KR102635019B1 - Molecular marker for discriminating early-flowering or late-flowering of Miscanthus sinensis cultivar and uses thereof - Google Patents

Abstract

The present invention relates to a molecular marker for identifying early-flowering or late-flowering silver grass varieties and its use. The molecular marker of the present invention has a very high correlation with the flowering-related traits of silver grass, so it can be used to identify early-flowering or late-flowering silver grass varieties. Since it can efficiently identify silver grass, it can be usefully used as a molecular marker that can dramatically shorten the growing period of silver grass varieties.

Description

Molecular marker for discriminating early-flowering or late-flowering of Miscanthus sinensis cultivar and uses thereof}

The present invention relates to molecular markers for identifying early-flowering or late-flowering silver grass varieties and their use.

Plants grow in volume and length during the vegetative growth stage, increasing biomass, but once they enter the reproductive growth stage, biomass no longer increases, so controlling the flowering time is an important key to directly controlling biomass. Miscanthus sinensis , which has recently been in the spotlight as a non-edible crop for bioenergy production, has a C4 photosynthetic circuit and is a perennial plant that generally reproduces underground. It is known that there are 17 species of Miscanthus plants, including Miscanthus sinensis and Miscanthus sacchariflorus , and the important Miscanthus species for bioenergy are diploid Miscanthus (2n=2x=38) and tetraploid Miscanthus (2n=2x=38). There is the miscanthus (2n=4x=76) and the triploid Miscanthus

The early flowering of silver grass is closely related to its biomass production, and silver grass with late flowering characteristics is known to have high biomass productivity. Therefore, in the development of silver grass cultivars for biomass, it is very important to evaluate early flowering using molecular markers.

Silver grass's beautiful plant shape and blooms provide excellent scenic value and are environmentally friendly. It is also easy to manage, so it can be used for landscaping, landscaping, and ecological restoration. Since the early onset of flowering is involved in the formation of the first growth of silver grass, it is also important to evaluate the early onset of flowering using molecular markers in the development of silver grass varieties for landscaping, landscaping, and ecological restoration.

In developing silver grass varieties, it is possible to develop silver grass varieties for various purposes, such as for biomass or landscaping, by controlling the flowering time. However, unlike other crops, research on reproductive growth such as emergence and flowering of silver grass is relatively insufficient. In particular, the flowering time of plants is a quantitative trait controlled by polygenes, so it has a very complex genetic pattern compared to qualitative traits controlled by a single gene. Silver grass is a perennial plant, and in order to confirm the flowering period of individuals propagated through seeds, accurate determination of the flowering period is possible only two years after sowing the seeds. Therefore, early identification of the flowering period is very important in the development of silver grass varieties.

Accordingly, the present inventors developed a SNP molecular marker combination that can distinguish early-flowering or late-flowering silver grass varieties, and created a primer set that can amplify the molecular markers to simply, quickly and accurately distinguish early-flowering or late-flowering silver grass varieties. I confirmed that it can be done.

Meanwhile, Korean Patent Publication No. 2013-0124746 discloses a 'method for identifying giant silver grass', but there is no description of molecular markers and their uses for identifying early-flowering or late-flowering silver grass varieties of the present invention.

The present invention was developed in response to the above-mentioned needs, and the present inventors used the genomic DNA of the F ₁ population obtained as a result of crossing SNU-M367 (mother copy), which has a slow flowering time, and SNU-M087 (study copy), which has a fast flowering time, as a standard. By mapping the genome, SNPs present in each chromosome were identified. A genetic map was created using the above SNP information, and a linkage analysis was performed using flowering-related trait data (first leaf development date, seeding start date, seeding 50%, first flowering start date, and flowering 50%), and then present on chromosome 5. SNP markers Msi_Flo_01, Msi_Flo_02, and Msi_Flo_03, as well as SNP markers Msi_Flo_04, located on chromosome 7, and Msi_Flo_05, located on chromosome 13, were developed. In addition, the present invention was completed by confirming that early-flowering or late-flowering silver grass varieties can be accurately identified through the combination of the above SNP markers.

In order to solve the above problems, the present invention provides an oligonucleotide primer set of SEQ ID NOs: 1 and 2; Oligonucleotide primer sets of SEQ ID NOs: 3 and 4; Oligonucleotide primer sets of SEQ ID NOs: 5 and 6; Oligonucleotide primer sets of SEQ ID NOs: 7 and 8; And oligonucleotide primer sets of SEQ ID NOs: 9 and 10; It provides a primer set composition for determining early-flowering or late-flowering Miscanthus sinensis varieties, including one or more primer sets selected from the group consisting of.

