TWI398522B - Method and kit for identifying hatchability performance in tsaiya ducks - Google Patents

Description

Method and set for identifying hatching rate of duck

The invention relates to a method and a kit for identifying the hatching rate of a duck, in particular to a method and a kit for identifying the hatching rate of a duck using the egg gypsum genotype.

Tsaiya duck is a type of duck that belongs to the egg-producing variety. The selection of the vegetable ducks has relied on the selection of individual body phenotypes or the use of traditional quantitative techniques.

The phenotypic selection is based on the performance and body phenotypic characteristics of individuals and their relatives. However, the body surface phenotype is a comprehensive manifestation of heredity and environment. If it is selected based on the individual's body phenotype, it is easy to cause mistakes.

However, if it is selected by traditional quantitative genetic techniques, it takes a long time and requires a large enough number of ethnic groups to maintain a high maintenance cost. When the performance improvement reaches a certain level, the selection difference becomes small, making it difficult to obtain effective. The amount of genetic improvement.

Therefore, the present invention provides a method and a kit for identifying the hatching rate of a duck, and can apply the identification method to screen a duck with a high hatching rate.

One aspect of the present invention provides a method for identifying a hatching rate of a duck, comprising analyzing a sequence of an ovomucoid gene, and when the sequence comprises SEQ ID NO: 7, a duck with a high hatching rate, when The sequence contains a duck with a low hatching rate when SEQ ID NO: 8.

Another aspect of the present invention provides a method for identifying a hatching rate of a duck, comprising polymerase chain reaction amplification of egg mammage DNA of duck with SEQ ID NO: 1 and SEQ ID NO: 2. When the augmented fragment is SEQ ID NO: 3, the duck is a high hatching rate, and when the augmented fragment is SEQ ID NO: 4, the duck is a low hatching rate.

A further aspect of the present invention provides a method for identifying hatching rate of a duck, comprising polymerase chain reaction amplification of egg mucin deoxyribonucleic acid of duck with SEQ ID NO: 5 and SEQ ID NO: 6. When the augmented fragment is the high hatching rate of the vegetable duck of SEQ ID NO: 7, when the augmented fragment is the low hatching rate of the vegetable duck of SEQ ID NO: 8.

According to an embodiment of the present invention, the fragment is further cut by using a restriction enzyme of the recognizable sequence 5'-AAATTC-3', and is a high hatching rate duck when the fragment of 168 base pairs is obtained; When a fragment containing 168 base pairs, 124 base pairs and 41 base pairs is obtained, it is also a high hatching rate of ducks; when a fragment having a length of 124 base pairs and 41 base pairs is obtained, Low hatching rate of duck.

A further aspect of the present invention provides a method for identifying the hatching rate of a duck, comprising extracting the genetic DNA of the duck to be tested, and increasing the amount of the sample to be tested with the primers SEQ ID NO: 5 and SEQ ID NO: 6. The egg mucin gene fragment sequence of the duck was cleavable by using a restriction enzyme 5'-AAATTC-3' restriction enzyme, and the fragment was amplified by agar gel electrophoresis. Because of the high hatching rate and low hatching rate, the egg mucin gene fragment of the duck has different maps after being cut, so the map can be used to identify the hatching rate of the duck.

According to an embodiment of the invention, wherein the restriction enzyme is Apo I.

According to an embodiment of the present invention, the duck can be selected as a breeding duck when the amplified fragment of the gene is cleaved by the restriction enzyme to obtain only a fragment of 168 base pairs in length.

Another aspect of the invention provides a kit for identifying hatching rates of ducks comprising primer pairs SEQ ID NO: 1 and SEQ ID NO: 2 or primer pairs SEQ ID NO: 5 and SEQ ID NO: 6.

According to an embodiment of the present invention, when the primer pair is SEQ ID NO: 1 and SEQ ID NO: 2, the amplified DNA fragment control SEQ ID NO: 3 and SEQ ID NO: 4 are further included. Deoxyribonucleic acid fragment.

According to another embodiment of the present invention, when the primer pair is SEQ ID NO: 5 and SEQ ID NO: 6, the deoxyribonucleic acid fragment of the reference SEQ ID NO: 7 and SEQ ID NO: 8 is further included.

