KR101784244B1 - SSR molecular markers for identifying citrus zygotic and nucellar individuals and uses thereof - Google Patents

Abstract

The present invention relates to SSR molecular markers for distinguishing plants derived from citrus plants and sirens, and uses thereof. The SSR markers of the present invention can be used to identify plants derived from the umbraculifera and gypsum in offspring plants obtained from citrus hybridization In order to effectively distinguish between the gymnosperm and the umbilical cord using SSR markers in the future, it is necessary to distinguish between effective and efficient citrus molecular breeding systems This is a very useful technique.

Description

[0002] SSR molecular markers for citrus and referee strains and their use [0002]

The present invention relates to SSR molecular markers for citrus hybrids and refolders and their uses.

Tangerines are the major fruit crops with the highest production worldwide (130 million tons in 2013-2014), and are cultivated extensively in tropical and subtropical regions. Citrus is not only a vitamin C, but also contain various functional substances including citron-specific flavonoids such as benzoethene and hesperidin, which are important foodstuffs to help people's health. Despite the economic value of citrus production, the majority of citrus fruit production is done through natural hybridization, selection from eye mutants, or simple introduction of varieties, rather than through a systematic targeted breeding program. Citrus is one of the most difficult cultivars to breed through traditional crossbreeding due to plant size, long juvenility, high heterozygosity, and unique reproductive and biological characteristics including nucellar embryony .

In most flowering plants, a zygotic embryo develops in the embryo sac. However, in many citrus cultivars, a nucellar embryo developed by sporophytic apomixis develops in a single backpack together with a crossbred, resulting in the development of several polyembryo in one seed . The umbilical cord begins to develop directly in the nucellar tissue of the mother surrounding the backpack with a developing bridge. Therefore, the umbilical cord is ultimately developed as an ovipositor with the same genotype as the mother, which often develops much more vigorously than the gypsy stem and degrades the stem. Apomixis is a genetic trait found in many citrus cultivars, and is one of the main factors that make it difficult to cultivate cultivars through crossing in citrus. Therefore, a systematic citrus breeding program requires a reproducible, objective, and cost-effective method to easily distinguish between a referee and a referee.

(ISSR-PCR) method, the RAPD analysis technique, the inter-simple sequence repeat polymerase chain reaction (ISSR-PCR), the isoenzymes analysis, the chromatographic method, Techniques have been developed, but objectivity, reproducibility, efficiency and labor intensity of selection have been raised. Simple sequence repeat (SSR) markers are DNA molecular markers that are used in a variety of fields, including breed identification due to their high degree of polymorphism, co-dominance, and wide and even distribution within the genome . The SSR markers have been successfully used to distinguish between the referee and the goat breed, but previous studies have used oil derived from a very limited number of breed combinations. The versatility of SSR markers used in previous studies has not been determined.

Korean Patent No. 0842432 discloses 'SSR primer derived from oranges and its use', Korean Patent No. 1603156 discloses 'a method for identifying a citrus variety using a supersatellite marker' The SSR molecular markers for citrus hybridization and refolding and their uses have not been disclosed.

The present invention has been made in view of the above-mentioned needs. In the present invention, 17 SSR markers showing polymorphism through comparative genomic analysis among citrus fruit varieties were obtained, and using the 17 SSR markers, The size of 17 alleles of each genetic source was determined by DNA fragment analysis on 101 genotypes of citrus collecting genome. In addition, by in a group derived from a F ₁ part crossbreeds with multi-stage isostery isostery and based on the determined genotype information to mating mobon it confirmed that it is possible to distinguish between T and giving japbae times, and completed the present invention.

In order to solve the above problems, the present invention provides a set of SSR primers for distinguishing between the umbilicus and the hybridized plants from citrus sarcoma comprising at least one SSR primer set selected from the group consisting of 17 SSR primer sets .

The present invention also provides a kit for distinguishing a stem from a stem and a stem from a stem of a citrus sarcoma comprising the SSR primer set.

The present invention also provides a method for distinguishing between a stem-line embryo and a hybrid stem-embryo-derived plant when the SSR primer set is used.

