CN103866004A - Molecular marker method for identifying fugu rubripes parentage as well as microsatellite and kit used for molecular marker method - Google Patents

Molecular marker method for identifying fugu rubripes parentage as well as microsatellite and kit used for molecular marker method Download PDF

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CN103866004A
CN103866004A CN201410033662.9A CN201410033662A CN103866004A CN 103866004 A CN103866004 A CN 103866004A CN 201410033662 A CN201410033662 A CN 201410033662A CN 103866004 A CN103866004 A CN 103866004A
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刘永新
周勤
张红涛
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BEIDAIHE CENTRAL EXPERIMENTAL STATION CHINESE ACADEMY OF FISHERY SCIENCES
China Aquatic Scientific Research Institute
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Abstract

The invention provides a method for identifying fugu rubripes parentage by using a molecular marker method as well as microsatellite primers and a kit used for the molecular marker method. Selected 44 microsatellite primers are stable in pure PCR amplification reaction, clear in amplified fragment and high in polymorphism. By using the technology, different families of mixed fugu rubripes individuals can be distinguished, so that different families of the fugu rubripes individuals can be quickly and accurately identified, and the technology has great application value for breed conservation, propagation and breeding selection of fugu rubripes.

Description

A kind of molecule marking method and micro-satellite and test kit of identifying Fugu rubripes (Temmincket Schlegel) parent child relationship
Technical field
The invention belongs to molecular marking technique field, relate to the method and micro-satellite primers and the test kit that utilize molecule marking method to identify the pure parent child relationship in red fin east.
Background technology
Red fin east pure (Takifugu rubripes) belongs to Osteichthyes (Osteichthyes), pure shape order (Tetraodontiformes), pure suborder (Tetraodontoidei), pure Superfamily (Tetraodntoidea), pure section (Tetraodontidae), sheerly (Takifugu) of east, be commonly called as " filefish ", mainly originate in Bohai and Yellow Seas and the East Sea of China, be mainly distributed in abroad the coastal and Korea peninsula of Japan.The pure fine and tender taste in red fin east, delicious flavour, high protein and low fat, contain abundant VITAMIN and trace element, have the good reputation of " king in fish ".Although red fin east is pure has a comparatively comprehensively good character, in present stage actual breeding process, can say without kind, about the selection breeding research that red fin east is pure also relatively less.Therefore,, by genetic improvement means, it is very important cultivating the dew kind in red fin east or the new lines that economic characters are good.
From current Research foundation analysis, improveing the pure Major Economic in red fin east, to adopt many familys to select breeding techniques are effective meanss comparatively.Many family selective breedings technology is mainly: set up multiple family full-sibses with male and female 1:1 combo, by cultivation contrast experiment, analyze the proterties such as the growth of each family, disease-resistant, degeneration-resistant, quality, for good character combination, select new variety or new lines that economic characters are better through too much.Cultivation to this experiment in, normally different familys are raised in tank independently, this just need to take breeding facility and the tank of large use.At present, also can be by individuality be carried out to electronic marker, then mix and raise in a large cement pit, to reduce the impact of environment on different familys, but also there is certain drawback in this method: carrying out electronic marker money at individuality still needs different familys to raise in independent tank 4-5 month, just can carry out electronic marker when whose body weight reaches 50g left and right.In addition the fry growth that, the fry of cultivation in independent tank cultivates in than great Chi is slow.In addition, the cost of individual electronic marker is also higher, and an average electronic marker board is wanted 20 yuan of left and right.
Therefore, in the situation that not affecting fish bulk-growth, search out a kind of precise Identification parent child relationship, distinguish fast and accurately different familys, will effectively promote the seed selection process of the dew kind in red fin east.
Summary of the invention
The present invention aims to provide the method and micro-satellite primers and the test kit that utilize molecule marking method to identify the pure parent child relationship in red fin east, contribute to fast, accurately to differentiate that the red fin east of different familys is pure, in the pure selection breeding in red fin east and hereditary conservation, have great using value.
One aspect of the present invention relates to a kind of micro-satellite primers of identifying the pure parent child relationship in red fin east, it is characterized in that, described primer is for the pure genomic different linkage groups in red fin east.
