CN103882144A - Method for evaluating breeding value of turbot family - Google Patents

Abstract

The invention discloses a method for evaluating a breeding value of a turbot family, belonging to the field of genetic breeding of seawater animals. The method comprises the steps: constructing the turbot family, performing individual data acquisition and DNA (Desoxvribose Nucleic Acid) extraction of the family, and performing genetic typing and data processing by a microsatellite site. Compared with a method for evaluating the breeding value by a physical family tree, the method is capable of improving the accuracy of a relationship coefficient among individuals and has advantages in terms of prediction of the breeding value and the accuracy. Therefore, the breeding work is well guided.

Description

A kind of turbot family breeding value appraisal procedure

Technical field

The invention belongs to seawater Animal Genetics field, relate to particularly a kind of method of turbot family being carried out to breeding value assessment that realizes

Background technology

Turbot (Scophthalmus maximus L.) is to originate in the seawater fish of dwelling of the European end, has that growth is rapid, low temperature resistant, unique flavor, personality docility, is suitable for the advantages such as intensive culture, and be one of the good sea farming kind on the ground such as Europe.Since 1999 break through productivity seeding raising technology, turbot aquaculture industry is developed rapidly in China, has become the coastal important fish farming kind of northern China.Because China is not the country of origin of turbot, breeding production is medium-term and long-term depends on the European turbot country of origin provides, supplements provenance.Meanwhile, because for many generations cultivation and inbreeding is more serious, generally there is germplasm degradation phenomena in domestic turbot industry.Main manifestations is the phenomenons such as growth Speed Reduction, albefaction rate increase, seedling rate reduction.Fine-variety breeding has become one of important topic of China's turbot aquaculture industry Sustainable development.Fine-variety breeding pattern based on complete physical pedigree record is one of important channel of carrying out brill fine-variety breeding.Complete, pedigree information not only can effectively instruct parent to select and remain accurately, avoids inbreeding depression; Utilize the individual coefficient of relationship that pedigree information is extrapolated is also the significant data for carrying out genetic parameter assessment simultaneously.But due to turbot sexual maturation cycle long (2-3), current most seed multiplication farm parent populations are possessed limited amount, say nothing of complete physics pedigree recorded information; Meanwhile, turbot belongs to introduction species, and the genetic background of colony is unclear, and this has also caused very large difficulty for carrying out turbot fine-variety breeding.Rebuild although utilize molecule marker can realize pedigree, this reconstruction is very limited, such as being reconstructed into a Parent generation, can only extrapolate the genotype combination of Parent, and cannot extrapolate the genotype of concrete male parent, female parent.In fine-variety breeding process, most important genetic parameter assessment is exactly the assessment of individuality/family breeding value.Therefore, at present in the urgent need to a kind of technology that just can carry out to family breeding value assessment without detailed physics pedigree record.

Summary of the invention

The technical problem to be solved in the present invention is to provide a kind of turbot family breeding value appraisal procedure, utilize to solve that background is unclear, physics pedigree incomplete recording or introduce turbot colony while carrying out fine-variety breeding, thereby the problem that cannot utilize individual coefficient of relationship that pedigree information is extrapolated to carry out genetic parameter assessment.

The present invention is achieved through the following technical solutions:

A kind of turbot family breeding value appraisal procedure, comprises turbot family structure, the collection of family individual data items and DNA extraction, microsatellite locus gene type and data processing;

Described family individual data items gathers: for the turbot of constructed family, in the time of 4 monthly age, each family is selected the individuality of body weight rank front 60 as core selective breeding family, measure weight data, get its mean value as family core selective breeding family mean value, simultaneously get 20 tail individualities from each family individuality of reserving seed for planting, extract DNA;

Described microsatellite locus gene type: utilize 12 SSR sites that turbot has been announced to carry out SSR-PCR amplification, ' end is mark 6-FAM respectively, HEX and ROX fluorescent mark for 12 pairs of primer positive sequences 5; Utilize mdk gene analyser to carry out the microsatellite locus gene type in 12 sites, finally utilize GeneMapper3.7 (Applied Biosystems) accurately to read and record the allelotrope peak value of each individuality on each site;

