A mnp marker site combination for identifying sweet potato varieties, a primer set, a kit and application thereof

Abstract

The disclosure provides a kind of identification sweet potato variety MNP marker site combination, primer group, kit and its application.The MNP marker site combination includes at least one of MNP-1~MNP-512;The primer group includes: at least one of the first primer pair to the 512th primer pair, each of the primer pair includes forward primer and reverse primer, 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 sequentially as in the sequence table SEQ ID NO:1 to SEQ ID NO:1024.The MNP marker site combination is multiple, polymorphism is high and variety distinguishing ability is strong, can satisfy the demand of sweet potato variety and its substantial derivative variety identification;The primer group does not interfere with each other, and identification accuracy is high, result reproducibility is strong, can satisfy the requirement of DNA fingerprint database construction.

Description

Technical Field

[0001] This disclosure relates to the field of biology, and in particular to a combination of MNP marker sites, primer sets, kits, and applications for identifying sweet potato varieties. Background Technology

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

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

[0004] Sweet potato breeding primarily involves hybridization, including controlled pollination and free pollination. However, natural mutation, artificial mutagenesis, and bio-breeding are also important methods, often resulting in essentially derived varieties (EDVs). On April 1, 2021, the agricultural industry standard "SSR Molecular Marker Method for Identifying the Authenticity of Sweet Potato Varieties" officially came into effect. However, using only seven SSR molecular marker loci is insufficient to meet the requirements for identifying essentially derived varieties. Therefore, there is an urgent need to discover accurate, efficient, and universal molecular marker loci to provide a standard method and guarantee for the identification of sweet potato varieties and their essentially derived varieties. Summary of the Invention

[0005] To address the problems of existing technologies, this disclosure provides an MNP marker site combination, primer set, kit, and application for identifying sweet potato varieties. The technical solution is as follows:

[0006] On the one hand, this disclosure provides a combination of MNP marker sites for identifying sweet potato varieties, the combination of MNP marker sites being used to identify sweet potato varieties, the combination of MNP marker sites including at least one of MNP-1 to MNP-512.

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

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

[0009] In another aspect, this disclosure provides an application of the above-mentioned MNP marker site combination, primer set, or kit, the application of which includes using the MNP marker site combination, primer set, or kit to identify 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 substantial derivative varieties of sweet potato.

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

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

[0014] The beneficial effects of the technical solution provided in this disclosure are as follows: This disclosure provides an MNP marker locus combination, primer set, kit, and application for identifying sweet potato varieties. The MNP marker locus combination has a large number of sites, high polymorphism, and strong variety differentiation ability, which can meet the needs of sweet potato variety and its substantial derivative varieties identification. The primer set does not interfere with each other, and has high identification accuracy and strong reproducibility, which can meet the requirements of DNA fingerprint database construction. It can be applied to large-scale sweet potato variety identification and has great application value in sweet potato product market supervision, intellectual property protection, sweet potato germplasm resource genetic diversity analysis, and sweet potato new variety breeding. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of this disclosure, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this disclosure. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 This is a distribution map of the MNP marker site combinations provided in Embodiment 1 of this disclosure on various chromosomes of sweet potato;

[0017] Figure 2 This is a distribution map of the detection rate of the MNP marker site combination provided in Embodiment 4 of this disclosure;

[0018] Figure 3 This is a distribution map of the number of alleles at the MNP marker sites provided in Embodiment 4 of this disclosure;

[0019] Figure 4 This is a distribution map of the proportion of differentially expressed MNP marker sites among sweet potato varieties provided in Embodiment 4 of this disclosure;

[0020] Figure 5 This is a kinship map of sweet potato varietal resources constructed using MNP marker genetic similarity, provided in Embodiment 7 of this disclosure. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this disclosure clearer, the embodiments of this disclosure will be described in further detail below with reference to the accompanying drawings.

[0022] Example 1

[0023] This disclosure provides a combination of MNP marker sites for identifying sweet potato varieties. The MNP marker site 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 site combination on various sweet potato chromosomes is shown in [reference needed]. Figure 1 .

