Sesbania SSR primer combination developed based on shallow genome sequence and application of sesbania SSR primer combination

Abstract

The invention discloses a sesbania SSR primer combination developed based on a shallow genome sequence and application thereof, and belongs to the technical field of molecular markers. The invention develops an SSR primer combination based on a shallow genome sequence of sesbania, and the SSR primer combination comprises 23 pairs of SSR primer pairs. The SSR primer pair developed by the invention has the advantages of high polymorphism, rich polymorphic information content, stable amplification result, high resolution, accuracy and the like, can quickly and accurately judge the source and variety of sesbania, is beneficial to the research of germplasm resource identification, germplasm resource genetic diversity analysis, genetic structure analysis and the like of sesbania, and has wide application prospects. The method has important significance in promoting conservation and utilization of sesbania germplasm resources and promoting industrial development.

Description

Technical Field

[0001] This invention relates to the field of molecular marker technology, and in particular to a sesquiter of guar SSR primers developed based on shallow genome sequences and their applications. Background Technology

[0002] Sesbania spp., a key leguminous green manure and forage crop, plays a vital role in agricultural production and ecological restoration. Its nitrogen-fixing properties enhance soil fertility, improve soil structure, and strengthen soil water and fertilizer retention capacity, thereby promoting sustainable agricultural development. Furthermore, sesbania spp. exhibits good adaptability to degraded land and plays a positive role in vegetation restoration and ecosystem reconstruction. However, against the backdrop of global climate change and shifts in agricultural production patterns, sesbania spp.'s genetic resources are facing severe challenges, including reduced genetic diversity and weakened resilience. Therefore, conducting systematic research on genetic diversity is particularly urgent.

[0003] Currently, plant genetic diversity research mainly relies on molecular marker technology. Among them, simple repeat sequence (SSR) markers are widely used in species genetic structure analysis, germplasm identification, and molecular breeding due to their advantages such as high polymorphism, co-dominance, wide distribution, and convenient detection. In recent years, scholars at home and abroad have used SSR markers to conduct in-depth research on the genetic diversity of various plants (such as Clematis chinensis, Cycas hainanensis, Daffodils, Sapindus mukorossi, and Marigold), providing important evidence for resource conservation and breeding. For example, Yang Le et al. used SSR markers to analyze Daffodil varieties, screened 17 pairs of highly polymorphic primers, and detected 85.14% of polymorphic loci; Gao Lei et al. used SSR markers in their study of Tilia amurensis and found that each locus contained an average of 12.6 alleles, confirming the reliability of the technology. In addition, the development of EST-SSR markers has further expanded the application scope of SSR. For example, Lü Xueqing developed EST-SSR primers for Quercus de Texas based on transcriptome data and successfully used them for genetic diversity assessment.

[0004] However, research on the development and application of SSR markers for sesbania is still relatively scarce. Traditional SSR marker development methods (such as genome library construction) are costly and time-consuming, while SSR development strategies based on shallow genome sequencing can significantly improve efficiency and reduce costs. In recent years, Gao Huixia et al. developed SSR markers for Astragalus membranaceus based on transcriptome data, and Guo Junwei discovered 10,182 SSR loci in his research on marigolds, both demonstrating the feasibility of combining high-throughput sequencing with bioinformatics analysis. In addition, Yang Jiawei et al. found in their association analysis between potato phenotype and genetic diversity that SSR markers can effectively reveal the genetic basis of phenotypic variation, providing precise guidance for breeding.

