CN109022590A - One kind SNP marker relevant to Macrobrachium rosenbergii body length and second step toe section length and its application - Google Patents
One kind SNP marker relevant to Macrobrachium rosenbergii body length and second step toe section length and its application Download PDFInfo
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- CN109022590A CN109022590A CN201810907429.7A CN201810907429A CN109022590A CN 109022590 A CN109022590 A CN 109022590A CN 201810907429 A CN201810907429 A CN 201810907429A CN 109022590 A CN109022590 A CN 109022590A
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Abstract
The invention discloses the one kind for belonging to molecular marker breeding technical field SNP marker relevant to Macrobrachium rosenbergii body length and second step toe section length and its applications.The SNP marker corresponds to the site Macrobrachium rosenbergii ND1 gene T658C, and base is T or C herein.SNP marker of the invention is related to Macrobrachium rosenbergii body length and second step toe section length, it is a new molecular labeling, early stage breeding is carried out by the genotype of determination Macrobrachium rosenbergii SNP site to be measured to which the body to Macrobrachium rosenbergii is long and second step toe section is long, production cost can effectively be saved and accelerate genetic progress, preferably serve the breeding of Macrobrachium rosenbergii, not only it had been able to satisfy the huge market demand, but also with very big Economic Application value and scientific research value.
Description
Technical field
The invention belongs to molecular marker breeding technical field, in particular to a kind of and Macrobrachium rosenbergii body length and second step toe
The long relevant SNP marker of section and its application.
Background technique
Molecular mark (Marker Assisted Selection, MAS) is using molecular labeling and to determine mesh
The characteristics of marking the gene close linkage of character is screened and important economic characters (such as weight, growth in animals and plants breeding process
Speed, resistance etc.) relevant molecular labeling, and building has the breeding population of related molecular marker in breeding process, with
Achieve the purpose that a kind of breeding method of selection target character, accuracy high feature strong with Objective can be significantly
Breeding efficiency is improved, the breeding time limit is shortened.
Mononucleotide loci polymorphism (Single Nucleotide Polymorphisms, SNP) be on genome by
The nucleotide variation that single nucleotide acid generates has the characteristics that distributed quantity is more, rich polymorphism, is after microsatellite molecular marker
Important molecular genetic marker another later, referred to as third generation molecular genetic marker.The label is widely used to animals and plants
The fields such as marker-assisted breeding, the assignment of genes gene mapping, genetic diversity Journal of Sex Research.
ND1 gene is the gene for encoding nadh dehydrogenase subunit 1,1 (NADH of nadh dehydrogenase subunit
Dehydrogenase subunit 1, ND1) gene be mtDNA protein coding gene, including cytochrome b, cytochromes
The coded sequence of 7 subunits of 3 subunits of oxidizing ferment, 2 subunits of ATP enzyme and nadh dehydrogenase is responsible for the egg of coding
White is the subunit I of NADH dehydrogenase.Nadh dehydrogenase is the main composition of electron transport chain complex I, by direct in respiratory chain
It participates in hydrogen and electron transmission and goes to participate in energetic supersession effect by oxidative phosphorylation generation ATP.Thus, it influences to a certain extent
The acquisition of animal exogenous nutrition substance, assimilation utilize and the activity of the vital metabolics such as transhipment and then the character of animal risen decisive
Effect.It is more about species ND1 gene diversity Journal of Sex Research both at home and abroad, such as three tail swallowtail butterflies (Bhutanitis thaidina), nothing
The discussion of the phylogeographies and genetic development such as web gecko (Gekko swinhonis), tree toad (Hylid).Currently, Hou Lingling
Have found that chicken NDl Gene A 4589G mutation and caecum length, 8 week old chest depths, 8 week old keel lengths and 8 week old shins length, leg flesh are long
Degree has significant related respectively to the influence of leg flesh width, glucose, lactic dehydrogenase, abdominal fat, abdominal fat and subcutaneous fat thickness
Property.
