CN102094022A - Gene Hoxc8 related to tassel head characters of chicken and application thereof - Google Patents
Gene Hoxc8 related to tassel head characters of chicken and application thereof Download PDFInfo
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- CN102094022A CN102094022A CN 201010266962 CN201010266962A CN102094022A CN 102094022 A CN102094022 A CN 102094022A CN 201010266962 CN201010266962 CN 201010266962 CN 201010266962 A CN201010266962 A CN 201010266962A CN 102094022 A CN102094022 A CN 102094022A
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Abstract
The invention relates to a gene Hoxc8 related to the tassel head characters of chickens. The invention can realize the detection of different phenotypic chicken breeds by using a microsatellite marker SSR5 (Simple Sequence Repeat) at the upstream of the Hoxc8 gene. The invention also relates to a molecular marker kit for detecting the tassel head characters of the chickens. The gene Hoxc8 can be used for the breeding of the chickens.
Description
Technical field
The present invention relates to the test kit of tassel head proterties in Hoxc8 gene relevant and the detection chicken colony with chicken tassel head proterties.
Background technology
Silk plumage Gallus Domesticus is as the distinctive indigenous chicken kind of China, has typical perfect proterties, its appearance has been compared evident difference with other chicken kinds, the silk plumage Gallus Domesticus of standard can be summarized as has ten big features, be called " perfect " proterties again, comprise: mulberry fruit hat, tassel head, green ear, beard, silk plumage, five pawls, dry foot, black skin, black meat, black bone.Wherein, tassel head proterties is meant and is present in the hair hat that a cluster length of plumage Gallus Domesticus head can reach eye that general hen is more obvious than cock.
Davenport mentioned tassel head proterties in being entitled as " Heredity and Mendel ' s Law " in 1907, pointed out that tassel head proterties is similar to silk plumage proterties, was a kind of dominant character.Studies show that afterwards that the tassel head proterties and the Frizzle linkage of characters with the white linkage of characters of dominance, and were constantly determined.F0 does not have the tassel head for A system (French star's fryer) all individualities in China Agricultural University's resource family, all individualities of C system (silk plumage Gallus Domesticus) all have the tassel head, F1 is the tassel head for individual phenotype,, there be separating than being 3: 1 (2324: 789) of tassel head individuality and no tassel head individuality in F2 generation, do not have difference between the reciprocal cross group.
Tassel head proterties is the feather morphological differences in the special zone of a class and the appearance proterties that causes.The feather of birds is similar to mammiferous hair, all belongs to the attached derivative of skin histology, and the growth course of hair follicle roughly is divided into three periods, i.e. vegetative period (anagen), catagen (catagen) and resting stage (telogen).Discover that in the process of hair follicle development, transcription factor family-Hox gene family that a class is important has a plurality of members to participate among the developmental regulation in these three periods.For example, at the hair follicle early period of origination, Hoxc12, Hoxc13, Hoxc8 are to the interior myelin of hair, and the growth of cortex projection reaches catagen Hoxb4, and Hoxc13 all plays regulating and controlling effect to the normal development of outer myelin of hair and matrix.
The animal embryo craniocaudal axis is made up of many dissimilar cells, has the genetic system of a complexity to take place by corresponding signal command embryo among the embryo.The center of this system by the similar genomic constitution of a nested structure be called homeotic gene (Homeotic gene, Hox), i.e. Hox gene.Discover that now the form of Hox gene and animal is closely related.Insect, onychophoran, nematode, priapulid, Polychaeta, leech, nemertinean, platyhelminth, Gastropoda, branchiopod guiding principle, snake, mouse, lancelet, sea urchin and human Hox gene have very high homology each other.