In addition, the present invention provides an oligonucleotide primer set of SEQ ID NOs: 1 and 2; Oligonucleotide primer sets of SEQ ID NOs: 3 and 4; Oligonucleotide primer sets of SEQ ID NOs: 5 and 6; Oligonucleotide primer sets of SEQ ID NOs: 7 and 8; And oligonucleotide primer sets of SEQ ID NOs: 9 and 10; It provides a primer set composition for determining early-flowering or late-flowering silver grass varieties, characterized in that it comprises three or more primer sets selected from the group consisting of.

In addition, the present invention provides a kit for determining early-flowering or late-flowering silver grass varieties containing the primer set composition.

In addition, the present invention includes the steps of isolating genomic DNA from a silver grass sample; Using the isolated genomic DNA as a template and performing an amplification reaction using the primer set composition to amplify a target sequence; and detecting the product of the amplification step. It provides a method for determining early-flowering or late-flowering silver grass varieties, including a step.

In addition, the present invention relates to a polynucleotide consisting of the base sequence of SEQ ID NO: 11 to 15, a polynucleotide consisting of 8 or more consecutive nucleotides including a SNP (single nucleotide polymorphism) base located at the 301st position in each base sequence. Provided is a SNP marker composition for identifying early-flowering or late-flowering silver grass varieties, which includes one or more polynucleotides selected from the group consisting of polynucleotides or complementary polynucleotides thereof.

In addition, the present invention provides a microarray for discriminating early-flowering or late-flowering silver grass varieties, including a polynucleotide containing the above SNP base or cDNA thereof.

The SNP molecular marker of the present invention has a very high correlation with the flowering-related traits of silver grass and can efficiently identify early or late-flowering silver grass varieties, making it a molecular marker that can dramatically shorten the cultivation period of silver grass varieties. It is expected that it will be useful.

Figure 1 is a frequency distribution chart of the flowering start date (the date when silver grass first flowered) _of the cross group (SNU-M367 The flowering date is calculated in Julian days (Julian day or Julian day number), and the difference in flowering date between the mother plant and the part is 29 days.
Figure 2 shows the location of flowering trait-related QTL (Quantitative Trait Loci) on the genetic map of SNU-M367 (parent), SNU-M087 (sub), and the cross group (SNU-M367×SNU-M087) F ₁ .
Figure 3 shows the LOD values obtained through linkage analysis between SNPs located on chromosome 7 of silver grass and flowering traits. Each line graph shows the date of leaf expansion, start of seeding, date of achieving 50% seeding, and flowering among the flowering-related traits. This refers to the analysis results of the start date and the date of achieving 50% flowering.

In order to achieve the object of the present invention, the present invention provides an oligonucleotide primer set of SEQ ID NOs: 1 and 2; Oligonucleotide primer sets of SEQ ID NOs: 3 and 4; Oligonucleotide primer sets of SEQ ID NOs: 5 and 6; Oligonucleotide primer sets of SEQ ID NOs: 7 and 8; And oligonucleotide primer sets of SEQ ID NOs: 9 and 10; It provides a primer set composition for determining early-flowering or late-flowering Miscanthus sinensis varieties, including one or more primer sets selected from the group consisting of.

The primer set composition of the present invention is the genomic DNA of the hybrid group F ₁ obtained using the Miscanthus sinensis line 'SNU-M367', which has a slow flowering time, as a model and the Miscanthus sinensis line, which has a fast flowering time, 'SNU-M087' as a copy. It was created based on SNPs present in QTL related to flowering traits present in each chromosome by mapping to the standard genome. The standard genome is a new genome sequence obtained by re-sequencing the SNU-M087 Miscanthus sinensis line used as a copy to the already known Miscanthus sinensis standard genome (Phytozome DB, Miscanthus sinensis v7.1), and is the SNP marker location of the present invention. The same can be confirmed in Phytozome DB's Miscanthus sinensis v7.1 (Mitros, et al., 2020).

That is, the primer set composition of the present invention is 16,328,527 bp, 18,264,190 bp, or 71,290,934 bp on chromosome 5 of the genome derived through the reference-guided assembly method of SNU-M087 and the Miscanthus sinensis standard genome (Phytozome DB, Miscanthus sinensis v7.1). SNPs present; SNP present on chromosome 7, 56,168,902 bp; Or, it was created based on a SNP present on chromosome 13, 8,845 bp.

In the present invention, the 'SNU-M087' silver grass line used as the model flowered for the first time on the 232nd day according to the Julian day (Julian day or Julian day number) calculation method, and 'SNU-M367' used as the model. The silver grass line first flowered on the 261st day according to the Julian day calculation method. In other words, the primer set composition of the present invention can distinguish early-flowering or late-flowering silver grass varieties whose flowering dates differ by about 28-31 days, but there may be differences in flowering times due to genetic or environmental factors. There is no particular limitation thereto.