Macrobrachium rosenbergii, also known as Malaysian prawn, fresh water prawn, big long-armed prawn are under the jurisdiction of Arthropoda, hapalonychia
Guiding principle, Decapoda, Palaemonidae, pond crayfish category are one of tropical fresh water shrimps largest in the world, and with growing, fast, feeding habits are wide, resistance to
The features such as high temperature, premunition are strong, the culture-cycle is short, mouthfeel is fresh and tender and rich in nutrition content and by consumers.It originates in
In the India Pacific region, various types of fresh water or salt-fresh water waters are lived in, is supported later by the shifting of many countries and regions numerous
It grows, becomes local main freshwater aquiculture shrimps.Currently, Macrobrachium rosenbergii is up to more than 60 ten thousand mu, but Roche in the cultured area of China
The breeding quantity of pond crayfish is far from meeting its huge industry size and huge seed demand, therefore, the early stage of Macrobrachium rosenbergii
Breeding seems very urgent and significant.
Summary of the invention
To overcome the above insufficient or defect in the prior art, the present invention provides a kind of and Macrobrachium rosenbergii body length and second
The long relevant SNP marker of step finger joint and its application, can by the genotype of determination Macrobrachium rosenbergii SNP site to be measured to
And second step toe section long progress early stage breeding long to the body of Macrobrachium rosenbergii, limits without age, the gender etc. by Macrobrachium rosenbergii,
The early stage breeding that can be effectively used for Macrobrachium rosenbergii promotes the breeding process of Macrobrachium rosenbergii.
The technical solution used in the present invention is:
A kind of SNP marker relevant to Macrobrachium rosenbergii body length and second step toe section length, the SNP marker position
In the site Macrobrachium rosenbergii ND1 gene T658C, base is T or C herein.
Application of the SNP marker in Macrobrachium rosenbergii early stage breeding.
A kind of method of the early stage breeding of Macrobrachium rosenbergii, for according to the genotype in the site Macrobrachium rosenbergii ND1 gene T658C
And second step toe section long progress early stage breeding long to the body of Macrobrachium rosenbergii, includes the following steps:
(1) design specific amplification contains the primer pair of the genetic fragment in the site Macrobrachium rosenbergii ND1 gene T658C；
(2) genomic DNA of Macrobrachium rosenbergii to be measured is extracted；
(3) PCR amplification, detection amplification institute are carried out with genomic DNA of step (1) the resulting primer pair to Macrobrachium rosenbergii
Obtain the genotype of the SNP site of genetic fragment；
(4) the long progress early stage breeding of body length and second step toe section based on the genotype of SNP site to Macrobrachium rosenbergii.
As a further improvement of the foregoing solution, the primer sequence of the PCR amplification is
ND1-F:5 '-GATAAAGTAGGCTATGCTGG-3 ' (SEQ ID № 1),
ND1-R:3 '-CCTCCTCTGTACTCGGTATT-5 ' (SEQ ID № 2).
As a further improvement of the foregoing solution, the amplification instrument that the PCR amplification uses is BIO-RAD T100 PCR
Instrument.
The beneficial effects of the present invention are: the present invention provides a kind of related to Macrobrachium rosenbergii body length and second step toe section length
SNP marker and its application, the SNP marker is located at the site Macrobrachium rosenbergii ND1 gene T658C, and base is T herein
Or C.The body of the nucleotide sequence of ND1 gene from 5 ' 658 bit base T or C of end and Macrobrachium rosenbergii are long and the in the Macrobrachium rosenbergii
The long significant correlation of two step finger joints, i.e. SNP marker on the site Macrobrachium rosenbergii ND1 gene T658C and Macrobrachium rosenbergii body are long
It is related to second step toe section length, be a new molecular labeling, by the genotype of determination Macrobrachium rosenbergii SNP site to be measured from
And early stage breeding is carried out to Macrobrachium rosenbergii, it can effectively save production cost and accelerate genetic progress, preferably serve Roche natural pond
The early stage breeding of shrimp had not only been able to satisfy the huge market demand, but also with very big Economic Application value and scientific research value.
Detailed description of the invention
Fig. 1 is the PCR products electrophoresis map of Macrobrachium rosenbergii ND1 Gene Partial segment；
Fig. 2 is 658 mutational site genotype mutations sequence alignment of Macrobrachium rosenbergii ND1 gene.
Specific embodiment
The present invention is specifically described below with reference to embodiment, in order to technical field personnel to of the invention
Understand.It is necessary to it is emphasized that embodiment is only intended to, the present invention will be further described herein, should not be understood as to this
The limitation of invention protection scope, fields person skilled in the art, according to foregoing invention content non-to made by the present invention
The modifications and adaptations of matter should still fall within protection scope of the present invention.Mentioned raw materials following simultaneously are unspecified,
For commercial product；The processing step or preparation method not referred in detail be processing step known to a person skilled in the art
Or preparation method.
