MNP marker site combination, primer group and kit for identifying sweet potato varieties and application of MNP marker site combination, primer group and kit

Abstract

The invention provides an MNP marker site combination, a primer group and a kit for identifying sweet potato varieties and application of the MNP marker site combination, the primer group and the kit. The MNP marker site combination comprises at least one of MNP-1 to MNP-512, and the number of the MNP-1 to the MNP-512 is at least one; the primer group comprises at least one of a first primer pair to a 512th primer pair, each primer pair comprises a forward primer and a reverse primer, and the forward primer of the first primer pair, the reverse primer of the first primer pair to the forward primer of the 512th primer pair and the reverse primer of the 512th primer pair are sequentially shown as SEQ ID NO: 1 to SEQ ID NO: 1024 in a sequence table. The MNP marker is large in site combination number, high in polymorphism and strong in variety distinguishing ability, and can meet the requirements of identification of sweet potato varieties and substantive derived varieties thereof; the primer group does not interfere with each other, is high in identification accuracy and high in result reproducibility, and can meet the requirements of DNA fingerprint database construction.

Description

Technical Field

[0001] The present disclosure relates to the biological field, and in particular to an MNP marker site combination, a primer set, a kit and applications thereof for identifying sweet potato varieties. Background Art

[0002] Sweet potato is one of the world's major food crops, as well as an important industrial raw material crop and feed crop. It is mainly distributed in Asia, America and Africa.

[0003] my country has always attached great importance to the cultivation of new sweet potato varieties, and hundreds of sweet potato varieties have been registered and protected. Sweet potato variety identification primarily relies on the DUS (Distinctness, Uniformity, and Stability) test based on phenotypic traits. DUS provides a reliable basis for determining new plant variety protection.

[0004] Sweet potato breeding is primarily based on hybridization, including controlled and uncontrolled pollination. However, natural mutation, artificial mutagenesis, and biological breeding are also important methods of sweet potato breeding. These breeding methods often produce essentially derived varieties (EDVs). On April 1, 2021, the agricultural industry standard "SSR Molecular Marker Method for Authentic Identification of Sweet Potato Varieties" was officially implemented. However, it only uses seven SSR molecular marker loci, which cannot meet the requirements for essentially derived variety identification. Therefore, there is an urgent need to explore accurate, efficient, and universal molecular marker loci to provide standard methods and guarantees for the identification of sweet potato varieties and their essentially derived varieties. Summary of the Invention

[0005] To address the problems of the prior art, the present disclosure provides a combination of MNP marker sites, primer sets, kits, and applications thereof for identifying sweet potato varieties. The technical solution is as follows:

[0006] In one aspect, the present disclosure provides an MNP marker locus combination for identifying sweet potato varieties, wherein the MNP marker locus combination is used to identify sweet potato varieties, and the MNP marker locus combination includes at least one of MNP-1 to MNP-512.

[0007] On the other hand, the present disclosure provides a primer set for identifying sweet potato varieties, the primer set comprising: at least one pair of the first primer pair to the 512th primer pair, each of the primer pairs comprising a forward primer and a reverse primer, the forward primer of the first primer pair, the reverse primer of the first primer pair to the forward primers of the 512th primer pair, and the reverse primer of the 512th primer pair are respectively as shown in SEQ ID NO: 1 to SEQ ID NO: 1024 in the sequence listing.

[0008] In yet another aspect, the present disclosure provides a kit for identifying sweet potato varieties, comprising the above primer set.

[0009] In yet another aspect, the present disclosure provides an application of the above-mentioned MNP marker site combination, the above-mentioned primer set or the above-mentioned kit, wherein the application comprises using the MNP marker site combination, the above-mentioned primer set or the above-mentioned kit for identifying sweet potato varieties.

[0010] Specifically, the application includes using the MNP marker site combination, the primer set or the kit to identify the authenticity of sweet potato varieties.

[0011] Specifically, the application includes using the MNP marker site combination, the primer set or the kit to identify substantially derived sweet potato varieties.

[0012] Specifically, the application includes using the MNP marker site combination, the primer set or the kit to construct a sweet potato variety DNA fingerprint library.

