CN110122411A - The construction method of one breeder inbred strais - Google Patents
The construction method of one breeder inbred strais Download PDFInfo
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- CN110122411A CN110122411A CN201910395006.6A CN201910395006A CN110122411A CN 110122411 A CN110122411 A CN 110122411A CN 201910395006 A CN201910395006 A CN 201910395006A CN 110122411 A CN110122411 A CN 110122411A
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- 238000010276 construction Methods 0.000 title claims abstract description 20
- 241000287828 Gallus gallus Species 0.000 claims abstract description 56
- 238000009399 inbreeding Methods 0.000 claims abstract description 44
- 238000000034 method Methods 0.000 claims abstract description 13
- 238000009395 breeding Methods 0.000 claims abstract description 6
- 230000001488 breeding effect Effects 0.000 claims abstract description 6
- 238000000465 moulding Methods 0.000 claims abstract description 4
- 235000013601 eggs Nutrition 0.000 claims description 35
- 238000012163 sequencing technique Methods 0.000 claims description 22
- 210000004369 blood Anatomy 0.000 claims description 18
- 239000008280 blood Substances 0.000 claims description 18
- 108090000790 Enzymes Proteins 0.000 claims description 14
- 102000004190 Enzymes Human genes 0.000 claims description 14
- 238000001914 filtration Methods 0.000 claims description 14
- 230000012447 hatching Effects 0.000 claims description 11
- 108090000623 proteins and genes Proteins 0.000 claims description 10
- 230000029087 digestion Effects 0.000 claims description 9
- 230000008021 deposition Effects 0.000 claims description 8
- 210000000582 semen Anatomy 0.000 claims description 8
- 238000004458 analytical method Methods 0.000 claims description 7
- 244000144977 poultry Species 0.000 claims description 7
- 108091008146 restriction endonucleases Proteins 0.000 claims description 7
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 6
- 238000012797 qualification Methods 0.000 claims description 6
- 244000144987 brood Species 0.000 claims description 5
- 238000001514 detection method Methods 0.000 claims description 5
- 230000007613 environmental effect Effects 0.000 claims description 5
- 238000010606 normalization Methods 0.000 claims description 5
- 241001465754 Metazoa Species 0.000 claims description 4
- 244000144972 livestock Species 0.000 claims description 4
- 238000007400 DNA extraction Methods 0.000 claims description 3
- 239000003146 anticoagulant agent Substances 0.000 claims description 3
- 229940127219 anticoagulant drug Drugs 0.000 claims description 3
- 238000005286 illumination Methods 0.000 claims description 3
- 230000009027 insemination Effects 0.000 claims description 3
- 238000007689 inspection Methods 0.000 claims description 3
- 210000005259 peripheral blood Anatomy 0.000 claims description 3
- 239000011886 peripheral blood Substances 0.000 claims description 3
- 238000012372 quality testing Methods 0.000 claims description 3
- 238000011084 recovery Methods 0.000 claims description 3
- 238000007619 statistical method Methods 0.000 claims description 3
- 238000009423 ventilation Methods 0.000 claims description 3
- 238000012795 verification Methods 0.000 claims description 3
- 102000012410 DNA Ligases Human genes 0.000 claims description 2
- 108010061982 DNA Ligases Proteins 0.000 claims description 2
- 230000002779 inactivation Effects 0.000 claims description 2
- 238000002347 injection Methods 0.000 claims description 2
- 239000007924 injection Substances 0.000 claims description 2
- 238000002205 phenol-chloroform extraction Methods 0.000 claims description 2
- 238000001228 spectrum Methods 0.000 claims description 2
- 230000001850 reproductive effect Effects 0.000 abstract description 4
- 230000035899 viability Effects 0.000 abstract description 3
- 230000002045 lasting effect Effects 0.000 abstract description 2
- 238000007726 management method Methods 0.000 description 8
- 230000009286 beneficial effect Effects 0.000 description 4
- 230000002068 genetic effect Effects 0.000 description 4
- 108091064702 1 family Proteins 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000004064 recycling Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 241000271566 Aves Species 0.000 description 1
- GJYPUXPGBAIWQC-UHFFFAOYSA-N C(Cl)(Cl)Cl.C1(=CC=CC=C1)O.C1=CC=CC=C1 Chemical compound C(Cl)(Cl)Cl.C1(=CC=CC=C1)O.C1=CC=CC=C1 GJYPUXPGBAIWQC-UHFFFAOYSA-N 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 241001269238 Data Species 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/02—Breeding vertebrates
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- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Animal Behavior & Ethology (AREA)
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- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses the construction methods of a breeder inbred strais, the present invention is set up by family individual, family expands procreation and records and analyzes, breeding chickling management, the establishment molding of chicken inbred strais and etc. successfully set up out the inbred strais chicken of the high coefficient of inbreeding, the invention also discloses the identification methods of the inbred strais chicken coefficient of inbreeding simultaneously, the present invention is by setting up chicken inbred strais, and carry out coefficient of inbreeding identification, solve in the complete situation of pedigree, the individual coefficient of inbreeding, affiliation inaccuracy and the coefficient of inbreeding it is low the problems such as；Matingplan is selected and remain by scientific and reasonable, the problem and inbred strais for effectively improving the slumps of disastrous proportions such as the growth performance, reproductive performance, viability of inbreeding offspring are difficult to the problems such as lasting.
