KR101770474B1 - Molecular marker for validating seed size of watermelon and use thereof - Google Patents

Abstract

The present invention relates to a molecular marker for determining the size of a watermelon seed, and more particularly to a medium-seed size (NS), a small-seed seed, a micro-seed size, SNP marker composition, a primer set, and a method for determining the size of a watermelon seed using the same.
By providing a molecular marker for determining the size of watermelon seeds according to the present invention, it is possible to predict the watermelon seed size in the seedling during the breeding process, and to determine whether the seedy-size hybrid is separated from the next generation (heterozygous) (Homozygous) can be judged. Accordingly, the marker associated with the TS size can be utilized for cultivating a variety of seeds having a very small seed size capable of simultaneous feeding with the flesh of the watermelon during the breeding of the watermelon by providing the marker of the watermelon seed size according to the present invention, The molecular markers developed in the present invention can be utilized even when the SS size is aimed at to maintain the seed productivity when cultivating watermelon without seeds.

Description

Molecular marker for validating seed size of watermelon and use thereof

The present invention relates to a molecular marker for determining watermelon seed size and its use, and more specifically, NS (medium-seed size), SS (small-seed seed), MS (micro-seed size), or TS (tamato- seed size), a SNP marker composition for discriminating a seed having a size of, a primer set, and a method of determining the size of a watermelon seed using the same.

Currently, with the development of sequencing methods and cost reduction, plant sequencing is rapidly progressing. In the case of the gourd family, the full-length nucleotide sequences of cucumber (2009), melon (2012), and watermelon (2013) have been recently disclosed, and high-density genetic maps based on genomic sequence are being created through genome sequencing. The securing of single nucleotide polymorphism (SNP) information not only distinguishes various plant lines and varieties, but also increases its utilization as a molecular marker. In particular, the problem of taking a long time from the conventional hybrid breeding method to phenotypic test has been successfully solved the disadvantages of improving breeding efficiency, shortening the breeding period, and reducing labor through the development of molecular markers related to traits.

On the other hand, the size of the seeds of watermelon (Citrullus lanatus, watermelon) varies according to the watermelon genetic source. What is characteristic is that the size of seeds produced in one fruit by lineage is very constant, and when targeting all genetic resources, GS (giant-seed size), BS (big-seed size), NS (medium-seed size), SS (small-seed size), MS (micro-seed size), TS (tamato-seed size) Classification according to is reported (Fig. 1). The size of watermelon seeds is also related to the productivity of seeds, so the smaller the size, the more the seed production per fruit tends to increase. In particular, the size of TS (smallest seed size range) is in the spotlight in Southeast Asia because it has very little refusal to eat even if it is eaten like pulp when eating watermelon, and if it can produce SS-sized watermelon seeds, triploid watermelon ( Seedless watermelon) can also improve the seed productivity. Therefore, genetic analysis on the size of seeds is very important in watermelon breeding and seeding, and when a molecular marker related to the seed size is secured, there is no cultivation period until the fruit of the watermelon is acquired, There is an advantage of being able to determine the seed size in advance with a marker.

As described above, the development of a molecular marker for determining the size of watermelon seeds is urgent, and research on this is being made, but it is still insufficient.

The present invention was conceived to solve the problems of the prior art as described above, and an object of the present invention is to analyze the QTL related to the size of watermelon seeds in an isolated group using TS3344 (TS size) and PI 189225 (GS size) as mating parents. It is to provide a molecular marker that can determine the size of watermelon seeds based on the obtained watermelon genomic region.

Another object of the present invention is to provide a method for determining the size of watermelon seeds using the molecular marker.

Another object of the present invention is to provide a completely genetically fixed watermelon line that no longer changes in seed size.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems that are not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the above object, the present invention

A polynucleotide sequence consisting of 8-100 consecutive bases including a single nucleotide polymorphism (SNP) located at the 553th base in the nucleotide sequence of SEQ ID NO: 1 or a complementary polynucleotide sequence thereof;

A polynucleotide sequence consisting of 8-100 consecutive bases including a single nucleotide polymorphism (SNP) located at the 552th base in the nucleotide sequence of SEQ ID NO: 2 or a complementary polynucleotide sequence thereof;

A polynucleotide sequence consisting of 8-100 consecutive bases including a single nucleotide polymorphism (SNP) located at the 553th base in the nucleotide sequence of SEQ ID NO: 3 or a complementary polynucleotide sequence thereof; And

A polynucleotide sequence consisting of 8-100 consecutive bases including a single nucleotide polymorphism (SNP) located at the 553th base within the nucleotide sequence of SEQ ID NO: 4 or a group consisting of a complementary polynucleotide sequence thereof It provides a SNP marker composition for determining watermelon seed size, comprising one or more polynucleotide sequences selected from or a complementary polynucleotide sequence thereof.

