CN104313135A - Evaluation method of individual breeding values of turbot - Google Patents

Abstract

The invention aims at providing an evaluation method of individual breeding values of turbot to solve the problems that a using background is not clear in source, a physical pedigree is incomplete or the individual kinship coefficient can not be calculated by utilizing physical pedigree information when an introduced turbot colony is used for breeding improved varieties, further solve the problem that evaluation of individual genetic parameters can not be performed and thus make up for deficiencies in the prior art. Compared with a method for evaluating the breeding value by relying on physical pedigree, the method provided by the invention can greatly reduce the time period for evaluation of the individual breeding values, namely that the evaluation of the individual breeding values can be completed by only one generation without performing multi-generation of culture of pedigree materials and other tedious steps.

Description

The individual breeding value appraisal procedure of a kind of turbot

Technical field

The invention belongs to seawater Animal Genetics field, relate to a kind of method realizing carrying out turbot individuality breeding value assessment particularly

Background technology

Turbot (Scophthalmusmaximus L.) to be dwelt seawater fish at the end originating in Europe, because its growth is rapid, low temperature resistant, personality is docile and be easy to the advantages such as intensive culture, it is one of the excellent sea farming kind on the ground such as Europe all the time.China started to introduce turbot in 1992, and broke through productive receivers technology before and after 1999 since, turbot aquaculture industry obtains fast development in China, has become the sea farming kind that northern China coastland is important.But the Bu Shi turbot country of origin of China, relies on the European turbot country of origin to provide for a long time, supplements provenance always.Meanwhile, owing to ignoring prevalent variety cultivation in domestic production, for many generations cultivation and inbreeding causes generally occurring kind of a matter degradation phenomena in aquaculture, and main manifestations is the phenomenons such as the speed of growth reduces, albefaction rate increases, seedling rate reduction.For the culture-cycle, when introducing the initial stage, the culturing time reaching commercial specification is 7-8 month, and generally extends to about 14 months now.Therefore, fine-variety breeding has become one of important research content of China's turbot breeding production Sustainable Healthy Development.Research shows that based on traditional fine-variety breeding pattern of complete physical pedigree record be one of important channel of carrying out turbot fine-variety breeding.Complete, accurately pedigree information not only can effectively instruct parent to select and remain, avoid inbreeding depression; The individual coefficient of relationship simultaneously utilizing pedigree information to extrapolate also is used to the significant data carrying out genetic parameter assessment.But due to turbot sexual maturation cycle long (2-3), individual mark requires strict, domestic at present almost do not have nursery to possess many generations, complete individual physics pedigree recorded information; And turbot belongs to introduction species, the genetic background of colony's individuality is unclear, and this also causes very large difficulty for carrying out turbot fine-variety breeding.Rebuild although molecule marker partly can realize physics pedigree, its finiteness outstanding behaviours, for can only be reconstructed into a Parent generation, can only extrapolate the genotype combination of Parent, and cannot extrapolate concrete male parent, maternal genotype etc.Then, in fine-variety breeding process, most important genetic parameter assessment is exactly the assessment of individuality/family breeding value.Therefore, in production in the urgent need to a kind of without the need to many technology just can carrying out breeding value assessment for detailed physical pedigree record to individuality.

Summary of the invention

The object of this invention is to provide the individual breeding value appraisal procedure of a kind of turbot, Background sources is utilized to fail to understand to solve, when physics pedigree is incomplete or utilize the turbot colony introduced to carry out fine-variety breeding, department of physics's spectrum information cannot be utilized to extrapolate individual coefficient of relationship, thus the problem of idiogenetics parameter evaluation can not be carried out, thus make up the deficiencies in the prior art.