The primer set composition of the present invention can evaluate and predict the flowering time of silver grass at an early stage, so early flowering or late flowering characteristics are evaluated and predicted for the genomic DNA of young plants or seeds at the beginning of the silver grass breeding stage. Breeding material capable of late flowering can be developed. In addition, it can be applied to various stages other than the initial stage in the breeding process of silver grass, and can be useful in cultivating new silver grass varieties with different flowering times by applying it to hybridization between various silver grass resources, lines, or varieties.

The primer set composition of the present invention preferably includes one or more primer sets selected from the above five primer sets, namely, the oligonucleotide primer sets of SEQ ID NOs: 1 and 2; Oligonucleotide primer sets of SEQ ID NOs: 3 and 4; Oligonucleotide primer sets of SEQ ID NOs: 5 and 6; Oligonucleotide primer sets of SEQ ID NOs: 7 and 8; and oligonucleotide primer sets of SEQ ID NOs: 9 and 10.

When using 3 of the 5 primer sets above, early-flowering or late-flowering silver grass varieties can be identified at a rate of 79~83%, and when using 4 primer sets, early-flowering or late-flowering silver grass varieties can be identified at a rate of 98~83%. It can be distinguished at a 100% level, and if all five primer sets are used simultaneously, early-flowering or late-flowering silver grass varieties can be distinguished at a 100% level (see Table 10).

The primer includes an oligonucleotide consisting of a fragment of 13 or more, 14 or more, 15 or more, 16 or more, 17 or more consecutive nucleotides in the sequence of SEQ ID NOs. 1 to 10, depending on the sequence length of each primer. can do. For example, the primer (26 oligonucleotides) of SEQ ID NO: 1 may include an oligonucleotide consisting of a segment of 22 or more, 23 or more, 24 or more, or 25 or more consecutive nucleotides in the sequence of SEQ ID NO. . In addition, the primer may also include sequences in which the base sequences of SEQ ID NOs: 1 to 10 are added, deleted, or substituted. Among the above sequence numbers, the odd-numbered oligonucleotide primer is a forward primer, and the even-numbered oligonucleotide primer is a reverse primer.

In the present invention, “primer” refers to a single-stranded oligonucleotide sequence complementary to the nucleic acid strand to be copied, and can serve as a starting point for the synthesis of a primer extension product. The length and sequence of the primers should allow for initiation of synthesis of the extension product. The specific length and sequence of the primer will depend on the complexity of the DNA or RNA target desired as well as the conditions under which the primer is used, such as temperature and ionic strength.

In the present specification, oligonucleotides used as primers may also include nucleotide analogues, such as phosphorothioates, alkylphosphorothioates or peptide nucleic acids, or May contain an intercalating agent.

The present invention also provides a kit for determining early-flowering or late-flowering silver grass varieties, including a primer set composition according to the present invention.

In the kit of the present invention, the primer set composition is the same as described above.

In one embodiment of the present invention, the kit may further include a reagent for performing an amplification reaction, and the reagent for performing the amplification reaction may include DNA polymerase, dNTPs, and a buffer. Not limited.

In one embodiment of the present invention, the kit may further include a user guide describing optimal reaction performance conditions. The guide is a printed material that explains how to use the kit, for example, how to prepare reverse transcription buffer and PCR buffer, and the suggested reaction conditions. Instructions include information leaflets in the form of pamphlets or leaflets, labels affixed to the kit, and instructions on the surface of the package containing the kit. Additionally, the guide includes information disclosed or provided through electronic media such as the Internet.

The present invention also,

Isolating genomic DNA from the silver grass sample;

Using the isolated genomic DNA as a template and performing an amplification reaction using the primer set composition of the present invention to amplify the target sequence; and

It provides a method for determining early-flowering or late-flowering silver grass varieties, including the step of detecting the product of the amplification step.

In the method according to one embodiment of the present invention, the primer set composition is the same as described above.

The method of the present invention includes the step of isolating genomic DNA from a silver grass sample. The silver grass sample is not limited to this, but may be a seed, pre-flowering leaf, or stem sample of a silver grass plant. Methods known in the art for isolating the genomic DNA may be used, for example, the CTAB method may be used, or the Wizard prep kit (Promega) may be used. The target sequence can be amplified by performing an amplification reaction using the isolated genomic DNA as a template and an oligonucleotide primer set according to an embodiment of the present invention as primers. Methods for amplifying target nucleic acids include polymerase chain reaction (PCR), ligase chain reaction, nucleic acid sequence-based amplification, and transcription-based amplification system. amplification system, strand displacement amplification or amplification via Qβ replicase or any other suitable method for amplifying nucleic acid molecules known in the art. Among these, PCR is a method of amplifying a target nucleic acid from a primer pair that specifically binds to the target nucleic acid using polymerase. These PCR methods are well known in the art, and commercially available kits can also be used.

