JP2014050329A - Method of determining chicken - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a technique of selecting and producing chickens that provide chicken meat or chicken eggs whose long-chain polyunsaturated fatty acid is adjusted without depending on an environmental factor such as formula feed, and without affecting an environmental factor unlike conventional genetic methods.SOLUTION: There is provided a method of determining a chicken which includes steps below: (1) a step of acquiring DNA from a sample derived from a chicken; (2) a step of acquiring at least one type of SNP information selected from the group consisting of A/T polymorphism in the 21490th base of a base sequence of EL5 gene, A/G polymorphism in the 586th base of a base sequence of D5D gene, and A/G polymorphism in the 25th base of a base sequence of D6D gene, regarding a base sequence of the acquired DNA; and (3) a step of estimating the content of long-chain polyunsaturated fatty acid in chicken meat and/or chicken eggs based on the SNP information.

Description

  The present invention relates to a chicken identification method, and more specifically, based on the genotype information of enzymes involved in the synthesis of long-chain highly unsaturated fatty acids in chicken or eggs, the long-chain height in chicken or eggs. The present invention relates to a method for identifying saturated fatty acids. The present invention further relates to a method for breeding chickens adjusted for long-chain highly unsaturated fatty acids, and a method for producing chickens or eggs adjusted for long-chain highly unsaturated fatty acids based on the results of the above-mentioned identification.

  Consumers are increasingly interested in food and health. Although Japanese consumers continue to have a high level of gourmet food, recently there is a growing demand for highly-preference foods in the ASEAN region, where economic development is remarkable. Therefore, the movement to export Japanese chicken and other products to the ASEAN region is accelerating mainly in the Kyushu region.

  Long-chain highly unsaturated fatty acids have attracted attention as substances that improve the nutritional value and taste of ingredients. Long-chain highly unsaturated fatty acids are roughly classified into ω-6 fatty acids and ω-3 fatty acids. The omega-6 fatty acid is a long-chain highly unsaturated fatty acid having two or more unsaturated bonds and having a first double bond at the sixth carbon atom from the methyl end. Arachidonic acid, which is a kind of omega-6 fatty acid, has a function of becoming a constituent material of a biological membrane constituting a cell, normalizing blood cholesterol level, and the like. Arachidonic acid also has the effect of improving the taste of the taste. For example, Patent Document 1 discloses a taste improver for foods and drinks comprising a long-chain highly unsaturated fatty acid and / or an ester thereof, and sterol and / or a sterol ester. Arachidonic acid (AA) is converted from linoleic acid (LA) by a series of enzymatic reactions shown in the biosynthetic circuit of FIG.

  The omega-3 fatty acid is a long-chain highly unsaturated fatty acid having two or more unsaturated bonds and having a first double bond at the third carbon atom from the methyl end. Of the omega-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are well known as physiologically important fatty acids. DHA is said to have actions such as brain development, memory improvement, anti-aging, anti-cancer, anti-allergy and the like. EPA is said to lower blood cholesterol and prevent adult diseases such as arteriosclerosis and cerebral thrombosis. These fatty acids are converted from α-linolenic acid (ALA) through a series of enzymatic reactions shown in the biosynthetic circuit of FIG.

  Long chain polyunsaturated fatty acids are not synthesized in the human body. Therefore, it must be taken daily from foods containing long chain polyunsaturated fatty acids. Chickens and eggs are promising as one of the food intake candidates. For this purpose, DHA eggs enriched with DHA are already on the market.

  The content of long-chain highly unsaturated fatty acids such as arachidonic acid in chicken is a phenotypic quality obtained by combining environmental factors and genetic factors. Inventors reported in nonpatent literature 1 that it can raise the arachidonic acid of a chicken as follows by giving a feed to which arachidonic acid high content fats and oils were added as an influence of an environmental factor. When the feed containing 0.625 to 2.5% of arachidonic acid-containing oil was given to broilers and Hachinai chickens, the content of arachidonic acid in the thigh was significantly increased as the rate of addition of the arachidonic acid content increased. At this time, the contents of glutamic acid and inosinic acid as other taste components were not significantly increased. In sensory evaluation, the 2.5% and 1.25% added groups were significantly stronger in aroma and taste than the control corn group.

  Thus, it is possible to improve chicken long-chain polyunsaturated fatty acid content by environmental factors. However, fats and oils containing a high amount of long-chain highly unsaturated fatty acids such as arachidonic acid are very expensive.

  Traditionally, statistical genetic techniques have been used to improve breeds of chickens using genetic factors. That is, after measuring the desired quantitative trait data (phenotype) from chickens, individuals with excellent results are mated to select excellent individuals.

  However, it takes a lot of time and effort to select between generations to improve meat quality. In addition, intergenerational selection is susceptible to environmental factors. Conventional selective breeding is not necessarily efficient, and it is desired to provide a technique that enables accurate and easy selection of individuals with excellent meat quality and a technique for stably producing excellent selected individuals.

Table 2010/004784

Poultry Science 90, 1817-1822, 2011

  The object of the present invention is to provide chicken and eggs with long chain polyunsaturated fatty acids adjusted without relying on environmental factors such as formula feed and without affecting environmental factors as in conventional genetic techniques. The object is to provide a technique for selecting and producing chickens.

  In a study by a part of the present inventors (Non-patent Document 1), the difference in the arachidonic acid content observed between broiler chickens and Hachinai chicken is genetic, and Furthermore, the arachidonic acid content varies, suggesting the presence of genes involved in the biosynthesis of long-chain highly unsaturated fatty acids such as arachidonic acid in the body.