In addition, the present invention provides a primer set for determining the size of watermelon seeds capable of amplifying the polynucleotide sequence or its complementary polynucleotide sequence.

In one embodiment of the present invention, the primer set, a primer set consisting of SEQ ID NOs: 5 and 6; A primer set consisting of SEQ ID NOs: 7 and 8; A primer set consisting of SEQ ID NOs: 9 and 10; And a primer set consisting of SEQ ID NOs: 11 and 12.

In addition, the present invention provides a PCR kit for HRM analysis for determining the size of watermelon seeds including the primer set.

In addition, the present invention

(1) performing PCR by mixing the PCR kit and the entire DNA sample extracted from watermelon seeds; And

(2) It provides a method of determining the size of a watermelon seed comprising the step of performing a high resolution melting (HRM) analysis using the amplified sample of step (1).

In addition, the present invention provides seeds of watermelon (KCTC18406P, KCTC18407P, KCTC18405P and KCTC18408P) having a property of fixing the seed size without changing the seed size of the posterior generation.

In one embodiment of the present invention, the seeds may have seed areas of 270.41-366.45mm2, 153.87-226.53mm2, 109.10-152.88mm2, and 49.64-74.93mm2, respectively.

By providing a molecular marker for determining the watermelon seed size according to the present invention, the watermelon seed size can be predicted in the young plant during the breeding process, and whether the target posterior hybrid species will separate the seed size trait in the next generation (hetero genotype), fixed and separated It is possible to judge whether or not (homo genotype). Accordingly, by providing a molecular marker for determining the size of a watermelon seed according to the present invention, a marker associated with the TS size can be used to cultivate a variety having a very small seed size that can be fed simultaneously with the flesh during the breeding process of watermelon. On the other hand, 3 The molecular marker developed in the present invention can be used even when the SS size is aimed to maintain seed productivity when growing pear seedless watermelon.

In addition, by providing a genomic region in which sequences associated with genes that determine the seed size of the watermelon genome exist, it is possible to continuously develop molecular markers.

Furthermore, the present invention provides four fixed strains for each seed size in which the seed size does not change in later generations during self-pollination, thereby enabling direct use in the development of commercial F1 varieties, molecular markers, and phenotype verification.

1 is a diagram showing the seed sizes of watermelon genetic resources classified into six types.
Fig. 2 is a diagram showing the seed size and variation of watermelon in F1 and F2 using PI 189225 and TS3344 as parental lines.
3 shows the result of seed size distribution produced by F2 individuals after crossing watermelon PI 189225 (P1) and TS3344 (P2).
Figure 4a shows the result of QTL analysis of the seed size phenotype obtained in F2:3 after crossing of watermelon PI 189225 (P1) and TS3344 (P2) (chromosome 2).
Figure 4b shows the results of QTL analysis of the seed size phenotype obtained in F2:3 after crossing of watermelon PI 189225 (P1) and TS3344 (P2) (chromosome 6).
Figure 5 shows the genomic regions of chromosomes 2 and 6 that determine the size seeds of watermelon in NT, SS, MS, and TS.
6 shows the nucleotide sequence of SEQ ID NO: 1 and the SNP position within the nucleotide sequence.
7 shows the nucleotide sequence of SEQ ID NO: 2 and the SNP position within the nucleotide sequence.
8 shows the nucleotide sequence of SEQ ID NO: 3 and the SNP position within the nucleotide sequence.
9 shows the nucleotide sequence of SEQ ID NO: 4 and the SNP position within the nucleotide sequence.
FIG. 10 is a result of HRM analysis using SEQ ID NO: 3 for four fixed systems, NT(NS)BC4F9, NT(SS)BC4F9, NT(MS)BC4F9, and NT(TS)BC4F9. Each graph represents a genotype, and the red graph represents individuals with the same base as the reference sequence [NT(NS)BC4F9, NT(MS)BC4F9], and the blue graph represents individuals with different bases from the reference sequence [NT(SS)BC4F9, NT (TS)BC4F9], the yellow graph represents hetero (H = Ref + Alt), and the genotype is heterogeneous F1 in which NT(NS) and NT(TS) are crossed, compared to homozygous lines It was further analyzed for.

