Sugar beet yellows virus disease resistance markers and their applications

Abstract

A technique for selecting sugar beet plants that are resistant to chlorosis virus disease is provided. [Solution] A method for determining whether or not a sugar beet plant has resistance to chlorosis virus disease includes a step of determining whether or not the sugar beet plant has resistance by identifying at least one of the gene loci involved in resistance to chlorosis virus disease located between 11.0 Mbp and 30.0 Mbp on chromosome 4 and between 8.0 Mbp and 38.0 Mbp on chromosome 7 in the sugar beet plant.

Description

[Technical Field]

[0001] The present invention relates to a sugar beet yellows virus disease resistance marker and its use. [Background technology]

[0002] Several pathogenic viruses have been reported worldwide to cause sugar beet yellows, a virus disease that infects and causes yellowing symptoms. These viruses are collectively known as virus yellows (VY). The main pathogenic viruses causing yellows virus diseases reported to date include Beet yellows virus (BYV, Japanese name: Beet yellows virus), Beet western yellows virus (BWYV, Japanese name: Beet western yellows virus), Beet mild yellowing virus (BMYV), Beet chlorosis virus (BChV), and Beet leaf yellowing virus (BLYV, Japanese name: Beet yellow leaf virus). In Japan, the diseases caused by BYV, BWYV, and BLYV are called beet yellows, western beet yellows, and beet yellowing, respectively. All of these diseases are known to be transmitted by the green peach aphid (GPA, English name: green peach aphid, scientific name: Myzus persicae).

[0003] Traditionally, highly effective neonicotinoid insecticides have been used to suppress the occurrence of green peach aphids, which transmit sugar beet yellows virus disease, in sugar beets. However, neonicotinoid insecticides have been banned worldwide due to their significant impact on other organisms. Therefore, one of the goals is to develop pesticides that can replace neonicotinoid insecticides. In addition, sugar beet yellows virus disease has repeatedly occurred in Hokkaido every 10 to 20 years since the 1950s, and when it does occur, it is difficult to control with insecticides. Therefore, the importance of planting varieties resistant to sugar beet yellows virus disease has been highlighted.

[0004] Non-Patent Document 1 describes the discovery that BMYV resistance can be introduced into cultivated species by backcrossing wild-derived BMYV resistance with cultivated species. The trait for resistance to yellows virus disease is thought to be inherited through complex polygenic control and is susceptible to fluctuations due to weather conditions, so attempts to select for resistance by cultivating in fields do not always work. To address these issues, a technique for selecting resistant plants using DNA polymorphisms is effective.

[0005] Non-Patent Document 1 describes that quantitative trait loci (QTL) analysis of plants inoculated with BMYV was performed to identify major QTLs associated with symptoms such as vein discoloration or mottled leaf color. Non-Patent Document 2 describes that a QTL associated with symptoms observed in leaves due to BMYV infection was found to be located on chromosome 4. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Grimmer MK, Bean KMR, Stevens AQM, Asher MJC (2008) The action of three Beet yellows virus resistance QTLs depends on alleles at a novel genetic locus that controls symptom development. Plant Breeding 127:391-397 [Non-patent document 2] James LC, Bean KMR, Grimmer MK, Barnes S, Kraft T, Stevens M (2012) Varieties of the future: Identification of 'broad spectrum' genetic resistance in sugar beet. International Sugar Journal 114:164-168. Summary of the Invention [Problem to be solved by the invention]

[0007] However, since the results of Non-Patent Document 1 are based on analysis using AFLP markers, it is difficult to obtain information about the chromosomal location of useful QTLs. Furthermore, Non-Patent Document 2 does not disclose information about markers that can be used to select for resistance.

[0008] The present invention has been made to solve the above-mentioned problems, and an object of the present invention is to provide a technique for selecting sugar beet plants that are resistant to yellows virus disease. [Means for solving the problem]

[0009] In order to solve the above problems, one embodiment of the present invention provides a method for determining whether or not a sugar beet plant has resistance to chlorosis virus disease, and includes a step of determining whether or not the sugar beet plant has resistance by identifying at least one of the gene loci involved in resistance to chlorosis virus disease located between 11.0 Mbp and 30.0 Mbp on chromosome 4 and between 8.0 Mbp and 38.0 Mbp on chromosome 7 in the sugar beet plant.

[0010] A production method according to one aspect of the present invention is a method for producing sugar beet plants resistant to chlorosis virus disease, and includes a selection step of selecting sugar beet plants resistant to chlorosis virus disease from sugar beet plants obtained by breeding or sugar beet plants of their progeny lineages using a determination method according to one aspect of the present invention.

[0011] A sugar beet plant according to one embodiment of the present invention is a hybrid of a sugar beet plant having resistance to chlorosis virus disease with another sugar beet plant, and has at least one of the loci involved in resistance to chlorosis virus disease located between 11.0 Mbp and 30.0 Mbp on chromosome 4 and between 8.0 Mbp and 38.0 Mbp on chromosome 7.

[0012] A determination kit according to one embodiment of the present invention is a kit for determining whether or not a sugar beet plant has resistance to yellows virus disease, and includes a primer set for amplifying at least one of the following regions (a) to (l): (a) a region sandwiched between a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1 and a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2; (b) a region sandwiched between a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 3 and a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 4; (c) a region sandwiched between a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 5 and a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 6; (d) a region sandwiched between a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 7 and a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 8; (e) a region sandwiched between a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 9 and a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 10; (f) a region sandwiched between a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 11 and a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 12; (g) a region sandwiched between a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 13 and a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 14; (h) a region sandwiched between a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 15 and a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 16; (i) a region sandwiched between a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 17 and a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 18; (j) a region sandwiched between a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 19 and a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 20; (k) a region sandwiched between a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 21 and a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 22; (l) a region sandwiched between a polynucleotide consisting of the base sequence shown in SEQ ID NO: 23 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 24; [Effects of the Invention]

[0013] According to one aspect of the present invention, a technique for selecting a sugar beet plant having resistance to yellows virus disease can be provided. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 is a graph showing the relationship between the yellowing index and the yellowing area rate (%) of the segregating generation (F3 line group) in an example. [Figure 2] FIG. 1 shows a linkage map created in the F2 generation between "C81-22" and "NK-323mm-O" in an example. [Figure 3] FIG. 10 is a diagram showing the distribution of ΔSNPs calculated based on the yellowing index by performing QTLseq analysis of yellows virus disease resistance on chromosome 1 of RefBeet-1.2.2 in an example. [Figure 4]FIG. 10 is a diagram showing the distribution of ΔSNPs calculated based on the yellowing area ratio by performing QTLseq analysis of yellowing virus disease resistance on chromosome 1 of RefBeet-1.2.2 in an example. [Figure 5] FIG. 10 is a diagram showing the distribution of ΔSNPs calculated based on the yellowing index by performing QTLseq analysis of resistance to yellows virus disease on chromosome 1 of EL10.2 in an example. [Figure 6] FIG. 10 shows the distribution of ΔSNP calculated based on the yellowing area ratio by performing QTLseq analysis of resistance to yellowing virus disease on chromosome 1 of EL10.2 in an example. [Figure 7] FIG. 10 is a diagram showing the distribution of ΔSNPs calculated based on the yellowing index by performing QTLseq analysis of yellows virus disease resistance on chromosome 4 of RefBeet-1.2.2 in an example. [Figure 8] FIG. 10 is a diagram showing the distribution of ΔSNPs calculated based on the yellowing area ratio by performing QTLseq analysis of resistance to yellowing virus disease on chromosome 4 of RefBeet-1.2.2 in an example. [Figure 9] FIG. 10 is a diagram showing the distribution of ΔSNP calculated based on the yellowing index by performing QTLseq analysis of resistance to yellows virus disease on chromosome 4 of EL10.2 in an example. [Figure 10] FIG. 10 shows the distribution of ΔSNP calculated based on the yellowing area ratio by performing QTLseq analysis of resistance to yellowing virus disease on chromosome 4 of EL10.2 in an example. [Figure 11] FIG. 10 is a diagram showing the distribution of ΔSNPs calculated based on the yellowing index by performing QTLseq analysis of yellows virus disease resistance on chromosome 7 of RefBeet-1.2.2 in an example. [Figure 12] FIG. 10 is a diagram showing the distribution of ΔSNPs calculated based on the yellowing area ratio by performing QTLseq analysis of resistance to yellowing virus disease on chromosome 7 of RefBeet-1.2.2 in an example. [Figure 13]FIG. 10 is a diagram showing the distribution of ΔSNPs calculated based on the yellowing index by performing QTLseq analysis of resistance to yellows virus disease on chromosome 7 of EL10.2 in an example. [Figure 14] FIG. 10 shows the distribution of ΔSNPs calculated based on the yellowing area ratio by performing QTLseq analysis of resistance to yellowing virus disease on chromosome 7 of EL10.2 in an example. [Figure 15] FIG. 10 is a diagram showing the distribution of ΔSNPs calculated based on the yellowing index by performing QTLseq analysis of yellows virus disease resistance on chromosome 8 of RefBeet-1.2.2 in an example. [Figure 16] FIG. 10 is a diagram showing the distribution of ΔSNPs calculated based on the yellowing area ratio by performing QTLseq analysis of resistance to yellowing virus disease on chromosome 8 of RefBeet-1.2.2 in an example. [Figure 17] FIG. 10 is a diagram showing the distribution of ΔSNPs calculated based on the yellowing index by performing QTLseq analysis of resistance to yellows virus disease on chromosome 8 of EL10.2 in an example. [Figure 18] FIG. 10 shows the distribution of ΔSNPs calculated based on the yellowing area ratio when QTLseq analysis of resistance to yellowing virus disease was performed on chromosome 8 of EL10.2 in an example. [Figure 19] FIG. 1 is a diagram showing the distribution of yellowing indexes of breeding lines in an example. DETAILED DESCRIPTION OF THE INVENTION

[0015] [Selection marker for plants resistant to chlorosis virus disease] The embodiments of the present invention are described below. However, the present invention is not limited thereto. All documents described in this specification are incorporated herein by reference. Furthermore, unless otherwise specified in this specification, "A to B" representing a numerical range means "A or more (including A and greater than A) and B or less (including B and less than B)."

