table 1.
The specifying information of each primer pair of reagent set of table 1, identification or auxiliary identification Beijing duck parent child relationship
2nd, identify or aid in the probability of exclusion of the reagent set of identification Beijing duck parent child relationship
The base of 96 Beijing ducks of Institute of Animal Sciences, Chinese Academy of Agricultural Sciences's Beijing duck conservation field is extracted respectively
Because of a group DNA, enter performing PCR amplification using each primer pair and annealing temperature using genomic DNA as template, PCR amplifications are using by excellent
Reaction condition (table 3) after the reaction system (table 2) of change and optimization.By PCR primer through ABI PRISM3730XL genetic analyzers
After carrying out electrophoresis and being sequenced, using GeneMapper V3.2 softwares, after setting molecular weight standard band, according to genetic analyzer
The color of collected electrophoresis fluorescence signal and position, the genotype of each microsatellite marker is calculated automatically.Fig. 1 with IAS07 mark and
Exemplified by IAS08 marks, IAS07-P (FAM, fluorescence green) is labeled as heterozygote in the figure, and clip size is (254bp, 276bp)；
IAS08-P (HEX, fluorescent blue) is labeled as homozygote, and clip size is (157bp, 157bp).
Table 2, PCR reaction systems
Note：In table 2,2 × Taq PCR MasterMix are Beijing Lei Chuan bio tech ltd product, constituent
Including：0.1units/ml Taq DNA Polymerase (recombinant), 4mM MgCl2, dNTPs (dATP, dCTP,
DGTP and dTTP, every kind of dNTP concentration are 0.5mM), other stabilizers and reinforcing agent；Blue dyes.
Table 3, PCR reaction cycle conditions
The Population Genetics analysis of microsatellite marker：
Using 3.07 software statistics of Cervus, 11 microsatellite marker genetic polymorphism parameters, including number of alleles, sight
Survey heterozygosity, it is expected that heterozygosity and polymorphism information content, statistical result are shown in Table 4.11 microsatellite marker number of alleles 4~
13, equal apparent altitude polymorphism；The observation heterozygosity respectively marked it is expected heterozygosity 0.585 between 0.551~0.859
Between~0.856, and the observation heterozygosity of each microsatellite marker and it is expected that heterozygosity difference is little, 0.004~0.138 it
Between, illustrate that the allele distributions of each microsatellite marker are reasonable, can accurately reflect the genetic structure of colony.What is respectively marked is more
It is higher to illustrate that this 11 microsatellite markers have in Beijing duck is identified between 0.545~0.834 for polymorphism information content (PIC)
Application value.
The genetic polymorphism of 4,11 microsatellite markers of table
| Mark | Number of alleles | Observe heterozygosity | It is expected heterozygosity | Polymorphism information content |
| IAS01 is marked | 7 | 0.641 | 0.614 | 0.571 |
| IAS03 is marked | 4 | 0.615 | 0.647 | 0.582 |
| IAS04 is marked | 5 | 0.551 | 0.673 | 0.627 |
| IAS05 is marked | 13 | 0.718 | 0.856 | 0.834 |
| IAS14 is marked | 5 | 0.564 | 0.585 | 0.545 |
| IAS07 is marked | 8 | 0.808 | 0.838 | 0.811 |
| IAS08 is marked | 7 | 0.615 | 0.745 | 0.708 |
| IAS12 is marked | 5 | 0.769 | 0.738 | 0.691 |
| IAS11 is marked | 8 | 0.859 | 0.835 | 0.807 |
| IAS06 is marked | 8 | 0.756 | 0.764 | 0.722 |
| IAS13 is marked | 4 | 0.718 | 0.714 | 0.66 |
According to the gene frequency of each microsatellite marker, the probability of exclusion for detecting mark and accumulation probability of exclusion are calculated
(Combined exclusion probabilities).Probability of exclusion (Probability of paternity
Exclusion, PE) it is an important indicator for evaluating genetic marker practical value size in paternity test, it is to utilize each mark
The gene frequency of note is not from natural father or the row of natural mother to calculate the allele of candidate parent mark
Except probability.Probability of exclusion calculates with accumulation probability of exclusion according to following three kinds of situations：
(1) when another parental information is unknown, exclusion filial generation is with the probability of exclusion P that candidate parent is parent child relationship：
The accumulation probability of exclusion CPE (1) of k microsatellite marker calculation formula is：
CPE (1)=1- (1-P1) (1-P2) (1-P3) ... (1-Pk), Pk is the probability of exclusion of k-th of microsatellite marker；
(2) when known to another parental information, exclusion filial generation is with the probability of exclusion R that candidate parent is parent child relationship：
The accumulation probability of exclusion CPE (2) of k microsatellite marker calculation formula is：
CPE (2)=1- (1-R1) (1-R2) (1-R3) ... (1-Rk), Rk is the probability of exclusion of k-th of microsatellite marker；
(3) the candidate father and mother of exclusion filial generation nothing to do with are to the probability of exclusion Q for being parent child relationship：
The accumulation probability of exclusion CPE (3) of k microsatellite marker calculation formula is：
CPE (3)=1- (1-Q1) (1-Q2) (1-Q3) ... (1-Qk), Qk is the probability of exclusion of k-th of microsatellite marker.