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

[0025]

[0026]

[0027]

[0028]

[0029]

[0030]

[0031]

[0032]

[0033]

[0034]

[0035]

[0036] Example 2

[0037] This disclosure provides a primer set for identifying sweet potato varieties. The primer set includes at least one pair 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 512th primer pair, and the reverse primer of the 512th primer pair are shown sequentially as SEQ ID NO: 1 to SEQ ID NO: 1024 in the sequence listing. These primer pairs do not conflict with each other, allowing for efficient amplification via multiplex PCR simultaneously, and exhibit high identification accuracy and strong reproducibility. In implementation, 5 μL of each primer is mixed to obtain the primer set.

[0038] Example 3

[0039] This disclosure provides a kit for identifying sweet potato varieties, which includes the primer set provided in Example 2.

[0040] Example 4

[0041] This disclosure provides an application of an MNP marker site as provided in Example 1, a primer set as provided in Example 2, or a kit as provided in Example 3, the application of which includes using the MNP marker site, primer set, or kit to identify sweet potato varieties.

[0042] We evaluated the MNP marker sites, primer sets, and kits using 140 different sweet potato varieties preserved in our institution, testing the detection rate, accuracy, and discrimination of the MNP marker sites. The 140 sweet potato samples are shown in Table 2.

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

[0044]

[0045]

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

[0047] Specifically as follows:

[0048] DNA samples were extracted from 140 sweet potato varieties, with a concentration greater than 5 ng / μL.

[0049] 140 DNA samples were subjected to the first round of PCR amplification to obtain the first round of amplification products. Each 30 μL 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×TMaster Mix. Water was used to make up the first amplification system to 30 μL, of which each primer in the primer set was 5 μL.

[0050] The first amplification program is as follows: 95℃ for 3 min; 95℃ for 20 sec, 14–20 cycles (20 cycles in this example), 60℃ for 4 min; 72℃ for 4 min; hold at 10℃. When the amount of DNA sample input is less than 50 ng, two more cycles can be added appropriately.

[0051] The first-round amplification product was purified to obtain the purified first-round amplification product.

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

[0053] The second amplification program is as follows: 95℃ for 3 min; each cycle includes: 95℃ for 15 sec, 58℃ for 15 sec, 70℃ for 5 min, for a total of 8 cycles; 72℃ for 5 min; and then hold at 10℃.

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

[0055] A 2% agarose gel was prepared. Tiangen Marker I or a similarly sized marker was used. The second-round amplification products were analyzed by electrophoresis to determine the fragment size and distribution of the high-throughput library, and the raw data were obtained. A qualified high-throughput library is approximately 400 bp in size with a relatively concentrated band distribution, and there are no primer dimer residues or non-specific amplification bands. The concentration of the high-throughput library was determined using Qubit; a normal high-throughput library concentration is above 10 ng / μL.

[0056] Data quality control was performed on the raw data obtained from sequencing to obtain high-quality clean data (valid data). The data quality control was as follows: the sequencing data of the sample was aligned to the marker sites of the reference genome using MLMNP variety identification software, and the average coverage fold C1 of the marker sites detected in the first test was calculated.

[0057] When C1 < 500, the amount of sequencing data of the sample is determined to be insufficient, and the steps of Example 4 are repeated until the average coverage of the marker sites in the first detection is C1 ≥ 500.

[0058] When C1≥500, the proportion of detected marker sites R1=T1 / T is further calculated, where T1 and T are the number of detected marker sites and the number of detected marker sites in the sample, respectively.

[0059] When R1 ≥ 95%, the sequencing data is considered acceptable;

[0060] When R1 < 95%, it is determined that the library construction may have failed, and the steps of Example 4 are repeated until the average coverage fold C2 of the marker sites in the second detection is ≥ 500.

[0061] When C2 ≥ 500, the proportion of marker sites detected in both the first and second detections is further calculated: R2 = T. 12 / (T1+T2), where T 12 T1 represents the number of marker sites detected together in the first and second tests, while T2 represents the number of marker sites detected in the first and second tests respectively.