[0016] As used herein, the term "polynucleotide" is used interchangeably with "nucleic acid" or "nucleic acid molecule," and refers to a polymer of nucleotides. Here, nucleic acids can exist in the form of DNA (e.g., cDNA or genomic DNA) or RNA (e.g., mRNA). DNA or RNA may be double-stranded or single-stranded. Single-stranded DNA or RNA may be a coding strand (sense strand) or a non-coding strand (antisense strand). As used herein, bases are represented by the single-letter symbols defined by IUPAC and IUB, as appropriate.

[0017] One aspect of the present invention relates to a technique for determining whether or not a sugar beet plant has resistance to chlorosis virus disease. One aspect of the present invention can be used to determine whether or not an individual sugar beet plant has resistance to chlorosis virus disease. One aspect of the present invention determines the genotype of a chlorosis virus disease resistance locus present in the genome of the sugar beet plant, thereby determining whether or not an individual sugar beet plant has resistance to chlorosis virus disease. One aspect of the present invention can be used to determine which sugar beet varieties or parent lines have resistance to chlorosis virus disease.

[0018] In one aspect of the present invention, the concept of resistance to chlorosis virus disease includes the ability to inhibit or suppress the onset and progression of disease caused by infection with a pathogen of chlorosis virus disease, or the degree of inhibition or suppression. For example, resistance to chlorosis virus disease may include the ability or degree of a sugar beet plant to prevent the onset of disease caused by infection with a pathogen of chlorosis virus disease, to stop the progression of disease that has occurred, and to suppress or inhibit the progression of disease that has occurred.

[0019] For example, sugar beet plants resistant to chlorosis virus disease have a higher ability to inhibit or suppress the onset and progression of disease caused by infection with a pathogen of chlorosis virus disease than sugar beet plants susceptible to chlorosis virus disease. Resistance to chlorosis virus disease can be evaluated by comparing the measured value of the degree of disease caused by infection with a pathogen of chlorosis virus disease measured in sugar beet plants with a predetermined threshold value or with the measured value of other sugar beet plants. Examples of indices representing the degree of disease damage include the yellowing index and the yellowing area ratio.

[0020] In this specification, an example of a "sugar beet plant" is sugar beet (Beta vulgaris ssp. vulgaris), which is classified in the genus Beta in the family Chenopodiaceae, and is also called sugar beet. Examples of parent lines of sugar beet include "C81-22," "CR951-210," "NK-323mm-O," "NK-237BRmm-O," "NK-195BRmm-O," and "NK-422mm-O."

[0021] The presence or absence of resistance to chlorosis virus disease varies depending on the sugar beet variety and parent line. For example, "C81-22" and "CR951-210" are resistant varieties to chlorosis virus disease, while "NK-323mm-O," "NK-237BRmm-O," "NK-195BRmm-O," and "NK-422mm-O" are susceptible varieties to chlorosis virus disease.

[0022] In this specification, the term "plant" may refer to a part or the whole of a plant body, such as propagation material (seeds, bulbs, etc.), scales, flowers, roots, etc.

[0023] In one embodiment of the present invention, the pathogenic viruses causing chlorosis virus diseases are beet yellow leaf virus and its related species. The main pathogenic viruses causing chlorosis virus diseases are all transmitted primarily by the green peach aphid (abbreviated as GPA, English name: green peach aphid, scientific name: Myzus persicae), but are taxonomically distinct viruses with different genetic characteristics. For example, BYV (beet yellows virus) belongs to the Closteroviridae family, while BWYV (beet western yellows virus), BMYV (beet mild yellowing virus), and BChV (beet chlorosis virus) belong to the Luteoviridae family and the Polerovirus genus. Incidence varies by country and region, with BMYV being more prevalent than BYV in Europe and BWYV being more prevalent than BYV in the United States. In the past, the existence of two types of causative viruses of yellows virus disease that occurred in Hokkaido was suspected based on sap inoculation tests using carborundum and inoculation tests using GPA, namely BWYV and BYV. However, in recent years, comparative analysis of the base sequences of virus genomes collected from various parts of Hokkaido has not confirmed the occurrence of BYV, and it has been reported that BWYV is different from BWYV and is BLYV (Beet Yellow Leaf Virus).

[0024] Yellows virus disease often appears in small patches in fields around July. When plants are infected with the virus, the leaves between the veins of the middle to lower leaves turn yellow, and the leaves become thick and brittle. The direct negative impact of the outbreak of yellows virus disease on the sugar beet industry is a reduction in the yield of sugar beets in raw material fields.

[0025] Field tests conducted in various parts of Europe between 1935 and 1955 reported that diseased plants had reduced stem and leaf weight by 0% to 61%, root weight by 21% to 69%, and sugar content by 28% to 72% compared to healthy plants. Furthermore, when the impact on sugar yields across the country was estimated based on the incidence rate of chlorosis virus disease, it was reported that sugar yields in European countries between 1973 and 1984 decreased by several percent to 26% due to the outbreak of chlorosis virus disease. Similarly, in the United States, it was reported that the outbreak of chlorosis virus disease reduced root weight by 14% to 53%, and actual sugar content by 0.4% to 2.2%.

[0026] In Hokkaido, sugar beet chlorosis virus occurred frequently in raw material fields throughout the prefecture around 2013, resulting in sugar production of 565,000 tons, 556,000 tons, and 551,000 tons from 2012 to 2014, respectively, far below the average of 639,000 tons recorded from 1986, when the sugar trading system was introduced, to 2015. While this decline is due to a combination of factors, including high summer temperatures and the occurrence of brown spot and black root disease, the occurrence of sugar beet chlorosis virus is also believed to be a contributing factor. The Hokkaido Agriculture Department conducted inoculation tests to analyze the effect of virus inoculation timing on sugar beet yield, and reported that early inoculation (late May to late July) resulted in a roughly 30% reduction in sugar yield compared to later inoculation (late August and late September).

[0027] Non-patent documents 1 and 2 are studies on resistance to BYV or BMYV, but do not disclose resistance to BLYV, the causative virus of yellows virus disease in Japan.

[0028] The present inventors have conducted extensive research into techniques for selecting varieties resistant to BLYV, with the aim of developing varieties that are resistant to yellows virus disease in sugar beet plants primarily grown in Japan, and have completed the present invention as a result.

[0029] [Sugar beet yellows virus disease resistance marker] A molecular marker according to one embodiment of the present invention is a chlorosis virus disease resistance marker that is involved in the presence or absence of resistance to chlorosis virus disease in sugar beet plants. The molecular marker can be used to identify loci involved in resistance to chlorosis virus disease (chlorosis virus disease resistance loci) on chromosomes 4 and 7 of sugar beet plants. The chlorosis virus disease resistance loci were identified by the present inventors and are located between 11.0 Mbp and 30.0 Mbp on chromosome 4 and between 8.0 Mbp and 38.0 Mbp on chromosome 7.

[0030] It should be noted that the chlorosis virus disease resistance loci may be present on any other chromosome instead of chromosome 4 and chromosome 7. For example, molecular markers can be used to identify the presence of loci corresponding to the chlorosis virus disease resistance loci on chromosomes 4 and 7 of sugar beet plants on any of the chromosomes of a sugar beet plant. The chlorosis virus disease resistance loci refer to quantitative trait loci or gene regions that affect resistance to chlorosis virus disease. Quantitative trait loci (QTL) generally refer to chromosomal regions involved in the expression of quantitative traits. QTLs can be defined using molecular markers that indicate specific loci on chromosomes. Techniques for defining QTLs using such molecular markers are well known in the art.

[0031] A molecular marker according to one embodiment of the present invention is a marker that identifies at least one of the chlorosis virus disease resistance loci, which are the region from 11.0 Mbp to 30.0 Mbp on chromosome 4 of a sugar beet plant and the region from 8.0 Mbp to 38.0 Mbp on chromosome 7. That is, the molecular marker may be a molecular marker that is a base or polynucleotide located within the chlorosis virus disease resistance locus or within a region near the chlorosis virus disease resistance locus and linked to the locus. The molecular marker is a DNA marker that indicates the presence of the chlorosis virus disease resistance loci on chromosomes 4 and 7 of a sugar beet plant.

[0032] The present inventors performed QTL analysis using "NK-323mm-O," a chlorosis virus disease-susceptible type that exhibits significant yellowing symptoms due to infection with a pathogenic virus, "C81-22," a chlorosis virus disease-resistant type that exhibits minimal yellowing symptoms, and progeny derived from a cross between these two parents, and found that chlorosis virus disease-resistance loci are present on chromosomes 4 and 7. The present inventors found that when the genotype of the chlorosis virus disease-resistance locus is the "C81-22" type, it exhibits less yellowing symptoms and is more resistant to chlorosis virus disease than the "NK-323mm-O" type.

[0033] A molecular marker according to one embodiment of the present invention may be a molecular marker that identifies the genotype of a yellows virus disease resistance locus, and may be a molecular marker that determines whether the genotype is resistant or susceptible.

