Whole genome SSR primer group-based radix paeoniae rubra population screening method and application

Abstract

The invention discloses a radix paeoniae rubra population screening method based on a whole genome SSR primer group and application, and the method comprises the following steps: sample collection and DNA extraction: collecting a fresh radix paeoniae rubra fibrous root sample, cleaning, carrying out dry ice preservation, extracting genome DNA, and detecting DNA concentration and quality through a micro spectrophotometer and 1% agarose gel electrophoresis; sSR primer design: based on red peony root whole genome data, scanning SSR loci with 2-6 base repetition by using software, screening loci with repetition times greater than or equal to 8 times, and designing SSR primers with primer length, amplification product length and annealing temperature, in the invention, 23 pairs of SSR primers obtained by two rounds of screening are designed for core loci with 2-6 base repetition times greater than or equal to 8 times; the average value of polymorphic information indexes reaches 0.725, the average number of effective alleles is 3.349, which is obviously higher than that of conventional SSR primers, the genetic variation of the radix paeoniae rubra genome can be accurately detected, the strip is clear, the amplification is stable, and the problems of non-specific amplification and towing are avoided.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and in particular to a method and application of red peony population screening based on a whole genome SSR primer set Background Art Red peony root has the effects of clearing heat, cooling blood, dispersing blood stasis and relieving pain. Its main source is the root of wild populations of red peony root, a plant of the Ranunculaceae family. According to the resource survey of red peony root, its wild resources are gradually decreasing. After half a century of indiscriminate mining since the late 1990s, wild red peony root has been depleted year by year and is already endangered.

[0002] Core germplasm usually refers to a collection of samples that use the least genetic redundancy to represent the greatest genetic diversity. Core germplasm screening is an important way to effectively utilize and protect genetic resource variation. It can remove duplications in original germplasm resources to the greatest extent possible and represent all or most of the genetic diversity of original germplasm resources with the least germplasm materials. The construction of core germplasm banks has greatly facilitated the efficient use of germplasm resources.

[0003] From the perspective of existing technologies, existing methods mostly rely on empirical designs or local genomic regions, and are not based on whole-genome data scanning, resulting in insufficient mining of key SSR sites with 2-6 base repeats and ≥8 repeats, especially incomplete coverage of compound repeat units and insertion repeat units; the screening process mostly uses single-round agarose gel electrophoresis, which is easy to miss primers that are unstable in amplification in some samples, and lacks precise polymorphism verification of capillary electrophoresis combined with genetic analysis software. It is difficult to ensure primer stability and specificity, and problems such as dragging, blurred or missing bands often occur; the existing primers have a generally low polymorphism information index and a small number of effective alleles, resulting in insufficient sensitivity in detecting observed heterozygosity and expected heterozygosity (He), making it difficult to accurately assess genetic variation within populations, lacking support from whole-genome SSR sites, and unclear subpopulation divisions, making it impossible to accurately reveal the association between geographic differentiation and genetic components. Summary of the invention

[0004] In order to overcome the above-mentioned defects of the prior art, the embodiments of the present invention provide a method and application for screening red peony populations based on a whole-genome SSR primer set, which solves the problems of lack of systematicity and precision in the development of SSR primers, technical limitations in genetic diversity assessment and population structure analysis, and insufficient efficiency in germplasm resource screening and protection. To achieve the above object, the present invention provides the following technical solutions: The method for screening red peony population based on the whole genome SSR primer set includes the following steps: S1: Sample collection and DNA extraction: Fresh root samples of red peony root were collected, and genomic DNA was extracted after washing and storage on dry ice. The DNA concentration and quality were tested by micro-spectrophotometer and 1% agarose gel electrophoresis. S2: SSR primer design: Based on the whole genome data of Paeonia lactiflora, the software was used to scan the SSR sites with 2-6 base repeats, and the sites with ≥8 repeats were screened to design SSR primers with primer length of 18-25 bp, amplification product length of 70-402 bp, and annealing temperature of 48.5-55.5℃; S3: Primer screening: Using red peony samples from different populations as templates, two rounds of PCR amplification screening were performed. In the first round, agarose gel electrophoresis was used to screen primers with clear bands. In the second round, capillary electrophoresis was performed using a fully automatic nucleic acid protein analysis system. The polymorphism and stability of the primers were verified by software, and 23 pairs of SSR primers were obtained. S4: Genetic diversity analysis: The genomic DNA of red peony samples was amplified by PCR using the 23 pairs of primers screened, and the effective allele number (Ne), Shannon information index (I), observed heterozygosity (Ho), expected heterozygosity (He) and polymorphism information index (PIC) were calculated.