Described data processing:

SSR site data are input in software and process according to the requirement of Coancestry1.0.1.2 software, obtain somatotype individuality molecular genetic relation conefficient (r between any two _xY);

According to somatotype individuality molecular genetic relation conefficient (r between any two _xY) calculate mean molecule genetic correlation coefficient (AMR), represent the genetic correlation between all individualities (comprising not somatotype individuality);

Recycling R3.0.2 software is converted into the result obtaining the form of molecule relationship matrix (Numerator Matrix); Utilize the make.positive.definite function of corpcor routine package in R3.0.2 software that above-mentioned matrix is carried out to positive definite (Bending), after positive definite, use again is.positive.definite function validates;

Utilize ASReml3.0 software according to linear mixed model estimation variance component and breeding value.

The present invention's beneficial effect compared with prior art: than the method that relies on physics pedigree assessment breeding value, present method can improve coefficient of relationship accuracy between individuality, is making the most of the advantage aspect breeding value prediction and accuracy thereof.In breeding process, be recurrent by pedigree latent faults of managing and other inevitable errors cause, especially higher to this fecundity of turbot fish colony.The molecule coefficient of relationship of therefore, being inferred by pedigree is not accurate enough often.So the genetic correlation that need to get between estimating individual from molecular level, obtains breeding value predictor accurately with this, better instructs seed selection work with this.

Embodiment

Embodiment

Below by embodiment, the technical solution of the present invention is further explained, but protection scope of the present invention is not subject to any pro forma restriction of embodiment.

A kind of turbot family breeding value appraisal procedure, comprises turbot family structure, the collection of family individual data items and DNA extraction, microsatellite locus gene type and data processing; Concrete steps are as follows:

One. turbot family builds

At the beginning of 2013 (January) candidate's turbot parent population is carried out to genital regulating by temperature control control light.When 4 the end of month, water temperature was applicable to, from candidate parent population, select gonadal maturation, build is complete, and body colour is normal, ingests actively, and without disability bouncing, 2 kilograms of above individualities of body weight carry out artificial insemination, build turbot family.Be specially: zygote is placed on inflation hatching in 80 × 60 × 60cm incubation net cage, and seawater temperature maintains 14～15 DEG C.Hatch and within 3 days, draw afterwards floating ovum according to 20ml/m ²the standard of (350/ml), is moved into (0.5m in the circular glass steel cylinder that mixes up in advance water temperature, salinity, PH and dissolved oxygen by hatchery ², volume 0.5m ³) micro-inflation, Lentic hatching is to emerging, and each family is all cultivated separately.After this cultivating process, with reference to turbot family conventional cultivation method, is cultivated 19 of turbot families altogether.

Two. family individual data items gathers and DNA extraction:

In the time of family individual growth to 4 monthly age, each family is selected the individuality of body weight rank front 60 as core selective breeding family, measures weight data, gets its mean value as family core selective breeding family, 1-19 family weight average value (g) is respectively 8.11,5.71,6.07,7.11,5.15,5.48,6.35,5.73,5.39,4.79,4.39,5.64,4.92,5.66,4.97,4.97,5.00,6.23,6.32.Simultaneously choose at random 20 tail individualities, the standby genes of individuals group DNA extraction of Qu Qi fin ray end flap (be similar to the hair tissue of getting animal, can not cause any harm to individuality) from each family individuality of reserving seed for planting.Extract genomic dna-75 DEG C cryopreservation with reference to ordinary method.