[0034] A molecular marker according to one embodiment of the present invention is at least one of the following regions (a) to (l): (a) a region sandwiched between a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1 and a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2; (b) a region sandwiched between a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 3 and a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 4; (c) a region sandwiched between a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 5 and a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 6; (d) a region sandwiched between a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 7 and a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 8; (e) a region sandwiched between a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 9 and a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 10; (f) a region sandwiched between a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 11 and a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 12; (g) a region sandwiched between a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 13 and a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 14; (h) a region sandwiched between a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 15 and a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 16; (i) a region sandwiched between a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 17 and a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 18; (j) a region sandwiched between a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 19 and a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 20; (k) a region sandwiched between a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 21 and a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 22; (l) A region sandwiched between a polynucleotide consisting of the base sequence shown in SEQ ID NO: 23 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 24.

[0035] The genotype of the chlorosis virus disease resistance locus can be identified by detecting at least one of the above regions (a) to (l) as a molecular marker. The above regions (a) to (f) are regions on chromosome 7, and the above regions (g) to (l) are regions on chromosome 4. The sequences of the above regions (a) to (e) and (g) to (l) are derived from the chlorosis virus disease-resistant variety "C81-22," and the sequence of the above region (f) is derived from the chlorosis virus disease-susceptible variety "NK-323mm-O."

[0036] A molecular marker according to one embodiment of the present invention may be a polynucleotide in the above regions (a) to (l), or a polynucleotide in linkage disequilibrium with the polynucleotide. Here, the linkage disequilibrium relationship is, for example, a relationship in which the linkage disequilibrium coefficient is 0.8 or more, preferably 0.9 or more, and more preferably 0.95 or more. Furthermore, a molecular marker according to one embodiment of the present invention may be a polynucleotide consisting of any of the nucleotide sequences set forth in SEQ ID NOS: 1 to 24, located at both ends of each of the above regions (a) to (l).

[0037] Examples of molecular markers according to one embodiment of the present invention include SNP markers, AFLP (amplified fragment length polymorphism) markers, RFLP markers, microsatellite markers, SCAR markers, and CAPS markers.

[0038] A molecular marker according to one embodiment of the present invention may be a base corresponding to any one of the following bases (i) to (iii), at least one base linked to the base, or a continuous polynucleotide containing the base: (i) the 1643rd base of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 25; (ii) the 742nd base of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 26; (iii) The 637th base of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 27.

[0039] A molecular marker according to one embodiment of the present invention detects genetic polymorphisms at chlorosis virus disease resistance loci on chromosomes 4 and 7. The genetic polymorphisms detected by the molecular marker are mutations (SNPs) at the chlorosis virus disease resistance loci on chromosomes 4 and 7, and the molecular marker may be an SNP marker. The molecular marker according to one embodiment of the present invention is an SNP marker newly identified by the present inventors, and those skilled in the art can identify the location of the SNP marker on the genome based on the nucleotide sequence of the SNP marker.

[0040] By using a molecular marker according to one embodiment of the present invention, when the base sequence of the molecular marker is detected in a sugar beet plant, the sugar beet plant can be determined to be resistant to yellows virus disease. For example, such a molecular marker can be detected using a primer pair specific to the molecular marker.

[0041] When at least one of the SNP markers (i) to (iii) or an SNP in linkage disequilibrium with the SNP marker is detected in a sugar beet plant, the sugar beet plant can be determined to have resistance to yellows virus disease. On the other hand, when these SNP markers are not detected in a sugar beet plant, the sugar beet plant can be determined to be susceptible to yellows virus disease. The presence or absence of resistance to yellows virus disease in a sugar beet plant may be determined by analyzing the SNP markers as a haplotype block consisting of any one of the SNPs (i) to (iii) and at least two SNPs in linkage disequilibrium with the SNP.

[0042] An SNP marker, which is a molecular marker according to one embodiment of the present invention, may be (1) the base corresponding to the SNP itself, (2) a consecutive polynucleotide containing the SNP, or (3) a consecutive polynucleotide containing two SNPs.

[0043] (1: The base corresponding to the SNP) In the SNP marker, the SNP is a single nucleotide polymorphism based on the genome sequence of the sugar beet plant. In the SNP marker, the single nucleotide polymorphism is a single nucleotide polymorphism based on the genome sequence of the sugar beet. The genome sequence of the reference plant sugar beet (Refbeet1-2-2) has been published on the website https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCF_000511025.2 / , and the genome sequence of sugar beet (EL10.2) has been published on the website https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCF_026745355.1 / .

[0044] "Any of the bases (i) to (iii)" refers to the SNP markers described in the present Examples. "A base corresponding to any of the bases (i) to (iii)" refers to an SNP marker that can be identified with the SNP markers described in the present Examples. As an example, the markers shown in Table 9 in the Examples can be used as molecular markers according to one aspect of the present invention. Information on the base sequences and locations of genetic polymorphisms of the markers shown in Table 9 can be obtained from a database on the website where the genome sequence of the above-mentioned reference plant is published, based on the gene names and mutation site information shown in Table 9.

[0045] "Any of the bases (i) to (iii)" is a mutation in a gene region on chromosome 4 and chromosome 7 that is related to resistance to yellows virus disease. The yellows virus disease resistance gene loci on chromosome 4 and chromosome 7 consist of a base sequence derived from a reference sugar beet plant. Other sugar beet plants may contain base sequence differences other than SNPs from the reference base sequence. The region on the genome where this SNP marker is located is conserved among many sugar beet plants, and therefore this SNP marker can be identified by a method such as a homology search.

[0046] That is, in other sugar beet plants, when chlorosis virus disease resistance loci on chromosome 4 and chromosome 7 (i.e., loci that are highly conserved among plants) exist, "a base corresponding to any one of bases (i) to (iii)" refers to a base on the chlorosis virus disease resistance loci on chromosome 4 and chromosome 7 that is determined to correspond to any one of bases (i) to (iii) by a method such as homology search. For example, the polynucleotides described in (i2) to (iii2) and the polynucleotides described in (i3) to (iii3) below are examples of chlorosis virus disease resistance loci on chromosome 4 and chromosome 7.

[0047] SNP (i) (hereinafter also referred to as SNP(i)) indicates a polymorphism of the 1643rd base of a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 25, or a base corresponding thereto. SNP (ii) (hereinafter also referred to as SNP(ii)) indicates a polymorphism of the 742nd base of a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 26, or a base corresponding thereto. SNP (iii) (hereinafter also referred to as SNP(iii)) indicates a polymorphism of the 637th base of a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 27, or a base corresponding thereto. SNP(i), SNP(ii), and SNP(iii) correspond to the SNPs at positions 8384102 on chromosome 7, 35415610 on chromosome 7, and 15265166 on chromosome 4, respectively, as shown in Table 11 in the Examples described below. The base sequence of the region surrounding SNP(i) is shown in SEQ ID NO: 28, the base sequence of the region surrounding SNP(ii) is shown in SEQ ID NO: 29, and the base sequence of the region surrounding SNP(iii) is shown in SEQ ID NO: 30.

[0048] In one embodiment of the molecular marker of the present invention, when the allele of the base corresponding to SNP (i) is homozygous for T or heterozygous for C / T, the sugar beet plant can be determined to be resistant to chlorosis virus disease. When the allele of the base corresponding to SNP (ii) is homozygous for G or heterozygous for A / G, the sugar beet plant can be determined to be resistant to chlorosis virus disease. Furthermore, when the allele of the base corresponding to SNP (iii) is homozygous for A or heterozygous for T / A, the sugar beet plant can be determined to be resistant to chlorosis virus disease.

[0049] (ii: Polynucleotide containing SNP) A molecular marker according to one embodiment of the present invention may be a contiguous polynucleotide containing SNP(i) (hereinafter also referred to as polynucleotide (i)), a contiguous polynucleotide containing SNP(ii) (hereinafter also referred to as polynucleotide (ii)), or a contiguous polynucleotide containing SNP(iii) (hereinafter also referred to as polynucleotide (iii)).

[0050] Polynucleotide (i) is (i1) a polynucleotide consisting of the base sequence of a region containing SNP(i) in the base sequence shown in SEQ ID NO: 25; (i2) a polynucleotide consisting of a base sequence in which one or more bases have been substituted, deleted, added or inserted relative to the base sequence other than SNP(i) in the base sequence of the polynucleotide (i1), and has the function of determining whether or not a sugar beet plant has resistance to yellows virus disease; or (i3) a polynucleotide consisting of a base sequence that is 90% or more identical to the base sequence other than SNP(i) in the base sequence of the polynucleotide (i1), and has the function of determining whether or not a sugar beet plant has resistance to yellows virus disease.

[0051] The polynucleotide (ii) is (ii1) a polynucleotide consisting of the base sequence of a region containing SNP(ii) in the base sequence shown in SEQ ID NO: 26; (ii2) a polynucleotide consisting of a base sequence in which one or more bases have been substituted, deleted, added or inserted relative to the base sequence other than SNP(ii) in the base sequence of the polynucleotide (ii1), and having the function of determining whether or not a sugar beet plant has resistance to yellows virus disease; or (ii3) a polynucleotide consisting of a base sequence that is 90% or more identical to the base sequence other than SNP(ii) in the base sequence of the polynucleotide (ii1), and having the function of determining whether or not a sugar beet plant has resistance to yellows virus disease.

