Molecular markers for identification of emilia sonchifolia and use thereof

Abstract

The present application relates to the field of molecular marker and biotechnology, more particularly to SNP molecular marker for distinguishing between Valencia orange and other common citrus varieties and application, wherein the molecular marker comprises SNP molecular marker Chr4:26265293; in the Valencia orange, Chr4:26265293 is G / A. The SNP marker of the present application can effectively identify Valencia orange.

Description

Technical Field

[0001] This invention relates to the fields of molecular markers and biotechnology, and more specifically to SNP molecular markers for identifying Yanming orange and their applications. Background Technology

[0002] Sweet orange (Citrus sinensis (L.) Osbeck), as one of the core varieties of the Citrus genus (Citrus L.) in the Rutaceae family, occupies an important position in the citrus consumer market. The 'Yanming Orange', independently bred in my country, is a bud mutation of the Newhall navel orange. After years of purification and observation by Hunan Agricultural University in China, it has been confirmed to possess stable cold-resistant characteristics and has been granted new variety rights protection (variety rights number: CNA20191001262). In the healthy development of the seed industry and the protection of seed intellectual property rights, the development of variety-specific molecular markers is a core technical means to prevent counterfeit and substandard germplasm from entering the market, and it is also the legal basis for new variety protection and rights enforcement.

[0003] Currently, the development of molecular markers for citrus mainly focuses on genetic polymorphisms such as single nucleotide polymorphisms (SNPs), insertions and deletions (InDels), and structural variations (SVs), employing traditional methods such as simple repeat (SSR) markers, amplified fragment length polymorphism (AFLP) markers, and restriction fragment length polymorphism (RFLP) markers. Although these traditional methods are simple and low-cost, they suffer from limitations in throughput, sensitivity, and time consumption. Some methods also require multiple amplification steps, making large-scale applications difficult.

[0004] In recent years, genotyping methods based on High Resolution Melt (HRM) analysis have gradually emerged. This method primarily relies on the property of saturated dyes binding to double-stranded DNA to detect SNPs or InDel mutations with high sensitivity, distinguishing between homozygous, heterozygous, and unknown mutations. Compared to traditional methods, HRM genotyping offers automation and high throughput, enabling simultaneous detection in 96-well or 384-well plates with short detection times and high accuracy. Furthermore, the entire process from PCR amplification to melting curve analysis is completed in sealed tubes, avoiding aerosol contamination and sample cross-contamination. The simplified operation reduces labor and consumable costs, making it particularly suitable for screening large-scale populations. Compared to TaqMan probe-based SNP detection methods, HRM technology does not rely on expensive sequence-specific probes, significantly reducing costs. Summary of the Invention

[0005] This disclosure utilizes next-generation sequencing data for SNP mining, providing a method for developing molecular markers using SNP sites and identifying the Yanming orange variety based on HRM technology. This disclosure employs PCR amplification and HRM analysis for SNP genotyping, effectively identifying the 'Yanming orange' variety and providing rapid and effective support for 'Yanming orange' variety protection and authenticity verification.

[0006] According to a first aspect of this disclosure, a molecular marker for the identification of Yanming orange is provided, the molecular marker comprising a first SNP molecular marker Chr4:26265293, wherein Chr4:26265293 is G / A in Yanming orange.

[0007] In some embodiments, the molecular markers further include a second SNP molecular marker Chr1:28425195 and / or a third SNP molecular marker Chr4:20168121, wherein in the Yanming Orange, Chr1:28425195 is A / C and Chr4:20168121 is A / C.

[0008] In some embodiments, the Yanming orange is a bud mutation of the Newhall navel orange. In some embodiments, the variety rights number of the Yanming orange is CNA20191001262.

[0009] In some implementations, the molecular marker is defined based on the sweet orange genome with NCBI accession number GCA_018104345.1.

[0010] In some embodiments, the first SNP molecular marker Chr4:26265293 corresponds to the 41st nucleotide of the nucleotide sequence shown in SEQ ID NO:11 or 12. In some embodiments, the second SNP molecular marker Chr1:28425195 corresponds to the 189th nucleotide of the nucleotide sequence shown in SEQ ID NO:7 or 8. In some embodiments, the third SNP molecular marker Chr4:20168121 corresponds to the 65th nucleotide of the nucleotide sequence shown in SEQ ID NO:9 or 10.