Three. microsatellite locus gene type:

PCR and product detect: select 12 SSR sites that turbot has been announced to carry out SSR-PCR amplification, site title is respectively YSKr271, YSKr262, YSKr108, YSKr231, YSKr80, YSKr244, YSKr197, YSKr115, YSKr221, YSKr124, YSKr218, YSKr259(table 1), ' end is mark 6-FAM, HEX and ROX fluorescent mark respectively for 12 pairs of primer positive sequences 5.PCR reaction system comprises: cumulative volume 25 μ l, wherein genomic dna 2 μ l (50ng/ μ l), TaqDNA polysaccharase 0.2 μ l (5U/ μ l), 10 × PCR Buffer2.5 μ l, Mg ²⁺2 μ l(2.5mmol/L), dNTP2 μ l (2.5mmol/L), the each 0.5 μ l(10 μ mol/L of primer), ddH ₂o15.3 μ l.Pcr amplification condition is: after 95 DEG C of denaturation 5min, enter 25 PCR circulations: 95 DEG C of sex change 40s, the annealing temperature of the each primer of annealing 1min(is in table 1), 72 DEG C are extended 1min, finally extend 5min, 4 DEG C of preservations in 72 DEG C.

The essential characteristic of table 1 micro-satellite primers

Gene type: utilize ABI3130 type mdk gene analyser to carry out micro-satellite somatotype in 12 sites.In each well of 96 orifice plates, add respectively (the deionized formamide: GeneScanTM-500LIZ Size Standard=7.9:0.1 of mark system in the above-mentioned pcr amplification product of 2 μ l and 8 μ l, described ratio is volume ratio) after abundant mixing, 95 DEG C of sex change 5min, finish that rear rapidly 96 orifice plates to be placed in to mixture of ice and water cooling.The 3130 genetic analysis instrument that cooled sample are placed in to Applied Biosystems, carry out fluorescence data collection and gene type.Finally utilize GeneMapper3.7 (Applied Biosystems) accurately to read and record the allelotrope peak value of each individuality on each site.

Four. data processing:

1. somatotype site data are input in software and process according to the requirement of Coancestry1.0.1.2 software, obtain somatotype individuality molecular genetic relation conefficient (r between any two _xY).

2. in order to utilize the molecular genetic relation conefficient between each family somatotype individuality to estimate the genetic correlation coefficient between all individualities, by the r having obtained _xYtransform, after conversion, the genetic correlation degree between all individualities all uses mean molecule genetic correlation coefficient (AMR, average molecular relatedness) to represent, AMR calculation formula is as follows:

In family ${\stackrel{\‾}{r}}_w=\mathrm{\Σ}{r}_w/n_w---\left(1\right)$

Wherein

represent the mean molecule genetic correlation coefficient between individuality in family, ∑ r _wand n _wrepresent respectively molecular genetic relation conefficient sum and number thereof between all somatotype individualities in family.

Between family $\stackrel{\‾}{r_b}=\mathrm{\Σ}{r}_b/n_b---\left(2\right)$

Wherein

represent the mean molecule genetic correlation coefficient between individuality between family, ∑ r _band n _brespectively molecular genetic relation conefficient sum and the number thereof between all somatotype individualities between family.AMR in 19 familys and between family is in table one.

Mean molecule genetic correlation coefficient in 19 familys of table one and between family

。Utilize R3.0.2 software according to formula (1) and (2), calculate the mean molecule genetic correlation coefficient between all individualities, and form a coefficient of relationship matrix.

3. utilizing the make.positive.definite function of corpcor routine package in R3.0.2 software that above-mentioned matrix is carried out to positive definite (Bending) is that its eigenwert is all greater than zero, uses is.positive.definite function validates after positive definite again.

4. according to ASReml3.0 software flow, matrix after positive definite is converted into .grm file, be input to ASReml3.0 software together with phenotypic data file and corresponding pedigree file, use AI-REML algorithm, utilize animal model (as follows) estimation variance component and family breeding value.

y＝Xb+Zu+e

Wherein y is phenotypic number vector, and X and Z are respectively the design matrix of b and u, b, and u and e are respectively fixed effect, and the vector of stochastic effect (additive genetic effect, maternal common environmental effect) and residual error, using age in days as concomitant variable, joins and in model, corrects body weight value.

According to Henderson(1959) mixture equations (as follows) solves animal model.

