CN105671170B - It is a kind of for identify chicken crawl character primer combination and its application - Google Patents

Abstract

The present invention relates to molecular biology field, specifically disclose it is a kind of crawl the primer combination and its application of character for identifying chicken, the primer combination includes SEQ ID No.1, SEQ ID No.2, primer sequence shown in SEQ ID No.3 and SEQ ID No.4.Being combined using the primer can be achieved the crawl identification of character of chicken and is combined using the primer, analyzed using multiple PCR method specially using the genomic DNA of test individual as template；If multiplexed PCR amplification has a kind of product, and sequence size is 224bp, then test individual is to crawl gene dominant homozygote (Cp/Cp), shows as lethal type；If multiplexed PCR amplification, there are two types of product, sequence size is respectively 438bp and 224bp, then test individual is genetic heterozygosis (Cp/+) of crawling, character of crawling is shown as；If multiplexed PCR amplification only has a kind of product, sequence size 438bp, then test individual is stealthy homozygote (+/+), shows as wild type.

Description

A kind of primer combination and its application for differentiating chicken prostrate character

technical field

The invention relates to the field of molecular biology, in particular to a combination of primers for identifying chicken prostrate traits and an application thereof.

Background technique

So far, eight dwarf genes have been found in chickens, which are respectively located on sex chromosomes and autosomes. Among them, the sex-linked dwarf gene dw has been studied more, and the gene has been precisely located and its mechanism of action has been clarified ( Agarwal, S.K., L.A. Cogburn, and J. Burnside. 1994. Dysfunctional growth hormone receptor in a strain of sex-linked dwarf chicken: evidence for a mutation in the intracellular domain. J. Endocrinol. 142:427-434.). There are many dwarf genes on autosomes, among which Cp gene (Cp: Creeper, creeping type) is an important dominant homozygous lethal gene on autosomes (Landauer, W., and L.C.Dunn.1930.Studies on the Creeperfowl.I.Genetics.J.Genet.23:397-413.), the gene controls the prostrate traits of chickens.

Cutler (Cutler, I.E. 1925. Reptilian fowls. J. Hered. 16:352-356.) was the first to publish the hypothesis that the creeping trait is affected by heterozygous genotypes. Landauer and Dunn (Landauer, W., and L.C. Dunn. 1930. Studies on the Creeper fowl. I. Genetics. J. Genet. 23: 397-413.) through hybridization experiments, found a cartilage abnormal development The dwarf chicken named creeper chicken (Creeper fowl), and confirmed that the genetic basis of the creeping trait is controlled by a single Mendelian dominant gene Cp, which is lethal under dominant homozygous (Cp/Cp) conditions, heterozygous The zygote (Cp/+) showed the prostrate type, and the recessive homozygote (+/+) showed the wild type. The mortality rate was 25% in the early incubation period, and the embryonic death time was concentrated on the fourth day of incubation.

Genetic studies have shown that the Cp gene is linked to the single crown or rose crown (Landauer, W.1932. Studies on the creeper fowl. V. The linkage of the genes for creeper and single-comb. J. Genet. 26:285-290. ; Landauer, W.1933. Creeper and single-comb linkage infowl. Nature 132:606.; Taylor, L.W.1934. Creeper and single-comb linkage in the fowl. J. Hered. 25:205-206.). Somes and Jr. located the Cp gene in linkage group I, the linkage distance with the rose crown gene (R) was 0.4cM, and the linkage distance with the tail fat gland segregation gene (U) was 30cM (Somes, R.G., and Jr.1973 . Linkage relationships in domestic fowl. J. Hered. 64:217-221.). Through linkage analysis and high-density SNP research, Imsland et al. showed that the 7.4Mb inversion on chromosome 7 caused a change in the position of the MNR2 gene, which resulted in a rose crown phenotype, further confirming that the rose crown gene (R) is located on chromosome 7 On (Imsland, F., C. Feng, H. Boije, B. Bed'hom, V. Fillon, B. Dorshorst, C. J. Rubin, R. Liu, Y. Gao, X. Gu, Y. Wang, D. Gourichon, M.C. Zody, W. Zecchin, A. Vieaud, M. Tixier-Boichard, X. Hu, F. Hallbook, N. Li, and L. Andersson. 2012. The Rose-comb mutation in chickens constitutes a structural rearrangement causing both altered comb morphology and defective sperm motility. PLoS Genet. 8:e1002775.). Xingyi bantam is one of the rare local bantam breeds in China. Due to its special crawling body shape, it has been highly valued and concerned by experts at home and abroad. The key gene controlling this trait is the Cp (Creeper) gene, which is an autosomal dominant homozygous lethal gene. The international research on the Cp gene has a history of more than 80 years (Cairns, J.M., and K.Gayer.1943. Identification of chick embryos homozygous for the Creeper factor. J. Exp. Zool. 92:229-242.; Landauer, W. 1944.Length of survival of homozygous creeper fowl embryos.Science100:553-554.;Dinner,B.1971.Chemical analysis of embryonic Creeper chickentibia.J.Dent.Res.50:704.;Fujio,Y.,and T. Shibuya.1974.Expression of lethality caused by Creeper gene in the chicken.Japan.J.Genet.49:87-91.), Xingyi, Miyi and other domestic local chicken breeds carry this gene, resulting in the hatching of these local chicken breeds The rate and regularity of growth and development are severely affected. Therefore, the research on the genetic law of the Cp gene controlling the creeping trait will help to protect, research, develop and utilize the resources of Xingyi Bantam and other local chicken breeds in my country. According to the conventional breeding method, the selection of the creeping trait is mainly determined according to the length of the shanks, and the cycle is longer and the cost is high. In addition, conventional morphological and cytological methods for detecting Cp homozygous lethal embryos and normal dead embryos have complex sampling operations, long analysis and measurement cycles, low automation, and insufficient detection accuracy. The use of molecular marker-assisted selection for molecular breeding can not only carry out direct selection, but also can be selected at an early stage, which greatly shortens the breeding cycle, improves the accuracy of breeding, and reduces the cost of breeding.