According to the present invention, the SSR markers of the present invention can be used to effectively distinguish early-stem and early-stem-stage plants from offspring plants obtained in a citrus hybrid combination, thereby significantly increasing the efficiency of citrus hybrid breeding and greatly reducing the cost of breeding Therefore, the distinction between the gypsum and the umbilical cord using the SSR marker will be a very useful technique for constructing an efficient tangerine molecular breeding system.

Figure 1 shows the results of genotyping analysis using BM-CiSSR-032 marker (A) and BM-CiSSR-100 marker (B) in 'Setoka' x 'only white milk' crossbreeding combination. Female, Zabang Chin ('Setoka'); Male, pollinated ('only white'); F ₁ zygotic hybrid, a hybrid with alleles and pollen alleles. Arrows indicate the detected allele peaks.
Figure 2 shows the results of genotyping using the BM-CiSSR-115b marker (A) and the BM-CiSSR-077 marker (B) in the 'Paige' x 'only white milk' crossbreeding combination. Female, Zip Chin ('page'); Male, pollinated ('only white'); F ₁ zygotic hybrid, a hybrid with alleles and pollen alleles. Arrows indicate the detected allele peaks.

In order to accomplish the object of the present invention, the present invention provides a method for distinguishing between a primitive embryo and a hybrid embryo-derived plant in a citrus sarcoma comprising at least one SSR primer set selected from the group consisting of 17 SSR (simple sequence repeat) primer sets Lt; RTI ID = 0.0 > SSR < / RTI >

The SSR primer set of the present invention specifically comprises SSR primer sets of SEQ ID NOS: 1 and 2; An SSR primer set of SEQ ID NOS: 3 and 4; An SSR primer set of SEQ ID NOS: 5 and 6; An SSR primer set of SEQ ID NOS: 7 and 8; An SSR primer set of SEQ ID NOS: 9 and 10; SSR primer sets of SEQ ID NOS: 11 and 12; An SSR primer set of SEQ ID NOS: 13 and 14; An SSR primer set of SEQ ID NOS: 15 and 16; An SSR primer set of SEQ ID NOS: 17 and 18; An SSR primer set of SEQ ID NOS: 19 and 20; An SSR primer set of SEQ ID NOS: 21 and 22; An SSR primer set of SEQ ID NOS: 23 and 24; An SSR primer set of SEQ ID NOS: 25 and 26; An SSR primer set of SEQ ID NOS: 27 and 28; An SSR primer set of SEQ ID NOs: 29 and 30; An SSR primer set of SEQ ID NOS: 31 and 32; And a set of SSR primers of SEQ ID NOS: 33 and 34. The SSR primer set of SEQ ID NO:

The SSR primer set of the present invention preferably comprises one or more, two or more, three or more, four or more, five or more, six or more, seven or more, At least 18, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16 primer sets, most preferably at least 17 primer sets, That is, SSR primer sets of SEQ ID NOS: 1 and 2; An SSR primer set of SEQ ID NOS: 3 and 4; An SSR primer set of SEQ ID NOS: 5 and 6; An SSR primer set of SEQ ID NOS: 7 and 8; An SSR primer set of SEQ ID NOS: 9 and 10; SSR primer sets of SEQ ID NOS: 11 and 12; An SSR primer set of SEQ ID NOS: 13 and 14; An SSR primer set of SEQ ID NOS: 15 and 16; An SSR primer set of SEQ ID NOS: 17 and 18; An SSR primer set of SEQ ID NOS: 19 and 20; An SSR primer set of SEQ ID NOS: 21 and 22; An SSR primer set of SEQ ID NOS: 23 and 24; An SSR primer set of SEQ ID NOS: 25 and 26; An SSR primer set of SEQ ID NOS: 27 and 28; An SSR primer set of SEQ ID NOs: 29 and 30; An SSR primer set of SEQ ID NOS: 31 and 32; And SSR primer sets of SEQ ID NOS: 33 and 34, respectively.

The SSR primers include SEQ ID NOs: 1 and 2; SEQ ID NOS: 3 and 4; SEQ ID NOS: 5 and 6; SEQ ID NOS: 7 and 8; SEQ ID NOS: 9 and 10; SEQ ID NOS: 11 and 12; SEQ ID NOS: 13 and 14; SEQ ID NOS: 15 and 16; SEQ ID NOS: 17 and 18; SEQ ID NOS: 19 and 20; SEQ ID NOS: 21 and 22; SEQ ID NOS: 23 and 24; SEQ ID NOs: 25 and 26; SEQ ID NOS: 27 and 28; SEQ ID NOs: 29 and 30; SEQ ID NOS: 31 and 32; And addition, deletion or substitution of the nucleotide sequences of SEQ ID NOS: 33 and 34.

