A SSR primer combination for identifying Sesbania dahurica and its application

Abstract

The present invention discloses an SSR primer combination for identifying sesbania and its application. The SSR primer combination of the present invention includes combination 1 and / or combination 2; wherein, combination 1 includes 12 pairs of SSR primer pairs obtained based on the chloroplast genome of sesbania, and the nucleotide sequences of the SSR primer pairs are shown in SEQ ID NO.1 to 24 in sequence; combination 2 includes 9 pairs of SSR primer pairs obtained based on the transcriptome of sesbania, and the nucleotide sequences of the SSR primer pairs are shown in SEQ ID NO.25 to 42 in sequence. Using the SSR primer combination of the present invention, the source and type of sesbania can be quickly and accurately determined, which is beneficial to the identification of sesbania germplasm resources, the analysis of genetic diversity of germplasm resources and the identification of hybrid offspring, and is also beneficial to the construction of fingerprint maps and genetic maps of sesbania germplasm resources.

Description

[0001] Invention Name

[0002] The invention belongs to the technical field of molecular markers, and particularly relates to an SSR primer combination for identifying sesbania dahurica and an application thereof. Background Art

[0003] Sesbania cannabina is an annual leguminous plant with a well-developed root system, many large nodules, strong nitrogen fixation ability, high fresh grass yield, rich nutrient content, good drought resistance, disease and insect resistance, and strong salt, waterlogging and barrenness tolerance. It is a pioneer plant for summer green manure and soil restoration and transformation of tidal flat saline-alkali land. At the same time, Sesbania cannabina also has high economic value and utilization value in animal husbandry, papermaking and chemical industries.

[0004] Although Sesbania truncatum is widely distributed in my country and has a wealth of application scenarios, the Sesbania truncatum varieties and resources currently used in production are numerous and complex, with problems such as unclear sources and low seed purity. Traditional phenotypic identification requires effective control of the environment, and requires multiple years of multi-point identification to accurately observe the variation level or expression stability of the identified traits. This identification method is not only time-consuming and labor-intensive, but also easily affected by the environment, has low reliability, high costs, and cannot meet market demand. Therefore, there is an urgent need for a method that can quickly and accurately identify Sesbania truncatum germplasm resources.

[0005] SSR (Simple sequence repeats) markers are stable, highly polymorphic, and require low DNA concentration DNA molecular markers, and are often used in variety identification, genetic analysis, and other aspects. Although there are reports on the identification of plant germplasm resources based on chloroplast genomes or transcriptomes, there are no SSR markers for Sesbania truncatula. For genomes of different genetic backgrounds, it is very difficult to screen suitable marker sites and design specific primer pairs. Although there are many SSR marker sites in the genome and transcriptome, there are many problems such as uneven distribution of molecular markers in chromosomes, complex genome structure, lack of molecular marker sites on some chromosomes, genome variation, and chain reactions between molecular markers. These problems will affect the identification efficiency of SSR primer pairs, and also make it difficult to quickly and accurately identify Sesbania truncatula germplasm resources based on SSR markers. Summary of the invention

[0006] The invention aims at the problems of various sesbania varieties and impure seeds in the market, lack of a method capable of efficiently and quickly identifying sesbania varieties, and provides an SSR primer combination for identifying sesbania and an application thereof.

[0007] The first object of the present invention is to provide an SSR primer combination for identifying Sesbania dahurica.

[0008] The second object of the present invention is to provide a product containing an SSR primer combination of Sesbania sesbania.

[0009] The third object of the present invention is to provide an SSR primer combination or a product containing the SSR primer combination for use in identifying sesbania germplasm resources or in preparing a kit for identifying sesbania germplasm resources.

[0010] The fourth object of the present invention is to provide an SSR primer combination or a product containing the SSR primer combination for use in identifying sesbania hybrid progeny or in preparing a kit for identifying sesbania hybrid progeny.

[0011] The fifth object of the present invention is to provide an application of an SSR primer combination or a product containing the SSR primer combination in the genetic diversity analysis of Sesbania germplasm resources or in the preparation of a kit for the genetic diversity analysis of Sesbania germplasm resources.

[0012] The sixth object of the present invention is to provide an application of an SSR primer combination or a product containing the SSR primer combination in constructing a fingerprint map of sesbania germplasm resources or in preparing a kit for constructing a fingerprint map of sesbania germplasm resources.

