KR100683068B1 - A new primer set and a selective method of hanwoo using said primer set - Google Patents

Abstract

A primer set and a selective method of Hanwoo by using the same primer set are provided to evaluate genetic ability of Hanwoo at any growth period precisely and conveniently by analyzing the presence of DNA marker related to economic traits of Hanwoo. A polynucleotide molecule specific to the economic traits of Hanwoo comprises the nucleotide sequence of SEQ ID NO:2 in the 5' terminal region and the nucleotide sequence of SEQ ID NO:3 in the 3' terminal region, contains a repetitive nucleotide sequence of (CA)15-23, and has total base number of 138-164bp. The primer set for detecting the gene specific to the economic traits of Hanwoo comprises the nucleotide sequences of SEQ ID NO:2 and SEQ ID NO:3, or complementary nucleotide sequences thereof. The Hanwoo having excellent economic traits is selected by performing PCR with the DNA obtained from the blood or cell of Hanwoo as a template using the primer set, and verifying whether the PCR product has the total base number of 138-164bp or not, wherein the economic traits of Hanwoo include daily weight gain, marbling score, backfat thickness and loin muscle area.

Description

A new primer set and a method of selecting Korean beef using the same {A NEW PRIMER SET AND A SELECTIVE METHOD OF HANWOO USING SAID PRIMER SET}

[Industrial use]

The present invention has excellent economic traits by analyzing the presence or absence of Hanwoo's unique microsatellite DNA sequence as a Hanwoo economy-associated gene marker, a primer set for searching the sequence, and the presence or absence of Hanwoo economy-associated gene marker. It relates to the method of screening Hanwoo.

[Private Technology]

Hanwoo (Hanwoo) is a unique prop species of Korea, and has been adapted to the Korean climate for thousands of years and has been fixed in the proper constitution. Korean beef is a product of Korean history that has been maintained for hundreds of years as a unique genetic source of Korea. Since 1970, the mechanization of agriculture and the industrial value of agriculture have diminished. Beef cattle play an important role. In recent years, the importance and value of Korean beef have increased as well as the maintenance and preservation of genetic resources as well as the opening of beef imports in response to changes in the international market economy of the WTO and FDA.

Most studies on cows in developed countries, which are exporters of beef, are mostly based on genetic research. The development of new genetic research techniques and the discovery of genes related to economic traits are carried out by biotechnology to improve domestic cattle. ought. In particular, it has been found that the repeating sequence of microsatellite DNA, which has about 40% of the nucleotide sequence in the bovine genome, has been studied a lot. Quantitative trait loci (QTL) are being performed (Ruyter-Spira et al., 1996; Fridolfsson et al., 1997; Yang et al., 1998, 1999; Lahbib-Mansais et al., 1999; Grosse et al., 2000).

Economic traits mean economically valuable traits among many traits of many domestic animals including Korean cattle, and in the case of Korean cattle, economic traits can be classified into three types: growth traits, carcass traits, and breeding traits. When subdivided in Korean cattle, growth traits include live weight, wean weight, 6 months old weight, 12 months old weight, 18 months old weight, factory weight and body shape. It can be divided into chest, chest, chest, rectangle, anterior tube, sciatic width, and horn width.The carcass shape includes carcass weight, backfat thickness, abdominal muscle cross-sectional area, intramuscular fat and carcass rate, and the fertilization shape contains fertilization rate, ovulation rate, The stillbirth rate, etc., and in addition, Hanwoo has a variety of traits. Since the traits of Hanwoo are expressed by the genes owned by the individual, identifying the genes for certain traits and selecting and using Hanwoo, which has a good ability, will have a great effect in improving the ability of the Korean beef. will be. However, although it is difficult to identify genes involved, the improvement of Hanwoo using genes is not an easy method at this time because several genes are involved at the same time.