Contents of the invention

In order to solve the problems in the prior art, the object of the present invention is to provide a primer combination for identifying chicken prostrate traits and its application.

In order to realize the object of the invention, the technical scheme of the present invention is as follows:

In a first aspect, the present invention provides a combination of primers for identifying chicken prostrate traits, said combination of primers comprising SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4 the primer sequence.

The primer combination is designed by Primer Premier 6.0 software. Under the premise of ensuring primer specificity, sensitivity and amplification efficiency, the desired target bands are obtained through multiple PCR experiments. The PCR products are purified, ligated, transformed and sequenced. , obtained after screening in a large population.

The primer combination has significant advantages in the development of molecular detection kits, poultry molecular breeding and development and utilization of local breed chicken resources, and can quickly, accurately and efficiently screen and identify chicken creeping traits (Creeper), with simple operation and Inexpensive. The primer combination has strong specificity, high sensitivity, low cost, simplicity and speed, and wide application range. The multiplex PCR method has its unique advantages, one reaction can screen individuals with three different genotypes, and can prepare to detect the presence or absence of creeping gene sequences. The combination of multiple primers is helpful for the protection, research, development and utilization of creeping chicken and local chicken breed resources in my country.

In the second aspect, the present invention provides the application of the combination of primers in identifying prostrate traits of chickens.

As well as the application of the primer combination in the preparation of reagents or kits for identifying chicken prostrate traits.

And the application of the combination of primers in assisted breeding of chicken creeping traits.

Further, the application is specifically:

Using the genomic DNA of the individual to be tested as a template, using the primer combination described above, the multiplex PCR method is used for analysis; if there is a product in the multiplex PCR amplification, and the sequence size is 224bp, the individual to be tested is a dominant homozygote for the creeping gene (Cp/Cp), showing a lethal type; if multiple PCR amplification has two products, and the sequence sizes are 438bp and 224bp respectively, then the individual to be tested is heterozygous for the creeping gene (Cp/+), showing a creeping trait; if Multiplex PCR amplification has only one product, and the sequence size is 438bp, so the individual to be tested is recessive homozygote (+/+), which is wild type.

As a preference, the present invention provides a multiplex PCR reaction system with the best amplification effect, specifically: genomic DNA 80-100ng, 10×PCR buffer (containing Mg ²⁺ ) 2.0 μL, dNTPs (2.5mM) 2.0 μL, Taq DNA Polymerase (2.5U/μL) 1.0 μL, delF and delR primers (10 μmol/l) each 0.2 μl, F and R primers (10 μmol/l) each 0.4 μl, and the reaction system was supplemented with ddH ₂ O to 20.0 μL.

As a preference, the present invention provides a multiplex PCR reaction condition with the best amplification effect, specifically: pre-denaturation at 94°C for 5 minutes; cycle flow: denaturation at 94°C for 30 s, annealing at 57°C for 30 s, extension at 72°C for 35 s, a total of 35 cycles; Finally, it was extended at 72°C for 10 minutes and stored at -20°C for a long time.

In a third aspect, the present invention provides a method for identifying prostrate traits of chickens. The method is to detect whether the individual lacks part of the Cp gene sequence and the 11.896kb deletion upstream and downstream sequences of chromosome 7 (chr 7:21798705-21810600).