Wherein, n represents the number of alleles of each microsatellite marker, and pi represents the frequency of i-th of microsatellite marker.
Paternity test is two philosophys based on Mendelian inheritance：Separation and independent assortment return rule.Two of filial generation
One, allele comes from father, and one comes from mother., can not if meeting mendel's law by interpretation of result
Exclusion has parent child relationship, otherwise can exclude its parent child relationship.According to mendel's law, the whole on offspring chromosome
Gene, half heredity is in its natural father, and half heredity is in its natural mother.Therefore, parent-offspring is carried out in application microsatellite marker
During identification, some marker genetype of (i) affirmative filial generation comes from one's own parent, and candidate parent does not carry this marker gene
Type, this has just run counter to mendel's law, can exclude the parent child relationship between filial generation and candidate parent；(ii) filial generation certainly
Some marker genetype come from one's own parent, and candidate parent also carries this marker genetype, then meets Mendelian inheritance and determine
Rule, therefore the affiliation between filial generation and candidate parent can not be excluded.
Each candidate parent is calculated in all microsatellite marker probability of exclusion (table 5), as shown in Table 5, the row of IAS05 marks
Except probability is up to 0.543, the probability of exclusion of IAS14, IAS01, IAS03 mark is relatively low.Excluded generally by calculating accumulation
Rate (table 6), as shown in Table 6, this 11 microsatellite markers under three kinds of different situations, accumulation probability of exclusion CPE (1), CPE (2),
CPE (3) is up to 0.9914,0.9997,0.999999 respectively, illustrates that this 11 microsatellite marker paternity test effect are high.
The probability of exclusion of 5,11 microsatellite markers of table
| Mark | P | R | Q |
| IAS05 is marked | 0.543 | 0.706 | 0.875 |
| IAS07 is marked | 0.496 | 0.667 | 0.842 |
| IAS11 is marked | 0.486 | 0.659 | 0.833 |
| IAS06 is marked | 0.365 | 0.543 | 0.728 |
| IAS08 is marked | 0.352 | 0.534 | 0.731 |
| IAS12 is marked | 0.326 | 0.504 | 0.689 |
| IAS13 is marked | 0.286 | 0.459 | 0.635 |
| IAS04 is marked | 0.262 | 0.439 | 0.63 |
| IAS03 is marked | 0.219 | 0.376 | 0.541 |
| IAS01 is marked | 0.215 | 0.388 | 0.579 |
| IAS14 is marked | 0.191 | 0.363 | 0.551 |
Table 6, microsatellite marker accumulation probability of exclusion
| Mark | CPE(1) | CPE(2) | CPE(3) |
| IAS05 is marked | 0.543 | 0.706 | 0.875 |
| IAS07 is marked | 0.769672 | 0.902098 | 0.98025 |
| IAS11 is marked | 0.881611 | 0.966615 | 0.996702 |
| IAS06 is marked | 0.924823 | 0.984743 | 0.999103 |
| IAS08 is marked | 0.951285 | 0.99289 | 0.999759 |
| IAS12 is marked | 0.967166 | 0.996474 | 0.999925 |
| IAS13 is marked | 0.976557 | 0.998092 | 0.999973 |
| IAS04 is marked | 0.982699 | 0.99893 | 0.99999 |
| IAS03 is marked | 0.986488 | 0.999332 | 0.999995 |
| IAS01 is marked | 0.989393 | 0.999591 | 0.999998 |
| IAS14 is marked | 0.991419 | 0.99974 | 0.999999 |
Note：In table 6, when the CPE (1) to be gone together with microsatellite marker is that another parental information is unknown, the line flag is with being somebody's turn to do