[0011] In some embodiments, the first SNP molecular marker is the 41st nucleotide from the 5' end of the amplification product obtained by PCR amplification using the nucleotide sequences shown in SEQ ID NO:5 and SEQ ID NO:6 as primers, as a template of the Yanming orange genomic DNA, which is G or A. In some embodiments, the second SNP molecular marker is the 189th nucleotide from the 5' end of the amplification product obtained by PCR amplification using the nucleotide sequences shown in SEQ ID NO:1 and SEQ ID NO:2 as primers, as a template of the Yanming orange genomic DNA, which is A or C. In some embodiments, the third SNP molecular marker is the 65th nucleotide from the 5' end of the amplification product obtained by PCR amplification using the nucleotide sequences shown in SEQ ID NO:3 and SEQ ID NO:4 as primers, as a template of the Yanming orange genomic DNA, which is A or C.

[0012] According to a second aspect of this disclosure, an isolated nucleic acid molecule from Yanming orange is provided, said nucleic acid molecule including the molecular marker of the first aspect of this disclosure.

[0013] In some embodiments, the first SNP molecular marker is the 41st nucleotide from the 5' end of the amplification product obtained by PCR amplification using the nucleotide sequences shown in SEQ ID NO:5 and SEQ ID NO:6 as primers, as a template of the Yanming orange genomic DNA, which is G or A. In some embodiments, the second SNP molecular marker is the 189th nucleotide from the 5' end of the amplification product obtained by PCR amplification using the nucleotide sequences shown in SEQ ID NO:1 and SEQ ID NO:2 as primers, as a template of the Yanming orange genomic DNA, which is A or C. In some embodiments, the third SNP molecular marker is the 65th nucleotide from the 5' end of the amplification product obtained by PCR amplification using the nucleotide sequences shown in SEQ ID NO:3 and SEQ ID NO:4 as primers, as a template of the Yanming orange genomic DNA, which is A or C.

[0014] In some embodiments, the nucleic acid molecule may include a first SNP molecular marker Chr4:26265293 corresponding to the Yanming orange, wherein the first SNP molecular marker Chr4:26265293 is G / A. In some embodiments, the nucleic acid molecule may include the nucleotide sequence shown in SEQ ID NO:11 and / or SEQ ID NO:12, or a nucleotide sequence having at least 85% sequence identity with it, wherein the 41st nucleotide is G / A.

[0015] In some embodiments, the nucleic acid molecule may further include a second SNP molecular marker Chr1:28425195 corresponding to the Yanming orange, wherein the second SNP molecular marker Chr1:28425195 is A / C. In some embodiments, the nucleic acid molecule includes the nucleotide sequence shown in SEQ ID NO:7 and / or SEQ ID NO:8, or a nucleotide sequence having at least 85% sequence identity with it, wherein the 189th nucleotide is A / C.

[0016] In some embodiments, the nucleic acid molecule further includes a third SNP molecular marker Chr4:20168121 corresponding to the Yanming orange, wherein the third SNP molecular marker Chr4:20168121 is A / C. In some embodiments, the nucleic acid molecule includes the nucleotide sequence shown in SEQ ID NO:9 and / or SEQ ID NO:10, or a nucleotide sequence having at least 85% sequence identity with it, wherein the 65th nucleotide is A / C.

[0017] According to a third aspect of this disclosure, a primer for the identification of Yanming orange is provided, said primer being capable of amplifying the molecular marker of the first aspect.

[0018] In some embodiments, the first SNP molecular marker is the 41st nucleotide from the 5' end of the amplification product obtained by PCR amplification using the nucleotide sequences shown in SEQ ID NO:5 and SEQ ID NO:6 as primers, as a template of the Yanming orange genomic DNA, which is G or A. In some embodiments, the second SNP molecular marker is the 189th nucleotide from the 5' end of the amplification product obtained by PCR amplification using the nucleotide sequences shown in SEQ ID NO:1 and SEQ ID NO:2 as primers, as a template of the Yanming orange genomic DNA, which is A or C. In some embodiments, the third SNP molecular marker is the 65th nucleotide from the 5' end of the amplification product obtained by PCR amplification using the nucleotide sequences shown in SEQ ID NO:3 and SEQ ID NO:4 as primers, as a template of the Yanming orange genomic DNA, which is A or C.

[0019] In some embodiments, the primers include a first primer set comprising nucleotide sequences as shown in SEQ ID NO:5 and SEQ ID NO:6, or nucleotide sequences having at least 85% sequence identity with them. In some embodiments, using the genome of the Yanming orange as a template, the first primer set can amplify an amplification product corresponding to the Chr4:26265293 site containing the Yanming orange.