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 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.

As used herein, an oligonucleotide used as a primer may also include a nucleotide analogue, such as phosphorothioate, alkylphosphorothioate, or peptide nucleic acid, or alternatively, And may include an intercalating agent.

The primer set includes Citrus sp.), and citrus varieties belonging to the genus Fortunella (sp.). Therefore, the primer set can be used to discriminate between the main stem and the cross stem in the case of the citrus variety breeding belonging to the genus in the genus Citrus. The primer set can be used, for example, to distinguish between a referee and a hybrid in crossbreeding combinations between citrus cultivars described in Table 1 below. Preferably, it can be used to distinguish crossbreeding from crossbreeding breeds with multiply breeds, for example, 'Setoka' or 'Harumi'.

In one embodiment of the present invention, the BM-CiSSR-032 marker of the present invention is used to distinguish crossbreed from crossbreeding of 'all white milks' used in pollen to 'multi-breed' , It is possible to identify 152 bp and 166 bp PCR bands in the selected _one of the 99 F ₁ bands, whereas only 152 bp PCR bands such as the umbrella ' 1A and Table 6).

In order to achieve still another object of the present invention,

Provided is a kit for distinguishing a stem from a stem and a stem from a stem of a citrus sarcoma, comprising a set of SSR primers according to the present invention and a reagent for carrying out an amplification reaction.

In the kit of the present invention, the reagent for carrying out the amplification reaction may include DNA polymerase, dNTPs, buffer and the like. In addition, the kit of the present invention may further include a user guide describing optimal reaction performing conditions. The manual is a printed document that explains how to use the kit, for example, how to prepare PCR buffer, the reaction conditions presented, and so on. The manual includes instructions on the surface of the package including a brochure or leaflet in the form of a brochure, a label attached to the kit, and a kit. In addition, the brochure includes information that is disclosed or provided through an electronic medium such as the Internet.

In order to achieve still another object of the present invention,

Isolating the citrus genomic DNA;

Amplifying the target sequence by performing amplification reaction using the separated genomic DNA as a template and using the SSR primer set according to the present invention; And

And detecting the amplification product. The present invention also provides a method for distinguishing a stem from a hybrid stem and a hybrid stem.

The method of the present invention comprises isolating genomic DNA from a citrus sample. A method known in the art may be used for separating the genomic DNA from the sample. For example, a CTAB method may be used, or a Wizard prep kit (Promega, USA) may be used. The target sequence can be amplified by performing amplification reaction using the separated genomic DNA as a template and SSR primer set according to an embodiment of the present invention as a primer. Methods for amplifying a target nucleic acid include polymerase chain reaction (PCR), ligase chain reaction, nucleic acid sequence-based amplification, transcription-based amplification system, Strand displacement amplification or amplification with Q [beta] replicase, or any other suitable method for amplifying nucleic acid molecules known in the art. Among them, PCR is a method of amplifying a target nucleic acid from a pair of primers that specifically bind to a target nucleic acid using a polymerase. Such PCR methods are well known in the art, and commercially available kits may be used.

In the method of the present invention, the amplified target sequence may be labeled with a detectable labeling substance. In one embodiment, the labeling material can be, but is not limited to, a fluorescent, phosphorescent or radioactive substance. Preferably, the labeling material is 6-FAM (6-Carboxyfluorescein), NED, VIC, PET or ROX. When the target sequence is amplified, 6-FAM, NED, VIC, PET or ROX is labeled at the 5'-end of the primer and PCR is carried out. The target sequence can be labeled with a detectable fluorescent labeling substance. Also, the labeling substance may include Cy-5 or Cy-3. When the radioactive isotope such as ³² P or ³⁵ S is added to the PCR reaction solution, the amplification product may be synthesized and the radioactive substance may be incorporated into the amplification product and the amplification product may be labeled as radioactive. The set of SSR primers used to amplify the target sequence is as described above.

