KR20160121931A - Gene composition for parentage testing in hanwoo - Google Patents
Gene composition for parentage testing in hanwoo Download PDFInfo
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- KR20160121931A KR20160121931A KR1020150051807A KR20150051807A KR20160121931A KR 20160121931 A KR20160121931 A KR 20160121931A KR 1020150051807 A KR1020150051807 A KR 1020150051807A KR 20150051807 A KR20150051807 A KR 20150051807A KR 20160121931 A KR20160121931 A KR 20160121931A
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Abstract
Description
More particularly, the present invention relates to a gene composition capable of detecting a SNP marker specific to Korean cattle, a DNA chip, and a method for distinguishing a Korean cattle using the same.
Hanwoo is one of the representative livestock in Korea, and it is essential to identify its lineage to improve its genetic capacity. Hanwoo 's genealogy can be used to preserve Hanwoo as an index of improvement of Hanwoo farmers, and it can increase the reliability of Hanwoo by providing accurate information to consumers in distribution process.
Accurate paternity identification of Hanwoo is essential for the success of the breeding program, which can lead to improved productivity and improve breeder 's expectation. However, in commercial breeding programs, pedigree problems can occur due to data loss, human error, or deliberate counterfeiting. In such cases, DNA-based paternity identification can help to identify the breed of Hanwoo and improve the breeding program.
In this regard, the International Society for Animal Genetics (ISAG) has proposed a set of single nucleotide polymorphism (SNP) markers for lineage testing of European tiger cattle ( Bos Taurus cattle). The SNP marker set consists of 100 core SNPs and an additional 100 markers (ISAG CMMPT. (2012) Cattle Molecular Markers and Parentage Testing Workshop. In: ISAG Conference , Cairns). However, since the core SNP is composed mainly of SNPs specific to European bull varieties, the marker set is not suitable for discrimination of breeders having no blood relationship with them, for example, Hanwoo (Werner FA et al. , (2004) Detection and characterization of SNPs useful for identity control and parentage testing in major European dairy breeds, Anim Genet 35 , 44-9).
Therefore, it is required to develop new SNP markers specialized in the identification of paternity and a gene composition capable of detecting the SNP markers.
Accordingly, an object of the present invention is to provide a gene composition and a DNA chip for detecting SNP markers, which can be used for identification of a parent of cattle.
Another object of the present invention is to provide a method for easily distinguishing a parent of a Korean bean using the DNA chip.
In order to accomplish the above object, the present invention provides a method for identifying a parental spermatogon of Hanwoo, comprising 20 to 200 consecutive polynucleotides or complementary polynucleotides thereof comprising the single nucleotide polymorphism (SNP) Lt; / RTI >
In order to achieve the above object, the present invention provides a method for identifying a parent of a Korean beef comprising 20 to 200 consecutive polynucleotides or complementary polynucleotides thereof comprising the single nucleotide polymorphism (SNP) Lt; / RTI >
In order to accomplish the above object, the present invention provides a DNA chip for identifying a parent of a Korean animal having the polynucleotide immobilized thereon.
Further, in order to accomplish the above other objects, the present invention provides a method for producing a nucleic acid sample, comprising the steps of: (i) obtaining a nucleic acid sample from a cattle; (ii) hybridizing the nucleic acid sample with the DNA chip described above; And (iii) analyzing the hybridization result.
The gene composition according to the present invention is composed of SNP markers specialized for Hanwoo, and has a high separation value and low false positive / false negative ratio for a Hanwoo group.
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph showing the separation values between the true father-child relationship and the false father-child relationship in the entire ISAG panel and the entire panel according to the present invention.
The present invention provides a gene composition for the identification of paternity in Hanwoo, which consists of 20 to 200 consecutive polynucleotides or complementary polynucleotides thereof containing the single nucleotide polymorphism (SNP) region shown in Table 2 herein.
The single nucleotide polymorphic sites listed in Table 2 are present in the autosomal chromosomes of Hanwoo, and are the markers selected based on the genotypic frequency among the SNP markers present in the 50K SNP chip panel (illumina Inc., USA) known in the art.