  There are qualitative traits and quantitative traits that are controlled by genes, but traits in which the amount of long-chain polyunsaturated fatty acids in chicken muscle fat continuously change from one individual to another are quantitative traits. Belongs. In order to elucidate genes involved in biosynthesis of long-chain highly unsaturated fatty acids in chicken, the present inventors conducted QTL (Quantitative trait loci) analysis of Hennai chickens. QTL analysis is an analysis method for the purpose of knowing the position on the chromosome of one or more loci that govern quantitative traits. A quantitative trait is often governed by multiple loci.

  To discover three genes involved in the biosynthesis of long-chain highly unsaturated fatty acids shown in FIG. 1, elongase 5 (EL5), delta 5 desaturase (D5D) and delta 6 desaturase (D6D) single nucleotide polymorphism (SNP) The base sequence of the gene was determined for the Hinouchi chicken. As a result, as shown in the examples described later, there are one single nucleotide polymorphism (SNP) and four types of haplotypes (combinations of SNPs) in the EL5, D5D and D6D gene expression control regions, respectively. discovered. In the biosynthetic circuit of FIG. 1, elongase is involved in the “elongation” that lengthens the molecule by incorporating carbon atoms, and desaturase is involved in the “desaturation” in which a new double bond is formed. Functions as an enzyme. And it discovered also that the amount of long-chain highly unsaturated fatty acids in chicken and eggs was significantly different between the haplotypes. Based on these findings, the following invention has been completed.

The present invention
(1) A step of obtaining DNA from a sample derived from chicken,
(2) Regarding the base sequence of the acquired DNA,
A / T polymorphism at the 21490th base of the base sequence of EL5 gene shown in SEQ ID NO: 1,
An A / G polymorphism at the 586th base of the base sequence of the D5D gene shown in SEQ ID NO: 3,
as well as,
An A / G polymorphism at the 25th base of the base sequence of the D6D gene shown in SEQ ID NO: 5;
Obtaining at least one kind of SNP information selected from the group consisting of:
(3) A chicken identification method including a step of estimating the content of a long-chain highly unsaturated fatty acid in chicken and / or eggs based on the SNP information.

  In the step (3), particularly when the polymorphism of SEQ ID NO: 1 is T, the polymorphism of SEQ ID NO: 3 is G, or the polymorphism of SEQ ID NO: 5 is G, a long chain It is estimated that the polyunsaturated fatty acid content will be higher.

  In the step (3), in particular, a haplotype composed of the combination of the SNPs, in the order of EL5-D5D-D6D, TAA is H1, TGA is H2, TGG is H3, And when AAA is defined as H4, based on the correlation between the maximum 10 types of genotypes formed from these haplotypes and the content of long-chain highly unsaturated fatty acids in chicken or eggs, Estimate the content of long chain highly unsaturated fatty acids in chicken and / or eggs.

  The long chain highly unsaturated fatty acid is preferably arachidonic acid.

The present invention is also a method for producing a practical chicken with a controlled long-chain highly unsaturated fatty acid content,
(A) A step of selecting a practical chicken having a specific genotype based on the result of the chicken identification method,
(B) a process of breeding a selected practical chicken seed,
as well as,
(C) The production method of the said practical chicken including the process of producing the said practical chicken in the breeding breeder is provided.

  In the present invention, a practical chicken means a chicken that provides chicken and / or eggs, and a breeding chicken means a chicken for the purpose of producing eggs that produce edible eggs and become practical chickens for meat. In the present specification, the term “seed chicken” is used in a sense that includes the original breed chicken that is the parent and the original breed chicken that is the parent.

  The practical chicken is preferably a first-generation hybrid.

  The step (B) preferably uses backcrossing.

  The present invention also provides a method for producing eggs having a controlled content of long chain polyunsaturated fatty acids, comprising breeding eggs produced by the method for producing practical chickens and producing eggs.

  Reports on genetic control of long-chain polyunsaturated fatty acids in chicken and eggs, and on the association of SNP information in EL5, D5D, and D6D genes with the content of long-chain polyunsaturated fatty acids in chicken Absent.

  In conventional selective breeding, target quantitative trait data is collected from chickens, and excellent offspring are obtained by mating individuals with excellent results. Since quantitative traits are susceptible to environmental factors, conventional selective breeding has not always been efficient. On the other hand, the present invention can select and breed chickens in which long chain polyunsaturated fatty acids are controlled based on gene mutations that affect the content of long chain polyunsaturated fatty acids in chicken and / or eggs. Since the present invention grasps a gene that governs a quantitative trait, it is possible to accurately and efficiently identify and select without being affected by the environment.

  The appraisal method of the present invention is to estimate the amount and polymorphism of long-chain highly unsaturated fatty acids in chicken and / or eggs based on genotype differences determined by EL5, D5D and / or D6D SNP information. Is possible. By this identification method, the nutritional value and taste of chicken or eggs can be estimated.

  The present invention can provide a method for selecting and breeding chickens and chicken eggs having excellent nutritional value and taste based on the results of the identification method. Foreign varieties called broilers are inferior in taste compared to local chicken (local special chicken) with excellent taste that Japanese prefer. One reason for this is that broiler chicken has a significantly lower arachidonic acid content than chicken. According to the breeding method of the present invention, the taste of conventional broilers can be improved.