[0005] Therefore, developing a set of highly polymorphic SSR primers suitable for sesquiterpene and applying them to its genetic diversity analysis and germplasm resource identification is of great theoretical and practical significance. Summary of the Invention

[0006] The purpose of this invention is to provide a sesquiterpene SSR primer pair developed based on shallow genome sequences and its application, thereby addressing the problems existing in the prior art. The SSR primer pair developed in this invention has advantages such as high polymorphism, rich polymorphic information content, stable amplification results, high resolution, and accuracy. It can quickly and accurately determine the origin and type of sesquiterpene germplasm, which is beneficial for research such as sesquiterpene germplasm resource identification, genetic diversity analysis, and genetic structure analysis. It is of great significance in promoting the protection and utilization of sesquiterpene germplasm resources and driving industrial development.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] This invention provides a primer set for sesquiterpene SSR developed based on shallow genome sequences, comprising 23 primer pairs numbered 1-23;

[0009] The nucleotide sequence of the primer pair numbered 1 is shown in SEQ ID NO. 1-2;

[0010] The nucleotide sequences of primer pair numbered 2 are shown in SEQ ID NO.3-4;

[0011] The nucleotide sequences of the primer pair numbered 3 are shown in SEQ ID NO.5-6;

[0012] The nucleotide sequences of the primer pair numbered 4 are shown in SEQ ID NO.7-8;

[0013] The nucleotide sequences of primer pair numbered 5 are shown in SEQ ID NO. 9-10;

[0014] The nucleotide sequences of the primer pair numbered 6 are shown in SEQ ID NO.11-12;

[0015] The nucleotide sequences of the primer pair numbered 7 are shown in SEQ ID NO.13-14;

[0016] The nucleotide sequences of the primer pair numbered 8 are shown in SEQ ID NO.15-16;

[0017] The nucleotide sequences of the primer pair numbered 9 are shown in SEQ ID NO.17-18;

[0018] The nucleotide sequences of the primer pair numbered 10 are shown in SEQ ID NO.19-20;

[0019] The nucleotide sequences of the primer pair numbered 11 are shown in SEQ ID NO.21-22;

[0020] The nucleotide sequences of the primer pair numbered 12 are shown in SEQ ID NO.23-24;

[0021] The nucleotide sequences of the primer pair numbered 13 are shown in SEQ ID NO.25-26;

[0022] The nucleotide sequences of the primer pair numbered 14 are shown in SEQ ID NO.27-28;

[0023] The nucleotide sequences of the primer pair numbered 15 are shown in SEQ ID NO.29-30;

[0024] The nucleotide sequences of the primer pair numbered 16 are shown in SEQ ID NO. 31-32;

[0025] The nucleotide sequences of the primer pair numbered 17 are shown in SEQ ID NO.33-34;

[0026] The nucleotide sequences of the primer pair numbered 18 are shown in SEQ ID NO.35-36;

[0027] The nucleotide sequences of the primer pair numbered 19 are shown in SEQ ID NO.37-38;

[0028] The nucleotide sequences of the primer pair numbered 20 are shown in SEQ ID NO.39-40;

[0029] The nucleotide sequences of the primer pair numbered 21 are shown in SEQ ID NO.41-42;

[0030] The nucleotide sequences of the primer pair numbered 22 are shown in SEQ ID NO.43-44;

[0031] The nucleotide sequence of primer pair numbered 23 is shown in SEQ ID NO.45-46.

[0032] The present invention also provides a product containing the aforementioned guar gum SSR primer combination.

[0033] Furthermore, the product is a gene chip or a reagent kit.

[0034] This invention also provides an application of the guar SSR primer combination or the product described herein in any of the following:

[0035] (1) Evaluation of genetic diversity of sesquiterpene;

[0036] (2) Genetic structure analysis of sesbania population;

[0037] (3) Research on the evolution and phylogenetic relationships of sesbania;

[0038] (4) Identification of sesame germplasm resources.

[0039] This invention also provides a method for using the aforementioned sesame SSR primer combination or the aforementioned product in the evaluation of sesame genetic diversity, analysis of population genetic structure, study of evolution and kinship, or identification of germplasm resources. The forward primer remains unchanged, and an M13 adapter without fluorescent label is added to the 5' end of the reverse primer. Then, a total of three primers with a fluorescent M13 primer are used to perform PCR amplification on the sample to be analyzed. After the amplification product passes the agarose gel electrophoresis test, it is detected by fluorescent capillary electrophoresis. The peak diagram is read, and the allele size of each sample is recorded for the evaluation of sesame genetic diversity, analysis of population genetic structure, study of evolution and kinship, or identification of germplasm resources.

