CN105087807B - A kind of method that genetic force estimation is carried out to clam mixed family - Google Patents
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Abstract
The invention belongs to seawater Animal Genetics field, more particularly to a kind of method that genetic force estimation is carried out to clam mixed family.The forward and reverse primer of clam microsatellite locus is specific as follows, acquires the character and DNA of 3 monthly age mixed breed clams, using SSR PCR and microsatellite locus Genotyping, realizes that the pedigree of family is rebuild, the genetic force of character to be determined is then estimated using ASREML.Compared to the method by family isolation cultivation estimated heritability, the method for the present invention can increase the quantity of assessment family, eliminate the influence of environmental effect, improve the accuracy of genetic force estimation, preferably inheritting and selecting work is instructed with this.MMB98, it is positive：AGCGAAGATTTTAACAAA, reversely：ATCTTCAACTCACCATCATCA；MM573, it is positive：TTAACAAACTTGCATTTCCTC, reversely：ATCATCATCTTCAACTCACCA；MM1031, it is positive：GGTTGTGAAAGACAATTGAGA, reversely：TGCCCATTAAAGACAAGACTA；MME2034, it is positive：CCACCAAGTCTACAGTCTTCA, reversely：CAAAAAGGACCAAAACAAAGT；MM6277, it is positive：GACCAATGAAATTCAGTCAAA, reversely：TAGAAAACATAAGGCACTGCT；MM9504, it is positive：TATGACTGACACACACGCATA, reversely：GACGATTTATTCACGTAAAGG.
Description
Technical field
The invention belongs to seawater Animal Genetics fields, and genetic force is carried out to clam mixed family more particularly to a kind of
The method of estimation.
Background technology
Clam (Meretrix meretrix) is imbedded and perched shellfish, mainly inhabits river mouth nearby or between littoral inner bay tide
In band, low tidal region or subtidal zone shallow sea, be distributed the north and south of China is coastal, be the important marine products economic shellfish in China it
One.The aquaculture production of clam obtains fast development in recent years, and China's clam total output is about 300,000 tons within 2009, accounts for the world
More than 90% clam yield.With the improvement of people's living standards, supply falls short of demand for domestic and international market, market potential is huge.
At present, cultivating clam mainly still breeds gained for wild or without selection and breeding cultivation parents, and fry quality is irregular, cultivates
The problem of reflecting in journey is also very prominent.It is mainly shown as that the speed of growth slows down, disease increases, product quality declines, supports
It grows difficulty to be gradually increased, culture efficiency is on a declining curve, affects stabilization and the sustainable development of industry.Fine-variety breeding into
One of important topic to develop in a healthy way for cultivating clam industry.Genetic force is an important heredity ginseng during fine-variety breeding
Number.Reliable heretability estimate value is the basis of rational hereditary and selection strategy, can effectively predict selection reaction.It is existing to grind
Study carefully and show that the premise that genetic force is accurately estimated is large-scale family quantity and the consistency of objective trait test environment.For text
For the intertidal shellfish such as clam, due to its special biological habit, individual mark or family label can not be carried out at present, it is meant that
Physically-isolated method can only be used, family is distinguished.In order to keep the consistency of growing environment so that cultivate family
Quantity is restricted, and physical isolation method can not completely eliminate the environmental difference between family.Factors above limits clam something lost
Pass the accuracy of parameter Estimation.Therefore genetic force estimation can be carried out to extensive mixed breed family there is an urgent need to a kind of at present
Technology.
Invention content
Present invention aims at provide a kind of method that genetic force estimation is carried out to clam mixed family.
