CN108642187A - Inbred male Macrobrachium rosenbergii analysis of genetic diversity method - Google Patents
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Abstract
The invention discloses inbred male Macrobrachium rosenbergii analysis of genetic diversity methods, include the following steps：Step 1, the inbred male Macrobrachium rosenbergii sample for acquiring various trait, and extract DNA；Step 2, selection microsatellite marker combination, and design Specific PCR primers group and expanded；Step 3, evaluation PCR product number, observe allelic gene typing and size；Number of alleles A, the allele of each male shrimp of step 4, record replicate number G, observation heterozygosity Ho, it is expected heterozygosity He, the precision runout value P based on Hardy's Weinberg equilibrium law, and analyze microsatellite locus difference；Gene flow Nm is calculated according to population gene frequency parameter；Gene spacing is measured with to gene Nei Y-factor method Ys.The present invention is based on microsatellite analysis methods, provide inbred male Macrobrachium rosenbergii analysis of genetic diversity method, are very suitable for studying different close relative's hero shrimp group genetic structures, are the high-quality male parent of Macrobrachium rosenbergii.
Description
Technical field
The present invention relates to gene engineering technology field more particularly to inbred male Macrobrachium rosenbergii analysis of genetic diversity
Method.
Background technology
Macrobrachium rosenbergii (Macrobrachium rosenbergii, De Man 1879) is a kind of large-scale fresh water shrimp, because of it
Wider change of water quality bear amplitude, easily cultivation, the features such as meat is soft, commercial value is high be increasingly becoming China, Southeast Asia or even
The important aquaculture kind in the world.Currently, growing Macrobrachium rosenbergii yield be still difficult to meet the consumption being gradually increasing and
The market demand, so the factor of limitation Macrobrachium rosenbergii output increased need to further be broken through to greatly improve yield.Due to male shrimp compared with
Female growth is fast, ripe head is big, cultivation quantum of output is high [1], therefore, mainly solves causing property in seed rearing and cultivation
Other and individual difference limiting factor.
People are to the just research of Macrobrachium rosenbergii genetic diversity, such as different groups analysis of genetic diversity.Different shape
Type hero shrimp has following reason：(1) the individual internal factor such as hereditary difference, abnormal age；(2) space and resource-constrained etc.
Environmental factor causes to compete；(3) social factor inside the populations such as Population Position grade, manor.In recent years, the microsatellite modern times point
Sub- labelling technique is widely used in Biology Breeding due to can solve subtle hereditary difference that equipotential zymotechnic cannot be found and heredity is ground
Study carefully, has many advantages, such as that polymorphism height, codominant inheritance, repeatability is high and easily detects.Currently, Macrobrachium rosenbergii is autonomous or artificial
Long-term inbred results in germplasm degeneration, and an urgent demand carries out identification differentiation to it.Along with Macrobrachium rosenbergii male shape is lost
Pass uncontrollable, it is insufficient to cause paternal inheritance difference assessment accuracy of electronic, has seriously affected fine-variety breeding, therefore utilize microsatellite mark
The individual growth genetic diversity that note technology finds out inbred morphotype hero shrimp helps to solve the realistic problem.
Currently, domestic and foreign scholars study Macrobrachium rosenbergii genetic diversity using microsatellite molecular marker technology,
But the genetic diversity of main research different population, heritage information obtains between the same gender of natural or artificial inbred acquisition
Know less.Wild Macrobrachium rosenbergii kind genetic diversity is studied using 6 microsatellite locus to find, the average equipotential base in each site
Factor is in great diversity, however, only obtaining this kind of gene diversity by the number of alleles of each microsatellite markers
It is limited.
Invention content
In order to solve the shortcoming present in above-mentioned technology, the present invention provides the Macrobrachium rosenbergii heredity of inbred male
Diversity analysis method.
