KR101805165B1 - Primer set for inspecting the combination purity or detecting the genotype of self-incompatibility ClassⅡ gene in Brassica oleracea and the inspection or detection method by using same - Google Patents

Primer set for inspecting the combination purity or detecting the genotype of self-incompatibility ClassⅡ gene in Brassica oleracea and the inspection or detection method by using same Download PDF

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KR101805165B1
KR101805165B1 KR1020167025811A KR20167025811A KR101805165B1 KR 101805165 B1 KR101805165 B1 KR 101805165B1 KR 1020167025811 A KR1020167025811 A KR 1020167025811A KR 20167025811 A KR20167025811 A KR 20167025811A KR 101805165 B1 KR101805165 B1 KR 101805165B1
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노일섭
박종인
양기웅
황인덕
정희정
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순천대학교 산학협력단
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Abstract

The present invention relates to a method for assaying a combination purity of a combinatorial purity of a cabbage self-incompatibility factor Class II or a combination of a primer set for classifying a genotype of Class II and a cabbage self-incompatibility factor Class II using the same, or a method for discriminating a genotype of Class II, Using the primer set for the combination purity assay of the cabbage self-incompatibility factor Class II of the present invention, it is possible to test whether the combination of the cabbage self-incompatibility factor Class II is homozygous or heterozygous, and when the cabbage self-incompatibility factor Class II The genotype of the SRK gene of Class II can be discriminated by using the SRK genotype discrimination primer set, thereby reducing the mating cost according to the self-incompatibility discrimination of the mating strain, and the breeding cost of the hybrid seed can be lowered by improving the efficiency of the hybrid seed .

Description

TECHNICAL FIELD The present invention relates to a primer set for identifying a combination purity of a cabbage self-incompatibility factor CLASSII or a genotype-identifying primer set, and a method for determining purity or genotyping using the primer set or the genotype identification method of the genus of Brassica oleracea and the inspecting method or detection method by using same}

The present invention relates to a method for assaying a combination purity of a combinatorial purity of a cabbage self-incompatibility factor Class II or a combination of a primer set for classifying a genotype of Class II and a cabbage self-incompatibility factor Class II using the same, or a method for discriminating a genotype of Class II, More specifically, the combination purity of the three factors, S2, S5, and S15, which are the S-locus receptor kinase (SRK) genes of the self-incompatibility factor ClassII of cabbage, is tested and the genotype of the SRK gene is determined, The present invention relates to a combination purity determination method and a genotype discrimination method of a cabbage self-incompatibility factor Class II capable of preventing moisture and fertilization failure due to crossing between cabbage lines having a synthetic gene.

Most of the major varieties of domestic vegetables are hybrids. Hybrid cultivation, self - incompatibility, and male sterility are mainly used for this kind of hybrid seed production. Among them, the cabbage has self - incompatibility, so it is essential to know the self - incompatibility of the cabbage used in the production of one -

Self - incompatibility is a phenomenon in which individuals who have the same self - incompatible gene do not get water and fertilization, and are inherent characteristics of the plant to avoid future weakness due to self - pollination. However, since this self - incompatibility phenomenon can be known only after pollen is pollinated to the stigma after flowering, self - incompatibility discrimination is very difficult for the breeding process.

Therefore, if the cabbage breeding breeder can easily identify the self-incompatible genotype, breeding between the same genotype can be avoided when establishing the breeding plan, and breeding between different genotypes can be greatly reduced. Private breeders can reduce breeding costs by up to 30 percent in an informal way. In addition, even in the case of one - to - one hybrid production, it is possible to reduce the unit price of the seed by using the self - incompatibility of the parents to cause hybridization. Currently, relative to the male sterility, relatively less, but the autogenous fluoride is still used in the F1 breed.

S-locus glycoprotein (SLG), S-locus receptor kinase (SRK), and S-locus protein / SCR (S-locus cys-rich protein ) Gene. ≪ / RTI > The self-incompatibility of the dorsal side is controlled by the expression of the SRK gene, and the autogenous disintegration of the pollen side is controlled by a gene called SP11 / SCR (Bateman, 1955; Nasrallah and Nasrallah, 1993; Takayama, 2003) .

To date, over 100 kinds of self-incompatible genotypes have been identified in cabbage ( Brassica oleracea ), cabbage ( B. campestris ) and radish ( Raphanus s ativus L.) (Ockendon, 1974; Nou et al., 1993; Sakamoto et al. , 1998; Lim et al., 2002). The PCR restriction fragment length polymorphism (PCR-RFLP) analysis of the SLG and SRK genes revealed the ability to identify different types of self-incompatible varieties of cabbage, cabbage, and radish. In this way, the classification of self-incompatible genotypes, which was possible by crossing various lines after blooming in the pavement, became possible in the laboratory shortly before flowering (Brace et al., 1993; 2002).

