A primer set for mango SSR molecular markers and its application and use method

Abstract

The invention provides a primer set of mango SSR molecular markers and its application and use method, belonging to the field of molecular detection technology. The primer set includes a G676 primer pair, a G1028 primer pair, a G1352 primer pair, a G1382 primer pair, a G1666 primer pair, a G1097 primer pair, a G1745 primer pair, a G1151 primer pair and a G1077 primer pair. The primer set can be used to effectively perform genetic diversity analysis and cluster analysis on different mango varieties and germplasm resources. The primer set has the advantages of rich polymorphism, stable amplification, good repeatability, etc. It is the first SSR molecular marker developed based on the whole genome of Alphonso mango; the use of the primer set for population genetic diversity analysis has laid a foundation for the protection of genetic diversity of mango germplasm resources and the screening of kinship research.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular detection, and in particular relates to a primer set of mango SSR molecular markers and an application and use method thereof. Background Art

[0002] Mango belongs to the genus Mangifera in the Anacardiaceae family. It is an important tropical and subtropical fruit and is known as the "King of Tropical Fruits". With the rapid development of economic globalization, the cultivation and introduction of some new and excellent mango varieties have made mango an important agricultural pillar industry. However, there is a serious phenomenon of homonyms or homonyms among the varieties and germplasm resources preserved by various research institutes. In order to reduce the repeated introduction of varieties or germplasm resources or the phenomenon of arbitrary naming after introduction, it is urgent to develop a set of molecular markers to protect the genetic diversity of mango germplasm resources.

[0003] Simple sequence repeats (SSRs) are simple repeat sequences composed of 1-6 nucleotides and are widely distributed throughout the genome of eukaryotic organisms. Although SSRs are distributed in different locations throughout the genome, the sequences at both ends are mostly conserved single-copy sequences. A pair of specific primers are designed based on the sequences at both ends, and the core SSR sequence is amplified by PCR technology. The length polymorphism can be obtained using electrophoresis analysis technology. SSR markers have been widely used in molecular assisted breeding, genetic diversity analysis, germplasm resource identification and other research due to their good co-dominance, ability to distinguish homozygous and heterozygous genes, high polymorphism, good repeatability and good stability.

[0004] In order to carry out molecular biology SSR marker-assisted identification of mango germplasm resources, promote the breeding of mango varieties, accelerate the development and utilization of germplasm resources, and protect the genetic diversity of mango germplasm resources, it is urgent to develop SSR primers for mango to provide a scientific basis for research such as mango germplasm resource evaluation, breeding of excellent varieties, and construction of core germplasm banks.

[0005] Regarding the development of mango SSR markers, traditional methods such as genomic libraries were used in the early stage. However, due to the complex operation, high technical difficulty, and the small number of mango germplasm resources used, the development and application of SSR markers were limited. With the rapid development of sequencing projects, transcriptome sequences were used to develop SSR primers, but the PCR amplification specificity of the obtained SSRs was poor and the polymorphism was relatively low, which limited the application of this method. However, the mining of SSR sites based on the chromosome sequence of the mango genome is limited. At the same time, the SSR markers that have been developed have defects such as lack of chromosome location information and uneven distribution of SSR markers. Therefore, it is necessary to develop mango SSR markers at the whole genome level, which can provide a basis for future research on mango germplasm resources. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide a primer set of mango SSR molecular markers and its application and use method. Based on the mango genome (Alphonso), based on the large amount of SSR information obtained, the present invention verifies and analyzes the polymorphism of selected SSRs through PCR detection, thereby obtaining universal polymorphic primers, laying a foundation for mango core germplasm screening, kinship research, and excellent variety breeding.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a primer set for mango SSR molecular markers, the primer set comprising a G676 primer pair, a G1028 primer pair, a G1352 primer pair, a G1382 primer pair, a G1666 primer pair, a G1097 primer pair, a G1745 primer pair, a G1151 primer pair and a G1077 primer pair;

[0009] The forward primer sequence of the G676 primer pair is shown in SEQ ID NO.1, and the reverse primer sequence is shown in SEQ ID NO.2;

[0010] The forward primer sequence of the G1028 primer pair is shown in SEQ ID NO.3, and the reverse primer sequence is shown in SEQ ID NO.4;

[0011] The forward primer sequence of the G1352 primer pair is shown in SEQ ID NO.5, and the reverse primer sequence is shown in SEQ ID NO.6;