The deletion detection of the Cp gene uses genomic DNA as a template and uses multiplex PCR (multiplex-PCRassay) method for analysis.

Specifically: use the genomic DNA of the individual to be tested as a template, use the primer combination, and use the multiplex PCR method for analysis; if there are two products in the multiplex PCR amplification, the sequence sizes are 438bp and 224bp respectively, then the individual to be tested is Heterozygous for the prostrate gene (Cp/+), showing prostrate traits.

The preferred multiplex PCR reaction system and multiplex PCR reaction conditions are the same as those described above.

The beneficial effects of the present invention are:

The invention provides a method of using multiple PCR methods, using chicken genome DNA as a template, and adopting special primer pairs to identify chicken creeping traits, and establishes a standard method for Cp genotype detection, which is helpful for improving the incubation of local chicken varieties in my country The rate and uniformity of growth and development provide an important reference for the protection, research, development and utilization of Xingyi bantam and other local chicken breed resources in my country. Experiments have proved that this method can carry out Cp genotype screening on chicken flocks by checking the Cp gene partial sequence and the upstream and downstream sequences of chromosome 7 deletion sequence (11.896kb, chr 7:21798705-21810600), so that it can accurately and quickly Individuals can be genotyped with Cp, and chickens can be selected early to shorten the selection time and save breeding costs. The invention provides a more accurate, fast, efficient, simple and cheap molecular genetic marker detection method for the screening and breeding of chicken prostrate Cp gene. Using the method and special primers provided by the invention to carry out molecular genetic marker-assisted selection for chicken prostrate traits can improve the accuracy of selection, and can perform early selection, greatly reducing breeding costs, and providing convenience for the breeding of chicken breeds with prostrate traits. It is of great significance to the protection and development of local chicken breeds. The detection method provided by the invention is simple in operation, high in accuracy, short in test time, cheap in price, and can realize automatic and intelligent detection. In addition, a standardized Cp genotype detection method can be established according to the method of the present invention, and a detection kit can be developed for screening individuals carrying the Cp gene, providing convenience for the breeding work of prostrate chickens, and protecting local chicken breeds and their Development and utilization are of great significance.

Description of drawings

Fig. 1 is a schematic diagram of primer design for Cp genotype detection of the present invention; wherein, the missing sequence (chr7:21798705-21810600), delF/delR is used to amplify the upstream and downstream sequences of the missing sequence, and the size of the amplified fragment is 224bp; F/R primers are used To amplify the partial gene sequence of the Cp gene, the size of the amplified fragment is 438bp.

Fig. 2 is the electrophoresis figure of screening and identification of multiple PCR primers in Example 1 of the present invention; Wherein, M is Marker (600bp); Swimming lane Swimming lane 1-12 is the PCR product of delF2/R2-F2/R2 primer combination; Swimming lane 13 -25 is the PCR product of delF1/R1-F2/R2 primer combination; Swimming lane 26-34 is the PCR product of delF1/R1-F1/R1 primer combination; Swimming lane 35-48 is the target fragment of delF3/R3-F1/R1 primer combination; Lanes 49-60 are PCR products of delF3/R3-F2/R2 primer combination.

Fig. 3 is the electrophoresis figure of different individual Cp genotyping by multiplex PCR method in Example 2 of the present invention; Wherein, swimming lane 1 (M) is the Marker of 600bp; Swimming lane 2-6 is homozygous genotype individual (Cp/Cp) ; Swimming lanes 7-11 are heterozygous genotype individuals (Cp/+); Swimming lanes 12-16 are recessive homozygous genotype (+/+) individuals.

Detailed ways

Preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are given for the purpose of illustration only, and are not intended to limit the scope of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. % in the following examples, unless otherwise specified, are mass percentages. Primer synthesis and sequencing were completed by Beijing Liuhe Huada Gene Technology Co., Ltd.

Design and screening of embodiment 1 primer combination

The design method of the primer combination is shown in Figure 1.

1.1 Primer design

The creeping genes used to design primers have been identified through high-throughput sequencing and bioinformatics analysis, which have important biological significance and potential application value. The gene sequence was also obtained through bioinformatics analysis. Due to the large missing sequence and multiple PCR reactions, in addition to the specificity, sensitivity, and amplification efficiency of the primers, the position of the primer design (the middle sequence of the missing fragment and the upstream and downstream sequences of the missing sequence), two PCR product size, Tm consistency of two pairs of primers, primer ratio, typing efficiency, etc. One multiplex PCR reaction can accurately distinguish individuals of different genotypes. This combination of primers simplifies the operation process, and the results are accurate and efficient.