The method of the present invention comprises detecting said amplification product. The detection of the amplification product can be performed through capillary electrophoresis, DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement or phosphorescence measurement, but is not limited thereto. As a method of detecting the amplification product, gel electrophoresis can be performed, and gel electrophoresis can be performed using acrylamide gel electrophoresis or agarose gel electrophoresis according to the size of the amplification product. In addition, capillary electrophoresis can be performed. Capillary electrophoresis can be performed, for example, using the ABI Genetic Analyzer. In the fluorescence measurement method, when a fluorescent dye is labeled at the 5'-end of the primer and PCR is performed, the target sequence is labeled with a fluorescent label capable of detecting the fluorescence. The fluorescence thus labeled can be measured using a fluorescence analyzer. In addition, in the case of performing the PCR, the radioactive isotope such as ³² P or ³⁵ S is added to the PCR reaction solution to mark the amplification product, and then a radioactive measurement device such as a Geiger counter or liquid scintillation counter The radioactivity can be measured using a liquid scintillation counter.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Materials and methods

Plant material and genomic DNA extraction

In order to isolate genomic DNA, leaf tissue was collected from 101 genetic resources of Jeju Special Self-Governing Province Agricultural Technology Center (Table 1). The collected leaf tissues were frozen in liquid nitrogen and stored at -80 ° C until genome DNA extraction. Genomic DNA was extracted using the Biomedic ^® Plant gDNA Extraction Kit (Biomedic Co., Korea). The amount and quality of extracted genomic DNA were determined by agarose gel electrophoresis with a DeNovix DS-11 + Spectrophotometer (DeNovix, USA). DNA quality for genome detoxification was further analyzed using Bioanalyzer 2100 (Agilent Technologies, USA).

Dielectric decryption

A paired-end DNA library for genome sequencing was generated using TruSeq DNA Library Prep Kits (Illumina, USA). Approximately 6 gigabytes of sequence information was obtained for each citrus variety using the Illumina HiSeq 2500 platform. The generated raw reads were trimming using the DynamicTrim and LengthSort software of the SolexaQA package (v.1.13; Cox et al. 2010). For the clean reads obtained after trimming, mapping was performed to the standard genome of haploid C. clementina 'Clemenules', and consensus sequence was obtained using the Burrows-Wheeler Aligner (BWA) program (0.6.1-r104) Respectively.

Secure SSR markers and primer design from genome information

MIcroSAtelitte (MISA) software (http://pgrc.ipk-gatersleben.de/misa/) was used to identify SSR sequences in consensus sequences. Only SSRs consisting of 2 to 10 nucleotide motifs were considered to determine the presence of SSRs. The minimum repeat unit of each SSR is as follows: 6 for two nucleotide motifs, 5 for 3 nucleotide motifs, 4 for 4 nucleotide motifs, 3 for 5 nucleotide motifs, 6 to 10 2 times for dog nucleotide motif. Feature analysis of SSR was obtained from statistical analysis of MISA files. The information obtained from the MISA analysis was used in primer design for amplification of SSR motifs. In-house script and Primer3 software (v2.3.5) (Untergrasser et al., 2012) were used for the design of the primer. Primer design was performed on the following criteria: primer length 18-24 bp (optimal 20 bp), GC% of primer 20-80% (optimal 50%), primer Tm value 55-65 ° C 60 ° C), the size of the PCR product is 150-500 bp.

SSR polymorphism in silico  analysis

In silico polymorphism analysis of SSR markers was performed using the virtual PCR method. That is, a pair of primer sequences derived from standard genomic information was mapped to the consensus sequence of each strain. If both sequences of the SSR motif have sequence lengths sufficient to allow primer design, they are candidates for SSR marker development. In addition, primers arranged in one place were selected as candidates for SSR markers. The amplicons produced by in silico were compared with the amplicon size predicted among the cultivars. When the amplicon size was at least 1 bp, it was classified as polymorphic SSR markers. When the amplicon size was the same, no polymorphism was identified.