[0020] In some embodiments, the primers further include:

[0021] (A1) A second primer set comprising the nucleotide sequences shown in SEQ ID NO:1 and SEQ ID NO:2, or a nucleotide sequence having at least 85% sequence identity with them; and / or

[0022] (A2) The third primer set includes the nucleotide sequences shown in SEQ ID NO:3 and SEQ ID NO:4, or nucleotide sequences that have at least 85% sequence identity with them.

[0023] In some implementations, using the genome of the Yanming orange as a template, the second primer set can amplify the amplification product corresponding to the Chr1:28425195 site containing the Yanming orange.

[0024] In some implementations, using the genome of the Yanming orange as a template, the third primer set can amplify the amplification product corresponding to the Chr4:20168121 site containing the Yanming orange.

[0025] According to a fourth aspect of this disclosure, a kit for the identification of Yanming orange is provided, the kit comprising reagents for detecting the molecular marker of the first aspect.

[0026] In some embodiments, the first SNP molecular marker is the 41st nucleotide from the 5' end of the amplification product obtained by PCR amplification using the nucleotide sequences shown in SEQ ID NO:5 and SEQ ID NO:6 as primers, as a template of the Yanming orange genomic DNA, which is G or A. In some embodiments, the second SNP molecular marker is the 189th nucleotide from the 5' end of the amplification product obtained by PCR amplification using the nucleotide sequences shown in SEQ ID NO:1 and SEQ ID NO:2 as primers, as a template of the Yanming orange genomic DNA, which is A or C. In some embodiments, the third SNP molecular marker is the 65th nucleotide from the 5' end of the amplification product obtained by PCR amplification using the nucleotide sequences shown in SEQ ID NO:3 and SEQ ID NO:4 as primers, as a template of the Yanming orange genomic DNA, which is A or C.

[0027] In some embodiments, the reagent includes the primers described in the third aspect.

[0028] In some embodiments, the reagent includes a probe for detecting the molecular marker described in the first aspect.

[0029] In some embodiments, the kit may further include standards. In some embodiments, the standards are nucleic acid molecules having the molecular markers of the first aspect of this disclosure.

[0030] In some embodiments, the kit includes a first standard, which may include a nucleotide sequence as shown in SEQ ID NO:11 and / or SEQ ID NO:12, or a nucleotide sequence having at least 85% sequence identity with it.

[0031] In some embodiments, the kit may further include a second standard. In some embodiments, the second standard may include a nucleotide sequence as shown in SEQ ID NO:7 and / or SEQ ID NO:8, or a nucleotide sequence having at least 85% sequence identity with it.

[0032] In some embodiments, the kit may further include a third standard. In some embodiments, the third standard may include a nucleotide sequence as shown in SEQ ID NO:9 and / or SEQ ID NO:10, or a nucleotide sequence having at least 85% sequence identity with it.

[0033] Those skilled in the art will understand that the first standard may include three standards simulating three genotypes (corresponding to nucleotides at position 41 of the nucleotide sequence shown in SEQ ID NO: 11 or 12 being G / G, A / A, and G / A). For example, to simulate three genotypes, the first standard may include one or more of the following standards: 1) a standard containing the nucleotide sequence shown in SEQ ID NO: 11, or a nucleotide sequence having at least 85% sequence identity with it; 2) a standard containing the nucleotide sequence shown in SEQ ID NO: 12, or a nucleotide sequence having at least 85% sequence identity with it; and 3) a standard containing the nucleotide sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12, or a nucleotide sequence having at least 85% sequence identity with them, preferably a 1:1 mixture of the nucleotide sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12. Similarly, the second standard and / or the third standard may each independently include three standards simulating three genotypes.

[0034] According to a fifth aspect of this disclosure, a method for identifying Yanming Orange is provided, the method comprising the following steps: (s1) detecting a first molecular marker in the genome of a sample, wherein the first SNP molecular marker is Chr4:26265293; and, when the first molecular marker is G / A, identifying the sample as Yanming Orange.

[0035] In some embodiments, the first SNP molecular marker is the 41st nucleotide from the 5' end of the amplification product obtained by PCR amplification using the nucleotide sequences shown in SEQ ID NO:5 and SEQ ID NO:6 as primers, as a template of the Yanming orange genomic DNA, which is G or A. In some embodiments, the second SNP molecular marker is the 189th nucleotide from the 5' end of the amplification product obtained by PCR amplification using the nucleotide sequences shown in SEQ ID NO:1 and SEQ ID NO:2 as primers, as a template of the Yanming orange genomic DNA, which is A or C. In some embodiments, the third SNP molecular marker is the 65th nucleotide from the 5' end of the amplification product obtained by PCR amplification using the nucleotide sequences shown in SEQ ID NO:3 and SEQ ID NO:4 as primers, as a template of the Yanming orange genomic DNA, which is A or C.