[0040] Furthermore, the sequences of the M13 primer and the M13 adapter are as shown in SEQ ID NO.47, and the fluorescent group is one of FAM, NED, VIC and ROX.

[0041] Furthermore, the PCR amplification reaction system is as follows: 2 μL of 10×PCR buffer, 1.6 μL of 2.5 mM MgCl2, 1.6 μL of 2.5 mM dNTPs, 0.5 μL each of 10 μM forward and reverse primers and fluorescent primers, 0.2 μL of 5 U / μL Taq DNA polymerase, 50-100 ng of template DNA, and ddH2O to make up to 20 μL.

[0042] Furthermore, the PCR amplification reaction program is as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 35 cycles; and finally 72℃ extension for 10 min.

[0043] The present invention discloses the following technical effects:

[0044] This invention develops an SSR primer set based on the shallow genome sequence of sesquiterpenes, comprising 23 SSR primer pairs. The SSR primer pairs developed in this invention possess advantages such as high polymorphism, rich polymorphic information content, stable amplification results, high resolution, and accuracy. They can quickly and accurately determine the origin and species of sesquiterpenes, which is beneficial for research on sesquiterpenes germplasm resource identification, genetic diversity analysis, and genetic structure analysis. This is of great significance in promoting the protection and utilization of sesquiterpenes germplasm resources and driving industrial development. Attached Figure Description

[0045] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0046] Figure 1 This is a UPGMA clustering diagram of the sessilis population based on Nei's genetic distance. Detailed Implementation

[0047] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0048] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0049] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0050] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0051] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0052] Example 1: Design and Screening of SSR Primers for the Guillarum Genome

[0053] 1. Design and screening of SSR primers for the sesquiterpene genome

[0054] DNA was extracted from sesbania samples (provided by the National Animal Husbandry Station) from different regions including Guangdong, Yunnan, and Fujian. Shallow genome sequencing was performed using the Illumina Mi Seq platform. After obtaining the raw sequencing data, SPAdes 3.6.1 software was used for quality control and contig assembly of the raw reads. SSR loci were then identified using MISA software (with a minimum dinucleotide repeat count of 5 and a minimum trinucleotide repeat count of 3). Ultimately, 237,370 SSR loci were successfully developed. Primers were designed based on the complementary sequences at both ends of the SSR loci, using the dinucleotide and trinucleotide repeat sites within the obtained SSR loci.

[0055] Using the guar gum genome sequence as a template, the designed SSR primers were screened using the TP-M13-SSR (simple sequence repeat with tailed primer M13) automated fluorescence detection technology.

[0056] The SSR-PCR amplification system contains three primers: a standard forward primer; a reverse primer with the M13 sequence (5'-CACGACGTTCTAAAACGAC-3', SEQ ID NO.47) added to the 5' end; and a third primer, a fluorescently labeled universal M13 primer (SEQ ID NO.47). The fluorescent labeling group can be FAM (blue), NED (yellow), VIC (green), or ROX (red), all of which are synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0057] The PCR amplification system consisted of 20 μL, including 2 μL of 10×PCR buffer, 1.6 μL of 2.5 mM MgCl2, 1.6 μL of 2.5 mM dNTPs, 0.5 μL each of forward and reverse primers and fluorescent primers (10 μM), 0.2 μL of Taq DNA polymerase (5 U / μL), 50-100 ng of template DNA, and the remainder was made up with ddH2O.

[0058] The PCR amplification program was set as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 35 cycles; and a final extension at 72℃ for 10 min. PCR products were sequenced by Sangon Biotech (Shanghai) Co., Ltd. using a 3730xl automated sequencer (ABI, USA). Analysis was performed using GeneMarker v2.2.0 software (SoftGenetics, State College, Pennsylvania, USA). After passing agarose gel electrophoresis, fluorescence capillary electrophoresis was performed to detect the peaks and record the allele size for each sample.