Citrus hybrid combination

Seto this study, with the isostery varieties bred mobon car (Citrus hybrid 'Setoka') x only white spirit (C. maxima 'Banbeiyu'), Harumi (Citrus hybrid 'Harumi') x only white spirit, Nova (C. reticulata ' Nova ') x million euros, page (C. reticulata' page ') x million euros, cheonggyeon (Citrus hybrid car used by Seto x paragraph (C. maxima) crossbreeds, however isostery bred varieties mobon' Kiyomi ') ( Citrus hybrid 'Seminole'), Ehime 28 ( Citrus hybrid 'Ehimekashi No. 28' x Citrus hybrid 'Orlando', Citrus hybrid 'Harehime' _One living entity and a sample and duplicate were used.

SSR marker polymorphism test

For selection of polymorphic SSR markers, genomic DNA of 11 citrus cultivars belonging to lemons, mandarins, sour oranges, citrons, oranges, and tantalum were respectively subjected to PCR. The PCR reaction consisted of 20 ng of genomic DNA, 5 의 of 2 x HS ^TM Taq mix (0.3 units / ㎕ HS Taq DNA polymerase, 3.2 mM MgSO ₄ , containing 0.4 mM dNTPs) (Dongsheng Biotech, China) A forward reaction was carried out using an ABI 2720 thermal cycler (Applied Biosystems, USA) to a reaction volume of 10 μl containing 0.2 μl of 10 pmol reverse primer. PCR amplification products were electrophoresed on 1.5% agarose gel to confirm amplification and polymorphism.

Genotyping using M13-tailed SSR markers

DNA fragment analysis for genotyping using SSR markers was performed using the M13-tailed PCR method. Genomic DNA was diluted to 10 ng / 에 in sterile distilled water (Biomedic) from which nucleic acid hydrolase was removed. The PCR reaction consisted of 20 ng genomic DNA, 5 2 2 x HS ^TM Taq mix (containing 0.3 unit / HS HS Taq DNA polymerase, 3.2 mM MgSO ₄ , 0.4 mM dNTPs) (Dongsheng Biotech), 0.2 쨉 l 10 pmol M13-tailed forward primer , 1 μl of 10 pmol reverse primer, 1 μl of 10 pmol 6-FAM labeled M13 primer (5'-6-FAM-TGTAAAACGACGGCCAGT-3 ' Applied Biosystems). The PCR primer set used in this experiment is summarized in Table 5. The conditions for the PCR amplification were as follows: Initial heat denaturation (once) -94 degrees 5 minutes, primary DNA amplification (total of 15 replicates) -94 degrees 30 seconds, 58 degrees 30 seconds, 72 degrees 30 seconds; Second DNA amplification (total 20 times) -94 degrees 30 seconds, 53 degrees 30 seconds, 72 degrees 30 seconds, final elongation reaction (once) -72 degrees 30 minutes. PCR amplification products were electrophoresed on 1.5% agarose gel to confirm amplification. DNA fragment analysis for PCR amplification products was performed as described above using ABI 3730 DNA Analyzer (Applied Biosystems) and the size of alleles was analyzed using GeneMapper software (ver. 4.0; Applied Biosystems).

Pear type test for citrus genetic circle

The number of seeds was measured by counting the total number of seeds in five ripe fruits. After measuring the number of seeds, 5 seeds were randomly selected to mix all the seeds collected from each variety and determine the type of embryo (single, multiple, mixed). Individual seeds were cut in half and the type of embryo was observed under a stereomicroscope. For the varieties showing hybrid varieties, the type of embryo was finally determined after further analysis of 5 seeds.

EXAMPLES Example 1. Collection of Citrus Genetic Resources

A total of 101 citrus genetic resources were collected for a systematic and targeted citrus breeding program. Plasmodium is a genetic trait found in many citrus cultivars and is a major obstacle to the cultivation of new varieties of citrus fruits by traditional cross breeding. Prior to the use of the collected citrus genetic resources in a full-scale breeding program, a multiclass test for mature seeds was conducted to examine whether any genetic material had a polymorphic phenotype caused by the umbilical cord. Of the 101 genotypes, 22 were monoplasic and 54 were polyploid phenotypes. The remaining 25 points showed mixed seeds with mixed seeds and mixed seeds in one fruit (Table 1). In summary, the results of multi-assays on citrus genetic resources collected so far show that the multiple traits are widely distributed in various citrus groups. According to previous reports, the incidence of umbilical embryo plants in major citrus groups decreased from 0 ('Kishiu' mandarin and 11 pumey varieties) to 100% ('Dancy' and 'Kara' mandarin ) Varies. All varieties belonging to the pumello group surveyed in this study were monoplasic , except for C. grandis . Not all seeds made by wood with the umbilical cord have several mature belly. Normal ovarian reproduction can also occur in genotypes with the trunk germ line. Genotypes with an umbilical cord can produce several different types of seeds: a seed with one mature stem, and a seed with one mature stem. Seeds with multiple mature jumbo pears, seeds with one mature pomace and one or more mature jumbo pears. Therefore, 25 citrus genetic resources showing mixed type can be classified as multiple species.