[0036] In some embodiments, the method may further include the steps of: (s2) detecting a second SNP molecular marker in the sample genome, wherein the second SNP molecular marker is Chr1:28425195; and, when the second SNP molecular marker is A / C, identifying the sample as Yanming Orange.

[0037] In some embodiments, the method may further include the steps of: (s3) detecting a third SNP molecular marker in the sample genome, wherein the third SNP molecular marker is Chr4:20168121; and, when the third SNP molecular marker is A / C, identifying the sample as Yanming Orange.

[0038] In some embodiments, the detection includes PCR. In some embodiments, the detection includes PCR amplification using a first primer set, wherein the first primer set includes nucleotide sequences as shown in SEQ ID NO:5 and SEQ ID NO:6, or nucleotide sequences having at least 85% sequence identity with them.

[0039] In an optional embodiment, the detection may further include: PCR amplification using a second primer set and / or a third primer set, wherein the second primer set comprises nucleotide sequences as shown in SEQ ID NO:1 and SEQ ID NO:2 or nucleotide sequences having at least 85% sequence identity with them, and the third primer set comprises nucleotide sequences as shown in SEQ ID NO:3 and SEQ ID NO:4 or nucleotide sequences having at least 85% sequence identity with them.

[0040] In a preferred embodiment, the detection includes quantitative real-time PCR, HRM detection, or sequencing.

[0041] In some embodiments, the detection in step (s1) may include: performing HRM detection and genotyping using a first primer set, wherein the first primer set comprises nucleotide sequences as shown in SEQ ID NO:5 and SEQ ID NO:6, or nucleotide sequences having at least 85% sequence identity with them. In some embodiments, the genotyping includes comparison with a first standard, which may include nucleotide sequences as shown in SEQ ID NO:11 and / or SEQ ID NO:12, or nucleotide sequences having at least 85% sequence identity with them.

[0042] In some embodiments, the detection in step (s2) may include: performing HRM detection and genotyping using a second primer set, wherein the second primer set comprises nucleotide sequences as shown in SEQ ID NO:1 and SEQ ID NO:2 or nucleotide sequences having at least 85% sequence identity with them. In some embodiments, the genotyping includes comparison with a second standard, which may comprise nucleotide sequences as shown in SEQ ID NO:7 and / or SEQ ID NO:8, or nucleotide sequences having at least 85% sequence identity with them.

[0043] In some embodiments, the detection in step (s3) may include: performing HRM detection and genotyping using a third primer set, wherein the third primer set includes the nucleotide sequences shown in SEQ ID NO:3 and SEQ ID NO:4, or nucleotide sequences having at least 85% sequence identity with them. In some embodiments, the genotyping includes comparison with a third standard, which may include the nucleotide sequences shown in SEQ ID NO:9 and / or SEQ ID NO:10, or nucleotide sequences having at least 85% sequence identity with them.

[0044] In some embodiments, the sample may be a polynucleotide-containing sample from sweet orange or citrus plants (e.g., but not limited to, Yanming orange, Newhall navel orange, Anglais orange, Washington navel orange, Vodka orange, 'CaraCara' red navel orange, and Big Red sweet orange). In some embodiments, the sample may be derived from or include leaves, roots, stems, flowers, fruits, seeds, cells, etc., of sweet orange or citrus plants. In some embodiments, the sample may be genomic DNA.

[0045] In some embodiments, the Yanming orange is a bud mutation of the Newhall navel orange. In some embodiments, the variety rights number of the Yanming orange is CNA20191001262.

[0046] In some implementations, the molecular marker is defined based on the sweet orange genome with NCBI accession number GCA_018104345.1.

[0047] According to the sixth aspect of this disclosure, the molecular marker of the first aspect, the nucleic acid molecule of the second aspect, the primer of the third aspect, and the kit of the fourth aspect are provided for their application in the identification or breeding of Yanming orange.

[0048] In some embodiments, the first SNP molecular marker is the 41st nucleotide from the 5' end of the amplification product obtained by PCR amplification using the nucleotide sequences shown in SEQ ID NO:5 and SEQ ID NO:6 as primers, as a template of the Yanming orange genomic DNA, which is G or A. In some embodiments, the second SNP molecular marker is the 189th nucleotide from the 5' end of the amplification product obtained by PCR amplification using the nucleotide sequences shown in SEQ ID NO:1 and SEQ ID NO:2 as primers, as a template of the Yanming orange genomic DNA, which is A or C. In some embodiments, the third SNP molecular marker is the 65th nucleotide from the 5' end of the amplification product obtained by PCR amplification using the nucleotide sequences shown in SEQ ID NO:3 and SEQ ID NO:4 as primers, as a template of the Yanming orange genomic DNA, which is A or C.

