Method for identifying s haplotype in brassicaceae

Abstract

Disclosed is a combination in which, when at least one target gene present at the S locus is amplified by PCR, a tail sequence that does not hybridize to the nucleic acid sequence of the target gene and does not affect the amplification reaction is added to the 5' side of at least one primer so that the S haplotype can be identified based on the size difference of the amplified DNA fragment, and a size difference occurs in the amplified DNA fragment. The haplotype can be identified through a simple method, such as electrophoresis, due to a size difference occurring in the amplified DNA fragment. The present invention makes it possible to provide a method for easily identifying the S haplotype that controls self-incompatibility in plants.

Description

S haplotype discrimination method in the Brassicaceae family

[0001] The present invention relates to a method for identifying haplotypes of the S allele that controls self-incompatibility in Brassicaceae plants.

[0002] Some plants have a property called self-incompatibility, in which pollen from the same flower or the same individual is rejected and does not germinate, or the pollen tube does not grow, leading to fertilization and no seeds being produced. Self-incompatibility plays a role in increasing genetic diversity within a species, but there are also F breeding methods that utilize self-incompatibility. １ Seed collection has been used for breeding, and many varieties have been created.

[0003] The S gene, which is responsible for self-incompatibility in the Brassicaceae family, is located at the S locus of the chromosome, and three genes related to self-incompatibility, SLG, SRK, and SCR / SP11, are closely linked to each other and this set is called an S haplotype. It is known that there are many self-incompatible S haplotypes in the Brassicaceae family.

[0004] Brassicaceae plants have many species and even varieties. For example, the species names of Chinese cabbage, turnip, and komatsuna are Brassica rapa, while the species names of cabbage, broccoli, and kale are Brassica oleracea. As can be seen, within a single species, there are many varieties with extremely diverse morphological and other characteristics. Furthermore, various varieties have been artificially created for each variety. Identifying S haplotypes is important not only for genetic research on Brassicaceae plants, but also for cultivar improvement.

[0005] Many Brassicaceae varieties have been crossbred using self-incompatibility (F1 hybrids). １ ) varieties are available. １ Generally, varieties are highly productive because they exhibit hybrid vigor. １ Seed purity of the variety (F １It is rare for the purity of the plant to be 100%. Self-incompatibility is not always complete, and it is known that there are differences in its strength and stability depending on the S haplotype and the line developed. Also, when for some reason the pollen of the other parent line becomes difficult to pollinate, it can lead to self-pollination or cross-pollination within the same line, resulting in fruit set. However, with the intensification of agriculture, producers are increasingly seeking F with higher purity. １ Demand for seeds is on the rise. １ Low-purity seeds from low lots are F １ This is because it is not possible to obtain the superior characteristics expected from a variety uniformly. １ The evaluation of purity is important, and a method for easily testing the purity is desired.

[0006] Patent Literature 1 discloses a primer set and a method for distinguishing the genotype of the self-incompatibility gene in Chinese cabbage, a member of the Brassicaceae family. Patent Literature 1 also discloses that S haplotypes are distinguished based on the SRK genotype, and that multiplex primers have been designed to distinguish SRK-22, -25, -46, -54, and -55, enabling simultaneous identification. Non-Patent Literature 1 discloses the development of a PCR method for distinguishing specific S haplotypes S-7, -33, and -45 in cabbage. Furthermore, one of the currently widely used S haplotype identification methods is the PCR-RFLP method of the SLG gene (Non-Patent Literatures 2 and 3). The PCR-RFLP method involves digesting DNA fragments amplified by PCR with restriction enzymes and classifying S haplotypes based on the resulting fragment length patterns. Non-Patent Documents 4 and 5, both papers by the present inventors, disclose a method for identifying a total of 87 S haplotypes in 42 Brassica rapa and 45 Brassica oleracea by dot blotting, utilizing the fact that SCR / SP11 has the highest diversity among S haplotypes. Non-Patent Document 6, also by the present inventors, discloses a method for identifying S haplotypes in the SRK gene by a similar method.