The source of the Macrobrachium rosenbergii cultured population of test: Guangdong (GD) adopts 47 samples；Adopt 76 samples in Guangxi 1 (GX1)
Product；Adopt 35 samples in Guangxi 2 (GX2)；44 samples are adopted in Hainan (HN), are provided by the seedling base of three water-white golden breeding companies,
Raised under identical environmental condition 106, in July, 2017 random acquisition at shrimp.
Embodiment
One, the detection of SNP marker
1. design primer: design specific amplification contains the primer of the genetic fragment in the site Macrobrachium rosenbergii ND1 gene T658C
It is right.
Using software Primer 5.0, with the ND1 gene (GenBank:NC_006880.1) of Macrobrachium rosenbergii in GenBank
The specificity amplification primer for making Reference Design, the primer sequence expanded are
Upstream F:5 '-GATAAAGTAGGCTATGCTGG-3 ' (SEQ ID № 1), 20bp；
Downstream R:3 '-CCTCCTCTGTACTCGGTATT-5 ' (SEQ ID № 2), 20bp.
Optimum temperature is 48.8 DEG C, product length 542bp, and by Guangzhou, Sheng Gong bioengineering Co., Ltd is synthesized.
2. the extraction of Macrobrachium rosenbergii genomic DNA
Use the whole blood kit of OMEGAD, the specific steps are as follows:
(1) each sample takes the blood sample of 150 μ L, and buffer Elution Buffer is added, and vortex mixes 10s；
(2) the Buffer GL lysate of 500 μ L is added in each sample, shakes up 20s manually, stands 20min；
(3) after the GL2 lysate of 100 μ L is added, there is white opacity in solution, after vortex mixes 60s, 13000 × g high speed
It is centrifuged 15min, the phenomenon that separation of solid and liquid after centrifugation；
(4) supernatant after centrifugation is transferred in adsorption column, 10000 × g high speed centrifugation, 1 min outwells absorption column tube
In waste liquid；
(5) the HB Buffer that 500 μ L are added binds liquid, after 10000 × g is centrifuged 1min, outwells useless in absorption column tube
Liquid；
(6) the Wash Buffer cleaning solution of 650 μ L is added, 10000 × g high speed centrifugation 1min is outwelled in absorption column tube
Waste liquid；
(7) (6) step is repeated；
(8) the Elution Buffer dilution of 70 DEG C of preheatings is added, 1000 × g is centrifuged after being incubated at room temperature 3min；
(9) liquid that centrifugation is obtained rejoins in adsorption column, and 1000 × g is centrifuged after being incubated at room temperature 3min, carries out pair
The label answered, -20 DEG C of preservations.
3. amplification and detection: carrying out PCR amplification, detection amplification with genomic DNA of the primer pair of design to Macrobrachium rosenbergii
The genotype of the SNP site of gained genetic fragment.
Take 40 samples at random in the genome extracted, every 4 samples take 2 μ L to mix, are divided into 10 groups, carry out PCR respectively
Amplification amounts to 10 samples.
Program: the PCR SuperMix of full formula gold is used.
System: DD H2O:22 μ L；Mix:25 μ L；
Upstream primer: 1 μ L；Downstream primer: 1 μ L；DNA:1 μ L.
Using BIO-RAD T100PCR instrument, wherein program setting: A, 94 DEG C of initial denaturation 5min；B, 94 DEG C of denaturation 30s；C,
48.8 DEG C of annealing 30s；D, 72 DEG C of 1 min of extension；E, B~D30 circulation；F:72 DEG C of extension 5min；G:4 DEG C of constant temperature saves.
With 0.8g BIOWEST agarose, 1.0% agarose is made in 1 × TAE liquid of 80ml and 8 μ L Goldview
It is solidifying.With 5 μ L product point samples, using the full formula gold DNA Marker DL2000 of 5 μ L as reference, in BIO-RAD Powerpac electrophoresis
In instrument, use 1 × TAE liquid as room temperature electrophoresis.(wherein, voltage: 150V；Electric current 400mA；Time: 20min.) after electrophoresis,
It observes and photographs to record under Tanon gel imaging system.Determine single and clearly bright purpose band occur (such as Fig. 1 institute
Show) after send to Guangzhou Sheng Gong biotech firm and be sequenced, Genotyping is carried out according to sequencing result, obtains Fig. 2.