The accumulation probability of exclusion of overlay mark is marked, it 0.543,0.543 is that IAS05 is marked such as to be with the CPE (1) that goes together of IAS05 marks
Probability of exclusion when another parental information is unknown, with the CPE (1) that goes together of IAS12 marks be 0.967166,0.967166 for
IAS05 marks, IAS07 marks, IAS11 marks, IAS06 marks, IAS08 marks and IAS12 marks when another parental information is unknown
The accumulation probability of exclusion of note, the CPE (1) such as to be gone together with IAS14 marks are 0.991419,0.991419 in another parental information
The accumulation probability of exclusion of this 11 microsatellite markers when unknown.CPE (2) and CPE (3) same to CPE (1).
11 microsatellite markers are ranked up according to the height of single marking probability of exclusion and (are sequentially shown in Table 5), Ran Houhua
It is divided into the mark combination of different number of labels, respectively containing 1-11 mark.In actual applications, (1≤N≤11) used containing N are individual
Microsatellite marker in the microsatellite marker combination of mark is according to the forward N number of mark of probability of exclusion sequence title, to ensure
In the case that number of labels is certain, the accumulation probability of exclusion of maximum is obtained.
As shown in Table 6, under different application scene, the identification effect of microsatellite marker is different.Microsatellite marker number
Single parent accumulates probability of exclusion (the accumulation probability of exclusion when another parental information is unknown) more than 0.99 when mesh is 11, and for
Second parental information is known and the identification of candidate father and mother couple, then be only respectively necessary for preceding 5 microsatellite markers (i.e. IAS05 marks,
IAS07 marks, IAS11 marks, IAS06 marks and IAS08 marks) and preceding 3 microsatellite markers (i.e. IAS05 marks, IAS07
Mark and IAS11 marks), you can accumulation probability of exclusion is more than 0.99；Therefore, it is not low according to paternity test accumulation probability of exclusion
In 0.99 standard, to reduce testing cost, in practical application, the microsatellite marker of required selection is respectively：(1) when another
Whether when one parental information is unknown, it is that parent child relationship need to apply all 11 microsatellite markers to detect filial generation to be measured with parent to be measured
(marked by IAS05, IAS07 mark, IAS11 mark, IAS06 mark, IAS08 mark, IAS12 mark, IAS13 mark,
The complete mark 3 that IAS04 marks, IAS03 marks, IAS01 marks and IAS14 marks form)；(2) when another parental information
Whether when knowing, it is that parent child relationship needs 5 microsatellite markers before applying (to be marked by IAS05 to detect filial generation to be measured with parent to be measured
Note, IAS07 marks, IAS11 marks, IAS06 marks and IAS08 mark the complete mark 2 of composition)；(3) exclude filial generation to be measured with
Father and mother to be measured to whether be parent child relationship when, then 3 microsatellite markers (are marked, IAS07 marks by IAS05 before only needing application
The complete mark 1 of note and IAS11 mark compositions).