[0013] Specifically, the application includes using the MNP marker site combination, the primer set or the kit to analyze the genetic relationship of sweet potato variety resources.

[0014] The technical solutions provided by the embodiments of the present disclosure have the following beneficial effects: the embodiments of the present disclosure provide an MNP marker site combination, a primer set, a kit, and applications thereof for identifying sweet potato varieties. The MNP marker site combination has a large number, high polymorphism, and strong variety differentiation capability, which can meet the needs of identifying sweet potato varieties and their substantially derived varieties; the primer sets do not interfere with each other, and have high identification accuracy and strong reproducibility, which can meet the requirements of building a DNA fingerprint database, can be applied to large-scale sweet potato variety identification, and have great application value in market supervision of sweet potato products, intellectual property protection, genetic diversity analysis of sweet potato germplasm resources, and breeding of new sweet potato varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present disclosure, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present disclosure. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0016] Figure 1 This is a distribution diagram of the MNP marker locus combination provided in Example 1 of the present disclosure on each chromosome of sweet potato;

[0017] Figure 2 This is a distribution diagram of the detection rate of the MNP marker site combination provided in Example 4 of the present disclosure;

[0018] Figure 3 is a distribution diagram of the number of allele types of the MNP marker site provided in Example 4 of the present disclosure;

[0019] Figure 4 This is a distribution diagram of the proportion of MNP marker sites that differ between sweet potato varieties provided in Example 4 of the present disclosure;

[0020] Figure 5 This is a phylogenetic relationship diagram of sweet potato variety resources constructed using MNP marker genetic similarity provided in Example 7 of the present disclosure. DETAILED DESCRIPTION

[0021] In order to make the objectives, technical solutions and advantages of the present disclosure more clear, the embodiments of the present disclosure will be further described in detail below with reference to the accompanying drawings.

[0022] Example 1

[0023] The present disclosure provides an MNP marker locus combination for identifying sweet potato varieties. The MNP marker locus combination is used to identify sweet potato varieties. The MNP marker locus combination includes at least one of MNP-1 to MNP-512 on the sweet potato genome GCA_002525835.2. The positions of MNP-1 to MNP-512 on the reference sequence are shown in Table 1. The distribution of the MNP marker locus combination on each chromosome of sweet potato is shown in Table 1. Figure 1 .

[0024] Table 1. Positions of MNP marker sites corresponding to primer set sequences

[0025]

[0026]

[0027]

[0028]

[0029]

[0030]

[0031]

[0032]

[0033]

[0034]

[0035]

[0036] Example 2

[0037] The disclosed embodiments provide a primer set for identifying sweet potato varieties. The primer set comprises at least one pair selected from MNP-1 to MNP-512, each primer pair comprising a forward primer and a reverse primer. The forward primer of the first primer pair, the reverse primer of the first primer pair, the forward primer of the first primer pair, the reverse primer of the 512th primer pair, and the reverse primer of the 512th primer pair are, respectively, as shown in the sequence listing as SEQ ID NO: 1 to SEQ ID NO: 1024. The primer pairs do not conflict with each other, can be efficiently amplified simultaneously by multiplex PCR, and have high identification accuracy and reproducible results. In practice, 5 μL of each primer is mixed in equal amounts to obtain the primer set.

[0038] Example 3

[0039] The disclosed embodiments provide a kit for identifying sweet potato varieties, which includes the primer set provided in Example 2.

[0040] Example 4

[0041] The disclosed embodiments provide an application of the MNP marker site provided in Example 1, the primer set provided in Example 2, or the kit provided in Example 3, which includes using the MNP marker site, primer set, or kit to identify sweet potato varieties.

[0042] The MNP marker loci, primer sets, and kit were evaluated using 140 different sweet potato varieties maintained by our institution to test the detection rate, accuracy, and discrimination of the MNP marker loci. The 140 sweet potato samples are listed in Table 2.

[0043] Table 2 140 sweet potato varieties involved in this embodiment

[0044]

[0045]

[0046] Multiplex PCR amplification and sequencing library construction were performed using the kit provided by the present invention, and multiplex amplification, second-generation high-throughput sequencing and data analysis were performed on the DNA samples of these 140 sweet potato varieties.

