KR20190055491A - A composition for cultivar discrimination in pear - Google Patents

Abstract

The present invention relates to a primer set for use for cultivar discrimination in pear; and uses thereof. The primer set of the present invention may be used to discriminate pear cultivars based on their maternal line. A primer set composition according to the present invention may include a primer set represented by a base sequence of SEQ ID NO:1 and SEQ ID NO:2, and a select pear cultivar and a cultivar bred from the same as a maternal line can be accurately distinguished using the primer set composition according to the present invention.

Description

[0001] The present invention relates to a composition for cultivar discrimination in pear,

The present invention relates to a primer set which can be used for identification of a pear variety and uses thereof.

The pear is the fruit of the pear, divided into western, Chinese and southern oriental pears, each of which has different appearance and taste. In South Korea, there are records of cultivation of Oriental Oriental Pear from mainland China. In addition, the abalone can be divided into early life such as fresh water, freshwater aquatic life, longevity, and other freshwater species such as Jangsilang, Fengsu, Youngsan Bay, Boat is the single item among agriculture and forestry exports, followed by ginseng, kimchi and paprika. In order to further cultivate and spread these domestic varieties, management and protection of varieties are important.

In addition, as a result of the UPOV's entry into the International Plant Conservation Alliance (UPOV), breeding of breeding species of breeders became necessary as the hatching was designated as a crop protection crop. Therefore, in order to protect the breed of the ship, the discrimination of the breed of the breed which is breeding in the domestic should be given priority.

A large amount of genetic resources are being collected every year from most major crops and materials used for breeding. On the other hand, the evaluation of genetic resources is not enough. Rapid identification of interspecies and subspecies of collected genetic resources is very important for the management and protection of accurate genetic resources. In addition to identification of varieties, identification and classification of genotypes of genotypes and identification of their relationship are very important for preservation of genetic resources, creation of new varieties and improvement of crops.

Accordingly, the present inventors have developed a gene and a primer set capable of identifying a gene that can identify a breed with good specific resources as a host, using the chloroplast genome of the embryo.

Patent Publication No. 2012-0069446

An object of the present invention is to provide a composition comprising a primer set for identification of a breed variety.

Another object of the present invention is to provide a method of identifying a pear variety.

Yet another object of the present invention is to provide a kit for identifying a breed variety.

The present invention provides a composition for identifying breeding variety comprising a primer set consisting of the nucleotide sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2.

The present invention also relates to a method for amplifying DNA comprising the steps of: (a) amplifying a chloroplast genomic DNA derived from a strain as a template by performing PCR with the primer set of claim 1;

(b) analyzing a size of the amplification product obtained in step (a) to determine a pear variety;

The method comprising the steps of:

The present invention also provides a kit for identifying a breed variety, comprising a primer set consisting of the nucleotide sequences shown in SEQ ID NOs: 1 and 2.

The use of the primer set composition according to the present invention makes it possible to accurately distinguish the specific pear variety and the breed which has been breeded therefrom, thereby contributing to the selection of excellent varieties of distribution seedlings and strengthening protection rights of the pear variety. In addition, it can be used as a breeding certification technology for distribution of domestic seedlings.

FIG. 1 shows a process of setting primers for amplifying regions having two SNPs, such as AA and TC in the ndhA gene of the embryo line and the original embryo (yellow notation).
Fig. 2 shows the result of band size analysis for 29 kinds of pears amplified by performing PCR with ndhA primer set and one apple.
FIG. 3 shows the result of comparing DNA sequences of the 29 strains amplified with the ndhA primer set and the DNA sequence of one apple against the DNASTAR program package using the CluterW method.
Fig. 4 shows the degree of the relationship between the 29 strains and one apple by the nucleotide sequence amplified with the ndhA primer.

The present invention provides a composition for identifying breeding variety comprising a primer set consisting of the nucleotide sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2.

Also, in the present invention, a "primer" refers to a single strand oligonucleotide sequence complementary to a nucleic acid strand to be copied, and may serve as a starting point for the synthesis of a primer extension product. The length and sequence of the primer should allow the synthesis of the extension product to begin. The specific length and sequence of the primer will depend on the primer usage conditions such as temperature and ionic strength, as well as the complexity of the desired DNA or RNA target.