[0052] The polynucleotide (iii) is (iii1) a polynucleotide consisting of the base sequence of a region containing SNP (iii) in the base sequence shown in SEQ ID NO: 27; (iii2) a polynucleotide consisting of a base sequence in which one or more bases have been substituted, deleted, added or inserted relative to the base sequence other than SNP (iii) in the base sequence of the polynucleotide (iii1), and has the function of determining whether or not a sugar beet plant has resistance to yellows virus disease; or (iii3) a polynucleotide consisting of a base sequence that is 90% or more identical to the base sequence other than SNP (iii) in the base sequence of the polynucleotide (iii1), and has the function of determining whether or not a sugar beet plant has resistance to yellows virus disease.

[0053] The polynucleotides (i1) to (iii1) can be obtained, for example, from a sugar beet plant resistant to chlorosis virus disease, based on the nucleotide sequence of the chlorosis virus disease resistance gene locus on chromosome 4 or chromosome 7.

[0054] The polynucleotides (i2) to (iii2) may have the base sequences of the polynucleotides (i1) to (iii1) conserved in the base sequences corresponding to SNPs (i) to (iii), but may contain modifications (substitutions, deletions, insertions, or additions) of several (for example, 1 to 10, preferably 1 to 5, and more preferably 1, 2, or 3) bases in the remaining base sequences. The base sequences of such polynucleotides will be clear to those skilled in the art, and can be determined by referring to the above-mentioned sugar beet genome sequence or by decoding the base sequence of the region adjacent to the SNP on the genome of a sugar beet plant resistant to yellows virus disease.

[0055] The polynucleotides (i3) to (iii3) may have, in the base sequence of the polynucleotides (i1) to (iii1), bases corresponding to SNPs (i) to (iii) conserved, and may have, for example, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the remaining base sequence. Such base sequence identity can be determined, for example, by aligning the two base sequences using analysis software such as BLAST or FASTA.

[0056] When the allele of the base corresponding to SNP (i) in polynucleotide (i) is homozygous for T or heterozygous for C / T, the sugar beet plant can be determined to be resistant to chlorosis virus disease. When the allele of the base corresponding to SNP (ii) in polynucleotide (ii) is homozygous for G or heterozygous for A / G, the sugar beet plant can be determined to be resistant to chlorosis virus disease. Furthermore, when the allele of the base corresponding to SNP (iii) in polynucleotide (iii) is homozygous for A or heterozygous for T / A, the sugar beet plant can be determined to be resistant to chlorosis virus disease.

[0057] (iii: Polynucleotide containing two SNPs) A molecular marker according to one embodiment of the present invention may be a continuous polynucleotide (hereinafter also referred to as polynucleotide (iv)) comprising at least two of SNPs (i) to (iii). Polynucleotide (iv) may comprise a region between at least two of SNPs (i) to (iii), together with at least two sites of SNPs (i) to (iii). Polynucleotide (iv) can be obtained, for example, by referencing a region between at least two of corresponding SNPs (i) to (iii) in a sugar beet plant resistant to yellows virus disease. The nucleotide sequence of polynucleotide (iv) at least partially coincides with the nucleotide sequence of the sugar beet plant resistant to yellows virus disease.

[0058] [Method for determining resistance to sugar beet yellows virus disease] A method for determining the presence or absence of resistance to chlorosis virus disease in a sugar beet plant according to one embodiment of the present invention includes a step of determining the presence or absence of resistance by identifying at least one of the chlorosis virus disease resistance loci located in the sugar beet plant between 11.0 Mbp and 30.0 Mbp on chromosome 4 and between 8.0 Mbp and 38.0 Mbp on chromosome 7. As an example, the method detects the presence of a locus on any of the chromosomes of the sugar beet plant that corresponds to the chlorosis virus disease resistance loci on chromosomes 4 and 7 of the sugar beet plant.

[0059] In one embodiment of the determination method of the present invention, the molecular marker is a molecular marker containing a polynucleotide in at least one of the regions (a) to (l) described above, or a molecular marker that is in linkage disequilibrium with the molecular marker, and the molecular marker is detected in a sugar beet plant as a molecular marker related to resistance to yellows virus disease.

[0060] Furthermore, in the determination method according to one aspect of the present invention, the molecular marker may be a molecular marker containing a base corresponding to any one of the bases (i) to (iii) described above and at least one of the bases linked to the base, or a consecutive polynucleotide containing the base, or a molecular marker in linkage disequilibrium with the molecular marker.

[0061] In one embodiment of the determination method of the present invention, the test sugar beet plant is a candidate plant for breeding material or a plant obtained through a breeding process. Examples of candidate plants for breeding material include parent plants used in crossbreeding and plants used in molecular breeding using genetic engineering. Examples of plants obtained through a breeding process include intraspecific hybrids of plants belonging to the above-mentioned sugar beet varieties, and their progeny lines. The test sugar beet plant may also be an interspecific hybrid, such as a hybrid between a sugar beet plant belonging to one variety and a sugar beet plant belonging to another variety, and their progeny lines. Furthermore, the test sugar beet plant may be, for example, a hybrid between two sugar beet plants resistant to chlorosis virus disease, a hybrid between a sugar beet plant resistant to chlorosis virus disease and another sugar beet plant, or a hybrid between two sugar beet plants susceptible to chlorosis virus disease, and their progeny lines. Furthermore, the subject sugar beet plant may be a hybrid plant obtained by crossbreeding sugar beet plants whose resistance to yellows virus disease is unknown, and its progeny lineage.

[0062] Furthermore, the subject sugar beet plants include sugar beet plants used in molecular breeding using recombinant DNA technology, and sugar beet plants obtained by molecular breeding.

[0063] A determination method according to one aspect of the present invention determines the presence or absence of resistance to chlorosis virus disease in a test sugar beet plant using the molecular marker according to one aspect of the present invention described above. The determination method according to one aspect of the present invention determines the presence or absence of resistance to chlorosis virus disease in a test sugar beet plant by identifying mutations in the genome of the test sugar beet plant using the molecular marker described above. According to the determination method according to one aspect of the present invention, the presence of chlorosis virus disease resistance gene loci on chromosomes 4 and 7 of a sugar beet plant can be detected by using the molecular marker according to one aspect of the present invention, and traits related to resistance to chlorosis virus disease in the sugar beet plant can be determined.

[0064] In one aspect of the determination method of the present invention, when the base sequence of the molecular marker is detected in the sugar beet plant in the determination step, the sugar beet plant is determined to be resistant to the yellows virus disease. On the other hand, in another aspect of the determination method of the present invention, when the base sequence of the molecular marker is not detected in the sugar beet plant in the determination step, the sugar beet plant is determined to be susceptible to the yellows virus disease.

[0065] For details of the molecular markers used in the determination method according to one aspect of the present invention, the above-mentioned description of the molecular markers according to one aspect of the present invention is incorporated herein by reference.

[0066] In the determination step, the method for detecting the molecular marker in the sugar beet plant is not particularly limited, and any known analytical method can be used, such as an analytical method that detects the region of the molecular marker in a PCR-amplified fragment of a test sugar beet plant. In one aspect of the determination method of the present invention, a primer that amplifies a region containing the molecular marker may be used to amplify the region in the DNA of the sugar beet plant.

[0067] The primer set for amplifying a region containing the molecular marker described above can be a determination kit for determining whether or not a sugar beet plant has resistance to yellows virus disease. That is, the determination kit for determining whether or not a sugar beet plant has resistance to yellows virus disease includes at least one of the primer sets for amplifying the regions (a) to (l) above. Furthermore, the determination kit for determining whether or not a sugar beet plant has resistance to yellows virus disease may include primers for amplifying a region containing a molecular marker containing at least one of the bases (i) to (iii) above, or a molecular marker that is in linkage disequilibrium with the molecular marker.

[0068] The region in the DNA of a test sugar beet plant can be amplified by polymerase chain reaction (PCR) using DNA extracted from the test sugar beet plant as a template and the above-mentioned primers. The nucleotide sequence (genotype) of the resulting amplified fragment is then determined, and the presence or absence of resistance to yellows virus disease in the sugar beet plant is determined based on data showing the relationship between the determined nucleotide sequence (genotype) and the presence or absence of resistance to yellows virus disease in the sugar beet plant. The genotype of the amplified fragment can be determined by any conventionally known method, such as DNA sequence analysis, agarose gel electrophoresis, or TaqMan analysis using real-time PCR.

[0069] The primer set used in PCR is not particularly limited as long as it can amplify a DNA fragment containing the target molecular marker. The primer set may be designed to shorten the length of the amplified fragment. For example, the primer set is designed so that the length of the primer-amplified fragment is preferably 500 base pairs (bp) or less, 400 bp or less, 3000 bp or less, 200 bp or less, 160 bp or less, 150 bp or less, 140 bp or less, 130 bp or less, 120 bp or less, or 100 bp or less. The primer set includes a forward primer and a reverse primer. The length of these primers may be, for example, 15 bp or more, 16 bp or more, 17 bp or more, 18 bp or more, 19 bp or more, 20 bp or more, 25 bp or more, or 30 bp or more, or 50 bp or less, 40 bp or less, or 35 bp or less.

[0070] By using a primer set that amplifies a region containing a molecular marker, the region containing the molecular marker can be specifically amplified, and the genotype can be determined by agarose electrophoresis.

[0071] The PCR in the determination step may be either singleplex PCR, which amplifies DNA fragments in a reaction system containing a single primer set, or multiplex PCR, which amplifies genes in a reaction system containing multiple primer sets. In the case of multiplex PCR, primer sets labeled with fluorescent substances having different wavelengths (e.g., NED, 6-FAM, VIC, PET) may be mixed.

