Primer set, kit and application thereof for identifying sunflower varieties

Abstract

The present disclosure provides a primer set, a kit and an application thereof for identifying sunflower varieties. The primer set includes: the first primer pair to the 552nd 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 primer of the 552nd primer pair and the reverse primer of the 552nd primer pair are shown in sequence as SEQ ID NO: 1 to SEQ ID NO: 1104 in the sequence table. The primer pairs do not interfere with each other, and the identification accuracy is high and the result reproducibility is strong. At the same time, the primer set can perform multiple PCR amplification, and the amplification product can be sequenced in combination with the second-generation sequencing platform. The detection of sunflower varieties has the characteristics of high throughput, high discrimination, high accuracy, etc., which can meet the needs of authenticity identification and substantial derivation identification of sunflower varieties, and realize the sharing and free comparison of variety data in the DNA fingerprint database.

Description

Technical Field

[0001] The present disclosure relates to the field of biotechnology, and in particular to a primer set and a kit for identifying sunflower varieties and applications thereof. Background Art

[0002] Sunflower (Helianthus annuus L.) is an annual herbaceous plant of the genus Helianthus in the Asteraceae family. As an important oil crop, sunflower plays an extremely important role in the agricultural economy.

[0003] At present, my country has registered nearly 2,000 sunflower varieties. With the expansion of the sunflower industry, the sunflower seed industry has problems such as insufficient utilization of germplasm resources and unclear variety rights. The Seed Law of the People's Republic of China, which was officially implemented on March 1, 2022, focuses on strengthening the protection of intellectual property rights in the seed industry, establishes a substantive derivative variety system, and expands the scope and protection links of new plant variety rights. As the main means of protecting new plant varieties, the variety DUS (Distinctness Uniformity Stability) test is based on the determination results of plant phenotypic traits. The International Union for the Protection of New Varieties of Plants uses SSR (Simple Sequence Repeat) and SNP (Single Nucleotide Polymorphism) molecular marker methods as auxiliary technical means for variety DUS testing, which plays a positive role in screening similar varieties and specificity determination. On April 1, 2021, the agricultural industry standard "SSR Molecular Marker Method for Authenticity Identification of Sunflower Varieties" was officially implemented, but because the sites of SSR molecular markers have low density on the genome and the allele type is relatively single. However, the original variety will give rise to multiple essential derived varieties (EDV). Except for the differences in traits caused by derivation, the essential derived varieties are the same as the original variety in expressing the basic traits produced by the genotype or genotype combination of the original variety. Therefore, the current SSR molecular marker method is difficult to meet the requirements of accurate identification of essential derived varieties.

[0004] Public Content

[0005] In order to solve the problems of the prior art, the present disclosure provides a primer set, a kit and an application thereof for identifying sunflower varieties. The technical solution is as follows:

[0006] On the one hand, the present disclosure provides a primer set for identifying sunflower varieties, the primer set comprising: the 1st primer pair to the 552nd primer pair, each of the primer pairs comprising a forward primer and a reverse primer, the forward primer of the 1st primer pair, the reverse primer of the 1st primer pair to the forward primer of the 552nd primer pair, and the reverse primer of the 552nd primer pair are respectively as shown in SEQ ID NO: 1 to SEQ ID NO: 1104 in the sequence listing.

[0007] In another aspect, the present disclosure provides a kit for identifying sunflower varieties, the kit comprising the primer set according to claim 1.

[0008] In yet another aspect, the present disclosure provides an application of a primer set for identifying sunflower varieties, the application comprising: using the primer set to identify sunflower varieties.

[0009] Specifically, the application includes: using the above primer set to identify the authenticity of sunflower varieties.

[0010] Specifically, the application includes: using the above primer set to identify substantially derived varieties of sunflower.

[0011] Specifically, the application includes: using the above primer set to construct a DNA fingerprint database of sunflower varieties.

[0012] The technical solution provided by the embodiments of the present disclosure has the following beneficial effects: the embodiments of the present disclosure provide a primer set, a kit and an application thereof for identifying sunflower varieties. The primer pairs do not interfere with each other, and the identification accuracy is high and the result reproducibility is strong. At the same time, the primer set can perform multiple PCR amplification and combine with the second-generation sequencing platform to sequence the amplified products. The detection of sunflower varieties has the characteristics of high throughput, high discrimination and high accuracy, which can meet the needs of authenticity identification and substantial derivative identification of sunflower varieties, realize the sharing and free comparison of variety data in the DNA fingerprint database, and meet the requirements for the construction of the DNA fingerprint database. The primer set can be applied to large-scale sunflower variety identification, and has great application value in market supervision of crop products, intellectual property protection, and new variety breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the technical solutions in the embodiments of the present disclosure, the drawings required for use in the description of the embodiments will be briefly introduced below. 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 paying any creative work.