[0005] Preferably, the sample storage method in step S1 is to transport the sample on dry ice and then place it in a -80°C ultra-low temperature refrigerator, the DNA extraction method is a kit method, and the DNA concentration and quality detection is completed by a micro-spectrophotometer and 1% agarose gel electrophoresis.

[0006] Preferably, the PCR reaction system in step S3 is 10 μL, including 5 μL 2×TaqMix, 0.5 μL 10 μM upstream primer, 0.5 μL 10 μM downstream primer, 1 μL DNA template and 3 μL ddH 2 O, the DNA template concentration is 20-50 ng / μL.

[0007] Preferably, the PCR reaction procedure is: pre-denaturation at 98°C for 5 min; denaturation at 98°C for 30 s, annealing at the corresponding primer annealing temperature for 30 s, extension at 72°C for 30 s, and 35 cycles; final extension at 72°C for 7 min, and storage at 4°C. The annealing temperature is set at 48.5-55.5°C according to the primer sequence.

[0008] Preferably, an SSR primer set for screening red peony populations comprises 23 pairs of SSR primers, wherein the primers target SSR sites with 2 to 6 base repeats in the red peony genome, with the number of repeats ≥ 8 times, the primer length is 18 to 25 bp, the amplification product length is 70 to 402 bp, and the annealing temperature is 48.5 to 55.5°C.

[0009] Preferably, the repeating units of the primers include the following types: Single repeating unit: The same sequence consisting of 2-6 bases is repeated at least 8 times, for example, (CT) is repeated 11 times and (CA) is repeated 16 times; Composite repeating unit: composed of two or more different 2-6 base repeating sequences in series, each repeating sequence is repeated no less than 6 times, and the total number of repeats is no less than 8 times, for example, (GT) is repeated 22 times, (TG) is repeated 16 times, and (AG) is repeated 17 times in series; Insertion repeat unit: A non-repetitive nucleotide fragment with a length of ≤10bp is inserted into a 2-6 base repeat sequence, where the repeat sequence is repeated no less than 8 times, for example, (TG) is repeated 19 times and then the ATC sequence is inserted.

[0010] Preferably, the polymorphism information index (PIC) of the 23 SSR loci in step S4 is 0.452-0.924, with an average value of 0.725, the average effective allele number (Ne) is 3.349, and the average Shannon information index (I) is 1.172.

[0011] Preferably, in step S3, the first round of screening excludes primers with dragging, disorder or missing amplification bands, and the second round of screening determines the polymorphism and stability of the primers by capillary electrophoresis and gene analysis software. The 23 pairs of primers finally obtained can stably amplify clear bands.

[0012] Preferably, the level of genetic diversity of the population is assessed by detecting the number of alleles, effective number of alleles, heterozygosity and polymorphism information index of the SSR loci in the target plant population.

[0013] Preferably, the genetic distance and genetic identity are calculated based on the primer amplification results, and the genetic groups of the plant population are divided using cluster analysis software to determine the number of subgroups and the proportion of genetic components.

[0014] The technical effects and advantages of the red peony population screening method and application based on the whole genome SSR primer set of the present invention are as follows: 1. This invention, through two rounds of screening, obtained 23 pairs of SSR primers, which were designed for core sites with 2-6 base repeats and ≥8 repeats. The average polymorphic information index reached 0.725, and the average number of effective alleles was 3.349, which was significantly higher than that of conventional SSR primers. It can accurately detect the genetic variation of the red peony genome, with clear bands and stable amplification, avoiding nonspecific amplification and dragging problems. The primer set contains single repeat units, composite repeat units and insertion repeat units, which comprehensively cover the complex SSR sites in the red peony genome, and is suitable for red peony populations with different genetic backgrounds, thereby improving the versatility and detection sensitivity of the primers.