In one embodiment of the present invention, the amplified target sequence may be labeled with a detectable label. The labeling material may be a material that emits fluorescence, phosphorescence, or radioactivity, but is not limited thereto. Preferably, the labeling substance is Cy-5 or Cy-3. When amplifying a target sequence, if the 5'-end of the primer is labeled with Cy-5 or Cy-3 and PCR is performed, the target sequence can be labeled with a detectable fluorescent labeling substance. In addition, labeling using a radioactive material may cause radioactivity to be incorporated into the amplification product as the amplification product is synthesized by adding a radioactive isotope such as ³² P or ³⁵ S to the PCR reaction solution during PCR, thereby causing the amplification product to be radioactively labeled. The oligonucleotide primer set used to amplify the target sequence was as described above.

In one embodiment of the present invention, the method for determining the early-flowering or late-flowering silver grass variety includes detecting the product of the amplification step, and the detection of the product of the amplification step is performed using a DNA chip, gel electrophoresis, This may be performed through, but is not limited to, HRM analysis, capillary electrophoresis, radioactivity measurement, fluorescence measurement, or phosphorescence measurement. As one of the methods for detecting amplification products, capillary electrophoresis can be performed. Capillary electrophoresis can be performed using, for example, the ABi Sequencer. Additionally, gel electrophoresis can be performed, and gel electrophoresis can use agarose gel electrophoresis or acrylamide gel electrophoresis depending on the size of the amplification product. In addition, the fluorescence measurement method is to perform PCR by labeling the 5'-end of the primer with Cy-5 or Cy-3, and the target sequence is labeled with a detectable fluorescent labeling substance, and the labeled fluorescence is measured using a fluorometer. can do. In addition, the radioactivity measurement method involves adding a radioisotope such as ³² P or ³⁵ S to the PCR reaction solution to label the amplification product when performing PCR, and then using a radioactivity measurement device, such as a Geiger counter or liquid scintillation. Radioactivity can be measured using a liquid scintillation counter.

The present invention also provides a polynucleotide consisting of 8 or more consecutive nucleotides including a SNP (single nucleotide polymorphism) base located at the 301st position in each nucleotide sequence, in the polynucleotide consisting of the base sequence of SEQ ID NO: 11 to 15. Provided is a SNP marker composition for identifying early-flowering or late-flowering silver grass varieties, which includes one or more polynucleotides selected from the group consisting of polynucleotides or complementary polynucleotides thereof.

In the SNP marker composition of the present invention, the contiguous nucleotides may be 8 to 100 contiguous nucleotides, but are not limited thereto.

In the present invention, 'nucleotide' is a deoxyribonucleotide or ribonucleotide that exists in single-stranded or double-stranded form, and includes analogs of natural nucleotides unless specifically stated otherwise.

The SNP marker composition of the present invention can be used to determine early-flowering or late-flowering silver grass varieties because the 301st base in the nucleotide sequence of SEQ ID NO: 11 is expressed differently as A or C, and the 301st nucleotide in the nucleotide sequence of SEQ ID NO: 12 is It appears differently as A or C, the 301st base in the nucleotide sequence of SEQ ID NO: 13 appears differently as A or G, the 301st base in the nucleotide sequence of SEQ ID NO: 14 appears differently as A or G, and the 301st base in the nucleotide sequence of SEQ ID NO: 15 appears differently as A or G. It is based on the fact that the 301st base appears differently as A or G. More preferably, if the 301st base in the base sequences of SEQ ID NOs: 11 to 15 is A, A, G, G, and A, it can be judged to be an early flowering silver grass variety, and the 301st base in the base sequences of SEQ ID NOs: 11 to 15 If are C, C, A, A, and G, respectively, it can be judged as a late-flowering silver grass variety. Silver grass has high heterozygosity, and the flowering characteristics of the plant are quantitative traits controlled by polygenes. Therefore, when the above five SNP markers are used in combination, early-flowering or late-flowering silver grass varieties can be effectively produced. It can be determined.

Specifically, as shown in Table 10 of the present invention, when five SNP markers (Tables 5 and 6) are used individually or two of the five SNP markers are selected and combined, early flowering or late flowering silver grass varieties Although the discrimination accuracy is low at around 1-21%, when 3 out of 5 SNP markers are selected and combined, the discrimination accuracy for early-flowering or late-flowering silver grass varieties is 79-83%, and when 4 are selected and combined, the discrimination accuracy is 79-83%. The discrimination accuracy for flowering or late-flowering silver grass varieties is 98 to 100%, and when all five are combined, the discrimination accuracy for early-flowering or late-flowering silver grass varieties is 100%. Therefore, if three or more SNPs are selected and combined among the five SNP markers of the present invention, early flowering or late flowering silver grass varieties can be more accurately identified.