[0007] Korean Patent No. 10-1413116

[0008] ChenW. et al., 2022, Plants, 11(10), 1372; https: / / doi.org / 10.3390 / plants11101372Nishio, T., et al., 1996, Theor Appl Genet,Vol.92, pp.388-394Nishio,T., et al., 1997, Theor Appl Genet, Vol.95, pp.335-342Oikawa, E. et al., 2010, Mol. Breeding, DOI10.1007 / s11032-010-9455-8Takuno etal., 2010, Theoretical Applied Genetics Vol.120, pp.1129-1138Haseyama, Y. et al., 2018, Mol Breeding, 38:116, https: / / doi.org / 10.1007 / s11032-018-0874-2

[0009] The methods described in Patent Document 1 and Non-Patent Document 1 are only capable of distinguishing between five or three S haplotypes, and identify only a very small number of S haplotypes in the Brassicaceae family, of which 50 to 100 haplotypes are said to exist per species. The methods described in Non-Patent Documents 2 and 3 are capable of distinguishing between many S haplotypes, but have the problem of requiring electrophoresis after cleavage with a restriction enzyme, which is time-consuming and labor-intensive, and requires the cost of purchasing the restriction enzyme. Furthermore, there are S haplotypes that do not have the SLG gene, and these cannot be distinguished. The methods described in Non-Patent Documents 4 to 6 are capable of distinguishing between many S haplotypes, but have the problem of requiring dot blotting, which is time-consuming. There has been a need for a method that can easily distinguish between many S haplotypes in a short period of time.

[0010] The present invention relates to the following identification methods and identification kits: (1) A method for identifying an S haplotype that controls self-incompatibility in a plant, comprising: performing a PCR reaction on a sample of genomic DNA contained in the plant using two or more pairs of primers that specifically hybridize to the nucleic acid sequence of at least one target gene present in the S locus; and identifying the S haplotype of the plant based on the size of the amplified DNA fragments obtained in the PCR reaction; the method is characterized in that at least one primer is provided with a tail sequence added to the 5' end thereof that does not hybridize to the nucleic acid sequence of the target gene and does not affect the amplification reaction, so that the amplified DNA fragments obtained in the PCR reaction can be distinguished based on their size difference. The SCR / SP11 gene exhibits high diversity between alleles, but many PCR products fall within the 100-200 bp range, making it difficult to easily distinguish by electrophoresis. The inventors have discovered that adding a tail sequence to the 5' end of a primer that does not hybridize to the nucleic acid sequence of the target gene and does not affect the amplification reaction makes it possible to easily identify S haplotypes.

[0011] (2) The method for distinguishing between S haplotypes according to (1), characterized in that the size difference is 8 bp or more and 80 bp or less. As a result of extensive research, the present inventors have found that a size difference of 8 bp or more makes it possible to distinguish between S haplotypes by microchip electrophoresis. By adding a tail so that a size difference of at least 20 bp or more occurs between S haplotypes, many S haplotypes can be distinguished by agarose electrophoresis. Furthermore, in designing PCR primers, it is not preferable to add a tail of more than 40 bases. Therefore, distinguishing between S haplotypes is possible if PCR primers are designed so that the size difference is 8 bp or more and 80 bp or less. Using this method, F １ The purity can be easily determined.

[0012] (3) Primers for distinguishing two or more S haplotypes, having 80% or more homology with the primers in Table 9, and at least one set of primers containing a tail portion whose difference from the GC content of the tail portion added to the primers described in Table 8-1 or Table 8-2 is within ±25%, and at least one set of primers containing 80% or more homology with the primers in Table 9, or at least one set of primers containing 80% or more homology with the primers in Table 12, and at least one set of primers containing a tail portion whose difference from the GC content of the tail portion added to the primers described in Table 11-1 or Table 11-2 is within ±25%, and at least one set of primers containing 80% or more homology with the primers in Table 12. A primer set for distinguishing S haplotypes of Brassica rapa or Brassica oleracea. The ability to distinguish S haplotypes, which have been difficult to distinguish easily until now, will be useful in genetic research, breeding, and F １ It can be used to determine purity.