[0059] A total of 23 pairs of SSR primers with high polymorphism and good stability were obtained, as shown in Table 1. These 23 pairs of SSR primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0060] Table 123 Primers for Tian Jing SSR

[0061]

[0062]

[0063] Example 2 Polymorphism Analysis of SSR Primers for the Guillarum Genome

[0064] The number of alleles (Na) was calculated using GenAlEx 6.5. This parameter reflects the number of alleles observed at a specific locus and is one of the basic indicators for measuring genetic diversity. The effective number of alleles (Ne) takes into account the frequency of alleles and can more accurately reflect the degree of genetic diversity in a population. The calculation formula is Ne = 1 / ∑(pi 2 ), where pi is the frequency of the i-th allele; Nei's genetic diversity index (H) is used to assess the level of genetic diversity within a population, with values ​​ranging from 0 to 1. A higher value indicates higher genetic diversity, and the calculation formula is H = 1 - ∑(pi) 2 The Shannon's Information Index (I) comprehensively considers both the number and frequency of alleles, providing a more complete reflection of genetic diversity. Its calculation formula is I = -∑(pi × ln(pi)). By calculating these parameters, the genetic diversity level of different sesbania populations can be comprehensively assessed, providing a data foundation for subsequent analysis and representing the PIC polymorphism information content. The genetic diversity of the 23 SSR primer pairs obtained in Example 1 is shown in Table 2.

[0065] Table 223 shows the genetic diversity of SSR primers.

[0066]

[0067]

[0068] Table 2 shows that 23 primer pairs exhibited high polymorphism. Among them, primers 2, 5, and 7 had the highest allele counts (Na), at 3.889, 3.444, and 4.000 respectively, indicating that these primers are highly efficient in detecting individual genetic variations and can reveal rich allele information, making them suitable as preferred molecular markers for subsequent studies. The overall average allele count (Na) was 1.686, the average effective allele count (Ne) was 1.394, and the Shannon Information Index (I) was 0.526. These data indicate that the overall genetic diversity of the sesbania population is at a moderate level. The average observed heterozygosity (Ho) was 0.546, higher than the expected heterozygosity (He) of 0.319, further confirming a significant heterozygous surplus in the population. This may be related to frequent gene exchange among individuals within the population and a low inbreeding coefficient. The polymorphism information content (PIC) of the 23 SSR primer pairs used in the experiment was high, with an average of 0.905, indicating that the selected primers could effectively reveal the genetic diversity of the sesbania population. They also show promising application prospects in germplasm resource identification, population genetic structure, evolution, and phylogenetic studies.

[0069] Example 3: Application of Guillarum genome SSR primers in genetic diversity analysis and population genetic structure.

[0070] 1. Experimental Materials

[0071] The sesame seed material used in this embodiment was provided by the National Animal Husbandry Station, and a total of 53 samples came from 9 provinces: Fujian, Guangdong, Hubei, Jiangsu, Sichuan, Yunnan, Guangxi Zhuang Autonomous Region, Hunan, and Jiangxi.

[0072] The above 53 sesame materials were cultured as seedlings in an artificial climate chamber. After 60 days of growth, healthy young leaves were collected, quickly frozen in liquid nitrogen, and stored in an ultra-low temperature freezer at -80℃ for later use.

[0073] 2. DNA extraction

[0074] Remove the sesame leaf samples from the -80℃ freezer, place them in a pre-cooled mortar, add liquid nitrogen and grind them quickly into powder. Transfer the powder to a centrifuge tube, add preheated CTAB extraction buffer, and incubate in a 65℃ water bath for 60-90 minutes, inverting the centrifuge tube every 15 minutes.

[0075] After incubation, add a chloroform:isoamyl alcohol (24:1, v / v) mixture, centrifuge at 12000 rpm for 15 min, and transfer the upper aqueous phase to a new centrifuge tube to avoid impurities.

[0076] Add isopropanol to the aqueous phase to precipitate DNA, let stand for 30 min, centrifuge at 12000 rpm for 10 min, discard the supernatant, wash the DNA precipitate with 70% ethanol, centrifuge at 8000 rpm for 5 min, discard the supernatant, air dry the DNA precipitate, add 50-100 μL of LTE buffer to dissolve the DNA, and store at 4℃ for later use.