The present invention also provides a microarray for discriminating early-flowering or late-flowering silver grass varieties, which includes a polynucleotide or cDNA thereof containing a SNP located at the 301st base of each sequence among the base sequences of SEQ ID NOs: 11 to 15.

The polynucleotide may preferably be fixed to a substrate coated with amino-silane, poly-L-lysine, or aldehyde active group, but is not limited thereto. Methods for immobilizing the polynucleotide on the substrate include, but are not limited to, micropipetting using a piezoelectric method or a method using a pin-shaped spotter. , various methods known in the art can be used. Additionally, the substrate may be a silicon wafer, glass, quartz, metal, or plastic, but is not limited thereto.

As described above, the microarray according to the present invention may further include one or more polynucleotides selected from the group consisting of polynucleotides consisting of the base sequences of SEQ ID NOs: 11 to 15, including SNP bases in the base sequence. . Since the polynucleotide contains SNP bases that exhibit polymorphism between early-flowering or late-flowering silver grass varieties, the microarray according to the present invention can be usefully used to determine early-flowering or late-flowering silver grass varieties.

Hereinafter, the present invention will be described in detail through examples. It should be noted that the specific examples and examples below include some embodiments of the invention and do not include all embodiments. It is obvious that the content of the invention disclosed by this specification is not limited to the specific embodiments described herein, and includes various implementations by those skilled in the art to which the present invention pertains. Therefore, it should be understood that the content of the invention disclosed herein is not limited to the specific embodiments described herein, and that modifications and other embodiments thereof are also included within the scope of the claims.

Example 1. Silver grass hybrid group F _One Securing and growing

F ₁ individuals were obtained by crossing SNU-M367, a Miscanthus line with late-flowering characteristics, as a model and SNU-M087, a Miscanthus line with early-flowering characteristics, as a parent. The above F ₁ 278 individuals were used, and experiments were conducted by growing the silver grass of this group for several years in an outdoor experimental field (Suwon Seoul National University College of Agriculture campus).

Example 2. Silver grass hybridization group F _One Analysis of flowering-related traits

During growth in the field, five indicators related to flowering were measured at regular intervals throughout the year, and the indicators related to flowering are as follows: (1) Date of first leaf development: The date when the flag leaf first fully developed, (2) Heading start date: The date when the uppermost tip of the ear can first be observed externally, (3) Heading 50%: The date when the ear first develops in more than 50% of the tillers of one silver grass individual, (4) Flowering First start date: The date when the silver grass flower first bloomed, (5) 50% flowering: The date when more than 50% of the spikes of a single silver grass individual flowered.

Each data was quantified using the Julian day calculation method at intervals of 1 to 2 days from late June to late October, which corresponds to the general flowering period of silver grass, and used for correlation analysis with base sequences. A survey of flowering-related traits was conducted on a total of 278 F ₁ cross groups. For example, in the case of flowering start date, the flowering start date of 278 individuals was distributed between 220 and 270 days, showing a distribution pattern that approximated a normal distribution, and QTL (Quantitative Trait) Loci) analysis was judged to be suitable (Figure 1).

Example 3. GBS-based high-density genetic map creation

Genomic DNA of the silver grass hybrid group (SNU-M367×SNU-M087) F ₁ was extracted using the Exgeme Plant SV mini (Jinall Biotechnology) extraction kit according to the manufacturer's protocol. Short reads data were obtained using extracted RNA and an Illumina Hiseq×ten device, and preprocessing such as demultiplexing, adapter removal, and sequence quality trimming was performed using cutadapt and SolexaQA package software. Cleaned reads that passed the preprocessing process were mapped to the reference guided assembly sequence of the standard genome using BWA software, and a SNP matrix was created through raw SNP detection. Among the read sequences, 3,193 SNPs were finally used to create a genetic map through filters such as missing rate <30%, missing within the individual <30%, and minimum depth≥3.

As a result, a total of 3,193 SNPs were used to create a genetic map, and a total of 1,628 unique loci were discovered, excluding markers mapped to the same region. The produced high-density genetic map formed a total of 19 chromosomes, with a total length of 1706.01 cM and an average length between markers of 1.109 cM (Table 1 and Figure 2).

Example 4. QTL analysis associated with early flowering of silver grass using genetic maps

Quantitative trait loci (QTL) analysis was performed based on five trait data related to flowering of the Miscanthus F ₁ population obtained through Example 2 and the genetic map created in Example 3. Association analysis was performed using GACD software, and the conditions were as follows: (1) mapping method: single marker analysis (SMA), (2) Missing phenotype: Deletion, (3) LOD threshlod: Permutation Times. =1,000, Type 1 Error=0.0500.