[0012] The forward primer sequence of the G1382 primer pair is shown in SEQ ID NO.7, and the reverse primer sequence is shown in SEQ ID NO.8;

[0013] The forward primer sequence of the G1666 primer pair is shown in SEQ ID NO.9, and the reverse primer sequence is shown in SEQ ID NO.10;

[0014] The forward primer sequence of the G1097 primer pair is shown in SEQ ID NO.11, and the reverse primer sequence is shown in SEQ ID NO.12;

[0015] The forward primer sequence of the G1745 primer pair is shown in SEQ ID NO.13, and the reverse primer sequence is shown in SEQ ID NO.14;

[0016] The forward primer sequence of the G1151 primer pair is shown in SEQ ID NO.15, and the reverse primer sequence is shown in SEQ ID NO.16;

[0017] The forward primer sequence of the G1077 primer pair is shown as SEQ ID NO.17, and the reverse primer sequence is shown as SEQ ID NO.18.

[0018] Preferably, all forward primers are connected to fluorescent markers; the fluorescent marker is one of FAM, HEX and ROX.

[0019] The invention provides application of the primer set in genetic diversity analysis of mango population.

[0020] The invention provides application of the primer set in analyzing the phylogenetic relationship of mango germplasm.

[0021] Preferably, the germplasm relationship is analyzed by UPMGA evolutionary tree.

[0022] The present invention also provides a method for analyzing the genetic diversity of a mango population, comprising the following steps:

[0023] (6.1) Extracting sample DNA;

[0024] (6.2) using the primer pairs in the primer set to perform PCR amplification on the DNA obtained in step (6.1) to obtain fluorescent PCR amplification products;

[0025] (6.3) performing capillary electrophoresis detection on the fluorescent PCR amplification product obtained in step (6.2), and counting the band detection results;

[0026] (6.4) Use the statistical band detection results in step (6.3) to analyze the genetic diversity of the mango population.

[0027] Preferably, the sample in step (6.1) is young mango leaves.

[0028] Preferably, the PCR amplification system in step (6.2) comprises the following components in 20 μL: 2 μL DNA template, 10 μL 2×Taq PCR Master Mix, 0.5 μL 10 pmol / μL forward fluorescent primer, 0.5 μL 10 pmol / μL reverse primer, and 7 μL ddH2O.

[0029] Preferably, the PCR amplification procedure in step (6.2) is as follows: pre-denaturation at 94°C for 30 s; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, for a total of 35 cycles; and finally extension at 72°C for 10 min.

[0030] Preferably, the specific steps of mango population genetic diversity analysis in step (6.4) are:

[0031] (10.1) According to the band detection results in step (6.3), record the amplified fragment size of each mango sample corresponding to each pair of primers;

[0032] (10.2) Arrange the amplified fragments, count the number of alleles, effective number of alleles, observed heterozygosity, expected heterozygosity, and Shannon information index of each site, and calculate the polymorphism information content.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] (1) The present invention develops a batch of mango SSR molecular markers, which solve the problems of lack of genetic background information of mango, limited number of EST-SSR markers developed, and lack of effectively available markers. It also provides sufficient information and available genomic SSR markers for the development of molecular markers for authenticity identification of mango hybrids.

[0035] (2) The prior art does not have SSR primer pairs developed for the whole genome of mango and multiple genetic resources. The present invention uses the whole genome of mango to design SSR primer pairs. The germplasm resources for genetic diversity analysis are large, and the sampled germplasm includes a small amount of wild germplasm. It has been verified that the primer pairs designed by the present invention have high accuracy, specificity, and strong stability, which lays a foundation for the protection of genetic diversity of mango germplasm resources, kinship research, and core germplasm screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Polyacrylamide gel electrophoresis for screening of SSR primers (primer pair G1382 and G1097) (1. Jinhuang, 2. Tainong 1, 3. Renong 1, 4. GeDong, 5. Keitt, 6. Sri Lanka 811, 7. Jinbaihua, 8. Yexiang, 9. Hongmang 6, 10. Haden, 11. Guiremang 82, 12. Qiumang);

[0037] Figure 2 The capillary electrophoresis diagram of G1382 on 12 mango varieties (1. Jinhuang, 2. Tainong No. 1, Keitt, 3. Renong No. 1, 4. GeDong, Haden, 5. Sri Lanka 811, Yexiang, 6. Jinbaihua, 7. Hongmang No. 6, 8. Guiremang No. 82, 9. Qiumang);