In order to solve the above technical problems, the technical solution adopted by the present invention is：
Inbred male Macrobrachium rosenbergii analysis of genetic diversity method, includes the following steps：
Step 1, the inbred male Macrobrachium rosenbergii sample for acquiring various trait, and extract DNA；
Step 2, selection microsatellite marker combination, and design Specific PCR primers group and expanded；
Step 3, evaluation PCR product number, observe allelic gene typing and size；
Number of alleles A, the allele of each male shrimp of step 4, record replicate number G, observation heterozygosity Ho, it is expected heterozygosis
He, the precision runout value P based on Hardy-Weinberg equilibrium law are spent, and analyzes microsatellite locus difference；According to population equipotential base
Because frequency parameter calculates gene flow Nm；Gene spacing is measured with to gene Nei Y-factor method Ys；
Wherein, the microsatellite marker combination in step 2, includes the complementary sequence of following microsatellite marker or its nucleotide sequence
Row：It is microsatellite marker 1, microsatellite marker 2, microsatellite marker 3, microsatellite marker 4, microsatellite marker 5, microsatellite marker 6, micro-
Satellite markers 7；Wherein,
The nucleotide sequence of the microsatellite marker 1 is as shown in SEQ ID NO.1；
The nucleotide sequence of the microsatellite marker 2 is as shown in SEQ ID NO.2；
The nucleotide sequence of the microsatellite marker 3 is as shown in SEQ ID NO.3；
The nucleotide sequence of the microsatellite marker 4 is as shown in SEQ ID NO.4；
The nucleotide sequence of the microsatellite marker 5 is as shown in SEQ ID NO.5；
The nucleotide sequence of the microsatellite marker 6 is as shown in SEQ ID NO.6；
The nucleotide sequence of the microsatellite marker 7 is as shown in SEQ ID NO.7.
Further, the Specific PCR primers group in the step 2, including：
The specific primer pair of microsatellite marker 1, nucleotide sequence is as shown in SEQ ID NO.8 and SEQ ID NO.9；
The specific primer pair of microsatellite marker 2, nucleotide sequence such as SEQ ID NO.10 and SEQ ID NO.11 institutes
Show；
The specific primer pair of microsatellite marker 3, nucleotide sequence such as SEQ ID NO.12 and SEQ ID NO.13 institutes
Show；
The specific primer pair of microsatellite marker 4, nucleotide sequence such as SEQ ID NO.14 and SEQ ID NO.15 institutes
Show；
The specific primer pair of microsatellite marker 5, nucleotide sequence such as SEQ ID NO.16 and SEQ ID NO.17 institutes
Show；
The specific primer pair of microsatellite marker 6, nucleotide sequence such as SEQ ID NO.18 and SEQ ID NO.19 institutes
Show；
The specific primer pair of microsatellite marker 7, nucleotide sequence such as SEQ ID NO.20 and SEQ ID NO.21 institutes
Show.
Further, the character of the inbred male Macrobrachium rosenbergii sample includes the long arm type of blue, orange long arm type
With small idiotype.
Further, genetic variation and genetic differentiation degree uses F statistical analyses, such as population gene frequency parameter FIS、FITAnd FST,
Wherein FISFor the average coefficient of inbreeding, the F of local groupITFor the average coefficient of inbreeding and F of entire groupSTFor with having affiliation
The average coefficient of inbreeding between Fang Qunti calculates gene flow Nm according to Wright methods, and calculation formula is：Nm=FST 0.25(1-
FST)/FST.FIS。
The present invention is based on microsatellite analysis methods, provide inbred male Macrobrachium rosenbergii analysis of genetic diversity side
Method, the results show that all microsatellite locus show the difference in height opposite sex, microsatellite marker combination is easy to identify, height is polymorphic, non-
It is adapted to study different close relative's hero shrimp group genetic structures, basic hereditary information is provided for the high-quality male parent selection and breeding of Macrobrachium rosenbergii,
Also there is definite meaning to the male Macrobrachium rosenbergii genetic development of regulation and control simultaneously, provide hereditary information for male parent selection and breeding, improve seed
Quality provides basis.
Specific implementation mode
The present invention will be further described in detail with reference to the specific embodiments.
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
【Embodiment 1】
1.1 sample collection
Guangxi population inbred bull Macrobrachium rosenbergii, 200 tails, by national (Nanning) Macrobrachium rosenbergii breeding
The heart provides, cultivation 4 months in the breeding central laboratory aquaculture pond (5 × 5 × 1.8m), based on color, behavior, growth characteristics and
Zigzag chela, point three kinds of long arm type (BCM) of blue, orange long arm type (OCM), small idiotype (SM) different shape type hero shrimps.3
Each 50 tail of morphotype, collects 150 tails altogether, BCM, OCM, SM weight be respectively 14.48 ± 1.05g, 10.04 ± 1.15g, 5.86 ±
0.66g。
1.2DNA extraction
Macrobrachium rosenbergii abdominal musculature extracting genome DNA (is purchased from U.S. according to Wizard genomic DNA purification kits
Promega companies of state) step extraction, using ultraviolet specrophotometer measure extracting solution 260nm and 280nm light absorption value
(OD), DNA concentration and purity are measured according to OD260nm/OD280nm ratios, with double steaming liquid constant volumes 1:40 to latter -20 DEG C preservations.