As a result of research efforts to develop an HRM marker capable of discriminating the watermelon seed size and the watermelon strain (Citrullus lanatus, watermelon) having a fixed seed size, the present inventors showed that TS3344 (TS size) X PI 189225 (GS size) F2 In the generation, the genomic region associated with the seed size is determined by QTL analysis, and the genome sequence of the four seed-sized watermelon strains produced based on this is compared with the reference sequence, and after securing the SNP, a primer that can identify the corresponding SNP is designed. As a result of the assay, it was confirmed that the primer set used showed a result in which both the associated marker and the phenotype were consistent, and the present invention was completed based on this.

Hereinafter, the present invention will be described in detail.

The present invention is a polynucleotide sequence consisting of 8-100 consecutive bases including a single nucleotide polymorphism (SNP) located at the 553th base within the nucleotide sequence of SEQ ID NO: 1 or a complementary polynucleotide sequence thereof ;

A polynucleotide sequence consisting of 8-100 consecutive bases including a single nucleotide polymorphism (SNP) located at the 552th base within the nucleotide sequence of SEQ ID NO: 2 or a complementary polynucleotide sequence thereof;

A polynucleotide sequence consisting of 8-100 consecutive bases including a single nucleotide polymorphism (SNP) located at the 553th base in the nucleotide sequence of SEQ ID NO: 3 or a complementary polynucleotide sequence thereof; And

A polynucleotide sequence consisting of 8-100 consecutive bases including a single nucleotide polymorphism (SNP) located at the 553th base within the nucleotide sequence of SEQ ID NO: 4 or a group consisting of a complementary polynucleotide sequence thereof It provides a SNP marker composition for determining watermelon seed size, comprising one or more polynucleotide sequences selected from or a complementary polynucleotide sequence thereof.

The nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 4 according to the present invention may be a base sequence present on chromosome 2 or 6 of watermelon, preferably 29,248,332-30,180,046 bp of chromosome 2 and 5,057,193- of chromosome 6 It may be a 5,927,079 bp region, more preferably 29,761,887-30,053,080 bp of chromosome 2 and 5,540,129-5,542,892 bp of chromosome 6.

In one embodiment of the present invention, the domestic watermelon cultivation line TS3344 (tomato-seed size) (flower parent: paternal) having the smallest seed size among watermelon genetic resources and the watermelon wild line PI having the largest seed size among genetic resources. After extracting the DNA of F2 generation individuals obtained through hybridization of 189225 (giant-seed size) (seed parent: maternal line), a genetic map was created through GBS analysis, and based on the results of the F2 generation seed size investigation, the quantitative trait locus ( Quantitative trait loci (QTL) analysis was performed to determine the regions 29,248,332-30,180,046 bp of chromosome 2 and 5,057,193-5,927,079 bp of chromosome 6 as seed size-related genomic regions.

In another embodiment of the present invention, each strain having a size of four seed sizes: medium-seed size (NS), small-seed seed (SS), micro-seed size (MS), and tamato-seed size (TS). After making them (NT-BC4F9, SS-BC4F9, MS-BC4F9, TS-BC4F9), based on the QTL region associated with the previously analyzed seed size, genomic sequence variations of four seed size lines within the range were determined. By comparative analysis, the previously analyzed QTL region could be further narrowed, and SNPs present in the region were secured.

The size of watermelon seeds can be determined as follows through the combination between the SNP markers according to the present invention:

■ NS (medium-seed size): Including the base sequence of SEQ ID NO: 1 to SEQ ID NO: 4 (same as the reference base sequence)

■ SS (small-seed seed): contains the nucleotide sequence of SEQ ID NO: 1 (same as the reference nucleotide sequence), and does not include the nucleotide sequence of SEQ ID NO: 2 to SEQ ID NO: 4 (different from the reference nucleotide sequence, that is, , The 552th base in the nucleotide sequence of SEQ ID NO: 2 is G, the 553th base in the nucleotide sequence of SEQ ID NO: 3 is deleted, and the 553th base in the nucleotide sequence of SEQ ID NO: 4 is T)

■ MS (micro-seed size): Does not include the nucleotide sequence of SEQ ID NO: 1 (differs from the reference nucleotide sequence, that is, the 553th base in the nucleotide sequence of SEQ ID NO: 1 is C), SEQ ID NO: 2 to sequence Includes the nucleotide sequence of number 4 (same as the reference nucleotide sequence)