[0014] Figure 1 is a distribution quantity diagram of the MNP markers provided in Example 1 of the present disclosure on the sunflower chromosome;

[0015] Figure 2 is a graph of the number of sequencing fragments and the detection rate of MNP marker sites provided in Example 3 of the present disclosure;

[0016] Figure 3 This is a distribution diagram of the differences in MNP marker ratios among sunflower varieties provided in Example 3 of the present disclosure. DETAILED DESCRIPTION

[0017] In order to make the objectives, technical solutions and advantages of the present disclosure more clear, the embodiments of the present disclosure will be described in further detail below.

[0018] Embodiment 1

[0019] The present disclosure provides a primer set for identifying sunflower varieties, the primer set comprising: the first primer pair to the 552nd primer pair, 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 552nd primer pair, and the reverse primer of the 552nd primer pair are respectively as shown in SEQ ID NO: 1 to SEQ ID NO: 1104 in the sequence listing.

[0020] Table 1 shows the sequences of 552 pairs of detection primers

[0021]

[0022]

[0023]

[0024]

[0025]

[0026]

[0027]

[0028]

[0029]

[0030]

[0031]

[0032]

[0033]

[0034] The screening of the primer set includes: collecting 25 sunflower varieties such as SH363, JK601, SH361, and X3939 for genome sequencing, obtaining sequencing data with more than 20X coverage for each variety, combining 290 sunflower whole genome sequencing data (PRJNA353001), and using the public sunflower genome sequence GCF_002127325.2 as a reference genome. First, Samtools (Version 1.2) and BCFtools (Version: 1.2) are used for sequence analysis to obtain SNP sites on the sunflower genome, and the SNP sites are compared and analyzed with the NT library of NCBI, and marker screening is performed according to the following principles: (1) The marker sequence is unique only to sunflower and does not appear in other species; (2) The sequence is a single copy in the genome; (3) There are at least three or more discontinuous SNP differences in the marker sequence; (4) The marker sequence length is less than 250bp. The discrimination of the candidate MNP markers screened out was analyzed by using the genome resequencing data of 25 sunflower varieties. Finally, 552 MNP marker loci were screened out as shown in Table 1. On average, each chromosome contained 16.2 marker loci. The number of marker loci on each chromosome was distributed as follows: Figure 1 shown.

[0035] Embodiment 2

[0036] The present disclosure provides a kit for identifying sunflower varieties, the kit comprising the primer set provided in Example 1. Take 5 μL of each primer in the primer set and mix them in equal amounts to prepare a primer mix.

[0037] The kit also includes: Multiplex PCR premix.

[0038] Embodiment 3

[0039] The present disclosure provides an application of a primer set for identifying sunflower varieties, and the application includes: using the primer set provided in Example 1 to identify sunflower varieties.

[0040] Specifically, the application includes: using the primer set provided in Example 1 to identify the authenticity of sunflower varieties.

[0041] Specifically, the application includes: using the primer set provided in the above-mentioned embodiment 1 to identify substantially derived varieties of sunflower.

[0042] Specifically, the application includes: using the primer set provided in Example 1 to construct a DNA fingerprint database of sunflower varieties.

[0043] This example uses 31 sunflower varieties for identification, and the specific process is as follows:

[0044] The samples to be tested in this embodiment are 31 sunflower varieties, and the whole genome DNA of 31 sunflowers is extracted respectively to obtain the whole genome DNA of 31 samples to be tested. In this embodiment, the whole genome DNA is extracted by a kit method, and the kit is Tiangen DP320 (column membrane method) DNAsecure new plant genome DNA extraction kit.

[0045] The primer set provided in Example 1 of the present invention was used to perform a multiplex PCR (Polymerase Chain Reaction) amplification reaction on the genomic DNA of 31 samples to be tested. In this example, the amplification system is shown in Table 2.

[0046] Table 2 shows the amplification system

[0047] Components volume MNPPrimerPanelMix 4μL DNA 50~200ng GenoPlexs3×TMasterMix 10μL <![CDATA[H2O]]> <![CDATA[Supplement the amplification system to 30 μL with H2O]]>

[0048] In this example, the amplification procedure is shown in Table 3.