Example 2. Detection of polymorphic SSR markers between genotypes and breeds

Genome decoys were performed to have genome coverage of 17.68, 16.42, 19.18, and 16.3 times for four citrus varieties, namely, Fina Sodea, Clementin Mandarin, Citrus, Citrus, 2). As a result of in silico analysis, polymorphic SSR markers were identified in the genome by using the decrypted genomic information. As a result, 521 polymorphic polymorphisms between mandarin and mandarin and 169 polymorphic polymorphic SSR markers were predicted (Table 3 and Table 4) ).

Example 3. Screening of SSR markers showing gene polymorphism with multiple traits

The first step to select polymorphic SSR markers that discriminate between citrus pear and gypsy or their derived plants is the use of 41 Expressed Sequence Tags (EST) -based SSRs and Ollitrault et al. (2008) reported by Luro et al. We used 77 SSR information from the bacterial artificial chromosome (BAC) end reported by the authors. To obtain more polymorphic SSR markers, we used 40 polymorphic candidate SSR markers obtained through in silico digestion based on standard genome-based resequencing and comparative genomic analysis of 'Fina Sodea' clementine mandarin and citrus fruit Respectively. Ten polymorphic candidate markers obtained from comparative genomic analysis for citrus and golden citrus were also used. Based on the results of agarose gel electrophoresis and DNA fragment analysis on these SSR markers, SSR markers polymorphic among eleven varieties belonging to several citrus group were selected (data not shown): lemon group - 'Lisbon' C. limon 'Lisbon lemon'; C. clementina , C. unshiu 'Miyagawa Wase', C. unshiu 'Okitsu Wase', C. unshiu 'Nichinan 1 gou'; Mandarin group - C. clementina ; Sauer orange group - C. natsudaidai ; Orange group - C. sinensis 'Sanggwinelli'; Citron group - C. sphaerocarpa , citron ( C. junos ); The group of trombels - bifurcation ( C. hybrida 'Shiranuhi'), bluegrass ( Citrus hybrid 'Kiyomi'). Finally, 17 polymorphic SSR markers were selected through the polymorphism test of SSR markers and named as BM-CiSSR-xxx (Table 5).

Example 4. Genotyping analysis of citrus genetic resources using selected polymorphic SSR markers

Genotyping of 101 citrus genetic resources was performed using the M13-tailed PCR method using 17 SSR markers selected from 11 polymorphous strains belonging to 6 citrus group (Table 6 ~ Table 11). At least 5 (BM-CiSSR-087) to 16 (BM-CiSSR-162) alleles were detectable for each marker in the resources used in the experiment. BM-CiSSR-162 markers have the highest polymorphism, and the size and number of each marker-specific allele are summarized in Tables 6-11.

Example 5 Identification of Plants Derived from Crosses in Crossbred Combinations Using the Polymorphic SSR Markers of the Present Invention

As a result of the multiple replicates of the citrus genetic resources collected in this study, the multiple traits were universally observed in various citrus groups except for pumice (Table 1). The SSR markers showing pollinated polymorphism were applied to investigate whether the plants derived from the F1 plant group derived from the crossbreeding combinations using the multipurpose varieties could be distinguished from the hybridized plants. We used 'Setoka', 'Harumi', 'Nova', 'Page', and 'Paekyu' and 'Paragraph' for pollinated varieties. Citrus hybrid 'Ehime Kashi 28 gou', 'Harehime', and 'Seminol' and 'Orlando' were used as the sweet potato varieties for comparison experiments. . BM-CiSSR-032, BM-CiSSR-100, and BM-CiSSR-100 were found to be polymorphic between the mating females and pollen females of each mating combination. 'Harumi' x 'only white' - BM-CiSSR-032, BM-CiSSR-073, BM-CiSSR-077; 'Nova' x 'only white' - BM-CiSSR-032, BM-CiSSR-159, BM-CiSSR-115b; 'Page' x 'only white' - BM-CiSSR-077, BM-CiSSR-115b; 'Setoka x' paragraph "- BM-CiSSR-032, BM-CiSSR-100; 'Blue eyes' x 'Seminol' - BM-CiSSR-013; 'Ehime 28' x 'Orlando' - BM-CiSSR-012, BM-CiSSR-087; 'Harehime' x 'Orlando' - BM-CiSSR-012, BM-CiSSR-013, BM-CiSSR-087.

