SRAP molecular marker primers for genetic diversity analysis of Pennisetum forage and genetic diversity analysis method thereof

Abstract

The application relates to SRAP molecular marker primers for genetic diversity analysis of pasture of the Puccinellia genus and a genetic diversity analysis method thereof; the SRAP molecular marker primers are composed of the following eight pairs of primers: ME1-EM5, ME2-EM3, ME3-EM5, ME4-EM3, ME5-EM2, ME6-EM7, ME7-EM2 and ME8-EM2. The eight pairs of SRAP molecular marker primers screened by the application have the advantages of rich amplification bands, rich polymorphism, large brightness and good repeatability, and can be used for genetic diversity analysis of the Puccinellia genus pasture varieties.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biological DNA marker technology and application, and particularly relates to an SRAP molecular marker primer for genetic diversity analysis of Pennisetum forage grass, an application thereof in genetic diversity analysis of Pennisetum forage grass, and a method for genetic diversity analysis of Pennisetum forage grass. Background Art

[0002] Sequence-related amplified polymorphism (SRAP) is a novel, dominant PCR-based marker system developed in 2001 by Drs. Li and Quiros of the Department of Vegetable Biology at the University of California, Berkeley, for Brassica crops. This marker system is characterized by simplicity, efficiency, high yield, high co-dominance, excellent reproducibility, ease of sequencing, and convenient cloning of target fragments. It has been successfully applied to crop genetic diversity analysis, genetic map construction, marker identification of important traits, and cloning of related genes.

[0003] Pennisetum forage grasses are one of the world's most important forage resources, primarily including elephant grass, Pennisetum, and various interspecific hybrids (hybrid Pennisetum and hybrid elephant grass). Pennisetum forage grasses are widely cultivated worldwide, and a growing number of new varieties are being developed, resulting in diverse genetic differentiation among varieties. Traditional taxonomy has made it difficult to identify these varieties. In recent years, researchers have begun using molecular markers to analyze the genetic diversity and phylogenetic relationships of Pennisetum forage grasses. However, to date, no studies have reported using the SRAP method to analyze the genetic diversity of Pennisetum forage varieties.

[0004] This study used SRAP to analyze the genetic diversity of 20 Pennisetum forage varieties using synthetic SRAP molecular marker primers. This study aims to provide molecular-level reference for the conservation and utilization of Pennisetum forage germplasm resources and the selection of new varieties (lines). Summary of the Invention

[0005] The object of the present invention is to provide a SRAP molecular marker primer system that can be used for genetic diversity analysis of Pennisetum forage grass and a method for genetic diversity analysis of Pennisetum forage grass.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A SRAP molecular marker primer set for analyzing the genetic diversity of Pennisetum forage grass comprises 8 pairs of primers, wherein the 8 pairs of primers are:

[0008] ME1-EM5, the nucleotide sequences of which are shown in SEQ ID NO. 1 and SEQ ID NO. 25;

[0009] ME2-EM3, the nucleotide sequences of which are shown as SEQ ID NO. 2 and SEQ ID NO. 23;

[0010] ME3-EM5, the nucleotide sequences of which are shown as SEQ ID NO. 3 and SEQ ID NO. 25;

[0011] ME4-EM3, the nucleotide sequences of which are shown as SEQ ID NO. 4 and SEQ ID NO. 23;

[0012] ME5-EM2, the nucleotide sequences of which are shown as SEQ ID NO. 5 and SEQ ID NO. 22;

[0013] ME6-EM7, the nucleotide sequences of which are shown as SEQ ID NO. 6 and SEQ ID NO. 27;

[0014] ME7-EM2, the nucleotide sequences of which are shown as SEQ ID NO. 7 and SEQ ID NO. 22;

[0015] ME8-EM2, the nucleotide sequences of which are shown as SEQ ID NO. 8 and SEQ ID NO. 22.

[0016] The present application synthesizes a plurality of SRAP primers and randomly selects 20 forward primers and 20 reverse primers (primer sequences are shown in Table 2) respectively, and optimizes, combines into 400 pairs of primer combinations, randomly selects 4 extracted DNA template samples to screen primer combinations, and finally screens out the above-mentioned 8 pairs of primers.