[0038] Figure 3 This is the UPMGA clustering result diagram of 221 individuals. DETAILED DESCRIPTION

[0039] The present invention provides a primer set for mango SSR molecular markers, the primer set comprising a G676 primer pair, a G1028 primer pair, a G1352 primer pair, a G1382 primer pair, a G1666 primer pair, a G1097 primer pair, a G1745 primer pair, a G1151 primer pair and a G1077 primer pair;

[0040] The forward primer sequence of the G676 primer pair is shown in SEQ ID NO.1, and is as follows:

[0041] 5'-CCCAGTTGAAAATATGGGTTCCC-3';

[0042] The reverse primer sequence of the G676 primer pair is shown in SEQ ID NO.2, and is as follows:

[0043] 5′-TCCACCGCTGAAACAACTCG-3′;

[0044] The forward primer sequence of the G1028 primer pair is shown in SEQ ID NO.3, and is as follows:

[0045] 5′-ATCCGGTGCTGATCATCAGG-3′;

[0046] The reverse primer sequence of the G1028 primer pair is shown in SEQ ID NO.4 and is as follows:

[0047] 5'-TCCTGGATGAAACCTTGGAGC-3';

[0048] The forward primer sequence of the G1352 primer pair is shown in SEQ ID NO.5, and is as follows:

[0049] 5′-GCTTGCAGCCATGAAGAACC-3′;

[0050] The reverse primer sequence of the G1352 primer pair is shown in SEQ ID NO.6, and is as follows:

[0051] 5'-AGCTGATAATCTGATGGAGCTGG-3';

[0052] The forward primer sequence of the G1382 primer pair is shown in SEQ ID NO.7, and is as follows:

[0053] 5′-TCCACGGTGGATGATGTAGC-3′;

[0054] The reverse primer sequence of the G1382 primer pair is shown in SEQ ID NO.8, and is as follows:

[0055] 5'-GAAGATTCAGCAAGCCAGACG-3';

[0056] The forward primer sequence of the G1666 primer pair is shown in SEQ ID NO.9, and is specifically as follows:

[0057] 5′-TATAACGCTGGACACGACGC-3′;

[0058] The reverse primer sequence of the G1666 primer pair is shown in SEQ ID NO.10, and is as follows:

[0059] 5'-GCCAAACCTTGACTTCATTACAGG-3';

[0060] The forward primer sequence of the G1097 primer pair is shown in SEQ ID NO.11, and is as follows:

[0061] 5′-TGGAAAGCCTTTGCAATCCC-3′;

[0062] The reverse primer sequence of the G1097 primer pair is shown in SEQ ID NO.12, and is as follows:

[0063] 5′-TCAATCTTGGGACCAGCACC-3′;

[0064] The forward primer sequence of the G1745 primer pair is shown in SEQ ID NO.13, and is as follows:

[0065] 5′-TGAGTGTGCCCAATGTTTGC-3′;

[0066] The reverse primer sequence of the G1745 primer pair is shown in SEQ ID NO.14, and is as follows:

[0067] 5′-CAAGCCCAAACCCAATCACC-3′;

[0068] The forward primer sequence of the G1151 primer pair is shown in SEQ ID NO.15, and is specifically as follows:

[0069] 5′-GTCAGATGTTGTCCGCATGC-3′;

[0070] The reverse primer sequence of the G1151 primer pair is shown in SEQ ID NO.16, and is as follows:

[0071] 5′-TCATCGTCCATTCCTGTGCC-3′;

[0072] The forward primer sequence of the G1077 primer pair is shown in SEQ ID NO.17, and is as follows:

[0073] 5′-AGTTTGGCACCTCTTGGAGC-3′;

[0074] The reverse primer sequence of the G1077 primer pair is shown in SEQ ID NO.18, and is as follows:

[0075] 5′-TTCCTCTTCACCCGAACTCG-3′.

[0076] In the present invention, all forward primers are connected to fluorescent markers; the fluorescent marker is one of FAM, HEX, and ROX, as shown in Table 1 below.

[0077] Table 1 Forward primer sequences for fluorescent labeling ligation

[0078]

[0079] The invention provides application of the primer set in genetic diversity analysis of mango population.

[0080] The invention provides application of the primer set in analyzing the phylogenetic relationship of mango germplasm.