[0013] The seventh object of the present invention is to provide an application of an SSR primer combination or a product containing the SSR primer combination in constructing a genetic map of Sesbania sesbania or in preparing a kit for constructing a genetic map of Sesbania sesbania.

[0014] The eighth object of the present invention is to provide a method for identifying hybrid progeny of Sesbania sesbania.

[0015] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0016] Based on the chloroplast genome and transcriptome data of Sesbania sesbania, the present invention obtains an SSR primer combination that can be used to identify Sesbania sesbania, and the SSR primer combination can be used to identify Sesbania sesbania germplasm resources, analyze the genetic diversity of Sesbania sesbania germplasm resources, construct a fingerprint map of Sesbania sesbania germplasm resources, identify Sesbania hybrid offspring, and construct a Sesbania genetic map. Therefore, the present invention claims protection for the SSR primer combination and related applications thereof.

[0017] The present invention claims a SSR primer combination for identifying Sesbania sesbania.

[0018] Specifically, the SSR primer combination includes combination 1 and / or combination 2.

[0019] Among them, combination 1 includes SSR primer pairs SCC7, SCC9, SCC10, SCC14, SCC19, SCC29, SCC31, SCC32, SCC33, SCC37, SCC53 and SCC65, and the SSR primer pairs are obtained based on the chloroplast genome of Sesbania truncatula;

[0020] The nucleotide sequence of the primer pair SCC7 is shown in SEQ ID NO. 1-2;

[0021] The nucleotide sequence of the primer pair SCC9 is shown in SEQ ID NO.3-4;

[0022] The nucleotide sequence of the primer pair SCC10 is shown in SEQ ID NOs. 5 to 6;

[0023] The nucleotide sequence of the primer pair SCC14 is shown in SEQ ID NOs. 7 to 8;

[0024] The nucleotide sequence of the primer pair SCC19 is shown in SEQ ID NOs. 9 to 10;

[0025] The nucleotide sequence of the primer pair SCC29 is shown in SEQ ID NOs. 11 to 12;

[0026] The nucleotide sequence of the primer pair SCC31 is shown in SEQ ID NOs. 13 to 14;

[0027] The nucleotide sequence of the primer pair SCC32 is shown in SEQ ID NOs. 15 to 16;

[0028] The nucleotide sequence of the primer pair SCC33 is shown in SEQ ID NOs. 17 to 18;

[0029] The nucleotide sequence of the primer pair SCC37 is shown in SEQ ID NOs. 19 to 20;

[0030] The nucleotide sequence of the primer pair SCC53 is shown in SEQ ID NOs. 21 to 22;

[0031] The nucleotide sequence of the primer pair SCC65 is shown in SEQ ID NOs. 23-24;

[0032] Combination 2 includes SSR primer pairs SCT3410, SCT8517, SCT12033, SCT15566, SCT27002, SCT37380, SCT51370, SCT51556, and SCT54839, which are obtained based on the Sesbania transcriptome;

[0033] The nucleotide sequence of the primer pair SCT3410 is shown in SEQ ID NOs. 25-26;

[0034] The nucleotide sequence of the primer pair SCT8517 is shown in SEQ ID NOs. 27 to 28;

[0035] The nucleotide sequence of the primer pair SCT12033 is shown in SEQ ID NOs. 29 to 30;

[0036] The nucleotide sequence of the primer pair SCT15566 is shown in SEQ ID NOs. 31 to 32;

[0037] The nucleotide sequence of the primer pair SCT27002 is shown in SEQ ID NOs. 33-34;

[0038] The nucleotide sequence of the primer pair SCT37380 is shown in SEQ ID NOs. 35-36;

[0039] The nucleotide sequence of the primer pair SCT51370 is shown in SEQ ID NOs. 37 to 38;

[0040] The nucleotide sequence of the primer pair SCT51556 is shown in SEQ ID NOs. 39 to 40;

[0041] The nucleotide sequences of the primer pair SCT54839 are shown in SEQ ID NOs. 41-42.

[0042] The present invention also claims to protect a product containing a SSR primer combination of Sesbania sesbania.

[0043] Specifically, the product is a gene chip or a kit.

[0044] The present invention also claims to protect the use of the SSR primer combination or a product containing the SSR primer combination in identifying sesbania germplasm resources or in preparing a kit for identifying sesbania germplasm resources.

