MNP Marker Loci, Primer Combinations and Kits for Osmanthus fragrans Variety Identification and Their Applications

Abstract

The present invention provides MNP marker loci for osmanthus variety identification. The MNP marker loci are genomic regions screened in the osmanthus genome and having multiple nucleotide polymorphisms within the osmanthus population. The MNP marker loci include MNP-1 to MNP-50. There is also provided a multiplex PCR primer composition for detecting the above MNP marker loci, including 50 primer pairs, and the nucleotide sequences of the primer pairs are as shown in SEQ ID NO:1 to SEQ ID NO:100. The MNP marker loci, primer composition and kit containing the primer composition of the present invention can identify osmanthus varieties, trace the origin of ancient osmanthus tree products, and have strong discrimination power, high identification throughput and accurate results.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular identification, and particularly relates to an MNP marker locus, a primer composition and a kit for identifying osmanthus fragrans varieties and their applications. Background Art

[0002] Osmanthus fragrans Lour. is one of the top ten traditional famous flowers and has a long cultivation history in China. Osmanthus fragrans resources are rich, with four main groups including Osmanthus fragrans var. semperflorens, Osmanthus fragrans var. latifolius, Osmanthus fragrans var. aurantiacus, and Osmanthus fragrans var. thunbergii, containing more than 150 varieties. Xianning in China, known as the "Hometown of Chinese Osmanthus", has more than 2,000 ancient osmanthus trees over a hundred years old. However, this unique local high-quality natural resource has not been fully developed and utilized. An important reason is that it is impossible to accurately identify the ancient osmanthus trees and the osmanthus produced. If the accurate correspondence between ancient osmanthus trees and the osmanthus produced can be achieved, it is of great significance for the protection, development and utilization of ancient osmanthus resources.

[0003] Currently, the variety identification of osmanthus fragrans mainly relies on morphological identification, DNA barcoding and SSR marker technology. Morphological identification is affected by the environment and depends on the experience of the identifier, so the identification accuracy and stability are poor and it is not suitable for product identification; DNA barcoding is mainly used for species identification and cannot identify taxonomic units below the species level, such as the identification between varieties; SSR markers have been widely used in variety identification due to their high polymorphism and simple operation. However, the SSR marker method can amplify no more than 5 loci in one PCR amplification, with a low throughput, and it is easy to generate slippage genotypes during DNA polymerase amplification, making it difficult to distinguish the slippage genotypes from the main genotypes of the samples. Therefore, SSR markers are not suitable for polyploid plants and mixed samples with multiple genotypes. In the current research on the variety identification of osmanthus fragrans, the work is mainly carried out based on a small number of osmanthus SSR markers. For example, in the paper "Development of EST-SSR Primers for Osmanthus fragrans and Their Application in Variety Identification" published by Li Jun et al. in 2018, only 19 EST-SSR loci were studied. Although the variety differentiation effect can be achieved, the identification results are in the form of graphs, making it difficult to realize the digital management of data. Therefore, developing new molecular markers with high polymorphism for the variety identification of osmanthus fragrans and their detection technology has become a technical problem to be solved urgently.

[0004] MNP markers refer to polymorphic markers generated by multiple nucleotide variations in a region of the genome. MNP markers have rich alleles and high polymorphism. Theoretically, there are 2 at a single MNP locus nAllele genotypes (n is the number of SNPs in the MNP marker), the MNP marker has strong variety discrimination ability. Due to the rich allele genotypes of the MNP marker, a large number of samples can be distinguished by using only a few marker loci. The MNP marker identification process is highly efficient. Hundreds or thousands of marker loci can be amplified simultaneously in a single PCR reaction. For example, 317 - 1042 MNP markers can be amplified simultaneously in the national standard GB / T 38551. The MNP marker identification has high accuracy. The PCR products of each MNP marker are sequenced hundreds of times through second-generation high-throughput sequencing, greatly reducing the genotyping errors caused by experimental errors. Based on the above advantages, the MNP marker technology has been widely applied in crops such as rice, corn, tomato, kiwifruit, etc. At present, there is no research report on MNP markers in the identification of osmanthus varieties, and the corresponding technology is also lacking. Summary of the Invention

[0005] To solve the problems in the background technology, the present invention provides an MNP marker locus, a primer composition and a kit for osmanthus variety identification, which can not only identify osmanthus varieties, but also trace the origin of ancient osmanthus tree products, and has the characteristics of strong discrimination ability, high identification throughput and accurate results.