1.2 Primer screening and identification

In order to carry out early screening and identification of individuals with creeping traits accurately and efficiently, Primer Premier 6.0 software was used to design primers according to the deletion sequence and Cp gene sequence, and 5 pairs of deletion sequence primers and 3 pairs of Cp gene primers were designed respectively. The sequence is as follows (5'→3'):

delF1:AGCCCCTCATTGTTGTCTCA (as shown in SEQ ID No.1);

delR1:TCGTTAAGCTGACACCTCCG (as shown in SEQ ID No.2);

delF2:GAACCCTCGCTCCTGAAA (355bp)

delF2:CTCGGTGGCTCCCTATTA

delF3:CCAGCCCCTCATTGTTGTCT (182bp)

delR3:CGCAAGGTGCCACTTATTTATT

delF4:GAACCCTCGCTCCTGAAA (255bp)

delF4:CACGCAAGGTGCCACTTAT

delF5:GAACCCTCGCTCCTGAAA (356bp)

delR5:GCTCGGTGGCTCCCTATT

F1: CTGCCTTGTGCGTTTCTCA (as shown in SEQ ID No.3);

R1: CAGGAAGTCGCTGTAGGTG (as shown in SEQ ID No.4);

F2: ACCTACAGCGACTTCCTG (455bp)

R2: TAGAGCAGCCCCGAGTAC

F3:AATTCCGCCCCACCTTTG (292bp)

R3:GAGTACCAGTGCACACCG

In order to obtain typing primers with good specificity, high sensitivity, convenience, quickness, and easy identification, the above deletion sequence primers were combined with Cp gene sequence primers in pairs to screen out the best primer combination.

Whole chicken embryos hatched for 4 days were collected and genomic DNA was extracted with a tissue extraction kit. Blood was collected from wing veins of chickens at 4 weeks old, DNA was extracted by traditional phenol imitation method, dissolved in ddH ₂ O, and stored at -20°C for later use.

Using the extracted genomic DNA as a template, use a multiplex PCR reaction system (20.0 μL): Chicken genomic DNA 80-100ng, 10×PCR buffer (containing Mg ²⁺ ) 2.0 μL, dNTPs (2.5mM) 2.0 μL, Taq DNA polymerization Enzyme (2.5U/μL) 1.0 μL, delF and delR primers (10 μmol/l) each 0.2 μl, F and R primers (10 μmol/l) each 0.4 μl, and the reaction system was supplemented with ddH ₂ O to 20.0 μL.

The multiplex PCR reaction conditions were: pre-denaturation at 94°C for 5 minutes; cycle process of denaturation at 94°C for 30 s, annealing at 57°C for 30 s, and extension at 72°C for 35 s, a total of 35 cycles; final extension at 72°C for 10 min, and long-term storage at -20°C.

After multiple PCR reactions and 2.0% agarose gel detection, the PCR products were sequenced and verified. The result is shown in Figure 2. It can be seen from Figure 2 that only five primer combinations amplified bands, namely delF2/R2-F2/R2, delF1/R1-F2/R2, delF1/R1-F1/R1, delF3/R3-F1/R1 and The primer combination of delF3/R3-F2/R2, other primer combinations did not obtain PCR products. Among them, M is Marker, and lanes 1-12 are the products of delF2/R2-F2/R2 primer combination. The amplification efficiency of this combination is not high, the size of the target fragment is similar, and it is not easy to perform typing screening. The delF1/R1-F2/R2 primer combination has low amplification efficiency, a small amount of primer dimers and non-specific amplification, and the typing bands are blurred (lanes 13-25). The delF1/R1-F1/R1 primer combination has good specificity, high amplification efficiency, appropriate band size, and easy typing (lanes 26-34). delF3/R3-F1/R1 (lanes 35-48) and delF3/R3-F2/R2 (lanes 49-60) showed primer dimers, low amplification efficiency, non-specific amplification, and unclear bands.

Therefore, the present invention obtains a superior primer combination (shown in SEQ ID No. 1-4) with good specificity, high sensitivity, convenience and quickness, and easy typing through molecular screening. The molecular detection method of traits is helpful to the conservation and improvement of creeping chickens and chicken breed resources in other places in my country.

Thus, the present invention obtains an advantageous primer combination with good specificity, high sensitivity, convenience and quickness, and easy typing, and applies for protection.