As a result, as shown in Figure 2, there were 134 quantitative trait loci (LOD > 5) that were significantly associated with five early flowering-related traits, and 34 of them were simultaneously associated with two or more flowering-related traits.

Among the new QTLs, QTLs with high LOD values and simultaneously linked to two or more flowering-related traits were preferentially selected as SNP marker candidates, and the LOD and PVE (phenotypic variance explained) values of these QTLs are shown in Tables 2 to 4 below. A total of 51 markers were selected, of which 28 were simultaneously associated with two or more flowering-related traits.

An LOD value of 5 or more means that the QTL is very likely to be actually related to the trait, and a PVE value of 7 to 10 means that the QTL determines 7 to 10% of flowering-related traits. . An LOD value of 5 or more means that there is a very high possibility that the QTL is actually related to the trait, so it is believed that it can be used as a marker not only for the F ₁ silver grass population but also for other silver grass populations.

Example 5. Development of SNP markers for identification of early-flowering or late-flowering silver grass varieties

To determine the usefulness of the new QTL obtained through Example 4, the following two indicators were first considered. (1) The LOD scores between SNPs and each trait were judged to be significant in descending order. (2) If several of the traits related to early flowering showed a high correlation with one SNP at the same time, the significance was judged to be very high. For example, Figure 3 of the present invention shows the LOD values between SNPs located on chromosome 7 of silver grass and early flowering-related traits, and among these, SNPs that show high LOD values in common in several early flowering-related traits are given priority. selected.

As a result, among the discovered QTL (Tables 2 to 4), Chr05_Flwr_01, Chr05_Flwr_04, Chr05_Flwr_07 located on chromosome 5; Chr07_Flwr_02 located on chromosome 7; and Chr13_Flwr_03 located on chromosome 13; SNP markers (Msi_Flo_01, Msi_Flo_02, Msi_Flo_03, Msi_Flo_04, and Msi_Flo_05) associated with 5 QTL were developed, and information on the corresponding markers is shown in Tables 5 and 6 below.

In the case of Msi_Flo_01 and Msi_Flo_02 markers, if the SNP position base is A, it can be identified as an early-flowering silver grass variety, and if C, it can be identified as a late-flowering silver grass variety. For Msi_Flo_03 and Msi_Flo_04 markers, if the SNP position base is G, it can be identified as an early-flowering silver grass variety, and if A, it can be identified as a late-flowering silver grass variety. It can be identified as a silver grass variety, and in the case of the Msi_Flo_05 marker, if the SNP position base is A, it can be identified as an early-flowering silver grass variety, and if it is G, it can be identified as a late-flowering silver grass variety.

A primer set based on the SNP of the present invention was produced using Primer3Plus (http://primer3plus.com/) software, and information on each primer set is shown in Table 7 below.

Example 6. Distinguishing early-flowering or late-flowering silver grass varieties using SNP molecular markers

To verify the applicability of SNP molecular markers, early-flowering silver grass lines (M131, M241, M138, M133, M130) and late-flowering silver grass lines (M036, M292, M295, Genotyping of M298, M038) was performed. The above silver grass line was not directly genetically related to the silver grass used in the development of the SNP marker, and the flowering start date of the early flowering line was distributed between 189 and 195 days, and the flowering start date of the late flowering line was distributed between 258 and 277 days. (Table 8).

The base sequence of the PCR product amplified using the primer set of the present invention (Table 7) was sequenced using an ABI 3730XL DNA analyzer (Thermofisher scientific, USA), and the results were analyzed using BioEdit software (Hall, T.A. 1999). .

The SNP genotype and allele frequencies of each silver grass lineage are shown in Table 9 below. In the case of early flowering silver grass lines, only the AA genotype was found in the Msi_Flo_02 marker, with the A allele frequency at 100%, the G allele frequency at 90% at the Msi_Flo_03 marker, and the A allele frequency at 80% at the Msi_Flo_05 marker.

On the other hand, in the late-flowering silver grass line, only the CC genotype was found in the Msi_Flo_01 marker, with a C allele frequency of 100%; in the Msi_Flo_04 marker, only the AA genotype was found, with an A allele frequency of 100%; and in the Msi_Flo_05 marker, the G allele frequency was 100%. It was found to be 80%.

However, as mentioned above, flowering time is a quantitative trait controlled by polygenes, and because silver grass has high heterozygosity, there are limitations in determining the flowering time of silver grass using only a single marker. Therefore, in the present invention, marker efficiency was improved through haplotype analysis of five SNP markers. There are a total of 32 haplotypes that can be created by combining each allele from the five SNP markers of Msi_Flo_01 to Msi_Flo_05. Among them, the allele frequency AAGGA type in Table 9 above is a SNP combination that appears specific to early flowering silver grass cultivars, and CCAAG type is a SNP combination that appears specifically in late-flowering silver grass varieties (Table 8).