To achieve the above object, the technical solution adopted by the present invention is：
A kind of clam microsatellite locus, the forward and reverse primer of clam microsatellite locus are：
MMB98, it is positive：AGCGAAGATTTTAACAAA, reversely：ATCTTCAACTCACCATCATCA
MM573, it is positive：TTAACAAACTTGCATTTCCTC, reversely：ATCATCATCTTCAACTCACCA；
MM1031, it is positive：GGTTGTGAAAGACAATTGAGA, reversely：TGCCCATTAAAGACAAGACTA
MME2034, it is positive：CCACCAAGTCTACAGTCTTCA, reversely：CAAAAAGGACCAAAACAAAGT；
MM6277, it is positive：GACCAATGAAATTCAGTCAAA, reversely：TAGAAAACATAAGGCACTGCT
MM9504, it is positive：TATGACTGACACACACGCATA, reversely：GACGATTTATTCACGTAAAGG.
A kind of method for being carried out genetic force estimation to clam mixed family using clam microsatellite locus, 3 monthly ages of acquisition are mixed
The character and DNA of cultivation clam are closed, using SSR-PCR and microsatellite locus Genotyping, realizes that the pedigree of family is rebuild, then
The genetic force of character to be determined is estimated using ASREML.
The forward and reverse primer of clam microsatellite locus is：
MMB98, it is positive：AGCGAAGATTTTAACAAA, reversely：ATCTTCAACTCACCATCATCA；
MM573, it is positive：TTAACAAACTTGCATTTCCTC, reversely：ATCATCATCTTCAACTCACCA；
MM1031, it is positive：GGTTGTGAAAGACAATTGAGA, reversely：TGCCCATTAAAGACAAGACTA；
MME2034, it is positive：CCACCAAGTCTACAGTCTTCA, reversely：CAAAAAGGACCAAAACAAAGT；
MM6277, it is positive：GACCAATGAAATTCAGTCAAA, reversely：TAGAAAACATAAGGCACTGCT；
MM9504, it is positive：TATGACTGACACACACGCATA, reversely：GACGATTTATTCACGTAAAGG.
5 ' end mark fluorescent labels of the microsatellite locus；Wherein, 5 ' end mark fluorescent marks of the upstream of every group of primer
Note, fluorescent marker FAM, HEX or ROX；
PCR reaction systems include：Total volume 20ul, wherein 40ng clams genomic DNA, Taq DNA polymerase 1U, 1 ×
PCR Buffer, Mg2+1.5mmol/L, dNTP 0.25mmol/L, primer 0.25umol/L, and it is added to 20 with distilled water complement system
μL；
SSR expands PCR programs：94 DEG C of pre-degeneration 5min, 94 DEG C of denaturation 45s, annealing temperature Ta annealing 45s, 72 DEG C extend
50s, 30 cycles, 72 DEG C of extension 10min, 4 DEG C of preservations.
Wherein, MMB98, MM573 flag F AM；MM1031, MME2034 mark HEX；MM6277, MM9504 mark ROX；
PCR reaction systems include：Total volume 20ul, wherein 40ng clams genomic DNA, Taq DNA polymerase 1U, 1 ×
PCR Buffer, Mg2+1.5mmol/L, dNTP 0.25mmol/L, primer 0.25umol/L, and it is added to 20 with distilled water complement system
μL；
SSR expands PCR programs：94 DEG C of pre-degeneration 5min, 94 DEG C of denaturation 45s, annealing temperature Ta annealing 45s, 72 DEG C extend
50s, 30 cycles, 72 DEG C of extension 10min, 4 DEG C of preservations.Specifically, it is carried out using ABI3130 type mdk genes analyzer micro-
Satellite parting adds in sample solution 10ul, the above-mentioned pcr amplification products of wherein 2ul and 8ul internal standards (deionization formyl in 96 orifice plates
Amine：GeneScan TM-500LIZ Size Standard；7.9: 0.1 volume ratios), after abundant mixing, after 95 DEG C of denaturation 5min,
96 orifice plates are put into mixture of ice and water rapidly.Sample after cooling is placed in analyzer, utilizes GeneMapper 3.7
(Appl ied Biosystms) carries out fluorescence data collection and the reading of allele peak value on each individual and each site.