[0017] The present application provides a method for analyzing the genetic diversity of Pennisetum forage grasses using the SRAP molecular marker primers, which amplifies, detects and SRAPs the DNA of the Pennisetum forage grass sample to be detected using the SRAP molecular marker primers. Specifically, the method comprises the following steps:

[0018] (1) Extracting the DNA of the sample to be detected using the CTAB method for standby;

[0019] (2) Amplifying the DNA sample extracted in step (1) using the SRAP molecular marker primers to obtain PCR amplification products respectively;

[0020] (3) Performing gel electrophoresis and imaging on the PCR amplification products obtained in step (2) to obtain corresponding band information;

[0021] (4) Performing cluster analysis on the samples according to the band information obtained in step (3) to generate a cluster diagram.

[0022] The reaction system for PCR amplification described in step (2) is specifically as follows: 2.5 μl 10X PCR buffer, 0.5 μl dNTPs, 0.5 μl Taq enzyme, 1 μl each primer and 2 μl template DNA, and ddH2O is added to a total volume of 25 μl;

[0023] The primers are any pair of ME1-EM5, ME2-EM3, ME3-EM5, ME4-EM3, ME5-EM2, ME6-EM7, ME7-EM2, and ME8-EM2.

[0024] The PCR amplification procedure was as follows: 94°C pre-denaturation for 5 minutes; 5 cycles of 94°C denaturation for 1 minute, 35°C annealing for 1 minute, and 72°C extension for 1 minute; 35 cycles of 94°C denaturation for 1 minute, 50°C annealing for 1 minute, and 72°C extension for 1 minute; and 72°C extension for 10 minutes after the cycle was completed. The amplification reaction was performed on a Bio-Rad PCR instrument.

[0025] The primers can be synthesized using fluorescent primers, and all amplified products are detected by fluorescence. The synthesis of fluorescent primers and fluorescence detection belong to conventional detection methods in the art and will not be described in detail here.

[0026] The specific method of step (4) is:

[0027] The sample gel images obtained by scanning the automatic sequencer 377 were analyzed using GENESCAN 3.1 software, and the results of the fragment sizes of the samples were extracted using Binthere software; the values ​​in the table that were not 0 were converted into 1 by EXCEL (i.e., the presence of a band at the same position on the electrophoresis map was recorded as 1, and the absence of a band at the same position was recorded as 0), and the values ​​0 were not converted, thereby generating an original matrix composed of "1" and "0"; data analysis was performed using NTSYSpc-2.11F software; the DICE similarity coefficient matrix was calculated for the original matrix using the SimQual program, and the similarity coefficient matrix was obtained; cluster analysis was performed using the UPGMA method in the SHAN program, and a cluster diagram was generated using the Tree plot module.

[0028] The SRAP molecular marker primers are used in the genetic diversity analysis of Pennisetum forage.

[0029] Compared to the prior art, the present invention has the following advantages: Multiple SRAP primers were synthesized, 20 forward primers and 20 reverse primers were randomly selected and optimized, and then paired to form 400 primer combinations. Four randomly extracted DNA template samples were then screened for primer combinations, ultimately resulting in eight pairs of SRAP molecular marker primers. These eight pairs of SRAP molecular marker primers amplified numerous bands, exhibited abundant polymorphisms, exhibited high brightness, and exhibited good reproducibility, making them suitable for genetic diversity analysis of Pennisetum forage varieties. PCR amplification of 20 Pennisetum varieties using these eight pairs of SRAP molecular marker primers yielded a total of 1,278 sites, of which 1,273 were polymorphic, accounting for 99.6% of the total. For example, primer combination ME1-EM5 amplified a maximum of 216 polymorphic sites, while primer combination ME3-EM5 amplified a minimum of 131 polymorphic sites. The average number of sites amplified was 159.8, and the average number of polymorphic sites amplified was 159.1. It can be seen that the eight selected SRAP primer combinations are extremely polymorphic in Pennisetum forage and can be used for genetic diversity analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The figure shows the electrophoresis results of PCR products of 20 samples using the ME1-EM5 primer combination.

[0031] Figure 2 The figure shows the electrophoresis results of PCR products of 20 samples using the ME2-EM3 primer combination.

[0032] Figure 3 The figure shows the electrophoresis results of PCR products of 20 samples using the ME3-EM5 primer combination.

[0033] Figure 4 The figure shows the electrophoresis results of PCR products of 20 samples using the ME4-EM3 primer combination.

[0034] Figure 5 The figure shows the electrophoresis results of PCR products of 20 samples using the ME5-EM2 primer combination.