On the other hand, microsatellite is a repetitive DNA group that repeats about 2 to 6 nucleotide sequences in a non-coding DNA sequence that is evenly distributed in the genome and exhibits very high polymorphism. equivalent (Zajc I and Sampson J (1999 ) Utility of canine microsatellites in revealing the relationships of pure bred dogs J Hered 90:. 104-107). Diversity is recognized between individuals according to the number of repeat units in a particular locus (Koreth J, O'Leary JJ and McGee JO (1996) Microsatellites and PCR genomic analysis. J Pathol 178: 239-248.) In the presence of hepatic polymorphism, polymerase chain reaction (PCR) is carried out using primers designed in adjacent regions, whereby polymorphisms are observed in the length of the PCR product and DNA polymorphism can be detected. Polymorphism markers using microsatellites are called microsatellite markers (O. Parnaud, X. Chen, SR McCouch, Development of microsatellite markers and characterization of simple sequence length polymorphism (SSLP) in rice (Oryza sativa L.) , Mol . Gen. Genet. 252: 597-607, 1996). Like other genes, microsatellites are delivered to children according to Mendel's genetic law and can be amplified using PCR, and co-dominant when electrophoresed.

In particular, microsatellite is small in size and can amplify two to eight primers simultaneously (Chamberlain JS, Gibbs RA, Ranier JE, Nguyen PN and Caskey CT (1988) Deletion screening of the Duchenne muscular dystrophy Nucleic Acids Res 16: 11141-11156) Reduces time and cost for analyzing polymorphism of genes, results in high sensitivity and accuracy, and uses PCR with primers recently attached (Hearne CM, Ghosh S and Todd JA (1992) Microsatellites for linkage analysis of genetic traits. Trends Genet 8: 288-294.) Thus, such microsatellites are highly valuable in the field of molecular breeding of livestock and can be used not only to prevent degeneration by inbreeding but also to select specific traits.

However, pure polymorphisms such as Korean cattle have rarely been subjected to genetic polymorphism analysis using the microsatellite method, and there have been no reports of genetic allele frequencies.

The present invention is to solve the above problems, an object of the present invention is to prepare a primer set that can search for the repeat base sequence of the unique microsatellite DNA of Hanwoo and economic quality of Hanwoo using the prepared primer set By analyzing the presence or absence of related gene markers, Hanwoo provides a selection method that can accurately and easily evaluate genetic ability at any time during the growth process of Hanwoo.

In order to achieve the above object, the present invention provides a novel primer set capable of searching for a repeat base sequence of a Hanwoo superior economic trait-associated polynucleotide molecule, a native microsatellite DNA, and an excellent economic trait of Hanwoo using the novel primer. Provide a selection method.

Hereinafter, the present invention will be described in detail.

The present invention provides polynucleotide molecules as gene markers. The polynucleotide includes a nucleotide sequence of SEQ ID NO: 2 at the 5 'end of the nucleotide sequence and a nucleotide sequence of SEQ ID NO: 3 at the 3' end, and comprises a repeating base sequence of (CA) ₁₅ to ₂₃ , wherein the total number of bases is 138 to It may be a polynucleotide molecule having an association with the Hanwoo superior economy of 164bp, preferably a polynucleotide molecule of SEQ ID NO: 1.

In the present invention, a BAC clone was prepared, and Hanwoo BAC DNA was separated therefrom, and amplified by PCR to obtain about 3.98 million DNA sequences of Korean cattle, which was repeatedMasker program (open-3.1.2, default mode run with cross_match version 0.990329). Microsatellite DNA sequences were analyzed using. Thus, a region having a dinucleotide repeat sequence in the microsatellite DNA of Hanwoo was secured. A polynucleotide molecule (SEQ ID NO: 1) containing the same was isolated and named as HWYU2CA-134.

In addition, in order to analyze the correlation between the polynucleotide molecules including the microsatellite sequences obtained as described above and the economic traits according to the genetic analysis through PCR and polymorphism of each microsatellite sequence, SAS Package (Version 9.1: SAS Inst., Inc., Cary, NC). This analysis model is as follows.