[0015] 2. This invention uses standardized procedures to improve detection accuracy: standardized operations of sample collection, DNA extraction, and PCR reaction systems, combined with the precise readings of Qsep100 capillary electrophoresis and software, ensure the accuracy of allele data, reduce manual interpretation errors, and provide a reliable data basis for genetic diversity analysis. The quality of primers is improved through a two-round screening strategy: the first round excludes primers with poor amplification effects, and the second round verifies primers with missing bands through capillary electrophoresis. The primers finally obtained can stably amplify the entire sample, solving the problem of high-quality primers being easily missed in traditional single-round screening. The primer efficiency screened out 23 pairs (23%) from the initial 100 pairs of designed primers, significantly improving the efficiency of primer screening.

[0016] 3. This invention accurately evaluates genetic diversity: by using 23 pairs of primers to detect wild red peony populations, the observed heterozygosity (Ho) averaged 0.861, the expected heterozygosity (He) averaged 0.588, and the Shannon information index (I) averaged 1.172, indicating that the red peony population is rich in genetic diversity, providing a quantitative basis for the protection of germplasm resources, and being able to identify populations with high genetic diversity and give priority protection.

[0017] 4. This invention clarifies the genetic structure and clustering relationship of the population: UPGMA clustering and Structure analysis divides 12 populations into 2 large groups or 12 subgroups, revealing the association between geographical differentiation and genetic components, providing a basis for population division for red peony genetic breeding, and assisting in parent selection and genetic distance assessment in hybrid breeding.

[0018] 5. This invention has high efficiency in constructing core germplasm: 104 core germplasms were constructed with a sampling ratio of 40% using the allele maximization simulated annealing algorithm, which retained 70.89% of the number of alleles (Na) and 82.43% of the effective number of alleles (Ne) of the original germplasm, and had no significant difference in genetic parameters with the original germplasm (P>0.05), greatly reducing the cost of germplasm preservation while fully preserving genetic diversity, facilitating subsequent resource utilization and genetic research.

[0019] 6. This invention has repeatability of experimental methods: the PCR reaction program, software parameters and data processing flow are disclosed in detail, so that technicians in this field can directly reproduce the experiment, accelerate the progress of related research, and promote technological progress in the field of plant molecular breeding and genetic protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The agarose gel electrophoresis images of primers SSR97, SSR98 and SSR61, SSR62 for 12 red peony root samples respectively; Figure 2 The capillary electrophoresis results of primer SSR98 for four red peony root samples (AD) were read respectively; Figure 3 It is the UPGMA cluster analysis diagram based on Nei's genetic distance; Figure 4 To determine the optimal number of populations based on the Structure software, a is the change in Lnp (D) value, and b is the change in DeltaK value; Figure 5 The analysis results of Structure software when K=6; Figure 6 This is the UPGMA cluster analysis diagram among different populations of red peony root; Figure 7 This is the principal coordinate distribution map of the core germplasm and original germplasm of wild red peony root. DETAILED DESCRIPTION

[0021] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device that includes a series of elements includes not only those elements, but also other elements that are not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of more restrictions, the elements defined by the sentence "include..." do not exclude the existence of other identical elements in the process, method, article or device that includes the elements. The present invention discloses a method and application of red peony population screening based on a whole genome SSR primer set, including the following contents: the research method used in the following embodiments includes: using ROX, NED, FAM, HEX four-fluorescence capillary electrophoresis technology to detect amplified bands, and using GeneMarkerv2.2 software to read data. The number of alleles (Number of alleles, Na), effective number of alleles (Effective number of alleles, Ne), observed heterozygosity (Observed heterozygosity, Ho), expected heterozygosity (Expected heterozygosity, He) and Shannon's diversity index (Shannon'snformation index, I) of each pair of primers are calculated using the GenAlEx6.503 plug-in; the polymorphic information content (Polymorphic information content, PIC) of primers is calculated using the PowerMarker software; the genetic similarity coefficient and genetic distance between wild red peony samples are obtained according to Nei's method, and a cluster diagram is constructed in a UPGMA manner using the MEGA11 software based on the genetic distance; and the population structure is analyzed using the Structure software. The above software all adopted default parameter settings.