[0035] Figure 6 This is the electrophoresis result of PCR products of 20 samples using the ME6-EM7 primer combination.

[0036] Figure 7 The figure shows the electrophoresis results of PCR products of 20 samples using the ME7-EM2 primer combination.

[0037] Figure 8 The figure shows the electrophoresis results of PCR products of 20 samples using the ME8-EM2 primer combination.

[0038] Figure 9 This is a table of genetic similarity coefficients of 20 materials marked by SRAP.

[0039] Figure 10 This is a clustering tree of 20 Pennisetum germplasm resources based on SRAP markers.

[0040] Figure 11 The figure shows the electrophoresis results of PCR products of 20 samples using other primer combinations.

[0041] Figure 12 The figure shows the electrophoresis results of PCR products of 20 samples using other primer combinations.

[0042] Figure 13 The figure shows the electrophoresis results of PCR products of 20 samples using other primer combinations.

[0043] Figure 14 The figure shows the electrophoresis results of PCR products of 20 samples using other primer combinations.

[0044] Figure 15 The figure shows the electrophoresis results of PCR products of 20 samples using other primer combinations.

[0045] Figure 16 The figure shows the electrophoresis results of PCR products of 20 samples using other primer combinations. DETAILED DESCRIPTION

[0046] The present invention is described in detail below with reference to the accompanying drawings and embodiments:

[0047] 1 Materials and Methods

[0048] 1.1 Test materials

[0049] This study collected 20 Pennisetum forage species, including elephant grass, Pennisetum, and interspecific hybrids such as hybrid Pennisetum and king grass. The sources of the test materials are detailed in the table below.

[0050] Table 1 Test materials

[0051]

[0052]

[0053] 1.2 DNA extraction

[0054] The 20 fresh tissue samples of young leaves of Pennisetum forage were used as materials, and DNA was extracted by CTAB method. The quality of genomic DNA was detected by 0.8% agarose electrophoresis. The final concentration of the sample was diluted to 100 ng / μl using the detection concentration and stored in a -20℃ refrigerator.

[0055] 1.3 SRAP primer screening

[0056] The present invention synthesized 20 forward primers and reverse primers (primer sequences are shown in Table 2), optimized, and combined into 400 primer pairs. Four randomly extracted DNA template samples were used for primer combination screening. Finally, 8 primer pairs, namely SRAP molecular marker primers, were selected for polymorphism detection of 20 Pennisetum forage varieties.

[0057] The method for primer combination screening is:

[0058] (1) PCR amplification was performed on four randomly selected template DNA samples using 400 primer pairs to obtain PCR amplification products;

[0059] (2) performing gel electrophoresis and imaging on the PCR amplification products obtained in step (1) to obtain corresponding spectral band information;

[0060] (3) Analyze the band information to obtain the total number of sites and the number of polymorphic sites corresponding to the primer combination, and combine the total number of sites and the number of polymorphic sites of the amplified products of all primer combinations to determine the optimal primer combination, namely the SRAP molecular marker primers.

[0061] The PCR amplification reaction system was as follows: 2.5 μl 10X PCR buffer, 0.5 μl dNTPs, 0.5 μl Taq enzyme, 1 μl each of forward primer and reverse primer, and 2 μl template DNA, and ddH2O was added to a total volume of 25 μl.

[0062] The PCR amplification procedure was as follows: initial denaturation at 94°C for 5 minutes; 5 cycles of denaturation at 94°C for 1 minute, annealing at 35°C for 1 minute, and extension at 72°C for 1 minute; 35 cycles of denaturation at 94°C for 1 minute, annealing at 50°C for 1 minute, and extension at 72°C for 1 minute; and finally, extension at 72°C for 10 minutes, followed by storage at 4°C. Amplification reactions were performed on a Bio-Rad PCR instrument.

[0063] The forward primer and reverse primer are the forward primer and reverse primer corresponding to any pair of primer combinations among the 400 pairs of primer combinations, such as the forward primer ME1 and reverse primer EM1 in the primer combination ME1-EM1.

