Methods for detecting fast and slow feathering and pure heterozygosity in chickens

Abstract

The present invention provides a method for detecting fast and slow feathering and homozygous variants in chickens, namely, a method for rapidly detecting fast and slow feathering and homozygous variants in chickens based on PCR and Sanger sequencing technology. The present invention also provides a set of specific primer combinations (SEQ ID NOs: 1-3) for detecting fast and slow feathering and homozygous variants in chickens. Compared with conventional methods, this method can quickly and accurately identify fast and slow feathering and homozygous variants without the need for testcrossing, shortening the time required to establish a fast and slow feathering self-sexed matching line, thereby reducing breeding costs.

Description

Technical Field

[0001] The invention belongs to the technical field of animal genetic breeding, and in particular relates to a method for detecting fast and slow feathers of chickens and their pure heterozygosity. Background Art

[0002] Hens and roosters have different production and economic values. In modern large-scale chicken production, sexing chicks is an important step. Currently, the main methods for sexing chicks are vent rotation identification and sex-linked trait identification (gold and silver feathers, fast and slow feathers, etc.). Vent rotation identification is based on the morphological characteristics of the reproductive protrusions of newborn chicks. The identifier needs to be professionally trained, and it is easy to cause damage to the chicks during the identification process. Sex-linked trait identification uses sex-linked genetic genes to cultivate special self-sexing matching lines for sexing, which is more intuitive and quick. Compared with gold and silver feather self-sexing, fast and slow feathers are not restricted by feather color and chicken breed, and can be applied to many breeds. It is currently the most widely used self-sexing method.

[0003] The fast and slow feathering traits are inherited along with the Z chromosome, slow feathering (K) is dominant, and fast feathering roosters (Z) are generally used in production. k Z k ) and slow-feathering hens (Z K W) hybridization, the offspring produced are all hens with fast feathers, and all roosters with slow feathers, thus realizing the self-differentiation of male and female. The phenotypic determination of fast and slow feathers is generally within 24 hours after the chick hatches. If its main wing feathers are longer than the covert main wing feathers by more than 2mm, it is a fast feather, otherwise it is a slow feather. Therefore, breeding a fast feather system and a slow feather system is the key to realizing the self-differentiation of male and female with fast and slow feathers. Fast feathers are recessive relative to slow feathers, and the establishment of a fast feather system can be directly determined based on phenotypic determination. There are two genotypes, homozygous and heterozygous, in slow feather roosters. Traditional establishment is generally identified by testcrossing, so it takes at least two generations to establish a slow feather pure system, which consumes a long time. Therefore, setting up a molecular biological method for quickly distinguishing fast and slow feathers and pure heterozygous thereof is of great significance to shortening the time for establishing a fast and slow feather system and saving breeding costs. Summary of the Invention

[0004] The present invention aims to provide a method for detecting fast and slow feathers and their homozygous in chickens, and in particular to a method for rapidly detecting fast and slow feathers and their homozygous in chickens by using specific primer pairs and based on PCR and Sanger sequencing technology.

[0005] In order to achieve the purpose of the present invention, in the first aspect, the present invention provides a set of primer combinations for detecting chicken fast and slow feathers and their pure heterozygous, the primer combination comprising two forward primers F1 and F2 and a reverse primer R, the nucleotide sequences of which are shown in SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 2, respectively.

[0006] The above primers were developed based on the repetitive sequence on chicken chromosome Z (chicken reference genome GRCg7b version Z: 11167618-11355728 bp) reported by Elferink et al. (2008) to identify fast and slow feathers and their homozygous counterparts in chickens. Based on the chicken reference genome GRCg7b version, primer F1 is located at Z: 11167565-11167588 bp, primer F2 is located at Z: 11355275-11355295 bp, and primer R is located at Z: 11167928-11167950 bp.

[0007] In a second aspect, the present invention provides a detection reagent or kit containing the primer combination.