Description
Technical field
The present invention relates to genetic arts, are specifically related to the construction method of a breeder inbred strais.
Background technique
Inbred strais, which refers to, carries out inbreeding by continuous parental generation and the modes such as filial generation or half sibs, full sibs, obtains
Phenotype tends towards stability consistent individual.Due to phenotype and the homozygosis relatively of genome and stablize, inbred strais is in new varieties (strain)
Cultivate, heterosis, hybrid vigor utilize and inbreeding depression molecular mechanism research, important economical trait related gene (chromosomal region) identification,
Deleterious gene rejecting etc. has important utility value.The inbreeding level of inbred strais generallys use the coefficient of inbreeding to indicate,
The pedigree coefficient of inbreeding generally is mostly used, recently as the development of molecular biology and bioinformatics technique, molecule inbred strais
Number is also applied.
The Local chicken breeds resource in China passes through long-term natural selection, and the overwhelming majority shows diversified outer in kind
Looks feature and biggish production performance difference have genetic diversity abundant.However, this characteristic but gives the foundation of inbred strais
Bring many difficulties.Due to the special oviparity modes of reproduction of chicken and shorter generation inteval (generally 1 year), using complete
(half sibs) mating pattern born of the same parents is the most effective technological means realizing the coefficient of inbreeding and quickling increase.Common practices is to establish house
System, then directly select and remain full sibs (half sibs) individual and mate, but there are many defects for this way: first is difficult to standard
The coefficient of inbreeding of apolegamy individual is really estimated, even if in the complete situation of pedigree, because the foundation of any one pedigree is all
Defaulting affiliation coefficient between the coefficient of inbreeding of initial underlying group individual, individual is zero, and pedigree record is not in production practices
Complete or even vicious phenomenon is generally existing；Second can ignore the genetic background of apolegamy individual, it is difficult to according to specific inbreeding mesh
Mark requires the inbreeding rate flexible operating reached.Third, not scientific and reasonable selects and remain matingplan, just not can avoid inbreeding
The problem of the slumps of disastrous proportions such as growth performance, reproductive performance, viability that offspring may occur, especially reproductive performance are to establish inbreeding
It is the factor considered first, inbred strais is difficult to continue.Therefore, a kind of quick, efficient, accurate chicken inbred strais construction method is established
The technical issues of being those skilled in the art's urgent need to resolve.
Summary of the invention
In view of this, the present invention provides the construction methods of a breeder inbred strais
To achieve the goals above, the present invention adopts the following technical scheme:
The construction method of chicken inbred strais, includes the following steps
(1) family individual is set up；
(2) family expands procreation and records and analyzes；
(3) breeding chickling management；
(4) the establishment molding of chicken inbred strais.
Further, above-mentioned family individual is set up method particularly includes:
By group based on the chicken group in peak age in days of laying eggs, the almost the same male and female chicken individuals of phenotypic characteristic are chosen,
Set up individual family；Each family is made of 1 cock and 10~12 hens, and it is 30 that described coefficient sets, which build quantity,
More than.