When specifically being detected, when another parental information is unknown, using the reagent set 3 in step 1 to parent to be measured
This is detected with progeny genome DNA to be measured, determines parent to be measured and 11 microsatellite markers in the complete mark of filial generation to be measured
Genotype, according to its genotype determine the parent to be measured whether be the filial generation to be measured parent：In 11 microsatellite markers
Have and do not meet Mendelian inheritance more than or equal to 2 marks, then the parent of the non-filial generation to be measured of the parent to be measured；Such as 11 microsatellites
There are less than 2 marks not meet Mendelian inheritance in mark, then the parent to be measured is the parent of the filial generation to be measured；
When known to another parental information, using the reagent set 2 in step 1 to parent to be measured and filial generation gene to be measured
Group DNA is detected, and the genotype of parent to be measured and complete this 5 microsatellite markers of mark 2 of filial generation to be measured is determined, according to its base
Because type determine the parent to be measured whether be the filial generation to be measured parent：As at least 1 mark is not met in 5 microsatellite markers
The parent of Mendelian inheritance, the then non-filial generation to be measured of the parent to be measured；Such as 5 microsatellite markers meet Mendelian inheritance, then
The parent to be measured is the parent of the filial generation to be measured；
When excluding the candidate father and mother of filial generation nothing to do with to being parent child relationship, using the reagent set 1 in step 1 to be measured
Each candidate parent and the progeny genome DNA to be measured of father and mother couple detects, and determines each candidate parent of father and mother couple to be measured
The genotype of this 3 microsatellite markers in mark 1 complete with filial generation to be measured, according to its genotype determine the father and mother to be measured to whether
For the parent of filial generation to be measured：For each parent to be measured of father and mother's centering to be measured, as having at least one in 3 microsatellite markers
Mark does not meet Mendelian inheritance, then the parent of the non-filial generation to be measured of parent to be measured of father and mother's centering to be measured；Such as 3 microsatellites
Mark all meets Mendelian inheritance, then the parent to be measured of father and mother's centering to be measured is the parent of filial generation to be measured.
3rd, the paternity test efficacy test of microsatellite marker combination
Cervus softwares can simulate (simulation) by parental right to assess confidence level.The purpose of simulation is to pass through ratio
The LOD value of relatively large random offspring, obtain substantial amounts of Delta distributions (simulation 10,000 time).By comparing most probable parent
Delta distributions and most probable parent are not real parents when (most-likely parent) is real parent (true parent)
The Delta distributions of this when, obtain Delta key values, if this key value is determined in particular trusted degree horizontal such as 95%, that
When analyzing the parental right relation of True Data, the LOD value of most probable parent more than Delta key values, its paternity identification can
Reliability is confidence level 95%.
Simulation parent is carried out to the genotyping result of 96 Beijing ducks of step 2 using 11 microsatellite markers above
Son identification, samples 10000 times.As a result show, when known to a parent, Delta key values are 0, and identification success rate reaches
99%, confidence level is more than 95%；When not having parental information, Delta key values are 1.05, and identification success rate reaches 87%, can
Reliability is more than 95%.
Randomly choosed from the Beijing duck colony of research institute of Institute of Animal Sciences, Chinese Academy of Agricultural Sciences Beijing duck conservation field
The accurate 6 couples of parent-offsprings of pedigree record to (E01 and D07, E03 and D03, E04 and D03, E06 and C02, E07 and D01, E09 with
A11, each parent-offspring are unknown to another parental information of the generation of neutrons) and 1 unrelated drake (F01), upper genome is extracted respectively
DNA, enter performing PCR amplification according to the method for embodiment 1 using the reagent set 3 of embodiment 1, in ABI PRISM3730XL heredity
Detected on analyzer, and determine the genotype of each microsatellite marker of each object.
By the analysis to their 11 microsatellite marker genotype, identify that the parent-offspring between them is closed using likelihood method
System.Qualification result is consistent with actual pedigree, and LOD value is from 3.64~8.32, and confidence level reaches 95% (table 7).And unrelated public affairs
When duck is as father candidate, LOD value is respectively less than 0, represents that it is unlikely to be the natural father of each filial generation.This result is further verified
Microsatellite marker combination can be accurately credible as the detection architecture of Beijing duck paternity test, testing result.
Table 7, LOD value
The reagent set of embodiment 2, identification or auxiliary identification Beijing duck parent child relationship is in Beijing duck parenthood determination
Application
3 are have selected from the Beijing duck colony of research institute of Institute of Animal Sciences, Chinese Academy of Agricultural Sciences Beijing duck conservation field
Beijing duck, numbering are respectively E03, E07 and C12, and it is the Beijing duck that numbering is C12 (public affairs) to have known Beijing duck that numbering is E07
Filial generation, the Beijing duck that numbering is E03 and E07 affinity-less relations (being unrelated father).Waited below using E03 and C12 as E07
Father is selected, the reagent set of embodiment 1 can be used to identify that Beijing duck parent child relationship is verified.