[0022] Example 1 This embodiment provides a method and application for screening red peony population based on a whole genome SSR primer set, which is used for sample collection and DNA extraction. The specific implementation content includes: A total of 256 test materials were taken from the wild red peony germplasm resource nursery in Bin County, Heilongjiang Province. Individual plants with good growth and no pests and diseases were selected, and fresh fibrous roots were taken; they were washed with distilled water, wiped dry, placed in dry ice and brought back to the laboratory, and stored in a -80℃ ultra-low temperature refrigerator for testing. The sample DNA was extracted using the kit method, and the DNA concentration and quality were detected using a microspectrophotometer and 1% agarose gel electrophoresis, respectively.

[0023] Table 1: Collection information of wild red peony germplasm resources

[0024] Example 2 This embodiment provides a method and application for screening red peony population based on a whole genome SSR primer set for primer design, and the specific implementation content includes: This embodiment uses Primer3.0 software to scan the whole genome sequence of wild red peony root to design primers, obtain 2-6 base repeat SSR sites, the number of repeats is not less than 8 times, set the primer length to 18-25bp, the amplification product length to 100-500bp, and the annealing temperature to 50-55°C. A total of 100 pairs of primers (SSR1-SSR100) were designed and synthesized. They were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0025] Twelve wild red peony samples from different populations (1-9, 2-5, 3-2, 4-19, 5-20, 6-2, 7-15, 8-10, 9-11, 10-1, 11-1, 12-1, as shown in Table 1) were selected to screen SSR primers with clear bands and stable amplification. A 10 μL PCR reaction system was used in the study, consisting of 5 μL 2× TaqMix (Beijing Qingke Biotechnology Co., Ltd.), 0.5 μL upstream primer (10 μM), 0.5 μL downstream primer (10 μM), 1 μL DNA template, 3 μL ddHO 2 O. Composition, PCR reaction procedures are shown in Table 2. 1% agarose gel electrophoresis was used to detect the amplified bands of PCR products. The electrophoresis was set to a constant voltage of 120v to screen primers with good amplification and clear bands. Capillary electrophoresis analysis was performed using the Qsep100 fully automatic nucleic acid protein analysis system, and primer polymorphism was analyzed by GeneMarkerv2.2 software reading analysis.

[0026] Table 2: PCR reaction program

[0027]

[0028] The effectiveness of 123 pairs of SSR primers was verified by using 12 wild red peony root samples. The agarose gel electrophoresis diagram of some primers is shown in Figure 1 As shown, lanes 1-12 are test samples, and from left to right are the gel electrophoresis results of primers SSR97, SSR98, SSR61, and SSR62 for 12 test samples. The results of the first round of primer screening showed that 65 pairs of primers amplified clear bands, but some samples failed to amplify; the remaining 58 pairs of primers had poor amplification effects, with problems such as dragging or messy bands, and these primers were discarded in the first round of screening. In the second round of primer screening, the 65 pairs of primers with missing bands were re-amplified, and capillary electrophoresis was used to detect that a total of 23 pairs of primers were able to amplify clear bands. According to the above experiments, after two rounds of primer amplification screening, a total of 23 pairs of SSR primers with good amplification effects and stability were obtained, and the primer sequence information is shown in Table 3 below.

[0029] Table 3: Sequence information of 23 pairs of SSR primers

[0030] Example 3 This embodiment provides a method and application for screening red peony population based on a whole genome SSR primer set for genetic diversity analysis of wild red peony. The specific implementation content includes: The 23 pairs of primers obtained by screening in Example 2 were used to perform PCR amplification on the genomic DNA of 256 wild red peony germplasm materials (the PCR reaction system and PCR reaction procedure are as shown in Example 2), and the PCR amplification results were analyzed, and the results are shown in Table 4 below. As shown in Table 4, the average value of the effective allele number (Ne) is 3.349, the average value of the Shannon information index (I) is 1.172, the average value of the observed heterozygosity (Ho) is 0.861, and the average value of the expected heterozygosity (He) is 0.588. The polymorphic information index (PIC) of the 23 loci is 0.452-0.924, with an average value of 0.725, which is higher than 0.500. It shows that the 23 pairs of SSR primers have high polymorphism and the genetic diversity of wild red peony resources is rich.