[0006] The technical solution of the present invention to solve the above technical problems is as follows:

[0007] On the one hand, the present invention provides an MNP marker locus for osmanthus variety identification. The MNP marker locus is a genomic region screened in the osmanthus genome with multiple nucleotide polymorphisms within the osmanthus population. The MNP marker locus includes MNP-1 to MNP-50, and the positions of MNP-1 to MNP-50 on the osmanthus genome GCA_019395295 are shown in the following table:

[0008]

[0009]

[0010]

[0011] The positions of MNP-1 to MNP-50 in other genomes of osmanthus are directly obtained through sequence alignment.

[0012] On the second hand, the present invention provides a multiplex PCR primer composition for detecting the above MNP marker locus, including 50 primer pairs, and the sequences of the 50 primer pairs are shown as SEQ ID NO:1 to SEQ ID NO:100.

[0013] On the third hand, the present invention provides a kit for detecting the above MNP marker locus, which contains the above multiplex PCR primer composition.

[0014] Fourthly, the present invention provides the application of the above-mentioned MNP marker locus or multiplex PCR primer composition or kit in the identification of osmanthus varieties.

[0015] Fifthly, the present invention provides the application of the above-mentioned MNP marker locus or multiplex PCR primer composition or kit in the traceability of ancient osmanthus tree products.

[0016] Sixthly, the present invention provides the application of the above-mentioned MNP marker locus or multiplex PCR primer composition or kit in the construction of an osmanthus variety DNA fingerprint database.

[0017] Seventhly, the present invention provides the application of the product for detecting the above-mentioned MNP marker locus in the identification of osmanthus varieties.

[0018] Eighthly, the present invention provides the application of the product for detecting the above-mentioned MNP marker locus in the traceability of ancient osmanthus tree products.

[0019] Ninthly, the present invention provides a method for identifying osmanthus varieties, using the above-mentioned MNP marker locus as a marker to identify the variety of the osmanthus sample to be tested.

[0020] Furthermore, based on the MNP marker locus, the genetic similarity coefficient between the sample to be tested and the control variety is judged. When the genetic similarity coefficient is greater than or equal to 99%, it is determined that the sample to be tested and the control variety are extremely similar varieties or the same variety. The calculation formula for the genetic similarity is:

[0021]

[0022] where GS is the genetic similarity coefficient between the sample to be tested and the control variety, n ij is the number of marker loci that are both detected in the sample to be tested and the control variety and have no genotype differences, and N ij is the number of marker loci that are both detected in the sample to be tested and the control variety.

[0023] The beneficial effects of the present invention are as follows: Through the MNP loci, primer sets and kits provided by the present invention, DNA fingerprint data of a sample to be tested can be obtained through multiplex amplification and high-throughput sequencing, and then a variety identification conclusion can be obtained through data analysis. The DNA fingerprint data is the base sequence obtained after sequencing, with a resolution reaching the single-base level, high data accuracy and digitalization degree. Hundreds of samples can be compared at one time through sequence analysis software to quickly obtain the variety identification conclusion. At the same time, based on the strategy of multiple marker detections, it has the characteristics of multiple targets, high throughput and high accuracy, which can greatly improve the accuracy and efficiency of the identification of osmanthus-derived products and provide a technical means for the traceability of ancient osmanthus tree products. The MNP marker loci, primer compositions and kits for osmanthus variety identification in this patent have good application value in application scenarios such as osmanthus product identification, DNA fingerprint database construction, and traceability of ancient osmanthus tree products, providing technical support for the molecular breeding and intellectual property protection of osmanthus varieties in China and promoting the healthy development of the industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a distribution diagram of the detected locus numbers of osmanthus MNP marker loci in Example 2 of the present invention;

[0025] Figure 2 It is a distribution diagram of the MNP marker difference ratio among osmanthus samples in Example 2 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0026] The principles and features of the present invention will be described below in conjunction with the accompanying drawings and specific embodiments. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0027] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood to have the meanings as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as the general understanding of those skilled in the art to which the embodiments of the present invention belong. In case of contradiction, this specification shall prevail. Unless otherwise specifically stated, various raw materials, reagents, instruments and equipment used in the embodiments of the present invention can be obtained through market purchase or can be prepared by existing methods.

[0028] The MNP marker technology has been widely applied in rice, corn, tomatoes, and kiwifruits. Currently, there is no research report on MNP markers in osmanthus, nor is there corresponding technology.