[0008] In a third aspect, the present invention provides the use of the primer combination or a detection reagent or kit containing the primer combination in identifying fast and slow feathers and their homozygous heterozygosity in chickens.

[0009] In a fourth aspect, the present invention provides a method for detecting fast and slow feathers and homozygous feathers in chickens, comprising the following steps:

[0010] (1) Extracting genomic DNA of the chicken species to be tested;

[0011] (2) using the DNA extracted in step (1) as a template and performing PCR amplification using the primer combination;

[0012] (3) Analyze the PCR amplification products.

[0013] Furthermore, the PCR amplification system was as follows: 2× Taq PCR Mix 10 μL, primers F1, R and F2 were 0.25 μL, 0.5 μL and 0.25 μL respectively, the concentration of each primer was 10 μM, 40-50 ng / μL DNA template 1 μL, and ddH2O 8 μL.

[0014] PCR amplification program: 95°C for 5 min; 95°C for 30 s, 61°C for 30 s, 72°C for 50 s, 26 cycles; 72°C for 5 min; storage at 4°C.

[0015] Furthermore, step (3) detects the PCR amplification product by agarose gel electrophoresis, identifies fast and slow feathers according to the number of bands in the agarose gel electrophoresis diagram, identifies pure heterozygotes according to the brightness of the bands, and the amount of DNA template of the chicken individuals to be tested is the same; specifically as follows:

[0016] 1) Fast-feathering individuals only have one band of 386 bp, while slow-feathering individuals have two bands of 386 bp and 786 bp;

[0017] 2) The brightness of the 386bp band in fast-feathering roosters is twice that in fast-feathering hens;

[0018] 3) The brightness of the 386bp band of slow-feathering hens is L, and the brightness of the 786bp band is L′;

[0019] The brightness of the 386bp band in the slow-feathering heterozygous rooster is 2L, and the brightness of the 786bp band is L′;

[0020] The brightness of the 386bp band in the slow-feathering homozygous rooster is 2L, and the brightness of the 786bp band is 2L′.

[0021] Preferably, the concentration of agarose gel is 1.5%, the sample volume is 6 μL; and the electrophoresis conditions are: 120 V, 30 min.

[0022] Furthermore, step (3) detects the PCR amplification product by Sanger sequencing, identifies the fast and slow feathers based on the Sanger sequencing peak diagram, and identifies the pure heterozygote based on the relative height of the peaks in the bimodal region; specifically as follows:

[0023] i) The single peak sequence of fast-feathering individuals is about 340 bp in size;

[0024] ii) The peak sequence of the slow feather individual is about 740 bp in size, with a double peak at about 300-350 bp;

[0025] The first double peak is recorded as the first position, and the double peaks at positions 17 and 31 are used as molecular markers to distinguish slow feathering homozygous individuals from slow feathering heterozygous individuals:

[0026] In slow feathering homozygous individuals, the height of the 17th T peak is equal to or greater than the C peak, and the height of the 31st T peak is greater than the G peak;

[0027] In the slow feathering heterozygous individuals, the height of the 17th T peak is smaller than the C peak, and the height of the 31st T peak is equal to or smaller than the G peak.

[0028] The present invention can extract DNA from chicken blood or tissue.

[0029] In a fifth aspect, the present invention provides the application of the method in sex identification and fast and slow feather identification of chickens.

[0030] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0031] (1) The present invention provides a specific primer pair and detection method for identifying the fast and slow feather genotypes of chickens. Compared with conventional methods, this method can quickly and accurately identify fast and slow feathers and their pure heterozygous without test crosses, shortening the time for establishing fast and slow feather self-differentiated male and female matching lines and saving breeding costs.

[0032] (2) Based on the repetitive sequence on the chicken Z chromosome, the present invention has developed a specific primer pair for identifying chicken fast and slow feathers and their pure heterozygous. The primers have good specificity and can identify fast and slow feathers and their pure heterozygous through PCR amplification. It has the advantages of easy operation, rapidity, high efficiency and high accuracy.