[0045] The present invention also claims to protect the use of the SSR primer combination or a product containing the SSR primer combination in identifying the hybrid offspring of Sesbania sesbania or in preparing a kit for identifying the hybrid offspring of Sesbania sesbania.

[0046] The present invention also claims to protect the use of the SSR primer combination or a product containing the SSR primer combination in the genetic diversity analysis of sesbania germplasm resources or in the preparation of a kit for the genetic diversity analysis of sesbania germplasm resources.

[0047] The present invention also seeks to protect the use of an SSR primer combination or a product containing the SSR primer combination in constructing a fingerprint map of sesbania germplasm resources or in preparing a kit for constructing a fingerprint map of sesbania germplasm resources.

[0048] The present invention also claims to protect the use of the SSR primer combination or a product containing the SSR primer combination in constructing a sesbania genetic map or in preparing a kit for constructing a sesbania genetic map.

[0049] Specifically, in the above application, the SSR primer combination includes 12 pairs of SSR primer pairs based on the chloroplast genome of Sesbania sesbania and / or 9 pairs of SSR primer pairs based on the transcriptome of Sesbania sesbania.

[0050] The present invention also claims a method for identifying hybrid progeny of Sesbania dahurica.

[0051] Specifically, DNA of the hybrid offspring of the tested Sesbania and its parents are extracted, PCR amplification is performed on the parents respectively using the SSR primer combination or a product containing the SSR primer combination, SSR primer pairs with differential bands in the parents are screened, and the hybrid offspring are amplified using the differential SSR primer pairs; if the hybrid offspring of the tested Sesbania have the characteristic bands of both parents, it is a true hybrid; if only the band of one of the parents can be detected, it is a false hybrid.

[0052] Specifically, the reaction procedure of the PCR amplification is pre-denaturation at 94°C for 3 min; 94°C for 45 s, 55°C for 40 s, 72°C for 1 min, 35 cycles; 72°C for 10 min.

[0053] The present invention has the following beneficial effects:

[0054] The present invention develops an SSR primer combination for identifying Sesbania sesbania, including 12 pairs of SSR primer pairs obtained based on Sesbania sesbania chloroplast genomes and / or 9 pairs of SSR primer pairs obtained based on Sesbania sesbania transcriptomes. The SSR primer pairs developed by the present invention have the advantages of stable amplification results, high resolution, accuracy, etc., can quickly and accurately determine the source and type of Sesbania sesbania, are conducive to the identification of Sesbania sesbania germplasm resources, genetic diversity analysis of germplasm resources and identification of hybrid offspring, and are also conducive to the construction of Sesbania sesbania germplasm resource fingerprints and genetic maps, which is of great significance in promoting the development of Sesbania sesbania breeding level and industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 This is the electropherogram of the screening of SCC16, SCC17, SCC18, SCC19 and SCC24 primer pairs using TJ2, TJ9, TJ24 and TJ41 sesbania germplasm resources.

[0056] Figure 2This is the electrophoresis diagram of the detection of TJ1-35 sesbania germplasm resources using primer pair SCC19.

[0057] Figure 3 This is a clustering diagram of 45 Sesbania sesbania germplasm resources based on the SSR primer pairs of the Sesbania sesbania chloroplast genome.

[0058] Figure 4 This is a clustering diagram of 45 Sesbania sesbania germplasm resources based on the SSR primer pairs of Sesbania sesbania transcriptome.

[0059] Figure 5 The results of population structure distribution of 45 sesbania germplasm resources; A in the figure is the K value distribution of SSR primer pairs based on the sesbania chloroplast genome, B in the figure is the K value distribution of SSR primer pairs based on the sesbania transcriptome, C in the figure is the population structure distribution of sesbania based on the SSR primer pairs based on the sesbania chloroplast genome, and D in the figure is the population structure distribution of sesbania based on the SSR primer pairs based on the sesbania transcriptome.

[0060] Figure 6 Figure 1 is a fingerprint of Sesbania sesbania based on the SSR primer pairs of the Sesbania sesbania chloroplast genome. The horizontal marks in the figure are the numbers of the bands of different sizes amplified by the 12 primer pairs, and the vertical marks are the types of bands amplified by the 12 primer pairs of 45 Sesbania sesbania germplasm resources. The green mark (0) in the figure indicates the presence of a band, the blue mark (1) indicates the absence of a band, and the red mark (2) indicates the absence of a band.