■ TS (tamato-seed size): Does not include the nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 4 (different from the reference base sequence, that is, the 553th base in the nucleotide sequence of SEQ ID NO: 1 is C, and SEQ ID NO: The 552th base in the nucleotide sequence of 2 is G, the 553th base in the nucleotide sequence of SEQ ID NO: 3 is deleted, and the 553th base in the nucleotide sequence of SEQ ID NO: 4 is T)

By using a primer set capable of amplifying the SNP marker as a molecular marker, the size of watermelon seeds can be predicted in a young plant during the breeding process. Accordingly, as another aspect of the present invention, the present invention provides the polynucleotide sequence or its complementary It provides a primer set for determining the size of watermelon seeds capable of amplifying the polynucleotide sequence, preferably a primer set consisting of SEQ ID NOs: 5 and 6; A primer set consisting of SEQ ID NOs: 7 and 8; A primer set consisting of SEQ ID NOs: 9 and 10; And a primer set consisting of SEQ ID NOs: 11 and 12.

In the present invention, the term "primer" is a nucleic acid sequence having a short free 3'hydroxyl group, which can form a base pair with a complementary template and functions as a starting point for template strand copying. It means a short nucleic acid sequence. Primers can initiate DNA synthesis in the presence of reagents for polymerization and four different nucleoside triphosphates at appropriate buffers and temperatures.

In another embodiment of the present invention, phenotypic and genotypic assays of four fixed lines (NT-BC4F9, SS-BC4F9, MS-BC4F9, TS-BC4F9) were performed using four primer sets. It was confirmed that the primer sets of the dogs showed the same results in both the association marker and the phenotype.

As described above, the primer set according to the present invention can be used to confirm the mutation in the nucleic acid sequence through HRM (High resolution melting) analysis. As another aspect of the present invention, the present invention comprises the primer set. A PCR kit for HRM analysis is provided to determine the size of watermelon seeds.

In the present invention, "HRM" changes the temperature of the amplified product DNA combined with a fluorescent reagent from about 60°C to 90°C according to the SNP on the DNA present in the PCR region, and changes the fluorescence intensity when denatured into single-stranded DNA. Means how to measure.

In addition, as another aspect of the present invention, the present invention

1) performing PCR by mixing the PCR kit with the entire DNA sample extracted from watermelon seeds; And

2) It provides a method of determining the size of watermelon seeds comprising the step of performing a high resolution melting (HRM) analysis using the amplified sample of step 1).

In addition, in the present invention, the fixed strains for each seed size (NS, SS, MS, TS) produced for the development of markers associated with the seed size are genetically completely fixed strains that do not change the seed size of later generations, and are used for commercial breeding. These are fixed systems that can also be used as systems.

Thus, as another aspect of the present invention, the present invention provides seeds of watermelon (KCTC18406P, KCTC18407P, KCTC18405P and KCTC18408P) having a property that the seed size is fixed without changing the seed size of the posterior generation.

Hereinafter, a preferred embodiment is presented to aid the understanding of the present invention. However, the following examples are provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[Example]

Example 1. QTL analysis to determine watermelon seed size

1-1. Genetic mapping and identification of the seed size of the F2 population

QTL analysis was performed to determine the region on the genome that determines the watermelon seed size. The domestic watermelon cultivation system TS3344 (tomato-seed size) (plant parent: paternal), which has the smallest seed size among watermelon genetic resources, and the wild watermelon strain PI 189225 (giant-seed size), which has the largest seed size among the genetic resources ( F1 obtained through hybridization of seed parent: maternal) was self-pollinated to secure 165 individuals in the F2 generation (FIG. 2), and GBS (genotyping by sequencing) analysis was performed to create a SNP-based genetic map.

More specifically, the genomic DNA extracted from each individual F2 is cut with the restriction enzyme ApeKI, and then the DNA sequence (barcode) that can identify each individual and the oligo DNA (collectively, the adaptor) synthesized for sequencing are ligated. Thereafter, PCR was performed once using a PCR primer complementary to an adapter sequence, followed by purification to prepare a sequencing library. The prepared library was analyzed for surrounding nucleotide sequences cut with ApeKI through NGS (next generation sequening), and the watermelon reference genome (http://www.icugi.org) and PI 189225 (maternal) and TS3344 (maternal) were analyzed. After comparing and analyzing the whole genome sequencing results of paternal), single nucleotide sequence polymorphism (SNP) was derived, and then based on the obtained SNP information, a related gene map was created using JoinMap (v. 4.1) software. I did.