Model; Y _{i -k} = u + YS _i + P _j + MS _k + bA _ijk + e _ijk

Where Y _{i -k} : individual observation for economic traits

(carcass traits)

μ: overall mean for economic traits (carcass traits)

YS _i : fixed effect of i ^th year-season

P _j : fixed effect of j ^th location

MS _k random effect of microsatellite

bA _ijk : regression coefficient for regression of Y _ijk

on age of k ^th calf

e _ijk : the error term

Polynucleotide molecules found to have excellent economic traits by the above analysis model may include a polynucleotide of SEQ ID NO: 2 at the 5 'end of the nucleotide sequence, and a polynucleotide of SEQ ID NO: 3 at the 3' end, (CA) The total number of nucleotide sequences may be 138 to 164 bp, including the repeating nucleotide sequence of ₁₅ to ₂₃ . In addition, the repeating sequence may be a polynucleotide molecule comprising a repeating nucleotide sequence of SEQ ID NO: 4, more preferably may be a polynucleotide molecule consisting of SEQ ID NO: 1 (HWYU2CA-134). Hanwoo, which has the polynucleotide molecule, has excellent economic traits, and in particular, they are composed of 138 to 164 bases, and sequences other than the above-specified sequences may consist of any.

The present invention also provides a primer set for the search of the polynucleotide molecule.

The primer set comprises a nucleotide sequence of SEQ ID NO: 2 and a nucleotide sequence of SEQ ID NO: 3, or a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 2, and a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 3 It may be a primer set for an association specific gene probe, and the primer set may be named as HWYU2CA-146.

A base sequence in which a part of the base sequence of the primer is replaced with another base, deleted, or the position and orientation of some base sequence are also included in the scope of the primer according to the present invention.

In addition, the present invention provides a method for screening Hanwoo having an excellent economic shape using the primer set.

The method for screening Hanwoo having the superior economic trait may include DNA of an individual taken from a cell or blood of Hanwoo, a base sequence of SEQ ID NO: 2 and a base sequence of SEQ ID NO: 3, or a base complementary to the base sequence of SEQ ID NO: 2 After PCR amplification by combining a primer set consisting of a sequence and a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 3, it may be to confirm that the binding pattern of the DNA and the primer set of the individual consists of 138 to 164 nucleotide sequences have.

In order to measure the binding pattern of the DNA and primer set of the individual, electrophoresis or sequencing may be used.

The excellent economic shape may be an excellent feature for at least one of the economic features consisting of daily gain, intramuscular fat, back fat thickness, and wick section.

Hereinafter, an Example demonstrates this invention in detail.

However, the following examples are merely to exemplify the present invention, and the contents of the present invention are not limited to the following examples.

< Example  1> Korean Native Microsatellite  DNA Identification

720 Korean-American Honorary Assistance Groups, which were conducted by the Hanwoo Breeding Department of the National Agricultural Cooperative Federation, Breeding for a day. As a result of the slaughter, the nuclei were separated from the blood of Hanwoo cattle, which were judged to have excellent meat and meat quality, and then a bacterial artificial chromosome (BAC) clone was produced by the following method.

1-1: BAC  How to make a clone

1) Production of bacteriophage library

In order to secure various genetic information necessary for the study of the Hanwoo Gene, in particular, to obtain genes related to meat mass and quality that are most interested as economic traits of the Korean cattle, the blood of Hanwoo cattle showed different results in slaughter and meat quality. Was extracted and used for the production of the bacteriophage genome library. Firstly, blood samples of Hanwoo individuals showed clear differences in carcass quality and meat grade. EDTA was added to prevent blood coagulation and refrigerated at 4 ° C. until used for chromosomal DNA extraction. In the blood DNA separation kit purchased from Genotein Co., Ltd. (Korea) and the protocol supplied by the company, the leukocyte cells were isolated from the blood, and the purified leukocyte DNA was purified. After purification, the DNA concentration was determined using a spectrophotometer. After the DNA was cut with a restriction enzyme, electrophoresis was performed on an agarose gel to confirm the quality of the DNA.