[0033] (3) The present invention provides a dual determination method based on gel images and sequencing peak images, which can reduce the determination error rate and has higher identification accuracy.

[0034] (4) The present invention effectively solves the problem of feather speed identification being limited by time.

[0035] (V) Compared with existing quantitative methods, the present invention does not require the establishment of reference ranges for each genotype first, and can be directly judged based on gel images and sequencing peak images, which is more intuitive and convenient.

[0036] (6) Compared with the enzyme cleavage method, the present invention directly performs PCR amplification across the repetitive sequence without the need to find and establish enzyme cleavage sites, which is simpler to operate and has a wider application.

[0037] (7) The present invention can provide effective identification for the fast and slow feathering genotypes of chickens, and also provide a reference for the identification of genotypes of other species and their pure and heterozygous genotypes. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Schematic diagram of the location of the repeat fragment on the chicken Z chromosome and the primer design position of the present invention.

[0039] Figure 2 and Figure 3 The agarose gel electrophoresis diagram of PCR amplification products at different concentrations and cycle numbers in a preferred embodiment of the present invention is shown in FIG1 , wherein 1 is a slow-feathering homozygous rooster, 2 is a slow-feathering heterozygous rooster, and 3 is a slow-feathering hen.

[0040] Figure 4 1 is an example diagram of agarose gel electrophoresis of PCR amplification products of different genotypes in a preferred embodiment of the present invention.

[0041] Figure 5-Figure 7 This is an agarose gel electrophoresis diagram of the genotype identification of some individuals in the preferred embodiment of the present invention.

[0042] Figure 8 This is a Sanger sequencing diagram of PCR amplification products of different genotypes in a preferred embodiment of the present invention.

[0043] Figure 9 This is an agarose gel electrophoresis diagram of the pure heterozygous identification of some slow-feathered roosters in the preferred embodiment of the present invention. DETAILED DESCRIPTION

[0044] The present invention aims to provide a method for rapidly detecting fast and slow feathering in chickens and their homozygosity based on PCR and Sanger sequencing technology. This method can quickly and accurately identify genotypes, shorten the time required to establish fast and slow feathering self-sexed matching lines, improve breeding efficiency, and reduce breeding costs.

[0045] The present invention adopts the following technical solutions:

[0046] In a first aspect, the present invention provides specific primer pairs for detecting fast and slow feathers and their homozygous heterozygous.

[0047] Specifically, the present invention uses the repetitive sequence on the chicken chromosome Z reported by Elferink et al. (2008) (chicken reference genome GRCg7b version Z: 11167618-11355728bp) as a molecular marker, and designs primers based on the repetitive sequence before the breakpoint (Z: 11167565-11167588), at the beginning of the segment (Z: 11167928-11167950bp) and at the end of the segment (Z: 11355275-11355295bp) ( Figure 1 ), the primer sequences are F1 (SEQ ID NO: 1), primer R (SEQ ID NO: 2), and primer F2 (SEQ ID NO: 3). The primers can specifically amplify, so that fast and slow feathers and their homozygous and heterozygous feathers can be identified based on the amplified products.

[0048] In a second aspect, the present invention provides a detection reagent or kit containing the primer pair.

[0049] In a third aspect, the present invention provides a method for identifying fast and slow feathers and their pure and heterozygous forms. Specifically, the method includes:

[0050] (1) Extraction of genomic DNA from the chicken species to be tested;

[0051] (2) Using the extracted DNA as a template, PCR amplification was performed using primers SEQ ID NO: 1-3;

[0052] (3) agarose gel electrophoresis and Sanger sequencing;

[0053] (4) Genotype determination (based on electrophoresis diagram and sequencing peak diagram).

[0054] Furthermore, the step of extracting the genomic DNA of the chicken species to be tested can be performed by using the traditional phenol-chloroform method or a kit.