[0061] Figure 7 Figure 1 is a fingerprint of Sesbania sesbania based on the SSR primer pairs of Sesbania sesbania transcriptome; the horizontal marks in the figure are the numbers of the bands of different sizes amplified by the 9 primer pairs, and the vertical marks are the types of bands amplified by the 45 Sesbania sesbania germplasm resources using the 9 primer pairs; the green mark (0) in the figure indicates the presence of a band, the blue mark (1) indicates the absence of a band, and the red mark (2) indicates the absence. DETAILED DESCRIPTION

[0062] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0063] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0064] Example 1 Obtaining Sesbania chloroplast genome SSR primers and transcriptome SSR primers

[0065] 1. The information of the Sesbania sesbania germplasm resources used in the present invention is shown in Table 1. The Sesbania sesbania germplasm resource numbers shown in Table 1 are the numbers in the National Tropical Plant Germplasm Resource Bank, and the germplasm resources are currently stored in the National Tropical Plant Germplasm Resource Bank in Danzhou, Hainan.

[0066] Table 1 Information on Sesbania truncatula germplasm resources

[0067]

[0068]

[0069] 2. Acquisition of Sesbania chinensis Chloroplast Genome and Transcriptome Data

[0070] The chloroplast genome (MN105118.1) and transcriptome dataset (GSE99532) of Sesbania truncatula were downloaded from the NCBI database (https: / / www.ncbi.nlm.nih.gov / ).

[0071] 3. Design and screening of SSR primer pairs for chloroplast genome of Sesbania truncatula

[0072] The present invention is based on the chloroplast genome data of Sesbania sesbania obtained from the NCBI database, from which 70 SSR sites are identified, and 68 pairs of SSR primers are designed for the identified SSR sites. First, the chloroplast genome sequence of Sesbania sesbania is used as a template, and the 68 pairs of SSR primers designed are screened using the E-PCR program to obtain 42 pairs of effective primers.

[0073] The 42 pairs of primers synthesized by the commissioned Shanghai Bioengineering Co., Ltd. were tested using four sesbania germplasm resources TJ2, TJ9, TJ24 and TJ41 (as shown in Table 1) with large phenotypic differences. The results showed that only 26 pairs of primers showed polymorphism, and the polymorphic primer comparison rate was 61.90%. As an example, the present invention uses SSR primer pairs SCC16, SCC17, SCC18, SCC19 and SCC24 to detect sesbania germplasm resources TJ2, TJ9, TJ24 and TJ41 respectively. The electrophoresis diagrams are as follows: Figure 1 As shown. Figure 1 It can be seen that among the SSR primer pairs SCC16, SCC17, SCC18, SCC19 and SCC24, only the SSR primer pairs SCC16 and SCC19 showed polymorphism.

[0074] The nucleotide sequences of the 26 pairs of primer pairs showing polymorphisms are shown in Table 2.

[0075] Table 2 Information of SSR primer pairs of Sesbania chloroplast genome

[0076]

[0077]

[0078] 4. Design and screening of Sesbania sesbania transcriptome SSR primer pairs

[0079] MISA software was used to search the transcriptome data set of Sesbania dahurica, and a total of 92,870 SSR marker loci were identified. Primer3.0 software was used to design primer pairs for SSR loci, and the effectiveness of primer pairs was tested using e-PCR program, and a total of 165 effective primer pairs were obtained.

[0080] The primer pairs synthesized by Sangon Biotech (Shanghai) Co., Ltd. were tested against 165 primer pairs screened out from four sesbania germplasm resources TJ2, TJ9, TJ24 and TJ41 with large phenotypic differences. It was found that 64 primer pairs showed polymorphism, and the polymorphic primer comparison rate was 38.79%.

[0081] The nucleotide sequences of the 64 pairs of primer pairs showing polymorphisms are shown in Table 3.

[0082] Table 3 Sesbania sesbania transcriptome SSR primer pair information

[0083]

[0084]

[0085]

[0086]

[0087] Example 2 Application of Sesbania chloroplast genome SSR and transcriptome SSR primer pairs

[0088] 1. Polymorphism analysis of Sesbania chinensis chloroplast genome SSR and transcriptome SSR primer pairs

[0089] (1) Detection of SSR primer pairs

[0090] The 45 sesbania germplasm resources in Table 1 were amplified using the 26 pairs of sesbania chloroplast genome SSR primer pairs (as shown in Table 2) and 64 pairs of transcriptome SSR primer pairs (as shown in Table 3) obtained in Example 1.