  Local special chickens are produced throughout the country, but their production costs are much higher than broilers. There is a strong demand from producers for further differentiation from broiler chickens in terms of taste. Even delicious chickens may vary from individual to individual. According to the present invention, it is possible to further improve the taste of regional special chickens. Moreover, even if it is the same kind, it becomes possible to always provide a certain quality. The present invention is expected to provide effective techniques in the fields of poultry farming (chicken breeding, breeding, breeding, improvement, etc.), chicken meat and egg production, and processing.

It is a figure which shows the biosynthetic circuit and synthesis | combination gene of a long-chain highly unsaturated fatty acid. It is a figure which shows the genotype distribution of D6D gene and EL5 gene. The genotype distribution of D6D gene and EL5 gene differs depending on the breed of chicken. It is an electrophoretic diagram which discriminate | determines SNP information of EL5, D5D, and D6D gene.

The chicken identification method of the present invention,
(1) A step of obtaining DNA from chicken or chicken egg,
(2) Check the base sequence of the obtained DNA,
A / T polymorphism at the 21490th base of the base sequence of EL5 gene shown in SEQ ID NO: 1,
An A / G polymorphism at the 586th base of the base sequence of the D5D gene shown in SEQ ID NO: 3,
as well as,
An A / G polymorphism at the 25th base of the base sequence of the D6D gene shown in SEQ ID NO: 5;
Obtaining at least one kind of SNP information selected from the group consisting of:
(3) including a step of estimating the content of long-chain highly unsaturated fatty acid in chicken and / or eggs based on the SNP information. The above steps will be described in order below.

(1) Step of obtaining DNA In step (1), DNA is obtained from chickens. Chickens can be classified from several perspectives. The chicken used in the method of the present invention may belong to any classification.

  Chicken uses are divided into meat-type, egg-type, or egg-meat-type. Examples of meat species are warfowl, brahma and cochin. Examples of egg seeds include white leghorn, black Minorca, Andalusian, australope, Isa brown, decalp warren sexual monkey, Harvard comet, high sex brown, and high line brown. Examples of egg-combined seeds include vertebrate prima rock, white plymouth rock, Rhode Island, New Hampshire, Nagoya Cochin, white cornish, rock horn, Wyen Red, and Orpington.

  Chickens are also divided into domestic and foreign chickens according to the country of origin. Foreign chickens are practical chickens that have been breeded and improved by overseas breeding companies, etc., and brought to Japan. An example of a foreign chicken is the broiler breeder for meat. Broiler is a collective term for young chickens for meat that has been improved to promote growth and improve meat quality in order to ship in the state of young chickens less than 3 months old.

  Domestic chickens are also known as brand chickens and local chickens, and have been breeded and improved by prefectures in Japan. In the midst of diversifying tastes, local special chickens, such as local chickens that have a reputation for meat taste, are produced throughout the country. In order to supply chickens to the market under the name of “local chickens”, it is necessary to satisfy the specific JAS standard (how to make JAS) certification conditions (pedigree, flat-breeding, breeding density, etc.) of local chicken meat. Examples of local chickens include Hinai chickens, Aizu chickens, Amami ancient chickens, Oita crown chickens, Amakusa Daio, Miyazaki chickens, Satsuma chickens, and Nagoya Cochin.

  The chicken may be either a fixed species that has been bred by self-breeding, or a primary hybrid that uses hybrid strength (hereinafter also referred to as F1 or hybrid). Most broiler chickens and chickens are first-generation hybrids. Most broilers are white cornish males and white plymouth rock females. Hinouchi chicken is a cross-breed of a cross between a male Hinouchi chicken and a female Rhode Island Red to improve productivity while taking over the features of Hinouchi chicken. The reason why the first-generation hybrids occupy the market is the expression of hybrid stress that yield characteristics, disease resistance, and other phenotypes are superior to their parents, the phenotype of chickens produced is uniform, and F2 obtained by crossing F1 Since the property of F1 is not inherited, the profit of the breeder provider is secured. Moreover, the breeding chicken used as the basis of production of a practical chicken may be sold only by one sex (male or female). Therefore, producers who introduce F1 chickens and raise chickens need to purchase chicks or eggs that hatch chicks from domestic and foreign hatchery and hatchery every time. Local chickens are not limited to primary hybrids, but may be produced by crossing three or four varieties, but in any case, the producers will receive chicks or chicks from a specific hatchery or hatchery. It is necessary to purchase eggs to beat each time.

  The part for obtaining DNA from the chicken is not particularly limited. Examples thereof include body fluids such as organs, tissues, blood, and cells obtained from chickens, chicken embryo-derived cells collected from amniotic fluid and chorioallantoic fluid, cells obtained by culturing the collected tissues, chicken eggs, chicken eggshells, and the like. Preferably, it is blood.

  The DNA may be genomic DNA or cDNA. cDNA is synthesized by reverse transcriptase after extracting and purifying mRNA from a test sample according to a conventional method. For extraction of DNA or RNA, methods known to those skilled in the art, for example, the phenol / chloroform method, a commercially available genomic DNA extraction kit, and the like are used.

(2) Step of examining SNP information The genes to which the identification method of the present invention is focused are EL5, D5D and D6D. These enzymes are involved in the biosynthesis of long-chain highly unsaturated fatty acids as shown in FIG. EL5 is an enzyme that makes essential fatty acids taken from feed longer in the body and increases unsaturated bonds. The entire genomic DNA sequence of the EL5 gene is shown in SEQ ID NO: 1. This base sequence is registered as chromosome 3 87,654,796-87,692,542 in Ensembl Chicken Genome Browser (http://www.ensembl.org/Gallus_gallus (chicken draft sequence November 2011 edition)). Has been. The amino acid sequence of EL5 is shown in SEQ ID NO: 2. This amino acid sequence is registered in the accession number ADO21498 in the protein database Entrez (http://www.ncbi.nlm.nih.gov/sites/entrez?db=protein) of the National Center for Biotechnology Information. Any sequence can be easily obtained by those skilled in the art.