[0055] Furthermore, the PCR amplification system includes: 10 μL of 2×Taq PCR Mix, 0.25 μL, 0.5 μL, and 0.25 μL of primers SEQ ID NO: 1-3, respectively (all at a concentration of 10 μM), 1 μL of DNA template (40-50 ng / μL), and 8 μL of ddH2O.

[0056] Furthermore, the PCR amplification program was as follows: 95°C for 5 min; 95°C for 30 s, 61°C for 30 s, 72°C for 50 s, 26 cycles; 72°C for 5 min; and storage at 4°C.

[0057] The agarose gel electrophoresis comprises: the concentration of the agarose gel is 1.5%, the sample volume is 6 μL, the electrophoresis voltage is 120 V, and the electrophoresis time is 30 minutes.

[0058] The Sanger sequencing can be performed by a commercial sequencing company.

[0059] Furthermore, genotype determination was performed based on the electrophoresis pattern, specifically including: imaging on a gel imaging instrument and adjusting the imaging until the bands were clear and easy to distinguish. Fast-feathering individuals had only one 386bp band amplified by the F1-R primer, while slow-feathering individuals had two bands, namely 386bp and 786bp bands amplified by the F1-R and F2-R primers, respectively. According to the dosage effect, fast-feathering hens amplified one 386bp band, fast-feathering roosters amplified two 386bp bands, slow-feathering hens amplified one 386bp band and one 786bp band, slow-feathering heterozygous roosters amplified two 386bp bands and one 786bp band, and slow-feathering homozygous roosters amplified two 386bp bands and two 786bp bands. Band brightness is consistent with the dosage. For the 386bp band, the following applies: fast female ≈ slow female ≈ 1 / 2 fast male ≈ 1 / 2 slow pure male ≈ 1 / 2 slow hybrid male. For the 786bp band, the following applies: slow female ≈ slow hybrid male ≈ 1 / 2 slow pure male. In individuals with slow feathering heterozygotes, the two bands are essentially identical in brightness. In individuals with slow feathering homozygotes, the 786bp band is approximately twice as bright as the 386bp band. Band brightness can be used to determine fast and slow feathering, pure heterozygotes, and sex differentiation.

[0060] Furthermore, genotype identification is performed based on the sequencing peak graph, specifically including: Sanger sequencing at a commercial sequencing company, and sequencing using primer R (SEQ ID NO: 2). After sequencing, a single peak sequence of about 340bp can be seen for the fast feather, and a peak graph sequence of about 740bp can be seen for the slow feather, presenting a double peak graph at about 300-350bp. The first double peak is counted as the 1st position, and a peak of about 43 can be seen. The 17th and 31st positions are used as molecular markers, and the height of the 17th T peak of the slow feather homozygous individual is equal to or greater than the C peak, and the height of the 31st T peak is greater than the G peak; the height of the 17th T peak of the slow feather heterozygous individual is less than the C peak, and the height of the 31st T peak is equal to or less than the G peak. Sequencing can distinguish between fast and slow feathers and their pure heterozygous, but cannot identify males and females.

[0061] Fourthly, other feasible technologies for extracting DNA from chicken blood or tissues can also be used in the present invention.

[0062] The method provided by the invention can be applied to the identification of sex and fast and slow feathers of chickens, can avoid the damage caused by anal inversion identification, and the identification is not limited to time and can be performed at any time of the chicken's growth, with a high identification accuracy rate.

[0063] The method provided by the present invention can be applied to chicken autogenous male-female pairs and genetic breeding. In the early stages of establishing fast-feather and slow-feather pairs in chickens, identification of fast-feather and slow-feather pairs, as well as pure heterozygotes, is necessary. The present invention eliminates the need for testcrosses and enables simple and rapid identification of pure heterozygotes, shortening the time required to establish fast-feather and slow-feather pairs and reducing breeding costs.