[0013] (1) Examination of the length of the tail added to the primer. Examination on a 3% agarose gel. (2) Examination of the length of the tail added to the primer. Examination on a 3% agarose gel. (3) Examination of the length of the tail added to the primer. Examination on a 3% agarose gel. (4) Examination of multiple S haplotypes simultaneously. Examination on a 3% agarose gel. (5) Examination of the length of the tail added to the primer. Examination on a 3% agarose gel. (6) Examination of the length of the tail added to the primer. Examination on a 3% agarose gel. (7) Examination of the length of the tail added to the primer. Examination on a 3% agarose gel. (8) Examination of multiple S haplotypes simultaneously. Examination on a 3% agarose gel. (9) Examination of the results of examining multiple S haplotypes simultaneously. Examination on a 3% agarose gel. (10) Examination of the length of the tail added to the primer. Examination on a 3% agarose gel. (11) Examination of the length of the tail added to the primer. Examination on a 3% agarose gel. (12) Examination of the results of examining multiple S haplotypes simultaneously. Examination on a 3% agarose gel. (13) Examination of the results of examining multiple S haplotypes simultaneously. Examination on a 3% agarose gel. (14) Examination of the results of examining multiple S haplotypes simultaneously. Examination on a 3% agarose gel. (15) Examination of the results of examining multiple S haplotypes simultaneously. Examination on a 3% agarose gel. (16) Examination of the results of examining multiple S haplotypes simultaneously. Examination on a 3% agarose gel. (17) Examination of the results of examining multiple S haplo

[0014] Conventionally, methods have been used in which genes within an S haplotype are amplified by PCR, cleaved with restriction enzymes, and the cleavage pattern is analyzed (Non-Patent Documents 2 and 3), or SCR / SP11 or SRK genes within an S haplotype are amplified by PCR, hybridized with probes specific to each haplotype, and the S haplotypes are distinguished (Non-Patent Documents 4 to 6). However, a region highly diverse between SRK alleles is located in a region called the S-domain, which may have high homology with SLG, making it difficult to design PCR primers and probes specific to each haplotype.

[0015] The SCR / SP11 gene exhibits even greater diversity between alleles, and primers specific to each haplotype can be designed based on the known base sequence. In this case, the S haplotype can be identified and distinguished by confirming the presence or absence of PCR amplification with each specific primer. However, since many PCR products fall within the 100-200 bp range, for example, when two S haplotypes are present, two PCR reactions are required to distinguish them from each other, and the development of a simpler method has been desired.

[0016] The inventors designed PCR primers with unique tail sequences added to each S haplotype so as not to affect the PCR amplification reaction. This made it possible to distinguish PCR products by electrophoresis even between combinations that were previously difficult to distinguish, thereby identifying and distinguishing S haplotypes.

[0017] Although the following examples are described for Brassica rapa and Brassica oleracea, the S haplotype can be easily identified using a similar method in other Brassicaceae vegetable species, such as Raphanus sativus (radish). Furthermore, even when a new S haplotype is discovered, the S haplotype can be identified using a similar method.

[0018] The present invention will be described below with reference to examples. [Investigation of the Size Difference of PCR Products Required for Discrimination] First, assuming that discrimination will be performed by electrophoresis, we investigated the size difference required for PCR products to be distinguishable. As described above, when discriminating S haplotypes based on the known nucleotide sequence diversity between SCR / SP11 alleles, using the primer sequences published in Non-Patent Document 4, many PCR products fall within the 100-200 bp range. Therefore, we conducted an investigation using a 3% agarose gel, which has a high resolution of sizes of approximately 100-200 bp. First, for Brassica rapa, PCR products from Non-Patent Document 4 in which the DNA amplified from the sequence had no difference in length or showed differences of 1 bp, 10 bp, or 15 bp were selected and analyzed (Figure 1A).