[0081] In the present invention, the germplasm phylogenetic relationship is analyzed by constructing a UPMGA evolutionary tree based on Nei's distance, and the software used is PowerMarker V3.25.

[0082] The present invention also provides a method for analyzing the genetic diversity of a mango population, comprising the following steps:

[0083] (6.1) Extracting sample DNA;

[0084] (6.2) using the primer pairs in the primer set to perform PCR amplification on the DNA obtained in step (6.1) to obtain fluorescent PCR amplification products;

[0085] (6.3) performing capillary electrophoresis detection on the fluorescent PCR amplification product obtained in step (6.2), and counting the band detection results;

[0086] (6.4) Use the statistical band detection results in step (6.3) to analyze the genetic diversity of the mango population.

[0087] In the present invention, DNA of a sample is extracted using a conventional commercially available kit; the sample is young mango leaves.

[0088] In the present invention, the primer pairs in the primer set are used to perform PCR amplification on the DNA obtained in step (6.1) to obtain fluorescent PCR amplification products. The PCR amplification system, in 20 μL, includes the following components: 2 μL DNA template, 10 μL 2×Taq PCRMasterMix, 0.5 μL 10 pmol / μL forward fluorescent primer, 0.5 μL 10 pmol / μL reverse primer, 7 μL ddH2O. The PCR amplification procedure is as follows: 94°C pre-denaturation for 30 s; 94°C denaturation for 30 s, 58°C annealing for 30 s, 72°C extension for 30 s, for a total of 35 cycles; and finally 72°C extension for 10 min.

[0089] In the present invention, the fluorescent PCR amplification product obtained in step (6.2) is subjected to capillary electrophoresis detection, and the band detection results are counted. The capillary electrophoresis data are read using GeneMarker V2.2.0 software.

[0090] In the present invention, the statistical band detection results in step (6.3) are used to perform genetic diversity analysis of mango populations. The specific steps of the genetic diversity analysis of mango populations are:

[0091] According to the band detection results in step (6.3), the size of the amplified fragment of each mango sample corresponding to each pair of primers is recorded;

[0092] The amplified fragments were sorted, and the number of alleles, effective number of alleles, observed heterozygosity, expected heterozygosity, and Shannon information index of each site were counted to calculate the polymorphism information content.

[0093] In the present invention, according to the band detection results in step (6.3), the amplified fragment size of each mango sample corresponding to each pair of primers is recorded. A DNA sequencer ABI3730XL is used for band detection, and GeneMarkerv2.2.0 is used to read the sample fragment size and the number of alleles.

[0094] In the present invention, the amplified fragments are sorted, the number of alleles, the effective number of alleles, the observed heterozygosity, the expected heterozygosity, and the Shannon information index of each site are counted, and the polymorphism information content is calculated. The number of alleles, the effective number of alleles, the observed heterozygosity, the expected heterozygosity, and the Shannon information index of each site are counted using GenALEx 6.2 software, and the polymorphism information content is calculated using PowerMarker V3.25. Wherein the greater the heterozygosity, the expected heterozygosity, and the polymorphism information content index, the higher the polymorphism, and the polymorphism information content value is greater than 0.6, indicating that the primer is a highly polymorphic primer.

[0095] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0096] Example 1

[0097] Using the existing mango (Alphonso) whole genome sequence, the Krait software (https: / / github.com / lmdu / krait / ) was used to scan and screen SSR sites, and a large number of SSR sites were obtained. The screening criteria were: mononucleotides were repeated at least 12 times, dinucleotides were repeated at least 7 times, and tri-, tetra-, penta-, and hexa-nucleotides were repeated at least 5 times to be identified as SSR sites. Primer 5 software was used to design primers for the obtained SSR sites, and the parameters were set as follows: primer length 18-27bp; annealing temperature 55-65℃; PCR product length range between 100bp-300bp; GC content between 40% and 60%.

[0098] In this way, 187 pairs of SSR primers were successfully designed. The amplification efficiency and polymorphism of primers were studied using 12 samples, and the screening criteria were: amplification efficiency higher than 80% and alleles ≥ 3. From the 187 pairs of primers, 35 pairs of primers that can stably amplify clear and polymorphic bands were screened out. The 35 pairs of primers were further used to verify 221 varieties, and finally 9 pairs of primers were screened out. The primer sequences are shown in Table 2 and were commissioned to be synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0099] Table 2 Primer sequences

[0100]

[0101]

[0102] Example 2

[0103] The SSR primers synthesized based on Example 1 can be applied to the genetic diversity analysis of mango population and the germplasm kinship analysis, and the analysis method comprises the following steps:

[0104] (1) Extract genomic DNA from mango samples to be tested; 221 mango samples (Table 3) were used to verify the effectiveness of the developed SSR markers. The samples were young mango leaves, and 5 individual plants were selected for each sample.