1.3PCR amplification
Prepared by PCR reaction solution, totally 50 μ L：10.0 μ L 5X PCR buffer, 5.0 μ L MgCl2(2.5mM), 1.0 μ L
DNTP (0.2mM), 2.0 μ L upstream and downstream primers (0.4 μM), the 24.75 sterile ddH of μ L2O2, 0.25 μ L Taq polymerases, 2.0 μ L
DNA profiling, sterile deionized water polishing.
The design of DAN micro-satellite primers is as shown in table 1.
The characteristic of the microsatellite locus of 1 Macrobrachium rosenbergii of table
F：Forward direction, R：Reversely
PCR response procedures are：95 DEG C of pre-degeneration 3min carry out 40 cycles, the reality of each pair of primer according to 95 DEG C of denaturation 30s
(48-60 DEG C) reaction 30s of border annealing temperature, 72 DEG C of extension 30s, last 72 DEG C of extensions 40s, 4 DEG C keep the temperature.
After pcr amplification product is detected through 2.5% agarose gel electrophoresis, red gel incarnadines, ultraviolet colour developing.Then
PCR product number is evaluated using polyacrylamide gel electrophoresis, the gel rubber system containing 1X tbe buffer liquids is added in reaction solution, in 75V items
Then swimming 8min under part is observed under gel imaging system (AlphaInnotech companies of the U.S.), extend DNA ladder type with 20bp
(Linza companies of the U.S.) is that reference standard ocular estimate observes allelic gene typing and size.
1.4 data analysis
Number of alleles (A), the allele of each male shrimp of record replicate number (G), observation heterozygosity (Ho), it is expected heterozygosis
(He), the precision runout value (P) based on Hardy-Weinberg equilibrium law are spent, and microsatellite position is analyzed using ARLEQUIN 3.11
Point difference.Genetic variation and genetic differentiation degree uses F statistical analyses, such as population gene frequency parameter (FIS, FIT, FST), according to Wright
Method calculates gene flow (Nm), formula：Nm=FST 0.25(1-FST)/FST.FIS.With measuring gene to gene Nei Y-factor method Ys
Away from, based on non-weighting pairing arithmetic mean method utilize POPGEN1.32 software building dendrograms.
【Embodiment 2】As a result with analysis
2.1 population genetic diversity
As shown in Table 2, other than the site of SM 5, other site hereditary information are in polymorphism, are wanted in all statistical analyses
Remove the site.The male shrimp species group of 3 kinds of morphotypes shows that high genetic difference, the number of alleles of BCM, OCM, SM are respectively
2.885,3.762 and 2.627, observation heterozygosity is respectively 0.5561,0.474 and 0.475.Hardy-Weinberg equilibrium law
Precision runout value (P) is assessed using Markov chain method, then by the multiple hypothesis experiment corrections of Bonferroni.The results show that
Most of site site significantly deviates.
2 three kinds of morphotype male Macrobrachium rosenbergii satellite site genetic diversities of table
G：Gene copy number, A：Number of alleles, Ho：Observe heterozygosity, He：It is expected that heterozygosity, P：Hardy-Weinberg is flat
Homeostasis restrains deviation, -- indicate that no polymorphism, * * indicate highly significant, ++ it indicates not notable.
2.2 sample genetic variation and genetic differentiations and relationship
As shown in Table 3, sample FST average values are 0.2877, illustrate that significant difference is presented in sample differentiation.Sample gene levelling
Mean value Nm is 0.7452, and 7 site Nm of site is maximum, is 1.1074, and 5 site Nm of site is minimum, is 0.2044.The F of whole samplesIT
And FISValue is in significant difference (P<0.05).As shown in Table 4, maximum Gene distance is located at BCM and SM, be 1.0369, minimum basis because
Distance is located at BCM and OCM, is 0.7497.