  D5D is an enzyme that desaturates the fifth position with the carboxyl group of the fatty acid as the first position. D5D is activated by the action of insulin and converts linoleic acid to arachidonic acid. The entire genomic DNA sequence of the D5D gene is shown in SEQ ID NO: 3. The said sequence is shown by the 5th chromosome 16,079,252-16,088,563 of the same Ensembl Chicken Genome Browser (chicken draft sequence November, 2011 edition). The amino acid sequence of D5D is shown in SEQ ID NO: 4. This sequence is shown in the accession number ADU05467 of the same Entrez.

  D6D is an enzyme having a function of converting linoleic acid to γ-linolenic acid. The entire genomic DNA sequence of the D6D gene is shown in SEQ ID NO: 5. The said sequence is shown by the 5th chromosome 16,094,554-16,111,784 of the same Ensembl Chicken Genome Browser (chicken draft sequence November, 2011 edition). The amino acid sequence of D6D is shown in SEQ ID NO: 6. This sequence is shown in the accession number ABR24806 of the same Entrez.

  The DNA base sequences of EL5, D5D, and D6D genes were determined and compared for Hinouchi, Rhode Island Red, and broilers involved in Hinouchi chicken production. As a result, SNPs were found in the expression control regions of each gene.

  In the base sequence shown in SEQ ID NO: 1 of EL5, the 21490th base (w) from the 5 'side is thymine (T) or adenine (A).

  In the base sequence shown in SEQ ID NO: 3 of D5D, the 586th base (r) from the 5 'side is adenine (A) or guanine (G).

  In the base sequence shown in SEQ ID NO: 5 of D6D, the 25th base (r) from the 5 'side is adenine (A) or guanine (G).

  The numbers given to the bases indicate the position corresponding to the first nucleotide in the genomic DNA sequence of the gene shown in SEQ ID NO: 1, 3 or 5.

At least one kind of SNP information of the above three genes of the sample to be examined is examined. The method for examining the SNP is not particularly limited, and a conventionally known method can be applied. Examples include MAMA method (allele-specific amplification method), PCR-RFLP (restriction fragment length polymorphism method) method, PCR-DGGE method, PCR-SSCP method, PCR-SSOP method, CR-RD method, PCR -MPH method, sequencing method, mini-sequencing method, RNase protection method, TaqMan PCR method, Invader method, single base extension method, SNaP shot ^TM method, Pyrosequencing method, Exonuclease Cycling Assay method, etc.

  In the PCR-RFLP method, a gene region containing a mutation site to be detected is amplified by the PCR method, the PCR product is digested with a restriction enzyme that recognizes the mutation site, and subjected to electrophoresis. It is a method of detecting. First, a DNA region containing SNP is amplified using genomic DNA or cDNA as a template. For example, PCR using a DNA sample as a template is performed using a primer that hybridizes to a DNA fragment containing the SNP site. The PCR method uses two oligonucleotide primers that are complementary to each other and preferentially hybridize with a template DNA, from heat denaturation of the DNA strand, annealing of the primer, and synthesis of the complementary strand by polymerase. The process which repeats the cycle which performs the primer extension synthesis which becomes 10 to 80 times is included. These operations can be easily carried out usually using a commercially available reagent kit containing a reaction solution and a heat-resistant polymerase, a commercially available PCR device, and the like. Regarding conditions such as PCR reaction conditions, reagents, primers, restriction enzymes, etc., optimum ones can be easily selected according to the manual of the apparatus, experiment, or experience.

  In the MAMA (Mismatch Amplification Mutation Assay) method, one primer was designed to be matched with a polymorphic site, and the other primer was designed to be matched with a region not containing the polymorphic site. A DNA amplification method is performed using a primer pair. If any allele corresponding to the polymorphism is present in the DNA sample, an amplification product is obtained, and if it is not present, the amplification product cannot be obtained. Therefore, the presence or absence of a specific allele can be determined by detecting the presence or absence of an amplification product.

  In the present invention, the haplotypes composed of combinations of three SNPs detected in EL5, D5D and D6D genes are theoretically eight types shown in Table 1.

A maximum of 36 (= 8 + 7 + 6 + 5 + 4 + 3 + 2 + 1) genotypes (genotypes) can be detected from the above eight haplotypes.

  Chicken genotype alleles are inherited from male and female parents. Some varieties may not have a specific haplotype. For example, as shown in FIG. 2, the ratio of the D6D gene has a G frequency of 0, and thus the ratio of the chicken does not have H3, H5, H6, and H8 haplotypes. In addition, the local chicken, the Satsuma, the Tosa chicken, and the Gifu chicken have an EL5 frequency of 0 and do not have H4, H6, H, and H8 haplotypes. Therefore, in a specific single breed and a group of chickens crossed with them, not all 8 types of haplotypes and all 36 types of genotypes may appear. For example, in the result of analysis of 94 female ratio domestic chickens, which will be described later, only four haplotypes (H1 to H4) shown in Table 6 are detected, and ten types of genotypes shown in Table 7 are detected. No haplotypes or genotypes were detected.