[0047] The details are as follows:

[0048] DNA samples of 140 sweet potato varieties were extracted, and the concentration of DNA samples was greater than 5 ng / μL;

[0049] 140 DNA samples were subjected to the first round of PCR amplification to obtain first-round amplification products. Each 30 μL of the first amplification system included: 4 μL of the primer set provided in Example 2, 50-200 ng of DNA sample, and 10 μL of GenoPlexs 3×T Master Mix. The first amplification system was made up to 30 μL with water, wherein each primer in the primer set was 5 μL.

[0050] The first amplification program is as follows: 95°C for 3 minutes; 95°C for 20 seconds, 14-20 cycles (20 cycles in this example), 60°C for 4 minutes; 72°C for 4 minutes; hold at 10°C. If the input DNA sample amount is less than 50 ng, two additional cycles may be added.

[0051] purifying the first-round amplification product to obtain a purified first-round amplification product;

[0052] The purified first-round amplification product was subjected to a second-round PCR amplification to obtain a second-round amplification product. Each 30 μL of the second amplification system included: 10 μL of GenoPlexs 3×T Master Mix, 4 μL of the purified first-round amplification product, 4 μL of the primer set provided in Example 2, and 16 μL of water.

[0053] The second amplification program was as follows: 95°C for 3 min; each cycle included: 95°C for 15 sec, 58°C for 15 sec, 70°C for 5 min, for a total of 8 cycles; 72°C for 5 min; and holding at 10°C.

[0054] The second-round amplification products were recovered for subsequent high-throughput sequencing or stored at -20°C.

[0055] Prepare a 2% agarose gel using Tiangen's Marker I or a marker of similar size. Run the second-round amplification products through electrophoresis to determine the fragment size and distribution of the high-throughput library and obtain raw data. A qualified high-throughput library should have a concentrated band around 400 bp, with no residual primer dimers or nonspecific amplification bands. Use Qubit to determine the concentration of the high-throughput library; the normal concentration is above 10 ng / μL.

[0056] The raw data obtained by sequencing were quality controlled to obtain high-quality clean data (valid data). The data quality control was as follows: the sequencing data of the samples were aligned to the marker sites of the reference genome using MLMNP variety identification software, and the average coverage factor C1 of the marker sites detected for the first time was calculated;

[0057] When C1 is less than 500, it is determined that the amount of sequencing data of the sample is insufficient, and the steps of Example 4 are repeated until the average coverage of the marker sites detected for the first time is C1 ≥ 500;

[0058] When C1 ≥ 500, the ratio of detected marker sites R1 = T1 / T was further calculated, where T1 and T were the number of detected marker sites and the number of detected marker sites in the sample, respectively;

[0059] When R1 ≥ 95%, the sequencing data was considered qualified;

[0060] When R1 is less than 95%, it is determined that the library construction may have failed, and the steps of Example 4 are repeated until the average coverage of the marker sites detected for the second time, C2, is greater than or equal to 500;

[0061] When C2≥500, the ratio of the marker sites detected in the first and second times is further calculated R2=T 12 / (T1+T2), where T 12 is the number of marker sites detected together in the first and second times, T1 and T2 are the numbers of marker sites detected in the first and second times respectively;

[0062] When R2≥95%, the sequencing data were judged to be qualified.

[0063] The clean data was then decomposed into target loci using BWA software, generating SAM-formatted alignments. The SAM-formatted files were then converted to BAM format using samtools. The reads in the BAM files were then sorted using SortSam in the Picard tool to generate the final BAM file. The genotypes of each target locus were then determined using GATK.

[0064] The primers and kit provided in this example were used to detect 512×140=71680 marker sites in one experiment, and the average sequencing coverage of each DNA sample was 1826.64, which shows the high efficiency of sweet potato MNP marker site detection.