[0049] Table 3 shows the amplification procedure.

[0050]

[0051] After the multiplex PCR amplification reaction is completed, the multiplex PCR amplification reaction product is obtained, and the multiplex PCR amplification reaction product is purified by magnetic beads. The multiplex PCR amplification reaction product is purified using DNA purification magnetic beads of Novozyme Biotechnology Co., Ltd., and the excess primers are removed to obtain the purified multiplex PCR amplification reaction product. For specific operations, refer to the instructions.

[0052] Multiplex PCR amplification and sequencing library construction were performed according to the kit provided in Example 2. Multiplex amplification, second-generation high-throughput sequencing and data analysis were performed on the 31 sunflower DNA test samples, achieving the detection of 552×31=17112 marker sites in one experiment. The average sequencing coverage of each test sample reached more than 20× times, showing the high efficiency of MNP marker site detection.

[0053] The distribution of the number of detected sites of sunflower MNP markers is shown in the figure below. Figure 2 As shown in Table 4, the number of MNP marker sites detected was counted in the sequencing data of the 31 samples to be tested, as shown in Table 4.

[0054] Table 4 shows the results of MNP molecular markers for 31 sunflower varieties

[0055]

[0056]

[0057] As shown in Table 4, an average of 531 MNP marker sites can be detected for each variety, the variety with the lowest marker site detection rate is 93.48%, and the average marker site detection rate is 96.23%, which shows that the sunflower MNP marker site detection rate provided in this embodiment is high and stable. The stable marker site detection rate can prevent the genetic similarity coefficient from differing greatly in different experiments due to the large variation of the detected marker sites, which ultimately leads to the problem of large deviation in the variety identification conclusion due to site sampling.

[0058] (2) Accuracy analysis

[0059] The number of marker loci 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 genotyping of marker loci. Since the true value is unknown, the absolute accuracy of any method is uncalculated. In practice, the accuracy is either calculated by assuming the reference value as the true value, or the accuracy is estimated using precision. Since the reference value of the genotype of the marker loci of the variety is also unknown, the precision is calculated using three reproducibility experiments, thereby calculating the accuracy of typing, and the accuracy = 1-(1-precision) / 2. Among them, precision refers to the proportion of marker loci with consistent typing results in two experiments to all marker loci, and reproducibility experiments refer to two independent experiments performed by different personnel, different batches of reagents, and different laboratories. Reproducibility experiments simulate the identification of different batches, and a high reproducibility rate means that the identification results of different laboratories can be accurately compared with each other.

[0060] In order to test the accuracy of the primer set, a reproducibility experiment was conducted on 8 varieties. The reproducibility experiment was completed in two units, Jianghan University and Bayannur Agricultural and Animal Husbandry Research Institute. Two libraries were constructed for each variety, numbered as library 1 and library 2; the sequencing typing results of the libraries from different laboratories were compared pairwise. The comparison results are shown in Table 5.

[0061] Table 5 shows the accuracy analysis of sunflower MNP marker sites

[0062]

[0063]

[0064] As shown in Table 5, a total of 4261 MNP marker sites were compared in the reproducibility test, the number of reproducible marker sites was 4254 MNP marker sites, and the accuracy of marker site typing was 99.92%. In one identification, the number of marker sites with incorrect typing did not exceed 552×(1-99.92%)=0.44, and compared with the true value of the genetic similarity coefficient, the deviation did not exceed 0.01%. It can be seen that the variety identification conclusions of different laboratories or different batches in the same laboratory using the primer set provided in the embodiment of the present disclosure are highly consistent, so there is no need to use parallel experiments to reduce experimental errors, which means that accurate comparisons can be made between DNA fingerprint data from different sources, which provides great convenience for variety identification.

[0065] (3) Discrimination

[0066] The core task of variety identification is to use the detected marker sites to distinguish the tested varieties from the control varieties. There are two important factors that affect the core task of variety identification. First, the stronger the distinguishing ability of a single marker site, the stronger the ability of the primer set to distinguish varieties; second, the more markers used, the stronger the variety distinguishing ability of the primer set. In recent years, the problems of homogenization and imitation of sunflower varieties have become increasingly serious. In this embodiment, 700 samples of sunflower varieties were clustered according to phenotypic traits to analyze the sunflower varieties participating in the DUS test, and representative varieties with relatively large phenotypic differences and varieties with extremely similar phenotypes that are difficult to distinguish (see Table 4) were screened out for MNP molecular labeling.