[0077] DNA quality was assessed using agarose gel electrophoresis and a nucleic acid protein assay instrument, selecting bands with clear, tailless, and high OD values. 260 / OD 280 High-purity DNA with a ratio between 1.8 and 2.0 was used for subsequent experiments.

[0078] 3. Genetic diversity analysis

[0079] Using DNA from 53 sesbania samples from different regions as templates, PCR amplification was performed using 23 primer pairs screened in Example 1. The amplification system and reaction conditions were the same as in Example 1. The genetic diversity of different sesbania populations was analyzed based on the amplification results, as shown in Table 3.

[0080] Table 3. Genetic diversity analysis based on different sesquiterpene populations

[0081]

[0082] Table 3 shows the genetic diversity indicators of different sesbania populations. The average number of alleles (Na) of the Guangxi population is 2.348, the effective number of alleles (Ne) is 1.837, and the Shannon index (I) is 0.703, all of which are the highest values ​​among all populations, indicating that the Guangxi population has the richest genetic diversity. This is related to the diverse habitat conditions, large population size, or historically less environmental disturbance in the region. In contrast, the indicators of the Sichuan population are extremely low, with Na at only 0.609, Ne at 0.609, and I at 0.211. In terms of heterozygosity, most populations have a higher Ho than He and a negative F value, indicating that there is a general surplus of heterozygotes in all populations, which may be due to gene flow between populations or their own reproductive methods.

[0083] 4. Cluster analysis

[0084] To visually represent the genetic relationships among different guarana materials, GenAlEx 6.5 principal coordinate analysis (PCoA) was used; and MEGA 7.0 was used to construct a UPGMA (unweighted group average) clustering tree based on Nei's genetic distance.

[0085] As shown in Table 4, in terms of genetic distance, the Sichuan population is relatively far from all other populations, with a distance of 1.115 from Fujian, indicating a high degree of genetic differentiation between the Sichuan population and other regional populations, possibly the result of long-term geographical isolation or independent evolution. In contrast, the genetic distance between Fujian and populations in Guangdong and Jiangsu is relatively small, all at 0.023, suggesting closer genetic relationships among these populations, possibly indicating a common ancestor or frequent historical gene exchange. Furthermore, the genetic distance between the Yunnan and Hunan populations is 0.224, showing a certain degree of genetic connection, possibly related to historical migration routes or ecological adaptations.

[0086] Table 4 Nei's genetic distance matrix

[0087]

[0088]

[0089] UPGMA cluster analysis, such as Figure 1 As shown, sesquiterpene germplasm from Sichuan clusters into a single branch, while sesquiterpene germplasm from other provinces clusters into another branch. Furthermore, sesquiterpene germplasm from Fujian, Guangdong, Jiangsu, Jiangxi, Hunan, and Guangxi Zhuang Autonomous Region collectively forms a single branch, indicating a high degree of similarity in their genetic background and relatively small genetic differences, which may suggest they belong to the same geographical or ecological region. Sesquiterpene germplasm from Hubei and Yunnan each form a separate branch.

[0090] The Hubei population exhibits a relatively moderate genetic distance from all other populations, neither overly close nor overly distant, which may reflect its unique position in terms of geographical distribution and genetic evolution. Although the Yunnan population shares some genetic links with the Hunan population, it remains clearly distinguishable in the UPGMA clustering tree, indicating that while there is some degree of genetic commonality between the two, sufficient genetic differences maintain their individual genetic uniqueness. Overall, the UPGMA clustering analysis not only reveals the genetic relationships among different sesbania populations but also provides valuable reference information for subsequent genetic improvement and germplasm resource utilization.