(3) Estimating the content of long-chain highly unsaturated fatty acids based on SNP information The long-chain highly unsaturated fatty acids estimated by the identification method of the present invention include γ-linolenic acid (C18: 3, GLA), dihomo-γ. Ω-6 acids of linolenic acid (C20: 3, DGLA) and arachidonic acid (C20: 4, AA), and stearidonic acid (C18: 4, SDA), eicosatetraenoic acid (C20: 4), eicosapentaene Acid (C20: 5, EPA), docosapentaenoic acid (C22: 5), tetracosapentaenoic acid (C24: 5), tetracosahexaenoic acid (C24: 6), and docosahexaenoic acid (C22: 6, DHA) ) -3 acids.

  Arachidonic acid improves the taste of chicken. Although there is a concern about excessive intake of cholesterol by eating eggs, EPA and DHA have a cholesterol-lowering effect. Therefore, the long chain polyunsaturated fatty acid is preferably arachidonic acid, EPA and DHA.

There is the following correlation between the allele of each gene and the arachidonic acid content in chicken.
(I) The allele of EL5 is thymine (T).
(II) the allele of D5D is guanine (G),
Or
(III) The allele of D6D is guanine (G).
In some cases, the arachidonic acid content is higher.

  The T allele in the EL5 gene, the G allele in the D5D gene, and the G allele in the D6D gene can be used as factors having a high arachidonic acid content. By identifying whether the SNP in the EL5 gene is T or A, the SNP in the D5D gene is G or A, and / or whether the SNP of the D6D gene is G or A, This makes it possible to predict chickens with high and low arachidonic acid content.

  T / T or T / A in the EL5 gene, G / G or A / G in the D5D gene, and G / G or A / G genotype in the D6D gene can be used as factors having a high arachidonic acid content.

  FIG. 2 shows a correlation diagram of the frequency of EL5 gene and D6D gene alleles depending on the breed of chicken. Referring to FIG. 2, the chickens that have been evaluated as delicious have a relatively high guanine frequency in the D6D gene and a high thymine frequency in the EL5 gene. In general, commercial broilers that are not well evaluated have low guanine frequency and EL5 thymine frequency in the D6D gene. Therefore, it can be expected that even commercially available broilers will produce more delicious meat-like broilers by changing the SNP information of (I) to (III).

  The quantitative trait of polyunsaturated fatty acid content is a combination of the three types of SNP information of the above genes. The expression traits of the taste and nutritional value of chicken and eggs depend on 8 haplotypes and up to 36 genotypes. Therefore, for more accurate prediction of the amount of long-chain highly unsaturated fatty acids, it is preferable to determine the correlation between haplotypes and long-chain highly unsaturated fatty acids in the variety to be evaluated.

In the case of 94 Hachinai chickens, the effect of the haplotype of 3 genes on arachidonic acid is estimated as shown in Table 2.

In Table 2, significant differences are recognized in H2>H1> H4 and H3>H1> H4. There is no significant difference between H2 and H3, but H3 tends to be high.

The correlation shown in Table 3 is estimated between the genotype of 3 genes and arachidonic acid.

  As described above, H3 / H3 and H4 / H4 homozygous individuals do not actually exist in the Hachinai chicken. However, the homozygote can be created by changing the genotype of the parent ratio chicken by backcross described below. The arachidonic acid content at that time is estimated to be the value in Table 3.

If the SNP information is used as a genetic marker, breeding chickens based on a desirable phenotype can be established more efficiently. A method for producing chickens that provides chicken and / or eggs with controlled long-chain highly unsaturated fatty acid content of the present invention,
(A) selecting a practical chicken having a specific genotype based on the results of the chicken identification method described above,
(B) a process of breeding a selected practical chicken seed,
as well as,
(C) including the step of causing the breeding chicken to lay the practical chicken. Each step will be described in detail below.

(A) The process of selecting the practical chicken which has a specific genotype Based on the result of the identification method of this invention, the practical chicken which has a desired genotype is selected. For example, with respect to the arachidonic acid content, the ratings for the 10 genotypes are as shown in Table 3. In Table 3, the individual with the highest arachidonic acid content is H3 / H3 homotype, and the individual with the lowest arachidonic acid content is H4 / H4 homotype. The higher the arachidonic acid content in terms of chicken taste, the better. The arachidonic acid content in the chicken is usually 0.5 to 5.0 mg / g, preferably 0.8 to 3.0 mg / g. This range combines high intake of arachidonic acid content and good taste. Therefore, in order to obtain an appropriate arachidonic acid content, individuals such as H1 / H4 heterotype, H1 / H1 homotype, and H2 / H4 heterotype may be selected.

  Practical chickens can be either fixed or hybrid. Since the industrial applicability of chickens and broilers whose arachidonic acid content is adjusted to a certain range is very high, a hybrid is preferable.

(B) Breeding the selected practical chicken seed chicken The genotype of the seed chicken is determined based on the selected practical chicken genotype. In addition to the seed chicken itself, the seed chicken may be an original seed chicken or an original seed chicken. For example, when genotype H3 / H3 is selected for a primary hybrid chicken for chicken and eggs, the genotype of the parent breeder is a H3 / H3 homozygote for both male and female genotypes. In addition, when the desired genotype of the primary hybrid is H2 / H3 heterozygous, the parent chicken genotype is H2 / H2 homozygous for one parent and H3 / H3 homozygous for the other parent. And

  Next, breeding chickens whose genotype is determined are bred. Specifically, selected genotypes are introduced into chickens by backcrossing, crossbreeding, crossbreeding, and the like.