[0019] In the figure, for example, S-38 / S-45 indicates that a sample containing S haplotypes 38 and 45 was subjected to PCR with primers amplifying S haplotypes 38 and 45. The numbers in parentheses below the combinations in Table 1 indicate the size of each PCR product amplified with primers without tails. In Figure 1, "-" indicates no tail, "+" indicates tail, and * indicates size marker. For example, in the case of S-38 / S-45, 15-base tails were added to the forward and reverse primers of the S-45 primer. In the range of 100 to 200 bp, it is difficult to distinguish between sequences with no difference in length (Figure 1A, top panel), or even with a 15-bp difference (Figure 1A, bottom panel). The primer sequences used are summarized in Table 1 below, and the sequence numbers and names listed in Table 1 are disclosed in Tables 8-1 to 8-9.

[0020] As shown in Figure 1A, even combinations that cannot be distinguished using primers without tails can be easily distinguished by adding a 15-base tail, i.e., a difference of 30 bp. This allows PCR to be performed at the same time using two sets of primers, and discrimination can be achieved with a single electrophoresis.

[0021]

[0022] A similar confirmation was performed for Brassica oleracea (Figure 1B). PCR products amplified with no difference in length or with a difference of 1 bp, 10 bp, or 15 bp were selected and confirmed on a 3% agarose gel. It was confirmed that even a 15 bp difference in the amplified PCR products was difficult to distinguish. In contrast, when a primer with a tail was used, i.e., when there was a size difference of 30 bp, distinction was possible. The primers used in Figure 1B are shown below.

[0023]

[0024] As shown above, when using a 3.0% agarose gel, S haplotypes can be distinguished with a size difference of approximately 30 bp. Agarose gels are often used at concentrations up to approximately 1.5%. Therefore, we investigated the size difference that can be distinguished using the commonly used 1.5% agarose gel (Figure 1C). When using a 1.5% agarose gel, a size difference of approximately 50-60 bp is sufficient for reliable discrimination. However, when the size difference is 40 bp or less, discrimination is sometimes possible, but it is difficult to say that reliable discrimination is possible. By adding a tail, discrimination is possible even with a 1.5% gel if the size difference is 50 bp or more. The primers used in Figure 1C are shown below.

[0025]

[0026] [Tail Length Study] Tail length was studied. The present inventors have already designed and published primers for amplifying the S haplotype (Non-Patent Documents 4 and 5). Utilizing the primers previously designed, the necessary length for adding a tail was investigated. In a study using a 3.0% agarose gel, a size difference of 30 bp was sufficient for discrimination. On the other hand, as shown in Figure 1C, it became clear that a size difference of approximately 30 bp can be difficult to discriminate using the widely used 1.5% agarose gel. Therefore, primers with a 30-base tail were designed, i.e., primers that increase the size difference of the amplified product by 60 bp compared to the original size. There were concerns that the longer tail portion would cause problems with amplification, but as shown in Figure 2A, amplification was successful without any problems. Note that Figure 2A shows the results of analysis using a 3% agarose gel. In Figure 2A, S haplotypes 8 and 46 were distinguished using the S-8 primer, and S haplotypes 12 and 56 were distinguished using the S-12 primer. A 30-base tail was added to each of the forward and reverse primers. It was shown that discrimination was possible if the size difference was 60 bp. The primers used were as follows:

[0027]