Preferably, described primer is selected from the 3-10 couple in following primer pair:
Figure BSA0000100743080000021
Figure BSA0000100743080000031
More preferably, described primer is made up of following primer pair:
Title Forward primer DNA sequence dna (5 '-3 ') Reverse primer DNA sequence dna (5 '-3 ')
f1360 ggacttcagctctggtcctg ggtgcgactgcttccatcta
f1407 caaagacgttcacccacaga accgtctctgcctttgacag
f333 tgtcctctggacctgtgttg ctcccacacatgaagacacg
f1752 ggatgtggaacaatctgctt gctgaagtcattcatgggaag
f1077 ctgaaagggaaaagcagcaa cacgtcagaagctgcgatta
The present invention relates to a kind of test kit of identifying the pure parent child relationship in red fin east on the other hand, comprises the micro-satellite primers of the pure parent child relationship in the red fin east of the evaluation described in first aspect present invention.
Further aspect of the present invention relates to a kind of molecule marking method of identifying the pure parent child relationship in red fin east, adopts following steps:
(1) select 44 pairs of special micro-satellite primers to carry out PCR reaction, the special micro-satellite primers title of this 44 couple and sequence list following (in table 1):
Table 144 is to micro-satellite primers
Figure BSA0000100743080000041
Figure BSA0000100743080000051
The selection of above-mentioned micro-satellite primers is based on following principle: for target sequence number of alleles many, there is height polymorphism, there is not each other linkage relationship, be distributed in multiple different linkage groups etc.
(2) gather the fin ray of family parent to be measured and filial generation, extract genomic dna.
Preferably, utilize TIANGEN marine animal genome DNA extracting reagent kit to carry out the extraction of genomic dna.
(3) be template with the genomic dna of step (2), the selected 44 pairs of micro-satellite primers of step (1) carry out respectively pcr amplification.
Preferably, in the time carrying out pcr amplification, PCR reaction cumulative volume is 25 μ L, comprising: 10 × Buffer2.5 μ L, Mg 2+(25mmol/L) 1 μ L, dNTPs (each 2mmol/L) 1 μ L, the each 1 μ L of forward and reverse primer (10 μ mol/L), masterplate DNA1 μ L (50ng/ μ L), Taq archaeal dna polymerase 1U, add appropriate ddH 2o.
Preferably, PCR response procedures is: 94 ℃ of denaturation 3min, and 30 circulations (72 ℃ are extended 30s for 94 ℃ of sex change 30s, annealing 30s), last 72 ℃ are extended 10min.
(4) pcr amplification product of step (3) is detected through 8% native polyacrylamide gel electrophoresis, after electrophoresis with 1% cma staining 10min, nitrite ion colour developing 10min.Gel is in the imaging of HP scanjet G4010 scanner.
(5), by the electrophoresis detection result digitized processing of PCR, analyze with genetic analysis software.
Preferably, electrophoretic band is carried out to digitized processing with the conventional genetic analysis software in this area and calculation formula, calculate the elimination factor (in table 2) of each microsatellite marker.According to elimination factor height, select the microsatellite marker (in table 3) of first 10 of sequence, the accumulation elimination factor scope of 3-10 microsatellite marker is 95.79%-99.99% (in table 4).Utilize 5 elimination factors preceding microsatellite marker that sorts to be used in combination, the parent child relationship that has completed family parent and filial generation is identified.
Preferably, in step (5), use gel analysis software Gel-Pro Analyzer4.5 and genetic analysis software CERVUS Version3.0 to analyze.
(6), according to the parent child relationship of parent and filial generation, just the pure family plot in Different Red fin east can be separated.
Title and the elimination factor of table 244 microsatellite marker
Title Elimination factor Linkage group Title Elimination factor Linkage group
f1176 0.542 1 f1407 0.647 1
f810 0.428 2 f791 0.298 2
f1064 0.054 3 f1055 0.307 3
f1732 0.359 4 f1012 0.537 4
f1752 0.62 5 f133 0.647 5
f1487 0.419 6 f112 0.595 6
f1062 0.566 7 f1408 0.467 7
f1102 0.535 8 f1316 0.527 8
f1202 0.366 9 f1077 0.615 9
f171 0.533 10 f1422 0.535 10
f663 0.358 11 f1641 0.533 11
f1367 0.565 12 f1061 0.324 12
f98 0.58 13 f809 0.563 13
f503 0.523 14 f573 0.56 14
f935 0.556 15 f637 0.563 15
f1235 0.534 16 f1680 0.306 16
f1326 0.565 17 f1066 0.611 17
f1356 0.531 18 f1385 0.575 18
f1129 0.384 19 f667 0.572 19
f1403 0.583 20 f665 0.598 20
f139 0.43 21 f1621 0.391 21
f840 0.358 22 f1360 0.662 22
0031title and the linkage group of front 10 microsatellite markers of table 3 elimination factor
Title Elimination factor Sequence Linkage group Title Elimination factor Sequence Linkage group
f1360 0.662 1 22 f1066 0.611 6 17
f1407 0.647 2 1 f665 0.598 7 20
f333 0.647 3 5 f112 0.595 8 6
f1752 0.62 4 5 f1403 0.583 9 20
f1077 0.615 5 9 f98 0.58 10 13
Table 4 microsatellite marker quantity and accumulation elimination factor
Site quantity Accumulation elimination factor Number of sites Accumulation elimination factor
3 95.79％ 7 99.90％
4 98.38％ 8 99.96％
5 99.38％ 9 99.98％
6 99.76％ 10 99.99％
The invention has the beneficial effects as follows:
Use this technology, the pure individuality in red fin east of the different familys that mix can be made a distinction, contribute to the pure conservation in red fin east, breeding combo and select breeding.The selected 44 pairs of micro-satellite primers of the present invention are stablized at the pure pcr amplification reaction in red fin east, amplified fragments is clear, polymorphism is high.