The bacteriophage library was constructed using Lamda Fix II / Xho I partial fill-in vector kit (Cat # 24811) and Xho I / Gigapack III gold packaging extract kit (Cat # 248612) purchased from Stratagene (California, USA). It was prepared according to the protocol provided by the company. First, purely isolated and purified chromosomal DNA from Hanwoo blood is treated with different amounts of BamH I enzyme, or the same amount of BamH I enzyme is used. Confirmed. Of course, under the conditions when selecting the most favorable conditions in the beef library production and therefore to determine the digestive conditions of the best enzymes in the library produced such a large amount of chromosomal DNA which shows the state of bovine chromosomal DNA is cut well in the most part BamH Partially cut using the enzyme I and only partially digested DNA is purified by phenol / chloroform extraction. To do this with BamH I partially cut DNA is Klenow (klenow) by giving maekkwo two of the four single nucleotide portion out of the end of using the enzyme formed by the BamH I cut the BamH I cut portion 2 nucleotides in man is a single nucleotide Ligation was prevented from ligation between partially cut DNA during ligation. After removal of the klenow enzyme by phenol / chloroform extraction, the purified DNA was ligation to Lamda FixII vector for 16 hours at 4 ℃.

Phage DNA containing each piece of Hanwoo chromosome was made into a complete bacteriophage clone using the phage DNA packaging kit mentioned above. The phage libraries thus produced were determined on the number of phage clones (independent clones) generated after infecting eligible cells and growing on LB / agarose plates. On average, LB / agar plates made from 100mm 150-mm Petri dishes were used in the process of creating a bacteriophage library from a single cattle.

2) Construction of Bacterial Artificial Chromosome Library

In order to construct a genome library of species having a giga-base genome, a plasmid library containing a large insertion site is essential. BAC or YAC vectors are used to produce large insertion libraries. In the case of YAC, genome rearrangement of the inserted DNA easily occurs, which is not suitable as a stable genomic library. In the case of BAC, this is possible because it has the origin of keeping the number of copies close to the number of single copies. It is difficult to isolate mega base DNA without damage. Therefore, genomic DNA of a high molecular weight type was extracted in agarose. The protocol for BAC library construction is as follows.

a) Isolation of nuclei from leukocytes in Hanwoo blood

100 mg of frozen blood samples are hybridized to 1 ml of hybridization solution (40 mM sodium citrate, 1% Triton X-100). After the tissue debris has subsided, take the supernatant. Centrifuge at 500 x g for 5 minutes and resuspend the pellet. Wash twice with wash buffer (containing 0.1% trizma base, 0.8M KCl, 0.1M EDTA, 10mM spermidine, 10mM spermine, pH9.5 or 0.5% Triton x-100 in 40mM sodium citrate). Store on ice until used for the manufacture of agarose embeded plug.

b) Preparation and enzymatic treatment of nucleus agarose plugs

Plug preparation: Damage to nuclear fusion is inevitable because high molecular weight DNA is vulnerable to mechanical shearing. Therefore, dissolution is carried out while the separated nucleus is contained in agarose gel. Place the plug mold purchased from Biorad on ice and heat the 1% low-melting point agarose in a microwave and place it in a 45 ° C water bath. The core is warmed in a 45 ° C. water bath, mixed with agarose and placed in a cold room with an ice bath for 30 minutes until it solidifies. Insert the plug into 50 ml of lysis buffer (0.5M EDTA, 1% SDS, 0.1mg / ml proteaseK) and incubate at 50 ° C for 24 hours. Discard the lysis buffer and dissolve for an additional 24 hours. Place the plug in fresh lysis buffer and store at 4 ° C. for one day.

c) size fraction

Size fractionation was made by pulse field gel electrophoresis. The markers are difficult to accurately identify above 200K using the general purpose low range PFG markers, but the approximate estimation is possible by extrapolating the log mobility above 200K. PFGE was performed at 5V / cm, initial 1, final 12 pulse field, 12 ° C for 16 hours. At least 500 kb intact genomic DNA was identified, followed by EcoR I digestion (pBACe3.6 vector), HindIII digestion (pIndigoBAC-5 vector) and size fractionation. By size fractionation, chromosomal DNA of approximately 150-250 Kb in size was ligated into the vector.

d) DNA isolation and ligation

Agarose gel fragments containing 150-250 Kb of Hanwoo chromosome DNA fragments were washed with electrophoresis buffer (plug 5 times with ultrapure water) and 1 μl of 50X gelase buffer was added to every 50 mg agarose at 70 ° C. After incubation for 3 minutes, transfer to 45 ° C. 1 unit per 200ml of GELase is treated for 45 minutes. In the final step, the DNA concentration is adjusted to 10 ng / μl. The ligation reaction is made of ligation mixture with the following composition, overnight at 16 ° C, and then inactivated at 65 ° C for 30 minutes.