According to the above, the method of the embodiment of the present invention is characterized in that only the genetic DNA of the duck is extracted, and a part of the gene fragment of the egg mucin is amplified by a polymerase chain reaction to limit the amplified fragment of the enzyme. Shearing can be carried out according to different shearing results to determine the hatching rate of the duck, and the breeding ducks can be screened accordingly.

Therefore, this selection method is faster and more accurate than the traditional selection method, and can be applied to the selection of ducks with high hatching rate performance.

The present invention first uses a complementary deoxyribonucleic acid (cDNA) microarray to analyze a molecular marker that may be associated with hatchability, and thereafter confirms the association of the molecular marker with hatchability performance, and uses a polymerase. The chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique was used to establish the genotypic identification method determined by the marker, and the hatching rate and high hatching of ducks were identified by this method. The rate of performance of the duck.

In the following, the application basis and feasibility of the embodiment of the present invention will be described in two parts. The first part is to reveal differentially expressed transcripts by using cDNA microarray to analyze the difference in the hatching rate between ducks and ducks. Techniques such as sequencing and PCR-RFLP analysis are used to find molecular markers related to the high hatching rate of ducks, so as to establish the application basis of egg genotypes for identification of duck hatching rate and selection of high hatching rate. Then, using the test results of the first part, the method of separating the genomic DNA from the blood of the duck is described in the examples, and the method of identifying the duck genotype by PCR-RFLP technology is provided for industrial application.

first part: (I), cDNA microarray analysis and confirmation of results

In the present embodiment, the ribonucleic acid (RNA) is extracted from the epithelial cells of the oviduct enlargement by the egg laying duck (hatching rate 44.00-81.82%; average 58.14±5.98%), and the commercial SMART cDNA Library Construction kit ( BD Biosciences Clontech) established a cDNA gene library. Proliferation cDNA fragment from a gene library, using the ^{OmniGrid Accent TM microarrayer (GeneMachine, Bethesda} , MD, USA) on a glass wafer made of points, 8736 points each. The cDNA of the laying ducks in the high hatching rate group (88.20 ± 1.21%) and the low hatching rate group (55.60 ± 1.04%) were hybridized with the probes on the wafer.

After microarray images were scanned, the data were analyzed using Avadis software (Strand Life Sciences, Redwood City, CA, USA). The results showed that when the fold change (Fc) was greater than 1.1, 27 differentially expressed transcripts were obtained; when the performance difference was greater than 1.5, 6 differentially expressed transcripts were obtained.

The sequence of the six differentially expressed transcripts was confirmed by bidirectional nucleotide sequencing, and the obtained sequence was aligned with the sequence in the NCBI (National Center for Biotechnology Information) gene database, and one of them was found to be egg sticky. Protein differences represent transcripts.

Please refer to Figure 1 for the analysis of the gene expression of egg yolk in high and low hatching rate. In order to confirm the results of microarray analysis, real-time PCR analysis showed that the egg adhesion protein ribonucleic acid (mRNA) expression in the low hatching rate group (1.00±0.20) was significantly higher than the high hatching rate. The group (0.56 ± 0.04), where p < 0.05, was statistically significant.

Further observation of the protein expression of egg mucin, Figure 2 is a graph showing the protein expression of egg yolk in high and low hatching rate. This figure was analyzed by Western blotting, in which actin was used as an internal control group to analyze the relative expression of ovomucin in the high and low hatching rate groups. The results showed that the protein expression of egg protein in the low hatching rate group was 8.42±2.44 AU (arbitrary unit), and the performance in the high hatching rate group was 2.38±0.80 AU.

In other words, from the results of Fig. 1 and Fig. 2, it can be seen that the egg adhesion protein of the low hatching rate group was significantly higher than that of the high hatching rate group (p<0.05). It indicates that the expression of egg adhesion gene is related to the hatching rate of duck.

(2) Polymerase chain reaction-restriction fragment length polymorphism technique to identify egg genotype of duck

The sequence of the expressed sequence tag (EST) of the egg mucin obtained from the expanded microarray of the vegetable duck showed a correlation with the hatching rate. After sequence alignment by nucleotide sequencing and NCBI database, the egg mucin gene fragment of the duckling high and low hatching rate group has a sequence similar to the chicken egg mucin gene intron G, so the following The egg mucin gene fragment of this duck is called "intron G". The exon sequences at the ends of the intron G of the duck egg mucin gene are also very similar to the exon 7 and exon 8 of the chicken egg mucin gene, so the following is also referred to as the egg mucin gene contained in the duck egg. The exon sequences at both ends of the sub-G are "exon 7" and "exon 8".