The markers are compared to the true sibilant-sibling relationship between the Hanwoo group and the false-sibilant relationship, as compared to the ISAG panel for the paternity discrimination of the previously known European Bosch Taurus cattle the separation value is high and the false-positive rate and the false-negative rate are low.
The gene composition according to the present invention may consist of 20 to 200 consecutive polynucleotides or complementary polynucleotides thereof containing the single nucleotide polymorphism (SNP) region described in Table 3 herein.
The single nucleotide polymorphic sites listed in Table 3 above additionally include additional single nucleotide polymorphic sites in the ISAG panel in addition to the single nucleotide polymorphic sites listed in Table 2 above. That is, the gene composition according to the present invention is a total of 195 full panels, consisting of 95 core panels as shown in Table 2 and an additional 100 markers in the ISAG panel.
The whole panel has a higher separation value and a lower false positive rate and a lower false negative rate than the core panel composed of the above-mentioned 95 markers.
Meanwhile, the present invention provides a DNA chip for identifying a parental strain of the Korean NK, to which the polynucleotide is immobilized. The DNA chip is not significantly different from the conventional DNA chip except that the polynucleotide described above is immobilized. The constitution and the production method of the DNA chip refer to conventionally known methods.
(I) obtaining a nucleic acid sample from a cattle; (ii) hybridizing the nucleic acid sample with the DNA chip described above; And (iii) analyzing the hybridization result.
Step (i) of the method according to the present invention is a process for obtaining nucleic acid samples from Hanwoo, which is well known in the art. For example, the nucleic acid samples can be obtained from the muscles, epidermis, blood, bones, organs of Hanwoo, and most preferably from muscle or blood. When the nucleic acid sample is genomic DNA, isolation of the genomic DNA can be carried out according to a conventional method known in the art (see Rogers SO and AJ Bendich, 1988, In Plant Molecular Biology Manual).
When the nucleic acid sample is an mRNA, the mRNA may be separated by isolating the total RNA according to a conventional method known in the art (see Sambrook, J. et al., Molecular Cloning. A Laboratory Biochem., 162: 156, 1987) and Chomczynski, P. et al., Anal. Biochem. The isolated total RNA can be synthesized using cDNA using reverse transcriptase. Since the total RNA is isolated from animal cells and has poly-A tail at the end of mRNA, the oligo dT primer and reverse transcriptase The cDNA can be easily synthesized using an enzyme.
In step (ii) of the method according to the present invention, a probe complementary to the sequence comprising the single base polymorphism of the present invention may be used. The probe is hybridized with the single base polymorphism site of the present invention, and the hybridization signal generated at this time can be detected to directly determine whether a single base polymorphism exists. As the probe to be used, a sequence completely complementary to the sequence including the single base polymorphism may be used, but a sequence substantially complementary may be used as long as it does not interfere with the specific hybridization. Preferably, the 3'-terminal or 5'-end of the probe has a base complementary to the single base polymorphic base. In general, the stability of the duplex formed by hybridization tends to be determined by the agreement of terminal sequences, so that a probe having a base complementary to a single base polymorphic base at the 3'-terminal or 5'- Such a duplex can be disassembled under stringent conditions.
Conditions suitable for hybridization can be determined by reference to the disclosure in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, 2001 and Haymes, BD, et al., Nucleic Acid Hybridization, A Practical Approach, 1985 . The stringent condition used for hybridization can be determined by controlling the temperature, the ionic strength (buffer concentration) and the presence of a compound such as an organic solvent, and the like. This stringent condition can be determined differently depending on the sequence to be hybridized.
The resultant analysis of step (iii) according to the present invention can be performed by applying known methods widely known in the art. For example, methods for analyzing single nucleotide polymorphisms include allele-specific probe hybridization, allele-specific amplification, sequencing, 5 ' (5 'nuclease digestion), molecular beacon assay, oligonucleotide ligation assay, size analysis or single-stranded polymorphism conformation polymorphism, and the like may be used, but the present invention is not limited thereto.
In one embodiment, identification of the gene composition may be performed by an allele-specific gene amplification method. When a single nucleotide polymorphism is applied to the gene amplification method, it is collectively referred to as SNAP (single nucleotide amplified polymorphism).