[0064] The SRAP molecular marker primers include 8 pairs of primers, and the 8 pairs of primers are respectively:

[0065] ME1-EM5, the nucleotide sequences of which are shown in SEQ ID NO. 1 and SEQ ID NO. 25;

[0066] ME2-EM3, the nucleotide sequences of which are shown in SEQ ID NO. 2 and SEQ ID NO. 23;

[0067] ME3-EM5, the nucleotide sequences of which are shown as SEQ ID NO. 3 and SEQ ID NO. 25;

[0068] ME4-EM3, the nucleotide sequences of which are shown as SEQ ID NO. 4 and SEQ ID NO. 23;

[0069] ME5-EM2, the nucleotide sequences of which are shown as SEQ ID NO. 5 and SEQ ID NO. 22;

[0070] ME6-EM7, the nucleotide sequences of which are shown as SEQ ID NO. 6 and SEQ ID NO. 27;

[0071] ME7-EM2, the nucleotide sequences of which are shown as SEQ ID NO. 7 and SEQ ID NO. 22;

[0072] ME8-EM2, the nucleotide sequences of which are shown as SEQ ID NO. 8 and SEQ ID NO. 22.

[0073] Table 2 SRAP primer table

[0074]

[0075]

[0076] 1.4 Genetic diversity analysis of 20 Pennisetum forage cultivars by using screened SRAP molecular marker primers

[0077] comprising the following steps:

[0078] (1) PCR amplification of the DNA samples extracted in step 1.2 by using the SRAP molecular marker primers screened in step 1.3, to obtain corresponding PCR amplification products;

[0079] (2) gel electrophoresis and imaging of the PCR amplification products obtained in step (1), to obtain corresponding band information;

[0080] (3) cluster analysis of the samples according to the band information obtained in step (2), to generate a cluster diagram and perform genetic similarity analysis.

[0081] The reaction system for the PCR amplification in step (1) is specifically as follows: 2.5 μl 10X PCR buffer, 0.5 μl dNTPs, 0.5 μl Taq enzyme, 1 μl of each primer, and 2 μl template DNA, with ddH2O added to a total volume of 25 μl;

[0082] The SRAP molecular marker primers are any pair of ME1-EM5, ME2-EM3, ME3-EM5, ME4-EM3, ME5-EM2, ME6-EM7, ME7-EM2, and ME8-EM2. Fluorescent primers are used for primer synthesis, and all amplified products are detected by fluorescence.

[0083] The PCR amplification program is as follows: PCR program: 94°C pre-denaturation for 5 min; 94°C denaturation for 1 min, 35°C annealing for 1 min, 72°C extension for 1 min, 5 cycles; 94°C denaturation for 1 min, 50°C annealing for 1 min, 72°C extension for 1 min, 35 cycles; after the cycle is completed, 72°C extension for 10 min, and storage at 4°C.

[0084] The specific method of step (3) is:

[0085] Sample gel images, obtained by scanning an automated sequencer 377, were analyzed using GENESCAN 3.1 software. Fragment size results were extracted using Binthere software. Excel conversion was used to convert non-zero values ​​to 1 (values ​​of 0 were not converted), thereby generating an original matrix composed of "1"s and "0"s. Data analysis was performed using NTSYSpc-2.11F software. The SimQual program was used to calculate the DICE similarity matrix for the original matrix and obtain the similarity matrix. Cluster analysis was performed using the UPGMA method in the SHAN program, and cluster plots were generated using the Tree Plot module.

[0086] 2 Results and Analysis

[0087] 2.1 SRAP primer screening

[0088] Four template samples were randomly selected to screen 400 primer combinations, of which 357 primer pairs were able to amplify bands. However, according to the amplification of polymorphic sites by the 400 primer combinations, as shown in some electrophoresis results ( Figures 11-16 ), not all primer pairs are suitable, and primer pairs with no amplified bands and few polymorphic sites need to be screened out. Finally, 8 primer pairs with multiple amplified bands, rich polymorphisms, high brightness, and good repeatability were screened out, namely SRAP molecular marker primers, which were used for genetic diversity analysis of 20 Pennisetum forage varieties. The SRAP molecular marker primers include 8 pairs of primers, which are:

[0089] ME1-EM5, the nucleotide sequences of which are shown in SEQ ID NO. 1 and SEQ ID NO. 25;

[0090] ME2-EM3, the nucleotide sequences of which are shown in SEQ ID NO. 2 and SEQ ID NO. 23;

[0091] ME3-EM5, the nucleotide sequences of which are shown in SEQ ID NO. 3 and SEQ ID NO. 25;

[0092] ME4-EM3, the nucleotide sequences of which are shown in SEQ ID NO. 4 and SEQ ID NO. 23;

[0093] ME5-EM2, the nucleotide sequences of which are shown in SEQ ID NO. 5 and SEQ ID NO. 22;

[0094] ME6-EM7, the nucleotide sequences of which are shown in SEQ ID NO. 6 and SEQ ID NO. 27;

[0095] ME7-EM2, the nucleotide sequences of which are shown in SEQ ID NO. 7 and SEQ ID NO. 22;

[0096] ME8-EM2, the nucleotide sequences of which are shown in SEQ ID NO.8 and SEQ ID NO.22.