[0031] Table 4: Genetic diversity analysis of 23 SSR loci

[0032] This embodiment also analyzes the genetic diversity of wild red peony germplasm materials of 12 populations, and the results are shown in Table 5 below. The Ne of the 12 populations is 2.289-3.794, with an average of 3.175; I is 0.837-1.350, with an average of 1.146; Ho is 0.652-0.957, with an average of 0.861; He is 0.449-0.662, with an average of 0.588. The data show that there are differences in the genetic diversity of the 12 populations. Based on He and I to measure the level of population genetic diversity, the genetic diversity of the P4 population is the highest, with I and He being 1.299 and 0.662 respectively; the genetic diversity of the P3 population is the lowest, with I and He being 0.837 and 0.449 respectively. It shows that the genetic diversity of the 12 wild red peony populations is rich.

[0033] Table 5: Genetic diversity analysis among wild red peony populations

[0034] Example 4 This embodiment provides a method and application of red peony population screening based on a whole genome SSR primer set for UPGMA cluster analysis within wild red peony species. The specific implementation content includes: The 23 pairs of primers obtained by screening in Example 2 were used to perform PCR amplification on the genomic DNA of 256 wild red peony germplasm materials (the PCR reaction system and PCR reaction procedure are as shown in Example 2), and the PCR amplification results were analyzed. The genetic distance between individuals was calculated according to Nei's method, as shown in Table 6, and UPGMA cluster analysis was performed using MEGA11 software. Figure 3 It can be clearly seen that wild red peony root is divided into two groups. The five populations of P1, P2, P3, P4 and P5 are clustered into one group, and the populations of P6, P7, P8, P9, P10, P11 and P12 are clustered into another group.

[0035] Table 6: Nei's genetic identity and genetic distance of wild red peony population

[0036] Note: The lower left part is the genetic distance, and the upper right part is the genetic identity.

[0037] Example 5 This embodiment provides a method and application for screening red peony population based on a whole genome SSR primer set for analyzing the intraspecific population structure of wild red peony. The specific implementation content includes: The 23 pairs of primers obtained by screening in Example 2 were used to perform PCR amplification on the genomic DNA of 256 wild red peony germplasm materials (the PCR reaction system and PCR reaction procedure are as shown in Example 2), and the PCR amplification results were analyzed. The population structure of wild red peony germplasm resources was further analyzed using Structure2.3.4 software, and the subpopulation number K was preset to 1 to 13, and repeated 10 times. Figure 4 As shown in a, the Lnp (D) value increases gradually with the increase of K value, first fast and then slow, and the number of wild red peony root subpopulations cannot be clearly determined; when K = 12, DeltaK is the maximum value, indicating that it is most appropriate to divide wild red peony root germplasm resources into 12 subpopulations, such as Figure 4 Medium b and Figure 5 shown.

[0038] When the wild red peony germplasm resources are divided into six subgroups, the first group is composed of the germplasm resources of Arxan Guomen (P6) in Arxan City, Amur Wild Red Peony Mountain (P8) in Mohe City, Heilongjiang Province, and Labdalin (P10) in Hulunbuir City; the second group is composed of the germplasm resources of Ulagai Wild Red Peony Valley (P7) in Inner Mongolia Autonomous Region and Khan Mountain (P12) in Holingol City; the third group comes from Chabanhe, Shibazhan, Huma County, Heilongjiang Province (P11) and Beihong Village, Mohe (P9); the fourth group comes from Dajuzi Forest Farm (P2) and Honghuaerjinuogannuoer Forest Farm (P5) in Chifeng City, Inner Mongolia Autonomous Region; the fifth group comes from Tiegongpaozi Village (P3) in Duolun County and Baiyinaobao (P4) in Chifeng City; and the sixth group comes from Yakeshi City (P1) in Inner Mongolia Autonomous Region.

[0039] Depend on Figure 4 It can be seen that most germplasms have mixed genetic components and complex genetic backgrounds, while a few germplasms have a relatively simple genetic background. The Gd of the 12 populations ranged from 0.1670 to 0.4801, and the Gi ranged from 0.2974 to 0.7704. Among them, the Gd between P3 and P11 populations was the largest (0.4801), and the Gd between P5 and P6 populations was the smallest (0.0169). The Gi between P8 and P10 populations was the largest (0.7704), and the Gi between P4 and P10 populations was the smallest (0.2974).