Further, above-mentioned family expands procreation and records and analyzes method particularly includes:
Individual record is done to the hen in each family, and artificial insemination is carried out to the hen in the individual family of establishment,
Mark each egg correspondence family and corresponding hen number；It is continuous collect hatching egg 12~15 days after, to obtained egg into
The hatching of row pedigree, obtains seedling chicken；Wing number is worn to the seedling chicken after hatching, and indicates corresponding family and corresponding hen.
It is using above-mentioned further beneficial effect, the present invention can accurately distinguish different familys by establishing pedigree
With (partly) complete in family compatriot's individual.
Further, above-mentioned breeding chickling management method particularly includes:
It is right in the closed cage henhouse that the environment such as temperature, humidity, ventilation, illumination automatically control and under normalization condition
Chick carries out feeding management, and the feeding management of the chicken divides brood time, finishing period and laying period three phases；By the mother of laying period
Cage on chicken does individual and lays eggs record according to " performance of poultry measure term and measure statistical method NY/T823-2004 ".
Further, above-mentioned standard condition be meet NYT388-1999 " livestock and poultry farm environmental quality standards " and
The condition that NYT1167-2006 " livestock and poultry farm environmental quality and sanitary control specification " is required.
Further, the brood time of above-mentioned chicken is 0~7 week, and finishing period is 8~17 weeks, and laying period is 18~66 weeks.
It is using above-mentioned further beneficial effect, feeding management, energy is carried out under normalization condition through the invention
Enough accurate performance datas for obtaining individual, and such environmental effects are reduced to the maximum extent, improve chicken inbred strais of the present invention
Set up success rate.
Further, above-mentioned chicken inbred strais establishment molding concrete operations the following steps are included:
Step 1: all upper cage hens of statistics start to lay eggs age in days, the weight that starts to lay eggs, start the weight of egg laid eggs, 43
Week old egg number, 43 week old egg sizes, 43 week old weight calculate average value, select and remain the health of average value bound 5~15%
Body, and phenotype consistency is taken into account,
Step 2: pedigree information verification being carried out to the healthy individuals selected and remain in step 1, and is included into place family.Count each family
Selected number of individuals in system, the family for being selected hen number of individuals > 10 are set up for inbred strais；Offspring hen is extremely in the family
Rare 1 full sibs hen, and at least from half with match hen；
Step 3: acquiring 0.5ml~1.5ml blood from the wing venous of selected hen individual, carry out letter after extracting DNA
Change gene order-checking, obtains genome mononucleotide polymorphic SNP variation；
Step 4: based on SNP variation information, the molecule coefficient of inbreeding of sequencing individual is calculated using biosoftware PLINK；Root
According to the molecule coefficient of inbreeding measured, in same family, individual and its full sibs of the molecule coefficient of inbreeding lower than 0.100 are rejected,
The full sibs male and female chicken individuals chosen according to the molecule coefficient of inbreeding, expand numerous, full sibs using full mode
Cock more than one, successively semen deposition, previous cock semen deposition is reserved seed for planting and is spaced 5-7 days after egg, then is continued with next cock
Semen deposition；
Step 5: the subculture expansion of inbred strais is numerous, persistently using full mode to get chicken inbred strais.