Accompanying drawing explanation
Fig. 1 is according to genetic distance, the sibship dendrogram of 5 familys;
Fig. 2 is according to genetic distance, the sibship dendrogram of 7 familys;
Fig. 3 is according to genetic distance, the sibship dendrogram of 8 familys.
Embodiment:
The present embodiment is invention preferred embodiment, and all its principles and basic structure are identical with the present invention, all belong in protection scope of the present invention.
Embodiment 1
A kind of molecule marking method of identifying the pure parent child relationship in red fin east of the present invention, adopts following technological step:
Pure 5 familys in red fin east totally 272 tail fishes, containing 5 couples of parents, adopt from center, China Aquatic Science Research Institute Bei Dai River experiment centre.Gather the belly fin ray of every tail fish, with TIANGEN marine animal genome DNA extracting reagent kit extraction genomic dna.Take got genomic dna as template, carry out respectively pcr amplification with the microsatellite marker of first 5 of elimination factor, PCR reaction cumulative volume is 25 μ L, comprising: 10 × Buffer2.5 μ L, Mg 2+(25mmol/L) 1 μ L, dNTPs (each 2mmol/L) 1 μ L, the each 1 μ L of forward and reverse trip primer (10 μ rnol/L), masterplate DNA1 μ L (50ng/ μ L), Taq archaeal dna polymerase 1U, add appropriate ddH 2o.PCR response procedures is: 94 ℃ of denaturation 3min, and 30 circulations (72 ℃ are extended 30s for 94 ℃ of sex change 30s, annealing 30s), last 72 ℃ are extended 10min.PCR product detects through 8% native polyacrylamide gel electrophoresis with amplified production, and after cma staining, colour developing, gel is in the imaging of HP scanjet G4010 scanner.By the electrophoresis detection result digitized processing of PCR, analyze with Gel-Pro Analyzer4.5 gel analysis software and CERVUS Version3.0, the parent child relationship that completes family filial generation and parent is identified, utilize the parent child relationship of parent and filial generation, distinguish the pure family in Different Red fin east (in table 5 and Fig. 1).
The paternity test result of table 55 family
Figure BSA0000100743080000101
Wherein microsatellite marker title and the sequence information of first 5 of elimination factor are as follows:
Title Forward primer DNA sequence dna (5 '-3 ') Reverse primer DNA sequence dna (5 '-3 ') The sequence of linkage group elimination factor
f1360 ggacttcagctctggtcctg ggtgcgactgcttccatcta 220.6621
f1407 caaagacgttcacccacaga accgtctctgcctttgacag 10.6472
f333 tgtcctctggacctgtgttg ctcccacacatgaagacacg 50.6473
f1752 ggatgtggaacaatctgctt gctgaagtcattcatgggaag 50.624
f1077 ctgaaagggaaaagcagcaa cacgtcagaagctgcgatta 90.6155
Embodiment 2
A kind of molecule marking method of identifying the pure parent child relationship in red fin east of the present invention, adopts following technological step:
Pure 7 familys in red fin east totally 313 tail fishes, containing 7 couples of parents, adopt from center, China Aquatic Science Research Institute Bei Dai River experiment centre.Gather the belly fin ray of every tail fish, with TIANGEN marine animal genome DNA extracting reagent kit extraction genomic dna.Take got genomic dna as template, carry out respectively pcr amplification with the microsatellite marker of first 5 of elimination factor, PCR reaction cumulative volume is 25 μ L, comprising: 10 × Buffer2.5 μ L, Mg 2+(25mmol/L) 1 μ L, dNTPs (each 2mmol/L) 1 μ L, the each 1 μ L of forward and reverse primer (10 μ mol/L), masterplate DNA1 μ L (50ng/ μ L), Taq archaeal dna polymerase 1U, add appropriate ddH 2o.PCR response procedures is: 94 ℃ of denaturation 3min, and 30 circulations (72 ℃ are extended 30s for 94 ℃ of sex change 30s, annealing 30s), last 72 ℃ are extended 10min.PCR product detects through 8% native polyacrylamide gel electrophoresis with amplified production, and after cma staining, colour developing, gel is in the imaging of HP scanjet G4010 scanner.By the electrophoresis detection result digitized processing of PCR, analyze with Gel-Pro Analyzer4.5 gel analysis software and CERVUS Version3.0, the parent child relationship that completes family filial generation and parent is identified, utilize the parent child relationship of parent and filial generation, distinguish the pure family in Different Red fin east (in table 6 and Fig. 2).