[0040] From the above results, it can be seen that the present invention has developed and screened 23 pairs of SSR primers with clear bands, high polymorphism and good repeatability based on the whole genome sequence of wild red peony. The number of alleles (Na), effective number of alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He), Shannon information index and polymorphic information content of 256 wild red peony germplasm materials were amplified by PCR using 23 pairs of high-quality SSR primers, and the average values ​​were 4.50, 3.18, 0.86, 0.59, 1.15 and 0.73, respectively. The genetic diversity of wild red peony germplasm resources is richer, with obvious geographical differentiation, and the main genetic variation comes from within the population.

[0041] Example 6 This embodiment provides a method and application for screening red peony population based on a whole genome SSR primer set for the construction of wild red peony core germplasm. The specific implementation content includes: PowerMarker v3.25 software was used to set four different sampling ratios (10%, 20%, 30%, and 40%), and the simulated annealing algorithm for allele maximization was used. (SANA) and simulated annealing algorithm based on maximizing allelic richness The core germplasm of wild red peony root was constructed by simulated annealing algorithm based on the maximizing genetic diversity (SAGD). After the core germplasm was screened, the differences in genetic diversity parameters between the core germplasm and the original germplasm were analyzed using t-test of SPSS software, and principal coordinate analysis (PCoA) was performed using GenAI Ex 6.5 software to evaluate the representativeness and heterogeneity of the core germplasm. The results showed that when the sampling ratio was 40%, the retention rate of Ne and I values ​​of the SANA method was the highest. Considering the genetic parameters and sampling ratio, 104 samples (40%) were extracted from 256 wild red peony root germplasms using the SANA sampling strategy as the core germplasm, and the retention rates of Na, Ne, I, Ho and He were 70.89%, 82.43%, 82.15%, 94.15% and 88.72%, respectively. The results of t-test showed that there were no significant differences in genetic diversity parameters between the constructed wild red peony root core germplasm and the original germplasm, and between the core germplasm and the reserved germplasm.

[0042] The principal coordinate analysis was performed on the constructed wild red peony root core germplasm and the retained backup germplasm ( Figure 7 ), verify its representativeness. Figure 7 It can be found that the core germplasm is distributed more dispersedly in the original population, with basically no overlap, and has similar geometric characteristics to the original population, indicating that the primary core germplasm can basically represent the genetic diversity of the original population.

[0043] The following is a comparison of the genetic diversity parameters of the core germplasm of wild red peony root under different construction strategies, see Table 7 for details; the following is a comparison of the genetic diversity of the core germplasm, reserved germplasm and original germplasm of wild red peony root, see Table 8 for details.

[0044] Table 7: Comparison of genetic diversity parameters of core germplasm of wild red peony root under different construction strategies

[0045] Table 8: Comparison of genetic diversity among core germplasm, reserved germplasm and original germplasm of wild red peony root

[0046] Note: t1 and t2 represent the t-test values ​​of each genetic parameter between core germplasm and original germplasm, and between core germplasm and reserved germplasm. *The significance level is 0.05.

[0047] It can be seen from the above Examples 1-6 that the red peony population screening method and application based on the whole genome SSR primer set provided by the present invention, through two rounds of screening (agarose gel electrophoresis primary screening + capillary electrophoresis verification), 23 pairs of SSR primers are obtained, designed for core sites with 2-6 base repeats and ≥8 repetitions, and the average polymorphism information index (PIC) is 0.725 (higher than 0.500), and the average effective allele number (Ne) is 3.349, which is significantly higher than conventional SSR primers. It can accurately detect the genetic variation of the red peony genome, with clear bands and stable amplification, avoiding nonspecific amplification and dragging problems.

[0048] The primer set contains single repeat units (such as (CT) 11, (CA) 16), compound repeat units (such as (GT) 22 (TG) 16 (AG) 17) and insertion repeat units (such as (TG) 19ATC), which comprehensively cover the complex SSR sites in the red peony genome and are suitable for red peony populations with different genetic backgrounds, thereby improving the versatility and detection sensitivity of the primers.

[0049] Standardized processes improve detection accuracy: standardized operations of sample collection, DNA extraction and PCR reaction system (10μL system contains 5μL2×TaqMix, 0.5μL upstream and downstream primers, etc.), combined with the precise readings of Qsep100 capillary electrophoresis and GeneMarkerv2.2 software, ensure the accuracy of allele data, reduce manual interpretation errors, and provide a reliable data basis for genetic diversity analysis.