[0091] PCR reaction system: 2×EasyTaq PCR SuperMix (Beijing Quanshijin Biotechnology Co., Ltd.) 10 μL, each pair of primers 1 μL (using concentration of 10 μM), template DNA 2 μL, ddH2O 6 μL.

[0092] PCR reaction amplification program: pre-denaturation at 94°C for 3 min; 94°C for 45 s, 55°C for 40 s, 72°C for 1 min, 35 cycles; 72°C for 10 min.

[0093] After the PCR reaction is completed, polyacrylamide gel electrophoresis is used for detection. The steps of polyacrylamide gel electrophoresis detection are as follows: add 1 / 2 volume of denaturant (a mixture of 0.25g / L xylene cyanol and bromophenol blue) to the PCR product, mix well, denature at 95℃ for 5 minutes, immediately cool on ice, then take 2μL of the product and use 6% denaturing polyacrylamide gel electrophoresis for detection, silver nitrate staining, development, scanning and preservation. The electrophoresis diagram of the TJ1-35 sesbania germplasm resources amplified using primer pair SCC19 is shown in Figure 2 As shown. Figure 2 It can be seen that SCC19 has differentially amplified bands in different Sesbania resources. These differential amplifications are the basis for using multiple SSR markers to identify germplasm resources and analyze genetic diversity.

[0094] (2) Data Analysis

[0095] For the detected stable and clear bands, the data were read according to the band presence as 0, the absence as 1, and the absence as 2. All the missing bands were verified by 3 independent PCRs. PowerMarker V3.25 software was used for data analysis.

[0096] The present invention uses 26 pairs of SSR primers obtained based on the chloroplast genome of Sesbania sesbania to detect 45 Sesbania sesbania germplasm resources, and a total of 81 sites are detected, with the range of marker amplification sites being 1 to 6, and the average being 3.11. The major gene frequency of the marker site ranges from 0.51 to 0.98, with an average of 0.69; the gene diversity ranges from 0.04 to 0.55, with an average of 0.39; the site PIC information content ranges from 0.04 to 0.48, with an average of 0.31; the PIC information content of the marker ranges from 0.37 to 0.90, with an average of 0.72.

[0097] The present invention uses 64 pairs of SSR primers obtained based on the transcriptome of Sesbania sesbania to detect 45 Sesbania sesbania germplasm resources, and a total of 164 sites are detected, with the range of marker amplification sites being 1 to 5, and the average being 2.56. The major gene frequency of the marker site ranges from 0.51 to 0.98, with an average of 0.80; the gene diversity ranges from 0.04 to 0.55, with an average of 0.26; the site PIC information content ranges from 0.04 to 0.49, with an average of 0.23; the PIC information content of the marker ranges from 0.20 to 0.86, with an average of 0.66.

[0098] The above results show that the developed SSR primer pairs have rich polymorphism, and the SSR primers based on the chloroplast genome of Sesbania sesbania have higher polymorphism than the SSR primers based on the transcriptome of Sesbania sesbania.

[0099] 2. Genetic diversity analysis of 45 Sesbania sesbania germplasm resources

[0100] The genetic distances among the 45 accessions of Sesbania sesbania were calculated using PowerMarker V3.25 software (Nei's, 1972). Cluster analysis was performed on the 45 accessions of Sesbania sesbania using MEGA V7.0 software.

[0101] Clustering diagram of 45 Sesbania sesbania germplasm resources based on 26 pairs of SSR primers in the chloroplast genome of Sesbania sesbania Figure 3 As shown. Figure 3 It can be seen that according to the genetic distance between the 45 sesbania germplasm resources, the 45 sesbania germplasm resources were clustered into two categories. The first category included 18 resources, including TJ2, TJ3, TJ7, TJ11, TJ12, TJ14, TJ15, TJ18, TJ21, TJ22, TJ23, TJ24, TJ29, TJ30, TJ31, TJ36, TJ39, and TJ43; the second category There are 27 resources in total, including TJ1, TJ4, TJ5, TJ6, TJ8, TJ9, TJ10, TJ13, TJ16, TJ17, TJ19, TJ20, TJ25, TJ26, TJ27, TJ28, TJ32, TJ33, TJ34, TJ35, TJ37, TJ38, TJ40, TJ41, TJ42, TJ44, and TJ45.