[0105] Healthy young leaves (young leaves) of 221 materials were selected, and their DNA was extracted using the CTAB Plant Genomic DNA Rapid Extraction Kit (purchased from Beijing Aidelai Biotechnology Co., Ltd.). The DNA concentration and purity of the test materials were detected using a Nanodrop nucleic acid analyzer and 1% agarose gel electrophoresis, and the qualified DNA samples were stored at -20°C for later use.

[0106] (2) According to the corresponding fluorescent labeling group in Table 2, the forward primer of each pair of primers is fluorescently labeled to obtain a forward fluorescent primer;

[0107] (3) Using the genomic DNA of the sample to be tested extracted in step (1) as a template, PCR amplification was performed with a reverse primer and a forward primer to obtain a fluorescent PCR amplification product; wherein the PCR amplification system is as follows: 2 μL genomic DNA, 10 μL 2×TaqPCRMasterMix, 0.5 μL 10 pmol / μL forward primer with a fluorescent modification group (ROX, HEX or FAM), 0.5 μL 10 pmol / μL reverse primer, 7 μL ddH2O. The PCR amplification reaction procedure is: 94°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 58°C annealing for 30 s, 72°C extension for 30 s, a total of 35 cycles; and finally 72°C extension for 10 min.

[0108] (4) The fluorescent PCR amplification product obtained in step (3) was detected by capillary fluorescence electrophoresis using a DNA sequencer ABI3730XL, and the sample fragment size and the number of alleles were read using GeneMarker v2.2.0, and the band detection results were counted.

[0109] (5) The sizes of the amplified fragments were comprehensively sorted out, and the number of alleles (Na), effective number of alleles (Ne), observed heterozygosity (Ho), and expected heterozygosity (He) at each site were counted using GenAlex6.2 software. The polymorphism information content (PIC) and the genetic distance between different mango varieties were calculated using PowerMarkerV3.25. A UPMGA evolutionary tree based on Nei's distance was constructed.

[0110] Table 3 Mango sample number and variety name

[0111]

[0112]

[0113]

[0114] Depend on Figure 1 It can be seen that primers G1382 and G1097 are polymorphic in 12 mango varieties.

[0115] Depend on Figure 2It can be seen that the G1382 primer detected 9 genotypes in 12 mango varieties. At the G1382 locus, the genotype of Jinhuang was 113 / 119, the genotype of Tainong No. 1 and Keitt was 113 / 116, the genotype of Renong No. 1 was 107 / 107, the genotype of GeDong and Haden was 110 / 113, the genotype of Sri Lanka 811 and Yexiang was 110 / 116, the genotype of Jinbaihua was 116 / 119, the genotype of Hongmang No. 6 was 107 / 110, the genotype of Guiremang No. 82 was 107 / 116, and the genotype of Qiumang was 116 / 116.

[0116] Table 4 Statistics of genetic polymorphism information of 221 mango materials using 9 pairs of SSR polymorphic primers

[0117]

[0118] As shown in Table 4, 9 pairs of primers amplified 65 polymorphic bands, the average number of alleles per primer pair was 7.22, and the effective allelic variation ranged from 3.19 to 4.92. Observed heterozygosity, expected heterozygosity, and polymorphic information content index are all indicators reflecting primer polymorphism. The larger the value, the higher the polymorphism. The average values ​​of the three indicators are 0.75, 0.74, and 1.49, respectively, indicating that the genetic diversity among mango varieties is very rich. The SSR primer set developed in the present invention can be applied to mango genetic diversity analysis and germplasm pedigree analysis. The PIC values ​​of the primers are between 0.63 and 0.77, and the PIC values ​​of all primers are greater than 0.6, indicating that these primers are highly polymorphic primers.

[0119] To further verify the application of this primer set in mango genetic diversity, cluster analysis was performed on 221 mango samples. Figure 3 The results of UPMGA cluster analysis showed that all mango samples could be divided into two categories: I and II according to different genetic distances. The first category (I) included some mango varieties from Thailand, Southeast Asia, and China. The second category (II) included some mango varieties from India, Australia, the United States, and China.