In fetal development, it expresses the position period at animal embryo, on genome, put in order into corresponding relation with it, promptly be positioned on the genome by the 3 ' family member who holds, when fetal development, more be inclined to and expressing the parallel function corresponding that makes near the zone of head, otherwise be more prone to express at caudad near the member of 5 ' end.For example, in the model animals fruit bat, Antp is responsible for controlling the growth of chest region, and Ubx then is responsible for the growth of mesothorax and uromere.And discover in recent years, Hox gene family member arranges with this collinearity principle in all tissues, particularly be positioned at the Hoxc12 in gene cluster downstream, members such as Hoxc13 are in mouse near head end processus hyoideus, expression in the beard hair follicle has more proposed new understanding to the collinearity theory that this was once thought.1998, the Hoxc13 mouse knocked out the model success, and its phenotype shows as at beard compared with the control, defectiveness in the growth of tongue hair follicle and body surface hair follicle.And calendar year 2001 is found, change the transgenic mice of Hoxc13 gene, body surface is changed by the growth of hair, can produce phenotype near nude mice, find by the gene chip analysis, along with Hoxc13 expression of gene amount increases, the special role of Hoxc13 in the mouse hair follicle development further determined in some variations that necessary keratin gene expression amount has produced significance in hair is formed.
Summary of the invention
The purpose of this invention is to provide and relevant Hoxc8 gene and the application of chicken tassel head proterties.
Another object of the present invention provides a kind of molecule marker test kit that is used for detecting chicken tassel head proterties.
In order to realize the object of the invention, the invention provides the application of Hoxc8 gene in the chicken breed seed selection.
The present invention also provides the Hoxc8 gene to have application in the chicken breed of tassel head proterties in seed selection.
The present invention also provides and the relevant microsatellite molecular marker SSR5 of chicken tassel head proterties, and it has the nucleotide sequence shown in the Seq ID No.1.
The present invention also provides above-mentioned molecule marker to have application in the chicken breed of tassel head proterties in seed selection.
The present invention's also be provided for increasing primer of SSR5 microsatellite molecular marker, it comprises forward primer F:5 '-CTGTCTCAGTGTGGCGGAGT-3 ' and reverse primer R:5 '-CGATCTCAAAGGCGTTGC-3 '.
The present invention also provides the detection kit that contains above-mentioned primer.
The present invention further provides the application of mentioned reagent box in detecting chicken tassel head proterties, concrete grammar is: the genomic dna with chicken is that template is carried out pcr amplification reaction, detects amplified production.Wherein, the annealing temperature of PCR reaction is 53.1 ℃.
The present invention utilizes the method for RT-PCR, has found a kind of gene Hoxc8 relevant with chicken tassel head shapes, and Genebank number is NM_204893.The primer is, forward primer F:5 '-CATCACACCACGTCCAAGA-3 ' and reverse primer R:5 '-GGGAACATGAGACTGGGAGA-3 ', and annealing temperature is 60 degree, expanding fragment length is 265bp.Forward and reverse primer all is arranged in first exon.The blank that does not add ThermoScript II is set to be polluted to eliminate genome.The result shows in the special skin histology of tassel head chicken kind silk plumage Gallus Domesticus and the white Leghorn of non-tassel head chicken kind, presents the varietY specificity differential expression of Hoxc8 gene.
The present invention utilizes the microsatellite marker of chicken tassel head trait related gene (Hoxc8) NM_204893 upstream, and the chicken kind of different phenotypes is carried out DNA detection.It is to utilize BAC to carry out the method for Solexa order-checking, the white Leghorn BAC clone who report is comprised differential expression genes checks order, obtained the scaffold of 86 different lengthss, amounted to the sequence of 175.975kb, the scaffold length that wherein comprises goal gene is 39.215kb.Utilize the method for PCR order-checking, find a microsatellite locus SSR5 who has polymorphism in China Agricultural University's resource family (CAU), expanding fragment length is 320bp, and annealing temperature is 53.1 degree.The PCR fragment that causes producing different lengths according to one section core tumor-necrosis factor glycoproteins (GA) quantity polymorphism in the amplified fragments.