In addition, as a result of examining the size of seeds produced by F2 generation individuals, it was confirmed that the distribution of quantitative traits was shown in FIG. 3.

1-2. Watermelon seed size correlation QTL analysis

A quantitative trait loci (QTL) analysis was performed based on the genetic map prepared in Example 1-1 and the result of the seed size investigation of the F2 generation.

As a result, as shown in FIG. 4, it was confirmed that the quantitative trait locus associated with the seed size determined through this was present in watermelon chromosomes 2 (qC2Sz1) and 6 (qC6Sz1). QTL on chromosome 2 had a dominant effect of 10.29 and QTL on chromosome 6 had a dominant effect of -9.85, indicating that the QTL of each chromosome affected the larger and smaller seeds. The analyzed QTL region was 29,248,332 to 30,180,046 bp for chromosome 2 and 5,057,193 to 5,927,079 bp for chromosome 6.

Example 2. Fabrication of fixed systems in four size ranges (NS, SS, MS, TS)

For the development of markers related to seed size, a line having four seed sizes: medium-seed size (NS), small-seed seed (SS), micro-seed size (MS), and tamato-seed size (TS). I made them.

More specifically, the F1 individuals obtained after hybridization of the commercial stationary system NT that produces medium-seed size seeds and the commercial stationary system TS3344 that produces tomato-seed size seeds are first hybridized to NT and TS3344, respectively. I did. At this time, seeds of the size of NS, SS, and MS appeared in the later generations of the NT and female hybrids, and the NS and SS sized plants were subjected to self-pollination of each NS and SS-sized line 8 times after female hybridization three more times. Fixed systems of different NS and SS sizes were fabricated.

On the other hand, when F1 was cross-crossed with TS3344, separate entities of MS and TS appeared, and after cross-crossing with TS three more times, each MS and TS-sized lines were self-pollinated eight times to separate different MSs and TSs. A fixed system of size was produced.

Four fixed systems that produce seeds of different sizes are no longer separated during self-pollination, and are provided as fixed systems (NT-BC4F9, SS-BC4F9, MS-BC4F9, TS-BC4F9) representing four different seed sizes. I did.

Example 3. Development of HRM markers for determining the size of watermelon seeds

3-1. Securing SNP to determine the size of watermelon seeds

After sowing the different fixed systems prepared in Example 2, DNA was extracted from the leaves, and the entire genome was subjected to next generation resequencing. The obtained nucleotide sequence was compared with the reference genome sequence of watermelon, and the mutant nucleotide sequence was analyzed.

In addition, based on the QTL region associated with the seed size analyzed in Example 1-2, as a result of comparative analysis of genomic sequence mutations of four seed size strains within the corresponding range, as shown in FIG. 5, the reference base Based on the sequence, in the 29,761,887- 30,053,080bp region of chromosome 2, the NS and SS seed size lines had the same sequence as the reference base sequence, and the MS and TS seed size lines had different sequences from the reference base sequence. Meanwhile, in the 5,540,129-5,542,892bp region of chromosome 6, NS and MS had the same sequence as the reference base sequence, and SS and TS had a different sequence from the reference base sequence.

Summarizing the above results, the 553 nucleotide in the nucleotide sequence of SEQ ID NO: 1, the 552 nucleotide in the nucleotide sequence of SEQ ID NO: 2, the 553 nucleotide in the nucleotide sequence of SEQ ID NO: 3, and the nucleotide sequence of SEQ ID NO: 4 The SNP located at the 553th base was obtained (see FIGS. 6 to 9).

3-2. Create a primer set

Primer 3 (http://primer3.ut.ee/), a web software published around the SNP obtained through Example 3-1, was used to prepare a primer set for each SNP, and for each primer set Specific information is shown in Table 1 below.