[0120] from Figure 3 According to the cluster analysis results, varieties such as 67 (Red Mango), 37 (Sensation), 161 (Xingre 2) and 104 (Hongmang 6) are clustered together. These varieties are all descendants of Haden and have a close relationship. The three varieties 184 (507), 182 (101) and 185 (Bamang) are clustered together, indicating that they are closely related; several Australian varieties such as 170 (KRS), 164 (Bambaroo), 11 (Kensington) and 191 (Spooner) are clustered together, indicating that they are closely related.

[0121] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A primer set for mango SSR molecular markers, characterized in that: The primer set includes a G676 primer pair, a G1028 primer pair, a G1352 primer pair, a G1382 primer pair, a G1666 primer pair, a G1097 primer pair, a G1745 primer pair, a G1151 primer pair and a G1077 primer pair; The forward primer sequence of the G676 primer pair is shown in SEQ ID NO.1, and the reverse primer sequence is shown in SEQ ID NO.2; The forward primer sequence of the G1028 primer pair is shown in SEQ ID NO.3, and the reverse primer sequence is shown in SEQ ID NO.4; The forward primer sequence of the G1352 primer pair is shown in SEQ ID NO.5, and the reverse primer sequence is shown in SEQ ID NO.6; The forward primer sequence of the G1382 primer pair is shown in SEQ ID NO.7, and the reverse primer sequence is shown in SEQ ID NO.8; The forward primer sequence of the G1666 primer pair is shown in SEQ ID NO.9, and the reverse primer sequence is shown in SEQ ID NO.10; The forward primer sequence of the G1097 primer pair is shown in SEQ ID NO.11, and the reverse primer sequence is shown in SEQ ID NO.12; The forward primer sequence of the G1745 primer pair is shown in SEQ ID NO.13, and the reverse primer sequence is shown in SEQ ID NO.14; The forward primer sequence of the G1151 primer pair is shown in SEQ ID NO.15, and the reverse primer sequence is shown in SEQ ID NO.16; The forward primer sequence of the G1077 primer pair is shown as SEQ ID NO.17, and the reverse primer sequence is shown as SEQ ID NO.

18.

2. The primer set according to claim 1, characterized in that All forward primers are connected to fluorescent markers; the fluorescent marker is one of FAM, HEX, and ROX.

3. Use of the primer set described in claim 1 or 2 in the analysis of genetic diversity of mango population.

4. Use of the primer set described in claim 1 or 2 in analyzing the genetic relationship of mango germplasm.

5. The use according to claim 4, characterized in that: The germplasm relationships were analyzed using the UPMGA phylogenetic tree.

6. A method for analyzing the genetic diversity of mango populations, characterized in that: The following steps are involved: (6.1) Extracting sample DNA; (6.2) using the primer pairs in the primer set of claim 2 to perform PCR amplification on the DNA obtained in step (6.1) to obtain fluorescent PCR amplification products; (6.3) performing capillary electrophoresis detection on the fluorescent PCR amplification product obtained in step (6.2), and counting the band detection results; (6.4) Use the band detection results counted in step (6.3) to analyze the genetic diversity of the mango population.

7. The method according to claim 6, characterized in that The sample described in step (6.1) is young mango leaves.

8. The method according to claim 6, characterized in that The PCR amplification system in step (6.2) is calculated as 20 μL and includes the following components: 2 μL DNA template, 10 μL 2×Taq PCR Master Mix, 0.5 μL 10 pmol / μL forward fluorescent primer, 0.5 μL 10 pmol / μL reverse primer, and 7 μL ddH2O.

9. The method according to claim 6, characterized in that The PCR amplification procedure in step (6.2) is as follows: pre-denaturation at 94°C for 30 s; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, for a total of 35 cycles; and finally extension at 72°C for 10 min.

10. The method according to claim 6, characterized in that The specific steps of mango population genetic diversity analysis in step (6.4) are: (10.1) According to the band detection results in step (6.3), record the size of the amplified fragment of each mango sample corresponding to each pair of primers; (10.2) Arrange the amplified fragments, count the number of alleles, effective number of alleles, observed heterozygosity, expected heterozygosity, and Shannon information index of each site, and calculate the polymorphism information content.

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