[0072] PCR reaction conditions can be appropriately set depending on the type of DNA polymerase and PCR device used, the length of the amplified fragment, etc. Cycling conditions include a three-step PCR method, in which one cycle consists of three steps: a denaturation step, an annealing step, and an extension step, and a two-step PCR method, in which one cycle consists of two steps: a denaturation step, an annealing step, and an extension step. An example of reaction conditions for three-step PCR is 10 to 40 cycles (e.g., 35 cycles) of a denaturation step at 90 to 100°C for 40 to 60 seconds (e.g., 95°C for 30 seconds), an annealing step for 10 to 60 seconds (e.g., 60 seconds), and an extension step at 65 to 80°C for 10 to 150 seconds (e.g., 72°C for 150 seconds). An example of the annealing temperature is a condition in which the temperature is gradually decreased in each predetermined cycle (e.g., decreased by 1°C per cycle) from an initial annealing temperature of 45 to 70°C (e.g., 61°C) to a final annealing temperature of 40 to 65°C (e.g., 45°C). PCR reaction conditions may be adjusted depending on the state of the template DNA in order to stably detect the molecular marker.

[0073] The PCR used in the determination step may be a real-time PCR such as TaqMan®-PCR, which amplifies and identifies molecular markers by PCR, or an intercalator method using SYBR® Green or the like. That is, a TaqMan® probe may be used to detect molecular markers contained in the amplified fragments amplified using a primer set. The use of real-time PCR can provide a high-throughput discrimination method. The molecular markers in the amplified fragments amplified by PCR may be identified by analyzing the nucleotide sequence of the amplified fragments using an automatic DNA sequencer or the like.

[0074] The method for extracting DNA to be amplified by PCR from a sugar beet plant specimen is not particularly limited, and known DNA extraction methods can be used. Alternatively, DNA may be extracted using a commercially available DNA extraction kit. Depending on the type of specimen and the amount of contaminants, appropriate pretreatment may be performed before the DNA extraction step. Furthermore, the DNA extracted from the specimen may be washed or purified as necessary for use as a template in a PCR reaction. Furthermore, the DNA extracted from the specimen may be digested with two restriction enzymes, and the resulting restriction enzyme fragments may be amplified by PCR.

[0075] According to the method of one aspect of the present invention, the presence or absence of resistance to chlorosis virus disease in a test sugar beet plant can be determined using molecular markers, and therefore, sugar beet plants and their progeny lines that are resistant to chlorosis virus disease can be selected based on the determination results.

[0076] According to the method of determining sugar beet resistance in accordance with one aspect of the present invention, it is possible to select sugar beet plants resistant to chlorosis virus disease, thereby significantly shortening the time required for breeding selection. Furthermore, by planting and growing only seedlings resistant to chlorosis virus disease selected at the seedling stage, it is possible to improve the efficiency of field utilization.

[0077] [Method for producing sugar beet plants resistant to yellows virus disease] A production method according to one aspect of the present invention is a method for producing a sugar beet plant resistant to chlorosis virus disease. The production method according to one aspect of the present invention includes a selection step of selecting a sugar beet plant resistant to chlorosis virus disease from a sugar beet plant obtained by breeding or a sugar beet plant of a progeny line thereof by the determination method according to one aspect of the present invention. The production method according to one aspect of the present invention may further include a hybridization step of intraspecifically crossing the sugar beet plant resistant to chlorosis virus disease with another sugar beet plant, and in the selection step, a sugar beet plant resistant to chlorosis virus disease may be selected from the sugar beet plant obtained by the hybridization step or a sugar beet plant of a progeny line thereof by the determination method according to one aspect of the present invention.

[0078] Therefore, the explanations regarding the molecular marker according to one aspect of the present invention and the determination method according to one aspect of the present invention are also used to explain the method for producing a sugar beet plant resistant to yellows virus disease.

[0079] In the production method according to one embodiment of the present invention, the sugar beet plant used as the parent plant for breeding may be, for example, a sugar beet plant resistant to chlorosis virus disease, a sugar beet plant susceptible to chlorosis virus disease, a sugar beet plant whose resistance to chlorosis virus disease is unknown, or a progeny line thereof.

[0080] Furthermore, the sugar beet plants used as parent plants for breeding may be sugar beet plants of a chlorosis virus disease-resistant type according to an aspect of the present invention described below. Furthermore, the sugar beet plants used as parent plants for breeding may be sugar beet plants of a chlorosis virus disease-resistant type selected by the determination method according to an aspect of the present invention. That is, the production method according to an aspect of the present invention may further comprise, before the crossbreeding step, a discrimination step of discriminating sugar beet plants of a chlorosis virus disease-resistant type from test sugar beet plants by the determination method according to an aspect of the present invention.

[0081] According to a production method of one aspect of the present invention, the presence or absence of resistance to chlorosis virus disease in a sugar beet plant can be determined using a molecular marker, and a sugar beet plant that is resistant to chlorosis virus disease can be produced by selecting the sugar beet plant based on the determination result. In one example of the production method of one aspect of the present invention, when producing a sugar beet plant that is resistant to chlorosis virus disease, such as "C81-22," the sugar beet plant that is resistant to chlorosis virus disease is selected using a molecular marker of one aspect of the present invention.

[0082] Furthermore, the present invention also encompasses a method for producing sugar beet plants that are resistant to chlorosis virus disease by determining whether or not a sugar beet plant has resistance to chlorosis virus disease using a molecular marker according to one embodiment of the present invention and selecting sugar beet plants that are resistant to chlorosis virus disease based on the determination results.

[0083] In a production method according to one aspect of the present invention, sugar beet plants resistant to yellows virus disease can be efficiently selected by utilizing information on DNA polymorphisms. According to the present invention, selection using information on DNA polymorphisms reduces the cultivation area required to test the resistance of sugar beet plants, thereby saving the labor required for variety development.

[0084] [Sugar beet plants resistant to chlorosis virus disease] A sugar beet plant according to one embodiment of the present invention is a hybrid of a sugar beet plant having resistance to chlorosis virus disease with another sugar beet plant, and has at least one of the gene loci involved in resistance to chlorosis virus disease located between 11.0 Mbp and 30.0 Mbp on chromosome 4 and between 8.0 Mbp and 38.0 Mbp on chromosome 7. That is, the sugar beet plant according to one embodiment of the present invention is a sugar beet plant resistant to chlorosis virus disease, and is a plant obtained by the production method according to one embodiment of the present invention described above.

[0085] Such a sugar beet plant has a mutation identified by the molecular marker according to one embodiment of the present invention and is a sugar beet plant of a chlorosis virus disease-resistant type. That is, the sugar beet plant of a chlorosis virus disease-resistant type according to one embodiment of the present invention has chlorosis virus disease-resistant gene loci on chromosomes 4 and 7 identified by a molecular marker containing at least one polynucleotide in the regions (a) to (l) above, a molecular marker containing a base corresponding to any of the bases (i) to (iii) above and at least one base linked to that base, or a consecutive polynucleotide containing that base, or a molecular marker in linkage disequilibrium with any of these molecular markers.

[0086] As shown in the determination method and production method described above, a sugar beet plant resistant to chlorosis virus disease according to one embodiment of the present invention can be obtained by selecting sugar beet plants resistant to chlorosis virus disease from sugar beet plants obtained by breeding and their progeny lines using a molecular marker according to one embodiment of the present invention.

[0087] The present invention may also include within its scope a sugar beet plant that has been genetically engineered to have a mutation identified by the above-mentioned molecular marker and is resistant to chlorosis virus disease. A sugar beet plant resistant to chlorosis virus disease according to one embodiment of the present invention has a chlorosis virus disease resistance gene or a homologous gene (including orthologs) on chromosomes 4 and 7. Such a chlorosis virus disease resistance gene or a homologous gene may be an endogenous gene of the sugar beet plant. A molecular marker according to one embodiment of the present invention may be a molecular marker on the above-mentioned chlorosis virus disease resistance gene or a homologous gene.

[0088] When the yellows virus disease resistance gene refers to a gene into which a mutation has been artificially introduced, the mutation may be artificially introduced using, for example, site-specific mutagenesis methods such as the Kunkel method (Kunkel, Proc Natl Acad Sci USA, 82: 488-492, 1985), mutagen treatment using drugs, mutagenesis methods using radiation (gamma rays, heavy ion beams, etc.), genome editing using site-specific nucleases, etc., or the mutation may be derived from a similar mutant polypeptide that exists in nature.

[0089] The yellows virus disease resistance gene may exist in the form of RNA (e.g., mRNA) or DNA (e.g., cDNA or genomic DNA). The DNA may be double-stranded or single-stranded. An example of a yellows virus disease resistance gene may be DNA consisting of a nucleotide sequence derived from the sugar beet variety "C81-22."

[0090] The method for obtaining (isolating) a yellows virus disease resistance gene is not particularly limited, but examples include a method of preparing a probe that specifically hybridizes with a portion of the base sequence of the yellows virus disease resistance gene and screening a genomic DNA library or a cDNA library.

[0091] Another method for obtaining a yellows virus disease resistance gene is to use an amplification method such as PCR. For example, primers are prepared from the 5' and 3' sequences (or their complementary sequences) of the DNA of the yellows virus disease resistance gene, and PCR or the like is performed using these primers and genomic DNA (or cDNA) as a template to amplify the DNA region between the two primers, thereby obtaining a large amount of a DNA fragment containing the yellows virus disease resistance gene.