[0028] To confirm whether it is possible to distinguish between three S haplotypes with small size differences, we investigated whether discrimination is possible with a 15-base tail, i.e., a 30-bp size difference, and a 30-base tail, i.e., a 60-bp size difference. The forward and reverse primers of the S-46 and S-8 primers were designed to have 15-base and 30-base tails, respectively, increasing the size by 30 bp or 60 bp compared to the original. Furthermore, the forward and reverse primers of the S-25 and S-12 primers were designed to have 15-base and 30-base tails, respectively, increasing the size by 30 bp or 60 bp compared to the original. The primers used to distinguish between S-8 and S-12 were the same as those listed in Table 4. As shown in Figure 2B, size differences of 30 bp and 60 bp enable easy discrimination. The primers used are as follows:

[0029]

[0030] As shown in Figure 2B, using primers with tails allows for simultaneous analysis of three or more S haplotypes, so we investigated whether more S haplotypes can be analyzed simultaneously. As shown in Figure 2C, by selecting the right primers, it is also possible to simultaneously analyze four S haplotypes. Depending on the S haplotype to be analyzed, it is possible to identify more S haplotypes at once by adjusting the length of the tail and the selection of primers. The primers used are as follows:

[0031]

[0032] These results indicate that a size difference of 30 bp to 60 bp allows for easy discrimination by agarose electrophoresis. Here, a tail of 15 or 30 bases in length was added to each forward and reverse primer, but discrimination is possible with any combination that results in a size difference of 30 bp to 60 bp. Furthermore, in designing PCR primers, it is not preferable to add a tail of more than 40 bases. Therefore, primers should be designed so that the size difference is 80 bp or less, preferably 60 bp or less.

[0033] [Haplotype Identification by Microchip Electrophoresis] Using microchip electrophoresis, which can distinguish even small size differences and trace amounts of sample, we investigated whether it is possible to distinguish S haplotypes with even smaller size differences (Figure 3). To determine whether the 8-bp size difference that could not be distinguished by agarose electrophoresis could be detected, we added a 4-base tail to each forward and reverse primer and performed analysis. Two peaks were observed, demonstrating that a size difference of 8 bp or more is possible. The primers used are as follows:

[0034]

[0035] From the above considerations, when using microchip electrophoresis, a size difference of 8 bp or more is sufficient for discrimination, and a size difference of 10 bp or more is preferable. When discriminating haplotypes simply by electrophoresis without using special equipment such as microchip electrophoresis, a size difference of more than 40 bp is required when using a 1.5% agarose gel, but when using a 3.0% agarose gel, it is advisable to add a tail to the PCR primer so that the size difference is 20 bp or more, more preferably 30 bp or more (see Figure 1C, lower table). Furthermore, electrophoresis using acrylamide gel, which is suitable for separating small DNA fragments in addition to agarose gel, also allows for sufficient discrimination.

[0036] Here, it is assumed that the discrimination is performed by electrophoresis, such as agarose electrophoresis, but any method that can discriminate the size of the amplified DNA may be used for discrimination. For example, after amplification using an intercalator such as SYBR Green, the two amplified PCR products can be analyzed by dissociation curve analysis.

[0037] Furthermore, the tail sequence to be added does not necessarily have to be identical to that disclosed herein. Any sequence may be used as long as it does not inhibit the PCR reaction and causes a size difference. Since it is believed that problems will not occur in the PCR reaction if the GC content of the tail sequence to be added is within a certain range, it is sufficient to use a tail sequence with a similar GC content. Specifically, even if the GC content differs by within 25%, preferably within 20%, and more preferably within 10% from the GC content of the tail sequence shown below, the PCR reaction can be carried out without problems. Depending on the sequence of the portion other than the tail, for example, if 8 of the 15-base tail are G or C (GC content 53%), 11 bases (GC content 73%, within a range of 20% or less of the GC content of the original sequence) or 5 bases (GC content 33%, within a range of 20% or less of the GC content of the original sequence) can be changed to a tail sequence with G or C. Furthermore, it is not necessary to add tails of equal length to the forward and reverse primers as long as a distinguishable size difference can be generated.