[0029] Based on the osmanthus reference genome and combined with the sequencing data of the main osmanthus varieties, through the marker screening rules developed by the inventors (see Example 1 for details), a set of osmanthus MNP marker loci with high polymorphism was screened. The marker loci are genomic regions screened on the osmanthus genome with multiple nucleotide polymorphisms within the osmanthus population, including the marker loci of osmanthus genome MNP-1 to MNP-50. The specific positions of MNP-1 to MNP-50 marker loci are shown in Table 1 of the specification. The start and end positions of the MNP markers marked in Table 1 are determined based on the sequence of the osmanthus reference genome GCA_019395295. The positions of the MNP marker loci in other osmanthus genomes can be obtained through sequence alignment.

[0030] The present invention will be described in detail below with reference to specific examples.

[0031] Example 1 Screening of MNP Marker Loci for Osmanthus Variety Identification and Design of Multiplex PCR Amplification Primers

[0032] The reduced-representation genome sequencing of the collected osmanthus varieties was carried out. Using the publicly released osmanthus genome sequence GCA_019395295 as the reference genome, first, Samtools (Version 1.2) and BCFtools (Version: 1.2) were used for sequence analysis to obtain the SNP loci on the osmanthus genome, and a comparative analysis was carried out with the NT library of NCBI. The MNP markers were screened according to the following principles: (1) The nucleic acid sequence of the marker is unique to osmanthus and does not appear in other species; (2) There are three or more discontinuous SNP differences on the marker sequence; (4) The length of the marker sequence is less than 250 bp. Further, the discrimination of the candidate MNP markers screened was analyzed using the reduced-representation sequencing data of the measured osmanthus varieties, and combined with the current breeding level of osmanthus trees in China, finally 50 candidate MNP marker loci with high polymorphism were determined, namely MNP-1 to MNP-50. The positions of MNP-1 to MNP-50 on the reference genome GCA_019395295 are shown in Table 1, and their positions in other osmanthus genomes can be obtained through sequence alignment.

[0033] According to the above MNP marker loci, a multiplex PCR primer set was designed. This primer set includes the 1st primer pair to the 50th primer pair. Each primer pair contains a forward primer and a reverse primer. The nucleotide sequences of the forward primer and the reverse primer of the 1st primer pair are shown in SEQ ID NO:1 and SEQ ID NO:2 respectively, and so on. The nucleotide sequences of the forward primer and the reverse primer of the 50th primer pair are shown in SEQ ID:99 and SEQ ID NO:100 respectively. These primers do not conflict with each other during the reaction and can simultaneously amplify the above 50 MNP marker loci through multiplex PCR, with high amplification efficiency and high identification accuracy, meeting the requirements for osmanthus variety identification and DNA fingerprint database construction. The identification accuracy is high. The detection markers corresponding to the primer set and the primer set information are shown in Table 1 below.

[0034] Table 1 Positions of 50 osmanthus MNP marker loci on the reference sequence and sequences of 50 pairs of detection primers

[0035]

[0036]

[0037]

[0038]

[0039] The above multiplex PCR primer composition can be used in a kit for detecting MNP marker loci. This primer composition and the kit can be applied to osmanthus variety identification, DNA fingerprint database construction, traceability of ancient osmanthus tree products, and other related fields.

[0040] Example 2 Evaluation of MNP markers, primer compositions, and kits for osmanthus variety identification. 50 pairs of primers were used to analyze the MNP markers of osmanthus. After synthesizing the 50 pairs of primers, 5 μL of each primer was taken and mixed in equal amounts to form a primer mix. 25 osmanthus varieties were randomly selected from the osmanthus samples collected by this unit to evaluate the developed MNP markers, primers, and kits, and to test the detection rate, accuracy, and discrimination of the MNP marker loci. The detailed information of the materials is shown in Table 2.

[0041] Table 2 Information of 25 osmanthus varieties

[0042]

[0043]

[0044] The specific experimental procedure is as follows:

[0045] DNA extraction to obtain the DNA of the sample to be tested.

[0046] Specifically, the leaf DNA of the above osmanthus varieties (test samples) was extracted using a plant genomic DNA extraction kit (manufacturer: Tiangen Biochemical Technology (Beijing) Co., Ltd., product number: DP320). The operation steps are detailed in the instruction manual of this kit. The DNA of the above 25 test samples was obtained. Then, 1 μL of the DNA of each test sample was taken and the DNA concentration of the test sample was measured using a Qubit fluorescence quantifier. The measured DNA concentrations of the test samples were all between 20 ng / μL and 50 ng / μL.