The vessels used in the present invention are Pyrus pyrifolia , Pyrus ussuriensis , Pyrus bretschnederi and Pyrus One or more of the calleryana can be selected, more specifically Pyrus pyrifolia .

The breed varieties that can be identified by the primer set according to the present invention may be those cultivated by breeding the breed, the declared embroidered embroidered or the embroidered embroidered embryo corresponding to the embryo's embryo. The varieties corresponding to the mother line used in the breeding of the embryo can be a thousand and one thousand, and the varieties cultivated using the embryo as a mother line may be gold, corn, persimmon, myth,

The primer set corresponding to SEQ ID NO: 1 and SEQ ID NO: 2 of the present invention can amplify the ndhA (putative NADH dehyd- yogenease A) gene region of the podroblast genome. The size of the product amplified using the primer set varies depending on the pear variety, and the cultivar cultivated as a model of the mother line of the pear blight, the declared pear, or the declared pear may be 864 bp.

By using the primer set of the present invention, it can be predicted that the cultivar is cultivated in the mother line through the primer set even in the case of the cultivar which can not confirm the sample other than the above-mentioned varieties.

Further, according to the present invention,

(a) performing amplification by performing PCR with the primer set of claim 1 using the chloroplast genomic DNA derived from the embryo as a template;

(b) analyzing a size of the amplification product obtained in step (a) to determine a pear variety;

The method comprising the steps of:

The method of extracting the chloroplast genomic DNA of the embryo in step a) can be carried out by a conventional method known in the art. For example, a CRAB method, a phenol / chloroform extraction method, an SDS extraction method, or the like, or a commercially available DNA extraction kit can be used.

In the step b), the primer set is a composition comprising a primer set consisting of the nucleotide sequences shown in SEQ ID NOs: 1 and 2. One or more sets of the primers set for identifying the cultivar species can be used, and if a plurality of molecular markers are used at the same time, the cultivars can be more accurately distinguished.

Also, the amplification of the chloroplast genome DNA in the step (a) can be performed by PCR (Polymerase Chain Reaction). PCR may be performed using PCR reaction mixtures containing components known to be required for PCR reactions in the art or using commercially available kits. The PCR reaction mixture may include chloroplast genomic DNA extracted from embryos, a primer set according to the present invention, an appropriate amount of DNA polymerase, dNTP, PCR buffer solution and water. The PCR buffer solution, but may include TrisHCl, MgCl _2, KCl, etc., without being limited thereto.

The DNA product amplified in the step (b) may be analyzed by a conventional method known in the art. For example, the DNA product may be analyzed by DNA chip, electrophoresis, radioactive measurement, fluorescence measurement or phosphorescence measurement .

Preferably, the amplified DNA product is analyzed using gel electrophoresis. Specifically, the amplification product is electrophoresed on an agarose gel or an acrylamide gel, and the resultant product is subjected to electrophoresis using ethidium bromide (EtBr), silver staining You can see the band by.

According to the identification method of the grape variety according to the present invention, the size of the amplification product of the grape variety can be determined by comparing the obtained amplification product.

The contents of the identifiable embryo variety and the chloroplast gene are overlapped with those described above, so that they are omitted and the above-mentioned description is directly quoted.

The present invention also provides a kit for identifying a breed variety, comprising a primer set consisting of the nucleotide sequences shown in SEQ ID NOs: 1 and 2.

The kit may include a DNA polymerase, dNTPs, and a buffer solution for PCR reaction, in addition to the above-described primer set for identifying breeds according to the present invention.

The kit for identifying a breed variety according to the present invention may further contain components necessary for electrophoresis to confirm whether or not the amplification of the PCR product is amplified.

The contents of the identifiable embryo variety and the chloroplast gene are overlapped with those described above, so that they are omitted and the above-mentioned description is directly quoted.

Hereinafter, the present invention will be described in more detail with reference to the following examples and comparative examples, but the scope of the present invention is not limited by the following examples.

Example 1 Chloroplast Genome Assembly of Pear Cultivars

We have developed a chloroplast genome assembly, one of the organelles that can analyze maternal genetic patterns.