[0038] Furthermore, the primer sequences of the portions other than the tail do not necessarily have to be identical. The regions shown here are selected as regions specific to the S haplotype, so it is possible to shift the position by several bases. For example, the positions may be shifted by approximately 3 bases toward the 5' or 3' side. Furthermore, although the primer sets shown below are each designed by selecting a sequence of 25 bases, it is also possible to design primers with longer sequences. Therefore, the sequences of the portions other than the tail may be 80% or more, preferably 85% or more, and more preferably 90% or more homologous. That is, a primer sequence that does not have a tail added can be a sequence that is 80% or more homologous to Table 9 (Brassica rapa) or Table 12 (Brassica oleracea), and a primer sequence that has a tail added can be a sequence that is 80% or more homologous to Table 9 or Table 12, and a tail sequence with a GC content within ±25% of the GC content of the tail portion sequences in Tables 8-1 and 8-2, or Tables 11-1 and 11-2 can be designed and used.

[0039] Tables 8-1 and 8-2 show the PCR primer sequences for Brassica rapa with a tail added, Table 9 shows the PCR primer sequences for Brassica rapa without a tail added, Tables 11-1 and 11-2 show the PCR primer sequences for Brassica oleracea with a tail added, and Table 12 shows the PCR primer sequences for Brassica oleracea without a tail added. Furthermore, Tables 10-1 to 10-13 show the S haplotypes of Brassica rapa that can be identified using Tables 8-1, 8-2, and 9, and Tables 13-1 to 13-10 show the combinations of S haplotypes of Brassica oleracea that can be identified using Tables 11-1, 11-2, and 12. In the sequence listings shown below, "Br" stands for B. In rapa, "Bo" indicates the species Brassica oleracea, "SCR" indicates that it is a primer created in the SCR / SP11 gene region, the number indicates the haplotype to be identified, "F" and "R" indicate forward and reverse primers, respectively, and the number before "bp" indicates the number of base pairs increased by the added tail.

[0040]

[0041]

[0042]

[0043]

[0044]

[0045]

[0046] For example, in the case of Brassica rapa, the S haplotype of which is unknown, the primers in Tables 8-1, 8-2, and 9 are used for PCR amplification, and in the case of Brassica oleracea, the primers in Tables 11-1, 11-2, and 12 are used for PCR amplification to identify the S haplotype. １ To check the purity, a combination of primers that amplify the S haplotype to be distinguished from the S haplotype of the parent strain can be selected and used for analysis. As shown in Tables 10-1 to 10-13 and 13-1 to 13-10, a large number of S haplotype combinations can currently be distinguished.

[0047] Currently, a wide variety of Brassicaceae plants are cultivated as vegetables. For example, the primer sets used to distinguish the genotypes of commonly available Brassicaceae B. oleracea vegetables, such as broccoli (var. italica), cabbage (var. capitata), cauliflower (var. botrytis), and B. rapa varieties such as Chinese cabbage (var. pekinensis), turnip (var. rapifera or rapa), komatsuna (var. perviridis), and bok choy (var. chinensis), are shown below. The primer sets shown in the table below are used to identify the S genotype of each variety and to identify the F genotype. １ This is useful as a primer set for checking purity.

[0048] Table 14 shows the results for broccoli. １ This paper summarizes the S haplotype information used in varieties and the genotypes predicted from it. The S genotypes 2b / 13, 2b / 28, 2b / 39, 2b / 18, 2b / 15, 15 / 18, 15 / 39, 15 / 64, 13 / 15, 13 / 18, and 13 / 39 are genotypes that have actually been confirmed in varieties.