[0102] Clustering diagram of 45 Sesbania sesbania germplasm resources based on 64 SSR primer pairs of Sesbania sesbania transcriptome Figure 4 As shown. Figure 4 It can be seen that according to the genetic distances between the 45 Sesbania sesbania germplasm resources, the 45 Sesbania sesbania germplasm resources were clustered into 2 categories. The division results were consistent with the clustering results of the 45 Sesbania sesbania germplasm resources based on the SSR primer pairs of the Sesbania sesbania chloroplast genome.

[0103] 3. Population structure analysis of 45 Sesbania sesbania germplasm resources

[0104] Structure V2.3.4 software was used to analyze the population structure of 45 Sesbania sesbania germplasm resources.

[0105] The population structure distribution analysis of 45 Sesbania sesbania germplasm resources based on 26 SSR primer pairs of Sesbania sesbania chloroplast genome or 64 SSR primer pairs of Sesbania sesbania transcriptome was performed. Figure 5 As shown, A in the figure is the K value distribution of the SSR primer pair based on the chloroplast genome of Sesbania sesbania, B in the figure is the K value distribution of the SSR primer pair based on the transcriptome of Sesbania sesbania, C in the figure is the distribution of the Sesbania sesbania population structure of the SSR primer pair based on the chloroplast genome of Sesbania sesbania, and D in the figure is the distribution of the Sesbania sesbania population structure of the SSR primer pair based on the transcriptome of Sesbania sesbania. Figure 5It can be seen that based on the chloroplast genome SSR or transcriptome SSR data of Sesbania sesbania, the 45 Sesbania sesbania germplasm resources can be divided into two groups when K = 2. The group division results based on the chloroplast genome SSR or transcriptome SSR primer pairs of Sesbania sesbania are basically consistent.

[0106] 4. Construction of fingerprints of 45 Sesbania sesbania germplasm resources

[0107] Based on the detection results of 45 sesbania germplasm resources using 26 pairs of SSR primer pairs obtained based on the chloroplast genome of sesbania or 64 pairs of SSR primer pairs obtained based on the transcriptome of sesbania, and combined with the differential banding patterns and amplification effects, the present invention screened out the minimum primer combination for identifying 45 sesbania germplasm resources, including 12 pairs of SSR primer pairs obtained based on the chloroplast genome of sesbania (SCC7, SCC9, SCC10, SCC14, SCC19, SCC29, SCC31, SCC32, SCC33, SCC37, SCC53 and SCC65), and / or 9 pairs of SSR primer pairs obtained based on the transcriptome of sesbania (SCT3410, SCT8517, SCT12033, SCT15566, SCT27002, SCT37380, SCT51370, SCT51556 and SCT54839).

[0108] The fingerprints of 45 sesbania germplasm resources were constructed using the 12 pairs of SSR primers obtained based on the chloroplast genome of sesbania. The fingerprint results are as follows: Figure 6 As shown, according to Figure 6 The fingerprint information of chloroplast SSR of each material can be quickly obtained.

[0109] The fingerprints of 45 sesbania germplasm resources were constructed using the 9 pairs of SSR primers obtained based on the sesbania transcriptome. The fingerprint results are as follows: Figure 7 As shown, according to Figure 7 The SSR fingerprint information of each material transcriptome can be quickly obtained.

[0110] Example 3 Application of Sesbania sesbania SSR primers in identification of hybrid offspring

[0111] The SSR primer pairs of the minimum primer combination obtained by the present invention are used to amplify and detect the DNA of the parents, and the SSR primer pairs with differential bands in the parents are screened, and the differential SSR primer pairs are used to amplify the hybrid offspring; if the tested field sesban hybrid offspring have the characteristic bands of both parents, it is a true hybrid; if only the band of one of the parents can be detected, it is a false hybrid.