[0092] Whether or not an isolated candidate gene for a chlorosis virus disease resistance gene has activity related to resistance to chlorosis virus disease can be evaluated by observing whether resistance to chlorosis virus disease is induced by expression of the candidate gene in the plant from which it is derived.

[0093] The chlorosis virus disease resistance gene can be used to elucidate the mechanism of resistance to chlorosis virus disease in sugar beet plants. Furthermore, the chlorosis virus disease resistance gene can be used to create a transformant by incorporating its sequence into an expression vector and introducing it into a plant or cells of a sugar beet plant. By cultivating a sugar beet plant into which the chlorosis virus disease resistance gene has been introduced, a sugar beet plant resistant to chlorosis virus disease can be obtained.

[0094] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention. [Example]

[0095] Materials and Methods (1:QTL analysis) QTL analysis was performed to understand the number of genomes involved, the magnitude of the effect, and an overview of the region. The analysis was performed using phenotypic and genotypic information obtained using the methods described below.

[0096] Phenotypic investigations were conducted over two years, in 2019 and 2020. Materials included "NK-323mm-O," which exhibits significant yellowing symptoms upon BLYV infection (BLYV susceptibility), and "C81-22," which exhibits minimal yellowing symptoms (BLYV resistance), as well as progeny derived from crosses between these two parents. The hybrid progeny included 146 F2 individuals and 146 F3 lines (F3 seeds) obtained by self-fertilizing the F2 individuals. "NK-323mm-O" was a seed parent developed by the Hokkaido Agricultural Research Center, and "C81-22" is a line available in the USDA Gene Bank (https: / / npgsweb.ars-grin.gov / gringglobal / taxon / taxonomydetail?id=7057).

[0097] In both years, seeds from 146 F3 phylogenetic groups were sown in early April, and seedlings were inoculated with BLYV-infected green peach aphids approximately 30 days after sowing. Inoculation was carried out over approximately 5 days under relatively cool temperature conditions (below 20°C). At the end of inoculation, the inoculated seedlings were treated with a 100x solution of thiamethoxam water-soluble solution by hand spray to kill the aphids. The inoculated seedlings were then planted in late May in an isolated experimental field at the Hokkaido Agricultural Research Center (Sapporo). Each phylogenetic group was planted in a randomized block design with 5 plants per plot, with 4 replicates. The planting density was 120 cm between rows and 22.5 cm between plants. Approximately 1 month after transplanting, leaf specimens were collected from all plants and tested for virus infection using Agdia's ELISA Reagent Set for Beet Western Yellows Virus (BEWV). Infection of the inoculated seedlings was confirmed.

[0098] Two phenotypic values were measured: the yellowing index and the percentage of yellowed area (%). The yellowing index was assessed for each plant every month after virus inoculation (June through September) using a four-point scale ranging from 0 to 3. A yellowing index of 0 indicates no symptoms, 1 indicates partial yellowing of mature leaves, 2 indicates widespread yellowing of mature leaves, and 3 indicates complete yellowing of mature leaves. The percentage of yellowed area (%) was calculated by processing aerial images of the entire field taken in late September, when yellowing symptoms become prominent. Specifically, the number of pixels in the entire leaf was counted by specifying a hue value range of 30 to 125 for the images. The number of pixels in the yellowed area was then counted by specifying a hue value range of 30 to 70. The percentage of pixels in the yellowed area relative to the total number of pixels in the entire leaf was calculated. Images were processed using the image analysis software ImageJ. Significance tests were performed using the Tukey-Kramer multiple test. Statistical processing was performed using Box-Cox transformed values for the yellowing index and arcsine transformed values for the yellowing area rate (%).

[0099] Genotypes were investigated using DNA extracted from leaves of the two parent lines and their F1 and 146 F2 individuals derived from their crosses. Genotype information for each line was collected by Genotyping by Random Amplicon Sequencing-Direct (GRAS-Di) analysis to identify DNA polymorphisms that could be distinguished between the parent lines. Using the genotype information, a linkage map was constructed using JoinMap ver. 5 software. Recombination frequencies in the linkage map were converted to map distances using the maximum likelihood method and the Kosambi mapping function, and a threshold logarithm of odds (LOD) score of greater than 3.0 was used.

[0100] QTL analysis was performed using MapQTL ver. 6 software, utilizing phenotypic and genotypic information. First, potential QTLs were estimated using interval mapping. Next, QTLs were identified using multiple QTL model (MQM) mapping. Markers close to the estimated QTL with the highest LOD score were set as cofactors, and the QTL location was refined by minimizing residual variance. The LOD score threshold was set based on a 5% significance level using 1000 permutation tests. From the final QTL model, the explained phenotypic variance (PVE) and additive effect parameters were derived. In this process, to estimate the chromosomal location of the discovered markers, a search for similar sequences was performed against two published reference genome sequences for sugar beet ["Refbeet1-2-2" (https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCF_000511025.2 / ) and "EL10.2" (https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCF_026745355.1 / )].

[0101] (2: QTLseq analysis) QTL-seq analysis was performed to refine the linkage map information from the above QTL analysis into information on a physical map, and useful mutation information was obtained using the method described below.

[0102] The materials used were DNA extracted from leaves of the F1 individuals derived from the two parent lines and their crosses, as well as 146 F2 individuals. Based on the results of the segregating generation evaluation, equal amounts of DNA from the F2 generation of 20 individuals with low and 20 with high yellowing index values were mixed to create two bulk DNA sets (Set 1: 2019 Bulk A and B; Set 2: 2020 Bulk A' and B'). Similarly, two bulk DNA sets were created for 20 individuals with low and 20 with high yellowing area percentage values (Set 3: 2019 Bulk C and D; Set 4: 2020 Bulk C' and D').

[0103] Whole-genome sequence information was obtained using HiseqX for all four sets, plus the parental lines and the first hybrid (F1) generation DNA (the amount of information analyzed was approximately 30 times that of the whole genome). QTL-seq analysis was performed using the whole-genome information obtained here, including each bulk set, the parental lines, and the F1 lines. The analysis results provided information on the chromosomal locations (gene mutation sites) of SNPs (ΔSNP-index, -1 to 1) whose frequencies differed significantly between the two types of bulk DNA. Furthermore, among all significant mutation sites, mutation sites predicted to affect genes were extracted using the analysis program snpEff (http: / / pcingola.github.io / SnpEff / ), and useful mutations were narrowed down.

[0104] (3: Mutation analysis using diverse breeding lines) The information from the above QTL and QTLseq analyses is valid for the parent lines actually used in the analysis, but it would be even more useful if it contained DNA polymorphisms that could be used for a variety of other materials. Therefore, mutation analysis using various breeding lines was carried out to find particularly useful mutations that could be used for a variety of purposes.

[0105] The materials used were 98 breeding lines and the control variety "Monohikari." The 98 lines were selected based on their diversity in terms of traits (seeds, roots, disease resistance, etc.) and population genetic structure. Because the resistance of these materials to chlorosis virus disease is largely unknown, phenotypic surveys were conducted in August and September over two years, 2021 and 2022, to attempt to classify the lines into resistant and susceptible groups. Virus inoculation, establishment of experimental cultivation plots, and phenotypic surveys were carried out using the same methods as described above. The resistance criteria used here were "C81-22" (strong resistance), "CR951-210" (strong resistance), "NK-210BR" (medium resistance), "Monohikari" (medium resistance), "NK-237BRmm-O" (weak resistance), and "NK-323mm-O" (weak resistance). Genotypic surveys were first conducted using HiseqX and other tools to obtain whole-genome analysis information. Next, the genome information was mapped to the existing reference genome sequence ('Refbeet1-2-2') using the analysis software bwa (https: / / bio-bwa.sourceforge.net). Comprehensive mutation information was output from the mapped data using the analysis software bcftools (http: / / www.htslib.org / doc / 1.8 / bcftools.html). Finally, examples of versatile and useful mutations specific to the resistant and susceptible lineages were extracted from the regions adjacent to the genomic regions detected by QTL analysis and QTLseq analysis.

[0106] 〔result〕 (1:QTL analysis) Regarding phenotypic investigations, ELISA tests showed that all inoculated plants planted in the test fields were positive, with an infection rate of 100%. Yellowing index investigations conducted from June to September showed that the disease progressed over the investigation period in both 2019 and 2020. The degree of progression was most pronounced in "NK-323mm-O," and clear differences in resistance were observed between the parent lines at each investigation period (Table 1). [Table 1] On the other hand, the 146 progeny lines (F3 lineages) derived from both parents showed a wide range of distribution, including the indices of both parents (Table 2). [Table 2] A survey of the yellowed area rate (%) conducted in September yielded results similar to those of the yellowing index (Table 3), and a positive correlation was observed between the two indices (Figure 1). Figure 1 shows the relationship between the yellowing index and the yellowed area rate (%) for 146 segregating generations (F3 lineage groups). In Figure 1, the rs value indicates the value of Pearson's rank correlation coefficient (p<0.01). [Table 3] In investigating the genotype, 288 mutations detected between the parents through GRAS-Di analysis were used as markers to determine the genotypes of 146 F2 individuals. As a result, a linkage map covering a total length of 574.2 cM, corresponding to sugar beet chromosomes 1 to 9, was constructed (Figure 2). Figure 2 shows the linkage map constructed for the F2 generation of "C81-22" and "NK-323mm-O." In Figure 2, the numbers on the left indicate the genetic distance (cM), and the horizontal lines on the chromosomes indicate the location of the markers.