Further, described in above-mentioned steps 3 simplify gene order-checking concrete operation method the following steps are included:
1) individual blood acquires
Selected hen wing venous 0.5~1.5ml of whole blood is acquired using disposable medical syringe, is injected rapidly after acquisition
Contain 1.5~2 μ L0.5mol/LNa2EDTA anti-coagulants without in enzyme pipe, then will be placed under 0~4 DEG C of environment and save without enzyme pipe
It is spare；
2) blood DNA extraction and quality testing
It is sucked out under room temperature without the 0.2~0.3ml of blood saved backup in enzyme pipe, is taken out using animal peripheral blood benzene phenol-chloroform
Formulation extracts individual blood DNA, and analyzes DNA integrity degree by agarose gel electrophoresis, detects the pure of DNA with spectrophotometer
Degree；
3) library construction and simplified gene order-checking
1. extracting 300~500ng of genomic DNA of quality inspection qualification, 0.4~0.6U EcoRI, T4 DNA connection is added
Enzyme, ATP and EcoRI connector react 2.5~3.0 hours at 35~37 DEG C, and 60~65 DEG C are annealed 0.5~1.0 hour, then
The end the restriction enzyme Reads1 end EcoRI, Reads2 NlaIII or Hin1IICATG and NlaIII connector is added to carry out double enzymes
It cuts, double digestion reacts 2.5~3.0 hours at 35~37 DEG C, places 20~30 points in 60~65 DEG C of PCR instruments after reaction
Clock inactivates restriction endonuclease
2. carrying out Piece Selection to connection product using agarose gel electrophoresis, 400~600bp recycling digestion is selected to produce
Object；
3. carrying out DNA to recovery product using Qubit3.0 to quantify, mixed in equal amounts sample；
4. carrying out DNA library building to mix products using IlluminaTruSeq kit；
The NlaIII connector can be by Gallus gallus.WASHU chicken with reference to restriction enzyme in genome sequence
The type and distribution of the restriction enzyme site of enzyme select other suitable enzymes to carry out digestion and build library；
4) original sequence data statistics and filtering
Sequencing steps 3) obtained in original series contain belt lacing, low-quality reads, in order to guarantee information analysis
Quality filters rawreads, obtains cleanreads for subsequent analysis；The step of data filtering, is as follows:
1. removing the paired-endreads of belt lacing；
2. needing to remove when the content of the N contained in single-ended sequencing read is more than the 10% of this read length ratio
This is to paired-endreads；
3. being needed when the low quality base number contained in single-ended sequencing read is more than the 50% of this read length ratio
This is removed to paired-endreads；
5) Cleanreads sequence data compares after filtering；
The effective sequencing data of the clean reads obtained after filtering, which is compared to Gallus gallus.WASHUC chicken, refers to base
Because of group；
6) mononucleotide polymorphic site SNP is identified；
According to comparison result, genome of the sample average depth 5 or more is chosen, and is defined as dd RAD label, is carried out
SNP detection inside group based on sample；
7) the molecule coefficient of inbreeding calculates
According to individual SNP qualification result, the molecule inbred strais of sequencing individual is calculated using science of heredity statistical software PLINK
Number.
Further, above-mentioned steps 3) in 1. described in EcoRI connector be containing the Index sequence for distinguishing sample
EcoRI connector.
It is using above-mentioned further beneficial effect, using identification genes of individuals group high density SNP marker, and then can
Accuracy measures the coefficient of inbreeding of each individual of initial population, solves because of no pedigree record, pedigree record generation number is very few or is
Spectrum record it is wrong and caused by can not calculate individual coefficient of inbreeding problem.
The beneficial effects of the present invention are: the present invention is by establishing pedigree, in conjunction with genome molecules labelling technique accuracy
The family individual coefficient of inbreeding is measured, matingplan is selected and remain using the full sibs of innovation, underlying group genetic background can effectively be avoided to interfere,
Inbred strais caused by overcoming because of inbreeding offspring's slump of disastrous proportions is difficult to continue problem, realizes quick, efficient, accurate group of chicken inbred strais
It builds.This method is suitable for all chicken kind resources, is also applied for the establishment of other resource of animal and birds inbred strais.
Detailed description of the invention
Fig. 1 is chicken inbred strais constructional flow figure of the present invention.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
Embodiment 1:
By taking langshan chicken as an example, the construction method of langshan chicken inbred strais includes the following steps
(1) family individual is set up
By group based on the langshan chicken group in 28~35 week old of peak age in days of laying eggs, black plumage, black shin, upright list are chosen
The almost the same cock 30 of hat, white skin isophenous feature, hen 300 set up 30 individual familys, and each family is by 1
Cock and 10 hen compositions, upper individual cage.
(2) family expands procreation and records and analyzes
To in the individual family of establishment hen carry out artificial insemination, mark each egg correspondence family and corresponding mother
Chicken number, if No. 1 family the 1st is all denoted as 01-01 with hatching egg that hen produces, the 2nd is all remembered with the produced hatching egg of hen
For 01-02, remaining hen and other familys and so on；After continuous collection hatching egg 10 days, pedigree is carried out to obtained egg
Hatching, obtains seedling chicken；Wing number is worn to the seedling chicken after hatching, and indicates corresponding family and corresponding hen；Specific full sibs is female
Chicken selection result is as shown in table 1.