[0064] The following examples are intended to illustrate the present invention but are not intended to limit the scope of the invention. Unless otherwise specified, the examples are based on conventional experimental conditions, such as those in Sambrook et al. Molecular Cloning: A Laboratory Manual (Sambrook J & Russell DW, Molecular Cloning: A Laboratory Manual, 2001), or the conditions recommended by the manufacturer's instructions. Example 1 Method for Identifying Chicken Fast and Slow Feathers and Their Homozygous

[0065] 1. Primer Design and Preparation

[0066] According to the repetitive sequence reported by Elferink et al. (2008), as a molecular marker, primers were designed using Primer 5 software before the breakpoint, at the beginning of the segment, and at the end of the repetitive sequence. The primers are F1, R, and F2, respectively, and their sequences are as follows:

[0067] Primer F1: 5'-GTTTGACCTGTGCTGTGGTTTGCT-3' (SEQ ID NO: 1)

[0068] Primer R: 5'-CTGTGCCCTTCCATCAGTGCTTC-3' (SEQ ID NO: 2)

[0069] Primer F2: 5'-GCCATCAGCCAGATCCGTCAG-3' (SEQ ID NO: 3)

[0070] The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0071] 2. Extraction of Genomic DNA from Chicken Blood

[0072] Genomic DNA was extracted using the blood / tissue / cell genomic extraction kit (#DP304-03) from Tiangen Biochemical Technology Co., Ltd. The extraction steps were performed according to the manufacturer's instructions.

[0073] 3. Exploration of PCR conditions

[0074] To optimize identification conditions, gradient testing and agarose gel electrophoresis were performed to test the annealing temperature, cycle number, and DNA template concentration, factors that influence PCR. A gradient test was conducted at 60-70°C, and the optimal annealing temperature was found to be 60-62°C. A gradient test of cycle numbers (26, 28, 30, and 32) and DNA template concentrations (40 ng / uL, 60 ng / uL, 80 ng / uL, 100 ng / uL, 150 ng / uL, and 200 ng / uL) was performed at 61°C. At 26 cycles, pure heterozygous identification was possible with all template concentrations; at 28 cycles, identification was possible with 40 ng / uL and 60 ng / uL template concentrations; and at 30 and 32 cycles, pure heterozygous identification was not possible with any of the four concentrations. Therefore, the optimal identification conditions are: annealing temperature 61°C, DNA template concentration 40-200 ng / uL (the template concentration of all individuals added should be basically the same during identification), reaction cycle number 26 ( Figure 2 and Figure 3 ).

[0075] The 2×Taq PCR Mix used for PCR condition exploration and the following genotype identification was from Beijing Huitian Oriental Technology Co., Ltd., with the product number HT201.

[0076] 4. PCR Amplification

[0077] The 20 μL PCR amplification system includes: 10 μL of 2× Taq PCR Mix, 0.25 μL, 0.5 μL, and 0.25 μL of primers F1 (SEQ ID NO: 1), R (SEQ ID NO: 2), and F2 (SEQ ID NO: 3), respectively (all at a concentration of 10 μM), 1 μL of DNA template (40-50 ng / μL), and 8 μL of ddH2O.

[0078] The PCR amplification program was as follows: 95°C for 5 min; 95°C for 30 s, 61°C for 30 s, 72°C for 50 s, 26 cycles; 72°C for 5 min; and storage at 4°C.

[0079] 5. Agarose Gel Electrophoresis Sanger Sequencing

[0080] Agarose gel electrophoresis includes: the concentration of agarose gel is 1.5%, the sample volume is 6 μL, the electrophoresis voltage is 120 V, the electrophoresis time is 30 minutes, and after electrophoresis, imaging is performed on a gel imager, and the imaging is adjusted until the bands are clear and easy to distinguish.

[0081] Sanger sequencing: Sanger sequencing was performed at Beijing Qingke Biotechnology Co., Ltd.