The genomic DNA of above-mentioned 3 Beijing ducks is extracted respectively, using the reagent set 3 of embodiment 1 according to embodiment 1
Method enters performing PCR amplification, is detected on ABI PRISM3730XL genetic analyzers, and determine each microsatellite marker of each sample
Genotype, it the results are shown in Table 8.
Table 8, testing result
In 11 detected microsatellite markers, in addition to IAS11 is marked, candidate's filial generation E07 and father candidate C12's
IAS01 marks, IAS03 marks, IAS04 marks, IAS05 marks, IAS14 marks, IAS07 marks, IAS08 marks, IAS12 marks
Note, IAS06 marks and IAS13 marks comply fully with Mendelian inheritance, according to foregoing criterion, E07 and father candidate C12
Parent child relationship be present；And E07 and father candidate E03 have 5 marks (IAS07, IAS08, IAS12, IAS13, IAS04)) it is not inconsistent
Mendelian inheritance is closed, therefore excludes filial generation E07 and father candidate E03 parent child relationship.The testing result and actual conditions are complete
It is complete consistent.
<110>Institute of Animal Sciences, Chinese Academy of Agricultural Sciences
<120>Identify the reagent set and method of Beijing duck parent child relationship
<160> 22
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 1
tctgtgtagt tgcggaatgc 20
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 2
aaagcatttt tagtgccccc 20
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 3
aattggctgt tcccaaagtg 20
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 4
tgcttcctca ctgcaacatc 20
<210> 5
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 5
agggcatttt gtgacgagac 20
<210> 6
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 6
cctgaagcgc ctattgaaac 20
<210> 7
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 7
aggcagaggt caactcctca 20
<210> 8
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 8
ccacctcact gaaagcaaca 20
<210> 9
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 9
tgggaatgga ggaacttgtc 20
<210> 10
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 10
ttggaccagg tgccttttag 20
<210> 11
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 11
ttgagggcat tgacttgttg 20
<210> 12
<211> 21
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 12
ccaacagaat cacaaagcac a 21
<210> 13
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 13
tccctgatct aaggtccctg 20
<210> 14
<211> 22
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 14
tcaaggcaac aaagaatcac tg 22
<210> 15
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 15
cagccaggat taaaaccaga 20
<210> 16
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 16
taggagatct gtcggccact 20
<210> 17
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 17
aaacactttg gggctttttg 20
<210> 18
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 18
ccacaccgat tttcatcaca 20
<210> 19
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 19
actagggaac aaggcagggt 20
<210> 20
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 20
ctctcccaca ccagaccaat 20
<210> 21
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 21
caactactcc gtggtcagca 20
<210> 22
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 22
agcaacgacc tctactccca 20
Claims (17)
1. identification or the reagent set of auxiliary identification Beijing duck parent child relationship, are reagent set 3, reagent set 2 or reagent set
1；
The reagent set 1 is made up of the primer pair that title is respectively IAS05-P, IAS07-P and IAS11-P；
The single stranded DNA that the IAS05-P is respectively IAS05-F and IAS05-R by title forms；
The IAS05-F is the single stranded DNA shown in sequence 1 in sequence table；
The IAS05-R is the single stranded DNA shown in sequence 2 in sequence table；
The single stranded DNA that the IAS07-P is respectively IAS07-F and IAS07-R by title forms；
The IAS07-F is the single stranded DNA shown in sequence 3 in sequence table；
The IAS07-R is the single stranded DNA shown in sequence 4 in sequence table；
The single stranded DNA that the IAS11-P is respectively IAS11-F and IAS11-R by title forms；
The IAS11-F is the single stranded DNA shown in sequence 5 in sequence table；
The IAS11-R is the single stranded DNA shown in sequence 6 in sequence table；
The reagent set 2 is made up of the reagent set 1 and reagent A；
The reagent A is entitled IAS06-P primer pair and/or entitled IAS08-P primer pair；
The single stranded DNA that the IAS06-P is respectively IAS06-F and IAS06-R by title forms；
The IAS06-F is the single stranded DNA as shown in sequence 7 in sequence table；