[0049] In some embodiments, the Yanming orange is a bud mutation of the Newhall navel orange. In some embodiments, the variety rights number of the Yanming orange is CNA20191001262.

[0050] This disclosure combines next-generation sequencing data with HRM technology to improve the accuracy, sensitivity, and efficiency of SNP marker screening. Through e-PCR pre-screening and IGV visualization verification, this disclosure reduces invalid primer design and improves primer amplification success rate. The selected SNP sites in this disclosure exhibit a haplotype distribution in Yanming oranges, clearly distinguishing them from varieties such as Newhall navel oranges and Dahong sweet oranges, demonstrating high specificity. SNP applications do not require fluorescent probes or complex equipment, shortening the detection cycle to 2 hours. Furthermore, the method of this disclosure is scalable; the protocol can be extended to the identification of Yanming oranges with other citrus varieties, providing molecular evidence for variety rights protection. Attached Figure Description

[0051] Figure 1 This is a high-resolution melting curve typing diagram based on primer 1-195 amplification.

[0052] Figure 2 This is a high-resolution melting curve typing diagram based on primer 4-121 amplification.

[0053] Figure 3This is a high-resolution melting curve typing diagram based on primer 4-293 amplification. Detailed Implementation

[0054] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0055] definition

[0056] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0057] The term "amplification" as used herein refers to nucleic acid fragments produced during primer-directed amplification reactions. Common methods for primer-directed amplification include polymerase chain reaction (PCR), ligase chain reaction (LCR), or strand displacement amplification (SDA). If the PCR method is chosen, the replication composition may contain components for nucleic acid replication, such as: nucleotide triphosphates, two (or more) primers with appropriate sequences, a thermostable polymerase, buffer, solute, and protein.

[0058] The term "primer" as used herein refers to a nucleic acid molecule with a specific nucleotide sequence that guides the synthesis of nucleotides at the initiation of nucleotide polymerization. Primers are typically two artificially synthesized oligonucleotide sequences: one primer is complementary to one DNA template strand at one end of the target region, and the other primer is complementary to the other DNA template strand at the other end of the target region. Their function is to serve as the initiation point for nucleotide polymerization. Artificially designed primers are widely used in polymerase chain reaction (PCR), qPCR, and sequencing. Primers can be of any length, for example, 5-200 bp, 10-100 bp, 20-800 bp, or 25-50 bp. The primers of this invention are used for the detection of SNPs.

[0059] The "Yanming Orange" described in this article belongs to the sweet orange (Citrus sinensis (L.) Osbeck), family Rutaceae, genus Citrus (Citrus L.), and possesses stable cold-resistant characteristics. In a specific embodiment, the Yanming Orange disclosed herein is a bud mutation of the Newhall navel orange line independently bred in my country, with variety right number CNA20191001262. The Yanming Orange disclosed herein is diploid, meaning that its somatic cells contain two complete sets of chromosomes.

[0060] The term "haplotype" as used herein refers to a combination of linked genes or genetic markers located on a chromosome or within a specific region. These markers tend to be passed on as a whole to offspring during inheritance. Haplotype distribution refers to the frequency and geographic / genetic distribution pattern of different haplotypes in a population, species, or specific region. It is of great significance in genetics, population biology, medicine, and evolutionary research. Haplotypes can result from linkage disequilibrium; adjacent genes or loci on a chromosome, due to their proximity, have low recombination rates and are often inherited as a whole. Haplotypes can also be constructed through genetic variations such as single nucleotide polymorphisms (SNPs), insertions / deletions, and microsatellite markers. The SNP loci provided in this disclosure are haplotype distributions, which can also be understood as heterozygous. For example, the first SNP molecular marker Chr4:26265293 in this disclosure is G / A, indicating that the Chr4:26265293 locus of Yanming Orange is either G or A.

[0061] The "SNP (single nucleotide polymorphism)" or "single nucleotide polymorphism" mentioned in this article refers to a class of molecular genetic markers, mainly referring to DNA sequence polymorphisms caused by variations in a single nucleotide at the genomic level. The polymorphisms exhibited by SNPs usually involve variations in only a single base, such as transitions, transversions, insertions, and deletions.