  A procedure for incorporating genotype H3 / H3 by backcrossing into a female specific internal chicken line as a breeding chicken of a specific hybrid domestic chicken having a genotype of H3 / H3 will be described below.

  A specific chicken having genotype H3 / H3 is crossed with a specific parent chicken (for example, H1 / H1) to obtain the F1 generation. The locus for the F1 generation is H1 / H3. 50% of the genetic background in the F1 generation is the same as the ratio chicken. This F1 generation is backcrossed with the ratio internal chicken to obtain the N2 generation. 75% of the genetic background in the N2 generation is the same as the ratio chicken. Regarding the gene locus, an individual having the H1 / H1 type and an individual having the H1 / H3 genotype are separated. Next, the N2 generation is again crossed back to the ratio inner chicken to obtain the N3 generation. At this time, an individual having an H3 allele, which is an N2 individual used for backcrossing to a ratio chicken, can be easily selected using a genetic marker. The N2 individuals thus selected are backcrossed to the nesting chicken to obtain the N3 generation. The genetic background of the N3 generation individuals is 87.5%, which is the same as that of the ratio chicken. As for the gene locus, as in the N2 generation, those having the H1 / H1 type and those having the H1 / H3 type are separated. In the same manner as in the case of the N2 generation, N3 individuals having H2 alleles are selected and backcrossed to the ratio internal chicken. In the obtained N4 individuals, 93.75% of the genetic background is the same as the ratio chicken. The gene locus is the same as that of the N2 and N3 generations, and the N5 generation and later are obtained by performing the same operation as described above. As the number of backcrosses increases, the genetic background of that generation approaches the genetic composition of the original ratio chicken, but the locus has a different H3 allele from the original ratio chicken.

  As long as backcrossing continues, the genotype of the locus remains H1 / H3 heterotype. When it is determined that the genetic background is sufficiently close to that of the Henchu chicken, heterozygous individuals are mated with each other, and a Hyogo chicken line having H3 / H3 homotype is finally established.

(C) The process of giving a breeding breeder a practical chicken A male and female breeder breeded at a process (B) is made to produce a practical chicken. The step (C) may be performed at the same or different place as the steps (A) and (B). Usually, the steps (A) and (B) are performed by a public institution or breeding company that owns breeding chickens, and the step (C) is performed at a seed chicken farm or a original breed chicken farm.

  In the method for producing a practical chicken of the present invention, it is not always necessary to perform the steps (A) and (B) before the step (C) as long as a breeding chicken line can be established in the steps (A) and (B). Therefore, it is included in the method of this invention to apply the breeding chicken established using process (A) and (B) to process (C).

  The present invention also provides a method for producing hen's eggs with a controlled content of long-chain highly unsaturated fatty acids, comprising breeding the hens produced by the above-described method for producing hens to lay eggs. In this case, the genotype of the female parent is passed on to the hen's egg as it is.

  The long chain polyunsaturated fatty acid whose content in the egg is regulated is preferably EPA and / or DHA.

  The present invention also provides a primer for specifically amplifying the gene region containing the SNP information by a gene amplification reaction. This primer consists of a forward primer and a reverse primer designed to sandwich the SNP on the chicken genomic DNA or cDNA sequence. The base sequence of the amplified DNA fragment includes the base sequence of SNP information.

  The primer is usually 10 mer to 100 mer, preferably 15 mer to 40 mer, more preferably 18 mer to 30 mer. The base length of the DNA fragment amplified by the primer is not particularly limited, but is usually 100 bp to 1,500 bp, preferably 200 bp to 700 bp.

  Those skilled in the art can easily design primers for detecting SNPs based on the nucleotide sequences of SEQ ID NOs: 1, 3 and 5. Examples of the primer for detecting the SNP of the EL5 gene include, but are not limited to, a primer pair including the nucleotide sequences represented by SEQ ID NOs: 7 and 8. Similarly, for example, SEQ ID NOS: 9 and 10 can be used as primers for detecting the SNP of the D5D gene, and SEQ ID NOS: 11 and 12 can be used as primers for detecting the SNP of the D6D gene.

  Conditions for hybridizing the primer to the genomic DNA can be appropriately selected by those skilled in the art. For example, prehybridize overnight at 42 ° C. in a hybridization solution containing 25% formamide (50% formamide under more severe conditions), 4 × SSC, 50 mM Hepes pH 7.0, 10 × Denhardt's solution, 20 μg / ml denatured salmon sperm DNA. After hybridization, a labeled probe is added and hybridization is performed by incubating overnight at 42 ° C. In the subsequent cleaning, the cleaning solution and temperature conditions were 1 × SSC, 0.1% SDS, 37 ° C., more severe conditions were 0.5 × SSC, 0.1% SDS, 42 ° C., and even more severe conditions were 0.2 × SSC, 0. Perform at 1% SDS at 65 ° C. The primer can be labeled with a fluorescent substance; a binding affinity substance such as biotin or digoxigenin.

  The present invention also provides an oligonucleotide used in the method for detecting a gene polymorphism of the above gene. Such an oligonucleotide specifically hybridizes to a region containing the SNP site of the gene. Here, “specific” means that the oligonucleotide hybridizes to a region containing the polymorphic site of the gene and does not hybridize to other regions under stringent conditions. Such hybridization conditions can be appropriately selected by those skilled in the art.

  Stringent conditions can be determined depending on the melting temperature Tm (° C.) of the duplex of the oligonucleotide and its complementary strand, the salt concentration of the hybridization solution, and the like. Sambrook, E .; F. Frisch, T .; Reference can be made to Maniatis; Molecular Cloning 2nd edition, Cold Spring Harbor Laboratory (1989).