Xenon DNA was extracted from the young leaves of 29 species and 1 apple sample using DNeasy Plant Mini Kit (Qiagen, CA, USA). Short - range sequence - based genomic sequencing was performed on eight abundant abundant resources of 29 abundant species, such as. Genome sequencing was performed using In-house Next-seq, an Illumina genome analyzer, and HiSeq 2500, 4000 service. A genomic library with a 350 bp insertion size was prepared according to the paired-end standard protocol recommended by the manufacturer. Each sample was tagged separately with a different index. Using the CLC-quality trim tool, the trimmed nucleotide sequence with a Phred score of 20 or less was assembled using a CLC genomic assembler (ver. 4.06 beta, CLC Inc, Rarhus, Denmark) with an overlap size of at least 200 to 600 bp Respectively. The main contig of representing chloroplast genomes is the Hosui Pear ( Pyrus pyrifollia , reference to NC_015996, the reference-guided chloroplast genome assembly pipeline was mounted on the vital part (support for the genome and information room). The pear variety used in the present invention is shown in Table 1 below.

The pear variety used in the present invention Serial Number Breed name English name Scientific name Mating combination Number of times Main characteristics One Declaration Niitaka P. pyrifolia 26-19516 Main cultivars 2 Golden boat Whangkeumbae P. pyrifolia Report x twentieth century Wood Mediocre, green skin 3 Zheng Sili Cheongshilli P. ussuriensis 26-19923 4 Cloth open air Amanogawa P. pyrifolia 5 Nectar Gamro P. pyrifolia Report * New Number 6 Prognosis Sunwhang P. pyrifolia Report * 7 mythology Shinhwa P. pyrifolia Report * Volcano 8 Youngsan ship Youngsanbae P. pyrifolia Report * 9 Hanam Hanareum P. pyrifolia Report x 9-0064 Early life 10 Anhydrous Wonwhang P. pyrifolia Early childhood * 11 Geomchonchu Imamuraaki P. pyrifolia 12 Geumchon Geumchonjosaeng P. pyrifolia Gimchonchu 13 Single Danbae P. pyrifoliax  P.ussuriensis Jangsilang x Cheongsil 26-19755 14 Chu Hwang Chuwhangbae P. pyrifolia Gimchonchu x twentieth century Wood Longevity 15 Gam-gil Okusankichi P. pyrifolia 16 Snow Seolwon P. pyrifolia * 17 Super Gold Supergold P. pyrifolia Chu Hwang Bae * 18 Jang-bil Chojuro P. pyrifolia 19 Round Doongkeulraebae P. ussuriensis 20 Cally Laurie Cheongdangrori P. ussuriensis 21 OPR125 OPR125 P. calleryana 22 OPR195 OPR195 P. calleryana 23 Thunder Yali P. bretschnederi 26-19595 Blackness susceptibility 24 Tangshan repairs Dangshansuli P. bretschnederi 26-19710 Chinese ship genome research material 25 5-1 Godang 5-1 26 Chang Sye Sul Cangxixueli 27 Actor Kozo 28 Bartlett Bartlett P. communis 11-2 Western blotting dielectric research material 29 Max red bartlett Max red bartlett P. communis 26-20083 Bartlett Azo mutation 30 Fuji Fuji Malus

< Example  2> Origin of chloroplast genome for 30 cultivars catlytic  subunits of the ndhA  amplification of protease complex (ndhA) gene, comparison of nucleotide sequence

Report times and times wonhwang chloroplast ndhA sequence comparison when the two SNPs ndhA forward primer 5'- to the area to be analyzed CAA TAA CCC GTT GGT TTA CA -3 '( SEQ ID NO: 1) and reverse primer 5'- CCT TGT TTT ndhA AGC GGA TCT CA-3 '(SEQ ID NO: 2) A bi-directional 20-mer primer set was outsourced. The predicted size of the PCR product was 1,003 bp (Fig. 1).