Primer Sequence mer Set 1
WSize-Ch2-1aF primer GGTGGGCATT TTCCAGCAGA TC (SEQ ID NO: 5) 22 WSize-Ch2-1aR primer CTCTTCTCCC CGCTCTTCAA (SEQ ID NO: 6) 20 Set 2
WSize-Ch6-1F primer ACACTACCCC GAGCTTTGAT (SEQ ID NO: 7) 20 WSize-Ch6-1R primer AGGTGAGCTT AACGACATAC AT (SEQ ID NO: 8) 22 Set 3
WSize-Ch6-2aF primer AGGGAGTCTC GCACACTTAG (SEQ ID NO: 9) 20 WSize-Ch6-2aR primer AGCACCTATG TTCCCCTGAA (SEQ ID NO: 10) 20 Set 4
WSize-Ch6-4F primer ATTGTGGTGG GGCTTAGGAT (SEQ ID NO: 11) 20 WSize-Ch6-4R primer ACCTCGACCA ACCCAAATCT (SEQ ID NO: 12) 20

Example 4. Validation of the applicability of HRM markers

First, the phenotypes of four fixed systems (NT-BC4F9, SS-BC4F9, MS-BC4F9, TS-BC4F9) prepared through Example 2 were tested. That is, the four fixed systems (NT-BC4F9, SS-BC4F9, MS-BC4F9, TS-BC4F9) were measured by dividing 20 grains for each 200 grains to obtain their average, maximum and minimum values (Table 2). In addition, each seed was photographed and the seed area was calculated with ImageJ software (Table 3).

ID NS SS MS TS Lineage NT(NS)BC4F9 NT(SS)BC4F9 NT(MS)BC4F9 NT(TS)BC4F9 Average (gram) 0.72425 0.42383 0.21905 0.11758 Maximum value (gram) 0.741 0.4334 0.2361 0.1247 Minimum value (gram) 0.6967 0.4123 0.2046 0.1105

ID NS SS MS TS Lineage NT(NS)BC4F9 NT(SS)BC4F9 NT(MS)BC4F9 NT(TS)BC4F9 Average(㎟) 314.53 185.61 124.08 62.73 Minimum value (㎟) 270.41 153.87 109.10 49.64 Maximum value (㎟) 366.45 226.53 152.88 74.93

Next, genotyping was performed on 20 individuals of each of the four fixed lines using the primer set prepared in Example 3. At this time, in the SNP-based HRM analysis, nucleotides identical to the sequence of the reference genome were expressed as Ref, and for other nucleotides, they were expressed as Alt, and the seed size was determined by four genotype combinations in chromosomes 2 and 6. As a result, as shown in Table 4 below, the four primer sets used had a result in which both the association marker and the phenotype were consistent.

In addition, HRM analysis was performed for each line of NS, SS, MS, and TS using SEQ ID NO: 3. As a result, as shown in FIG. 10, the phenotypic result and the analyzed genotype result were consistent.

Additionally, genotype and phenotypic assays of the F2 isolated generation obtained after crossing of the NS and TS size fixed lines were performed. More specifically, as a result of performing the HRM marker assay for the seed size in the F2 isolated generation obtained after crossing the NS size and the TS size fixed line [NT(NS)BC4F9 x NT(TS)BC4F9], as shown in Table 5 below Likewise, all of the association markers and phenotypes were consistent with the exception of NS/TS F2-22.

Therefore, it was demonstrated that NS, SS, MS, and TS seed sizes can be distinguished through the combination of the HRM markers presented through the present invention.

The above description of the present invention is for illustrative purposes only, and those of ordinary skill in the art to which the present invention pertains will be able to understand that other specific forms can be easily modified without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and are not limiting.

Microbial Resource Center, Korea Research Institute of Bioscience and Biotechnology KCTC18406P 20150930 Microbial Resource Center, Korea Research Institute of Bioscience and Biotechnology KCTC18407P 20150930 Microbial Resource Center, Korea Research Institute of Bioscience and Biotechnology KCTC18405P 20151006 Microbial Resource Center, Korea Research Institute of Bioscience and Biotechnology KCTC18408P 20151006