[0049]

[0050] In Table 15, for cabbage, F １ This article summarizes the S haplotype information used in varieties and the genotypes predicted from it. The following S genotypes have actually been confirmed in varieties: 1 / 2b, 2b / 5, 2b / 6, 2b / 7, 2b / 8, 2b / 14, 2b / 15, 2b / 18, 2b / 28, 2b / 33, 2b / 45, 2b / 51, 2b / 68, 5 / 15, 5 / 18, 5 / 45, 6 / 14, 6 / 15, 6 / 28, 6 / 33, 6 / 45, 6 / 51, 7 / 14, 7 / 15, 7 / 28, 7 / 68, 14 / 15, 15 / 45, 15 / 68, 18 / 33, 28 / 45, 33 / 45, 51 / 57, and 51 / 68.

[0051]

[0052] In Table 16, for cauliflower, F １ This paper summarizes the S haplotype information used in varieties and the genotypes predicted from it. The S genotypes 2b / 12, 2b / 15, and 15 / 45 are genotypes that have actually been confirmed in varieties.

[0053]

[0054] In Table 17, for Chinese cabbage, F １ This paper summarizes the S haplotype information used in varieties and the genotypes predicted from it. The S genotypes 12 / 22, 22 / 60, 25 / 40, 25 / 44, 25 / 46, 25 / 54, 25 / 60, 40 / 46, 40 / 54, 40 / 60, 40 / 99, 46 / 54, 46 / 60, 54 / 60, 54 / 99, and 60 / 99 are genotypes that have actually been confirmed in varieties.

[0055]

[0056] In Table 18, for turnips, F １ This article summarizes the S haplotype information used in varieties and the genotypes predicted from it. The S genotypes 8 / 25, 8 / 41, 8 / 44, 8 / 56, 8 / 60, 22 / 40, 22 / 44, 22 / 47, 25 / 40, 25 / 44, 25 / 54, 25 / 60, 27 / 52, 27 / 60, 29 / 44, 29 / 53, 29 / 56, 40 / 47, 40 / 53, 44 / 53, 44 / 56, 53 / 56, and 54 / 60 are genotypes that have actually been confirmed in varieties.

[0057]

[0058] Table 19 shows the results of F １This article summarizes the S haplotype information used in varieties and the genotypes predicted from it. The S genotypes 8 / 60, 22 / 29, 22 / 44, 22 / 60, 25 / 29, 25 / 45, 25 / 53, 25 / 54, 25 / 60, 26 / 41, 27 / 41, 27 / 45, 27 / 56, 29 / 53, 41 / 45, 41 / 60, 44 / 45, 44 / 53, 45 / 56, 45 / 60, 53 / 60, and 56 / 60 are genotypes that have actually been confirmed in varieties.

[0059]

[0060] Table 20 shows the results of F for bok choy. １ This paper summarizes the S haplotype information used in varieties and the genotypes predicted from it. The S genotypes 8 / 46, 9 / 44, 9 / 60, 12 / 44, 12 / 60, 22 / 44, 22 / 46, 22 / 60, 25 / 27, 25 / 55, 25 / 60, 27 / 55, 29 / 60, 34 / 60, 44 / 46, 44 / 60, and 46 / 60 are genotypes that have actually been confirmed in varieties.

[0061]

[0062] To distinguish between these varieties, use the table of combinations above. １ Purity testing can also be easily performed using the above primers. Specifically, the primer set in Table 14 can be used to identify the genotype of broccoli, the primer set in Table 15 to identify the genotype of cabbage, the primer set in Table 16 to identify the genotype of cauliflower, the primer set in Table 17 to identify the genotype of Chinese cabbage, the primer set in Table 18 to identify the genotype of turnip, the primer set in Table 19 to identify the genotype of Komatsuna, and the primer set in Table 20 to identify the genotype of Bok Choy. Genotypes can be easily identified by using the primer set in Table 14, the primer set in Table 15 to identify the genotype of cabbage, the primer set in Table 16 to identify the genotype of cauliflower, the primer set in Table 17 to identify the genotype of Chinese cabbage, the primer set in Table 18 to identify the genotype of turnip, the primer set in Table 19 to identify the genotype of Komatsuna, and the primer set in Table 20 to identify the genotype of Bok Choy.１ A kit for examining purity may be a set of all primers listed in the table for each variety, which can be used to identify the genotype of each variety, or a kit containing primers for identifying genotypes actually confirmed in the variety. Furthermore, a kit may be prepared containing a set of primers for identifying multiple S haplotypes, for example, two, preferably three, and more preferably 17 S haplotypes, for each variety.