[0050] The two-round screening strategy improved primer quality: the first round eliminated primers with poor amplification effects (58 pairs were eliminated due to dragging or disorder), and the second round verified primers with missing bands through capillary electrophoresis (23 pairs out of 65 pairs passed). The primers finally obtained were all able to stably amplify the entire sample, solving the problem of traditional single-round screening that high-quality primers are easily missed. The primer efficiency was 23 pairs (23%) screened out from the initial 100 pairs of designed primers, significantly improving the efficiency of primer screening.

[0051] Accurate assessment of genetic diversity: 23 pairs of primers were used to detect wild red peony populations, with an average observed heterozygosity (Ho) of 0.861, an average expected heterozygosity (He) of 0.588, and an average Shannon information index (I) of 1.172, indicating that the red peony population is rich in genetic diversity, providing a quantitative basis for the protection of germplasm resources, and identifying high genetic diversity populations (such as the P4 population He=0.662) and giving priority to their protection.

[0052] Clarify the population genetic structure and clustering relationship: UPGMA clustering and Structure analysis divided the 12 populations into 2 large groups or 12 subgroups, revealing the association between geographical differentiation and genetic components (such as P1-P5 clustered into one category, P6-P12 clustered into another category), providing a basis for population division for red peony genetic breeding, and assisting in parent selection and genetic distance assessment in hybrid breeding (such as the genetic distance between P3 and P11 populations is the largest, which is conducive to the cultivation of hybrid advantages).

[0053] High efficiency of core germplasm construction: 104 core germplasms were constructed with a sampling ratio of 40% using the allele maximization simulated annealing algorithm (SANA), which retained 70.89% of the number of alleles (Na) and 82.43% of the effective number of alleles (Ne) of the original germplasm, and had no significant difference in genetic parameters with the original germplasm (P>0.05), greatly reducing the cost of germplasm preservation (from 256 to 104), while fully preserving genetic diversity to facilitate subsequent resource utilization and genetic research.

[0054] Potential applications across plant species: Primer design strategies (2-6 base repeats, number of repeats ≥ 8 times) and screening methods can be extended to closely related species of the genus Paeonia (such as tree peony and paeonia lactiflora). The superordinate expressions of "plant genetic diversity assessment" and "plant population structure analysis" in the claims make it suitable for research on a variety of plant germplasm resources, broadening the application field of the technology.

[0055] Repeatability of experimental methods: The PCR reaction program (98℃ pre-denaturation for 5 minutes, 35 cycles, etc.), software parameters (Primer3.0, Structure2.3.4 default parameters) and data processing flow are disclosed in detail, so that technicians in this field can directly reproduce the experiment, accelerate the progress of related research, and promote technological progress in the field of plant molecular breeding and genetic protection.

[0056] The above embodiments may be implemented in whole or in part through software, hardware, firmware or any other combination. When implemented by software, the above embodiments may be implemented in whole or in part in the form of a computer program product.

[0057] A person of ordinary skill in the art will appreciate that the modules and algorithm steps of each example described in conjunction with the embodiments disclosed herein can be implemented in electronic hardware, or a combination of computer software and electronic hardware. Whether these functions are performed in hardware or software depends on the specific application and design constraints of the technical solution. Professional and technical personnel can use different methods to implement the described functions for each specific application, but such implementation should not be considered to be beyond the scope of this application.

[0058] In addition, each functional module in each embodiment of the present application may be integrated into one processing module, or each module may exist physically separately, or two or more modules may be integrated into one module.

[0059] The above description is only a specific implementation manner of the present application, but the protection scope of the present application is not limited thereto. Any technician familiar with the technical field can easily think of changes or substitutions within the technical scope disclosed in the present application, which should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

[0060] Finally: 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 principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for screening red peony population based on a whole genome SSR primer set, characterized in that: The following steps are involved: S1: Sample collection and DNA extraction: Fresh root samples of red peony root were collected, and genomic DNA was extracted after washing and storage on dry ice. The DNA concentration and quality were tested by micro-spectrophotometer and 1% agarose gel electrophoresis. S2: SSR primer design: Based on the whole genome data of Paeonia lactiflora, the software was used to scan the SSR sites with 2-6 base repeats, and the sites with ≥8 repeats were screened to design SSR primers with primer length of 18-25 bp, amplification product length of 70-402 bp, and annealing temperature of 48.5-55.5℃; S3: Primer screening: Using red peony samples from different populations as templates, two rounds of PCR amplification screening were performed. In the first round, agarose gel electrophoresis was used to screen primers with clear bands. In the second round, capillary electrophoresis was performed using a fully automatic nucleic acid protein analysis system. The polymorphism and stability of the primers were verified by software, and 23 pairs of SSR primers were obtained. S4: Genetic diversity analysis: The genomic DNA of red peony samples was amplified by PCR using the 23 pairs of primers screened, and the effective allele number (Ne), Shannon information index (I), observed heterozygosity (Ho), expected heterozygosity (He) and polymorphism information index (PIC) were calculated.