<110> Chung-Ang University Industry-Academy Cooperation Foundation <120> Molecular marker for validating seed size of watermelon and use thereof <130> MP15-160 <160> 12 <170> KoPatentIn 3.0 <210> 1 <211> 1106 <212> DNA <213> Citrullus lanatus <400> 1 tcagtggttc tgtagcaccc agacggattc atctggcggc gttgccgtac cccggcagag 60 gccacatcaa cgctctcatg aatctctgca agcttctctc tctcaaaaac cccaacattc 120 tcatctcctt catcgtcacc gacgagtggc tcaccttcct cgccgccgat cccaaacccc 180 aaaacatccg tttcgccact ttccccaatg ttatcccctc tgagctcggc cgcgccaacg 240 acttcaccgg tttcctccga tccatccaca cccatatgga ggctcccgtt gagactctac 300 tccatcgcct cgacccgccg ccgactgcca ttctcgccga tgccttcgtc acttgggctg 360 tccagttggg gaaacgcctc aatgttccgg tcgcttcact ctggcccatg tcggctacgg 420 ttttctccat cctttaccat ttcgaccttc tcaaggaaaa tgggcatttt ccagcagatc 480 tctcaggtaa ctttaaaatc taattgatgg aaattacatt tttggaatca ggatttaagt 540 tctttcttac tctgttttga agagcgggga gaagagattg tcgattactt ccccggagtg 600 tcgaagattt gtcttgcaga tttgccgtct ttcttttctg gcgatggtct ccaaagcgtc 660 gaatccgccg tgaactccgc ccgttccgtc gacaaatccc aatttttcat ctccacctct 720 gtttacgagc ttgaatcctc tgttatcgac gccttaaaag caaaatttcc cttcccgatt 780 tacaccatcg gacccagtac tccatatttc gagctagaat gctccgcccc aaatggcggc 840 accgacgact atttccggtg gctggactcc caagcagagg gctctgtttt gtacatttca 900 cagggcagtt atctttcagt ttctagcgcc caaatagacg agatcgtcgc cggggtgaaa 960 gccagcggcg ttcggttctt gtgggtggtg cgtggagatg acggccggtt gaaggacgtg 1020 gacagagaaa ctgggatggt ggttggatgg tgcgatcaat tgaaggttct gtgccacaga 1080 tccgtgggag ggttttggac tcacgg 1106 <210> 2 <211> 1106 <212> DNA <213> Citrullus lanatus <400> 2 aaaaacagaa tgaaatgcac tgctctcttc cacgtttcta aattgaatgg taactcctct 60 ggaatgagat ttctgtttta aaagcaaaac tattcaatga cgcgagcaag aacagggctt 120 gattcttcaa caagttaaat tgtacagaat tacagatact gaattaccat tgcagagaca 180 gatttgtaag ggaagcaaca cataagaaat gacaaagttg atcaaaatgg tgccataaat 240 ttttttttaa gagcatgcaa acatttcttc acaatgtcgt agttattagc atgagactac 300 cattcttacc tcttcgctgg catcgccttc tgaaatgcaa actgactgaa cagggcctaa 360 ccgaaatatc tgtagacgtg aaaagaaata taaaattaat catattacat tctctaaatt 420 gatcacacca tcgattcatc gctaacaaat tctagcaaat accaacacgt aaacaattga 480 tcaatatgtc atatacgaca gtgcaagagt tcaacacact accccgagct ttgatactat 540 agtaaatcat cattgagcta aaagctaggt atctaggtta tggaacattt aaactattat 600 aagaacggaa aatgtatgtc gttaagctca cctgtaaagc aaaacgagca ttcttactat 660 gcctcactat ttctcatttt gaacaatgca gaaccaaaaa acaaaataaa gaaaacaaaa 720 aatagttctc ttttttctct cttctttatt cgttctaaac tctccaacac actagacaac 780 aatcacgata cgcaaattca ttacaaatga aacagaacct gagaagtccg aagatcgaaa 840 gcaaggtcat cggaatcagc ctcctgccgg aaccgaagct ctgccgcgcc ggaatcgcca 900 ctgaaccgcg caaccaccgg cgcggacgaa gaggacgagg aggaagacga agaagcaagc 960 tcagaaatcg aaacaagagt gaactcctga ggtttgtgct tttgccctaa ggagccttcc 1020 attaatgcaa aacctgaagc cgaaaatgta tcttcactcc aaaaaaaatg gaaaaatata 1080 gagtcgaagt ttcaatagtg caaagc 1106 <210> 3 <211> 1106 <212> DNA <213> Citrullus lanatus <400> 3 tatatttatt ttaaaatcat ggttggttac ttaatgggtt ggctttgtgt tgtctcaaat 60 cttgtcccaa cggttgtctc accgggtgtt gattatgcag aatattttat ttccttatta 120 tgcatctcat ttcttaattg tccgcctttc ttgactacgg tttaccgact ttatttgagg 180 gattataatt tccatatttt tgttgtgcta tttatgagca agaagtgatc ttgttatggt 240 tgtcgcattt gcttttaaga ggttgttcat tgtgttcccg acatagaaca ttttgtgggt 300 tttatgtttg ttgaacaata agttttctct ttattggcag cttgttctat tattgcatgt 360 tcaatgatta