[0112] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. An SSR primer combination for identifying Sesbania sesbania, characterized in that: Includes combination 1 and / or combination 2; Among them, combination 1 consisted of SSR primer pairs SCC7, SCC9, SCC10, SCC14, SCC19, SCC29, SCC31, SCC32, SCC33, SCC37, SCC53, and SCC65; The nucleotide sequence of the primer pair SCC7 is shown in SEQ ID NO. 1-2; The nucleotide sequence of the primer pair SCC9 is shown in SEQ ID NO.3-4; The nucleotide sequence of the primer pair SCC10 is shown in SEQ ID NOs. 5 to 6; The nucleotide sequence of the primer pair SCC14 is shown in SEQ ID NOs. 7 to 8; The nucleotide sequence of the primer pair SCC19 is shown in SEQ ID NOs. 9 to 10; The nucleotide sequence of the primer pair SCC29 is shown in SEQ ID NOs. 11 to 12; The nucleotide sequence of the primer pair SCC31 is shown in SEQ ID NOs. 13 to 14; The nucleotide sequence of the primer pair SCC32 is shown in SEQ ID NOs. 15 to 16; The nucleotide sequence of the primer pair SCC33 is shown in SEQ ID NOs. 17 to 18; The nucleotide sequence of the primer pair SCC37 is shown in SEQ ID NOs. 19 to 20; The nucleotide sequence of the primer pair SCC53 is shown in SEQ ID NOs. 21 to 22; The nucleotide sequence of the primer pair SCC65 is shown in SEQ ID NOs. 23-24; Combination 2 consisted of the SSR primer pairs SCT3410, SCT8517, SCT12033, SCT15566, SCT27002, SCT37380, SCT51370, SCT51556, and SCT54839; The nucleotide sequence of the primer pair SCT3410 is shown in SEQ ID NOs. 25-26; The nucleotide sequence of the primer pair SCT8517 is shown in SEQ ID NOs. 27 to 28; The nucleotide sequence of the primer pair SCT12033 is shown in SEQ ID NOs. 29 to 30; The nucleotide sequence of the primer pair SCT15566 is shown in SEQ ID NOs. 31 to 32; The nucleotide sequence of the primer pair SCT27002 is shown in SEQ ID NOs. 33-34; The nucleotide sequence of the primer pair SCT37380 is shown in SEQ ID NOs. 35-36; The nucleotide sequence of the primer pair SCT51370 is shown in SEQ ID NOs. 37 to 38; The nucleotide sequence of the primer pair SCT51556 is shown in SEQ ID NOs. 39 to 40; The nucleotide sequences of the primer pair SCT54839 are shown in SEQ ID NOs. 41-42.

2. A product containing the SSR primer combination according to claim 1.

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

4. Use of the SSR primer combination of claim 1 or the product of any one of claims 2 to 3 in the identification of Sesbania sesbania germplasm resources or in the preparation of a kit for the identification of Sesbania sesbania germplasm resources.

5. Use of the SSR primer combination of claim 1 or the product of any one of claims 2 to 3 in identifying hybrid progeny of Sesbania tatarica or in preparing a kit for identifying hybrid progeny of Sesbania tatarica.

6. Use of the SSR primer combination of claim 1 or the product of any one of claims 2 to 3 in genetic diversity analysis of Sesbania sesbania germplasm resources or in the preparation of a kit for genetic diversity analysis of Sesbania sesbania germplasm resources.

7. Use of the SSR primer combination of claim 1 or any one of the products of claims 2 to 3 in constructing a fingerprint map of Sesbania sesbania germplasm resources or in preparing a kit for constructing a fingerprint map of Sesbania sesbania germplasm resources.

8. Use of the SSR primer combination of claim 1 or the product of any one of claims 2 to 3 in constructing a genetic map of Sesbania sesbania or in preparing a kit for constructing a genetic map of Sesbania sesbania.

9. A method for identifying hybrid offspring of Sesbania sesbania, characterized in that: Extract DNA from the hybrid offspring of the tested Sesbania sesbania and its parents, perform PCR amplification on the parents respectively using the SSR primer combination described in claim 1 or the products described in any one of claims 2 to 3, screen SSR primer pairs with differential bands in the parents, and amplify the hybrid offspring using the differential SSR primer pairs; if the hybrid offspring of the tested Sesbania sesbania has the characteristic bands of both parents, it is a true hybrid; if only the band of one of the parents can be detected, it is a false hybrid.

10. The method according to claim 9, characterized in that: The reaction procedure of the PCR amplification is pre-denaturation at 94°C for 3 min; 94°C for 45 s, 55°C for 40 s, 72°C for 1 min, 35 cycles; 72°C for 10 min.

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