By technique scheme, the present invention has following advantage and beneficial effect at least:
(1) the present invention is applied to the Hoxc8 gene in the chicken breed that seed selection has tassel head proterties first;
(2) utilize little satellite detection kit provided by the invention can realize rapid detection to tassel head proterties individuality in the chicken colony.
Description of drawings
Fig. 1 is different phenotype chicken kind different tissues differential expression results in the preferred embodiment 1 of the present invention, wherein, 1 is white Leghorn skin of head, and 2 is white Leghorn skin of back, and 3 is white Leghorn heart, 4 is silk plumage Gallus Domesticus skin of head, 5 is a silk plumage Gallus Domesticus skin of back, and 6 be a silk plumage Gallus Domesticus heart, and 7 is to be the negative control of template with water, GAPDH contrasts as housekeeping gene, and-RT representative does not add the RNA template of ThermoScript II.
Fig. 2 detects the polymorphism distribution situation of SSR5 microsatellite locus in Different Individual for the 3130xl genetic analyzer of ABI in the preferred embodiment 2 of the present invention.
Embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
Experimental technique in following examples if no special instructions, is ordinary method; Used test materials if no special instructions, is the commercial goods; % among the following embodiment if no special instructions, is the quality percentage composition; The PCR instrument uses the east victory-Long PCR instrument of eastern KingMax new company.
1, experiment material
From Leghorn kind egg, silk plumage Gallus Domesticus kind egg is all from ranchette of China Agricultural University.
2, experiment reagent
The RNA enzyme inhibitors is available from magnificent biotech firm, and RNA extracts test kit available from Qiagen company, and RNA ThermoScript II test kit is available from Promege company.
DEPC water: add 0.1%DEPC in the deionized water, 37 ℃ are spent the night autoclaving.
RNA operates used rifle head, centrifuge tube spends the night through 0.1%DEPC water logging bubble, and deionized water rinsing 2-3 time, autoclaving.
75% ethanol that the RNA operation is used: prepare with the water that DEPC handles.
3, laboratory apparatus
Brooder is available from Beijing Hai Jiang brooder Manufacturing Co., Ltd.
4, experimental procedure:
(1) gather skin of head in the 12nd day white Leghorn of hatching and the silk plumage Gallus Domesticus chicken embryo respectively, skin of back, three tissues of heart freeze in liquid nitrogen.
(2) utilize the RNA of Qiagen to extract test kit (article No.: 74104) cryopreserved tissue is carried out RNA and extract and purifying, finally with the DEPC water dissolution of 0.1% concentration and measure concentration.
(3)RT-PCR
2 μ g RNA are joined in the centrifuge tube of no RNase, and the amount that adds 0.5 μ g oligo (dT) primer according to per 1 μ g RNA adds oligo (dT) 18 primers.In system less than 15 μ l, 70 ℃ of heating 5min, of short duration centrifugal and collection.
Respectively each component is added in the above-mentioned mixture of annealed in the following order:
M-MLV 5X reaction buffer 5 μ l
dNTP 1.25μl
RNase inhibitor 0.75 μ l
M-MLV ThermoScript II 1 μ l
Adding DEPC water to final volume is 25 μ l, mixing gently, and 42 ℃ are extended 60min.The cDNA that reverse transcription is obtained places-20 ℃ of preservations.
With cDNA is that template is carried out pcr amplification reaction, and the primer is, forward primer F:5 '-CATCACACCACGTCCAAGA-3 ' and reverse primer R:5 '-GGGAACATGAGACTGGGAGA-3 ', and reaction conditions is as follows:
94 ℃ of pre-sex change 5min; 94 ℃ of sex change 30 seconds, 60 ℃ of annealing 30 seconds, 72 ℃ were extended 30 seconds, and carried out 35 circulations altogether; 72 ℃ are extended 7min, 4 ℃ of preservations.
Getting 4 μ lPCR products at last detects with 2% agarose gel electrophoresis.