In the present invention, the size of the polynucleotide is limited to 20 to 200 bp, but this is merely limited to a technical specificity at a reproducible level by the present hybridization technique. It can be used as a size deviating from the above range depending on the technique to be used.
The method of distinguishing paternity of Hanwoo according to the present invention is superior in accuracy to the method using a conventionally known ISAG panel, and thus can be usefully used in a breeding program of Hanwoo.
EXAMPLES Hereinafter, the present invention will be described in detail with reference to Examples, but the following Examples are intended to illustrate the present invention without limiting the scope of the present invention.
Example
1: SNP for identification of Korean parents
Marker
Selection
<1-1> SNPs for core panels Marker Selection
257 SNP markers specific to Hanwoo varieties were firstly selected through comparative analysis of genotype frequencies from a conventionally known 50K SNP chip panel (illumina Inc., USA). The SNP markers were selected from a group of 265 animals, and the selection consisted of high call rate (GC score), SNP at Hardy-Weinberg equilibrium (to minimize the likelihood of future marker fixation) In order to achieve individual differentiation, it was based on marker diversity indicated by high minimum allele frequency and heterozygosity, and markers uniformly distributed throughout the chromosome were selected to reduce the inter-marker association.
The selected 257 SNP markers are shown in Table 1 below.
95 SNP markers for the core panel were selected in consideration of the chromosomal location, the distance between the markers, and the genotypic frequency from the selected 257 SNP markers.
The selected 95 SNP markers are shown in Table 2.
<1-2> Production of whole panel
In addition to the 95 SNP markers selected in Example 1 as core panels, 100 additional SNP markers proposed by the International Society for Animal Genetics (ISAG) (http://www.isag.us/committees. asp? autotry = true & ULnotkn = true).
Table 3 shows the SNP markers constituting the whole panel for discriminating the parents of the Hanwoo.
Example
2:
Marker
Panel efficiency comparison
The efficiency of the core panel and the entire panel manufactured in Example 1 was compared with conventionally known panels. As a panel for comparison, an ISAG entire panel consisting of an ISAG core panel consisting of 100 markers and an additional 100 markers added to the ISAG core panel (total 200) (http://www.isag.us/committees. asp? autotry = true & ULnotkn = true).
The efficiency of the marker panels was tested against a pure Korean Hanwoo (326) group. The Hanwoo group is a group that includes genotypes of the father of a semi-sibling group.
<2-1> Genotype analysis of Hanwoo group
The genotypes of the Hanwoo group were analyzed using a 700K Bovine HD BeadChip (Illumina, Inc.). As a result, the group consisted of 36 sires and 290 offspring (6 to 9 horses Of children). No information was available about the relationship between the sows, but it was expected that the siblings would be related to some extent due to the small size of these varieties.
<2-2> Exclusion power ( exclulsion power calculation
As a primary evaluation for predicting how effectively the core panel and the entire panel according to the present invention will work, the panel is described in Jamieson A. & Taylor S.C. (1997) Animal Genetics 28, 397-400) to calculate one exclusionary parental scenario exclusion. For comparison, an ISAG core panel and an ISAG full panel were used.
As a result of the exclusion power calculation, the core panel of the present invention exhibited an exclusion force of 0.99 and the entire panel of the present invention exhibited an exclusion power of 1, while the ISAG core and the entire panel showed a total exclusion power of 0.99 in the Hanwoo group .
The above results show that the panel and ISAG panel according to the present invention will work well for identifying false Abi-child assignments, and in particular demonstrate that the entire panel according to the invention is better than the ISAG full panel.
<2-3> Separation value Calculation
For each group, the number of allelic conjugates in each animal was calculated using the marker genotype (Ferdosi M. H., Kinghorn B. B. P., Werf J. H. J. V. D. & Gondro C. (2014) Genetics, Selection, Evolution, 46, 11). An homozygous conjugate for any given marker is defined as one homozygous for one allelic variant and the other homozygous for the alternate allele. This is an easy way to confirm Mendel's inconsistency, which should not occur in true father-child relationships except for genotyping errors or unlikely mutations. On the other hand, the homozygous conjugate should occur more frequently among unrelated animals and may be used to eliminate lineage relationships (Hayes B.J. (2011) Journal of Dairy Science 94, 2114-7).