The autotrophic genotypes of cabbage and crops are classified into class I and class II by their nucleotide sequence (Nasrallah et al., 1991). In general, the genotype belonging to class I is dominant in the pollen side than the genotype belonging to class II, It has been reported that the class II genotype is weak in vitality or is not constant in its expression (Hatakeyama et al., 1998; Nasrallah and Nasrallah, 1993; Thompson and Taylor, 1966). The genotype of this weakly expressed phenotype is often found in Japanese hybrids of cabbage or broccoli, and these genotypes, which adversely affect seed purity, are used for the production of hybrids It has been suggested that it is difficult to isolate, either in association with or very close to the class II gene.

Therefore, the present inventors have focused on the fact that the SRK gene of the Class II group involved in the cabbage autolysis accumulates a large number of mutations over a long period of time. Using these genetic information, a primer capable of discriminating other strains And the present invention has been completed on the basis thereof.

Accordingly, a technical object of the present invention is to provide a primer capable of testing the purity of a combination F1 of the autofluorescence factor Class II of cabbage.

Another object of the present invention is to provide a primer capable of discriminating the genotype of the SRK gene of the self-incompatibility factor Class II of cabbage.

Another technical problem to be solved by the present invention is to provide a method for testing the purity of a combination F1 varieties of cabbage autofluorescence factor ClassII.

Another technical problem to be solved by the present invention is to provide a method for determining the genotype of the SRK gene of the cabbage self-incompatibility factor ClassII.

In order to solve the above-mentioned technical problems, the present invention provides a primer set for combination purity assay of a cabbage autolysin factor Class II comprising a forward primer having a nucleotide sequence of SEQ ID NO: 1 and a reverse primer having a nucleotide sequence of SEQ ID NO: 2 do.

In addition, the present invention provides a primer set for genotyping a cabbage autolysin class II, which is composed of a forward primer having the nucleotide sequence of SEQ ID NO: 3 and a reverse primer having the nucleotide sequence of SEQ ID NO: 4.

The present invention also provides a method for preparing a recombinant vector, comprising the steps of: performing PCR (polymerase chain reaction) using a primer set for a combination purity assay of a cabbage autolysin factor Class II comprising the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2; And a step of determining the identity of the cabbage self-incompatibility factor Class II combination by high resolution melting (HRM) analysis of the PCR amplification product to determine the combination purity of the cabbage self-incompatibility factor Class II .

Also, the present invention provides a method for detecting a polymorphism comprising the steps of: performing PCR using a primer set for discriminating the genotype of the cabbage autolysing factor Class II comprising the forward primer of SEQ ID NO: 3 and the reverse primer of SEQ ID NO: 4; And determining the genotypic identity of the cabbage self-incompatibility factor ClassII by analyzing HRM of the PCR amplification product. The present invention also provides a method for determining the genotype of the cabbage self-incompatibility factor ClassII.

The present invention focuses on the fact that the SRK gene of the Class II group, which is involved in the incompatibility of cabbage autolysis, accumulates a large number of mutations over a long period of time. Using these genetic information, a primer capable of discriminating other lines of autofluorescence Respectively.

The inventors compared SRK gene information obtained from the genetic resources of different cabbages with autofluoscence (Figs. 1A and 1B).

First, the SRK gene of the cabbage self-incompatibility factor ClassII obtained by NCBI was compared to identify differences among the genes, and a primer for each gene was devised by using these differences.

The base sequence of the primer set for testing the purity of the combination (S2, S5, S15) X (S2, S5, S15) of SRK genes of the cabbage autolysing factor ClassII according to one preferred embodiment of the present invention is shown in the following table 1 < / RTI >

Base sequence SEQ ID NO: classII-BoSRK-SNP-F1 CAG TTA AAT GTC TTT GGA AAT SEQ ID NO: 1 classII-BoSRK-SNP-R1 TCA TCC CGT CCA AAG ATC CT SEQ ID NO: 2

PCR was carried out using the above primer set and HRM (high resolution melting) analysis of the PCR amplification product was performed to determine whether the combination of the cabbage autolysin class II was a homozygous gene combination or a heterozygous gene combination The purity can be assayed.

The nucleotide sequences of SRK genotyping primer sets of the cabbage autolysing factor ClassII according to one preferred embodiment of the present invention are shown in Table 2 below.

Base sequence SEQ ID NO: classII-BoSRK-F2 GAA GCT GAC ACG AGG AAG GT SEQ ID NO: 3 classII-BoSRK-R2 CGT TCA TCG CAT ATT CTG GAGE SEQ ID NO: 4

PCR is performed using the above primer set, and the obtained PCR amplification products are analyzed by HRM to determine genotypes of the SRK genes S2, S5 and S15 of the cabbage autolysin factor ClassII.

The high resolution melting (HRM) analysis used in the present invention is an analytical method capable of showing the difference in base sequence of DNA fragments by distinguishing a melting temperature which is changed by one nucleotide sequence difference without performing a base sequence analysis. It shows that it can be used as a polymorphic primer by showing differences in the melting curve between two DNA samples.

According to one embodiment of the present invention, the combination of the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2 obtained from the SRK gene DNA of the self-fluorinating factor ClassII of cabbage and the combination purity of the cabbage autolysing factor ClassII Performing PCR using a primer set for assay; And determining the identity of the SRK gene combination of the cabbage self-incompatibility factor ClassII by performing HRM analysis on the PCR amplification product.

In accordance with another embodiment of the present invention, SRK genotyping of the cabbage autolysin Factor Class II comprising the forward primer of SEQ ID NO: 3 and the reverse primer of SEQ ID NO: 4 obtained by template SRK gene DNA of the self- Performing PCR using a primer set for the primers; And determining the identity of the SRK genotype of the cabbage self-incompatibility factor ClassII by analyzing the PCR-amplified product by HRM, thereby providing a genotyping method for the autolysin factor ClassII of cabbage.

In this way, the purity of the SRK gene combination of the self-incompatibility factor ClassII of cabbage is assayed, the genotype of the SRK gene is determined, and the genotypes are subjected to moisture and / or fertilization between different cabbage species, Moisture and / or unmodifiable intrinsic properties can be overcome.

As described above, it is possible to test whether the combination of the cabbage self-incompatibility factor Class II is homozygous or heterozygous using the primer set for the combination purity test of the cabbage self-incompatibility factor ClassII of the present invention, Identification of genotype of SRK gene of ClassII using SRK genotype identification primer set of synthesis factor ClassII can reduce mating cost according to self-incompatibility discrimination of mating person and increase efficiency of hybrid seed species to increase breeding cost Can be lowered.

1A and 1B show the nucleotide sequences of SRK regions of the S-gene of the cabbage autolysing factor ClassII.
FIG. 2 is a graph showing the results of HRM (high resolution melting) analysis of a PCR product amplified using a primer set for assaying the combinatorial purity of the cabbage self-incompatibility factor ClassII of the present invention.
FIG. 3 is a graph showing the results of HRM analysis of the PCR product amplified using the primer set for discriminating the genotype of the cabbage self-incompatibility factor ClassII of the present invention.

Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.

<Example 1> Primer design

A large number of cabbage lines were collected, PCR amplified the SRK gene region, and PCR products were cloned and sequenced to distinguish autofluorescence genotype. Among them, S-genotype of Class II of cabbage registered in Gene Bank (NCBI) DNA base of SRK2 (S2), SRK5 (S5), SRK15 (S15) belonging to Class II group in which DNA base was confirmed by sequencing The oligonucleotides of about 20 bp as shown in Table 3 were synthesized using the kinase domain region of SRK, which is a factor of the incompatible factor, and securing the respective SRK gene sequences (FIGS. 1A and 1B).

Namely, the DNA base of the three cabbage strains (SRK2 (S2), SRK5 (S5), and SRK15 (S15)) in which the autofluorescence genotype was confirmed was used as a standard DNA base and the cabbage strain having the nucleotide sequences of SEQ ID NOs: &Lt; / RTI &gt; ClassII combination primer for purity assay; And S-genotyping primers of Class II of cabbage having the nucleotide sequences of SEQ ID NOS: 3 and 4 were synthesized.

Base sequence SEQ ID NO: classII-BoSRK-SNP-F1 CAG TTA AAT GTC TTT GGA AAT SEQ ID NO: 1 classII-BoSRK-SNP-R1 TCA TCC CGT CCA AAG ATC CT SEQ ID NO: 2 class II-BoSRK-F2 GAA GCT GAC ACG AGG AAG GT SEQ ID NO: 3 classⅡ-BoSRK-R2 CGT TCA TCG CAT ATT CTG GAGE SEQ ID NO: 4

&Lt; Example 2 > Purity determination of the combination of the cabbage autologous gene Class II

Class F1 (S2, S5, S15) X class II (S2, S5, S15) combination was used as a material after confirming the class combination of commercially available F1 cabbage varieties.

HRM (high resolution melting) analysis was performed using the forward primer of SEQ ID NO: 1 synthesized in Example 1 and the reverse primer of SEQ ID NO: 2 as follows.

HRM analysis PCR was the conditions shown below by using the using the LightCycler 480 ⓡ (Roche Diagnostics, Penzberg , Germany) , and a reaction composition 25mM MgCl 2 in 2X High Resolution Melting Master ⓡ, DNA 5ug, primer (5pM) Table 4 Respectively.

Target (° C) Acquisution mode Hold (hh: mm: ss) 95 None 0:10:00 1Cycles Amplification 95 None 0:00:10 40Cycles 57 None 0:00:30 72 Single 0:00:30 Melting Curve 95 None 0:01:00 1Cycles 40 None 0:01:00 65 None 0:00:01 95 Continuous (5R / ° C) 0:00:01

FIG. 2 is a graph showing the results of HRM analysis of PCR products amplified using a primer set for assaying the combinatorial purity of the cabbage self-incompatibility factor ClassII of the present invention.

As shown in FIG. 2, HRM analysis using a primer set consisting of a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2 revealed that a melting curve between a homozygous gene combination and a heterozygous gene combination sample and the difference in melting peak. From this, the combination purity of the cabbage self-incompatibility factor ClassII (S2, S5, S15) X ClassII (S2, S5, S15) can be verified using the primer set for the combination purity test of the cabbage self- .

&Lt; Example 3 > Genotype discrimination of cabbage self-incompatible gene ClassII

The Asian cabbage and Korean cabbage were distributed from the Korean cabbage.

Using the forward primer of SEQ ID NO: 3 and the reverse primer of SEQ ID NO: 4 synthesized in Example 1, HRM analysis was performed as follows.

HRM analysis PCR was the conditions shown below by using the using the LightCycler 480 ⓡ (Roche Diagnostics, Penzberg , Germany) , and a reaction composition 25mM MgCl 2 in 2X High Resolution Melting Master ⓡ, DNA 5ug, primer (5pM) Table 5 Respectively.

Target (° C) Acquisution mode Hold (hh: mm: ss) 95 None 0:03:00 1Cycles Amplification 95 None 0:00:10 55Cycles 60 None 0:00:20 72 Single 0:00:20 Melting Curve 95 None 0:01:00 1Cycles 38 None 0:02:00 85 Continuous (5R / ° C) 0:00:01

FIG. 3 is a graph showing the results of HRM analysis of the PCR product amplified using the primer set for discriminating the genotype of the cabbage self-incompatibility factor ClassII of the present invention.

As shown in FIG. 3, HRM analysis using a primer set consisting of a forward primer of SEQ ID NO: 3 and a reverse primer of SEQ ID NO: 4 showed that differences in melting curve and melting peak between samples of S2, S5 and S15 genotypes Respectively. From this, it can be seen that the primers set for the genotyping of the cabbage self-incompatibility factor ClassII of the present invention can be used to discriminate the genes S2, S5 and S15 of the cabbage self-incompatibility factor ClassII.

<110> Industry-academic cooperation foundation of sunchon national university <120> Primer set for inspecting the combination purity or detecting the          genotype of self-incompatibility Class II gene in Brassica          oleracea and the inspection or detection method by using same <130> PA-14-0214 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 21 <212> DNA <213> Class II-BoSRK-SNP-F1 <400> 1 cagttaaatg tctttggaaa t 21 <210> 2 <211> 20 <212> DNA <213> classII-BoSRK-SNP-R1 <400> 2 tcatcccgtc caaagatcct 20 <210> 3 <211> 20 <212> DNA <213> class II-BoSRK-F2 <400> 3 gaagctgaca cgaggaaggt 20 <210> 4 <211> 22 <212> DNA &Lt; 213 > class II-BoSRK-R2 <400> 4 cgttcatcgc atattctgga ga 22

Claims (4)

A primer set for combination purity assay of a forward primer having a nucleotide sequence of SEQ ID NO: 1 and a reverse primer having a nucleotide sequence of SEQ ID NO: 2 and a cabbage autolysing factor ClassII. A primer set for discriminating genotypes of the cabbage autolysing factor Class II consisting of a forward primer having the nucleotide sequence of SEQ ID NO: 3 and a reverse primer having the nucleotide sequence of SEQ ID NO: 4. Performing PCR (polymerase chain reaction) using a primer set for the combination purity assay of the cabbage autolysin factor Class II comprising the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2 according to claim 1; Determining the identity of the cabbage self-incompatibility factor Class II combination by HRM (high-resolution melting) analysis of the PCR amplification product; and determining the combination purity of the cabbage self-incompatibility factor Class II. Performing PCR using a primer set for discriminating the genotype of the cabbage autolysing factor Class II comprising the forward primer of SEQ ID NO: 3 and the reverse primer of SEQ ID NO: 4 according to claim 2; Determining the genotype identity of the cabbage self-incompatibility factor Class II by analyzing the PCR amplification product by HRM; and determining the genotype of the cabbage self-incompatibility factor Class II.
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