The individual breeding value appraisal procedure of turbot of the present invention, includes following step:

1) first turbot male and female individuality to be detected is built family, described family is maternal family half sibs group and/or family full-sibs;

2) to build family in offspring individual after cultivation, choose the measurement that all or part of individuality carries out growth traits; Obtain the observed value of individual growth traits;

3) DNA extracting the female male parent building family carries out SSR Genotyping; Record the allelotrope peak-data of each individuality on each SSR site;

Utilize disclosed 12 turbot SSR sites to carry out SSR-PCR amplification, 12 pairs of primer forward sequence 5 ' ends mark 6-FAM, HEX or ROX fluorescent mark respectively; Utilize mdk gene analyser to carry out the SSR gene type in 12 sites, finally utilize GeneMapper3.7 (Applied Biosystems) accurately read and record each individuality allelotrope peak-data on each SSR site.

4) utilize Coancestry1.0.1.2 software to calculate SSR typing data and try to achieve coefficient of parentage coancestry between parent individual and inbreeding coefficient F;

Coefficient of parentage between family progeny individuality is tried to achieve according to formula (1) by the coefficient of parentage between its Parent, wherein individuality is obtained by formula (2) with the coefficient of parentage of itself, and calculate the Genetic correlation coefficient between offspring individual by formula (3), i.e. molecular genetic relation conefficient;

${\mathrm{\θ}}_{\mathrm{PQ}}=\frac{1}{4}({\mathrm{\θ}}_{\mathrm{AC}}+{\mathrm{\θ}}_{\mathrm{AD}}+{\mathrm{\θ}}_{\mathrm{BC}}+{\mathrm{\θ}}_{\mathrm{BD}})---\left(1\right)$

${\mathrm{\θ}}_{\mathrm{AA}}=\frac{1}{2}(1+F_A)---\left(2\right)$

r _PQ＝2θ _PQ (3)

Wherein A and B, C and D represent the Parent of P and Q respectively, and θ represents coefficient of parentage, and F represents inbreeding coefficient, and r represents Genetic correlation coefficient;

5) R3.0.2 software is then utilized coefficient of parentage between Parent to be converted into molecular genetic correlation matrix between its offspring individual; Finally utilize the individual breeding value of ASReml computed in software:

y＝Xb+Zu+e

Wherein y represents the observed value of turbot family offspring individual, and bi represents fixed effect, and u represents stochastic effect, and e represents residual error effect.

Heretability estimate value is tried to achieve again according to narrow-sense heritability formula:

$h^2={\mathrm{\σ}}_a^2/{\mathrm{\σ}}_p^2$

with represent additive genetic variance and performance variance respectively.

Compared to the method relying on physics pedigree assessment breeding value, the method of present method has following advantage: the time limit 1) significantly can saving the assessment of individual breeding value, namely without the need to through being too much commissioned to train tedious steps such as educating a based material, a generation is only needed can to complete the assessment of individual breeding value; 2) tolerance range can increase.In turbot breeding process, by poor management and the pedigree latent fault that causes of other reason be recurrent, the fish long for this sexual maturation cycle of turbot, reproductivity is high are more common.Therefore, the coefficient of relationship of being inferred by pedigree is often not accurate enough, and molecule coefficient of relationship, then without the need to being limited to this, namely can be used for idiogenetics parameter evaluation by means of only the molecule coefficient of relationship between direct analysis parent.

Embodiment

Under method of the present invention can meet the condition without any physics pedigree record, carry out the work of turbot fine-variety breeding, its for Breeding Traits be mainly the growth traitss such as body length, body weight.Especially be applicable to introducing the work that colony or the basic population just built carry out turbot fine-variety breeding.The individual breeding value assessment of any growth phase turbot can be met.

Genetic parameter is the substance of quantitative genetics, comprises genetic correlation three basic parameters between heritability, repetition rate, proterties.The relation of heritability reflection character inheritance and environment, repetition rate reflects the relation between same proterties each metric, and genetic correlation shows the genetic affinity between proterties and proterties.This is most important three kinds of relations in the various relation of quantitative character.Had the parameter of these three kinds of relations, in quantitative genetics, various calculating has just had framework, is therefore the basis of various relevant calculation.

Coefficient of parentage is defined as: on a locus, the probability that two one of them individual random allele individual are same with another individual random allele descendant.