[0062] The "sequence identity percentage" or "identity percentage" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by sequences within a comparison window, taking into account additions or deletions (i.e., vacancies) that must be introduced for optimal alignment of the two sequences. A matching position is any location where the same nucleotide or amino acid is present in both the target and reference sequences. Vacancies are not nucleotides or amino acids and are not counted in the target sequence. Similarly, vacancies in the reference sequence are not counted because nucleotides or amino acids from the target sequence are included, but those from the reference sequence are excluded.

[0063] The percentage of sequence identity can be calculated as follows: determine the number of positions in both sequences where the same amino acid residue or nucleic acid base appears (the number of matching positions), divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the percentage of sequence identity. Sequence comparison and determination of the percentage of sequence identity between two sequences can be accomplished using software that is readily available online and downloadable. Suitable software programs are available from various sources for protein and nucleotide sequence alignment. A suitable program for determining the percentage of sequence identity is bl2seq, which is part of the BLAST program suite available from the National Center for Biotechnology Information (NCBI) website (blast.ncbi.nlm.nih.gov). Bl2seq uses either the BLASTN or BLASTP algorithm for comparing two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Institute of Bioinformatics (EBI) at www.ebi.ac.uk / Tools / psa.

[0064] Those skilled in the art will understand that "at least 85% sequence identity" between a nucleic acid sequence or amino acid sequence described herein and another nucleic acid sequence or amino acid sequence means that the sequence has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity with the other nucleic acid sequence or amino acid sequence.

[0065] The term "HRM (High-Resolution Melting Curve Analysis)" used in this article refers to a molecular detection technique based on the melting characteristics of nucleic acids, widely used in gene mutation detection, genotyping, methylation analysis, and other fields. During heating, the DNA double strand dissociates into single strands due to the breakage of hydrogen bonds between base pairs. Its melting temperature (Tm value) is mainly determined by sequence length, GC content, and base pairing stability. If the two DNA strands have sequence differences (such as single nucleotide polymorphisms (SNPs), insertion / deletion mutations, etc.), the double strands have different stability, leading to differences in the rate of fluorescence signal change and temperature during melting. By monitoring the fluorescence signal of the double-stranded DNA in real time during heating using a high-precision quantitative PCR instrument, melting curves can be plotted, and software analysis of curve morphology differences can determine sequence variations between samples. HRM technology supports simultaneous detection in 96-well / 384-well plates, processing nearly 100 samples in a single run, significantly improving efficiency compared to traditional methods.

[0066] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0067] Example

[0068] Example 1. Obtaining the SNP molecular marker of Yanming Orange

[0069] (1) Identification and screening of SNP sites in Yanming orange:

[0070] Two to three young leaves of Yanming orange (variety rights number CNA20191001262) were collected, cleaned, and dust-free. DNA was extracted using the High-Efficiency Plant Genomic DNA Extraction Kit from Tiangen Biotech (Beijing) Co., Ltd., following the kit's instruction manual. Genome sequencing was performed on the Illumina PE150 platform to obtain second-generation whole-genome resequencing data for Yanming orange (variety rights number CNA20191001262). Using the second-generation whole-genome resequencing data of Yanming orange, and with the second-generation sweet orange reference genome (NCBI accession number: GCA_018104345.1) as a control, BWA was used for mapping and alignment to generate a BAM file. SNP sites were then identified using the variant detection software GATK4.0. The filtering criteria were: QD < 2.0, MQ < 40.0, FS > 60.0, SOR > 3.0, MQRankSum < -12.5, and ReadPosRankSum < -8.0. Finally, 3529 high-confidence SNP loci were obtained through screening.

[0071] (2) SNP validation and screening:

[0072] By loading the BAM file using IGV software, manually verifying the SNP site polymorphism and ensuring its authenticity, three SNP sites were finally selected. The information of each site is shown in Table 1.

[0073] Table 1 SNP locus information

[0074] SNP name Location of SNP sites Second-generation sweet orange reference base REF Yanming Orange Mutant Base ALT SNP_1 Chr1:28425195 A C SNP_2 Chr4:20168121 A C SNP_3 Chr4:26265293 G A

[0075] Example 2. Validation of the SNP molecular marker of Yanming Orange

[0076] (1) Primer design optimization and electronic PCR simulation:

[0077] Primers near SNP sites were designed using the Primer3 online website, and primers meeting the following requirements were screened: ① The upstream or downstream primer has an SNP site at its terminal 1-5 bp; ② The primer Tm value is 60±2℃; ③ The primer GC content is 40%–50%; ④ The primer amplification product length ranges from 100–250 bp; ⑤ The primer dimer score is ≤3.0. Primer amplification was verified using electronic PCR (e-PCR) to exclude interference from repetitive sequences and polymorphic regions. Three pairs of primers were finally selected, and detailed primer information is shown in Table 2.