(3) breeding chickling management
It is right in the closed cage henhouse that the environment such as temperature, humidity, ventilation, illumination automatically control and under normalization condition
Chick carries out feeding management, and the feeding management of the chicken divides brood time 0~7 week, finishing period 8~17 weeks and laying period 18~66
All three phases；By the upper cage of the hen of laying period, according to " performance of poultry measures term and measure statistical method NY/T823-
2004 ", individual is done to lay eggs record.
The establishment of 2 chicken inbred strais of embodiment forms
Step 1: count cage offspring hen in all familys start to lay eggs age in days, the weight that starts to lay eggs, start the chicken laid eggs
Egg size amount, 43 week old egg numbers, 43 week old egg sizes, 43 week old weight calculate average value, select and remain average value bound 5~15%
Healthy individuals, and take into account phenotype consistency,
Step 2: pedigree information verification being carried out to the healthy individuals selected and remain in step 1, and is included into place family.Count each family
Selected number of individuals in system, the family for being selected hen number of individuals > 10 are set up for inbred strais；
Step 3: acquiring 1.5ml blood from the wing venous of selected hen individual, carry out simplifying genome after extracting DNA
Sequencing obtains genome mononucleotide polymorphic SNP variation；
Step 4: based on SNP variation information, the molecule coefficient of inbreeding of sequencing individual is calculated using biosoftware PLINK；Root
According to the molecule coefficient of inbreeding measured, in same family, individual and its full sibs of the molecule coefficient of inbreeding lower than 0.100 are rejected,
The full sibs male and female chicken individuals chosen according to the molecule coefficient of inbreeding, expand numerous, full sibs using full mode
Cock more than one, successively semen deposition, previous cock semen deposition is reserved seed for planting and is spaced 5-7 days after egg, then is continued with next cock
Semen deposition；
Step 5: the subculture expansion of inbred strais is numerous, persistently using full mode to get chicken inbred strais.
Full sibs hen selects in 1 family of table
Embodiment 3 carries out coefficient of inbreeding test to obtained chicken inbred strais offspring
1) individual blood acquires
Selected hen wing venous whole blood 1.5ml is acquired using disposable medical syringe, injection contains rapidly after acquisition
1.5μL0.5mol/LNa2EDTA anti-coagulants without in enzyme pipe, then will be placed under 4 DEG C of environment and save backup without enzyme pipe；
2) blood DNA extraction and quality testing
It is sucked out under room temperature without the blood 0.3ml saved backup in enzyme pipe, is mentioned using animal peripheral blood phenol-chloroform extraction method
Individual blood DNA is taken, and DNA integrity degree is analyzed by agarose gel electrophoresis, with the purity of spectrophotometer detection DNA；
3) library construction and simplified gene order-checking
1. extract quality inspection qualification genomic DNA 500ng, addition 0.6U EcoRI, T4 DNA ligase, ATP and
EcoRI connector reacts 3.0 hours at 37 DEG C, and 65 DEG C are annealed 0.5 hour, then plus restriction enzyme Reads1 end EcoRI,
The end Reads2 NlaIII and NlaIII connector carry out double digestion, and double digestion reacts 2.5 hours at 37 DEG C, after reaction 65
30 minutes inactivation restriction endonucleases are placed in DEG C PCR instrument；
2. carrying out Piece Selection to connection product using agarose gel electrophoresis, 400~600bp digestion products is selected to carry out
Recycling；
3. carrying out DNA to recovery product using Qubit3.0 to quantify, mixed in equal amounts sample；
4. carrying out DNA library building to mix products using IlluminaTruSeq kit；
4) original sequence data statistics and filtering
Sequencing steps 3) obtained in original series contain belt lacing, low-quality reads, in order to guarantee information analysis
Quality carries out data filtering to reads, obtains cleanreads for subsequent analysis；
The step of data filtering, is as follows:
1. removing the paired-endreads of belt lacing；
2. needing to remove when the content of the N contained in single-ended sequencing read is more than the 10% of this read length ratio
This is to paired-endreads；
3. being needed when the low quality base number contained in single-ended sequencing read is more than the 50% of this read length ratio
This is removed to paired-endreads；
5) Cleanreads sequence data compares after filtering；
The effective sequencing data of the clean reads obtained after filtering, which is compared to Gallus gallus.WASHUC chicken, refers to base
Because of group；
6) mononucleotide polymorphic site SNP is identified；
According to comparison result, genome of the sample average sequencing depth 5 or more is chosen, dd RAD label is defined as, and
Carry out SNP detection inside the group based on sample；
7) according to individual SNP qualification result, the molecule inbred strais of sequencing individual is calculated using science of heredity statistical software PLINK
Number；Each offspring's coefficient of inbreeding testing result such as table 2.