e) BAC colony picking and glycerol stock preparation

After the ligation was completed, the ligation mixture was transformed into bacterial competent cells, and 100 µl of the transformed cells were spread so that 2,000-7,000 colonies appeared on a square plate. 2X LB medium was inoculated in a 96 deep well plate containing 1ml and incubated for 20 hours. 10 μl of glycerol and 40 μl of bacterial culture were mixed and stored in a deep freezer.

f) BAC DNA pure separation

Eight colonies were randomly selected per media test, and cultured in LB, 2LB, 2YT, and TB, respectively, to extract DNA. Cultured in LB had the least genomic DNA contamination, but the efficiency was too low and 2YT had the highest efficiency, but the genomic DNA contamination was the most severe. The best sequencing results were 2X LB culture and 2YT culture.

The Hanwoo BAC clone was incubated in a 96 deep-well culturing block containing 1.4 ml of 2X LB media (including 12.5 µg / ml chloramphenicol) and incubated for 24 hours at 320 rpm in a 37 ° C shaking incubator. Hanwoo BAC DNA was isolated using Montage BAC96 miniprep kit (Millpore), and electrophoresis of the extracted plasmid was performed to confirm DNA integrity and concentration, and then prepared for sequencing reaction and PCR using thermocycler. Sequencing was performed. Primers used for the DNA sequencing are T7 and M13R primers.

After the thermosaticling sequencing is terminated, capillary sequencing is performed using an ABI 3730 DNA sequencer, and the obtained DNA sequencing is deleted from the DNA sequence corresponding to the vector using an analysis program. Only validated high quality sequences were systematically organized and stored.

A total of 4,024 clones (7,441 BAC end-) using two universal sequencing primers (M13R priemr: 5'-GCGGATAACAATTTCACACAGG-3 'and T7 primer: 5'- AATACGACTCACTATAG-3' (synthesized by Macrogen)) As a result of sequencing of the 5 'and 3' ends of the sequence, the nucleotide sequence of the Hanwoo chromosome of about 3.98 million base pairs (3.89 Giga base) was revealed.

The obtained Hanwoo sequencing was analyzed using a computer program in the Bioinformatics Laboratory at the Illinois State University's Institute of Biotechnology. As a result, the region having a repeat base sequence of (CA) _{n in} the obtained microsatellite DNA of Hanwoo was identified, and the correlation analysis with the economic trait according to polymorphism of each microsatellite sequence and genetic analysis through PCR was performed. GLM analysis of SAS Package (Version 9.1: SAS Inse., Inc., Cary, NC) was used.

As a result of the analysis as described above, microsatellite DNA of Hanwoo having excellent economic morphology commonly includes a repeat base sequence of (CA) ₁₈ , and the polynucleotide molecule including the microsatellite DNA is 5 'terminal. Equipped with the base sequence of SEQ ID NO: 2 and the 3 'end in the common sequence including the base sequence of SEQ ID NO: 3. In addition, it can be seen that the length of the polynucleotide molecule is composed of 138 to 164 bases, the length of the sequence is important in the case of polynucleotide molecules commonly possessed by Korean cattle with excellent economic traits, the sequence specified above It was found that the type of nucleotide sequences other than the two were not important.

< Example  2> unique to Korean cattle Microsatellite  DNA primer  making

For genetic analysis through PCR and correlation analysis with economic traits according to polymorphism of nucleotide sequences of each microsatellite DNA, only nucleotide sequences present around the region having the microsatellite DNA (CA) _n repeat base sequence are recognized. PCR primers were prepared using the Primer3 program (http://www.broad.mit.edu/cgi-bin/primer/ primer3_www.cgi). The base sequence of the primer is represented by SEQ ID NO: 5 and SEQ ID NO: 6. Table 1 below shows the DNA base sequence (HWYU2CA-134) including the (CA) ₁₈ repeat base sequence identified using the primer. At this time, italicized is the prepared primer sequence, and the underlined base sequence means (CA) ₁₈ repeat base sequence.

