KR102359027B1 - Single Nucleotide Polymorphism Molecular Marker Related to fruit acidity Characteristic in Apple and uses thereof - Google Patents

Abstract

The present invention relates to a single nucleotide polymorphic marker associated with the malic acidity trait and uses thereof. The single nucleotide polymorphism marker associated with the apple acidity trait according to the present invention has a high genetic similarity, so that it is possible to accurately discriminate a variety according to the acidity trait and acidity that does not show a significant difference in the phenotype. This means that when the marker of the present invention is used, the acidity of apples can be discriminated only by leaf leaves without apple fruit. .

Description

Single Nucleotide Polymorphism Molecular Marker Related to fruit acidity Characteristic in Apple and uses thereof

The present invention relates to a single nucleotide polymorphism marker associated with the malic acidity trait and uses thereof.

Plant breeding is being greatly accelerated by marker-assisted selection (MAS). Pedigree-based quantitative trait loci (QTL) mapping and genome wide association study (GWAS) are powerful tools to map both qualitative and quantitative traits. Recently, BSA-seq, a QTL mapping strategy based on next-generation sequencing (NGS), has succeeded in rapidly mapping genes in several crops and vegetables. When MapQTL and BSA-seq were combined, ARN6.1 and Ef1.1 genes, which are AAA ATPase genes for water tolerance and early flowering, respectively, in cucumber ( Cucumis sativus ); and ABA1/ZEP gene for heat resistance of Korean lettuce ( Lactuca sativa ); were efficiently identified. Nevertheless, MAS is still difficult to apply in practice to the breeding program of perennial plants such as apple ( Malus domestica Borkh.).

On the other hand, apple ( Malus domestica Borkh.) is an economically important temperate crop because of its high production worldwide. Because apples have a large plant size, self-incompatibility, high heterozygosity, and a relatively long juvenile period, it is difficult to identify fruit characteristics compared to annual crops. There is this. Apples are also considered as model species for comparative and functional genome studies of plants of the Rosaceae family.

The main objective of the apple breeding program is to combine fruit quality with disease resistance. Studies on existing varieties have reported that quality characteristics, including titratable acidity (TA), soluble solids content (SSC), and firmness of fruit quality, have a significant effect on consumer preference. . This quality characteristic is the main goal of consumer-oriented plant breeding in apples. Apple breeders are taking advantage of the wide range of genetic and phenotypic diversity to meet consumer demand for new, higher fruit quality apples.

Accordingly, the present inventors developed a single nucleotide polymorphism marker for discriminating the acidity trait of apples, and through this, confirmed that the acidity trait could be discriminated only from the leaves of the apple, thereby completing the present invention.

Therefore, an object of the present invention is the 1332163th position of the apple chromosome 16 (NC_024254.1); Or a composition for determining the apple acidity trait comprising an agent capable of detecting a single nucleotide polymorphism marker at the locus of 1545939th position of the chromosome 16 of apple (NC_024254.1), and an apple acidity trait discrimination kit and method using the same is to provide

In order to achieve the above object, the present invention provides an apple chromosome 16 (NC_024254.1) at the 1332163th position; or 1545939th position of chromosome 16 of apple (NC_024254.1); provides a composition for identifying apple acidity traits comprising an agent capable of detecting a single nucleotide polymorphism marker of a locus.

The present invention also provides an apple acidity characterization kit comprising the composition for discriminating the malic acidity trait.

In addition, the present invention comprises the steps of (a) extracting the DNA of the apple; (b) using the DNA of step (a) as a template, and performing a polymerase chain reaction using the composition for determining the malic acidity; and (c) analyzing the polymerase chain reaction product to determine the acidity trait of the apple.

The single nucleotide polymorphism marker associated with the apple acidity trait according to the present invention has a high genetic similarity, so that it is possible to accurately discriminate a variety according to the acidity trait and acidity that does not show a significant difference in the phenotype. This means that when the marker of the present invention is used, the acidity of apples can be discriminated only by leaf leaves without apple fruit. .

1 is a diagram showing the results of selecting significant SNPs through genotyping by sequencing (GBS) analysis.
2 is a diagram showing the results of group structure analysis (a, b), principal component analysis (PCA) results (c), and phylogenetic trees (d) of apple genetic resources.
3 is a diagram showing a QQ-plot prepared based on the GWAS results according to the acidity trait of apple genetic resources.
4 is a diagram showing a manhattan plot prepared based on the GWAS results according to the acidity trait of apple genetic resources.
5 is a diagram showing the results of determining the acidity of apples using the primer set C16S63 according to the present invention through HRM (High Resolution Melting) analysis (A: melting curve, B: difference graph).
6 is a diagram showing the result of determining the acidity of apples using the primer set C16S39B according to the present invention through HRM analysis (A: melting curve, B: difference graph).