The establishment of embodiment 2 multiplex PCR method and Cp genotyping

1. Analysis of Cp genotypes in different individuals

1. Experimental materials. The experimental flock construction and sample collection were completed in the China Agricultural University Poultry Genetic Resources and Breeding Experimental Base. Xingyi bantam chicken is a valuable poultry genetic resource, with special prostrate traits, which are controlled by a single dominant gene Cp, which is a dominant homozygous lethal gene on autosomal, gene heterozygosity leads to prostrate traits, embryonic death was concentrated on the fourth day after hatching. In this experiment, the progeny of prostrate roosters and prostrate hens were selected as the experimental subjects.

2. Genomic DNA extraction

Whole chicken embryos hatched for 4 days were collected and genomic DNA was extracted with a tissue extraction kit. Blood was collected from wing veins of chickens at 4 weeks old, DNA was extracted by traditional phenol imitation method, dissolved in ddH ₂ O, and stored at -20°C for later use.

3. Multiplex PCR amplification

Using the genomic DNA extracted in step 2 as a template, the specific primer pair obtained in Example 1 was used for Cp genotyping.

Multiplex PCR reaction system (20.0 μL): chicken genomic DNA 80-100ng, 10×PCR buffer (containing Mg ²⁺ ) 2.0 μL, dNTPs (2.5mM) 2.0 μL, Taq DNA polymerase (2.5U/μL) 1.0 μL, 0.2 μl each of delF and delR primers (10 μmol/l), 0.4 μl each of F and R primers (10 μmol/l), and supplement the reaction system with ddH ₂ O to 20.0 μl.

The multiplex PCR reaction conditions were: pre-denaturation at 94°C for 5 minutes; cycle process of denaturation at 94°C for 30 s, annealing at 57°C for 30 s, and extension at 72°C for 35 s, a total of 35 cycles; final extension at 72°C for 10 min, and long-term storage at -20°C.

4. PCR product detection

4.5 μL of the PCR product was taken for detection by 2.0% agarose gel electrophoresis (see FIG. 3 ).

All detected individuals can be divided into 3 types according to the electrophoretic strips:

Type 1: There is only one band, the size is 224bp, it is a dominant homozygous individual (Cp/Cp), and the individual is lethal;

Type 2: There are two bands, the size is 224bp and 438bp respectively, it is a heterozygous genotype individual (Cp/+), and the individual presents a creeping type;

Type 3: There is only one band with a size of 438bp, which is a recessive homozygous individual (+/+), and the individual is wild type.

2. Sequencing verification

The multiplex PCR products of each sample in step 3 were sequenced. The result shows: the nucleotide sequence of the multiplex PCR amplification product of Cp/Cp genotype individual is as shown in SEQ ID No.5; The nucleotide sequence of the PCR amplification product of +/+ genotype individual is as SEQ ID No. 6; the multiplex PCR amplification product of the Cp/+ genotype individual is a hybrid of the DNA shown in SEQ ID No.5 and SEQ ID No.6.

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

Claims (6)

1. a kind of crawl the primer combination of character for identifying chicken, which is characterized in that primer combination includes SEQ ID No.1, SEQ ID No.2, primer sequence shown in SEQ ID No.3 and SEQ ID No.4.

2. primer combination described in claim 1 identifies the application in the reagent or kit that chicken crawls character in preparation.

3. primer described in claim 1 combination is crawled the application in character assistant breeding reagent in preparation chicken.

The method of character 4. a kind of identification chicken of non-disease diagnostic purpose crawls, which is characterized in that with the genome of test individual DNA is template, is combined using primer described in claim 1, is analyzed using multiple PCR method；If multiplexed PCR amplification There are two types of product, sequence size is respectively 438bp and 224bp, then test individual is genetic heterozygosis (Cp/+) of crawling, and is shown as It crawls character；If multiplexed PCR amplification only has a kind of product, sequence size 438bp, then test individual is allozygote (+/+), show as wild type；The test individual is 4 week old chickens.

5. according to the method described in claim 4, it is characterized in that, multi-PRC reaction system are as follows: genomic DNA 80- 2.0 2.0 μ L, Taq archaeal dna polymerase of μ L, dNTPs of 100ng, 10 × PCR buffer, 1.0 μ L, SEQ ID No.1, SEQ ID Each 0.4 μ L of primer shown in each 0.2 μ L, SEQ ID No.3 of primer shown in No.2, SEQ ID No.4, uses ddH₂O supplements reactant It is to 20.0 μ L.

6. method according to claim 4 or 5, which is characterized in that multi-PRC reaction condition are as follows: 94 DEG C of initial denaturation 5min； Circulation process is 94 DEG C of denaturation 30s, 57 DEG C of annealing 30s, 72 DEG C of extension 35s, and totally 35 recycle；Last 72 DEG C of extensions 10min.