Inbreeding coefficient (inbreeding coefficient) refers to that two gene sources, in germanus probability, are applicable to body one by one on the arbitrary site of body one by one, the degree of sibship between the parent representing this individuality.

Family full-sibs: the offspring of two identical parents is referred to as family full-sibs

Maternal family half sibs group: refer to that multiple familys that a female parent and multiple male parent breed generation form a maternal family half sibs group.

Below method of the present invention is further described.

The individual breeding value appraisal procedure of turbot of the present invention, comprises turbot family structure, the collection of family individual data items and DNA extraction, microsatellite locus gene type and data processing step; Specific as follows:

One. turbot family builds

At the beginning of 2013 (January) by temperature control control light, genital regulating is carried out to candidate's turbot parent fish.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, and ingest actively, without disability bouncing, the individuality that body weight is more than 2 kilograms carries out artificial insemination, builds turbot family.Be specially: manually squeeze ovum to the male and female parent population picked out respectively and squeeze essence, mixing fertilization in beaker, then zygote be placed on inflation hatching in 80 × 60 × 60cm incubation net cage, seawater temperature maintains about 14.5 DEG C.Hatch and draw floating ovum after 100 hours and according to the standard of 10-30ml/m2 (350/ml), move into (0.5m in the circular glass steel cylinder mixing up water Inversion phenomenon, PH and dissolved oxygen in advance by hatchery ², volume 0.5m ³) micro-inflation, Lentic hatching is to emerging, and each family is cultivated all separately.After this cultivating process is with reference to turbot family conventional cultivation method, cultivates turbot family 39 altogether, relates to parent and comprise 31 tail milters and 16 tail rauns, comprise 9 maternal family half sibs groups and 6 family full-sibses.

Two. family individual data items gathers and DNA extraction:

When family individual growth to 3 monthly age, each family selects the individuality of before body weight rank 60 as core selective breeding family, and it measures weight data, 1-39 family Weight averages (g) is respectively 10.27, 10.66, 9.93, 9.64, 10.62, 9.62, 9.92, 9.28, 9.84, 9.86, 11.43, 10.69, 11.71, 7.61, 8.11, 5.71, 6.07, 7.11, 5.15, 6.44, 5.79, 5.95, 5.47, 5.48, 6.35, 5.73, 5.39, 4.79, 4.39, 5.35, 5.63, 5.64, 4.92, 5.66, 4.97, 4.97, 5.00, 6.23, 6.32.The parent simultaneously related to each family gets the standby genes of individuals group DNA extraction of its fin ray end flap (be similar to the hair tissue getting animal, can not cause any harm to individuality).Genomic dna is extracted ,-75 DEG C of cryopreservation with reference to ordinary method.

Three. microsatellite locus gene type:

PCR and product detect: SSR-PCR amplification is carried out in 12 the SSR sites selecting turbot to announce, and site title is respectively YSKr271, YSKr262, YSKr108, YSKr231, YSKr80, YSKr244, YSKr197, YSKr115, YSKr221, YSKr124, YSKr218, YSKr259 (table 1), 12 pairs of primer positive sequence 5 ' ends mark 6-FAM, HEX and ROX fluorescent mark respectively.PCR reaction system comprises: cumulative volume 25 μ l, wherein genomic dna 2 μ l (50ng/ μ l), Taq DNA polymerase 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), each 0.5 μ l of primer (10 μm of ol/L), ddH ₂o15.3 μ l.Pcr amplification condition is: enter 25 PCR circulations after 95 DEG C of denaturation 5min: 95 DEG C of sex change 40s, annealing 1min (annealing temperature of each primer is in table 1), 72 DEG C extend 1min, finally extend 5min in 72 DEG C, 4 DEG C of preservations.