[0082] 6. Genotype determination

[0083] (1) Genotype determination based on agarose gel electrophoresis

[0084] Fast-feathering individuals had only one 386bp band amplified by the F1-R primer; slow-feathering individuals had two bands: a 386bp band amplified by the F1-R and a 786bp band amplified by the F2-R primers. According to the dosage effect, fast-feathering hens amplified one 386bp band, fast-feathering roosters amplified two 386bp bands, slow-feathering hens amplified one 386bp band and one 786bp band, slow-feathering heterozygous roosters amplified two 386bp bands and one 786bp band, and slow-feathering homozygous roosters amplified two 386bp bands and two 786bp bands (Table 1). Band brightness results were generally consistent with dosage: for the 386bp band, the following relationship was true: fast hens ≈ slow hens ≈ 1 / 2 fast hens ≈ 1 / 2 slow-homogeneous hens ≈ 1 / 2 slow-homogeneous hens; for the 786bp band, the following relationship was true: slow hens ≈ slow-homogeneous hens ≈ 1 / 2 slow-homogeneous hens. The brightness of the two bands of the slow feathering heterozygous individual is basically the same, and the brightness of the 786bp band of the slow feathering homozygous individual is about twice that of the 386bp band. According to the number of bands, fast feathering or slow feathering can be determined, and according to the brightness of the bands, pure heterozygous and male and female can be determined. Figure 4-Figure 7 ).

[0085] Table 1 Number of bands amplified by PCR for different genotypes

[0086]

[0087] (2) Genotype determination based on sequencing peak graph

[0088] Sequencing was performed using primer R (SEQ ID NO: 2). After sequencing, a 340bp single-peak sequence can be seen for the fast feather, and a 740bp peak sequence can be seen for the slow feather, showing a double-peak peak at about 300-350bp. The reasons for the slow feather peak: from the R primer to the breakpoint position, the sequences of the fast and slow feathers are consistent, and a single peak is shown after sequencing; because the slow feather contains repetitive sequences, the sequences amplified by the fast and slow feathers are different after the breakpoint, so a double peak will appear until the sequence reaches the primer F1 position; after passing the F1 primer, only the slow feather can amplify the F2-R fragment, and the peak graph shows a single peak until the F2 primer. Due to the dosage effect, the relative difference in the concentration of pure heterozygous bases at the double peak leads to the difference in the height of the peak graph. The first double peak in the double peak segment after the breakpoint is counted as the first position, and a peak of about 43 can be seen. The site with obvious height difference of the pure heterozygous peak graph is found as a molecular marker. The present invention mainly uses the 17th and 31st positions as molecular markers. The height of the T peak at position 17 of the slow feather homozygous individual is equal to or greater than the C peak, and the height of the T peak at position 31 is greater than the G peak; the height of the T peak at position 17 of the slow feather heterozygous individual is less than the C peak, and the height of the T peak at position 31 is equal to or less than the G peak. According to the sequencing peak diagram, the fast and slow feathers and their homozygous and heterozygous ( Figure 8 ).

[0089] 7. Comparison of Agarose Gel Electrophoresis and Sanger Sequencing

[0090] Under the same PCR amplification conditions, both agarose gel electrophoresis and Sanger sequencing can identify fast and slow feathers and their pure heterozygotes, but agarose gel electrophoresis can also identify male and female based on the brightness of the bands.