[0097] 2.2 Genetic diversity analysis of 20 Pennisetum forage varieties using the selected SRAP molecular marker primers

[0098] 2.2.1 PCR amplification results

[0099] PCR amplification of 20 Pennisetum varieties using eight primer combinations yielded a total of 1,278 sites (as shown in Table 3), of which 1,273 were polymorphic, accounting for 99.6% of the total. Primer combination ME1-EM5 amplified the most polymorphic sites, 216, while primer combination ME3-EM5 amplified the fewest, 131. The average number of sites amplified was 159.8, and the average number of polymorphic sites was 159.1. This indicates that the selected SRAP primer combinations are extremely polymorphic in Pennisetum forages, making them suitable for genetic diversity analysis.

[0100] The gel electrophoresis results of PCR products of 20 samples using primer combinations ME1-EM5, ME2-EM3, ME3-EM5, ME4-EM3, ME5-EM2, ME6-EM7, ME7-EM2, and ME8-EM2 are shown in the figure. Figure 1-8 shown.

[0101] Table 3 Selected primer combinations to amplify site polymorphisms

[0102]

[0103] 2.2.2 Genetic similarity analysis

[0104] DICE genetic similarity coefficient (such as Figure 9 The genetic similarity coefficients for the 20 SRAP-marked accessions shown in the table above show that the similarity coefficients for the studied materials ranged from 0.74 to 0.84, indicating a certain degree of genetic variation. The lowest similarity coefficient was 0.7402 for Purple Elephant Grass and Guangxi King Grass, while the highest was 0.8459 for Pennisetum 2 and Pennisetum 4.

[0105] 2.2.3UPGMA cluster analysis

[0106] From the tree diagram (such as Figure 10 ) shows that the 20 Pennisetum varieties clustered into four groups at a DICE similarity coefficient of 0.79. The first group consisted of Minmu 6, Guimu 1, Jujuncao, Nanmu 1, and Wangcao. The second group consisted of Tainong 1, hybrid Pennisetum, slender hybrid Pennisetum, Taiwan sweet elephant grass, dwarf elephant grass, Pennisetum 2, Pennisetum 3, Pennisetum 4, new varieties of Wangcao, South African Pennisetum, and sugarcane. Guangxi Wangcao formed a separate third group. The fourth group included red elephant grass, purple elephant grass, and Pennisetum 1. Within this second largest group, four subgroups emerged: slender hybrid Pennisetum and Pennisetum 3 formed a single subgroup, dwarf elephant grass and hybrid Pennisetum each formed a separate subgroup, and the remaining varieties were grouped together in a single subgroup.

[0107] 3 Conclusion and Discussion

[0108] This study used the SRAP method to analyze the genetic diversity of 20 Pennisetum forage varieties. The results showed that the eight selected primer combinations detected a total of 1,278 loci, of which 1,273 were polymorphic, with a polymorphism ratio of 99.6%. These eight SRAP molecular marker primer pairs amplified numerous bands, exhibited rich polymorphisms, were bright, and had good reproducibility, making them suitable for genetic diversity analysis of Pennisetum forage varieties. Compared with the results of previous studies, the polymorphism ratio was significantly higher.

[0109] The DICE genetic similarity coefficient ranged from 0.7402 to 0.8459. Cluster analysis revealed that the top five genetic relationships, ranked from closest to least distant, were: Pennisetum 2 and Pennisetum 4, Red Elephant Grass and Pennisetum 1, the new King Grass variety and forage sugarcane, South African Pennisetum and forage sugarcane, and Guangxi King Grass and forage sugarcane. These relationships are relatively close, making interspecific hybridization studies unsuitable. These results can provide molecular insights for establishing Pennisetum forage resource nurseries and cultivating new varieties.