The IAS06-R is the single stranded DNA shown in sequence 8 in sequence table；
The single stranded DNA that the IAS08-P is respectively IAS08-F and IAS08-R by title forms；
The IAS08-F is the single stranded DNA shown in sequence 9 in sequence table；
The IAS08-R is the single stranded DNA shown in sequence 10 in sequence table；
The reagent set 3 is made up of the reagent set 2 and reagent B；
The reagent B is that title is respectively drawing for IAS12-P, IAS13-P, IAS04-P, IAS03-P, IAS01-P and IAS14-P
Six kinds of thing centering, it is wantonly five kinds, wantonly four kinds, wantonly three kinds, any two or it is any；
The single stranded DNA that the IAS12-P is respectively IAS12-F and IAS12-R by title forms；
The IAS12-F is the single stranded DNA shown in sequence 11 in sequence table；
The IAS12-R is the single stranded DNA shown in sequence 12 in sequence table；
The single stranded DNA that the IAS13-P is respectively IAS13-F and IAS13-R by title forms；
The IAS13-F is the single stranded DNA shown in sequence 13 in sequence table；
The IAS13-R is the single stranded DNA shown in sequence 14 in sequence table；
The single stranded DNA that the IAS04-P is respectively IAS04-F and IAS04-R by title forms；
The IAS04-F is the single stranded DNA shown in sequence 15 in sequence table；
The IAS04-R is the single stranded DNA shown in sequence 16 in sequence table；
The single stranded DNA that the IAS03-P is respectively IAS03-F and IAS03-R by title forms；
The IAS03-F is the single stranded DNA shown in sequence 17 in sequence table；
The IAS03-R is the single stranded DNA shown in sequence 18 in sequence table；
The single stranded DNA that the IAS01-P is respectively IAS01-F and IAS01-R by title forms；
The IAS01-F is the single stranded DNA shown in sequence 19 in sequence table；
The IAS01-R is the single stranded DNA shown in sequence 20 in sequence table；
The single stranded DNA that the IAS14-P is respectively IAS14-F and IAS14-R by title forms；
The IAS14-F is the single stranded DNA shown in sequence 21 in sequence table；
The IAS14-R is the single stranded DNA shown in sequence 22 in sequence table.
2. identification or the system of auxiliary identification Beijing duck parent child relationship, including the reagent set described in claim 1.
3. identification or the complete molecular labeling of auxiliary identification Beijing duck parent child relationship, are complete mark 3, complete mark 2 or complete
Mark 1；
The complete mark 1 is made up of the molecular labeling that title is respectively IAS05, IAS07 and IAS11；
The IAS05 expands to enter performing PCR using IAS05-P described in claim 1 as template using Beijing Duck genome DNA
The DNA molecular arrived；
The IAS07 expands to enter performing PCR using IAS07-P described in claim 1 as template using Beijing Duck genome DNA
The DNA molecular arrived；
The IAS11 expands to enter performing PCR using IAS11-P described in claim 1 as template using Beijing Duck genome DNA
The DNA molecular arrived；
The complete mark 2 is made up of the complete mark 1 and complete mark A；
The complete mark A is entitled IAS06 molecular labeling and/or entitled IAS08 molecular labeling；
The IAS06 expands to enter performing PCR using IAS06-P described in claim 1 as template using Beijing Duck genome DNA
The DNA molecular arrived；
The IAS08 expands to enter performing PCR using IAS08-P described in claim 1 as template using Beijing Duck genome DNA
The DNA molecular arrived；
The complete mark 3 is made up of the complete mark 2 and complete mark B；
The complete mark B is that title is respectively in IAS12, IAS13, IAS04, IAS03, IAS01 and IAS14 molecular labeling
Six kinds, it is wantonly five kinds, wantonly four kinds, wantonly three kinds, any two or it is any；
The IAS12 expands to enter performing PCR using IAS12-P described in claim 1 as template using Beijing Duck genome DNA
The DNA molecular arrived；
The IAS13 expands to enter performing PCR using IAS13-P described in claim 1 as template using Beijing Duck genome DNA
The DNA molecular arrived；
The IAS04 expands to enter performing PCR using IAS04-P described in claim 1 as template using Beijing Duck genome DNA
The DNA molecular arrived；
The IAS03 expands to enter performing PCR using IAS03-P described in claim 1 as template using Beijing Duck genome DNA
The DNA molecular arrived；
The IAS01 expands to enter performing PCR using IAS01-P described in claim 1 as template using Beijing Duck genome DNA
The DNA molecular arrived；
The IAS14 expands to enter performing PCR using IAS14-P described in claim 1 as template using Beijing Duck genome DNA
The DNA molecular arrived.