[0047] Multiplex PCR amplification of MNP marker loci was performed to obtain multiplex PCR amplification products.

[0048] Specifically, in the amplification reaction of each test sample, 4 μL of the primer set provided in the embodiment of the present invention, 4 μL of the DNA of the test sample, 10 μL of GenoPlexs 3×T Master Mix (manufacturer: Shijiazhuang Boreidy Biotechnology Co., Ltd.), and 12 μL of water were added and mixed well by oscillation to obtain a mixture, and this mixture was used for multiplex PCR amplification. Multiplex PCR amplification program: 95°C for 3 min; (95°C for 20 sec, 60°C for 4 min) × 17 cycles; 72°C for 4 min.

[0049] Then, DNA purification magnetic beads (manufacturer: Nanjing Novoprotein Scientific Co., Ltd.) were used to purify the amplification products after the reaction, and the method was referred to the product instruction manual.

[0050] Construct a high-throughput sequencing library

[0051] Specifically, the following reaction reagents were added to the purified multiplex PCR amplification products: 10 μL of GenoPlexs 3×T Master Mix, 2 μL of an illumina sequencing adapter primer with a concentration of 5 μM, and 16 μL of water. PCR reaction was carried out according to the following program: 95°C for 3 min; (95°C for 15 s, 58°C for 15 s, 70°C for 30 s) × 8 cycles; 72°C for final extension for 5 min, and the reaction ended at 16°C.

[0052] After the reaction ended, a high-throughput sequencing library of the test sample was obtained. Then, DNA purification magnetic beads were used to purify the high-throughput sequencing library to obtain a purified high-throughput sequencing library, and the purification method was referred to the instruction manual of this product.

[0053] Library sequencing

[0054] The high-throughput sequencing library was sequenced using an illumina NextSeq550 sequencer to obtain the sequencing data of the test sample. The detailed sequencing steps are referred to the instruction manual of this sequencer. After the sequencing ended, the sequencing data was copied to a mobile hard drive.

[0055] Sequencing data analysis

[0056] Using the data alignment software Bowtie2 (version 2.1.0), the sequencing data of the test samples was aligned to the Osmanthus fragrans reference genome to obtain the DNA sequences of MNP markers for each test sample. The alignment results were saved in SAM (The Sequence Alignment / Map format) format.

[0057] (1) Detection rate of MNP markers

[0058] According to the kit of the present invention, multiplex PCR amplification and construction of sequencing libraries were carried out, and multiplex amplification, second-generation high-throughput sequencing and data analysis were performed on these 25 Osmanthus fragrans DNA samples. All of these 50 markers could be detected in these 25 samples. Among them, the highest number of detected markers in the Lianzi Dangui variety was 49, and an average of 46.4 MNP markers could be detected in each variety, with a detection rate of 92.88%. The distribution of the detected loci of Osmanthus fragrans MNP markers is as Figure 1 shown.

[0059] (2) Accuracy analysis of Osmanthus fragrans MNP markers

[0060] In order to test the accuracy of Osmanthus fragrans MNP markers, a reproducibility experiment was carried out on 10 Osmanthus fragrans varieties (referring to two independent experiments conducted by different personnel, different batches of reagents, and different instruments). The two experimental data of each sample were compared and analyzed, and the genotyping accuracy was calculated according to the formula: accuracy = 1 - (1 - precision) / 2. Among them, precision refers to the proportion of marker loci with consistent genotyping results in the two experiments among all marker loci. The statistical results are shown in Table 2. The results show that a total of 472 MNP markers were compared, and the number of non-reproducible loci was 1, and the genotyping accuracy was 99.89%. The high marker accuracy indicates that DNA fingerprint data collected by different laboratories or at different times can be accurately compared with each other, providing technical support for the sharing of DNA fingerprint data.

[0061] Table 2. Information table for accuracy evaluation of 50 Osmanthus fragrans MNP marker loci

[0062]

[0063] (3) Variety discrimination of Osmanthus fragrans MNP markers

[0064] All detected MNP marker genotypes of these 25 Osmanthus fragrans samples were compared pairwise. Based on the principle that at least 1 SNP difference in the allelic genotypes of the same MNP marker locus in different varieties was judged as different, the number of different MNP markers in the pairwise comparison of these 25 samples was counted. A total of 300 pairs of comparison results were obtained, with an average of 40.4 marker loci different for each pair of samples, and the difference ratio was 90.55%. The distribution of the difference ratio is asFigure 2 As shown, the screened markers can significantly distinguish any osmanthus variety.