[0062] When R² ≥ 95%, the sequencing data is considered acceptable.

[0063] Then, BWA software was used to break down the clean data into individual target loci, obtaining alignment results in SAM format. The SAM files were then converted to BAM format using samtools software. Finally, the reads in the BAM file were sorted using SortSam in Picard, yielding the final BAM file. GATK can then be used to determine the genotype of each target locus.

[0064] Using the primers and kits provided in this embodiment, a single experiment was able to detect 512 × 140 = 71680 marker sites, with an average sequencing coverage of 1826.64 for each DNA sample, demonstrating the high efficiency of sweet potato MNP marker site detection.

[0065] A high marker detection rate can prevent differences in genetic similarity coefficients due to variations in detected sites across different experiments, and avoid significant biases in variety identification conclusions caused by sampling issues at MNP marker sites. The distribution of sweet potato MNP marker detection rates is shown below. Figure 2 As shown, the number of MNP marker sites detected in the sequencing data of these 140 DNA samples was counted. Figure 3As shown, an average of 494 (96.57%) MNP markers were detected in each variety. Among the 140 varieties, only 18 varieties had a detection rate below 95%, with the lowest detection rate being 90.63%. This indicates that the sweet potato MNP marker site detection rate provided in this embodiment is high and stable.

[0066] Accuracy analysis of sweet potato MNP labeling method

[0067] The number of marker loci with both differences and identical genotypes among varieties is the basis for determining the conclusions in variety identification. Therefore, the accuracy of variety identification ultimately depends on the accuracy of marker locus genotyping. Since the true value is unknown, the absolute accuracy of any method cannot be calculated. In practice, either the reference value is assumed to be the true value when calculating the accuracy, or precision is used to assess the accuracy. Since the reference value for the genotype of the marker loci of a variety is also unknown, this embodiment uses two reproducibility experiments to calculate precision, and then calculates the accuracy of genotyping: accuracy = 1 - (1 - precision) / 2. Here, precision refers to the proportion of marker loci whose genotyping results are consistent in the two experiments out of all marker loci. A reproducibility experiment refers to two independent experiments performed by different personnel, different batches of reagents, and different laboratories. The reproducibility experiment simulates identification of different batches, and a high reproducibility rate means that the identification results of different laboratories can be accurately compared with each other.

[0068] To verify the accuracy of the sweet potato MNP marker method, a reproducibility experiment was conducted on 18 sweet potato varieties out of 140 varieties. The reproducibility experiment was conducted independently by different researchers at different times. Pairwise comparisons were performed on the common marker sites between the two batches of data for each variety, and the accuracy of the genotyping was calculated. Accuracy = 1 - (1 - precision) / 2. The results are shown in Table 3.

[0069] Table 3. Accuracy of sweet potato marker genotyping results

[0070]

[0071]

[0072] Table 3 shows that a total of 7574 MNP markers were compared, with a non-repeating rate of 0.42% and a genotyping accuracy of 99.79%. This demonstrates that the sweet potato MNP marker combination provided in this embodiment exhibits high accuracy. This means that the variety identification results using the marker combination provided in this embodiment are highly consistent across different laboratories or different batches within the same laboratory, eliminating the need for parallel experiments to reduce experimental errors. It also implies that precise comparisons can be made between DNA fingerprint data from different sources, greatly facilitating variety identification.

[0073] Sweet potato MNP marker variety differentiation

[0074] The core task of variety identification is to distinguish between the tested variety and the control variety using the detected marker sites. Two important factors affect the core task of variety identification: First, the stronger the distinguishing power of a single marker, the stronger the ability of the marker method to distinguish varieties; second, the more markers used, the stronger the ability of the marker method to distinguish varieties.