An intuitive approach to assessing the efficiency of a lineage marker panel is to determine the minimum number of allelic conjugates found in all false-parent relationships (ie, all pairwise combinations except for the actual parent-child pair) (Hayes BJ (2011) Journal of Dairy Science 94, 2114-7). ≪ / RTI > This difference can be divided by the total number of SNPs in the panel for comparison between different panel sizes. This difference was referred to as a separation value. Intuitively, the larger the value, the better the panel's pedigree assignment, and if the value is zero or negative, a true father-child relationship and a false father-son relationship can not be perfectly separated. It should be noted that since the number of allelic conjugates is panel, population and animal specific, the actual separation values are only meaningful within the data set.
The result of the separation value measurement is shown in Fig. As shown in FIG. 1, the entire panel according to the present invention exhibited a higher separation value than the ISAG full panel, demonstrating that the entire panel of the present invention is superior to the ISAG overall panel.
<2-4> Calculation of false positives and false negatives
The false-positive rate was then calculated as the ratio of mis-assigned child-child relationships from the total number of relationships identified from all pairs of child-child pairs. We also calculated the false-negative rate as the ratio of child-child relationships that were misdirected from the total number of possible relationships. In accordance with ISAG guidelines and to accommodate genotyping errors, a maximum of one allelic conjugate was allowed in the allowed lineage.
The calculation results are shown in Table 4.
(# Father / child)
(36/290)
MAF: Average minor allele frequency of all markers
He: average heterozygosity of all markers
fp: false positive rate
fn: false negative rate
As shown in Table 4, the false positive rate of the ISAG core panel was 3.01%, and the false negative rate was 0%. In addition, the false positive rate of the entire ISAG panel was 0%, and the false negative rate was 2%.
On the other hand, the core panel according to the present invention showed a false positive rate of 2.03% and a false negative rate of 0%, and the entire panel according to the present invention showed a false positive rate of 0% and a false negative rate of 0%.
The above results demonstrate that the core panel and the entire panel according to the present invention are more effective than conventional ISAG panels in discriminating parents of Hanwoo.
Claims (4)
<Table 2>
<Table 3>
(ii) hybridizing the nucleic acid sample with the DNA chip according to claim 3; And
(iii) analyzing the hybridization result
Wherein the method comprises the steps of:
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR102083675B1 (en) * | 2018-12-13 | 2020-03-02 | 대한민국 | Method for identification of Chikso breed using single nucleotide polymorphism markers |
| KR20200100980A (en) * | 2019-02-19 | 2020-08-27 | 전북대학교산학협력단 | Method and apparatus for paternity test using the microsatellite marker composition in dogs |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20200064401A (en) | 2018-11-29 | 2020-06-08 | 주식회사나우리 | Parent identification method using Hanwoo gene composition |
| KR20200064406A (en) | 2018-11-29 | 2020-06-08 | 주식회사나우리 | Correction of Parental Error of Paternity Disagreeing Individuals Using Hanwoo Gene Composition |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR100933223B1 (en) | 2006-11-14 | 2009-12-22 | 대한민국 | SNP marker combination for Korean beef |
| KR101351990B1 (en) | 2011-12-02 | 2014-01-16 | 서강대학교산학협력단 | Single Nucleotide Polymorphisms for Individual Identification of Hanwoo and Use Thereof |
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Non-Patent Citations (2)
| Title |
|---|
| ISAG CMMPT. (2012) Cattle Molecular Markers and Parentage Testing Workshop. In: ISAG Conference, Cairns |
| Werner F.A. et al., (2004) Detection and characterization of SNPs useful for identity control and parentage testing in major European dairy breeds. Anim Genet 35, 44-9 |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR102083675B1 (en) * | 2018-12-13 | 2020-03-02 | 대한민국 | Method for identification of Chikso breed using single nucleotide polymorphism markers |
| KR20200100980A (en) * | 2019-02-19 | 2020-08-27 | 전북대학교산학협력단 | Method and apparatus for paternity test using the microsatellite marker composition in dogs |
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