Table 2 sets up the selection of male and female chicken individuals and the coefficient of inbreeding for inbred strais
It is proved by experimental data above, the present invention successfully sets up out the higher chicken inbred strais of the coefficient of inbreeding, to solve
Solve in the complete situation of pedigree, the individual coefficient of inbreeding, affiliation inaccuracy and the coefficient of inbreeding it is low the problems such as；Pass through section
Reasonably select and remain matingplan, effectively improves the slumps of disastrous proportions such as the growth performance, reproductive performance, viability of inbreeding offspring
Problem and inbred strais are difficult to the problems such as lasting.
Claims (9)
1. the construction method of a breeder inbred strais, which is characterized in that include the following steps
(1) family individual is set up；
(2) family expands procreation and records and analyzes；
(3) breeding chickling management；
(4) the establishment molding of chicken inbred strais.
2. the construction method of a breeder inbred strais according to claim 1, which is characterized in that the tool that the family individual is set up
Body method are as follows:
By group based on the chicken group in peak age in days of laying eggs, the almost the same male and female chicken individuals of phenotypic characteristic are chosen, are set up
Individual family；Each family is made of 1 cock and 10~12 hens, described coefficient sets build quantity be 30 with
On.
3. the construction method of a breeder inbred strais according to claim 1, which is characterized in that the family expands procreation and note
Record analysis method particularly includes:
Individual record is done to the hen in each family, and artificial insemination, label are carried out to the hen in the individual family of establishment
The correspondence family of each egg and corresponding hen number；After continuous collection hatching egg 12~15 days, to obtained egg system
Spectrum hatching, obtains seedling chicken；Wing number is worn to the seedling chicken after hatching, and indicates corresponding family and corresponding hen.
4. the construction method of a breeder inbred strais according to claim 1, which is characterized in that the tool of the breeding chickling management
Body method are as follows:
To chick in the closed cage henhouse that the environment such as temperature, humidity, ventilation, illumination automatically control and under normalization condition
Feeding management is carried out, the feeding management of the chicken divides brood time, finishing period and laying period three phases；It will be on the hen of laying period
Cage does individual and lays eggs record according to " performance of poultry measure term and measure statistical method NY/T823-2004 ".
5. the construction method of a breeder inbred strais according to claim 4, which is characterized in that the normalization condition is to meet
NYT388-1999 " livestock and poultry farm environmental quality standards " and NYT1167-2006 " livestock and poultry farm environmental quality and sanitary control specification " are wanted
The condition asked.
6. the construction method of a breeder inbred strais according to claim 4, which is characterized in that the brood time of the chicken is 0~7
Week, finishing period are 8~17 weeks, and laying period is 18~66 weeks.