3 three groups of Macrobrachium rosenbergii microsatellite locus F statistical values of table
Nm：Gene flow valuve
Gene spacing of the table 4 based on Macrobrachium rosenbergii microsatellite locus Nei data
3 discuss
Microsatellite molecular marker technology is widely used in aquatic products as a kind of neutral molecule marking tool of full blast and supports
Grow field.Currently, domestic and foreign scholars study Macrobrachium rosenbergii genetic diversity using microsatellite molecular marker technology, but
The genetic diversity of main research different population, heritage information is known between the same gender of natural or artificial inbred acquisition
It is less.In this research, using easy to identify, highly polymorphic Macrobrachium rosenbergii microsatellite locus as standard, studies 3 close relative's hero shrimp groups and lose
Pass structure.The results show that all microsatellite locus show the difference in height opposite sex；In addition, BCM, OCM, SM respectively contain 24,28,
22 not iso-alleles show that the allele in 3 groups of hero shrimps has higher diversity and abundance.Utilize 6 microsatellites
Site is studied wild Macrobrachium rosenbergii kind genetic diversity and is found, the average number of alleles in each site is in great diversity, so
And it is limited only to obtain this kind of gene diversity by the number of alleles of each microsatellite markers.
Gene heterozygosity is also known as gene diversity, is the optimal parameter for indicating gene difference.It is by experimental result it is found that all
Heterozygosity (Ho) average value is observed in set of samples and is less than desired heterozygosity (He) average value, shows the heterozygosity of male Macrobrachium rosenbergii
Decline.Observation heterozygosity average value ranges in 3 groups of samples are 0.474-0.5561.
FISIndicate that the extent of deviation of gene random pair, value indicate that heterozygosis has significantly deficiency to be positive, be worth for negative indication
There are excessive heterozygotes.F in this researchISIt is -0.0189, indicates that heterozygote is superfluous, and superfluous negligible amounts.Except site 1 and position
Outside point 2, the F of all sitesISValue is negative, illustrates that these sites have excessive heterozygote, it may be possible to amorph,
Caused by Genotyping mistake, inbreeding or Wahlund effects.
Card side (χ2) and likelihood ratio (G2) be used to examine to deviate significantly from (P on gene loci<0.05) Hardy-Weinberg equilibrium
The group of law.In this research, other than 3 gene locis of OCM, all gene locis and Hardy-Weinberg equilibrium law
There are deviations, it may be possible to due to the presence of amorph.In addition, the gene spacing range of 3 groups of samples is 0.7497-
1.0369, illustrate that 8 microsatellite markers have high otherness, shows the influence of hereditary variation of the environment between sample sets in same
Domain distributivity.BCM and SM numerical value maximums both illustrate that gene spacing is maximum, and BCM and OCM gene spacing be small both to be shown genetically
It is close or homologous.
Most in recent years, researcher to the gene diversity of the cultivation kind of the Macrobrachium rosenbergii of country variant or region and wild species into
Research is gone.This research is based on the basis of same population inbred, is chosen 3 kinds of different shape type hero shrimps and is carried out genetic diversity
Research, and the unknown single-minded locus gene for causing 3 kinds of morphotype difference, this species diversity may be due to the genes such as heredity because
Caused by the environmental factors and therein group factor etc. such as element, food source and living space.The significance difference of male Macrobrachium rosenbergii
Different not only show can illustrate in the form of its heritability, be also embodied in liver index and biochemical character.Result of study is aobvious
Show, after cultivation in 4 months, BCM average weights are 23.34 ± 1.26g, and OCM average weights are 18.39 ± 1.37g, and SM is averaged body
Weight is 9.06 ± 0.53g, accounts for 21%, 62.5%, the 16.5% of research sample populations respectively.Therefore, their existing forms, solution
Cut open and physiological difference.
Microsatellite molecular marker in the research of Macrobrachium rosenbergii population genetic diversity using more, and to inbred hero shrimp
Hereditary information have not been reported, since male Macrobrachium rosenbergii is uncontrollable in hereditary form, study its genetic diversity performance be father
This selection and breeding provides hereditary information, improves fry quality.From the foregoing discussion, it should be apparent that 3 kinds of morphotype male sieve for passing through inbred
Family name pond crayfish is there are hereditary difference, this hereditary difference existing forms, physiologically, and therefore, which can be that Macrobrachium rosenbergii is excellent
Matter male parent selection and breeding provide basic hereditary information, while also having definite meaning to the male Macrobrachium rosenbergii genetic development of regulation and control.