2. The method for screening red peony population based on a whole genome SSR primer set as claimed in claim 1, characterized in that: The sample storage method in step S1 is to transport the sample on dry ice and then place it in a -80°C ultra-low temperature refrigerator. The DNA extraction method is a kit method. The DNA concentration and quality detection are completed by a micro-spectrophotometer and 1% agarose gel electrophoresis.

3. The method for screening red peony population based on the whole genome SSR primer set according to claim 1, characterized in that: The PCR reaction system in step S3 is 10 μL, including 5 μL 2×TaqMix, 0.5 μL 10 μM upstream primer, 0.5 μL 10 μM downstream primer, 1 μL DNA template and 3 μL ddH2O, and the concentration of the DNA template is 20-50 ng / μL.

4. The method for screening red peony population based on the whole genome SSR primer set according to claim 1, characterized in that: The PCR reaction procedure is: pre-denaturation at 98°C for 5 min; denaturation at 98°C for 30 s, annealing at the corresponding primer annealing temperature for 30 s, extension at 72°C for 30 s, and 35 cycles; final extension at 72°C for 7 min, and storage at 4°C. The annealing temperature is set at 48.5-55.5°C according to the primer sequence.

5. An SSR primer set for screening red peony population, characterized in that: The invention comprises 23 pairs of SSR primers, wherein the primers are directed to the SSR sites with 2 to 6 base repeats in the red peony genome, the number of repeats is ≥ 8 times, the primer length is 18 to 25 bp, the amplification product length is 70 to 402 bp, and the annealing temperature is 48.5 to 55.5°C.

6. The method for screening red peony population based on a whole genome SSR primer set as claimed in claim 5, characterized in that: The repeating units of the primers include the following types: Single repeating unit: The same sequence consisting of 2-6 bases is repeated at least 8 times, for example, (CT) is repeated 11 times and (CA) is repeated 16 times; Composite repeating unit: composed of two or more different 2-6 base repeating sequences in series, each repeating sequence is repeated no less than 6 times, and the total number of repeats is no less than 8 times, for example, (GT) is repeated 22 times, (TG) is repeated 16 times, and (AG) is repeated 17 times in series; Insertion repeat unit: A non-repetitive nucleotide fragment with a length of ≤10bp is inserted into a 2-6 base repeat sequence, where the repeat sequence is repeated no less than 8 times, for example, (TG) is repeated 19 times and then the ATC sequence is inserted.

7. The method for screening red peony population based on the whole genome SSR primer set according to claim 1, characterized in that: The polymorphism information index (PIC) of the 23 SSR loci in step S4 was 0.452-0.924, with an average value of 0.725, an average value of the effective number of alleles (Ne) of 3.349, and an average value of the Shannon information index (I) of 1.

172.

8. The method for screening red peony population based on whole genome SSR primer set according to claim 1, characterized in that: In step S3, the first round of screening excluded primers with dragging, disordered or missing amplified bands. The second round of screening determined the polymorphism and stability of the primers by capillary electrophoresis and gene analysis software. The 23 pairs of primers finally obtained were all able to stably amplify clear bands.

9. The use of the SSR primer set according to claim 5 in plant genetic diversity assessment, characterized in that: The level of genetic diversity of the population was evaluated by detecting the number of alleles, effective number of alleles, heterozygosity and polymorphism information index of SSR loci in the target plant population.

10. Use of the SSR primer set according to claim 6 in analyzing the genetic structure of a plant population, characterized in that: Genetic distance and genetic consistency were calculated based on the primer amplification results, and cluster analysis software was used to divide the genetic groups of the plant population and determine the number of subpopulations and the proportion of genetic components.

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