Experimental result as shown in Figure 1.Wherein, 1 is white Leghorn skin of head, 2 is white Leghorn skin of back, 3 is white Leghorn heart, and 4 is silk plumage Gallus Domesticus skin of head, and 5 is silk plumage Gallus Domesticus skin of back, 6 is silk plumage Gallus Domesticus heart, 7 for being the negative control of template with water, and GAPDH contrasts as housekeeping gene, and-RT representative does not add the RNA template of ThermoScript II.The result shows that the Hoxc8 gene has the silk plumage Gallus Domesticus and not having in the white Leghorn of tassel head proterties of tassel head proterties, presents the kind specifically expressing difference of Hoxc8 gene in the special skin histology skin of head that tassel head proterties takes place.
Experiment material: CAU resource family: being used to of setting up in 1998 studying the location by the Li Ning of China Agricultural University professor seminar influence chicken important economical trait QTL, seeks the experimental population of cloning major gene, wherein F0 selects 277 chickens for use altogether for the parent, be respectively A system, B system, C system, wherein A is French star's fryer, 15 ♂, 43 ♀; B is the brown laying hen of agricultural university that China Agricultural University cultivates, 21 ♂, 70 ♀; C is the Chinese calm and peaceful silk plumage Gallus Domesticus of drawing from Gallus Domesticus field, Beijing, 11 ♂, 117 ♀.CAU resource family adopts the outbreeding F2 of colony design.F0 is positive and negative mating (according to male and female ratio 1: 7) with C respectively for A system, B system, produces two classes, four groups of F1 for colony; Every group of F1 is final segregating population for avoiding half sibs traversed by (according to male and female ratio 1: 5) to produce F2 generation in the colony.F2 for the segregating population scale is: ACAC, and------702 individualities of 9 familys, BCBC---2506 individualities of 21 familys, CBCB---1239 individualities of 9 familys add up to F2 generation 6496 individualities for 2049 individualities of 24 familys, CACA; The present invention selects 4 family half sibses (51 familys, 56 familys, 66 familys, 89 familys) from the CAU family, amount to F0, F1, F2 generation totally 278 individualities.
CAU resource family reference: Deng, X.M., Li, J.Y., Li, N and Wu, C.X. (2001) .[Genetic analysis of important growth trait based on F-2 resource population in chicken] .Yi Chuan Xue Bao 28,801-807.
1, the extraction of genomic dna
The wing venous blood collection, the back cracking is handled in anti-freezing, behind protease K digesting, with the imitative extracting of phenol, TE dissolving-20 ℃ of preservations in back.
2, PCR reaction
According to the BAC sequencing result, design primer, forward primer F:5 '-CTGTCTCAGTGTGGCGGAGT-3 ' and reverse primer R:5 '-CGATCTCAAAGGCGTTGC-3 ' with the goal gene upstream apart from initiator codon 361bp with apart from the core tumor-necrosis factor glycoproteins (GA) between the initiator codon 681bp.With Different Individual DNA is that template is carried out pcr amplification reaction, and Qiagen Hotstart test kit is adopted in reaction, and system is as follows:
DNA 1μl(20ng)
10X damping fluid 1.5 μ l
dNTP 1μl
Forward primer 0.2 μ l
Reverse primer 0.2 μ l
Q-Solution 3μl
HotStart?Taq 0.1μl
ddH
2O 8μl
Cumulative volume 15 μ l
The PCR reaction conditions with 53.1 ℃ as annealing temperature, the extension time is 30 seconds.
3. genotype detection
Last press proof product preparation: mark dilution in mark reagent+985 μ l deionized formamide reagent carry out in the 15 μ l, after mark mixes in 1 μ l PCR product+9 μ l dilution, place on the PCR instrument, 94 ℃ of 5min place on ice then, after the cooling, last machine testing.