[0110] The new kinggrass variety under study is most closely related to forage sugarcane, and clusters with Tainong No. 1, hybrid pennisetum, slender hybrid pennisetum, Taiwan sweet elephant grass, dwarf elephant grass, and pennisetum 2. It is not in a large group with kinggrass and Guangxi kinggrass. The new kinggrass variety shares the same growing environment and climate as forage sugarcane, but differs significantly from kinggrass and Guangxi kinggrass. This phenomenon may be due to the fact that kinggrass and hybrid pennisetum are interspecific hybrids, and their genetic material is not very stable. Long-term growth in different environments and climates may lead to new genetic differentiation. Therefore, hybrids of different pennisetum varieties may tend to converge under the same environment and climate, while different environments and climates may cause genetic divergence within the same hybrid.

Claims

1. A SRAP molecular marker primer combination for analyzing the genetic diversity of Pennisetum forage grasses, characterized by: It consists of the following 8 pairs of primers: Primer pair 1: ME1-EM5, the nucleotide sequences of which are shown in SEQ ID NO. 1 and SEQ ID NO. 25; Primer pair 2: ME2-EM3, the nucleotide sequences of which are shown in SEQ ID NO. 2 and SEQ ID NO. 23; Primer pair 3: ME3-EM5, the nucleotide sequences of which are shown in SEQ ID NO. 3 and SEQ ID NO. 25; Primer pair 4: ME4-EM3, the nucleotide sequences of which are shown in SEQ ID NO. 4 and SEQ ID NO. 23; Primer pair 5: ME5-EM2, the nucleotide sequences of which are shown in SEQ ID NO. 5 and SEQ ID NO. 22; Primer pair 6: ME6-EM7, the nucleotide sequences of which are shown in SEQ ID NO. 6 and SEQ ID NO. 27; Primer pair 7: ME7-EM2, the nucleotide sequences of which are shown in SEQ ID NO. 7 and SEQ ID NO. 22; and Primer pair 8: ME8-EM2, the nucleotide sequences of which are shown in SEQ ID NO.8 and SEQ ID NO.

22.

2. A method for analyzing the genetic diversity of Pennisetum forage grasses using the SRAP molecular marker primer combination according to claim 1, characterized in that: The eight pairs of primers in the SRAP molecular marker primer combination according to claim 1 are used to amplify and analyze the DNA of the Pennisetum forage sample to be detected.

3. The method according to claim 2, wherein: The following steps are involved: (1) Extract the DNA of the sample to be tested using the CTAB method; (2) performing PCR amplification on the DNA extracted in step (1) using the eight pairs of primers to obtain PCR amplification products; (3) performing gel electrophoresis and imaging on the PCR amplification products obtained in step (2) to obtain corresponding band information; (4) Performing cluster analysis on the samples to be tested based on the spectral band information obtained in step (3) to generate a cluster diagram.

4. The method according to claim 3, wherein: The reaction system for PCR amplification in step (2) is specifically: 2.5 μL 10× PCR buffer, 0.5 μL dNTPs, 0.5 μL Taq enzyme, 1 μL each of the forward primer and the reverse primer of any one of the 8 pairs of primers, and 1 μL template DNA, and ddH2O is added to a total volume of 25 μL.

5. The method according to claim 4, characterized in that: The PCR amplification reaction procedure is as follows: pre-denaturation at 94°C for 5 min; 5 cycles of denaturation at 94°C for 1 min, annealing at 35°C for 1 min, and extension at 72°C for 1 min; 35 cycles of denaturation at 94°C for 1 min, annealing at 50°C for 1 min, and extension at 72°C for 1 min; and extension at 72°C for 10 min after the cycle is completed, followed by storage at 4°C.

6. The method according to claim 3, wherein: The specific method of step (4) is: The sample gel images obtained by scanning an automatic sequencer were analyzed using GENESCAN 3.1 software, and the fragment sizes of the samples were extracted using Binthere software. Values ​​other than 0 in the table were converted to 1 using Excel, while values ​​0 were not converted, thereby generating an original matrix composed of "1" and "0". Data analysis was performed using NTSYSpc-2.11F software. The DICE similarity coefficient matrix was calculated using the SimQual program for the original matrix, and a similarity coefficient matrix was obtained. Cluster analysis was performed using the UPGMA method in the SHAN program, and a cluster plot was generated using the Tree plot module.

7. Use of the SRAP molecular marker primer combination according to claim 1 in genetic diversity analysis of Pennisetum forage grasses.

8. A kit for analyzing the genetic diversity of Pennisetum forage grasses, characterized by: The invention comprises the SRAP molecular marker primer combination according to claim 1.

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