PCR reaction was performed by adding 13 μl of qPCR 2X PreMix with SYBR Green (Enzynomics, RT500M), 10 pmole (1 μl each) of bidirectional primer and 25 ng (5 μl, 5 ng / Were reacted with 96 well plates (Bio-Rad, BRHSP-9601) to a total of 26 μl. The PCR reaction instrument was an Applied Biosystems PCR system 97000 instrument. PCR was performed by treating the PCR reaction at 94 ° C. for 5 minutes once, at 94 ° C. for 30 seconds, at 60 ° C. for 30 seconds, at 72 ° C. for 1 minute, and at the final reaction at 72 ° C. for 10 minutes. The prepared primer set was amplified with the ndhA primer set (SEQ ID NOS: 1 and 2) for the 29 species and one apple shown in Table 1 (Fig. 2). Electrophoresis was performed by injecting 2 [mu] l of the PCR product in 2.5% agarose gel, and developed for 10 hours at 100 volts. A 1 Kb DNA ladder marker (Enzynomics, DM003) was used to confirm PCR product size.

As a result, the band size of 886bp was observed in the cultivated varieties of gold, maize, persimmon, myth, yongsan pear, In addition, the band size of 864 bp, which is the same as that of the filaments, was observed in the blue silk, white, snowy, cedar lily and sari sully.

< Example  3> Chloroplast genome origin of 29 kinds of pear and 1 apple variety catlytic  Analysis of polymorphisms in subunits of the ndhA protease complex (ndhA) gene