[0075] This example analyzed the genetic distance among 140 sweet potato varieties (genetic distance = number of different MNP loci among varieties / number of common MNP loci among varieties × 100%), obtaining a total of 9730 comparison results. Only 20 pairs of samples had a genetic distance within 10%, and all other sweet potato varieties differed by at least 243 marker loci, with an average genetic distance of 87.51% among varieties. (Details are as follows...) Figure 4 As shown. Combined with Figure 4 It can be seen that the MNP marker combination provided in this embodiment has a strong ability to distinguish varieties, and can distinguish 99.88% of any variety combination. The relationships between the varieties with the smallest differences 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] Identification of substantial derivative varieties requires a sufficient number of marker loci to accurately calculate the genetic similarity coefficient (genetic similarity coefficient = 1 - genetic distance). In this embodiment of the invention, 512 marker loci combinations are used, which can be employed for the identification of substantial derivative varieties.

[0079] The International Seed Federation (ISF) uses SSR markers to identify substantial derivatives of species such as maize and soybean, often using a genetic similarity coefficient of around 90% as the threshold. However, in actual sweet potato breeding, hybridization is frequently used, but natural mutation, induced mutation, and transgenic breeding techniques are also employed, leading to the creation of substantial derivative varieties. This example uses a 90% threshold to analyze the genetic similarity coefficients of the widely promoted Pushu 32 variety and eight other sweet potato materials. Of 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, respectively. Specific results are shown in Table 4.

[0080] Table 4. Determination of Substantial Derivative Varieties of Pushu 32

[0081]

[0082] Table 4 shows that C122, C123, and C126 share a genetic similarity coefficient of over 98.81% with Pushu 32, indicating a suspected substantial derivation relationship. In contrast, the highest genetic similarity coefficient among the other five varieties and Pushu 32 is only 27.45%, falling outside the category of substantial derivation varieties. This aligns with actual conditions. Therefore, the method provided in this embodiment accurately identifies substantial derivation varieties, effectively protecting the interests of the original breeders and suppressing imitation breeding.

[0083] Example 6

[0084] Constructing a standard DNA fingerprint library of sweet potatoes using MNP core marker primer combinations

[0085] Following the method provided in Example 4, MNP marker analysis was performed on the DNA of 140 sweet potato samples. The genotyping of each sample at 512 marker sites was statistically analyzed, forming the sweet potato MNP fingerprint database. This database can be used for services such as variety authenticity identification, market supervision and anti-counterfeiting, variety management, inferring the identity of unknown varieties, and screening similar varieties. Specifically, the method involves detecting the MNP fingerprint of the variety to be tested and comparing it with data in the fingerprint database.

[0086] Example 7

[0087] Analysis of the phylogenetic relationships of sweet potato cultivars using MNP markers

[0088] The 512 pairs of sweet potato MNP primers provided in this invention can be used for phylogenetic analysis of sweet potato varietal resources. The specific method is as follows: The genetic distances of MNP markers among different sweet potato samples in Example 4 are organized into a matrix, input into MAGE11 software, and a clustering tree is constructed using the neighbor-joining method. The 140 sweet potato varieties involved in this example are divided into 5 different groups. See [link to relevant documentation]. Figure 5 Varieties clustered in the same group are more closely related.

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

Claims

1. An application of an MNP marker locus combination for identifying sweet potato varieties, characterized in that, The application includes using the MNP marker locus combination for sweet potato variety identification. The MNP marker locus combination includes 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 the table below:

2. The application according to claim 1, characterized in that, The application includes using the combination of the MNP marker sites to identify the authenticity of sweet potato varieties.

3. The application according to claim 1, characterized in that, The application includes using the combination of the MNP marker sites to identify substantial derivative varieties of sweet potato.

4. The application according to claim 1, characterized in that, The application includes combining the MNP marker sites to construct a DNA fingerprint library of sweet potato varieties.

5. The application according to claim 1, characterized in that, The application includes combining the MNP marker sites to analyze the phylogenetic relationships of sweet potato varietal resources.

6. A primer set for identifying sweet potato varieties, characterized in that, The primer set includes primer pairs 1 to 512, each primer pair including 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 primer 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.

7. A kit for identifying sweet potato varieties, characterized in that, The kit includes the primer set as described in claim 6.

Images