The family tree is rebuild and Trait heritability is estimated as：Using software Pedigree 2.2 to individual microsatellite position
Point typing data carries out family tree reconstruction, re-establishes compatriot between individual, half sibs relationship.Phenotypic data, which combines, to be rebuild
Pedigree data, using ASReml-R softwares using animal model carry out phenotypic character genetic force estimate.
The advantageous effect of the present invention compared with prior art：Molecular labeling is based under mixed breeding family pattern provided by the invention
The method accurately estimated of genetic force, compared with tradition is by department of physics's Power estimation genetic force method, the method for the present invention is not by family
It is the limitation of scale, ensure that homogenization and the consistency of objective trait test environment, overcoming traditional family physical isolation needs
The hardware requirements such as a large amount of breeding facility, place are wanted, simplify the workload of family management, improve the accurate of genetic force estimation
Property can preferably instruct the inheritting and selecting work of clam.The method of the present invention can increase the quantity of assessment family, eliminate Environmental Effect
The influence answered improves the accuracy of genetic force estimation, preferably inheritting and selecting work is instructed with this.
Specific embodiment
Below by embodiment, the technical solution of the present invention is further explained, but protection scope of the present invention not by
The limitation of embodiment in any form.
The method of clam genetic force can meet and be raised together in family, under conditions of any physical markings, pass through molecule
Label realizes that pedigree is rebuild, and realizes the accurate estimation of genetic parameter.In terms of its character analyzed is mainly growth, including body
Again, shell is long, shell is wide, shell is high etc., the clam genetic force for the arbitrary growth phase for meeting DNA extraction and analysis can be assessed.
The present invention is applied to shellfish fine-variety breeding field, suitable for having the mixed breeding group of family structure.Mainly for property
Shape is growth traits, is a kind of genetic force Accurate Estimation Method being not required under the conditions of being physically separated to family.
Embodiment 1
1. the structure of clam family
Acquire gonadal maturation in June, 2014, the close shellfish of the full health of shell mould carries out artificial insemination, establishes clam man
System.Specially：Close shellfish is dried in the shade after placement 4~8 hours, is cleaned body surface and is individually placed in the beaker of the seawater containing 800ml and hastens parturition
The smart ovum of acquisition.10 male shellfishes and 30 female shellfishes are chosen, take the 1 male nest-mode mating desigh female to 3, its smart ovum is pressed into proper proportion
After mixing, it is positioned in the culture bucket of 10L and is fertilized.Ocean temperature maintains 27 degree, salinity 20 ‰.After fertilization 30 minutes, by structure
The 30 familys mixing built, which is placed in the circular glass steel cylinder of 1000L, hatches culture.After fertilization 3 days, there is eyespot in floating larvae
And foot, at this time bucket bottom sand, seedling enters the settlement and metamorphosis phase.When double water pipes occurs in larva, juvenile mollusk growth phase is initially entered,
Hereafter cultivating process according to clam conventional cultivation method.
It is extracted 2. clam individual data items are acquired with DNA
When clam family individual growth to 6 monthly age raised together, take that 450 bulk measurement shells are long, shell is high, shell is wide at random and
Weight data, while its mantle tissue is taken to be extracted for genes of individuals group DNA, pay attention to avoiding cross contamination between individual.Reference
After conventional method extraction genomic DNA, -75 DEG C of Cord bloods.
3. microsatellite locus Genotyping
PCR and product detection：6 clam SSR sites of this development in laboratory is selected to carry out SSR-PCR amplifications, site sequence
Row and title are shown in Table 1.The end of primer 5 ' difference flag F AM, HEX and ROX fluorescent marker.PCR reaction systems include：Total volume
20ul, wherein 40ng clams genomic DNA, Taq DNA polymerase 1U, 1 × PCR Buffer, Mg2+1.5mmol/L, dNTP
0.25mmol/L, primer 0.25umol/L, and it is added to 20mL with distilled water complement system.SSR expands PCR programs：94 DEG C of pre-degenerations
5min, 94 DEG C of denaturation 45s, the optimum annealing temperature Ta annealing 45s of each primer optimization, 72 DEG C of extension 50s, 30 recycle, 72 DEG C
Extend 10min, 4 DEG C of preservations.