According to the expressed sequence tag sequence of egg mucin obtained from the cDNA microarray wafer of the duck expansion section, the primer pairs of SEQ ID NO: 1 and SEQ ID NO: 2 were designed, and the genetic DNA of duck duck with different hatching rate was used as a template. a fragment of SEQ ID NO: 3 or SEQ ID NO: 4 may be amplified, comprising a portion of exon 7 of the duck egg egg mucin gene, an intron G (SEQ ID NO: 9 or SEQ ID NO: 10) and Part of the sequence of exon 8. It was found that the ducks in the high hatching rate group had a complete intron G sequence (see SEQ ID NO: 9). In the egg mucin gene of the low hatching group, there is a TAT deletion between the 371th base pair and the 375th base pair of the intron G sequence (see SEQ ID NO: 10). (In the case of SEQ ID NO: 3 and SEQ ID NO: 4, the above-mentioned "TAT" deletion is located between the 409th base pair and the 413th base pair of the sequence of SEQ ID NO: 3.)

The restriction enzyme of the recognizable sequence 5'-AAATTC-3' can distinguish such polymorphisms, such as the restriction enzyme Apo I. Those with a "TAT" deletion can be cleaved by the above restriction enzymes; the entire sequence cannot be cleaved by the restriction enzyme described above.

Next, PCR-RFLP was performed to identify the genotype of the duck. Within the amplified fragment, primer pairs SEQ ID NO: 5 and SEQ ID NO: 6 were designed. For the high hatching rate of the duck, a fragment of 168 base pairs in length can be amplified, and the gene sequence is SEQ ID NO: 7, and there is no deletion of "TAT". For ducks with low hatching rate, a fragment of 165 base pairs in length can be amplified, and the gene sequence is SEQ ID NO: 8, which has a deletion of "TAT". As a result of PCR-RFLP analysis, the genotypes of the test ducks can be divided into +/+, +/- and -/- three types.

Figure 3 is the result of electrophoretic separation of agarose gel, where M is the molecular weight marker of deoxyribonucleic acid. In Figure 3, the fragment of SEQ ID NO: 7 (168 base pairs) was unable to be cleaved by Apo I, and only one 168 base pair fragment was present by electrophoresis, which was a +/+ genotype; SEQ ID NO A fragment of 8 (165 base pairs), which is cleaved by Apo I, will produce two bases of 124 base pairs and 41 base pairs, which are -/- genotypes; if they are heterozygous individuals, 168 base pairs are visible. The three fragments of 124 base pairs and 41 base pairs are +/- genotypes.

(3) Correlation between genotype and traits

The genetic DNA of the duck is subjected to a polymerase chain reaction of SEQ ID NO: 5 and SEQ ID NO: 6 with a primer. The product of the increase, after cleavage with the restriction enzyme Apo I, can distinguish three genotypes. Table 1 shows that the hatching rate of the fertilized egg produced by the -/- gene duck is significantly lower than that of the +/+ or +/- genotype (p<0.05), but the fertilization rate, the number of days of fertilization, and the egg weight For the total number of eggs, there was no significant difference between the ducks of all genotypes (p>0.05). From the genotype frequency distribution, the +/+, +/- and -/- genotypes were 0.39, 0.45 and 0.16 respectively; the alleles (+) and "-" were 0.61 and 0.39. In terms of hatching rate, the dual gene "+" is a favorable gene.

the second part:

Therefore, in one of the following examples, the method of extracting blood from a duck and separating the genomic DNA, and using the PCR-RFLP technique to identify the hatching rate of the duck to be tested is described.

Example:

In this example, the genomic DNA is isolated from the duck in the egg. First, blood was collected from the duck, 80 ml of blood was taken out, rinsed with physiological saline, and resuspended in 3 ml of lysis buffer (10 mM) of Tris-HCl, 150 mM sodium chloride (NaCl), 10 mM Ethylenediaminetetraacetic acid (EDTA) and 300 μl of 10% ammonium chloride (NH ₄ Cl) were uniformly mixed and thawed twice. Next, add 75 μl of proteinase K (10 mg/ml), 25 μl of collagenase (3.8 IU/ml) and 200 μl of dodecyl sulfate at a concentration of 100 g/L. Sodium (sodium dodecyl sulfate; SDS) was incubated at 55 ° C for 24 hours with agitation. Deoxyribonucleic acid was extracted with phenol and chloroform by a conventional method, and then precipitated with isopropanol, and then washed with 70% alcohol, and the alcohol was removed and purified to obtain deoxyribonucleic acid. The deoxyribonucleic acid is dissolved in deionized water for use in a polymerase chain reaction.