4. identification or the method for auxiliary identification Beijing duck parent child relationship, including：Detect filial generation north to be measured respectively using reagent set
Capital duck and the molecular labeling of parent's Beijing duck to be measured, filial generation Beijing duck to be measured is treated with described according to the molecular markers for identification
Survey whether parent's Beijing duck has parent child relationship；
The reagent set is the reagent set of the identification or auxiliary identification Beijing duck parent child relationship described in claim 1；
The molecular labeling is the complete molecular labeling of the identification or auxiliary identification Beijing duck parent child relationship described in claim 3.
5. according to the method for claim 4, it is characterised in that：The filial generation to be measured according to the molecular markers for identification
Beijing duck and parent's Beijing duck to be measured whether have parent child relationship be according to the filial generation Beijing duck to be measured with it is described to be measured
Filial generation Beijing duck to be measured described in the genotype or Sequence Identification of molecular labeling described in parent's Beijing duck and described parent Beijing to be measured
Whether duck has parent child relationship.
6. the method according to claim 4 or 5, it is characterised in that：It is described to detect filial generation to be measured respectively using reagent set
The molecular labeling of Beijing duck and parent's Beijing duck to be measured, for using the reagent set respectively to the filial generation Beijing duck to be measured and
The genomic DNA of parent's Beijing duck to be measured enters molecular labeling described in performing PCR augmentation detection.
7. according to the method for claim 6, it is characterised in that：It is the IAS05-P, described when carrying out PCR amplification
It is IAS07-P, the IAS11-P, the IAS06-P, the IAS08-P, the IAS12-P, the IAS13-P, described
IAS04-P, the IAS03-P, the IAS01-P and the IAS14-P annealing temperature are 55-60 DEG C.
8. according to the method for claim 7, it is characterised in that：When carrying out the PCR amplifications, the IAS05-P's moves back
Fiery temperature is 60 DEG C；
And/or the annealing temperature of the IAS07-P is 58.5 DEG C；
And/or the annealing temperature of the IAS11-P is 57.5 DEG C；
And/or the annealing temperature of the IAS06-P is 58 DEG C；
And/or the annealing temperature of the IAS08-P is 58.5 DEG C；
And/or the annealing temperature of the IAS12-P is 57.5 DEG C；
And/or the annealing temperature of the IAS13-P is 58 DEG C；
And/or the annealing temperature of the IAS04-P is 57.5 DEG C；
And/or the annealing temperature of the IAS03-P is 57.5 DEG C；
And/or the annealing temperature of the IAS01-P is 58.5 DEG C；
And/or the annealing temperature of the IAS14-P is 58.5 DEG C.
9. application of the reagent set in identification or auxiliary identification Beijing duck parent child relationship product is prepared described in claim 1.
10. application of the reagent set in identifying or aiding in identification Beijing duck parent child relationship described in claim 1.
11. application of the reagent set in Beijing duck breeding described in claim 1.
12. application of the system in identification or auxiliary identification Beijing duck parent child relationship product is prepared described in claim 2.
13. application of the system in identifying or aiding in identification Beijing duck parent child relationship described in claim 2.
14. application of the system in Beijing duck breeding described in claim 2.
15. application of the complete molecular labeling in identifying or aiding in identification Beijing duck parent child relationship described in claim 3.
16. application of the complete molecular labeling in Beijing duck breeding described in claim 3.