[0091] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A sesame SSR primer combo developed based on shallow genome sequences, characterized in that, This includes 23 primer pairs numbered 1-23; The nucleotide sequence of the primer pair numbered 1 is shown in SEQ ID NO. 1-2; The nucleotide sequences of primer pair numbered 2 are shown in SEQ ID NO.3-4; The nucleotide sequences of the primer pair numbered 3 are shown in SEQ ID NO.5-6; The nucleotide sequences of the primer pair numbered 4 are shown in SEQ ID NO.7-8; The nucleotide sequences of primer pair numbered 5 are shown in SEQ ID NO. 9-10; The nucleotide sequences of the primer pair numbered 6 are shown in SEQ ID NO.11-12; The nucleotide sequences of the primer pair numbered 7 are shown in SEQ ID NO.13-14; The nucleotide sequences of the primer pair numbered 8 are shown in SEQ ID NO.15-16; The nucleotide sequences of the primer pair numbered 9 are shown in SEQ ID NO.17-18; The nucleotide sequences of the primer pair numbered 10 are shown in SEQ ID NO.19-20; The nucleotide sequences of the primer pair numbered 11 are shown in SEQ ID NO.21-22; The nucleotide sequences of the primer pair numbered 12 are shown in SEQ ID NO.23-24; The nucleotide sequences of the primer pair numbered 13 are shown in SEQ ID NO.25-26; The nucleotide sequences of the primer pair numbered 14 are shown in SEQ ID NO.27-28; The nucleotide sequences of the primer pair numbered 15 are shown in SEQ ID NO.29-30; The nucleotide sequences of the primer pair numbered 16 are shown in SEQ ID NO. 31-32; The nucleotide sequences of the primer pair numbered 17 are shown in SEQ ID NO.33-34; The nucleotide sequences of the primer pair numbered 18 are shown in SEQ ID NO.35-36; The nucleotide sequences of the primer pair numbered 19 are shown in SEQ ID NO.37-38; The nucleotide sequences of the primer pair numbered 20 are shown in SEQ ID NO.39-40; The nucleotide sequences of the primer pair numbered 21 are shown in SEQ ID NO.41-42; The nucleotide sequences of the primer pair numbered 22 are shown in SEQ ID NO.43-44; The nucleotide sequence of primer pair numbered 23 is shown in SEQ ID NO.45-46.

2. A product containing the guar SSR primer combination of claim 1.

3. The product according to claim 2, characterized in that, The product in question is a gene chip or a reagent kit.

4. The use of the guar SSR primer combination of claim 1 or the product of claim 2 or 3 in any of the following: (1) Evaluation of genetic diversity of sesquiterpene; (2) Genetic structure analysis of sesbania population; (3) Research on the evolution and phylogenetic relationships of sesbania; (4) Identification of sesame germplasm resources.

5. A method of using the sesquiterpene SSR primer combination of claim 1 or the product of claim 2 or 3 in the evaluation of genetic diversity, analysis of population genetic structure, study of evolution and phylogenetic relationships, or identification of germplasm resources of sesquiterpene, characterized in that, The forward primer remains unchanged, but an M13 adapter without fluorescent label is added to the 5' end of the reverse primer. Then, a fluorescent M13 primer is used for a total of three primers to amplify the sample to be analyzed. After the amplification products pass the agarose gel electrophoresis test, fluorescence capillary electrophoresis is performed to detect the peaks, record the allele size of each sample, and conduct genetic diversity evaluation, population genetic structure analysis, evolution and kinship studies, or germplasm resource identification of sesbania.

6. The method of use according to claim 5, characterized in that, The sequences of the M13 primer and the M13 adapter are as shown in SEQ ID NO.47, and the fluorescent group is one of FAM, NED, VIC and ROX.

7. The method of use according to claim 5, characterized in that, The PCR amplification reaction system consisted of: 2 μL of 10×PCR buffer, 1.6 μL of 2.5 mM MgCl2, 1.6 μL of 2.5 mM dNTPs, 0.5 μL each of 10 μM forward and reverse primers and fluorescent primers, 0.2 μL of 5 U / μL Taq DNA polymerase, 50-100 ng of template DNA, and ddH2O to make up to 20 μL.

8. The method of use according to claim 5, characterized in that, The PCR amplification reaction program was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 35 cycles; and finally 72℃ extension for 10 min.

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