[0107] QTL analysis revealed that four QTLs, qBLYV1, qBLYV4, qBLYV7, and qBLYV8, were detected for the yellowing index and the percentage of yellowed area (%) (Table 4, LOD score ≥ 3.5, p < 0.05). Therefore, selection using the four detected QTLs as indicators can be used to select plants with low values (high resistance) for both the yellowing index and the percentage of yellowed area (%). [Table 4]

[0108] Of these, two types, qBLYV4 and qBLYV7, had relatively high and stable contribution rates and were detected in both years for both the yellowing index and yellowing area rate (%) traits (qBLYV7 (contribution rate 11.0%-21.5%), qBLYV4 (contribution rate 4.0%-8.3%). For both of these QTLs, the values for both traits were low in the homozygous genotype (mu_A) whose nearest locus was the same as that of "C81-22," while the values were high in the homozygous genotype (mu_B) whose nearest locus was the same as that of "NK-323mm-O." The heterozygous genotype (mu_H) tended to show values intermediate between the two genotypes, characterizing the difference in resistance to chlorosis virus disease between "C81-22" and "NK-323mm-O."

[0109] On the other hand, qBLYV1 and qBLYV8 were detected in a single year or for only one of the traits. The contribution rate was relatively low (4.0%-5.8%), and they exhibited the opposite effect to the above QTLs. That is, the values for both traits tended to be high in the homozygous genotype (mu_A) of "C81-22," while the values tended to be low in the homozygous genotype (mu_B) of "NK-323mm-O." This is different from the difference in resistance to chlorosis virus disease between "C81-22" and "NK-323mm-O," suggesting that there is a region that contributes to susceptibility in "C81-22" or a region that contributes to resistance in "NK-323mm-O."

[0110] A total of 15 markers were present within the confidence interval of the QTL detected based on the MQM mapping method (Table 5). [Table 5] Using the sequences of each marker as a query, we searched for the chromosomal location of the two published reference genome sequences for sugar beet, and found that all markers were located on the chromosomes identified by the linkage analysis (Table 6). Therefore, utilizing the base sequence information of these markers is thought to be useful for developing varieties resistant to yellows virus disease. [Table 6]

[0111] In particular, selection using qBLYV4 and qBLYV7 as indicators, which had a relatively high contribution rate and were detected in both years, is thought to be efficient. Looking at the values of the yellowing index and yellowing area percentage by genotype near the qBLYV4 and qBLYV7 QTLs, the lowest was the same type (A1 / A2) as ``C81-22'', the highest was the same type (B1 / B2) as ``NK-323mm-O'', and the other genotypes tended to show values between these (Table 7). [Table 7]

[0112] Specifically, by using the same type (A1 / A2) as "C81-22," the yellowing index as of September over the two years was 0.82 to 1.16, and the yellowing area rate was 21.6% to 29.5%, whereas by using the same type (B1 / B2) as "NK-323mm-O," the yellowing index was 2.39 to 2.53, and the yellowing area rate was 54.8% to 61.7%.

[0113] (2: QTLseq analysis) By analyzing two bulk sets with different yellowing indices (2019: Set 1, 2020: Set 2) and two bulk sets with different yellowing area rates (2019: Set 3, 2020: Set 4), we searched for mutation sites with large absolute ΔSNP-index values (bias in SNP frequency was observed between the two bulk sets) that may be involved in resistance to chlorosis virus disease. As a result, although significant ΔSNP-index values were observed across all chromosomes, there was a tendency for changes in ΔSNP-index values to be observed near the four QTLs detected in the QTL analysis. Therefore, we focused on the regions near the four QTLs for more detailed analysis.

[0114] The results are shown in Figures 3 to 18. In Figures 3 to 18, light and dark gray lines indicate the 5% and 1% levels, respectively. Figures 3 and 4 show the distribution of ΔSNPs calculated by QTL-seq analysis of chlorosis virus disease resistance on chromosome 1 of the reference sequence (Refbeet-1-2-2). Figures 5 and 6 show the distribution of ΔSNPs calculated by QTL-seq analysis of chlorosis virus disease resistance on chromosome 1 of the reference sequence (EL10.2). Figures 7 and 8 show the distribution of ΔSNPs calculated by QTL-seq analysis of chlorosis virus disease resistance on chromosome 4 of the reference sequence (Refbeet-1-2-2). Figures 9 and 10 show the distribution of ΔSNPs calculated by QTL-seq analysis of chlorosis virus disease resistance on chromosome 4 of the reference sequence (EL10.2). Figures 11 and 12 show the distribution of ΔSNPs calculated by QTL-seq analysis of chlorosis virus disease resistance on chromosome 7 of the reference sequence (Refbeet-1-2-2), Figures 13 and 14 show the distribution of ΔSNPs calculated by QTL-seq analysis of chlorosis virus disease resistance on chromosome 7 of the reference sequence (EL10.2), Figures 15 and 16 show the distribution of ΔSNPs calculated by QTL-seq analysis of chlorosis virus disease resistance on chromosome 8 of the reference sequence (Refbeet-1-2-2), and Figures 17 and 18 show the distribution of ΔSNPs calculated by QTL-seq analysis of chlorosis virus disease resistance on chromosome 8 of the reference sequence (EL10.2).

[0115] As shown in Figures 3 to 6, a ΔSNP with a negative value that met the significance level was found near 34.0 Mbp to 35.0 Mbp on chromosome 1. As shown in Figures 7 to 10, a ΔSNP with a positive value that met the significance level was found near 11.0 Mbp to 30.0 Mbp on chromosome 4. As shown in Figures 11 to 14, a ΔSNP with a positive value that met the significance level was found near 8.0 Mbp to 38.0 Mbp on chromosome 7. As shown in Figures 15 to 18, a ΔSNP with a negative value that met the significance level was found near 32.5 Mbp to 34.5 Mbp on chromosome 8.

[0116] A positive ΔSNP value indicates that the same genotype as "C81-22" has the effect of suppressing yellowing (the same genotype as "NK323mm-O" has the effect of promoting yellowing), while a negative ΔSNP value indicates that the same genotype as "C81-22" has the effect of promoting yellowing (the same genotype as "NK323mm-O" has the effect of suppressing yellowing). The positive and negative effects of the ΔSNP on each chromosome were confirmed to be consistent with the results of QTL analysis. Therefore, it is thought that genes controlling qBLYV1, qBLYV4, qBLYV7, and qBLYV8 exist near the same region.

[0117] The mutations detected in the candidate regions of qBLYV1, qBLYV4, qBLYV7, and qBLYV8 were 0–25, 663–9692, 2424–9154, and 39–1461, respectively (Table 8). [Table 8] To narrow the candidate regions, we limited the search to mutations common to two or more of the four bulk sets (mutations predicted to affect genes). The results were 0, 34 (35 genes) to 41 (35 genes), 171 (107 genes) to 302 (152 genes), and 6 (6 genes) to 10 (10 genes). Analysis of mutations predicted to affect genes revealed no candidate genes for qBLYV1. However, for other QTLs, the mutations may be responsible for yellows virus disease resistance, making them useful as selection markers (Tables 9 and 10). Table 9 uses RefBeet-1.2.2 as the reference genome, and Table 10 uses EL10.2 as the reference genome.

[0118] [Table 9] JPEG0007724484000010.jpg136170JPEG0007724484000011.jpg130170JPEG0007724484000012.jpg139170JPEG0007724484000013.jpg70170 [Table 10] JPEG0007724484000015.jpg133170JPEG0007724484000016.jpg137170JPEG0007724484000017.jpg143170JPEG0007724484000018.jpg132170JPEG0007724484000019.jpg136170JPEG0007724484000020.jpg133170(3: Mutation analysis using diverse breeding lines) Phenotypic surveys conducted over two years, 2021 and 2022, showed that the inoculated plants planted in the test fields generally tested positive in ELISA tests. A few negative plants were identified, but these were excluded from analysis to avoid any impact on the test results. Yellowing index surveys conducted in August and September revealed clear differences in resistance between the lines used as the resistance benchmark at each survey time point in both years. Wide variations in resistance were observed between lines, confirming similar results across the two years (Figure 19). Figure 19 shows the distribution of yellowing indexes for 98 breeding lines. The rs value in Figure 19 indicates Pearson's rank correlation coefficient (p<0.01). In Figure 19, open circles indicate strong resistance, open squares indicate moderate resistance, and open triangles indicate weak resistance.

[0119] Based on the distribution of yellowing index values at the time of the final survey in September, the strains were broadly divided into a resistant group with an index value of around 1.0 or lower, and a susceptible group with an index value of higher than that (Table 11). [Table 11] The resistant line group included six lines, including those with strong resistance. Five of these were chlorosis virus disease-resistant lines obtained from the USDA gene bank or lines developed using those lines. Therefore, considering that the USDA-derived lines exhibit resistance to Japanese viruses and that the virus species in Japan and the United States are different, it is possible that they possess common resistance to multiple types of viruses that cause chlorosis virus disease.

[0120] A total of 1,645,056 mutations were extracted from the whole genome information, and no significant bias was observed in the number of mutations per chromosome (124,712 to 276,004). Three highly versatile and useful mutations specific to resistant and susceptible lines were extracted from the neighboring regions of the genome detected by QTL analysis and QTLseq analysis (Table 12). Assuming that the mutations in "C81-22" contain resistance mutations, the targets of extraction were two of the four QTLs detected by QTL analysis: qBLYV4 and qBLYV7. qBLYV1 and qBLYV8 were excluded from the analysis because the "C81-22" type was susceptible. [Table 12] For a SNP on chromosome 4 (mutation site 15265166, gene name: XM_010676655.2), 83.3% of resistant lines were homozygous for A or T / A, while 68.5% of susceptible lines were homozygous for T. For a SNP on chromosome 7 (mutation site 8384102, gene name: XM_010685763.2), 66.7% of resistant lines were heterozygous for C / T, while 89.1% of susceptible lines were homozygous for C. For a SNP on chromosome 7 (mutation site 35415610, gene name: XM_010686933.2), 66.7% of resistant lines were heterozygous for A / G, while 84.8% of susceptible lines were homozygous for A. Therefore, the mutations that differ in frequency between these resistant and susceptible lineages are useful for breeding selection as they provide information that can be distinguished across a larger number of breeding materials. [Industrial Applicability]

[0121] The present invention can be used to efficiently select sugar beets with less yellowing symptoms by utilizing information on genetic polymorphisms, and can therefore be used to reduce the labor required for variety development.