Gene type assay:
Adopt ABI Genemapper4.1 software to analyze.Pcr amplified fragment size is judged to be A for the allelotrope of 314bp, is clip size that the allelotrope of 320bp is judged to be B, by statistics, individual 57 of total AA, individual 120 of AB, individual 82 of BB does not detect 19 of individualities.
Carry out association analysis to declaring type The data EpiSNP2 software.
The Statistic analysis models that is adopted is as follows:
Y＝μ+Genotype+e
Y: individual phenotypic character observed value;
μ: the mean value of colony;
Genotype: genotype is to the effect value of phenotypic character;
E: corresponding to the random residual effect of observed value.
The association analysis result is as shown in table 1:
The correlation analysis result of table 1 SSR5 site and chicken tassel head proterties
| ?Locus | The P value | Geno | G_Effect | G_Freq | Geno | G_Effect | G_Freq | Geno | G_Effect | G_Freq |
| ?ssr5 | 5.53E-33 | AB | 1.33E-01 | 4.73E-01 | BB | -1.15E-01 | 3.14E-01 | AA | -1.25E-01 | 2.13E-01 |
In the table 1, Geno is a genotype, and G_Effect is the genotype effect, and G_Freq is a genotype frequency.As can be seen from the results, this microsatellite locus SSR5 and tassel head proterties cognation P value are 5.53E-33, reach utmost point conspicuous level.Wherein, contain the allelic individuality of B and mostly be individuality greatly with tassel head.
This result shows the microsatellite locus SSR5 sequence that is positioned at the differential expression candidate gene Hoxc8 upstream relevant with tassel head proterties, and its sequence polymorphism and tassel head proterties have extremely significant dependency.Therefore, this microsatellite marker can be applied in the breed breeding at the relevant special phenotype chicken kind of tassel head proterties.
Though above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (9)
1.Hoxc8 the application of gene in the chicken breed seed selection.
2.Hoxc8 gene has application in the chicken breed of tassel head proterties in seed selection.
3. with the relevant microsatellite molecular marker of chicken tassel head proterties, it is characterized in that having the nucleotide sequence shown in the SeqID No.1.
4. the described molecule marker of claim 3 has application in the chicken breed of tassel head proterties in seed selection.
5. be used to the to increase primer of the described molecule marker of claim 3 is characterized in that it comprises forward primer F:5 '-CTGTCTCAGTGTGGCGGAGT-3 ' and reverse primer R:5 '-CGATCTCAAAGGCGTTGC-3 '.
6. the detection kit that contains the described primer of claim 5.
7. the application of the described test kit of claim 6 in detecting chicken tassel head proterties.
8. application as claimed in claim 7, concrete grammar is: the genomic dna with chicken is that template is carried out pcr amplification reaction, detects amplified production.
9. application as claimed in claim 8, wherein the annealing temperature of PCR reaction is 53.1 ℃.
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| CN116042841A (en) * | 2022-08-05 | 2023-05-02 | 华南农业大学 | Molecular marker related to leg hair follicle size of yellow-feather broiler and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101343632A (en) * | 2007-07-10 | 2009-01-14 | 李宁 | Chicken silk feather character gene and uses thereof |
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| CN101343632A (en) * | 2007-07-10 | 2009-01-14 | 李宁 | Chicken silk feather character gene and uses thereof |
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| 《自然科学进展》 20070430 高宇 基于中国丝羽乌骨鸡和白洛克肉鸡资源家系的遗传连锁图谱的构建与分析 全文 1-9 , 第4期 * |
| 《遗传》 20070831 柳晓峰 利用鸡F_2资源群体构建1号染色体遗传连锁图谱 全文 1-9 , 第8期 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116042841A (en) * | 2022-08-05 | 2023-05-02 | 华南农业大学 | Molecular marker related to leg hair follicle size of yellow-feather broiler and application thereof |
| CN116042841B (en) * | 2022-08-05 | 2023-08-11 | 华南农业大学 | Molecular marker related to leg hair follicle size of yellow-feather broiler and application thereof |
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