The above embodiment is not limitation of the present invention, and the present invention is also not limited to the example above, this technology neck
The variations, modifications, additions or substitutions that the technical staff in domain is made within the scope of technical scheme of the present invention, also belong to this hair
Bright protection domain.
Sequence table
<110>Guangxi Zhuang Autonomous Region aquatic science research institute
<120>Inbred male Macrobrachium rosenbergii analysis of genetic diversity method
<130> 1
<160> 21
<170> SIPOSequenceListing 1.0
<210> 1
<211> 78
<212> DNA
<213>Macrobrachium rosenbergii (Macrobrachium rosenbergii)
<400> 1
gcgcggagca tagagaacgg gaatctcgtg ggcactcaca gatgagtggg agtggccggg 60
gtgacagcag gatgaatg 78
<210> 2
<211> 33
<212> DNA
<213>Macrobrachium rosenbergii (Macrobrachium rosenbergii)
<400> 2
gtgggagtgg ccggggtgac agcaggatga atg 33
<210> 3
<211> 53
<212> DNA
<213>Macrobrachium rosenbergii (Macrobrachium rosenbergii)
<400> 3
aatcggatgg gagcgcggag catagagaac gggaatctcg tgggcactca cag 53
<210> 4
<211> 87
<212> DNA
<213>Macrobrachium rosenbergii (Macrobrachium rosenbergii)
<400> 4
caacagctgc tgcttggaag cttaatcctg caggcaaatg caagcgcttg gatgtgcctg 60
ctgctgctgc tgatatacaa aatctga 87
<210> 5
<211> 155
<212> DNA
<213>Macrobrachium rosenbergii (Macrobrachium rosenbergii)
<400> 5
atttcatccg gttttgttcc agtattgctt aatcatgaaa agaaacaaca agagcgataa 60
taatgatgat gatgatgatg atgatgaggg taaaaatcta tgattcaagt ataaatgtac 120
atttaaaaat agacatactt aaatatacat ttata 155
<210> 6
<211> 100
<212> DNA
<213>Macrobrachium rosenbergii (Macrobrachium rosenbergii)
<400> 6
cattaaagtt ttgtttcgtt tttatttcat ccggttttgt tccagtattg cttaatcatg 60
aaaagaaaca acaagagcga taataatgat gatgatgatg 100
<210> 7
<211> 50
<212> DNA
<213>Macrobrachium rosenbergii (Macrobrachium rosenbergii)
<400> 7
agggtaaaaa tctatgattc aagtataaat gtacatttaa aaatagacat 50
<210> 8
<211> 19
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 8
ggctctctcc aggaasgtc 19
<210> 9
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 9
agttccacct gcattcatcc 20
<210> 10
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 10
aggatgaatg caggtggaac 20
<210> 11
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 11
caaaacaaga cgtccccttc 20
<210> 12
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 12
atttgcggaa ggatgtgttc 20
<210> 13
<211> 19
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 13
tatgcttccg gctcgtatg 19
<210> 14
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 14
tatcagcagc agcagagaag 20
<210> 15
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 15
gtctgtttgt gcaggtggag 20
<210> 16
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 16
ggcattccgt tgttgttg 18
<210> 17
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 17
cgctcttgtt gtttcttctc 20
<210> 18
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 18
atttcatccg gttttgttcc 20
<210> 19
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 19
agctcggatc cactagtaac g 21
<210> 20
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 20
ttgttatccg tccacaattc c 21
<210> 21
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 21
attaccgcct ttgagtgagc 20
Claims (4)
1. inbred male Macrobrachium rosenbergii analysis of genetic diversity method, which is characterized in that include the following steps：
Step 1, the inbred male Macrobrachium rosenbergii sample for acquiring various trait, and extract DNA；
Step 2, selection microsatellite marker combination, and design Specific PCR primers group and expanded；
Step 3, evaluation PCR product number, observe allelic gene typing and size；
Number of alleles A, the allele of each male shrimp of step 4, record replicate number G, observation heterozygosity Ho, it is expected heterozygosity
He, the precision runout value P based on Hardy-Weinberg equilibrium law, and analyze microsatellite locus difference；According to population allele
Frequency parameter calculates gene flow Nm；Gene spacing is measured with to gene Nei Y-factor method Ys；
Wherein, the microsatellite marker combination in step 2, includes the complementary series of following microsatellite marker or its nucleotide sequence：
Microsatellite marker 1, microsatellite marker 2, microsatellite marker 3, microsatellite marker 4, microsatellite marker 5, microsatellite marker 6, Wei Wei
Asterisk note 7；Wherein,
The nucleotide sequence of the microsatellite marker 1 is as shown in SEQ ID NO.1；
The nucleotide sequence of the microsatellite marker 2 is as shown in SEQ ID NO.2；
The nucleotide sequence of the microsatellite marker 3 is as shown in SEQ ID NO.3；
The nucleotide sequence of the microsatellite marker 4 is as shown in SEQ ID NO.4；
The nucleotide sequence of the microsatellite marker 5 is as shown in SEQ ID NO.5；
The nucleotide sequence of the microsatellite marker 6 is as shown in SEQ ID NO.6；
The nucleotide sequence of the microsatellite marker 7 is as shown in SEQ ID NO.7.