Claims

1. A method for determining whether or not a sugar beet plant has resistance to yellows virus disease, comprising the steps of: a determining step of determining the presence or absence of resistance by identifying at least one of gene loci involved in resistance to yellows virus disease located between 11.0 Mbp and 30.0 Mbp on chromosome 4 and between 8.0 Mbp and 38.0 Mbp on chromosome 7 in the sugar beet plant, In the determination step, a molecular marker linked to the locus is detected. Identify the gene locus, The molecular marker is selected from at least one of the following regions (a) to (l): (a) a region sandwiched between a polynucleotide consisting of the base sequence shown in SEQ ID NO: 1 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 2; (b) a region sandwiched between a polynucleotide consisting of the base sequence shown in SEQ ID NO: 3 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 4; (c) a region sandwiched between a polynucleotide consisting of the base sequence shown in SEQ ID NO: 5 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 6; (d) a region sandwiched between a polynucleotide consisting of the base sequence shown in SEQ ID NO: 7 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 8; (e) a region sandwiched between a polynucleotide consisting of the base sequence shown in SEQ ID NO: 9 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 10; (f) a region sandwiched between a polynucleotide consisting of the base sequence shown in SEQ ID NO: 11 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 12; (g) a region sandwiched between a polynucleotide consisting of the base sequence shown in SEQ ID NO: 13 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 14; (h) a region sandwiched between a polynucleotide consisting of the base sequence shown in SEQ ID NO: 15 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 16; (i) a region sandwiched between a polynucleotide consisting of the base sequence shown in SEQ ID NO: 17 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 18; (j) a region sandwiched between a polynucleotide consisting of the base sequence shown in SEQ ID NO: 19 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 20; (k) a region sandwiched between a polynucleotide consisting of the base sequence shown in SEQ ID NO: 21 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 22; (l) a region sandwiched between a polynucleotide consisting of the base sequence shown in SEQ ID NO: 23 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 24; or a polynucleotide of The molecular marker is a base or base sequence corresponding to the base or base sequence of any one of the mutation sites in Table 9 or Table 10 in the Examples, and at least one of the bases or base sequences linked to the base or base sequence, or a continuous polynucleotide containing the base or base sequence: The molecular marker comprises a base corresponding to any one of the following bases (i) to (iii) and at least one base linked to the base: (i) the 1643rd base of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 25; (ii) the 742nd base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 26; (iii) the 637th base of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 27; or a continuous polynucleotide containing the base, In the DNA of the sugar beet plant, the sugar beet plant is determined to be resistant to yellows virus disease when the base or base sequence corresponding to the base or base sequence at the mutation site shown in Table 9 or Table 10 is the mutant base or base sequence shown in the column "C81-22"; The method for determining that the sugar beet plant is resistant to yellows virus disease is as follows: for the molecular markers (i) to (iii), when, in the bases of the molecular marker, an allele of the 1643rd base of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 25 or a base corresponding thereto is a homozygous T type or a heterozygous C / T type, when an allele of the 742nd base of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 26 or a base corresponding thereto is a homozygous G type or a heterozygous A / G type, or when an allele of the 637th base of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 27 or a base corresponding thereto is a homozygous A type or a heterozygous T / A type.

2. The method for determining whether the yellowing virus disease is caused by a pathogenic virus such as beet yellow leaf virus or a closely related species thereof, according to claim 1 .

3. 1. A method for producing a sugar beet plant resistant to yellows virus disease, comprising: a step of selecting sugar beet plants having resistance to yellows virus disease from sugar beet plants obtained by breeding or their progeny lines by the method of claim 1 or 2; A manufacturing method comprising:

4. a sugar beet plant having resistance to chlorosis virus disease and another sugar beet plant are used as parents for crossing, and the plant has at least one of the gene loci involved in resistance to chlorosis virus disease located between 11.0 Mbp and 30.0 Mbp on chromosome 4 and between 8.0 Mbp and 38.0 Mbp on chromosome 7, the yellows virus disease resistance loci on chromosome 4 and chromosome 7 are identified by molecular markers linked to the loci or molecular markers in linkage disequilibrium with either of these molecular markers; The molecular marker comprises at least one polynucleotide selected from the following regions (a) to (l): A molecular marker containing at least one base or base sequence corresponding to the base or base sequence of any of the mutation sites in Table 9 or Table 10 in the Examples, or a continuous polynucleotide containing said base or base sequence; or A sugar beet plant resistant to yellows virus disease, wherein the molecular marker comprises at least one base corresponding to any one of the bases (i) to (iii) below or a consecutive polynucleotide containing said base: (a) a region sandwiched between a polynucleotide consisting of the base sequence shown in SEQ ID NO: 1 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 2; (b) a region sandwiched between a polynucleotide consisting of the base sequence shown in SEQ ID NO: 3 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 4; (c) a region sandwiched between a polynucleotide consisting of the base sequence shown in SEQ ID NO: 5 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 6; (d) a region sandwiched between a polynucleotide consisting of the base sequence shown in SEQ ID NO: 7 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 8; (e) a region sandwiched between a polynucleotide consisting of the base sequence shown in SEQ ID NO: 9 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 10; (f) a region sandwiched between a polynucleotide consisting of the base sequence shown in SEQ ID NO: 11 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 12; (g) a region sandwiched between a polynucleotide consisting of the base sequence shown in SEQ ID NO: 13 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 14; (h) a region sandwiched between a polynucleotide consisting of the base sequence shown in SEQ ID NO: 15 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 16; (i) a region sandwiched between a polynucleotide consisting of the base sequence shown in SEQ ID NO: 17 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 18; (j) a region sandwiched between a polynucleotide consisting of the base sequence shown in SEQ ID NO: 19 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 20; (k) a region sandwiched between a polynucleotide consisting of the base sequence shown in SEQ ID NO: 21 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 22; (l) a region sandwiched between a polynucleotide consisting of the base sequence shown in SEQ ID NO: 23 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 24; (i) the 1643rd base of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 25; (ii) the 742nd base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 26; (iii) The 637th base of a polynucleotide consisting of the base sequence shown in SEQ ID NO:

27.

5. A kit for determining whether or not a sugar beet plant has resistance to yellows virus disease, comprising: A primer set that amplifies at least one of the following regions (a) to (l) as a molecular marker, or at least one of the regions containing a continuous polynucleotide containing at least one base or base sequence corresponding to the base or base sequence of any of the mutation sites in Table 9 or Table 10 in the Examples below, or at least one of the regions containing at least one base corresponding to any of the bases in (i) to (iii) below: (a) a region sandwiched between a polynucleotide consisting of the base sequence shown in SEQ ID NO: 1 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 2; (b) a region sandwiched between a polynucleotide consisting of the base sequence shown in SEQ ID NO: 3 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 4; (c) a region sandwiched between a polynucleotide consisting of the base sequence shown in SEQ ID NO: 5 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 6; (d) a region sandwiched between a polynucleotide consisting of the base sequence shown in SEQ ID NO: 7 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 8; (e) a region sandwiched between a polynucleotide consisting of the base sequence shown in SEQ ID NO: 9 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 10; (f) a region sandwiched between a polynucleotide consisting of the base sequence shown in SEQ ID NO: 11 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 12; (g) a region sandwiched between a polynucleotide consisting of the base sequence shown in SEQ ID NO: 13 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 14; (h) a region sandwiched between a polynucleotide consisting of the base sequence shown in SEQ ID NO: 15 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 16; (i) a region sandwiched between a polynucleotide consisting of the base sequence shown in SEQ ID NO: 17 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 18; (j) a region sandwiched between a polynucleotide consisting of the base sequence shown in SEQ ID NO: 19 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 20; (k) a region sandwiched between a polynucleotide consisting of the base sequence shown in SEQ ID NO: 21 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 22; (l) a region sandwiched between a polynucleotide consisting of the base sequence shown in SEQ ID NO: 23 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 24; The base or base sequence of any of the mutation sites in Table 9 or Table 10 in the Examples; (i) the 1643rd base of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 25; (ii) the 742nd base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 26; (iii) the 637th base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 27; Including, determining that the sugar beet plant is resistant to yellows virus disease when the base or base sequence at the mutation site shown in Table 9 or Table 10 in the PCR amplified fragment in the sugar beet plant DNA using the primer set is the mutant base or base sequence shown in the "C81-22" column; The kit determines that the sugar beet plant is resistant to yellows virus disease when, for the molecular markers (i) to (iii) in the PCR amplified fragment in the DNA of the sugar beet plant using the primer set, the allele of the 1643rd base of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 25 or a base corresponding thereto is homozygous for T or heterozygous for C / T, the allele of the 742nd base of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 26 or a base corresponding thereto is homozygous for G or heterozygous for A / G, or the allele of the 637th base of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 27 or a base corresponding thereto is homozygous for A or heterozygous for T / A.

Images