  Hybridization conditions include, for example, low stringency conditions. The low stringent conditions are, for example, conditions of 42 ° C., 5 × SSC, 0.1% SDS, and preferably 50 ° C., 2 × SSC, 0.1% SDS in washing after hybridization. is there. More preferable hybridization conditions include highly stringent conditions. High stringent conditions are, for example, 65 ° C., 0.1 × SSC, and 0.1% SDS. Factors affecting the stringency of hybridization include a plurality of factors such as temperature and salt concentration, and those skilled in the art can realize the same stringency by appropriately selecting these factors.

  The oligonucleotide need not be completely complementary to the DNA region as long as it hybridizes to the DNA region containing the SNP. For example, there may be substitution with another base of about several bp on the 5 'terminal side, and an arbitrary base may be added on the 5' terminal side.

  The oligonucleotide chain length is not particularly limited as long as it hybridizes to the DNA region containing the SNP. Preferably, it is 10mer-100mer, More preferably, it is 15mer-40mer, More preferably, it is 18mer-30mer.

  The oligonucleotide may be immobilized on a solid phase. Examples of the solid phase include cellulose derivatives such as cellulose and nitrocellulose, sepharose, agarose, metal, glass, ceramics, and resins. The shape and material of the solid phase are not particularly limited. Oligonucleotides can be easily obtained by using a commercially available oligonucleotide synthesizer and a synthetic oligonucleotide contract service.

The oligonucleotide can be used as a probe for detecting the SNP and an adsorption ligand for purifying the DNA containing the SNP. The probe may be labeled for easy detection. Examples of labeling means include spectroscopic, optical, biochemical, immunological, enzymatic chemistry, and radiochemical means. Specific examples of the labeling means include enzymes such as peroxidase and alkaline phosphatase, isotopes such as ³² P, biotin, fluorescent dyes, luminescent substances, and coloring substances.

  The said primer and / or oligonucleotide can be used as a test reagent for SNP detection and also for evaluating the content of long-chain highly unsaturated fatty acids in chicken or eggs. Therefore, the present invention provides a gene polymorphism detection kit containing the primer and / or oligonucleotide. The kit appropriately includes other reagents and instruments, for example, various reagents for use in the method of the present invention, restriction enzymes, reaction solutions, control samples, reaction containers, operation instruments, and the like.

  In particular, the present invention provides a DNA chip or DNA microarray using a plurality of the above oligonucleotides. The DNA chip performs SNP typing by hybridizing DNA of a test sample and scanning the substrate to detect the presence or absence of a hybridization signal. Responses to multiple probes can be observed simultaneously. The probe attached to the DNA chip may be either a synthetic oligonucleotide or cDNA. It may be a complementary sequence of the base sequence. The length of the nucleotide is preferably 20 to 30 mer.

Hereinafter, the present invention will be described in detail by showing examples of the present invention. However, the present invention is not limited to the following examples.
[Example 1]
The SNP information on the three genes of genomic DNA prepared from Hennai chicken was examined by the following procedure.

1. Obtaining DNA 94 females of Hinouchi chicken (Hainai chicken and Rhode Island Red breed first breed) were obtained and fed the same diet for 22 weeks to acclimatize to the environment. Blood was collected from this inland chicken, and DNA was extracted and purified by the following method. Drip chicken blood on FTA card and air dry. Punch with a punch (1.2 mm diameter) and place one disc per well of a 96 well PCR plate. FTA purification reagent (80668019, Whatman), DNAzolBD reagent (DN129, Molecular Research Center), followed by washing twice with sterilized distilled water, 100 μl of sterilized distilled water was added, and the mixture was heated at 90 ° C. for 10 minutes. DNA was recovered.

2. Determination of SNP Using the above DNA, SNP information of EL5, D5D and D6D genes was determined by MAMA method. The composition of the PCR reaction solution was as follows.
1 to 10 ng of genomic DNA,
Emerald Amp PCR master mix (RP300A, Takara Bio) 4 μl,
Forward primer (200 μM) 0.0125 μl,
as well as,
Reverse primer (200 μM) 0.0125 μl
Was made up to 8 μl by adding ultrapure water to the above composition.

Table 4 shows the sequences of the forward primer and the reverse primer.

  PCR reaction conditions were heat denaturation (98 ° C., 10 seconds), annealing (X ° C., 30 seconds), and extension reaction (72 ° C., 30 seconds) for 30 cycles. When PCR was performed with the combinations of sequences shown in Table 5 and the annealing temperature, genotyping was performed based on whether or not a DNA band of an assumed size was detected by agarose gel electrophoresis.

The fragment after the reaction was subjected to gel electrophoresis. The electrophoresis conditions were 2.5% agarose gel, 100 V, 30 minutes.
(1) In the case of EL5, in the electrophoresis diagram of FIG. 3, when only the 247 bp band of the T primer is detected, T / T, when only the 248 bp band of the A primer is detected, A / A, The time of detection was determined as T / A.
(2) In the case of D5D, in the electrophoretic diagram of FIG. 3, A / A when only the 195 bp band of the A primer is detected, G / G when only the 195 bp band of the G primer is detected, and both When the band was detected, it was determined as G / A.
(3) In the case of D6D, in the electrophoretic diagram of FIG. 3, when only the 274 bp band of the A primer is detected, A / A, when only the 397 bp band of the G primer is detected, G / G, and both When the band was detected, it was determined as G / A.