[0065] Example 3: Traceability and Identification of Ancient Osmanthus Tree Products

[0066] The osmanthus samples of ancient osmanthus trees, ancient osmanthus-1, ancient osmanthus-2 and ancient osmanthus-3, collected from Yangloudong Primary School in Yangloudong Village, Zhaoliqiao Town, Chibi City, Xianning and Guihuayuan Scenic Area were tested according to the experimental process described in Example 2, and then compared with the DNA fingerprint data of the above 25 samples to obtain the genetic similarity coefficient. The results are shown in Tables 3, 4 and 5. With reference to the existing national standard "MNP Marking Method for Plant Variety Identification", the genetic similarity coefficient is used as the basis for the conclusion judgment during variety identification. When the GS is greater than or equal to 99%, the sample to be tested and the control sample are judged to be "very similar varieties or the same variety". As shown in the results, the number of difference sites between the osmanthus collected from each ancient tree and the corresponding ancient tree is 0, and the GS value reaches 100%; while the number of difference sites between the osmanthus samples and other osmanthus samples is significant (greater than or equal to 37 sites), the GS value is extremely low (less than or equal to 21.28%), and they are determined to be different varieties; the results show that the present invention can accurately identify which ancient osmanthus tree the osmanthus comes from, and can be applied to the traceability of ancient osmanthus tree products.

[0067] Table 3 Identification results of Guguihua-1 (only the top 10 varieties with the highest genetic similarity are listed)

[0068]

[0069] Table 4 Identification results of Guguihua-2 (only the top 10 varieties with the highest genetic similarity are listed)

[0070]

[0071] Table 5 Identification results of Guguihua-3 (only the top 10 varieties with the highest genetic similarity are listed)

[0072]

[0073]

[0074] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A multiplex PCR primer composition for detecting MNP marker loci for osmanthus variety identification, characterized in that It includes 50 primer pairs, and the nucleotide sequences of the 50 primer pairs are shown as SEQ ID NO:1 to SEQ ID NO:100; the MNP marker loci are genomic regions screened in the osmanthus genome with multiple nucleotide polymorphisms within the osmanthus population. The MNP marker loci include MNP-1 to MNP-50, and the positions of MNP-1 to MNP-50 on the osmanthus genome GCA_019395295 are shown in the following table: The positions of MNP-1 to MNP-50 in other genomes of osmanthus are directly obtained through sequence alignment.

2. A kit for detecting MNP marker loci for osmanthus variety identification, characterized in that The kit includes the multiplex PCR primer composition described in claim 1.

3. Use of the multiplex PCR primer composition according to claim 1 or the kit according to claim 2 in osmanthus variety identification.

4. Use of the multiplex PCR primer composition according to claim 1 or the kit according to claim 2 in tracing the origin of ancient osmanthus tree products.

5. Use of the multiplex PCR primer composition according to claim 1 or the kit according to claim 2 in constructing a DNA fingerprint database of osmanthus varieties.

6. A method for identifying osmanthus varieties, characterized in that Using the MNP marker loci for osmanthus variety identification as markers, identify the variety of the osmanthus sample to be tested. The MNP marker loci are genomic regions screened in the osmanthus genome with multiple nucleotide polymorphisms within the osmanthus population. The MNP marker loci include MNP-1 to MNP-50, and the positions of MNP-1 to MNP-50 on the osmanthus genome GCA_019395295 are shown in the following table: The positions of MNP-1 to MNP-50 in other genomes of osmanthus are directly obtained through sequence alignment.

7. According to the method for identifying osmanthus varieties according to claim 6, characterized in that Based on the MNP marker loci, judge the genetic similarity coefficient between the sample to be tested and the control variety. When the genetic similarity coefficient is greater than or equal to 99%, it is determined that the sample to be tested and the control variety are extremely similar varieties or the same variety. The calculation formula for genetic similarity is: Among them, GS is the genetic similarity coefficient between the sample to be tested and the control variety, n ij is the number of marker loci that are detected in both the sample to be tested and the control variety but have no genotype differences, and N ij is the number of marker loci that are detected in both the sample to be tested and the control variety.

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