The paternity test result of table 67 family
Wherein microsatellite marker title and the sequence information of first 5 of elimination factor are as follows:
Title Forward primer DNA sequence dna (5 '-3 ') The sequence of reverse primer DNA sequence dna (5 '-3 ') linkage group elimination factor
0055?
f1360 ggacttcagctctggtcctg ggtgcgactgcttccatcta 220.6621
f1407 caaagacgttcacccaeaga accgtctctgcctttgacag 10.6472
f333 tgtcctctggaectgtgttg ctcccacacatgaagacacg 50.6473
f1752 ggatgtggaacaatctgctt gctgaagtcattcatgggaag 50.624
f1077 ctgaaagggaaaagcagcaa caegtcagaagctgcgatta 90.6155
Embodiment 3
A kind of molecule marking method of identifying the pure parent child relationship in red fin east of the present invention, adopts following technological step:
Pure 8 familys in red fin east totally 359 tail fishes, containing 8 couples of parents, adopt from center, China Aquatic Science Research Institute Bei Dai River experiment centre.Gather the belly fin ray of every tail fish, with TIANGEN marine animal genome DNA extracting reagent kit extraction genomic dna.Take got genomic dna as template, carry out respectively pcr amplification with the microsatellite marker of first 5 of elimination factor, PCR reaction cumulative volume is 25 μ L, comprising: 10 × Buffer2.5 μ L, Mg 2+(25mmol/L) 1 μ L, dNTPs (each 2mmol/L) 1 μ L, the each 1 μ L of forward and reverse primer (10 μ mol/L), masterplate DNA1 μ L (50ng/ μ L), Taq archaeal dna polymerase 1U, add appropriate ddH 2o.PCR response procedures is: 94 ℃ of denaturation 3min, and 30 circulations (72 ℃ are extended 30s for 94 ℃ of sex change 30s, annealing 30s), last 72 ℃ are extended 10min.PCR product detects through 8% native polyacrylamide gel electrophoresis with amplified production, and after cma staining, colour developing, gel is in the imaging of HP scanjet G4010 scanner.By the electrophoresis detection result digitized processing of PCR, analyze with Gel-Pro Analyzer4.5 gel analysis software and CERVUS Version3.0, the parent child relationship that completes family filial generation and parent is identified, utilize the parent child relationship of parent and filial generation, distinguish the pure family in Different Red fin east (in table 7 and Fig. 3).
The paternity test result of table 78 family
Figure BSA0000100743080000131
Wherein microsatellite marker title and the sequence information of first 5 of elimination factor are as follows:
Title Forward primer DNA sequence dna (5 '-3 ') Reverse primer DNA sequence dna (5 '-3 ') The sequence of linkage group elimination factor
f1360 ggacttcagctctggtcctg ggtgcgactgcttccatcta 220.6621
f1407 caaagacgttcacccacaga accgtctctgcctttgacag 10.6472
f333 tgtcctctggacctgtgttg ctcccacacatgaagacacg 50.6473
f1752 ggatgtggaacaatctgctt gctgaagtcattcatgggaag 50.624
f1077 ctgaaagggaaaagcagcaa cacgtcagaagctgcgatta 90.6155
Above embodiment is only unrestricted for the present invention is described, the present invention is also not limited only to above-mentioned giving an example, and all do not depart from technical scheme and the improvement thereof of the spirit and scope of the present invention, and it all should be encompassed in protection scope of the present invention.