Table 1: the essential characteristic of micro-satellite primers

Gene type: utilize ABI3130 type mdk gene analyser to carry out micro-satellite somatotype in 12 sites.Add after the above-mentioned pcr amplification product of 2 μ l fully mixes with mark system in 8 μ l (deionized formamide: GeneScanTM-500LIZ Size Standard=7.9:0.1) in each well of 96 orifice plates respectively, 95 DEG C of sex change 5min, terminate rapidly 96 orifice plates to be placed in mixture of ice and water and to cool afterwards.Cooled sample is placed in 3130 Genetic Analyser of Applied Biosystems, carries out fluorescence data collection and gene type.GeneMapper3.7 (Applied Biosystems) is finally utilized accurately to read and record the allelotrope peak value of each individuality on each site.

Four, statistical study:

1. the estimation of Genetic correlation coefficient

Utilize Coancestry1.0.1.2 software to process SSR typing data, calculate the coefficient of parentage (coancestry) and inbreeding coefficient (F) of trying to achieve between parent individual.

Coefficient of parentage between offspring individual can be tried to achieve according to formula (1) by the coefficient of parentage between its Parent, wherein individuality is obtained by formula (2) with the coefficient of parentage of itself, when obtaining coefficient of parentage between individuality, the twice equaling coefficient of parentage according to Genetic correlation coefficient (r) between individuality directly can calculate the relevant i.e. formula (3) of molecular genetic between offspring individual.

${\mathrm{\θ}}_{\mathrm{PQ}}=\frac{1}{4}({\mathrm{\θ}}_{\mathrm{AC}}+{\mathrm{\θ}}_{\mathrm{AD}}+{\mathrm{\θ}}_{\mathrm{BC}}+{\mathrm{\θ}}_{\mathrm{BD}})---\left(1\right)$

${\mathrm{\θ}}_{\mathrm{AA}}=\frac{1}{2}(1+F_A)---\left(2\right)$

r _PQ-2θ _PQ (3)

Note: A and B, C and D are the Parent of P and Q respectively, and θ representative organizes coefficient altogether, and F represents inbreeding coefficient, and r represents Genetic correlation coefficient.

Utilize R3.0.2 software according to above-mentioned principle and formula, coefficient of parentage between Parent is converted into the molecular genetic correlation matrix (see table 2) between its offspring individual, according to physics Genetic correlation coefficient matrix (see table 3) between the offspring individual that complete pedigree (three generations's pedigree) calculates.

Table 2: molecular genetic correlation matrix, wherein the first row and first row are family number, identical with between family of the Genetic correlation coefficient between offspring individual, so place shows is the estimated value of family level.

Table 3: physics Genetic correlation coefficient matrix, the first row and first row are family number, identical with between family of the Genetic correlation coefficient between offspring individual, so place shows is the estimated value of family level.

In family level, the Pearson correlation coefficient between Computational Physics coefficient of heredity and molecular genetic coefficient.

Result shows, and the Pearson's coefficient between these two kinds of relation conefficients is 0.872 (P<0.01).

2. the estimation of coefficient of heredity

Estimate the heritability of turbot colony according to animal model, predict individual breeding value.

y _ijk＝μ+cb _i+a _j+d _k+e _ijk

Y represents the body weight value of turbot individuality, and μ represents average, and bi represents i-th individual age in days, and as concomitant variable, c is corresponding regression coefficient, and aj represents additive genetic effect, and dk represents maternal instinct common environmental effect, and e represents residual error effect.

Variance component structure:

A, d and e represent additive genetic effect respectively, maternal instinct common environmental effect and residual error effect, with

Their variance respectively.

Concrete computation process is completed by ASReml3.0 software, requirement according to ASReml software arranges pedigree file and data file, wherein pedigree file arranges according to physics Genetic correlation coefficient and molecule relation conefficient respectively, then above-mentioned animal model is input in ASReml software, finally utilizes AI-REML algorithm to try to achieve the estimated value of each point of difference component and breeding value respectively according to two kinds of pedigree file iteration.Heretability estimate value is tried to achieve again according to narrow-sense heritability formula (as follows).

$h^2={\mathrm{\&sigma;}}_a^2/{\mathrm{\&sigma;}}_p^2$

with represent additive genetic variance and performance variance respectively.