 Results of polymorphism analysis No Variety 10? 30 (14 bp C? T;
16bPa? T; 18bpA? T) 36.58 (38bPa? C;
54 bp A → T; 56bp T? A) One Niitaka AATTCTATATTAATTAATGAA ACACAAATTATATATCTATTTAT 2 Whangkeumbae AATTCTATATTAATTAATGAA ACACAAATTATATATCTATTTAT 3 Cheongshilli AATTCTATATTAATTAATGAA ACACAAATTATATATCTATTTAT 4 Amanogawa AATTCTATATTAATTAATGAA ACCCAAATATATATCTATTAAT 5 Gamro AATTCTATATTAATTAATGAA ACACAAATTATATATCTATTTAT 6 Sunwhang AATTCTATATTAATTAATGAA ACACAAATTATATATCTATTTAT 7 Shinhwa AATTCTATATTAATTAATGAA ACACAAATTATATATCTATTTAT 8 Youngsanbae AATTCTATATTAATTAATGAA ACACAAATATATATCTATTAAT 9 Hanareum AATTCTATATTAATTAATGAA ACACAAATTATATATCTATTTAT 10 Wonwhang AATTCTATATTAATTAATGAA ACACAAATATATATCTATTAAT 11 Imamuraaki AATTCTATATTAATTAATGAA ACACAAATTATATATCTATTTAT 12 Geumchonjosaeng AATTCTATATTAATTAATGAA ACACAAATTATATATCTATTTAT 13 Danbae AATTCTATATTAATTAATGAA ACACAAATTATATATCTATTTAT 14 Chuwhangbae AATTCTATATTAATTAATGAA ACACAAATTATATATCTATTTAT 15 Okusankichi AATTCTATATTAATTAATGAA ACACAAATATATATCTATTAAT 16 Seolwon AATTCTATATTAATTAATGAA ACACAAATTATATATCTATTTAT 17 Supergold AATTCTATATTAATTAATGAA ACACAAATATATATCTATTAAT 18 Chojuro AATTCTATATTAATTAATGAA ACACAAATTATATATCTATTTAT 19 Doongkeulraebae AATTCTATATTAATTAATGAA ACACAAATTATATATCTATTTAT 20 Cheongdangrori AATTCTATATTAATTAATGAA ACACAAATTATATATCTATTTAT 21 OPR125 AATTCTATATTAATTAATGAA ACCCAAATTATATATCTATTTAT 22 OPR195 AATTCTATATTAATTAATGAA ACCCAAATTATATATCTATTTAT 23 Yali AATTCTATATTAATTAATGAA ACACAAATTATATATCTATTTAT 24 Dangshansuli AATTCTATATTAATTAATGAA ACACAAATTATATATCTATTTAT 25 Godang 5-1 AATTTTTATATAATTAATGAA ACACAAATTATATATCTTTTTAT 26 Cangxixueli AATTCTATATTAATTAATGAA ACACAAATTATATATCTATTTAT 27 Kozo AATTTATATATATATAATGAA ACCCAAATATATATCTATTAAT 28 Bartlett AATTTTTTTTAATTAATGAA ACCCAAATTATATATTTTTTTAT 29 Max red bartlett AATTTTTTTTAATTAATGAA ACCCAAATTATATATTTTTTTAT 30 Fuji AATTTTTTTTTTTTTTTGAA ACCCAAATTTTTTTTTTTTTTAA No Variety 75.119 (77 bp A? T; 83 bp A? T; 81 bp A? C;
83 to 115 bp Insertion 23 bp 'TAAGGATTAAAGTAACAAAGGAT') One Niitaka ATTAAAGTTACAAAGGAT ------------------------ AAAT 2 Whangkeumbae ATAAAAGTTACAAAGGAT ------------------------ AAAT 3 Cheongshilli ATAAAAGTTACAAAGGAT ------------------------ AAAT 4 Amanogawa ATAAAAGTTACAAAGGAT ------------------------ AAAT 5 Gamro ATTAAAGTTACAAAGGAT ------------------------ AAAT 6 Sunwhang ATAAAAGTTACAAAGGAT ------------------------ AAAT 7 Shinhwa ATAAAAGTTACAAAGGAT ------------------------ AAAT 8 Youngsanbae ATAAAAGTTACAAAGGAT ------------------------ AAAT 9 Hanareum ATAAAAGTTACAAAGGAT ------------------------ AAAT 10 Wonwhang ATAAAAGTAACAAAGGATTAAGGATTAAAGTAACAAAGGATAAAT 11 Imamuraaki ATAAAAGTAACAAAGGATTAAGGATTAAAGTAACAAAGGATAAAT 12 Geumchonjosaeng ATAAAAGTAACAAAGGATTAAGGATTAAAGTAACAAAGGATAAAT 13 Danbae ATTAAAGTAACAAAGGAT ------------------------ AAAT 14 Chuwhangbae ATAAAAGTAACAAAGGATTAAGGATTAAAGTAACAAAGGATAAAT 15 Okusankichi ATAAAAGTAACAAAGGATTAAGGATTAAAGTAACAAAGGATAAAT 16 Seolwon ATAAAAGTTACAAAGGAT ------------------------ AAAT 17 Supergold ATAAAAGTAACAAAGGATTAAGGATTAAAGTAACAAAGGATAAAT 18 Chojuro ATAAAAGTAACAAAGGATTAAGGATTAAAGTAACAAAGGATAAAT 19 Doongkeulraebae ATAAAAGTAACAAAGGATTAAGGATTAAAGTAACAAAGGATAAAT 20 Cheongdangrori ATTAAAGTAACAAAGGAT ------------------------ AAAT 21 OPR125 ATAAAAGTAACAAAGGAT ------------------------ AAAT 22 OPR195 ATAAAAGTAACAAAGGAT ------------------------ AAAT 23 Yali ATTAAAGTAACAAAGGATTAAGGATTAAAGTAACAAAGGATAAAT 24 Dangshansuli ATTAAAGTAACAAAGGATTAAGGATTAAAGTAACAAAGGATAAAT 25 Godang 5-1 ATTAAAGTAACCAAGGAT ------------------------ AAAT 26 Cangxixueli ATTAAAGTAACAAAGGAT ------------------------ AAAT 27 Kozo ATTAAAGTAACAAAGGATTAAGGATTAAAGTAACAAAGGATAAAT 28 Bartlett ATTAAAGTTACCAAGGAT ------------------------ AAAT 29 Max red bartlett ATTAAAGTTACCAAGGAT ------------------------ AAAT 30 Fuji ATTAAAGTTACACATTTT -------------------- AAAAAAAAG No Variety 390..402 (396 bp A → C) One Niitaka TCCTTTCCATATGT 2 Whangkeumbae TCCTTTCCATATGT 3 Cheongshilli TCCTTTCCATATGT 4 Amanogawa TCCTTTCCATATGT 5 Gamro TCCTTTCCATATGT 6 Sunwhang TCCTTTCCATATGT 7 Shinhwa TCCTTTCCATATGT 8 Youngsanbae TCCTTTCCATATGT 9 Hanareum TCCTTTCCATATGT 10 Wonwhang TCCTTTACATATGT 11 Imamuraaki TCCTTTACATATGT 12 Geumchonjosaeng TCCTTTACATATGT 13 Danbae TCCTTTCCATATGT 14 Chuwhangbae TCCTTTACATATGT 15 Okusankichi TCCTTTACATATGT 16 Seolwon TCCTTTCCATATGT 17 Supergold TCCTTTACATATGT 18 Chojuro TCCTTTACATATGT 19 Doongkeulraebae TCCTTTACATATGT 20 Cheongdangrori TCCTTTCCATATGT 21 OPR125 TCCTTTACATATGT 22 OPR195 TCCTTTACATATGT 23 Yali TCCTTTACATATGT 24 Dangshansuli TCCTTTACATATGT 25 Godang 5-1 TCCTTTACATATGT 26 Cangxixueli TCCTTTCCATATGT 27 Kozo TCCTTTACATATGT 28 Bartlett TCCTTTACATATGT 29 Max red bartlett TCCTTTACATATGT