Figure ISA0000100743100000021
Figure ISA0000100743100000031
Figure ISA0000100743100000041
Figure ISA0000100743100000051
Figure ISA0000100743100000061
Figure ISA0000100743100000071
Figure ISA0000100743100000081
Figure ISA0000100743100000091
Figure ISA0000100743100000101
Figure ISA0000100743100000111
Figure ISA0000100743100000121
Figure ISA0000100743100000131
Figure ISA0000100743100000141
Figure ISA0000100743100000151
Figure ISA0000100743100000161
Figure ISA0000100743100000171
Figure ISA0000100743100000181
Figure ISA0000100743100000191
Figure ISA0000100743100000201
Figure ISA0000100743100000211
Figure ISA0000100743100000221
Figure ISA0000100743100000241
Figure ISA0000100743100000251
Figure ISA0000100743100000271
Figure ISA0000100743100000281
Figure ISA0000100743100000291
Figure ISA0000100743100000301

Claims (10)

1. a micro-satellite primers of identifying the pure parent child relationship in red fin east, is characterized in that, described primer is for the pure genomic different linkage groups in red fin east.
2. micro-satellite primers according to claim 1, is characterized in that, described primer is selected from the 3-10 couple in following primer pair:
。
3. micro-satellite primers according to claim 1 and 2, is characterized in that, described primer is made up of following primer pair:
Title Forward primer DNA sequence dna (5 '-3 ') Reverse primer DNA sequence dna (5 '-3 ') f1360 ggacttcagctctggtcctg ggtgcgactgcttccatcta f1407 caaagacgttcacccacaga accgtctctgcctttgacag
f333 tgtcctctggacctgtgttg ctcccacacatgaagacacg f1752 ggatgtggaacaatctgctt gctgaagtcattcatgggaag f1077 ctgaaagggaaaagcagcaa cacgtcagaagctgcgaRa
。
4. a test kit of identifying the pure parent child relationship in red fin east, is characterized in that, comprises claim 1 -the micro-satellite primers of the pure parent child relationship in evaluation red fin east in 3 described in any one.
5. a molecule marking method of identifying the pure parent child relationship in red fin east is characterized in that adopting following steps:
(1) in option table 1,44 pairs of special micro-satellite primers carry out PCR reaction;
(2) gather the fin ray of different family parents and offspring individual, extract genomic dna;
(3) be template with the genomic dna of step (2), the selected 44 pairs of micro-satellite primers of step (1) carry out pcr amplification;
(4) the PCR product of step (3) is detected through 8% native polyacrylamide gel electrophoresis with amplified production, after cma staining, colour developing, gel is in the imaging of HP scanjet G4010 scanner;
(5), by the electrophoresis detection result digitized processing of PCR, analyze with genetic analysis software;
(6) obtain the elimination factor of each microsatellite marker, the accumulation elimination factor of 3-10 mark of statistics; Utilize the marker combination of first 5 of elimination factor to use, the parent child relationship that completes family filial generation and parent is identified;
(7) utilize the parent child relationship of parent and filial generation, just the pure family plot in Different Red fin east can be separated.
6. method according to claim 5, is characterized in that, in step (3), in the time carrying out pcr amplification, PCR reaction cumulative volume is 25 μ L, comprising: 10 × Buffer2.5 μ L, the Mg of 25mmol/L 2+1 μ L, the dNTPs1 μ L of each 2mmol/L, the each 1 μ L of forward and reverse primer of 10 μ mol/L, the masterplate DNA1 μ L of 50ng/ μ L, Taq archaeal dna polymerase 1U, adds appropriate ddH 2o.
7. method according to claim 5, is characterized in that, in step (3), PCR response procedures is: 94 ℃ of denaturation 3min, and 94 ℃ of sex change 30s, the 30s--72 ℃ of extension 30s that anneal carries out 30 circulations, and last 72 ℃ are extended 10min.
8. method according to claim 5, is characterized in that, in step (5), the genetic analysis software of use is Gel-Pro Analyzer4.5 gel analysis software and CERVUS Version3.0.
9. method according to claim 5, is characterized in that, in step (6), electrophoretic band is carried out to digitized processing with genetic analysis software, calculates the elimination factor of each microsatellite marker; According to elimination factor height, select the microsatellite marker of first 10 of sequence, the accumulation elimination factor scope of 3-10 microsatellite marker is 95.79%-99.99%; Utilize 5 elimination factors sort preceding microsatellite marker completed family parent and filial generation parent child relationship identify.
10. method according to claim 5, is characterized in that, in step (6), microsatellite marker title and sequence information that elimination factor is first 5 are as follows:
Title Forward primer DNA sequence dna (5 '-3 ') Reverse primer DNA sequence dna (5 '-3 ') Linkage group f1360 ggacttcagctctggtcctg ggtgcgactgcttccatcta 22 f1407 caaagacgttcacccacaga accgtctctgcctttgacag 1 f333 tgtcctctggacctgtgttg ctcccacacatgaagacacg 5 f1752 ggatgtggaacaatctgctt gctgaagtcattcatgggaag 5 f1077 ctgaaagggaaaagcagcaa cacgtcagaagctgcgatta 9
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