Hereinafter, the present invention will be described in detail.

According to an aspect of the present invention, the present invention relates to an apple chromosome 16 (NC_024254.1) at position 1332163; or 1545939th position of chromosome 16 of apple (NC_024254.1); provides a composition for identifying apple acidity traits comprising an agent capable of detecting a single nucleotide polymorphism marker at the locus .

In the present invention, single nucleotide polymorphism (SNP) refers to a genetic change or mutation showing a difference in one nucleotide sequence (A, T, G, C) from a DNA nucleotide sequence, and DNA sequence polymorphism ( polymorphism) is the most common form.

In the present invention, polymorphism refers to a case in which two or more alleles exist at one locus, and among polymorphic sites, only a single base differs from one person to another is called single nucleotide polymorphism. . Preferred polymorphic markers have two or more alleles that exhibit an incidence of at least 1%, more preferably at least 10% or 20% in a selected population.

In the present invention, an allele refers to several types of a gene present on the same locus of a homologous chromosome. Alleles are also used to indicate polymorphism, for example, SNPs have two types of alleles.

In the present invention, a marker refers to a nucleotide sequence used as a reference point when identifying a genetically unspecifically related genetic locus, and the location of the marker on a genetic map may be indicated as a genetic locus.

In an embodiment of the present invention, the single nucleotide polymorphism marker is a single nucleotide polymorphism marker in which the base at position 1332163 of the apple chromosome 16 (NC_024254.1) is AA, AG or GG; or a single nucleotide polymorphism marker in which the base at position 1545939 of chromosome 16 of apple (NC_024254.1) is AA, AG or GG;

In a preferred embodiment of the present invention, when the base at position 1332163 of chromosome 16 of the apple is AA or AG, the acidity may be determined to be 1.0% or more, and if the base is GG, the acidity may be determined to be less than 1.0%. In addition, when the base at position 1545939 of chromosome 16 of the apple is GG, the acidity can be determined to be 1.3% or more, and when the base is AA or AG, the acidity can be determined to be less than 1.3%.

Furthermore, based on the result of determining the acidity trait, the variety of the apple may be determined. For example, if the base at position 1332163 of chromosome 16 is AA or AG, the apple variety has an acidity of 1.0% or higher, i.e., Asiro8, Akita Tare, Roberts Crab, Asiro3, Dolgo, Spy, Lodi, Jonathan, Noya. , Humboldt, Parkland, and M7 can be identified as one or more selected from the group consisting of, and in the case of GG, cultivars with an acidity of less than 1.0%, that is, Jonared, Jiguan, Red well, Crimson King, Virginia Gold, Sansa, Hongro, Granny Smith, Fuji , Tsugaru, Picnic and Yoko can be identified as one or more selected from the group consisting of. In addition, when the base at the 1545939th position of the apple chromosome 16 is GG, it can be identified as one or more selected from the group consisting of cultivars with an acidity of 1.3% or higher, that is, Asiro8, Asiro3, Akita Tare, Ottawa8, Maypole and Purple wave. In the case of AA or AG, it can be determined as one or more selected from the group consisting of varieties with an acidity of less than 1.3%, that is, Ginger gold, Crimson king, Mayqueen, Alps Otome, Hongro, Gamhong, Tallmans sweet and Britegold.

In an embodiment of the present invention, the agent capable of detecting the single nucleotide polymorphism marker comprises: a polynucleotide comprising 10 to 100 consecutive DNA sequences comprising a single nucleotide polymorphism marker site; Or a complementary polynucleotide thereof; and a polynucleotide that specifically hybridizes (hybridization) is preferred, but is not limited thereto.

In a preferred embodiment of the present invention, the agent capable of detecting the single polymorphic marker may be a polynucleotide, which is selected from the group consisting of primers, probes, and combinations thereof capable of detecting the single polymorphic marker. may be more than one species.

In a more preferred embodiment of the present invention, the polynucleotide may be a primer, which may be such that the 3' last base is complementary to a single nucleotide polymorphism site.

In the present invention, the primer is a nucleic acid sequence having a short free 3' hydroxyl group and can form a base pair with a template of a complementary nucleic acid, and is used for strand copying of the nucleic acid template. It refers to a short nucleic acid sequence that serves as a starting point. Primers are capable of initiating DNA synthesis in the presence of reagents for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in appropriate buffers and temperatures.

When designing the primer, there are various restrictions such as the A, G, C, and T content ratio of the primer, prevention of primer dimer formation, and prohibition of repeating the same nucleotide sequence more than 3 times. (template) Conditions such as DNA amount, primer concentration, dNTP concentration, Mg ²⁺ concentration, reaction temperature, and reaction time must be appropriate.

The above primers may incorporate additional features that do not change the basic properties. That is, the nucleic acid sequence can be modified using a number of means known in the art. Examples of such modifications include methylation, encapsulation, substitution of a nucleotide with one or more homologues and uncharged linkages such as phosphonates, phosphotriesters, phosphoroamidates or carbamates or phosphorothioates or phosphorodithioates. Modification of nucleotides into charged linkages such as In addition, the nucleic acid may include one or more of nucleases, toxins, antibodies, signal peptides, proteins such as poly L lysine, intercalating agents such as acridine or psoralen, chelating agents such as metals, radioactive metals, iron oxidizing metals, and alkylating agents. It may have additional covalently attached residues.

In addition, the primer sequence of the present invention can be modified using a label capable of directly or indirectly providing a detectable signal. The primer may include a label that can be detected using spectroscopic, photochemical, biochemical, immunochemical or chemical means. Useful labels include proteins for which ³² P, fluorescent dyes, electron-dense reagents, enzymes (commonly used in ELISAs), biotin or haptens and antisera or monoclonal antibodies are available.

The primers of the present invention are cloning and restriction enzyme digestion of an appropriate sequence, phosphotriester method such as Narang (1979, Meth, Enzymol. 68:90-99), diethyl phosphoramidi such as Beaucage. Chemical synthesis may be accomplished using any other well-known method, including direct chemical synthesis methods, such as the method of chemotherapy (1981, Tetrahedron Lett. 22: 1859-1862), and the solids supported method of US Pat. No. 4458066.

In an embodiment of the present invention, the composition for determining the apple acidity trait can determine the acidity trait of an apple from a fruit, leaf, stem, root or seed sample, and determining the apple acidity trait from a young leaf sample Preferably, but not limited thereto.

The single nucleotide polymorphism marker associated with the apple acidity trait according to the present invention has a high genetic similarity, so that it is possible to accurately discriminate a variety according to the acidity trait and acidity that does not show a significant difference in the phenotype. This means that when the marker of the present invention is used, the acidity of apples can be discriminated only by leaf leaves without apple fruit. .

According to another aspect of the present invention, there is provided an apple acidity characterization kit comprising the composition for discriminating the apple acidity trait.

In the present invention, the discrimination kit can detect the single nucleotide polymorphism marker to discriminate the acidity trait of apples. The malic acidity characterization kit may include polynucleotides, primers, and probes for detecting a single nucleotide polymorphism marker, and may include one or more other components, solutions or devices suitable for the analysis method.

For example, the malic acidity characterization kit of the present invention may be a kit including essential elements for performing an allele-specific polymerase chain reaction. The allele-specific polymerase chain reaction kit includes a test tube or other suitable container, reaction buffer (pH and magnesium concentration vary), dNTP (DeoxyriboNucleotide), in addition to a polynucleotide, primer, or probe specific for the mononucleotide polymorphic marker of the present invention. TriPhosphate), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNAse inhibitors, DEPC-water and sterile water, and the like.

According to another aspect of the present invention, the present invention provides a method for determining malic acidity trait.

The method for determining the apple acidity trait comprises the steps of (a) extracting apple DNA; (b) using the DNA of step (a) as a template, and performing a polymerase chain reaction using the composition for determining the malic acidity; and (c) analyzing the polymerase chain reaction product to determine the acidity trait of the apple.

In an embodiment of the present invention, in step (a), the acidity trait of an apple can be determined from a fruit, leaf, stem, root or seed sample, and it is preferable to extract the apple DNA from the leaf sample, but is not limited thereto. does not

In the present invention, polymerase chain reaction (PCR) refers to an experimental method for amplifying only a desired part of a DNA chain in large quantities, for example, polymerase chain reaction (Polymerase Chain Reaction), allele-specific polymerization Allele Specific Polymerase Chain Reaction, Nested-Polymerase Chain Reaction, multiplex Polymerase Chain Reaction, multiplex PCR, competitive Polymerase Chain Reaction ), real-time Polymerase Chain Reaction, Real-time Quantitative Polymerase Chain Reaction, DNA chip, and loop-mediated isothermal amplification. It may be one or more methods selected from the group, preferably an allele-specific polymerase chain reaction, but is not limited thereto.

In an embodiment of the present invention, in the determination of the apple acidity trait in step (c), when the base at position 1332163 of chromosome 16 of apple is AA or AG, the acidity can be determined to be 1.0% or more, and in the case of GG, the acidity is 1.0% less can be identified. In addition, when the base at position 1545939 of chromosome 16 of the apple is GG, the acidity can be determined to be 1.3% or more, and when the base is AA or AG, the acidity can be determined to be less than 1.3%.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1. Confirmation of apple genetic resources for GWAS (genome-wide association study) analysis

reference set; A part of the core collection selected in the 1st stage of the Next-Generation BioGreen 21 project; and a portion of domestic apple genetic resources; samples for GWAS analysis were selected. The selected apple genetic resources are 308, and are shown in Table 1.

No. Sample No. Sample No. Sample No. Sample One Adam's Crab 78 Kile 155 red well 232 Sinano-red 2 Akane 79 Kingstone Black 156 Reinette du Canada 233 Sinano-gold 3 Akita 80 Kinrei 157 Reinette Grace 234 Tsugaru 4 Akita Tare 81 Lady Williams 158 Rescue 235 Arisoo 5 Alome Mills 82 Landsberger Reinette 159 Rev.w Wilks 236 Aikahyang 6 Alps Otome 83 Law Red Delicious 160 Ribston 237 Anhaeng Tare 7 Amere 84 Laxton's Superb 161 Rome beauty 238 Yeohong 8 Amur Naliv 85 Local 10 (907583) 162 Rossoshanskoje Polosatoje 239 Yongdonghaedang 9 Anna 86 Local 11 (907584) 163 Roter Boskoop 240 Chicheng 10 Antonovka 87 Lura Red 164 Royal Jonathan 241 Zaohuang 11 Antonovka karmen 88 M.Darth Manta 165 Royale d'Angleterre 242 Zhongqiu 12 Antonovka Shafran 89 M.prunifolia Sikora 166 Russian 243 Ginger-gold 13 Aori No.13 90 M. transitoria 167 Sakada Tsugaru 244 champion fuji 14 Arlet 91 M.Brevipes SI-22-40 168 Samar Candskoe rannee 245 Chukwang 15 Arthur Turner 92 M.floribunda Sieb 169 Scarlet Sentinel M.11 apple 246 Shuka Tsugaru 16 Asiro3 93 M. John Downie 170 Sekaiichi 247 Cameo 17 Beautiful Arcade 94 M. mandshurica 171 Sensacion 248 Pink Lady 18 Belle De Boskoop 95 M.robusta var electa 88075 172 Senshu 249 Honggeum 19 Beninomai 96 M.sikkimensis 173 SH-6 250 Hongso 20 Bhrens 97 M. Taurensii 174 Shaphran Letnij 251 Hwangtaepyeong 21 Blairmont 98 M3 175 Shengli 252 Fukutami 22 Brettacher 99 M7 176 Smokehouse 253 Himekami 23 Britegold 100 Macfree 177 Smootee golden 254 Asiro8 24 Calvoic Sansover 101 Magnolia Gold 178 Somerset Red Streak 255 Ben Davis 25 Carroll 102 Manshu 179 Spartan 256 Chouyou 26 Champion 103 Marin Onfroy 180 Spigold 257 Cox's orange pippin 27 Chiver's Delight 104 Marshall McIntosh 181 Spur Earliblaze 258 Geneva Black 28 Columbia 105 Maypole 182 Spur Golden Delicious 259 Geneva Early 29 Crimson King 106 Mayqueen 183 Spur Starking 260 Harcourt 30 Dab 325 107 McIntosh 184 Spy 261 Hibernal 31 Dayton 108 McIntosh spur 185 Stafford 262 Humboldt 32 Demir 109 Melrose 186 Succary 263 Idajon 33 Diana 110 Merton Pearmain 187 Summer Rambo 264 Jon Grimes 34 DIR 68T 492 111 Mislimka 188 Sun Gold 265 Jonagold Daliguy 35 Dolgo 112 Misuzu Tsugaru 189 sun rise 266 King Cole 36 Dorothea 113 Mobb's royal 190 Sundale Sturdee Spur 267 Lodi 37 Dorsett Golden 114 Namangan Skoe Krasnoe 191 Sunlight 268 M.arnoldiana Sarg 38 Double Red Delicious 115 Nico 192 Suntan 269 Merton Ace 39 Dunkerton late 116 Nishtani Jonathan 193 Surpasse Frequin 270 Michelin 40 E559-13 117 Noiphing 194 Sweet Coppin 271 Mollie's Delicious 41 Eikou 118 Norda 195 Sweet Delicious 272 Newtwon pippin 42 Eley purple crab 119 Norfolk Royal 196 Sweet Jonathan 273 Odin 43 Empire 120 Novosibirsk sweet 197 Tale Sweet 274 parkland 44 Empress spur G.D. 121 Nugget 198 Tallman's Sweet 275 pilot 45 Falla Water 122 Obilnoye 199 Telamon 276 Roberts Crab 46 fireside 123 Oswego 200 Totem 277 Rome 47 Frequin 124 Ottawa 201 Transcendent 278 Webster 48 Friandise 125 Ottawa 8 202 Treco Red Gala 279 Winter Banana 49 Frostproof 126 Ottawa546 203 Trent 280 Xingiangtianguo 50 Galaxy Gala 127 Parkian Crab 81090HT 204 Virginia Crab K-6 281 Kwansang1 51 George Cave 128 Pearmain de clark 205 Virginia Gold 282 Noya 52 Golden Delicious 129 Pederstrup 206 Wakapingguo 283 Summer Dream 53 Golden Delicious Klon.Bis 130 Pethyre 207 white winter pearl 284 Suhongsaekbingwa 54 Gravenstein 131 Peypring Cerueuko 208 Wickson 285 Shinano Sweet 55 Hanyae Hanakaidou 132 Piervomaiskoie 209 Williams Favorite 286 Picnic 56 Heyer #12 133 pink pearl 210 Winesap 287 Green Ball 57 Hillwell Red Braeburn 134 Pink Wood 211 Xanthocarpa 288 Jonathan 58 Hokuto 135 Pixie 212 Yantaishaguo 289 Granny Smith 59 Holstein 136 Pomme Cloche 213 Yellow Bell flower 290 Fuji 60 Hordapfel 137 Pomme Royale 214 Yellow Delicious 291 Gamhong 61 Huidobro 138 Prairie Spy 215 Yellow spur golden delicious 292 Ralls Janet 62 Hunter Melba (4*) 139 PRI 17-1 216 Yellow Transparent 293 Sansa 63 Hunter Spartan (4*) 140 67PRI 1279-9 (27-55) 217 Yoko 294 Hwahong 64 Idared I 141 Priscilla 218 Young America 295 Gala 65 Ikorocavka Alaja 142 Prof. Grebnicka Renete (Berzi) 219 Zuboff 296 Hongan 66 Indian Magic Crab 143 Pumpkin Sweet 220 Kunmamyeongwol 297 Hongro 67 Indian Summer Crab 144 Puritan 221 Kumhong 298 Indo 68 Ingrid Marie 145 purple wave 222 Kiou 299 Hwangok 69 Iwakami 146 Quinte 223 Ryoka 300 Summer King 70 Jacquin 147 Radiant 224 Manbok 301 Delicious 71 Jadernicka 148 Radoux 225 Biko Tugaru 302 Vance Delicious 72 James Grieve-2 149 Red Apple 226 Bingwa 303 Orin 73 Jiguan 150 Red Bow 227 Sandongbingwa 304 red gold 74 Jonafree 151 Red cox's poruona 228 Seokwang 305 Toki 75 Jonared 152 Red Delicious 229 Seohong 306 Nero Rome 76 Jumbo 153 Red Dougherty 230 Sunhong 307 Starking 77 Kamsolmolez 154 Red King 231 Seopsa Tsugaru 308 Beverly Hills

In Examples to be described later, DNA extracted from leaves of 308 apple genetic resources selected above was used as a sample.

Example 2. Single nucleotide polymorphism (SNP) selection using GBS (genotyping by sequencing)

GBS analysis of 308 apple genetic resources of Example 1 was performed. Specifically, genomic DNA of apple leaves was extracted with DNeasy Plant Mini Kit (QIAGEN, Germany). GBS of the extracted genomic DNA was performed. The GBS was analyzed using an Illumina Hiseq 2000 system, paired-end. The results of GBS analysis are shown in FIG. 1 .

As shown in FIG. 1 , for raw reads, an average of 3,863,262,097 reads were sequenced from a minimum of 88,768 to a maximum of 20,569,116. After an average of 92.8% of the trimming process, an average of 66% of reads were mapped to the ‘Golden Delicious’ genome (Velasco et al., 2010) used as a reference genome. Finally, reads are distributed in an average of 15.64 depth, 63,380 genome regions, and have coverage of about 2% of the reference genome. As a result of selecting SNPs by variety compared to the reference gnome based on this, 'Roberts Crab' had the highest number of total SNPs and heterozygous SNPs with 143,966 and 29,270, and homozygous SNPs and other SNPs with 'Asiro8' had the most with 94,379 and 28,606, respectively. . On average, 43,721 SNPs were selected, 27,481 homozygous SNPs, 7,929 heterozygous SNPs, and 8,312 other SNPs were identified. Here, for homozygous SNPs, more than 90% of the reads were identified as SNPs, for heterozygous SNPs 40 to 60%, and other SNPs were classified as other elusive ones. This was filtered under the following conditions.

[Condition A]

- SNP loci of biallelic, missing rate ＜ 10%

- minor allele frequency > 5%

[Condition B]

- SNP loci of biallelic, missing rate ＜ 20%

- minor allele frequency > 5%

SNPs selected through filtering were 12,608 (condition A) and 51,434 (condition B), respectively.

Example 3. Genetic diversity, PCA and Population structure analysis of apple genetic resources

3-1. Population structure analysis

The population structure of 308 apple genetic resources was analyzed using the SNPs (12,608) selected under condition A in Example 2 above. The population structure analysis is STRUCTURE v.2.3.4. The program was used, and the analysis conditions were as follows (Pritchard et al., 2000).

- Bayesian model-based approach (MCMC: markov chain monte cario)

- Burn-in period: 10,000

- Number of MCMC Reps : 10,000

- Ancestry Model : Admixture model

- Allele frequency model: correlated allele frequency

- Number of populations (K): 1 to 10

- Number of iterations : 2

The optimal population K was calculated using the analysis results from the structure harvester website (Evanno et al., 2005). The group structure analysis results are shown in FIGS. 2A and 2B .

As shown in FIGS. 2A and 2B , it was confirmed that the appropriate population K was 4.

3-2. PCA

Based on the value of the appropriate population K confirmed in Example 3-1, PCA analysis was performed. Principal component analysis was performed using sequence information (12,608 SNPs) of 308 samples of apple genetic resources and TASSEL ver.5.2.60 software (Bradbury et al., 2007). The PCA analysis result is shown in FIG. 2c.

As shown in Fig. 2c, the 308 apple genetic resources were divided into 4 groups by the PC1 axis or up to 6 groups by the 2 axes of PC1 and PC2. PC1 was the classification criterion for Malus domestica and Malus sp ., Malus hybrid and crabapples (PCA-I, crabapples such as Malus floribunda , Malus sikkimensis , and Malus arnoldiana ; PCA-II, crabapple, Malus hybrid and Malus sp .; PCA -Ⅲ, PCA-IV, Malus domestica ).

3-3. Genetic diversity analysis

A phylogenetic tree was prepared using sequence information of 308 samples of apple genetic resources and MEGA ver.6.0 program. The conditions for creating a phylogenetic tree are as follows (Tamura et al., 2013).

- Statistical method: Neighbor-joining

- Substitution model/method : Maximum composite likelihood

- Gaps/Missing data treatment: Pairwise deletion

The prepared phylogenetic tree is shown in FIG. 2D.

As shown in FIG. 2D , it was confirmed that the 308 apple genetic resources samples were diverse individuals with little correlation with each other. In addition, it was confirmed that the 308 apple genetic resources were grouped into Tsugaru, Golden Delicious, Gala, Fuji, Jonathan, Delicious, McIntosh, and Daemok, and the varieties included in each group are as follows.

- Tsugaru Group: Beninomai, Migwang Tsugaru, Sakada Tsugaru and Miszu Tsugaru, etc.

- Golden Delicious group: Golden Delicous, Spur Golden Delicious, Empress Spur Golden Delicious, etc.

- Gala groups: Galaxy Gala, Treco Red Gala, Sansa, etc.

- Fuji Group: Champion Fuji, Nugget, Iwakami, etc.

- Jonathan Groups: Rome beauty, Rome, Alps Otome, Jonared, etc.

- Delicious group: Vance Delicious, Red King, Red Delicious, Double Red Delicious, etc.

- McIntosh Group: Totem, Macfree, Maypole, Marshall McIntosh, etc.

- Root group: M3, M.Tauresii, M.Pruifolia Sikora, M.John Downie, etc.

Example 4. GWAS analysis of fruit useful traits targeting apple genetic resources

In this example, an experiment was conducted on 301 points with phenotypic data out of 308 apple genetic resources.

4-1. GWAS analysis_QQ plot according to acidity trait of 301 apple genetic resources

(i) Comparison of acidity trait phenotype and genotype distribution of 301 apple genetic resources using SNPs (51,434) selected under condition B in Example 2; and (ii) GWAS analysis;

(i) Comparison of pH trait phenotype and genotype distribution

Prior to GWAS analysis, the distribution of phenotypes and genotypes for acidity traits in apple genetic resources was compared. Samples for comparison were taken every year and observed for 4 years.

As a result, it was confirmed that in the first year, fruits with an acidity of 0.0 to 0.9% accounted for the most at 70.9%, and in the second year, fruits with an acidity of 0.3 to 1.3% accounted for the most at 66.6%. In the 3rd year, apple fruits with an acidity of 0.3 to 1.0% accounted for 65.4%, and in the 4th year, fruits with an acidity of 0.3 to 1.1% accounted for 73.7%.

(ii) GWAS analysis

GWAS was performed on 301 apple genetic resources with phenotypic data. For the analysis method, GAPIT (Genomic Association and Prediction Integrated Tool) and PLINK program based on R for acidity trait were used (Lipka et al. 2012, Purcell et al. 2007). GWAS results were visualized by QQ-plot. If the null hypothesis that there is no association between SNP and acidity trait in QQ-plot is correct, then all p-values follow a uniform distribution. However, if it is determined that there is a relationship between the SNP and the acidity trait, this distribution, i.e., the unipolar distribution, is deviated. A QQ-plot prepared based on the GWAS results is shown in FIG. 3 .