17. application of any described method in Beijing duck breeding in claim 4-8.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710072905.3A CN106755516B (en) | 2017-02-10 | 2017-02-10 | Identify the reagent set and method of Beijing duck parent child relationship |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710072905.3A CN106755516B (en) | 2017-02-10 | 2017-02-10 | Identify the reagent set and method of Beijing duck parent child relationship |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106755516A CN106755516A (en) | 2017-05-31 |
| CN106755516B true CN106755516B (en) | 2018-02-23 |
Family
ID=58955850
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710072905.3A Active CN106755516B (en) | 2017-02-10 | 2017-02-10 | Identify the reagent set and method of Beijing duck parent child relationship |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106755516B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108315436B (en) * | 2018-03-23 | 2023-01-31 | 黑龙江八一农垦大学 | Method applied to goose paternity test |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1179044C (en) * | 2001-02-15 | 2004-12-08 | 华中农业大学 | Hog microsatellite DNA mark suitable for use in classifying hog breeds |
| CN1858251A (en) * | 2006-03-10 | 2006-11-08 | 江苏省家禽科学研究所 | Fowl variaty SSR finger print determining technology |
| CN102465181A (en) * | 2010-11-19 | 2012-05-23 | 同济大学 | Paternity test method for goats and microsatellite primer and kit thereof |
-
2017
- 2017-02-10 CN CN201710072905.3A patent/CN106755516B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN106755516A (en) | 2017-05-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Hohenlohe et al. | Next‐generation RAD sequencing identifies thousands of SNPs for assessing hybridization between rainbow and westslope cutthroat trout | |
| CN110257547B (en) | Corn core SNP marker developed based on KASP technology and application thereof | |
| CN103866043B (en) | A kind ofly identify silver carp, flathead hybridization and the microsatellite marker of Introgression In Hatchery Stocks individuality and Auele Specific Primer and application | |
| CN106048042B (en) | For identifying single nucleotide polymorphism site, primer, kit and the application of Peach fruits color traits | |
| CN106191240B (en) | For identifying single nucleotide polymorphism site, primer, kit and the application of Peach fruits epidermal hair character | |
| CN105925721B (en) | For identifying single nucleotide polymorphism site, primer, kit and the application of Peach fruits epidermis coloring character | |
| CN108424958A (en) | A kind of relevant SNP marker of Larimichthys crocea genetic sex and its primer and application | |
| KR20210082127A (en) | Genetic marker for parentage and thereof in Limanda yokohamae | |
| CN109868323A (en) | A kind of kind duck egg laying performance associated gene SMAD3 method for screening molecular markers | |
| Bian et al. | Development of a multiplex PCR assay for parentage assignment of the redclaw crayfish (Cherax quadricarinatus) | |
| CN110452969A (en) | A kind of rat quality of heredity monitoring SNP marker classifying method and kit based on KASP | |
| CN104789648B (en) | Identify molecular labeling and its application of the section haplotypes of rice CMS restoring genes Rf 1 | |
| CN106755516B (en) | Identify the reagent set and method of Beijing duck parent child relationship | |
| Badr et al. | Molecular genetic diversity and conservation priorities of Egyptian rabbit breeds | |
| KR102226016B1 (en) | Marker for identification of chloroplast derived from Ilex integra and Primer set for identification of Ilex x wandoensis | |
| CN117089644A (en) | MNP (MNP) marking site for identification of arundo donax varieties, primer composition, kit and application of MNP marking site | |
| CN104342489A (en) | Method for detecting genotype of chicken beard | |
| KR102646426B1 (en) | Snp marker set for identifying cucurbita maxima cultivars and method for identifying cucurbita maxima cultivars using the same | |
| CN115961054A (en) | Genetic marker for south China tiger individual identification and/or paternity test and application | |
| CN111607654B (en) | Zhuwei satellite polymorphic site, screening method, primer pair combination and amplification method | |
| CN108416189A (en) | A kind of variety of crops Heterosis identification method based on molecular marking technique | |
| CN101016565A (en) | Method of identifying chicken variety by DNA strip shape coding | |
| CN109943639A (en) | For selecting hen to store up the SNP marker and its application of smart ability | |
| Shi et al. | Highly informative single-copy nuclear microsatellite DNA markers developed using an AFLP-SSR approach in black spruce (Picea mariana) and red spruce (P. rubens) | |
| KR20110030808A (en) | Microsatellite marker and methods for identification of dogs |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |