KR101165586B1 - Primer sets of detecting cytoplasmic male sterility genotype in brassicaceae family and detection method of cytoplasmic male sterility genotype using them - Google Patents

Abstract

The present invention provides a primer set for screening cytoplasmic male sterility genotype of cabbage crops, a kit for screening cytoplasmic male sterility genotype of cabbage crops comprising the primer set and a cytoplasmic male sterility genotype of cabbage crops using the primer set. It relates to a method for screening.

Chinese cabbage, Cytoplasmic male sterility gene, CMS

Description

Primer sets of detecting cytoplasmic male sterility genotype in brassicaceae family and detection method of cytoplasmic male sterility genotype using them}

The present invention provides a primer set for screening cytoplasmic male sterility genotype of cabbage crops, a kit for screening cytoplasmic male sterility genotype of cabbage crops comprising the primer set and a cytoplasmic male sterility genotype of cabbage crops using the primer set. It relates to a method for screening.

Male sterility caused by cytoplasmic factors is called 'cytoplasmic male sterility' (CMS '), which is a cytoplasmic factor that mitochondria do not perform their normal functions and produce abnormal pollen, which does not have the ability to self-fertilize. I can't speak. CMS is a maternal inheritance, and any fertility crosses 100% infertility, so male sterility is very easy to maintain, and it is very convenient to apply to crops using nutrients such as leaves and stems.

For this reason, attempts have been made to use CMS for hybrid seed production. To this end, molecular research on the CMS of many plants has been conducted. As a result, many cellular factors causing CMS have been found. Currently, in the Brassica family crops, CMS has a CMS and is known in Brassica sp.Polima, Ogura, Napus, Anand, and Raphanus. sp.) Ogura and Kosena are known. In the case of the first type cabbage, Williams announced the Ogura CMS, which transferred radish's Ogura CMS to cabbage through various routes (Yoon et al. 1981). Okawa (Ohkawa) (1984, 1985) is to obtain a cytoplasmic male sterility of Chinese cabbage in the later hybridization of the New Zealand wild turnip (B. campestris ssp. Rapifera) and Japanese cabbage (B. campestris). Earl et al. Of Cornell University developed the Ogura CMS strain of broccoli, which does not show yellowing at low temperatures due to cell fusion (1997). The CMS strain was grown (1998). Vetmy (2000) cultivated five varieties of Chinese cabbage in rapeseed polyma CMS to cultivate the polyma CMS strains of cabbage. Hirat (2001) of Japan combined cabbage rubies with cabbage rubies on board and obtained chimeric plants, which later discovered cellular male infertility after hybridizing litters.

Here, the correct determination of the male fertility (N-) cytoplasm and the male infertility (S-) cytoplasm of plants, including edible crops, is very important for the breeding system. Purity and cytoplasmic genotype of hybrid seeds are typically measured by GOT (grow-out test). The GOT method is based on evaluating morphological and floral features (different hybrids) from well-developed representative plant specimens. For example, cabbage plants require several months to develop, and seeds must be stored under suitable conditions because the seeds cannot be marketed until they have been made available. Moreover, substantial retardation occurs in the first growing season after the production of hybrid seeds absorbed by GOT, or is the preferred time for hybrid culture. In this case, the seed must be stored for at least one year before it is marketed, for example until the next developmental time. Therefore, hybrid seeds are stored in stored form for extended periods to wait for GOT results through the vendor. A disadvantage of other GOT methods is that they can be altered by environmental influences in the expression of morphological properties. Furthermore, adverse climatic conditions (such as strong winds or rain, high temperatures, droughts) can destroy or adversely affect crops and make it difficult to collect data. To solve this problem, techniques using sequences associated with CMS, such as DNA markers, have been readily developed to identify cytoplasmic male infertility.

Therefore, the present invention was intended to develop a method for easily identifying a specific CMS, and as a result, the present invention was completed.

One object of the present invention is to provide a primer set for screening cytoplasmic male sterility genotype of the Brassica family crops.

Another object of the present invention is a primer set; And it provides a kit for screening the cytoplasmic male sterility genotype of the Brassica family crops containing a reagent for performing the amplification reaction.

Another object of the present invention is to provide a method for screening cytoplasmic male sterility genotype of the Brassica family crops using the primer set.

In one aspect, the present invention provides a primer set for screening cytoplasmic male sterility genotype of the Brassica family crops.

In the present invention, the Brassicaceae family crops include Chinese cabbage, turnips, cabbage, mustard, mustard, rapeseed, broccoli, and the like, preferably cabbage, turnips and cabbage, and more preferably cabbage.

In the present invention, the cytoplasmic male sterility (CMS) gene refers to a gene that generates a male infertility mutation in the cytoplasmic component of the genome. The cytoplasmic male infertility gene is maternally inherited and is known to be due to mutations in mitochondrial DNA. In the present invention, the cytoplasmic male infertility gene preferably includes a gene whose function has been characterized by the present inventors. In the present invention, the gene whose function has been identified by the present inventors is designated as SNU-3 cytoplasmic male infertility gene or SNU-3 CMS gene, which is represented by the nucleotide sequence of SEQ ID NO: 1.

As used herein, the term "primer set" refers to a short nucleic acid sequence that can form a base pair with a complementary template and that serves as a starting point for template strand copying. The primer set provided in the present invention is a forward and reverse primer designed to amplify the SNU-3 cytoplasmic male sterility gene in the Brassica family crop. In a specific embodiment of the present invention, primer sets having nucleic acid sequences of SEQ ID NOs: 2 and 3 were used. The primer of SEQ ID NO: 2 is represented by BrmatK-F for convenience, and the primer of SEQ ID NO: 3 is represented by BrmatK-R for convenience.

The primer set of the present invention is designed for screening cytoplasmic male infertility genotype in cabbage crops. In one specific implementation, DNA extraction from cabbage crops using the primer sets of SEQ ID NOs: 2 and 3 confirmed that amplification of only the SNU-3 CMS whose function was newly identified by the present inventors. Thus, the primer set of the present invention is useful for screening cytoplasmic male infertility genotype, especially SNU-3 CMS, in cabbage crops.

In another aspect, the invention provides a primer set for screening cytoplasmic male sterility genotype of the Brassica family crops; And it provides a kit for screening the cytoplasmic male sterility genotype of the Brassica family crops comprising a reagent for performing the amplification reaction.

In the kit of the present invention, the primer set is as described above, the reagent for performing the amplification reaction may include DNA polymerase, dNTPs, buffers and the like. In addition, the kit of the present invention may further include a user manual describing the conditions for performing the optimal reaction.

In another aspect, the present invention provides a method for producing genomic DNA, comprising the steps of: separating genomic DNA from a sample of a Brassica family crop; Amplifying a target sequence by using the isolated genomic DNA as a template and performing an amplification reaction using the primer set according to the present invention; And it provides a method for screening the cytoplasmic male sterility genotype of Chinese cabbage crop comprising the step of detecting the amplification product.

The method of the present invention comprises the step of separating genomic DNA from a sample of the Brassica family crop. The sample may be any part of the cruciferous crop, for example roots, stems, leaves, flowers and the like. In addition, the method for separating genomic DNA from the sample may be based on genomic DNA known in the art as a template, and amplifying a target sequence by performing an amplification reaction using a primer set according to an embodiment of the present invention. Methods for amplifying target nucleic acids include polymerase chain reaction (PCR), ligase chain reaction, nucleic acid sequence-based amplificatoins, transcription-based amplification systems, There is any other suitable method other than amplification via strand displacement amplification or Qβ replication or amplification of nucleic acid molecules known in the art. Among them, PCR is a method of amplifying a target nucleic acid from a primer pair that specifically binds to the target nucleic acid using a polymerase. Such a PCR method may take conventional conditions. As an example, initial denaturation is performed at 94 ° C. for 3 minutes, followed by denaturation (30 seconds at 94 ° C.), and annealing at 53 ° C. 30 seconds), extension (60 seconds at 72 ° C.) a total of 40 times, and finally the extension (5 minutes at 72 ° C.). In addition, such PCR methods may use commercially available kits.

In the method of the present invention, the amplified target sequence may be labeled with a detectable labeling substance. As one specific example, the labeling material may be a material that emits fluorescence, phosphorescence, or radiation, but is not limited thereto. Preferably the labeling substance is Cy-5 or Cy-3. When amplifying the target sequence, PCR is performed by labeling Cy-5 or Cy-3 at the 5′-end of the primer to allow the target sequence to be labeled with a detectable fluorescent labeling substance. In addition, the label using a radioactive substance may be added to the PCT reaction solution by adding a radioisotope such as ³² P or ³⁵ S to the PCT reaction solution when PCR is performed, and the radiation product may be incorporated into the amplification product and the amplification product may be labeled with radiation. . The primer set used to amplify the target sequence is as described above.

The method of the present invention includes detecting the amplification product. Detection of the amplification product may be carried out through DNA chip, gel electrophoresis, radiation measurement, fluorescence measurement or phosphorescence measurement, but is not limited thereto. As one of the methods for detecting amplification products, gel electrophoresis can be performed. Gel electrophoresis may use agarose gel electrophoresis or acrylamide gel electrophoresis depending on the size of the amplification product. In addition, in the fluorescence measurement method, PCR is performed by labeling Cy-5 or Cy-3 at the 5′-end of a primer, and a target sequence is labeled with a detectable fluorescent labeling material, and the labeled fluorescence is measured using a fluorimeter. can do. In addition, the radiation measuring method is to add a radioisotope, such as ³² P or ³⁵ S to the PCR reaction solution to label the amplification product when performing the PCR, and then a radiation measuring device, such as a Geiger counter or liquid Radiation can be measured using a liquid scintillation counter.

By the method of the present invention, the presence of amplified fragments indicates that the crop is SNU-3 cytoplasmic male infertility, and the absence of amplified fragments indicates that the crop is SNU-3 cytoplasmic male fertility.

By using a primer set for screening cytoplasmic male infertility genes of the Brassica family crop of the present invention, a kit comprising the primer set and a screening method using the primer set, cytoplasmic male infertility genes, particularly in cabbage crops The present inventors can reduce the breeding cost and increase the efficiency of hybrid seed cultivation according to the presence or absence of the newly identified SNU-3 CMS, it is possible to lower the cost of breeding.

Hereinafter, the present invention will be described in more detail with reference to the following Examples and Experimental Examples. However, the following Examples and Experimental Examples are only examples of the present invention, and the scope of the present invention is not limited thereto.

Example 1 CMS Line Analysis of SNU-3 CMS

1-1. SNU-3 CMS Screening

In order to find a specific part of the chloroplast matK gene of Chinese cabbage and crops, NCBI was searched for a previously published crop-specific matK gene. Entrez program was used to collect the entire sequence of the gene. As a search method, matK was selected as a keyword. Among the collected matK genes, the base sequences of Chinese cabbage, such as Chinese cabbage, were selected, and the polymorphisms of the base sequences were found through the BLAST program, and the characteristics of these sequences were studied. Representative nucleotide sequence of the nucleotide sequence is represented by SEQ ID NO: 1, it was named SNU-3 CMS.

1-2. DNA Extraction and PCR Conditions

To confirm the PCR amplification and type of male GF1, MF2, SNU-2-, SNU-1-, SNU-3- and Saco CMS, total DNA was extracted from the leaf tissue of Chinese cabbage by CTAB method. PCR reaction was performed under the following conditions. PCR reaction was performed with Perkin-elmer PE-9700, pre-denaturation at 94 ° C for 5 minutes, denaturation at 94 ° C for 1 minute, annealing at 55 ° C for 1 minute, and extension at 72 ° C for 30 minutes for 1 minute. Reacted. Post-extension was performed at 72 ° C. for 7 minutes. PCR amplification reaction solution was prepared with 2.5 μl 10x reaction buffer (500 mM KCl, 100 mM Tris-HCl, PH 8.3, 15 mM MgCl ₂ ), 2.5 μl 2.5 mM dNTP, 2 μl Genomic DNA 50 ng, 100 μm Primer 0.1 μl, 0.5 unit Taq polymerase 0.25 μl (TaKaRa , Japan) and sterile water using Total Vol. It was made up to 25 μl. The reaction solution was separated by 1.0% agarose gel electrophoresis and confirmed on a UV transilluminator.

1-3. Identification of SNU-3 CMS Type Using Ogura Type CMS Specific Marker

By the present inventors SNU-3 CMS is Sako (Saco), but the CMS introduced ability by breeding with other lines, such as CMS, SNU-3 CMS is little more chlorosis at low temperature than Sako CMS hybrids F ₁ It was found that the strength was better and markedly better as CMS. Thus, the present inventors performed PCR using a primer for selection of the Ogura CMS cabbage strain to determine whether the SNU-3 CMS cabbage strain is the same strain as the conventionally known CMS cabbage strain or a new CMS strain.

Specifically, PCR was performed using the orf 138 primer, which is a specific marker of Ogura type CMS, in the same manner as in Example 2-1. At this time, the same cabbage strain as in Example 2-1 was used as a control.

The results are shown in FIG. As shown in FIG. 1, PCR products (350 bp) were amplified in SNU-3 CMS and Saco CMS by performing PCR using orf 138 primer, which is a specific marker of Ogura type CMS. Therefore, these results confirm that SNU-3 CMS and SACO CMS are Ogura type CMS.

Example 2: Development of Markers Specific to SNU-3 CMS

In order to develop a marker specific to the SNU-3 CMS of Example 1, the base sequence of the matK gene collected in Example 1-1 was selected from Chinese cabbage, which is a cabbage plant, and a portion showing polymorphism of the nucleotide sequence was BLAST. After the program was found, primers were prepared around the part. The prepared primers are shown in SEQ ID NOs: 2 and 3 as BrmatK-F and BrmatK-R primers.

As a result of PCR of the DNA extracted from the leaf tissue of the Chinese cabbage using the prepared primer, only the PCR product of 891bp was amplified (Fig. 2). The base sequence of these amplified PCR products was analyzed by Solgent Co., Ltd. (Korea). As a result, the PCR product of Example 2 was confirmed to be the same as the nucleotide sequence of SEQ ID NO: 1. Therefore, it was confirmed that Ogura type CMS is distinguished from SNU-3 CMS, and the BrmatK-F (SEQ ID NO: 2) and BrmatK-R (SEQ ID NO: 3) primers may be useful as SNU-3 CMS specific markers.

1 is a diagram showing the Ogura type CMS specific band amplified by PCR product (350bp) in SNU-3 and Saco CMS using orf138 synthesized in the mitochondrial region (M: 100bp DNA ladder, 1: MF1, 2: SNU-2 CMS, 3: SNU-1 CMS, 4: SNU-3 CMS, 5: Saco CMS, 6: MF2).

Figure 2 shows the results of electrophoresis using primer sets (BrmatK-F / BrmatK-R) of SEQ ID NOS: 2 and 3 synthesized from chloroplast DNA Figure shows the SNU-3 CMS specific bands (M: 100bp DNA ladder, 1: MF1, 2: SNU-2 CMS, 3: SNU-1 CMS, 4: SNU-3 CMS CMS, 5: Saco CMS, 6) : MF2).

<110> INDUSTRY ACADEMY COOPERATION CORPS OF SUNCHON NATIONAL UNIVERSITY <120> Primer sets of detecting cytoplasmic male sterility genotype in          brassicaceae family and detection method of komatsuna cytoplasmic          male sterility genotype using them <130> PA090105 <160> 3 <170> Kopatentin 1.71 <210> 1 <211> 891 <212> DNA <213> Brassica rapa <220> <221> gene (222) (1) .. (891) <223> SNU-3 CMS <400> 1 ctcagtaatc ttagtcttac tgcttactgt atgaacattt aataatagaa ataaatgact 60 tttgataata caaaataatt aatttttttg ttatctccgc attccgttta cgttctgata 120 aattttgatt taatttatgg agcctcagaa ccccattatt catgattgac caaatcatta 180 agataaagaa tatccaaata ccaaacccgc actcgatata atcttttaga agcataatca 240 cttcttggga agattaaaga aagaacttgg tcttcccccg taaggaattc ttctaataaa 300 cccgagccca acctttttaa aaaagcgcgt acagtacttt tgtgtttacg agccaaagtt 360 ttaacacaac aaagacgaag tatatatttt attcgataca aattcttttt gtttgaagat 420 ccgctgtaat aatgtgaaat atttctggat atacgcacaa atcggttgag aatatcagaa 480 tctgatgaat ccgtccaggt cgctttacta atgggatgcc ctaatacatt acaaaattta 540 tctttagcca acgacccaat aatagaaaaa attggaatgt tgctatccaa ttttattcta 600 acattatcta ttagaaatga gttttctagc atttgactac gtaccactaa aggatttagt 660 cgcaaacttg agagataacc cagaaattct aaattatctt tagataattg atttatatta 720 accttttgcg attgaaacca tacggaaaaa taacattgcc ataaattaac aaaataaaat 780 ttccatttat tcatcagaag tggcgtatcc tttgttgcca gaatgtattt tccatgatat 840 ctaacataat gtaggaaagg atccttgaac aaccctaaaa gcgccggaaa t 891 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of SNU-3 CMS <400> 2 ctcagtaatc ttagtcttac tg 22 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of SNU-3 CMS <400> 3 atttccggcg cttttagggt tgttc 25  

Claims (10)

A primer set for screening the SNU-3 CMS genotype in a Brassicaceae family crop comprising a nucleotide set of SEQ ID NO: 2 and SEQ ID NO: 3. The primer set according to claim 1, wherein the cabbage crop is any one selected from the group consisting of cabbage, turnip, cabbage, mustard, mustard, rapeseed, and broccoli. delete A primer set of claim 1; And a kit for screening SNU-3 CMS genotypes in the Brassicaceae family crops comprising reagents for performing amplification reactions. The kit according to claim 4, wherein the cabbage crop is any one selected from the group consisting of cabbage, turnip, cabbage, mustard, mustard, rapeseed and broccoli. delete Separating genomic DNA from a sample of the Brassica family crops; Amplifying a target sequence by using the isolated genomic DNA as a template and performing an amplification reaction using the primer set according to claim 1; And A method for screening SNU-3 CMS genotypes in Chinese cabbage crops comprising the step of detecting the amplified products. 8. The method of claim 7, wherein the amplified target sequence is labeled with a substance that exhibits fluorescence, phosphorescence, or radiation. 8. The method of claim 7, wherein the cabbage crop is any one from the group consisting of cabbage, turnip, cabbage, mustard, mustard, rapeseed and broccoli. delete

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