[0078] Table 2 Primer nucleotide sequences

[0079]

[0080] (2) Genomic DNA extraction from Yanming orange and other sweet oranges:

[0081] For each of the following varieties—Yanming Orange, Newhall Navel Orange, Dark Willow Orange, Washington Navel Orange, Fulingxia Orange, 'CaraCara' Red Navel Orange, and Da Hong Sweet Orange—2–3 young leaves were collected. After washing and removing dust, DNA was extracted using the High-Efficiency Plant Genomic DNA Extraction Kit from Tiangen Biotech (Beijing) Co., Ltd. The extraction procedure was performed according to the kit's instruction manual. Two biological replicates were used for each variety.

[0082] (3) PCR amplification and HRM typing based on orange genomic DNA:

[0083] • Prepare the PCR reaction solution according to the following PCR reaction system: The total volume of the PCR reaction system is 10 μL, which includes 5 μL of 2×Precision Melt Supermix (BIO-RAD) reaction solution, 0.2 μL each of upstream and downstream primers (10 μmol / L) and 50 ng DNA template.

[0084] The PCR amplification system is as follows: 95℃ pre-denaturation for 2 min; 40 cycles (95℃ denaturation for 10 s, 57.5℃ annealing for 30 s, 72℃ extension for 30 s).

[0085] • Perform HRM typing according to the following procedure: extend at 95°C for 30 seconds, hold at 60°C for 1 minute, and then increase the temperature to 95°C at 0.2°C / 10 seconds.

[0086] The PCR amplification products were sent to a sequencing company for sequencing. The sequences of the Yanming orange amplification products are shown in Table 3, where the thickened bases are polymorphic sites.

[0087] Table 3 shows the nucleotide sequences of the amplification products using three pairs of primers.

[0088]

[0089] HRM typing results showed that, based on the three pairs of primer markers designed, different varieties could be genotyped by the difference in melting curve peak shape. Figure 1 , Figure 2 and Figure 3 Furthermore, Yanming oranges can be distinguished from other varieties based on the SNP molecular marker Chr4:26265293 or its combination with the SNP molecular markers Chr1:28425195 and / or Chr4:20168121.

[0090] Figure 1 , Figure 2 and Figure 3 The graphs show high-resolution melting curves used to identify different citrus varieties using three pairs of core primers, including Yanming orange, Newhall navel orange, Anglais orange, Washington navel orange, Vodka orange, 'CaraCara' red navel orange, and Dahong sweet orange. Each band in the graph represents one citrus sample, with two biological replicates for each citrus variety.

[0091] Table 4 shows the identification results based on amplification and genotyping using three pairs of primers: According to primer 1-195, Newhall navel oranges were classified into genotype 1 (A / A), Yanming oranges, Anliu oranges, Washington navel oranges, Fulingxia oranges, and Dahong sweet oranges into genotype 2 (A / C), and 'CaraCara' red-fleshed navel oranges into genotype 3 (C / C); according to primer 4-121, Newhall navel oranges, Anliu oranges, and Washington navel oranges were classified into genotype 1 (A / A), Yanming oranges, Fulingxia oranges, and Dahong sweet oranges into genotype 2 (A / C), and 'CaraCara' red-fleshed navel oranges into genotype 3 (C / C); according to primer 4-293, Yanming oranges were classified into genotype 1 (G / A), Newhall navel oranges, Anliu oranges, Washington navel oranges, Fulingxia oranges, and Dahong sweet oranges into genotype 2 (G / G), and 'CaraCara' red-fleshed navel oranges into genotype 3 (A / A). Furthermore, the Yanming orange can be distinguished from seven other common citrus varieties simply by using primer 4-293.

[0092] Table 4. HRM typing results based on amplification using 3 pairs of primer markers.

[0093]

[0094] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. Use of a molecular marker for identifying Flaminorange or in breeding Flaminorange, characterized in that, The molecular marker includes a first SNP molecular marker, which is located at position 41 of sequences SEQ ID NO: 11 and SEQ ID NO: 12 in the Yanming Orange, and its base type is G / A.

2. Use according to claim 1, characterized in that, The molecular marker further includes a second SNP molecular marker and / or a third SNP molecular marker. In the Yanming Orange, the second SNP molecular marker is located at position 189 of SEQ ID NO: 7 and SEQ ID NO: 8, and its base type is A / C. The third SNP molecular marker is located at position 65 of SEQ ID NO: 9 and SEQ ID NO: 10, and its base type is A / C.