2. according to the method described in claim 1, it is characterized in that：Specific PCR primers group in the step 2, including：
The specific primer pair of microsatellite marker 1, nucleotide sequence is as shown in SEQ ID NO.8 and SEQ ID NO.9；
The specific primer pair of microsatellite marker 2, nucleotide sequence is as shown in SEQ ID NO.10 and SEQ ID NO.11；
The specific primer pair of microsatellite marker 3, nucleotide sequence is as shown in SEQ ID NO.12 and SEQ ID NO.13；
The specific primer pair of microsatellite marker 4, nucleotide sequence is as shown in SEQ ID NO.14 and SEQ ID NO.15；
The specific primer pair of microsatellite marker 5, nucleotide sequence is as shown in SEQ ID NO.16 and SEQ ID NO.17；
The specific primer pair of microsatellite marker 6, nucleotide sequence is as shown in SEQ ID NO.18 and SEQ ID NO.19；
The specific primer pair of microsatellite marker 7, nucleotide sequence is as shown in SEQ ID NO.20 and SEQ ID NO.21.
3. according to the method described in claim 1, it is characterized in that：The character packet of the inbred male Macrobrachium rosenbergii sample
Include the long arm type of blue, orange long arm type and small idiotype.
4. according to the method described in claim 1, it is characterized in that：Genetic variation and genetic differentiation degree uses F statistical analyses, such as population equipotential
Gene frequency parameter FIS、FITAnd FST, wherein FISFor the average coefficient of inbreeding, the F of local groupITFor the average inbreeding of entire group
Coefficient and FSTTo there is the average coefficient of inbreeding between affiliation local group to calculate gene flow Nm according to Wright methods, calculate
Formula is：Nm=FST0.25(1-FST)/FST.FIS。
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110016510A (en) * | 2019-04-22 | 2019-07-16 | 宁波大学 | A kind of molecular labeling for Macrobrachium rosenbergii hereditary and selection |
| CN111118174A (en) * | 2020-01-10 | 2020-05-08 | 浙江省农业科学院 | Macrobrachium rosenbergii sex identification method based on PCR and sequencing technology |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106434974A (en) * | 2016-11-22 | 2017-02-22 | 河北大学 | Microsatellite primer for macrobrachium nipponensis diversity analysis and application thereof |
-
2018
- 2018-05-17 CN CN201810471046.XA patent/CN108642187A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106434974A (en) * | 2016-11-22 | 2017-02-22 | 河北大学 | Microsatellite primer for macrobrachium nipponensis diversity analysis and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| L MIN SEE等: "Development of microsatellite markers from an enriched genomic library for the genetic analysis of the Malaysian giant freshwater prawn, Macrobrachium rosenbergii", 《BIOCHEMICAL GENETICS》 * |
| 吕敏等: "异型雄性罗氏沼虾遗传多样性的微卫星分析", 《水产科学》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110016510A (en) * | 2019-04-22 | 2019-07-16 | 宁波大学 | A kind of molecular labeling for Macrobrachium rosenbergii hereditary and selection |
| CN110016510B (en) * | 2019-04-22 | 2022-06-07 | 宁波大学 | Molecular marker for genetic breeding of macrobrachium rosenbergii |
| CN111118174A (en) * | 2020-01-10 | 2020-05-08 | 浙江省农业科学院 | Macrobrachium rosenbergii sex identification method based on PCR and sequencing technology |
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