A partial fragment of the egg mucin gene was then amplified by polymerase chain reaction with SEQ ID NO: 5 and SEQ ID NO: 6 using primers. The reactants and reagents were carried out in the following ratios: 10 mM Tris-HCl, pH 8.8, 1.5 mM magnesium chloride (MgCl ₂ ), 50 mM potassium chloride (KCl), 0.1% Triton X-100, 200 μM dNTPs, 300 The nM primer pair, 100 ng of the gene deoxyribonucleic acid template and 1 U of DyNAzymes (Finnzymes Inc.) were added to sterile deionized water to adjust the total volume to 25 μl, and placed in a polymerase chain reaction reactor for polymerase chain reaction. The reaction conditions were 95 ° C for 2 minutes, and the following 30 cycles were carried out: a reaction at 94 ° C for 30 seconds, a reaction at 60 ° C for 30 seconds, and a reaction at 72 ° C for 45 seconds, followed by a reaction at 72 ° C for 7 minutes.

The amplified product was cleaved with the restriction enzyme Apo I, and the deoxyribonucleic acid molecule was cleaved according to the protocol provided by the restriction enzyme manufacturer. After appropriate amount of DNA sample, restriction enzyme buffer and sterile deionized water are mixed uniformly, then add appropriate amount of restriction enzyme to mix evenly, and react at the optimal reaction temperature of the enzyme.

The sheared DNA fragment was separated by 3% agarose gel electrophoresis. The electrophoresis gel was stained with ethidium bromide (EtBr) and placed under UV light. The shearing results were observed to analyze the gene of the duck. type.

As a result of electrophoresis, only a 168 base pair band is present, which is a +/+ genotype, which is a high hatching rate of duck. This duck can also be used as a breeding duck; it has 124 base pairs and 41 base pairs. The band is --- genotype, which is a low hatching rate of ducks; if it is a heterozygous individual, 168 base pairs, 124 base pairs and 41 base pairs are visible as +/- genotypes. Is a duck with a high hatching rate.

In addition, in the application of the present invention, the primer pair SEQ ID NO: 1 and SEQ ID NO: 2 may constitute a kit for identifying the hatching rate of the duck, which may further comprise SEQ ID NO: 3 and SEQ ID NO: 4. The oligoribonucleic acid fragment is used as a control; the primer pair SEQ ID NO: 5 and SEQ ID NO: 6 may also constitute a kit for identifying the hatching rate of the duck, and may further comprise SEQ ID NO: 7 and SEQ ID NO: 8. Deoxyribonucleic acid fragments were used as controls.

Although the present invention has been disclosed in the above embodiments, it is not intended to limit the present invention, and the present invention can be modified and modified without departing from the spirit and scope of the present invention. The scope is subject to the definition of the scope of the patent application attached.

<110>National Chung Hsing University

<120> A method and a set for identifying the hatching rate of ducks

<160>10

<210>SEQ ID NO: 1

<211>20

<212>DNA

<213>Artificial sequence

<220>primer

<223> Carrying out a PCR amplification of a part of the egg mucin gene fragment (forward)

<400>1

<210>SEQ ID NO: 2

<211>22

<212>DNA

<213>Artificial sequence

<220>primer

<223> Performing PCR amplification of partial ovomucin gene fragment (reversed)

<400>2

<210>SEQ ID NO: 3

<211>603

<212>DNA

<213> Anas platyrhynchos

<223> Partial gene sequence of ovomucin without "TAT" deletion

<400>3

<210>SEQ ID NO: 4

<211>600

<212>DNA

<213> Anas platyrhynchos

<223> Partial ovarian protein sequence missing from "TAT"

<400>4

<210>SEQ ID NO: 5

<211>21

<212>DNA

<213>Artificial sequence

<220>primer

<223> Carrying out a PCR amplification of a part of the egg mucin gene fragment (forward)