[0067] The primer set provided in this example was used to authenticate 31 sunflower varieties. On average, 531 MNP markers were detected in each variety. The ratio of MNP markers that differed between each pair of sunflower varieties was analyzed, and a total of 465 pairs of comparison results were obtained. The ratio of MNP markers that differed between each pair of varieties is called the inter-variety distance, which directly shows the ability of the primer set to distinguish varieties. The results showed that the average inter-variety distance reached 70.39%, as shown in the following figure. Figure 3 As shown, this primer set has a strong ability to distinguish varieties and can distinguish 99.99% of any variety combination.

[0068] In the pairwise comparison of MNP markers of 31 sunflower varieties, there are 2 pairs of varieties with similar phenotypes, namely the well-known main cultivated varieties XRK0009 and variety XRK0017. This pair of varieties has 0 difference in 40 trait codes among the 43 basic traits of DUS test, and they are similar varieties. Through MNP molecular markers, there are 5 difference sites among the 552 MNP sites between variety XRK0009 and variety XRK0017 in phenotype and variety XRK0017, and the genetic similarity is 99.06%. According to the conclusion of GB / T38551-2020 "MNP Marking Method for Plant Variety Identification", they are similar varieties. The other pair is the main cultivated variety XRK0001 and variety XRK0018, which has 0 difference in 40 trait codes among the 43 basic traits of DUS test, 32 difference sites among the 552 MNP marker sites, and the genetic similarity is 93.9%. The MNP marker results of these two varieties are consistent with the screening results of similar varieties in the DUS test. In 2023, 7 pairs of modified and improved varieties were collected and the varieties were marked with MNP molecules. The specific results are shown in Table 6.

[0069] Table 6 shows the MNP results of substantially derived varieties

[0070]

[0071]

[0072] Varieties 6 and 7 in Table 6 are sister lines selected by conventional breeding methods, with genetic similarity between 80% and 85%. Varieties 1 to 3 are transgenic broomrape-resistant varieties bred in a company's laboratory. Field tests have shown that there are no significant phenotypic differences between the three pairs of varieties except for resistance. They meet the definition of substantially derived varieties. Varieties 5 (s23-4) is derived from the mutation of its control variety (s23-5), and the maternal parent of variety 4 (23SJ18) is the same as that of its control variety (23SJ19). The paternal parent of variety 4 (23SJ18) is derived from the mutation of the paternal parent of its control variety (23SJ19). It can be seen that this primer set can be used for rapid screening of similar varieties between plant varieties and identification of substantially derived varieties.

[0073] The identification of substantially derived varieties requires the calculation of genetic similarity coefficients, so the number of marker loci used must be sufficient to accurately calculate the genetic similarity coefficients. The International Seed Federation (ISF) requires the use of more than 300 SSR markers for the determination of substantially derived varieties. It has been proven that the polymorphism of MNP markers is higher than that of SSR markers in rice varieties, and the standard number used in the present invention reaches 552, so it is also sufficient for the determination of substantially derived varieties.

[0074] The above description is only an optional embodiment of the present disclosure and is not intended to limit the present disclosure. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present disclosure shall be included in the protection scope of the present disclosure.

Claims

1. A primer set for identifying sunflower varieties, characterized in that: The primer set includes: the 1st primer pair to the 552nd primer pair, each of the primer pairs includes a forward primer and a reverse primer, the forward primer of the 1st primer pair, the reverse primer of the 1st primer pair to the 552nd primer pair, and the reverse primer of the 552nd primer pair are respectively as shown in SEQ ID NO: 1 to SEQ ID NO: 1104 in the sequence listing.

2. A kit for identifying sunflower varieties, characterized in that: The kit comprises the primer set as claimed in claim 1.

3. An application of a primer set for identifying sunflower varieties, characterized in that: The application includes: using the primer set as claimed in claim 1 to identify sunflower varieties.

4. The use according to claim 3, characterized in that: The application includes: using the primer set as claimed in claim 1 to identify the authenticity of sunflower varieties.

5. The use according to claim 3, characterized in that: The application includes: using the primer set as claimed in claim 1 to identify substantially derived varieties of sunflower.

6. The use according to claim 3, characterized in that: The application comprises: using the primer set as claimed in claim 1 to construct a DNA fingerprint database of sunflower varieties.

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