Genotyping：Microsatellite parting is carried out using ABI3130 type mdk genes analyzer, is added in 96 orifice plates
The above-mentioned pcr amplification product of sample liquid 10ul, wherein 2ul (is inside designated as deionized formamide by volume with 8ul internal standards：
GeneScan TM-500LIZ Size Standard=7.9: 0.1), after abundant mixing, after 95 DEG C of denaturation 5min, rapidly by 96
Orifice plate is put into mixture of ice and water.Sample after cooling is placed in analyzer, utilizes 3.7 (Applied of GeneMapper
Biosystms fluorescence data collection and the reading of allele peak value on each individual and each site) are carried out.
4. pedigree is rebuild
The microsatellite locus typing data of above-mentioned acquisition is input to by 2.2 requirement forms of Pedigree in software and is handled,
It is divided into analysis and obtains 30 Full-sib partition, 10 Kin partition, compatriot, half have been re-established between individual
The relationship of compatriot.Genealogical relationship between the family rebuild is shown in Table 2.
5. genetic force is estimated
According to ASReml-R software requirements, build phenotypic data file and pedigree file, partial data are shown in Table 3.Using
REML algorithms carry out the estimation of genetic force using animal model：
Y=Xb+Za+e
Wherein, y is phenotypic number vector, and b, a and e are respectively fixed effect, stochastic effects (individual animals additive genetic effect
Vectorial, maternal common environmental effect vector) and residual error vector.X, Z represent the relational matrix of corresponding vector.Growth traits heredity
The estimation of power uses unisexuality shape animal model, and genetic force calculation formula is：WhereinWithIt represents to add respectively
Property hereditary effect variance component and residual error effect variance component.The growth traits genetic force of estimation is shown in Table 4.
The 16 microsatellite marker of clam information for the recombination of clam pedigree of table
The pedigree and the scale of 30 familys that table 2. is re-established using microsatellite marker
The pedigree and phenotype fileinfo of 3 ASReml-R software analyses of table
| ID | Sire | Dam | FAMILY | SL | SH | SW | TW |
| 1 | S-1 | A-1 | 1 | 23.04 | 19.02 | 11.43 | 2.9 |
| 2 | S-1 | A-1 | 1 | 19.89 | 16.35 | 9.66 | 1.9 |
| 3 | S-1 | A-1 | 1 | 18.27 | 15.43 | 8.61 | 1.4 |
| 4 | S-1 | A-1 | 1 | 21.62 | 18.24 | 10.86 | 2.7 |
| 5 | S-1 | A-1 | 1 | 22.6 | 17.98 | 11.75 | 2.8 |
| 6 | S-1 | A-1 | 1 | 22.32 | 18.24 | 11.07 | 2.8 |
| 7 | S-1 | A-1 | 1 | 18.48 | 15.27 | 8.92 | 1.5 |
| 8 | S-1 | A-1 | 1 | 17.97 | 15.24 | 8.97 | 1.5 |
| 9 | S-1 | A-1 | 1 | 19.05 | 15.05 | 9.27 | 1.6 |
| 10 | S-1 | A-1 | 1 | 16.21 | 13.38 | 7.82 | 1 |
| 11 | S-1 | A-1 | 1 | 19.11 | 16.24 | 9.21 | 1.7 |
| 12 | S-1 | A-1 | 1 | 20.91 | 17.64 | 9.98 | 2.2 |
| 13 | S-1 | A-1 | 1 | 24.12 | 19.8 | 11.39 | 3.15 |
| 14 | S-1 | A-1 | 1 | 20.33 | 16.67 | 9.67 | 2.1 |