<400>5

<210>SEQ ID NO: 6

<211>24

<212>DNA

<213>Artificial sequence

<220>primer

<223> Performing PCR amplification of partial ovomucin gene fragment (reversed)

<400>6

<210>SEQ ID NO: 7

<211>168

<212>DNA

<213> Anas platyrhynchos

<223> Partial gene sequence of ovomucin without "TAT" deletion

<400>7

<210>SEQ ID NO: 8

<211>165

<212>DNA

<213> Anas platyrhynchos

<223> "TAT" missing egg mucin partial gene sequence

<400>8

<210>SEQ ID NO: 9

<211>451

<212>DNA

<213> Anas platyrhynchos

<223> Partial gene sequence of ovomucin without "TAT" deletion

<400>9

<210>SEQ ID NO: 10

<211>448

<212>DNA

<213> Anas platyrhynchos

<223> "TAT"-deficient egg mucin partial gene sequence

<400>10

The above and other objects, features, advantages and embodiments of the present invention will become more apparent and understood.

Figure 1 shows the gene expression analysis of egg yolk in high and low hatching rate.

Figure 2 shows the protein expression of egg yolk protein in high and low hatching rate.

Figure 3 is the electrophoresis results of the polymerase chain reaction-restriction fragment length polymorphism.

Claims (10)

A method for identifying a hatching rate of a duck, comprising: analyzing a sequence of an egg mucin gene, a duck with a high hatching rate when the sequence comprises SEQ ID NO: 7, and a low hatching when the sequence comprises SEQ ID NO: The rate of duck. A method for identifying the hatching rate of a duck comprises: polymerase chain reaction amplification of the egg mucin deoxyribonucleic acid of the duck with the primers SEQ ID NO: 1 and SEQ ID NO: 2, wherein the amplified fragment is SEQ ID NO: The vegetable duck of 3 is a high hatching rate vegetable duck, and the vegetable duck with the increased fragment of SEQ ID NO: 4 is a duck with low hatching rate. A method for identifying the hatching rate of a duck, comprising: polymerase chain reaction amplification of the egg mucin deoxyribonucleic acid of the duck with the primers SEQ ID NO: 5 and SEQ ID NO: 6, when the amplified fragment is SEQ ID NO: The vegetable duck of 7 is a high hatching rate of the vegetable duck, and when the augmented fragment is the duck of SEQ ID NO: 8 is a duck with low hatching rate. The method for identifying the hatching rate of a duck according to claim 3, further comprising: cutting the amplified fragments by using a restriction enzyme 5'-AAATTC-3', to obtain a fragment of 168 base pairs in length. For high hatching rate, ducks with a length of 168 base pairs, 124 base pairs and 41 base pairs are also high hatching rate ducks, when obtained with a length of 124 base pairs and 41 bases When the fragment is paired, it is a duck with low hatching rate. A method for identifying a hatching rate of a duck comprises: (a) extracting a genetic DNA of a duck; (b) increasing the egg adhesion protein of the duck with the primers SEQ ID NO: 5 and SEQ ID NO: 6. Fragment sequence; (c) cleavage of the fragment by restriction enzyme 5'-AAATTC-3' restriction enzyme; and (d) identification of the fragment after agarose gel electrophoresis, high hatching rate and low The hatching rate of the egg mucin gene fragment of the duck has different patterns after being cut. A method for identifying a hatching rate of a duck according to claim 5, wherein the restriction enzyme is Apo I. A method for identifying a hatching rate of a duck according to claim 6, wherein the duck is selected as a breeding duck when the fragment is cut to obtain a fragment of 168 base pairs in length. A kit for identifying the hatching rate of a duck, comprising: primer pair SEQ ID NO: 1 and SEQ ID NO: 2 or primer pair SEQ ID NO: 5 and SEQ ID NO: 6. The kit for identifying the hatching rate of the duck as described in claim 8, when the primer pair is SEQ ID NO: 1 and SEQ ID NO: 2, further comprises the deoxygenation of SEQ ID NO: 3 and SEQ ID NO: 4. A ribonucleic acid fragment. The kit for identifying the hatching rate of the duck as described in claim 8, when the primer pair is SEQ ID NO: 5 and SEQ ID NO: 6, further comprises the deoxygenation of SEQ ID NO: 7 and SEQ ID NO: 8. A ribonucleic acid fragment.