Two, SNP site genotype and the association analysis that Macrobrachium rosenbergii body is long and second step toe section is long
The relevance of linear analogue analysis SNP site genotype and character is carried out using SAS9.0 software Mixed program,
In, it is long with body when analysis and second step toe section length represents phenotypic number, as follows using model:
Model: Yijkx=p+Bj+Gk+Eijkl+Fx+bjk:
YijkxFor the observation of Carcass Traits, p is group's mean, BjIt is sex-effects, GkFor genotype effects.
EijklFor random error.FxFor family stochastic effects.bjkFor the regression coefficient for influencing slaughter traits.With GLM program
The least squares means and standard error of different genotype slaughter trait are calculated, while carrying out significance test.
The association analysis that the gene frequency and body of its SNP site are long and second step toe section is long is as shown in table 1 below.
The association analysis that the gene frequency and body of 1 SNP site of table are long and second step toe section is long
As shown in Table 1, which is that the body of the Macrobrachium rosenbergii of TT type is long and second step toe section length is significantly higher than CC type
(P<0.05), TC type be not significant (P>0.05) to TT, CC type difference.The nucleotide sequence holds 658 bit base T or C and sieve from 5 '
Family name's pond crayfish body is long significant related to second step toe section length.
Above-described embodiment is the preferred embodiment of the present invention, all with similar technique of the invention and made equivalence changes,
It should belong to protection category of the invention.
SEQUENCE LISTING
<110>Foshan Science &. Technology College
<120>a kind of SNP marker relevant to Macrobrachium rosenbergii body length and second step toe section length and its application
<130> 2018.08.01
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213>artificial sequence
<400> 1
gataaagtag gctatgctgg 20
<210> 2
<211> 20
<212> DNA
<213>artificial sequence
<400> 2
cctcctctgt actcggtatt 20
Claims (6)
1. a kind of SNP marker relevant to Macrobrachium rosenbergii body length and second step toe section length, which is characterized in that the SNP
Molecular labeling is located at the site Macrobrachium rosenbergii ND1 gene T658C, and base is T or C herein.
2. a kind of application of SNP marker relevant to Macrobrachium rosenbergii body length and second step toe section length, which is characterized in that will
SNP marker described in claim 1 is applied in Macrobrachium rosenbergii early stage breeding.
3. a kind of method of Macrobrachium rosenbergii early stage breeding, which is characterized in that the genotype of SNP site according to claim 1
And second step toe section long progress early stage breeding long to the body of Macrobrachium rosenbergii.
4. according to the method described in claim 3, it is characterized by comprising the following steps:
(1) design specific amplification contains the primer pair of the genetic fragment in the site Macrobrachium rosenbergii ND1 gene T658C；
(2) genomic DNA of Macrobrachium rosenbergii to be measured is extracted；
(3) PCR amplification, detection amplification gained genetic fragment are carried out with genomic DNA of the primer pair of step (1) to Macrobrachium rosenbergii
SNP site genotype；
(4) the long progress early stage breeding of body length and second step toe section based on the genotype of SNP site to Macrobrachium rosenbergii.
5. according to the method described in claim 4, it is characterized in that, the sequence of the primer pair of the PCR amplification such as SEQ ID № 1
Shown in 2.
6. according to the method described in claim 4, it is characterized in that, the amplification instrument that uses of the PCR amplification is BIO-RAD
T100PCR instrument.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103141421A (en) * | 2013-03-13 | 2013-06-12 | 广西壮族自治区水产研究所 | Character identification method for macrobrachium rosenbergii |
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- 2018-08-10 CN CN201810907429.7A patent/CN109022590A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103141421A (en) * | 2013-03-13 | 2013-06-12 | 广西壮族自治区水产研究所 | Character identification method for macrobrachium rosenbergii |
Non-Patent Citations (3)
| Title |
|---|
| AGARWAL ET AL.: "SNP mining in transcripts and concomitant estimation of genetic variation in Macrobrachium rosenbergii stocks", 《CONSERVATION GENETICS RESOURCES》 * |
| THANH ET AL.: "Single nucleotide polymorphisms in the actin and crustacean hyperglycemic hormone genes and their correlation with individual growth performance in giant freshwater prawn Macrobrachium rosenbergii", 《AQUACULTURE》 * |
| 陈雪峰等: "罗氏沼虾缅甸野生原种rDNA-ITS区序列分析", 《动物学杂志》 * |
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