[0065] A higher detection rate of marker sites can prevent the difference in genetic similarity coefficients caused by the variation of detection sites in different experiments, and avoid the problem of large deviations in variety identification conclusions due to the sampling of MNP marker sites. Figure 2 As shown in the figure, the number of MNP marker sites detected in the sequencing data of these 140 DNA samples is counted, as shown in the figure. Figure 3As shown, an average of 494 (96.57%) MNP markers were detected for each variety, and only 18 of the 140 varieties had a detection rate below 95%, with the lowest marker detection rate being 90.63%. This indicates that the sweet potato MNP marker site detection rate provided in this example is high and stable.

[0066] Accuracy Analysis of Sweet Potato MNP Labeling Method

[0067] The number of marker sites with different and identical genotypes between varieties is the basis for the conclusion judgment during variety identification. Therefore, the accuracy of variety identification ultimately depends on the accuracy of marker site genotyping. Since the true value is unknown, the absolute accuracy of any method is not calculable. In practice, either the reference value is assumed to be the true value to calculate the accuracy, or the accuracy is evaluated using precision. Since the reference value of the marker site genotype of the variety is also unknown, this embodiment uses two reproducibility experiments to calculate the accuracy, and then calculates the accuracy of typing, accuracy = 1-(1-precision) / 2. Among them, precision refers to the proportion of marker sites with consistent typing results in the two experiments to all marker sites, and reproducibility experiments refer to two independent experiments performed by different personnel, different batches of reagents, and different laboratories. The reproducibility experiment simulates the identification of different batches, and a high reproducibility means that the identification results of different laboratories can be accurately compared with each other.

[0068] To test the accuracy of the sweet potato MNP tagging method, a reproducibility experiment was conducted on 18 of the 140 sweet potato varieties. The reproducibility experiments were conducted independently by different experimenters at different times. The common marker sites between the two batches of data for each variety were compared and analyzed pairwise, and the typing accuracy was calculated. Accuracy = 1-(1-precision) / 2. The results are shown in Table 3.

[0069] Table 3 Accuracy of sweet potato marker loci typing results

[0070]

[0071]

[0072] As shown in Table 3, a total of 7,574 MNP markers were compared, with a non-recurring proportion of 0.42% and a typing accuracy of 99.79%. This demonstrates that the sweet potato MNP marker locus combination provided in this example exhibits high accuracy. This means that using the marker locus combination provided in this example, variety identification results are highly consistent across different laboratories or across different batches within the same laboratory, eliminating the need for parallel experiments to reduce experimental error. This also allows for accurate comparison of DNA fingerprint data from different sources, greatly facilitating variety identification.

[0073] Discrimination of sweet potato varieties by MNP markers

[0074] The core task of variety identification is to distinguish between the test variety and the control variety using the detected marker loci. Two important factors influence this core task: first, the stronger the discriminating power of a single marker, the stronger the ability of the marker method to distinguish between varieties; second, the greater the number of markers used, the stronger the ability of the marker method to distinguish between varieties.

[0075] This example analyzed the genetic distances between 140 sweet potato varieties (genetic distance = number of different MNP loci between varieties / number of MNP loci detected in common between varieties × 100%), and obtained a total of 9730 pairs of comparison results. Only 20 pairs of samples had genetic distances within 10%, while at least 243 marker loci differed between any other sweet potato varieties, with an average genetic distance of 87.51%. Figure 4 As shown. Combined Figure 4 The MNP marker combination provided in this example has a strong ability to distinguish varieties, and can distinguish 99.88% of any variety combination. The varieties with the smallest difference ratios in these 20 pairs of samples are synonyms, mutants and wild types, or transgenic lines and recipient varieties.

[0076] Example 5

[0077] Used for identification of substantial derivative varieties of sweet potato

[0078] The identification of substantially derived varieties requires a sufficient number of marker loci to accurately calculate the genetic similarity coefficient (genetic similarity coefficient = 1-genetic distance). The number of marker loci combinations used in the embodiment of the present invention is 512, which can be used for the identification of substantially derived varieties.