Claims

1. A method for identifying the S haplotype that controls self-incompatibility in a plant, comprising: performing a PCR reaction using two or more sets of primers that specifically hybridize to the nucleic acid sequence of at least one target gene present in the S locus, with genomic DNA contained in the plant as a sample; and identifying the S haplotype of the plant from the size of the amplified DNA fragments obtained in the PCR reaction, wherein a tail sequence that does not hybridize to the nucleic acid sequence of the target gene and does not affect the amplification reaction is added to the 5' side of at least one or more primers so that the amplified DNA fragments obtained in the PCR reaction can be identified based on their size difference.

2. The method of claim 1, wherein the plant is a member of the Brassicaceae family.

3. The method according to claim 2, wherein the Brassicaceae plant is Brassica rapa, Brassica oleracea, or Raphanus sativus.

4. The method of any one of claims 1 to 3, characterized in that the size difference is 8 bp or more and 80 bp or less.

5. The method of claim 4, wherein, when the Brassicaceae plant is Brassica rapa, the sequence portion of Brassica rapa is such that the primer has 80% or more homology with the primer in Table 9, and the difference in GC content of the tail portion is within ±25% of the GC content of the tail portion in Tables 8-1 and 8-2; and when the plant is Brassica oleracea, the sequence portion of Brassica oleracea is such that the primer has 80% or more homology with the primer in Table 12, and the difference in GC content of the tail portion is within ±25% of the GC content of the tail portion in Tables 11-1 and 11-2.

6. F characterized by using the method of claim 5 １ Varietal purity testing methods.

7. A primer set for discriminating S haplotypes of Brassica rapa or Brassica oleracea, comprising primers for discriminating between two or more S haplotypes, the primer set comprising: at least one primer set having 80% or more homology with the primers in Table 9 and containing tail portions whose GC content differs by ±25% or less from the GC content of the tail portions added to the primers in Table 8-1 or Table 8-2; and at least one primer set having 80% or more homology with the primers in Table 9; or at least one primer set having 80% or more homology with the primers in Table 12 and containing tail portions whose GC content differs by ±25% or less from the GC content of the tail portions added to the primers in Table 11-1 or Table 11-2; and at least one primer set having 80% or more homology with the primers in Table 12.

8. A kit for identifying the S haplotype of Brassica rapa or Brassica oleracea, comprising the primer set of claim 7 and reagents necessary for PCR amplification.

9. A primer set for identifying the S haplotype of Brassica rapa or Brassica oleracea, comprising the primers in Table 8-1, Table 8-2, and Table 9, or Table 11-1, Table 11-2, and Table 12.

10. A primer set for identifying an S haplotype of Table 14 selected from the primer sets of claim 9 for identifying an S haplotype of broccoli.

11. A primer set for identifying an S haplotype in Table 15, selected from the primer sets of claim 9, for identifying an S haplotype in cabbage.

12. A primer set for identifying an S haplotype of Table 16 selected from the primer sets of claim 9 for identifying an S haplotype of cauliflower.

13. A primer set for identifying an S haplotype of Table 17 selected from the primer sets of claim 9 for identifying the S haplotype of Chinese cabbage.

14. A primer set for identifying an S haplotype of Table 18 selected from the primer sets of claim 9 for identifying the S haplotype of turnip.

15. A primer set for identifying an S haplotype of Table 19 selected from the primer sets of claim 9 for identifying the S haplotype of Komatsuna.

16. A primer set for identifying an S haplotype of Table 20 selected from the primer sets of claim 9 for identifying the S haplotype of bok choy.

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