3. Determination of haplotypes When haplotypes were identified, four haplotypes were found (Table 6).

4). For 94 genotypes, the number and frequency of each genotype was detected (Table 7).

5. Fatty acid composition Thirty-two out of 94 birds were systematically picked up and the fatty acid composition was investigated. The test method is as follows.

(Measurement of arachidonic acid content)
4.5 ml of PBS (-) was added to 0.5 g of thigh minced meat, and homogenized with Ultratax T-10 (IKA, S10D-7G-KS-110 shaft generator) at 300 rpm for 30 seconds. 1 ml of a thigh meat suspension, 3 ml of a chloroform / methanol (1: 2 v / v) mixed solution, 900 μl of chloroform, 100 μl of an internal standard solution (C23: 0 methyl was dissolved in chloroform, about 0.01 g / ml), PBS (−) 0. Lipid was extracted by adding 5 ml and homogenizing again. After the solvent of the extract was distilled off to obtain lipids, fatty acid methyl esters were prepared according to the standard oil and fat analysis test method (2.4.1.2-1996, reference 3.2.3-1996), and gas chromatographic method was used. The arachidonic acid content was analyzed by

(Separation conditions)
Column: Capillary column DB-23 Inner diameter 0.25 mm × 30 m (manufactured by Agilent Technologies)
Carrier gas: helium head pressure: 126 kPa (linear velocity 35.4 cm / sec)
Column temperature: heating mode 80-230 ° C
Detector: FID

The relationship between the genotype of each gene and arachidonic acid was as follows.
EL5: T / T> T / A (P <0.05)
Of the two alleles of the EL5 gene, T / T-type chickens have a higher arachidonic acid content than T / A-type chickens.

D5D: G / G> A / A, A / G> A / A (P <0.05)
Of the two alleles of the D5D gene, G / G and G / A chickens have a higher arachidonic acid content than A / A chickens. There was no significant difference between G / G and A / G of D5D.

D6D: A / G> A / A (P <0.05)
Of the two alleles of the D6D gene, A / G type chickens have a higher arachidonic acid content than chickens carrying A / A type.

The relationship between the 3 gene SNPs and arachidonic acid was examined (Table 8).

Significant differences were observed between D5D and D6D SNPs. Although no significant difference was observed between T and A of EL5, T tended to be large. By examining the genotypes of the three genes, it was shown that it was possible to determine the arachidonic acid content of chickens and the resulting good taste.

  The effect of each haplotype of 3-genes on arachidonic acid was expressed as Thesia (Tregouet DA, Garelle V. A new JAVA interface implementation of THE 10 at 10 eSs. Estimated using The results are shown in Table 9.

  H2> Hl> H4 and H3> H1> H4, and a significant difference was observed between each haplotype. There was no significant difference between H2 and H3, but H3 tended to be high (P = 0.053). To explain clearly, the arachidonic acid of the H4 / H4 type individual is estimated to be 0.76 × 2 = 1.52% as shown in Table 10, and the arachidonic acid of the H3 / H3 type individual is 1.55. It is estimated that x2 = 3.10%.

  From the above results, by utilizing the SNP combination (haplotype) information of EL5, D5D, and D6D genes, arachidonic acid of chicken can be genetically regulated, and practical chickens that meet market demands can be produced.

Claims (8)

(1) A step of obtaining DNA from a sample derived from chicken,
(2) Regarding the base sequence of the acquired DNA,
A / T polymorphism at the 21490th base of the base sequence of EL5 gene shown in SEQ ID NO: 1,
An A / G polymorphism at the 586th base of the base sequence of the D5D gene shown in SEQ ID NO: 3,
as well as,
An A / G polymorphism at the 25th base of the base sequence of the D6D gene shown in SEQ ID NO: 5;
Obtaining at least one kind of SNP information selected from the group consisting of:
(3) A chicken identification method including a step of estimating the content of a long-chain highly unsaturated fatty acid in chicken and / or eggs based on the SNP information.   When the polymorphism of SEQ ID NO: 1 is T, the polymorphism of SEQ ID NO: 3 is G, or the polymorphism of SEQ ID NO: 5 is G in the step (3), The chicken identification method according to claim 1, wherein the saturated fatty acid content is estimated to be higher.   In the step (3), the haplotypes composed of the combinations of the SNPs are arranged in the order of EL5-D5D-D6D, T-A-A is H1, T-GA is H2, T-G-G is H3, and A -When AA is defined as H4, based on the correlation between up to 10 genotypes formed from these haplotypes and the content of long-chain highly unsaturated fatty acids in chicken or eggs, 2. The chicken identification method according to claim 1, wherein the content of a long-chain highly unsaturated fatty acid in the egg is estimated.   The chicken identification method according to claim 1, wherein the long-chain highly unsaturated fatty acid is arachidonic acid. A method for producing practical chickens with controlled long-chain highly unsaturated fatty acid content,
(A) selecting a practical chicken having a specific genotype based on the results of the chicken identification method of claim 1;
(B) a process of breeding a selected practical chicken seed,
as well as,
(C) The production method of the said practical chicken including the process of producing the said practical chicken in the breeding breeder.   6. The chicken production method according to claim 5, wherein the practical chicken is a first-generation hybrid.   The method for producing chickens according to claim 6, wherein the step (B) uses backcrossing.   6. A method for producing eggs having a controlled content of long-chain highly unsaturated fatty acids, comprising breeding eggs by producing a practical chicken produced by the method for producing a practical chicken according to claim 5.