7. the construction method of a breeder inbred strais according to claim 1, which is characterized in that the group of the chicken inbred strais is built up
Type concrete operations the following steps are included:
Step 1: all upper cage hen Age at first laying of statistics, open produce weight, open weight of laying eggs, 43 week old egg numbers, 43 week old egg sizes,
43 week old weight calculate average value, select and remain the healthy individuals of average value bound 5~15%, and take into account phenotype consistency；
Step 2: pedigree information verification being carried out to the healthy individuals selected and remain in step 1, and is included into place family.It counts in each family
Selected number of individuals, the family for being selected hen number of individuals > 10 are set up for inbred strais；
Step 3: acquiring 0.5ml~1.5ml blood from the wing venous of selected hen individual, carry out simplifying base after extracting DNA
Because of a group sequencing, genome mononucleotide polymorphic SNP variation information is obtained；
Step 4: based on SNP variation information, the molecule coefficient of inbreeding of sequencing individual is calculated using biosoftware PLINK；According to survey
The molecule coefficient of inbreeding obtained rejects individual and its full sibs of the molecule coefficient of inbreeding lower than 0.100 in same family,
The full sibs male and female chicken individuals chosen according to the molecule coefficient of inbreeding, expand numerous, full sibs cock using full mode
More than one, successively semen deposition, previous cock semen deposition is reserved seed for planting and is spaced 5~7 days after egg, then is continued with next cock defeated
Essence；
Step 5: the subculture expansion of inbred strais is numerous, persistently using full mode to get chicken inbred strais.
8. the construction method of a breeder inbred strais according to claim 7, which is characterized in that simplify gene described in step 3
Group sequencing concrete operation method the following steps are included:
1) individual blood acquires
Selected hen wing venous whole blood 0.5-1.5ml is acquired using disposable medical syringe, injection contains rapidly after acquisition
1.5~2 μ L 0.5mol/LNa2EDTA anti-coagulants without in enzyme pipe, then will be placed in without enzyme pipe under 0~4 DEG C of environment save it is standby
With；
2) blood DNA extraction and quality testing
It is sucked out under room temperature without the 0.2~0.3ml of blood saved backup in enzyme pipe, using animal peripheral blood phenol-chloroform extraction method
Individual blood DNA is extracted, and DNA integrity degree is analyzed by agarose gel electrophoresis, with the purity of spectrophotometer detection DNA；
3) library construction and simplified gene order-checking
1. extracting 300~500ng of genomic DNA of quality inspection qualification, 0.4~0.6U EcoRI, T4 DNA ligase, ATP is added
It is reacted at 35~37 DEG C 2.5~3.0 hours with EcoRI connector, 60~65 DEG C are annealed 0.5~1.0 hour, then plus restricted
The end the restriction endonuclease Reads1 end EcoRI, Reads2 NlaIII or Hin1IICATG and NlaIII connector carry out double digestion, and double digestion exists
It is reacted at 35~37 DEG C 2.5~3.0 hours, places 20~30 minutes inactivation inscribes in 60~65 DEG C of PCR instruments after reaction
Enzyme；
2. carrying out Piece Selection to connection product using agarose gel electrophoresis, 400~600bp digestion products is selected to be returned
It receives；
3. carrying out DNA to recovery product using Qubit3.0 to quantify, mixed in equal amounts sample；
4. carrying out DNA library building to mix products using IlluminaTruSeq kit；
4) original sequence data statistics and filtering
Sequencing steps 3) obtained in original series contain belt lacing, low-quality reads, in order to guarantee information analysis matter
Amount carries out data filtering to reads, obtains cleanreads for subsequent analysis；
The step of data filtering, is as follows:
1. removing the paired-endreads of belt lacing；
2. it is right to need to remove this when the content of the N contained in single-ended sequencing read is more than the 10% of this read length ratio
paired-endreads；
3. needing to remove when the low quality base number contained in single-ended sequencing read is more than the 50% of this read length ratio
This is to paired-endreads；
5) Cleanreads sequence data compares after filtering；
The effective sequencing data of the clean reads obtained after filtering, which is compared to Gallus gallus.WASHUC chicken, refers to gene
Group；
6) mononucleotide polymorphic site SNP is identified；
According to comparison result, genome of the sample average sequencing depth 5 or more is chosen, is defined as dd RAD label, and carry out
SNP detection inside group based on sample；
7) the molecule coefficient of inbreeding calculates
According to individual SNP qualification result, the molecule coefficient of inbreeding of sequencing individual is calculated using science of heredity statistical software PLINK.
9. the construction method of a breeder inbred strais according to claim 7, which is characterized in that in step 3) 1. described in
EcoRI connector is the EcoRI connector containing the Index sequence for distinguishing sample.
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