| 15 | S-1 | A-1 | 1 | 13.97 | 12.12 | 6.6 | 0.6 |
| 16 | S-1 | A-1 | 1 | 17.81 | 14.11 | 8.78 | 1.4 |
| 17 | S-1 | A-1 | 1 | 18.06 | 15.32 | 9.01 | 1.5 |
| 18 | S-1 | A-1 | 1 | 19.36 | 16.29 | 9.36 | 1.8 |
| 19 | S-1 | A-2 | 2 | 17.76 | 15.43 | 8.53 | 1.4 |
| ... | ... | ... | ... | ... | ... | ... | ... |
| 444 | S-10 | J-3 | 30 | 17.23 | 14.8 | 8.93 | 1.26 |
| 445 | S-10 | J-3 | 30 | 22.91 | 19.04 | 10.97 | 2.76 |
| 446 | S-10 | J-3 | 30 | 22.9 | 18.44 | 11.19 | 2.69 |
| 447 | S 10 | J 3 | 30 | 18.77 | 15.81 | 9.04 | 1.51 |
| 448 | S 10 | J 3 | 30 | 19.68 | 16.1 | 9.16 | 1.72 |
| 449 | S 10 | J 3 | 30 | 19.44 | 16.18 | 9.78 | 1.91 |
| 450 | S-10 | J-3 | 30 | 17.51 | 14.07 | 8.62 | 1.34 |
Table 6 monthly age of 4 clam growth traits heretability estimate value
Claims (2)
- A kind of 1. method for carrying out genetic force estimation to clam mixed family using clam microsatellite locus, it is characterised in that：It adopts Collect the character and DNA of 3 monthly age mixed breed clams, using SSR-PCR and microsatellite locus Genotyping, realize the pedigree of family It rebuilds, the genetic force of character to be determined is then estimated using ASREML；The forward and reverse primer of clam microsatellite locus is：MMB98, it is positive：AGCGAAGATTTTAACAAA, reversely：ATCTTCAACTCACCATCATCA；MM573, it is positive：TTAACAAACTTGCATTTCCTC, reversely:ATCATCATCTTCAACTCACCA；MM1031, it is positive：GGTTGTGAAAGACAATTGAGA, reversely：TGCCCATTAAAGACAAGACTA；MME2034, it is positive：CCACCAAGTCTACAGTCTTCA, reversely：CAAAAAGGACCAAAACAAAGT；MM6277, it is positive：GACCAATGAAATTCAGTCAAA, reversely：TAGAAAACATAAGGCACTGCT；MM9504, it is positive：TATGACTGACACACACGCATA, reversely：GACGATTTATTCACGTAAAGG.
- 2. the method as described in claim 1 for carrying out genetic force estimation to clam mixed family using clam microsatellite locus, It is characterized in that：5 ' end mark fluorescent labels of the microsatellite locus；PCR reaction systems include：20 μ l of total volume, wherein 40ng clams genomic DNA, Taq DNA polymerase 1U, 1 × PCR Buffer,Mg2+1.5mmol/L, dNTP 0.25mmol/L, 0.25 μm of ol/L of primer, and it is added to 20 μ L with distilled water complement system；SSR expands PCR programs：94 DEG C of pre-degenerations 5min, 94 DEG C of denaturation 45s, annealing temperature Ta annealing 45s, 72 DEG C of extension 50s, 30 cycles, 72 DEG C of extension 10min, 4 DEG C of preservations.
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| CN104313135A (en) * | 2014-09-30 | 2015-01-28 | 中国水产科学研究院黄海水产研究所 | Evaluation method of individual breeding values of turbot |
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Non-Patent Citations (2)
| Title |
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| Mining of EST-SSR markers in clam Meretrix meretrix larvae from 454 shotgun transcriptome;Hongxia Wang et al.;《Genes Genet. Syst.》;20111231(第86期);第197-205页 * |
| 长牡蛎成体生长性状的遗传参数估计;王庆志 等;《中国水产科学》;20120731;第19卷(第4期);第700-706页 * |
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