When a combination of 5 SNP markers is used to identify varieties based on the haplotype, the accuracy of species discrimination according to the number of matches with the allele combination in distinguishing early-flowering and late-flowering varieties is shown in Table 10 below. For example, if the haplotype of a specific variety was AAGGA, the probability that it was an early-flowering variety was 100%, and if the haplotype was CCAAG, the probability that it was a late-flowering variety was also 100%. In addition, when three or more haplotype alleles matched, the discrimination accuracy of early-flowering varieties was 92.53%, and the discrimination accuracy of late-flowering varieties was 89.62%.

Therefore, it was found that early-flowering and late-flowering silver grass varieties can be effectively discriminated through the combination of SNP markers presented through the present invention.

<110> Seoul National University R&DB Foundation <120> Molecular marker for discriminating early-flowering or late-flowering of Miscanthus sinensis cultivar and uses it <130> PN21305 <160> 15 <170> KoPatentIn 3.0 <210> 1 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 atcccaatgc aattctgatt gacaat 26 <210> 2 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 catcattatt tcgccaagga aaacac 26 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ccggtatttt tgttagctaa ccaact 26 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 gatgcagtgg aataaactag tgactg 26 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 gaaagtactc gtatcattgg caatagg 27 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 atttgtgaag ctagtgtttg attgcta 27 <210> 7 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 caacctagta gctgatagat gtctgc 26 <210> 8 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 aatttacaat ttctgcagtc tctctcc 27 <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 gctgctgtaa aactaatggc tctac 25 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 gatggagccg tagtggaaga tcttg 25 <210> 11 <211> 601 <212> DNA <213> Unknown <220> <223> Miscanthus sinensis <400> 11 ggatcaagtt tacagaaaat aatcaaatat ataaaacatat acaataccaa gtaaatgcac 60 tatcgaatat atttcatgtt gtatctaatc aatcttgttt tatgttgtag ataagttagc 120 attgtttgac acaggagcca gtaaggacag atctaaaatt acttataatt tggaacagag 180 gaagtataag ccaggccatc ccaatgcaat tctgattgac aatatgtcta aagtagatgc 240 aaatgcgcag tcggacaaca gcatagcagc caaatgaact gagaccattc atgtgataaa 300 acaggtcata acagcttggc atttcagcat aaactttcat gttcagcggg aaaacaaaaca 360 gtactgctgc ttctacccaa cttactaaaa tattgtcgga gaaaaaatgat tacaagcgat 420 gtgttttcct tggcgaaata atgatgtaaa tgattgacaa accgactgga gattggagaa 480 atgtaaggca gtggagaaag attcatgcat gccgccagcc aagtccagac tggatgacac 540 aacaaaattc ggtgtagatg ctatgtgtag gatccactgc agcacggtct ggaaaaatat 600 g 601 <210> 12 <211> 601 <212> DNA <213> Unknown <220> <223> Miscanthus sinensis <400> 12 cttgtaggtg gcttgcatat atcaacgatg gtgtacgtcc atgtcggcga ccatgcagcc 60 agcaacccgg aggccaggat cctcctccgc agcggcggcg acgttcgtgc tcctgatcga 120 tgtgcatgca catgccgcat gcgatgagac tggatctcgg acgggtcaaa gggcatctgg 180 cggcagtgct gcgactgtac agggaggcag catcgatccg gacatccggt atttttgtta 240 gctaaccaac tgtatatatg taggtcatgt gatgagcaca agatctgaag atctatctcg 300 acgtctagct gcttacctaa gaatttaatt taaatggtac aattagggcc tgcatgcatc 360 tgctgaagtg atgatcagct agagcagtga ggagtccttt cagtcactag tttatccac 420 tgcatctgga gtaattttgc tgcacagtat tatattgtaa ataaacataa cacattagtg 480 agggattaga ttagtatgtt cttgaattta ccttagtcat gctaactgta agatattaag 540 ctgtcaatta ctcaccttag gcgcaaatga agtgatgctt tggatgcact tagcccctta 600 t601 <210> 13 <211> 601 <212> DNA <213> Unknown <220> <223> Miscanthus sinensis <400> 13 acagtacatg taatgggcag aatgaattat gctacggacg aggcccaggg tattcttccg 60 cttgcataca gattattaca gttgaagctt tcatattctt atttacattt ggaagaactc 120 attatagatt aatcctttaa tttaaatttt gcttgttgca gatatcctga tgaatttgaa 180 agtactcgta tcattggcaa taggcaattc ctcaaggctg tcgatctttt ccattcagct 240 tctgaagaaa tacagggcag cgttgattac aggcatacct acctagattt ctcgcaactc 300 gaagtgaatg tttctgcaag tacaggaggt cagcatccga cggtgaaaac atgcccagca 360 gccatggggt ttgcctttgc tgctggaacc acagatggcc ctggagcttt tgattttaaa 420 caaggagatg tcaaggttgg tcctactgta gcaatcaaac actagcttca caaatttcgt 480 tggcgtgcta ttgactgttt ttttacgtaa tccttcgtta gggaaaccca ttctggtggt 540 tagtacgaga tgtaattagg ccacccggcc cagagcaagt gaaatgtcag gctccgaaac 600 c 601 <210> 14 <211> 601 <212> DNA <213> Unknown <220> <223> Miscanthus sinensis <400> 14 gcccatgggc acgactgcac gagcacgacg gccggcaaaa cacaaaagcg acgctacagt 60 ggagtccatt cgagcccctg ctcccggcgc gacacacgta gtagtacgta aatgacgcag 120 tccgcccggg cgcagtgcag tggaaataat gcagcactgg cactagcagg atatcgagct 180 agctagtgag cgcaacctag tagctgatag atgtctgcac acactggagg ccaatcgaaa 240 ggagcgcggg aaagctgctt ggacggagga gagaaataaa ctgctgcgac agggagttgg 300 gcgaagtggc tgaggacgaa ctgctatcga tctttatatc cacgtacgca tccaagcact 360 gcattgcatc cactgatcga ccgcacgcac agatagcagc agcaaaaggg gagcactgct 420 gcacatccac taggaaagaa aacatggaca tgggatcgat ggagagagac tgcagaaatt 480 gtaaattcag agagcgagag gagagagccg ctctgcatgc cttcacttgg tggtgctgtt 540 gaagtgttga atcgctagga tcatagatct agtcggggtg cttgatgaaa caaagaccta 600 g 601 <210> 15 <211> 601 <212> DNA <213> Unknown <220> <223> Miscanthus sinensis <400> 15 agataaaaaaa aacactcttt ttttaacctt acttattaat cactaagcaa tgtacgttat 60 gtaattttcc cacctgctct gatattatta tactaacatg gaggaagcca aaacttgttg 120 gctcatctct gattatagat taacacactc cattcatagt ctgcacagta ctctactatg 180 tttttttttg gtccttcgat tcaactgcat tgtttgctgc tactatgctg ctgtaaaact 240 aatggctcta ctccactgcc ctgcagttcg gcgacgacga gttcgggcac atgctggtca 300 acatcctgaa gcaaaacaac gtcaactccg aaggctgcct cttcgaccag cacgcgcgca 360 ccgcgctggc cttcgtcacc ctcaagcaca acggggagcg cgagttcatg ttctaccgga 420 acccgagcgc ggacatgctg ctcacggagg cggagctgaa cctggacctg atccggcgcg 480 ccaagatctt ccactacggc tccatctcgc tcatcgcgga ccccgtccgg tcggcgcacc 540 tggccgccat gcgcgccgcc aaggccgccg gcatcctctg ctcctacgac cccaacgtcc 600 g 601