3. A nucleic acid molecule isolated from Yanming orange, characterized in that, The nucleic acid molecule includes a first nucleic acid molecule, which includes a site corresponding to the first SNP molecule marker as described in claim 1. The nucleotide sequence of the first nucleic acid molecule is shown in SEQ ID NO: 11 and SEQ ID NO: 12, wherein the 41st nucleotide is G / A.

4. The nucleic acid molecule of claim 3, wherein, The nucleic acid molecule further includes a second nucleic acid molecule, the second nucleic acid molecule including a site corresponding to the second SNP molecule marker as described in claim 2. The nucleotide sequence of the second nucleic acid molecule is shown in SEQ ID NO: 7 and SEQ ID NO: 8, wherein the nucleotide at position 189 is A / C, and / or The nucleic acid molecule further includes a third nucleic acid molecule, which includes a site corresponding to the third SNP molecule marker described in claim 2. The nucleotide sequence of the third nucleic acid molecule is shown in SEQ ID NO: 9 and SEQ ID NO: 10, wherein the 65th nucleotide is A / C.

5. A primer for identification of Erythromycin, characterized in that, The primers are capable of amplifying the molecular markers of claim 1 or 2, and the primers comprise a first primer set, the sequences of which are shown in SEQ ID NO: 5 and SEQ ID NO:

6.

6. The primer of claim 5, wherein The primers also include: (A1) A second primer set, the sequences of which are shown in SEQ ID NO: 1 and SEQ ID NO: 2; and / or (A2) The third primer set, the sequence of which is shown in SEQ ID NO: 3 and SEQ ID NO:

4.

7. A kit for the identification of Erythromycin, characterized in that, The kit contains the primers as described in claim 5 or 6.

8. The kit of claim 7, wherein The kit also includes standards selected from one or more of the following: The first standard comprises the nucleotide sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12; The second standard comprises the nucleotide sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8; and The third standard comprises nucleotide sequences as shown in SEQ ID NO: 9 and SEQ ID NO:

10.

9. A method of identifying Flamingo Orange, characterized by, The method includes the following steps: (s1) detecting the genotype of the first SNP molecular marker in the sample genome, wherein the first SNP molecular marker is located at position 41 of sequences SEQ ID NO: 11 and SEQ ID NO: 12, and its base type is G / A. When the genotype of the first SNP molecular marker is GA, the sample is identified as Yanming Orange.

10. The method according to claim 9, characterized in that, The method further includes the following steps: (s2) Detect the genotype of the second SNP molecular marker in the sample genome, wherein the second SNP molecular marker is located at position 189 of SEQ ID NO: 7 and SEQ ID NO: 8, and its base type is A / C. When the genotype of the second SNP molecular marker is AC, the sample is identified as Yanming Orange, and / or (s3) Detect the genotype of the third SNP molecular marker in the sample genome, wherein the third SNP molecular marker is located at position 65 of SEQ ID NO: 9 and SEQ ID NO: 10, and its base type is A / C. When the genotype of the third SNP molecular marker is AC, the sample is identified as Yanming Orange. The detection methods include quantitative real-time PCR, HRM detection, or sequencing.

11. The method of claim 9, wherein, The detection method is HRM detection. The detection in step (s1) includes: performing HRM detection and genotyping using a first primer set, wherein the sequences of the first primer set are shown in SEQ ID NO: 5 and SEQ ID NO: 6, and the genotyping includes comparison with a first standard, the first standard comprising the nucleotide sequences shown in SEQ ID NO: 11 and SEQ ID NO:

12. The detection in step (s2) includes: performing HRM detection and genotyping using a second primer set, wherein the sequences of the second primer set are shown in SEQ ID NO: 1 and SEQ ID NO:

2. The genotyping includes comparison with a second standard, which comprises nucleotide sequences shown in SEQ ID NO: 7 and SEQ ID NO:

8. The detection in step (s3) includes: performing HRM detection and genotyping using a third primer set, wherein the sequences of the third primer set are shown in SEQ ID NO: 3 and SEQ ID NO: 4, and the genotyping includes comparison with a third standard, wherein the third standard comprises nucleotide sequences shown in SEQ ID NO: 9 and SEQ ID NO:

10.

12. The method according to claim 9, wherein the sample is a polynucleotide-containing sample from a citrus plant.

13. The use of the nucleic acid molecule of claim 3 or 4, the primer of claim 5 or 6, or the kit of claim 7 or 8 in the identification of Yanming orange or in the breeding of Yanming orange.

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