cttcatcctt gagaagcttt attcgttctt aggggagcct aaggataagt 420 tcattgaaaa tggattctct gacttagggg gagtctaagc cttccattaa aggaaagcta 480 atacaagaga agtgctcttc aatatgagag ggagtctcgc acacttaggg ggagtctaag 540 tgctttttca ttgatacgca tatacttaga gggagcctaa gtttgatgaa gaagaattct 600 cacatttcag gggaacatag gtgctacgtg tgtcgttgta ttcgttattt atattacata 660 taagtacaat gttgtaaatg cttgatttta ttatattagt taatataatt tcttccctta 720 gcacactacc caacagacgt aggtgtattt caccgaacta ggtcaacaaa ctttaagtga 780 tattatctct ctttattgct tgttgtccta atgtgtgata aagcgttgta tgtgacatct 840 gtgtggtatg tatcagattc tcctctattt ttcaatatat atatatatat atatatacct 900 aagacattta aattattatt attattttgt ttttaaaaaa tatattgtta tttagaattt 960 aataaattta aaattaaagt tatcttagtt ggagatattc tcattttgat cacttattta 1020 taaaattata caagtaaaac gcaacgtttt tttctttttt ttaaaggtaa attgtttatt 1080 tattaattct accattacat tgcgtg 1106 <210> 4 <211> 1106 <212> DNA <213> Citrullus lanatus <400> 4 ataacaaagt ttagatatgt tttttttttt cttttttctt taaataaaag gcatagttgt 60 gaacttcttt cttaatagtt tagctaataa aataatatga aacagtggta taggcaaact 120 actcgaagtc agtgatacta tagtataaaa tttaaattaa atattgatca aggcttatgt 180 gattcgacat tcaagtgagg gagtttgagc ttttgcataa atctgcgtta ttgttaaata 240 aacaatgaag aacaaatgat aatattgatg gtaaaattag gataaggaaa ctctttgaac 300 ttcatattct ttccacatta aatttataat tttggggaag gaagacgact aacaagataa 360 aaagagagaa catcgttgtg gtacctaact cataacctcc tatacttaaa caagtattta 420 gcaagatgtt agagcaagtt agaaagaagt aaaagaactt acttttgggt gtgcttgtga 480 ccccatattt ttattgtggt ggggcttagg atccaaacct aaagaagatt aagaaaagtt 540 gattcgggtc ggcctatgag gctaagattt gggttggtcg aggtacgcga gttgagttgt 600 tcggctcaac tcccttcttc ctcttggtct tttcctccct tggcctgttg gcatttctct 660 aattcttgcg tgacgataat ttttttgaac ttggaattct tgattagaga cgatctataa 720 tagataatat ttaaaataaa tttataataa acattagttt ttttatggaa aaaaaattat 780 aaatcaaaac aacattttga acaatttttt tagtacaata aaacgtgaga gattcaaatc 840 acaaaccttt taattactaa ttcatatttt atgtattgaa ctaggctcac cctaccaaat 900 tttgctttat taataaaaat atttgaataa agagcttaat aaaaacaaaa gttgaataat 960 aaaaataggt tttagactta cattacatgc accttttccc ccattttttc atatagaatg 1020 aaataaatgt gactatttga aaattaatat acaatcgatt ttatcaggtg cttaaaacgg 1080 ttaaaatgtt tctaacttag ttttac 1106 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> WSize-Ch2-1aF primer <400> 5 ggtgggcatt ttccagcaga tc 22 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> WSize-Ch2-1aR primer <400> 6 ctcttctccc cgctcttcaa 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> WSize-Ch6-1F primer <400> 7 acactacccc gagctttgat 20 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> WSize-Ch6-1R primer <400> 8 aggtgagctt aacgacatac at 22 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> WSize-Ch6-2aF primer <400> 9 agggagtctc gcacacttag 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> WSize-Ch6-2aR primer <400> 10 agcacctatg ttcccctgaa 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> WSize-Ch6-4F primer <400> 11 attgtggtgg ggcttaggat 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> WSize-Ch6-4R primer <400> 12 acctcgacca acccaaatct 20

Claims (2)

Seed of watermelon deposited with deposit number KCTC18405P, which has the property that the seed size is fixed without changing the seed size.
The method according to claim 1,
Wherein the seed has a seed area of 109.10-152.88 mm &lt; 2 &gt;.

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