$\left[\begin{array}{cc}{X}^{\′}X& X^{\′}Z\\ Z^{\′}X& Z^{\′}Z+A^{-1}\mathrm{\λ}\end{array}\right]\left[\begin{array}{c}b\\ \hat{a}\end{array}\right]=\left[\begin{array}{c}{X}^{\′}y\\ Z^{\′}y\end{array}\right]$

Wherein X ' and Z ' are respectively X and Z transpose of a matrix; A ^-1it is A inverse of a matrix matrix;

with

it is respectively the estimated value of fixed effect and stochastic effect;

5. breeding value and the standard error thereof of estimation extract from the .pvc file obtaining.For the method for only utilizing pedigree estimation family breeding value, do not need to calculate .grm file, directly phenotypic data file and known pedigree file are imported to ASReml file and utilize animal model estimation breeding value.According to mean molecule genetic correlation coefficient (AMR) and physics genetic correlation coefficient, the family breeding value of estimation is in table two.

The estimated breeding value of 19 familys of table two

6. utilize cross validation (cross-validation) relatively pRwith the difference of AMR in breeding value accuracy.Utilize R3.0.2 software that the weight data recording is divided into impartial 10 parts at random, wherein 9 parts as training set (training set), remaining a as checking collection (validation set).According to training set data, in ASReml3.0, predict checking collection phenotypic number with animal model.Calculate breeding value accuracy according to following formula

$r(a,\hat{a})=r(y,\hat{y})/\sqrt{h^2}$

itsin

represent breeding value accuracy;

represent to verify the Pearson degree of correlation between predictor and the observed value collecting; h ² represent according to the heritability of pedigree and weight data estimation.Carry out cross validation according to PR and AMR respectively, and repeat 500 times, average as net result.Result show the breeding value accuracy that AMR method estimates (

0.660 refers to the heritability of estimating according to PR, below 0.660 identical with it) will be higher than the accuracy of department of physics's spectral method estimated breeding value

$(r=0.449/\sqrt{0.660}=0.553).$

Technical indicator

A kind of turbot family estimation of breeding value method can meet under the condition without any physics pedigree record, carries out the work of turbot fine-variety breeding, its for Breeding Traits be mainly growth aspect, comprise that body is long, body weight.Especially be applicable to carrying out with the basic population of introducing colony or just build the work of turbot fine-variety breeding.It can meet the turbot breeding value assessment of carrying out family level, any growth phase.

Range of application

This technology is mainly used in turbot fine-variety breeding field, especially introduces colony and physics pedigree and records the imperfect breeding population even lacking.Being growth traits mainly for proterties, is that a kind of breeding value of family level is accurately estimated.

Claims (2)

1. a turbot family breeding value appraisal procedure, is characterized in that described method comprises turbot family structure, the collection of family individual data items and DNA extraction, microsatellite locus gene type and data processing;

Described family individual data items gathers: for the turbot of constructed family, in the time of 4 monthly age, each family is selected the individuality of body weight rank front 60 as core selective breeding family, measure weight data, get its mean value as family core selective breeding family mean value, extract each family individual DNA that reserves seed for planting simultaneously;

Described microsatellite locus gene type: utilize 12 SSR sites that turbot has been announced to carry out SSR-PCR amplification, ' end is mark 6-FAM respectively, HEX and ROX fluorescent mark for 12 pairs of primer positive sequences 5; Utilize mdk gene analyser to carry out the microsatellite locus gene type in 12 sites, finally utilize GeneMapper3.7 accurately to read and record the allelotrope peak value of each individuality on each site;

Described data processing:

SSR site data are input in software and process according to the requirement of Coancestry1.0.1.2 software, obtain somatotype individuality molecular genetic relation conefficient between any two;

According to somatotype individuality molecular genetic calculation of correlation mean molecule genetic correlation coefficient between any two, represent the genetic correlation coefficient between all individualities;

Recycling R3.0.2 software is converted into the result obtaining the form of molecule relationship matrix; Utilize the make.positive.definite function of corpcor routine package in R3.0.2 software that above-mentioned matrix is carried out to positive definite, after positive definite, use again is.positive.definite function validates;

Utilize ASReml3.0 software according to linear mixed model estimation variance component and breeding value.

2. a kind of turbot family breeding value appraisal procedure according to claim 1, is characterized in that described family individual data items acquisition step, extracts 20 individual DNA of each family, and 20 described individualities are for choosing at random.