As shown in FIG. 3 , as a result of confirming the prepared QQ-plot, it was confirmed that a uniform distribution was not followed, which means that SNP and acidity traits are related.

4-2. GWAS analysis according to the acidity trait of 301 apple genetic resources_manhattan plot

The positions of related SNPs were visualized with a manhattan plot. The horizontal axis of the manhattan plot means the chromosome number and physical location, and the vertical axis means -log10(P) value. This value is the value obtained by replacing the p-value obtained by the F-test with -log10(P) under the null hypothesis that there is no association between the SNP and the acidity trait. That is, it is judged that the higher this value, the higher the probability that there is a correlation between the SNP and the acidity trait. The prepared manhattan plot is shown in FIG. 4 .

As shown in Figure 4, it was confirmed that the highest p-value in chromosome 16. This result means that the SNP present on chromosome 16 is highly correlated with the acidity trait.

Example 5. Acidity trait SNP selection

Based on the above-described GWAS results, SNPs with the highest significance for acidity traits were selected. The selected acidity trait SNPs are shown in Table 2.

Trait Marker SNP Alleles P value Genic/Intergenic Annotation acidity
(Titratable Acidity, TA) chr16: 1466019 C/T 8.62×10 ^-9 LOC103402725 [Exon] methionine aminopeptidase 1B chr16: 1466001 A/G 9.56×10 ^-9 LOC103402725 [Exon] methionine aminopeptidase 1B chr16: 1350941 G/T 2.36×10 ^-8 LOC103402717 [Exon] unknown chr16: 1378405 C/T 1.72×10 ^-7 LOC103402719 [Exon] CTL-like protein DDB_G0274487 chr16: 1545939 A/G 9.37×10 ^-7 LOC103402728 [Exon] unknown chr16: 1348804 A/G 1.31×10 ^-6 LOC103402717 [Exon] unknown chr16: 1450841 A/C 2.23×10 ^-6 Intergenic chr16: 1316991 A/C 2.59×10 ^-6 LOC103402710 [Exon] ACD11 homolog protein chr16: 1313364 C/T 4.59×10 ^-6 LOC103402711 [Exon] dihydrolipoyllysine-residue acetyltransferase component 4 of pyruvate dehydrogenase complex chr16: 1332163 A/G 6.00×10 ^-6 LOC103402712 [Exon] aluminum-activated malate transporter 4-like

Example 6. Determination of acidity of apples using selected acidity trait SNPs

The acidity of apples was determined using the acidity trait SNPs shown in Table 2 above. First, a primer set for the acidity trait SNP described in Table 2 was designed. In this example, the acidity determination results using C16S63 and C16S39B among the designed primer sets were disclosed.

(i) Determination of acidity using primer set C16S63

In the primer set C16S63, the size of the amplification product was 107 bp, and the annealing temperature was 60°C. In addition, the primer set C16S63 is for discriminating the SNP at position 1335263 of chromosome 16. Using the primer set C16S63, the acidity of the apple gene source was discriminated, ie, the SNP marker was confirmed. Additionally, High Resolution Melting (HRM) analysis was performed. The acidity determination result is shown in Table 3, and the HRM result is shown in FIG. 5 (A: melting curve, B: difference graph).

Acidity (%) kind Discrimination criteria (%) 1.0 - 3.6 Asiro8, Akita Tare, Roberts Crab, Asiro3, Dolgo, Spy, Lodi, Jonathan, Noya, Humboldt, Parkland, M7, etc. 1.0 or higher 0.1 - 0.9 Jonared, Jiguan, Red well, Crimson King, Virginia Gold, Sansa, Hongro, Granny Smith, Fuji, Tsugaru, Picnic, Yoko, etc. less than 1.0

As shown in FIG. 5 , as a result of HRM analysis, differentiation was confirmed based on 1.0% in the melting curve.

As shown in Table 3, varieties were discriminated based on acidity of 1.0%. It was confirmed that the varieties having an acidity of 1.0 to 3.6% include Asiro8 and Akita Tare, and the varieties having an acidity of 0.1 to 0.9% include Jonared, Jiguan, Red well, Crimson King, and the like. As a result of analyzing the SNPs of the above varieties, it was confirmed that the SNP at position 1335263 of chromosome 16 was AA or AG in varieties with an acidity of 1.0% or higher, and GG in varieties with an acidity of less than 1.0%.

(ii) Determination of acidity using primer set C16S39B

The primer set C16S39B had an amplification product size of 125 bp and an annealing temperature of 60°C. In addition, the primer set C16S39B is for discriminating the SNP at position 1545939 of chromosome 16. Using the primer set C16S39B, the acidity of the apple gene source was determined, that is, the SNP marker was confirmed. Additionally, HRM analysis was performed. The acidity determination result is shown in Table 4, and the HRM result is shown in FIG. 6 (A: melting curve, B: difference graph).