Claims (7)

Oligonucleotide primer sets of SEQ ID NOs: 1 and 2; Oligonucleotide primer sets of SEQ ID NOs: 3 and 4; Oligonucleotide primer sets of SEQ ID NOs: 5 and 6; Oligonucleotide primer sets of SEQ ID NOs: 7 and 8; And a primer set composition for determining early flowering or late flowering Miscanthus sinensis cultivars, comprising an oligonucleotide primer set of SEQ ID NOs: 9 and 10. delete A kit for determining early-flowering or late-flowering silver grass varieties containing the primer set composition according to claim 1. Isolating genomic DNA from the silver grass sample;
Using the isolated genomic DNA as a template and performing an amplification reaction using the primer set composition of claim 1 to amplify a target sequence; and
A method for determining early-flowering or late-flowering silver grass varieties, including the step of detecting the product of the amplification step. The method of claim 4, wherein the detection of the amplification product is performed through gel electrophoresis, HRM analysis, DNA chip, radioactivity measurement, fluorescence measurement, or phosphorescence measurement. In the polynucleotide consisting of the base sequences of SEQ ID NOs: 11 to 15, a polynucleotide consisting of 8 or more consecutive nucleotides containing a SNP (single nucleotide polymorphism) base located at the 301st position in each base sequence, or a complementary polynucleotide thereof A SNP marker composition for determining early-flowering or late-flowering silver grass varieties, including. A microarray for discriminating early-flowering or late-flowering silver grass varieties, comprising a polynucleotide or cDNA thereof containing the SNP (single nucleotide polymorphism) base described in claim 6.