[0091] It should be noted that (1) due to the differences in the use of PCR instruments and Taq enzymes, differences in amplification efficiency may occur, resulting in the optimal PCR conditions being different from those of the present invention. In practical applications, it is necessary to first explore the Tm value and find the optimal Tm value so that the band brightness is as close to the theoretical state as possible (the state that conforms to the dose effect), otherwise the judgment error will be large. (2) When identifying genotypes through gel images, it is necessary to ensure that the total amount of template added to all individuals to be tested in PCR amplification is basically the same, otherwise only fast and slow feathers can be identified, and pure heterozygotes cannot be identified; during sequencing identification, the total amount of template amplified by PCR can be inconsistent, and pure heterozygote identification is less affected by the amount of template. (3) Under different PCR amplification efficiencies, the brightness of agarose gel electrophoresis bands and the height of Sanger sequencing peak graphs will also vary. During identification, the amplification efficiency, band brightness and peak graph may not be exactly the same as in this example. According to the judgment ideas of the present invention, similar band brightness patterns and peak graph markers can be found to complete genotype identification. (4) During the identification process, if the samples to be tested are of the same sex, there is no need to set a reference sample. Fast-feathered, slow-feathered, and pure and heterozygous identification can be performed based on agarose gel electrophoresis and Sanger sequencing. If samples of different sexes are to be identified, a reference sample needs to be set. It is recommended that the reference sample be a slow-feathered hen. After PCR amplification, the genotype and gender can be identified by the brightness of the agarose gel electrophoresis band, or the genotype can be identified by sequencing.

[0092] Example 2 Identification of fast and slow feathers of Shouguang chicken

[0093] Utilize the method for embodiment 1, 34 fast-feather Shouguang roosters, 48 ​​slow-feather Shouguang roosters, 74 fast-feather Shouguang hens, 76 slow-feather Shouguang hens of known phenotype are carried out genotype identification, all fast-feather chickens all amplify a 386bp band, all slow-feather chickens all amplify two bands (386bp and 786bp), and the fast-feather and slow-feather identification accuracy rate is 100%. Genotype identification of 48 slow-feather Shouguang roosters is homozygous 19, heterozygous 29, and is tested and verified with dwarf chicken fast-feather hens. If the offspring hatches more than 5 slow-feather chicks and 0 fast feathers, it is determined that this individual is homozygous. If the offspring has both fast feathers and slow feathers, it is determined that this individual is heterozygous. The test cross result shows that, except for 1 rooster offspring identified as homozygous, there are 13 slow-feather chicks and 1 fast-feather chick, and the others all meet the genotype identification result. The genotype of the fast-feathering chick was identified, and it was found that the individual and its father did not conform to Mendel's law of inheritance. It was speculated that the individual was not the offspring of a homozygous rooster, which may be caused by factors such as taking the wrong eggs or writing the wrong number during the egg collection or incubation process. Therefore, the test cross result is consistent with the genotype identification result ( Figure 9 ).

[0094] Example 3 Identification of Pure Heterozygous Local Chicken Breeds

[0095] Using the method of Example 1, 59 Beijing oily chickens, 27 Tibetan chickens, 17 green-shelled chickens, and 1 fighting cock were genotyped. The results showed that 37 oily chickens were homozygous and 22 were heterozygous; 3 Tibetan chickens were homozygous and 24 were heterozygous; 3 green-shelled chickens were homozygous and 14 were heterozygous; and 1 fighting cock was heterozygous. After typing, the slow-feathered roosters and fast-feathered hens were test-crossed. If the offspring produced more than 5 slow-feathered chicks and 0 fast-feathered chicks, the individual was determined to be homozygous. If the offspring had both fast and slow feathers, the individual was determined to be heterozygous. The test-cross results showed that, except for one green-shelled rooster whose genotype was identified as homozygous, the offspring produced 20 slow-feathered chicks and 1 fast-feathered chick. The other 103 slow-feathered roosters were consistent with the genotype identification results, and the accuracy rate of pure heterozygous identification was greater than 99%. Genotyping of the fast-feathering chick revealed that the individual and its father did not conform to Mendel's laws of inheritance. It is speculated that this individual is not the offspring of a homozygous green-shelled rooster, possibly due to factors such as incorrect egg collection or incubation, or incorrect numbering. Therefore, the testcross results are consistent with the genotyping results.

[0096] The above results show that the identification method of the present invention can identify the fast-feathering and slow-feathering genotypes of chicken breeds with high accuracy.