Acidity (%) kind Discrimination criteria (%) 1.3 - 3.9 Asiro8, Asiro3, Akita Tare, Ottawa8, Maypole, Purple wave, etc. 1.3 or higher 0.1 - 1.3 Ginger gold, Crimson king, Mayqueen, Alps Otome, Hongro, Gamhong, Tallmans sweet, Britegold, etc. less than 1.3

As shown in FIG. 6 , as a result of HRM analysis, differentiation was confirmed based on 1.3% in the melting curve.

As shown in Table 4, varieties were discriminated based on acidity of 1.3%. It was confirmed that varieties with acidity of 1.4 to 3.9% or more include Asiro8, Asiro3, Akita Tare, and the like, and varieties with acidity of 0.1 to 1.3% include Ginger gold, Crimson king, Mayqueen, and the like. As a result of analyzing the SNPs of the above varieties, it was confirmed that the SNP at position 1545939 of chromosome 16 was GG in varieties with an acidity of 1.3% or higher, and AA or AG in varieties with an acidity of less than 1.3%.

Overall, when the present inventors use the selected SNP marker of the present invention and the primer set to amplify it, it is possible to accurately discriminate varieties according to acidity traits and acidity that do not show a significant difference in phenotype due to high genetic similarity. This means that when the SNP marker of the present invention is used, the acidity of an apple can be determined only with leaves without apple fruit, and the SNP marker of the present invention can be used in various ways in the field of apple breeding.

Above, a specific part of the present invention has been described in detail, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Accordingly, it is intended that the substantial scope of the present invention be defined by the appended claims and their equivalents.

<110> Kyungpook National University Industry-Academic Cooperation Foundation <120> Single Nucleotide Polymorphism Molecular Marker Related to fruit acidity Characteristic in Apple and uses thereof <130> 1-326P <150> KR 10-2019-0071213 <151> 2019-06-17 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 201 <212> DNA <213> Malus domestica <400> 1 ctaatgaagt agcaactccc ttgaaattct tcacwaccaa gttatgcaga tcctctcccg 60 accatatcgg atatatgcat atgttaacag ctaaacaaac rgcagctccc actgcaataa 120 gaaccagtcg agtcacaact gcctcattgt actcccttgt tctgttccca gcaaccatta 180 ggatgcagta tgtcaacacg a 201 <210> 2 <211> 201 <212> DNA <213> Malus domestica <400> 2 atagatatac ttgagacgat gacatgttga gaacattcca aactaccaac aaataggttc 60 ttttaaacgc tgaatatacc tctaagtgca taaagagtag ratctggaaa atgattagag 120 gaactttcat aatatgccca ccgatatcct gcaggctgca tactactgtc tcgtcttgat 180 cttcatctga agaaatgact a 201

Claims (10)

A composition for determining malic acidity, comprising an agent capable of detecting a single nucleotide polymorphism (SNP) marker of the 101st base in the polynucleotide consisting of SEQ ID NO: 1. According to claim 1,
The single nucleotide polymorphic marker is
In the polynucleotide represented by the nucleotide sequence of SEQ ID NO: 1, the 101st base is AA, AG or GG, which is a single nucleotide polymorphism marker, a composition for identifying malic acid characteristics. According to claim 1,
The agent capable of detecting the polymorphism marker includes: a polynucleotide comprising 10 to 100 consecutive DNA sequences including a single polymorphism marker site; Or its complementary polynucleotide; and a polynucleotide that specifically hybridizes with, a composition for identifying malic acid characteristics. 4. The method of claim 3,
The polynucleotide is at least one selected from the group consisting of a primer capable of detecting a single nucleotide polymorphism marker, a probe, and a combination thereof, a composition for identifying malic acidity. 5. The method of claim 4,
In the primer, the 3' last base is complementary to the single nucleotide polymorphism site, the composition for determining malic acidity. According to claim 1,
The composition is to discriminate the malic acidity trait in young leaves samples. Claims 1 to 6, comprising the composition according to any one of the malic acidity characterization kit. (a) extracting the DNA of the apple;
(b) using the DNA of step (a) as a template, and performing a polymerase chain reaction using the composition according to any one of claims 1 to 6; and
(c) analyzing the polymerase chain reaction product to determine the acidity trait of the apple; 9. The method of claim 8,
The step (a) is to extract the DNA of the apple from the sample of the young leaves of the apple, the apple acidity characterization method. 9. The method of claim 8,
The polymerase chain reaction of step (b) is an allele-specific polymerase chain reaction, malic acidity characterization method.

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