CGACATCGACAAGGATGGCATGCTGGATGATGAGGAGTTCGCACTGGCCAACCACCTCATCAAGGTCAAGCTGGAGGGGCATGAGCTGCCCAACGAGCTGCCCGCCCACCTTGTACCCCCCTCCAAAAGGAAGGCCACCGAGTGATGGGAGGCAGGGAGGTC CAGAGCAGGCAGGTGTTAGA AGAGATGGGAGAGGCAAT CACACACACACGCACACACACACACACACACACACACACACACACACA GATGTTGAGACTGGCACTGCAGATTAAGAGG GAACAAGGATCCCCAGGTTT CCAGGATACATCTCCAAGTGCCCAGTATTGGCTGAAACAGAAGCACCCAGAGTCCCAGGAATCATGTCTCAGTCTCCATCCCTCTTTCATTCCCTGGTCCCACTGCCCGGAGACCCACCTTCTCCTGTGGAGGCCGTTCACTCGGCCACAGATGGCCCACCCACCTCCAGATTCCCTGGACCCAGAGCAGACGGAAAAGGCTTTTCACTTAATAGACAATCCACTTTTTTCTATGCTTTATCCCCACCTCTTTGACTTGCTAACATGAAATCTGCTCTGGGGCCAAGAGATGGGAGGGGAGGGGGCTTCAGCAGGGAGAGGAGTGAAGGGGGAAGTGTCCTCAGGGAACCAGACCCCAGACTTGAAGAGACGTAGCCATAGCCGTGAGCCCTCTTCTTTCCCATGCTGAGGCTCTGACTGCCTCGGCAGACCTGGCTTTAAACTTGCTTGCATTTCCTTTCA

< Experimental Example  1> How to Select Korean Beefs with Excellent Economic Quality

DNA polymorphism of Korean cattle obtained using the primers prepared in Example 2 was measured, and the group with the lowest economic performance and the group with the highest economic performance in the Hanwoo population showing normal distribution were each 15. A DNA pattern that tests the association with economic traits by selecting two, where the allele is a candidate DNA marker related to the economic traits of Hanwoo, if the distribution of alleles is shown between the lowest and highest populations. marker).

As shown in FIG. 1, a genetic marker in which alleles expressed at 138 to 164 bp among multiple alleles is associated with intramuscular fat, daily weight gain, back fat thickness, and loin area, which are important economic traits of Hanwoo It was confirmed.

In addition, the information of the allele for the microsatellite DNA primer sequence obtained in Example 2 was obtained in the following manner and shown in Table 2. The Tm value of allele information was selected by using a gradient-capable PCR machine, and the temperature of which band appeared most clearly was adopted. As a result of electrophoresis, eight kinds of Allele appeared. The H value could be obtained using POPGENE u1.31.

designation Primer set sequence Temperature (℃) Allele number Sequence number of the allele (bp) H HWYU2CA-146 F CAGAGCAGGCAGGTGTTAGA R AAACCTGGGGATCTTGTTC 58 8 138 to 164 81.3

* H; Heterozyosity

* F: Forward primer sequence, R: Reverse primer sequence

As shown in Table 2, the alleles of the primers of SEQ ID NO: 2 and SEQ ID NO: 3 obtained in Example 2 can be synthesized at 58 ℃, the range of alleles were 8 to 138 to 164bp. The heterozygosity indicated by H means that the alleles of the locus located on the chromosome are different. If the heterozygosity is high, there may be many variations of the microsatellite allele so that it can be used as a suitable DNA marker for mapping. do. The heterozygosity rate of the primer of the present invention is 81.3% as shown in Table 2, which seems suitable for use as a marker for association mapping.

In the next step, the average score between the allele and the group was selected and compared with the average score between 15 alleles and 15 lesser grades. In this case, the economic trait was judged according to whether the polynucleotide molecule having a length of 156 bp among the polynucleotide molecules related to the superior economic trait was present.

Allele (bp) Number Economic Intramuscular fat map (1-21) Daily weight gain (㎏) Back fat thickness (mm) Fillet cross section (㎠) 156 9 10.6 0.69 8.2 75.2 None 21 9.4 0.66 6.8 75.3

As shown in Table 3, for the 156 bp allele for the primers of SEQ ID NOs: 2 and 3, the mean difference between the group with the 156 bp allele and the group without the 156 bp allele was 1.2 in the intramuscular fat, and the 30g grade difference in the daily gain. Appeared. In the back fat thickness, there was a 1.4mm grade difference.