As a result of the genotyping analysis to distinguish between hybridization and embryo embryos using polymorphic SSR markers for each hybridization combination, it was found that among the five hybridization combinations using the multipurpose varieties, Three hybridization strains were identified in the combination of 1, 'page' x 'only white' hybridization (Table 12; FIGS. 1 and 2). However, in the case of 'Harumi' x 'Only White', 'Nova' x 'Only White', and 'Setoka x' paragraphs, no markers could be identified in all the markers used (Table 7). On the other hand, all of the F ₁ plants derived from the three crossbreeding combinations of 'Chung Dog' x 'Seminol', 'Ehime 28' x 'Orlando', 'Harehime' x 'Orlando' (Table 12). It was confirmed that there was a hybridization with the alleles and pollinated alleles simultaneously for the genetic locus (Table 12).

The results of genotyping with BM-CiSSR-115b marker showed that all pollen allele peaks were low (37, < RTI ID = 0.0 > 45) only the pollen allele was detected (# 40). Genetic analysis was performed with BM-CiSSR-077 marker to re-test whether these three plants were derived from gypsum. As a result, in the case of plants 40 and 45, I could confirm. On the other hand, in the case of the 37th plant, one of the two alleles detected coincided with the allele of the zebrafish, while the other allele was a new allele that did not exist in the zebrafish and pollen. In the genotypic analysis of the 40th plant using the BM-CiSSR-115b marker, only the pollen allele was detected, while the BM-CiSSR-077 marker detected all the wild-type and pollinated alleles (FIG. 2). These results indicate that only one allele is detected in the wild-type relative to the BM-CiSSR-115b genetic locus (marker), which can be judged as homozygote (Fig. 2A), but in reality the allele is heterozygous ) State. In other words, the BM-CiSSR-115b genetic locus in the sputum is considered to be a null allele of one homologous chromosome. Therefore, F1 plants derived from fertilization with gametes with null alleles only detect pollen-related alleles for BM-CiSSR-115b genetic loci (markers). A genotype analysis of the 37th plant using the BM-CiSSR-077 marker revealed a new allele that did not exist in the wild-type and pollen-free alleles. BM-CiSSR-077 is a repetitive SSR marker with two bases (AT) (Table 5). The newly emerged allele in the 37th plant is longer or shorter than the pollinated or wild-type allele (4 bases 2B). The SSR mutation occurs at a rate of 10 ^-2 to 10 ^-6 per genetic locus per generation, which is known to be very high compared to the point mutation rate in the protein coding region. The main mechanism of SSR mutation (increase or decrease of repeating unit) is the slippage of DNA strand and the recombination between DNA strands during DNA replication process. . BM-CiSSR-077 Genetic loci The emergence of new alleles in plant # 37 appears to be caused by one of these two mechanisms in gametogenesis.

The genotypic analysis using the SSR marker for the F1 plants derived from crossbreeding using various replicate cultivars showed that the multiplication effect by the umbilical cord significantly reduced the efficiency of breeding, . Therefore, the distinction between the hybrid and the umbilical cord using polymorphic SSR markers is a very useful technique for constructing an efficient molecular breeding system.