[0079] The International Seed Federation (ISF) uses SSR markers to identify substantive derivatives of species such as corn and soybean, often using a genetic similarity coefficient of around 90% as the threshold for determination. In the actual breeding process of sweet potatoes, hybrid breeding is often used, but breeding techniques such as natural mutation, induced mutation, and transgenics have also been used, which can lead to the generation of substantive derivatives of sweet potato. This example used a 90% threshold for determination to analyze the genetic similarity coefficients of the widely used Pushu 32 and eight sweet potato materials. Among the eight samples, samples C122 and C123 are transgenic materials of Pushu 32, sample C126 is a natural mutant of Pushu 32, and samples C160, C37, C85, C27, and C83 are Xushu 18, Zhenghong 23, Sushu 28, Yanshu 25, and Sushu 25. The specific results are shown in Table 4.

[0080] Table 4 Determination of the substantial derivatives of Pushu 32

[0081]

[0082] As shown in Table 4, the genetic similarity coefficients of C122, C123, and C126 with Pushu 32 exceed 98.81%, indicating that they are suspected of being substantially derived from Pushu 32. However, the highest genetic similarity coefficient between the other five varieties and Pushu 32 is only 27.45%, meaning none of them fall into the category of substantially derived varieties, which is consistent with the actual situation. This demonstrates that the method provided in this example accurately identifies substantially derived varieties, effectively protecting the interests of original breeders and curbing copycat breeding.

[0083] Example 6

[0084] Construction of a standard DNA fingerprint library for sweet potato using MNP core-labeled primer combinations

[0085] Following the method provided in Example 4, 140 sweet potato DNA samples were analyzed for MNP markers. The typing results for each sample at 512 marker loci were statistically analyzed to create a sweet potato MNP fingerprint database. This sweet potato fingerprint database can be used for services such as variety authenticity verification, market supervision and anti-counterfeiting efforts, variety management, inferring the identity of unknown varieties, and screening for similar varieties. The specific method involves detecting the MNP fingerprint of the test variety and comparing it with the data in the fingerprint database.

[0086] Example 7

[0087] Analysis of the genetic relationship of sweet potato cultivar resources using MNP markers

[0088] The 512 pairs of sweet potato MNP primers provided by the present invention can be used for phylogenetic analysis of sweet potato variety resources. The specific method is as follows: the genetic distances of the MNP markers between different sweet potato samples in Example 4 are organized into a matrix, input into the MAGE11 software, and a cluster tree is constructed using the neighbor-joining method. The 140 sweet potato varieties involved in this example are divided into 5 different groups, see Figure 5 , varieties clustered in the same group have closer genetic relationships.

[0089] The above description is merely an optional embodiment of the present disclosure and is not intended to limit the present disclosure. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present disclosure shall be included in the scope of protection of the present disclosure.

Claims

1. A combination of MNP marker sites for identifying sweet potato varieties, characterized in that: The MNP marker locus combination is used to identify sweet potato varieties. The MNP marker locus combination includes at least one of MNP-1 to MNP-512. The positions of MNP-1 to MNP-512 on the reference sequence are shown in the following table:

2. A primer set for identifying sweet potato varieties, characterized in that: The primer set includes: at least one pair of the first primer pair to the 512th primer pair, each of the primer pairs includes a forward primer and a reverse primer, and the forward primer of the first primer pair, the reverse primer of the first primer pair to the forward primers of the 512th primer pair, and the reverse primer of the 512th primer pair are shown in sequence as SEQ ID NO: 1 to SEQ ID NO: 1024 in the sequence listing.

3. A kit for identifying sweet potato varieties, characterized in that: The kit comprises the primer set according to claim 2.

4. A use of the MNP labeling site combination according to claim 1, the primer set according to claim 2, or the kit according to claim 3, characterized in that: The application includes using the MNP marker site combination, the primer set or the kit for identifying sweet potato varieties.

5. The use according to claim 4, characterized in that The application includes using the MNP marker site combination, the primer set or the kit to identify the authenticity of sweet potato varieties.

6. The use according to claim 4, characterized in that The application includes using the MNP marker site combination, the primer set or the kit to identify substantially derived sweet potato varieties.

7. The use according to claim 4, characterized in that The application includes using the MNP marker site combination, the primer set or the kit to construct a sweet potato variety DNA fingerprint library.

8. The use according to claim 4, characterized in that The application includes using the MNP marker site combination, the primer set or the kit to analyze the genetic relationship of sweet potato variety resources.

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