The polymorphism in the nucleotide sequences of the 29 strains and 1 apple strain amplified with the ndhA primer set (SEQ ID NO: 1 and SEQ ID NO: 2) was carried out by comparing the sequences with the CluterW method in the DNASTAR program package. The results are shown in Table 2 and FIG.

A polymorphism between four sequences of 29 strains amplified with the ndhA primer set (SEQ ID NO: 1 and SEQ ID NO: 2) and one nucleotide sequence of apple was investigated.

 In particular, there is an insertion / deletion polymorphism of the base sequence corresponding to 23 bp from 115 bp to 83 bp in the 75 bp to 119 bp comparison region among the compared regions, and a 23 bp deletion in the cultivars cultivated as a mother line, And it was distinguished from other varieties. Among the 390bp to 402bp comparison regions, the A → 'C' SNP mutation was observed at 396bp, which was observed in the cultivars cultivated as a maternal line.

    Using the above SNPs and insert / markers, we further confirmed that the cultivars cultivated as examples of declared and declared embryos and the cultivars used in cultivation of embryos can be distinguished from other cultivars.

   In addition, A → T or A → C SNPs were identified in 10 bp and 30 bp and 36 bp and 58 bp regions, respectively. These SNP markers could be used to identify pear cultivars.

Based on these polymorphic sequences, the relationship between the strains of 29 strains and 1 apple was analyzed and shown in FIG. 29 species and apples were the least flexible, and the next two strains were distant from Bartlett and Max Red Bartlett, two species of western species, and 5-1, And P. calleryana was located between Western and Oriental Pear. P. ussuriensis species were mixed in a relationship that is also flexible split times to step report of P. pyrifolia species and reporting systems and ship mobon wonhwang and geumchonchu and Geumchon mobon, the Chinese ship P. bretschnederi species are located in the Report and baegye Geumchon Fall contacts Respectively.

<110> Republic of Korea <120> A composition for cultivar discrimination in pear <130> P17R12C1050 <160> 2 <170> KoPatentin 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ndhA forward primer <400> 1 caataacccg ttggtttaca 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ndhA reverse primer <400> 2 ccttgtttta gcggatctca 20

Claims (8)

A primer set comprising a nucleotide sequence represented by SEQ ID NO: 1 and SEQ ID NO: 2. The method according to claim 1,
Wherein said cultivar is a cultivar which is cultivated by looking at a variety of cultivars corresponding to the mother line of the cultivar, a declared pear and a declared pear. The method according to claim 1,
Wherein the primer set amplifies the ndhA (putative NADH dehydongase A) gene in the chloroplast genome. The method of claim 3,
Wherein the product amplified by the primer set has a size of 864 bp. (a) performing amplification by performing PCR with the primer set of claim 1 using the chloroplast genomic DNA derived from the embryo as a template;
(b) analyzing a size of the amplification product obtained in step (a) to determine a pear variety;
&Lt; / RTI &gt; 6. The method of claim 5,
Wherein said pear cultivar is a cultivar derived from a model, a declared pear and a declared pear corresponding to the mother line of the declared pear. 6. The method of claim 5,
Wherein the step of determining the cultivar is to determine that the cultivar is a cultivar which is cultivated as a breed, a declared pear or a declared pear corresponding to the mother line of the declared pear when the size of the obtained amplification product is 864 bp. A kit for identifying a pear variety, comprising the composition of any one of claims 1 to 4.

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