Result shows: the heritability utilizing physics Genetic correlation coefficient and molecular genetic relation conefficient to estimate is respectively 0.5470 (± 0.2161) and 0.5220 (± 0.1336).Utilize t-test to check the significance of difference between two kinds of heritability, t value is 0.098, therefore difference is not remarkable.Between the individual breeding value of two kinds of method predictions, the Pearson degree of correlation is 0.878.

3. cross validation

This experiment utilizes cross validation method to compare two kinds of method predictive abilities and predicts the accuracy of individual breeding value.Method is as follows: whole data sets is divided into ten parts at random, a copy of it is as checking collection, all the other nine parts as training set, namely predict the breeding value of checking collection with training set Modling model at every turn, to verify that the Pearson degree of correlation between the actual phenotypic number of collection and prediction breeding value evaluates two kinds of method predictive ability sizes, weigh the accuracy of prediction breeding value with this.

Result: two kinds of method predictive abilities according to physics Genetic correlation coefficient and molecular genetic relation conefficient are respectively 0.8135 and 0.8136, therefore illustrate that the predictive ability difference of two kinds of methods is very little.

No matter in heritability estimating or individual breeding value prediction, the progeny molecule Genetic correlation coefficient utilizing Parent SSR site to derive carries out the method for genetic evaluation and the method no significant difference according to complete pedigree (three generations's pedigree), for turbot unexpected mass incident improves a kind of good alternative method.

Claims (4)

1. the individual breeding value appraisal procedure of turbot, it is characterized in that, described method includes following step:

1) first turbot male and female individuality to be detected is built family,

2) to build family in offspring individual after cultivation, choose the measurement that all or part of individuality carries out growth traits; Obtain the observed value of individual growth traits;

3) DNA extracting the female male parent building family carries out SSR Genotyping; Record the allelotrope peak-data of each individuality on each SSR site;

4) utilize Coancestry1.0.1.2 software to calculate SSR typing data and try to achieve coefficient of parentage coancestry between parent individual and inbreeding coefficient F;

Coefficient of parentage between family progeny individuality is tried to achieve according to formula (1) by the coefficient of parentage between its Parent, wherein individuality is obtained by formula (2) with the coefficient of parentage of itself, and calculate the Genetic correlation coefficient between offspring individual by formula (3), i.e. molecular genetic relation conefficient;

${\mathrm{\&theta;}}_{\mathrm{PQ}}=\frac{1}{4}({\mathrm{\&theta;}}_{\mathrm{AC}}+{\mathrm{\&theta;}}_{\mathrm{AD}}+{\mathrm{\&theta;}}_{\mathrm{BC}}+{\mathrm{\&theta;}}_{\mathrm{BD}})---\left(1\right)$

${\mathrm{\&theta;}}_{\mathrm{AA}}=\frac{1}{2}(1+F_A)---\left(2\right)$

r _PQ＝2θ _PQ (3)

Wherein A and B, C and D represent the Parent of P and Q respectively, and θ represents coefficient of parentage, and F represents inbreeding coefficient, and r represents Genetic correlation coefficient;

5) R3.0.2 software is then utilized coefficient of parentage between Parent to be converted into molecular genetic correlation matrix between its offspring individual; Finally utilize the individual breeding value of ASReml computed in software:

y＝Xb+Zu+e

Wherein y represents the observed value of turbot family offspring individual, and bi represents fixed effect, and u represents stochastic effect, and e represents residual error effect;

Heretability estimate value is tried to achieve again according to narrow-sense heritability formula:

$h^2={\mathrm{\&sigma;}}_a^2/{\mathrm{\&sigma;}}_p^2$

with represent additive genetic variance and performance variance respectively.

2. the method for claim 1, is characterized in that, described step 1) in family be maternal family half sibs group and/or family full-sibs.

3. the method for claim 1, is characterized in that, described step 3) in the micro-satellite primers sequence information used by SSR Genotyping as follows:

4. the method for claim 1, is characterized in that, described step 3) described in SSR Genotyping be utilize mdk gene analyser to carry out SSR gene type.