Based on this, in order to analyze the correlation with the economic trait of the allele of microsatellite DNA, the economic traits of 476 30 to 35th National Honor Test Groups were measured and shown in Table 4 below.

effect Number Economic Intramuscular fat map (1-21) Daily weight gain (㎏) Back fat thickness (mm) Fillet cross section (㎠) LSMEAN ± SE LSMEAN ± SE LSMEAN ± SE LSMEAN ± SE P <0.05 P = 0.072 P = 0.844 P = 0.463 LSMEAN 476 5.54 ± 0.31 0.75 ± 0.01 7.49 ± 0.24 75.66 ± 0.63 156 48 6.28 ± 0.59 0.74 ± 0.02 7.27 ± 0.45 76.04 ± 1.19 None 428 4.81 ± 0.20 0.76 ± 0.01 7.7 ± 0.15 75.29 ± 0.4 Year-season - P = 0.487 P <0.05 P = 0.212 P = 0.08 Place - P = 0.331 P <0.001 P <0.01 P = 0.395

Table 4 shows the least squares mean values for Hanwoo with alleles of 156 bp in the primer set of the present invention applied to the National Honor Test Group. Unlike Table 3, after fixing environmental factors, the genetic factors were shown. In intramuscular fat score, the difference between the group with 156bp allele and the group without was significantly 1.47. However, in Table 3, there was no significant difference in backfat thickness and fillet area showing high grade differences, indicating that economic characteristics were affected by other environmental factors not considered in the statistical model.

In addition, Table 5 below shows the distribution of intramuscular fat of the population having an allele of 156 bp. HWYU2CA-146's 156bp allele had a 7.5% higher prevalence rate than the 13.8% prevalence rate among the 476 two 30-35th national black cattle.

Allele (bp) Number Intramuscular fat grade Rate 1 or higher (1+ and 1) 1+ (16-21) 1 (10-15) 2 (4-9) 3 (1-3) 156 48 3 (6.4%) 7 (14.9%) 22 (46.8%) 15 (31.9%) 10 (21.3%) None 476 11 (4.1%) 26 (9.7%) 106 (39.6%) 125 (46.6%) 37 (12.8%)

As such, by using a primer set designed to search for the unique microsatellite of the present invention, the DNA marker that appears in the 156 bp allele can be precisely and easily improved at any time during the growth process of the cattle. As it is possible to select Hanwoo with economic qualities, it is considered that it can be used as a supplementary tool in identifying excellent individuals, selecting excellent breeders, and selecting stock management for high-quality meat groups.

Attach sequence list electronic file

Claims (5)

5 'nucleotide sequence of the nucleotide sequence of SEQ ID NO: 2 and 3' end of the nucleotide sequence of SEQ ID NO: 3, including the repeat base sequence of ₁₅ to ₂₃ , the total base number of 138 to 164bp excellent Hanwoo Economic traits specific polynucleotide molecules. Hanwoo excellent economy including the nucleotide sequence of SEQ ID NO: 4, the nucleotide sequence of SEQ ID NO: 2 at the 5 'end of the nucleotide sequence, and the nucleotide sequence of SEQ ID NO: 3 at the end of the nucleotide sequence 3', and the total base number of 138 to 164bp Associative polynucleotide molecules. Hanwoo excellent economy associated specific polynucleotide molecule consisting of SEQ ID NO: 1. Hanwoo excellent economic related gene probe consisting of the nucleotide sequence of SEQ ID NO: 2 and the nucleotide sequence of SEQ ID NO: 3, or the nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 2, and the nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 3 Primer set for. After PCR amplification using the DNA of the individual obtained from the Hanwoo cells or blood as a template and the primer set of claim 4 as a primer, it was confirmed that the product obtained by the PCR amplification consists of 138 to 164 nucleotide sequences. Thus, the method for selecting a Hanwoo beef having excellent characteristics for at least one of the economic trait criteria consisting of daily gain, intramuscular fat, back fat thickness and loin sectional area.