[0097] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A primer combination for detecting chicken fast and slow feathers and pure heterozygous thereof, characterized in that: The primer combination includes two forward primers F1 and F2 and one reverse primer R, and the nucleotide sequences are shown in SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 2, respectively.

2. A detection reagent or kit containing the primer combination according to claim 1.

3. Use of the primer combination according to claim 1 or the detection reagent or kit according to claim 2 in identifying fast and slow feathers and their homozygous and heterozygous in chickens.

4. A method for detecting fast and slow feathers in chickens and their pure heterozygosity, characterized in that: The following steps are involved: (1) Extracting genomic DNA of the chicken species to be tested; (2) using the DNA extracted in step (1) as a template and performing PCR amplification using the primer combination described in claim 1; (3) Analyze PCR amplification products; Step (3) detecting the PCR amplification product by agarose gel electrophoresis, identifying fast and slow feathers according to the number of bands in the agarose gel electrophoresis diagram, identifying pure heterozygotes according to the brightness of the bands, and the amount of DNA template of the chicken individuals to be tested is the same; specifically as follows: 1) Fast-feathering individuals only have one band of 386 bp, while slow-feathering individuals have two bands of 386 bp and 786 bp; 2) The brightness of the 386bp band in fast-feathering roosters is twice that in fast-feathering hens; 3) The brightness of the 386bp band in slow-feathering heterozygous roosters is twice that of the 386bp band in slow-feathering hens; the brightness of the 786bp band in slow-feathering heterozygous roosters is equivalent to that of the 786bp band in slow-feathering hens; The brightness of the 386bp band in slow-feathering homozygous roosters is twice that of the 386bp band in slow-feathering hens; the brightness of the 786bp band in slow-feathering homozygous roosters is twice that of the 786bp band in slow-feathering hens.

5. A method for detecting fast and slow feathers in chickens and their pure heterozygosity, characterized in that: The following steps are involved: (1) Extracting genomic DNA of the chicken species to be tested; (2) using the DNA extracted in step (1) as a template and performing PCR amplification using the primer combination described in claim 1; (3) Analyze PCR amplification products; Step (3) detects the PCR amplification product by Sanger sequencing, identifies the fast and slow feathers based on the Sanger sequencing peak diagram, and identifies the pure heterozygote based on the relative height of the peaks in the bimodal region; specifically as follows: i) The single peak sequence of the fast-feathering individual corresponds to 340 bp; ii) The peak sequence of the slow feather individual corresponds to 740 bp, in which a double peak appears at 300-350 bp; The first double peak is recorded as the first position, and the double peaks at positions 17 and 31 are used as molecular markers to distinguish slow feathering homozygous individuals from slow feathering heterozygous individuals: In slow feathering homozygous individuals, the height of the 17th T peak is equal to or greater than the C peak, and the height of the 31st T peak is greater than the G peak; In the slow feathering heterozygous individuals, the height of the 17th T peak is smaller than the C peak, and the height of the 31st T peak is equal to or smaller than the G peak.

6. The method according to claim 4, characterized in that PCR amplification system: 2× Taq PCR Mix 10 μL, primers F1, R, and F2 0.25 μL, 0.5 μL, and 0.25 μL, respectively, each primer concentration is 10 μM, 40-50 ng / μL DNA template 1 μL, ddH2O 8 μL; PCR amplification program: 95°C for 5 min; 95°C for 30 s, 61°C for 30 s, 72°C for 50 s, 26 cycles; 72°C for 5 min; storage at 4°C.

7. The method according to claim 4, characterized in that The concentration of agarose gel was 1.5%, the sample volume was 6 μL, and the electrophoresis conditions were: 120 V, 30 min.

8. The method according to any one of claims 4 to 7, characterized in that: Extract DNA from chicken blood or tissue.

9. Use of the method according to any one of claims 4, 6 to 8 in sexing and fast and slow feather identification of chickens.

10. Use of the method according to any one of claims 5, 6 to 8 in identifying fast and slow feathers of chickens.

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