<110> Biomedic Co., LTD <120> SSR molecular markers for identifying citrus zygotic and nucellar          individuals and uses thereof <130> PN16409 <160> 35 <170> Kopatentin 2.0 <210> 1 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 tgtaaaacga cggccagtgg gctcagttct tctctactc 39 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 gcattaggct tctctcatac c 21 <210> 3 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 tgtaaaacga cggccagtgg tggcatacat acatacatac a 41 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 gcaacatctg gaactactca 20 <210> 5 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 tgtaaaacga cggccagtgc ctgagtttct ttgttatg 38 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 cattccatcg tctcctattg t 21 <210> 7 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 tgtaaaacga cggccagtat tagtgcgggt aagatgaa 38 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 aaggatttgg tgtaggaagt aa 22 <210> 9 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 tgtaaaacga cggccagtcg gacaaggaga tgaagatag 39 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 ttctaacagc accaagcag 19 <210> 11 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 tgtaaaacga cggccagttg tatttatttc tgactacgac c 41 <210> 12 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 atgcgtttgg tgtgtgtt 18 <210> 13 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 tgtaaaacga cggccagtac ctgagccctt tttggttt 38 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 gccagatcaa ggctcaaatc 20 <210> 15 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 tgtaaaacga cggccagtca gatcctattg cagaggca 38 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 gcccatttgt attgccattt 20 <210> 17 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 tgtaaaacga cggccagtcc ccctcttctt tcacacaa 38 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 ggtgagcagc catcttcttc 20 <210> 19 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 tgtaaaacga cggccagtga attgggagga cgaactga 38 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 cgagccctag acagagatgg 20 <210> 21 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 tgtaaaacga cggccagtgt tttcagctgg attcgagg 38 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 cacgtgtcct cctggaactt 20 <210> 23 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 tgtaaaacga cggccagtcc gatacagcac aaagcaaa 38 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 24 tggaaagaga gaagccaagc 20 <210> 25 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 25 tgtaaaacga cggccagtcg gtgtgtattg ggtacacg 38 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 26 gctttttcga aagcgtcaag 20 <210> 27 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 27 tgtaaaacga cggccagtgc aacgtgtact gacgcttg 38 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 28 gctcgtatct gaagctcgcc 20 <210> 29 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 29 tgtaaaacga cggccagtat gacctcaaac ggtgagca 38 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 30 cttccacatc cgaaccgaca 20 <210> 31 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 31 tgtaaaacga cggccagtgc tagggttcca gacttccag 39 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 32 gatttggccg atcgaaagcc 20 <210> 33 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 33 tgtaaaacga cggccagtag caacttaagg tccttcacga 40 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 34 ttctctgctc tgctgtgcat 20 <210> 35 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 35 tgtaaaacga cggccagt 18

Claims (6)

An SSR primer set of SEQ ID NOS: 1 and 2; An SSR primer set of SEQ ID NOS: 3 and 4; An SSR primer set of SEQ ID NOS: 5 and 6; An SSR primer set of SEQ ID NOS: 7 and 8; An SSR primer set of SEQ ID NOS: 9 and 10; SSR primer sets of SEQ ID NOS: 11 and 12; An SSR primer set of SEQ ID NOS: 13 and 14; An SSR primer set of SEQ ID NOS: 15 and 16; An SSR primer set of SEQ ID NOS: 17 and 18; An SSR primer set of SEQ ID NOS: 19 and 20; An SSR primer set of SEQ ID NOS: 21 and 22; An SSR primer set of SEQ ID NOS: 23 and 24; An SSR primer set of SEQ ID NOS: 25 and 26; An SSR primer set of SEQ ID NOS: 27 and 28; An SSR primer set of SEQ ID NOs: 29 and 30; An SSR primer set of SEQ ID NOS: 31 and 32; And an SSR primer set of SEQ ID NOS: 33 and 34, respectively. delete The SSR primer set of claim 1, wherein the citrus fruit belongs to Citrus sp. Or Fortunella sp.. The SSR primer set is used to distinguish between a stem and a stem. A kit for distinguishing between a primate embryo and a hybrid embryo-derived plant comprising a SSR primer set according to claim 1 and a reagent for carrying out an amplification reaction. 5. The kit according to claim 4, wherein the reagent for carrying out the amplification reaction comprises a DNA polymerase, dNTPs and a buffer. Isolating the genomic DNA from the citrus;
Amplifying the target sequence by performing amplification reaction using the separated genomic DNA as a template and using the SSR primer set according to claim 1; And
And detecting the amplification product. 2. A method for distinguishing between a primitive embryo and a transgenic embryo-derived plant in the case of citrus sarcoma.

Images