Areca InDel marker and application thereof in provenance detection

Abstract

The invention relates to the technical field of molecular biology, in particular to a betel nut InDel marker and application thereof in provenance detection. According to the invention, on the basis of the results of re-sequencing of one betel nut germplasm resource and comparative analysis of a reference genome sequence, InDel sites are excavated to develop molecular markers, 46 pairs of InDel molecular marker primer pairs with high polymorphism are obtained, the variation range of Shannon's diversity index is 0.361-1.713, and the variation range of polymorphism information content is 0.201-0.752. The clustering analysis result shows that the genetic similarity coefficient is 0.36, 211 parts of areca-nut materials are divided into four groups, the InDel marker pair provided by the invention can be effectively used for detecting and analyzing the genetic background of areca-nut germplasm resources, the genetic relationship between the tested areca-nut materials is accurately identified, and the method has the advantages of high specificity, high accuracy and high accuracy. And a foundation is laid for genetic diversity analysis of betel nut germplasm resources and detection of seed fruit and seedling sources.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular biology, in particular to a Areca InDel marker and its application in provenance detection. BACKGROUND

[0002] Areca has a long history of cultivation in Hainan, and is a perennial palm tree. It takes 4-5 years to flower and bear fruit after cultivation. In recent years, with the development of the Areca industry, the market demand for Areca seedlings has increased. Since the local Areca in Hainan has good fresh fruit quality and high market price, there are fewer seed fruits left, and the price of seed fruits is high. Areca seedling production is in a spontaneous state, and the source of seedlings cannot be monitored. In the case of a serious shortage of seed fruits and seedlings, seed fruits and seedlings from neighboring Southeast Asian countries such as Vietnam and Cambodia flow into the domestic seedling market through various channels. Such seed fruits and seedlings are cheap, and the fruits do not meet the characteristics of Hainan commodity fruits, seriously disrupting the Hainan Areca seedling market. It is difficult to identify different varieties of Areca seedlings, and after a few years of planting, it is found that the fruits do not meet the requirements of commodity fruits, and can only be used as secondary fruits or no one will buy them, seriously damaging the interests of farmers. In this study, an InDel marker was developed to detect the genotypes of Areca germplasm resources, and genetic clustering analysis of Areca germplasm resources from domestic and foreign sources was carried out to provide technical support for early detection of the source of Areca seedlings.

[0003] InDel markers (Insertion-Deletion) are developed on the basis of whole genome resequencing, and are polymorphic variations produced by the insertion or deletion of a short sequence at the allelic site. Two samples have a certain number of differences in the insertion or deletion of nucleotides in the whole genome. According to the insertion or deletion site in the genome, PCR primers that amplify these sites are designed, which are InDel markers. Compared with SSR markers, the amplification product of InDel markers has clear and simple banding, and the stability and product separation effect are better than those of SSR markers. InDel markers based on whole genome resequencing have the characteristics of wide distribution in the genome, strong polymorphism, easy detection, simple typing system, and genetic stability. They have been successfully applied to the fields of plant and animal population genetic analysis, construction of molecular marker genetic maps, and genetic diversity analysis. InDel molecular markers have been applied in the genetic research and variety identification of crops such as rice, corn, and cotton, and there are few reports on the application of InDel molecular markers in Areca germplasm resource-related research. SUMMARY

[0004] Therefore, the present application solves the technical problem of providing an Areca InDel marker and its application in provenance detection. The present application provides an InDel molecular marker primer set for Areca based on whole genome resequencing.

[0005] The application provides an InDel molecular marker primer set of Areca catechu, which has the following characteristics:

[0006] (1) a nucleotide sequence as shown in any of SEQ ID NO: 1-SEQ ID NO: 92; and / or

[0007] (2) a nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases of the nucleotide sequence shown in (1); and / or

[0008] (3) a sequence having at least 80% homology with the nucleotide sequence shown in (1); and / or

[0009] (4) a complementary sequence of the sequence shown in (1), (2) or (3).

[0010] In some embodiments, the amplification primer set comprises at least one of the following combinations:

[0011] Combination X:

[0012] (5) the upstream primer has a nucleotide sequence as shown in SEQ ID NO: (2X-1), and the downstream primer has a nucleotide sequence as shown in SEQ ID NO: (2X); or

[0013] (6) a nucleotide sequence obtained by substituting, deleting or adding one or more bases of the nucleotide sequence shown in (5), and a nucleotide sequence having the same or similar function as the nucleotide sequence shown in (5); or

[0014] (7) a nucleotide sequence having at least 80% homology with the nucleotide sequence shown in (5) or (6);

[0015] wherein X is selected from any integer from 1 to 46.

[0016] In some specific embodiments, the InDel molecular marker primer set of Areca catechu comprises combinations 1-combinations 46.

[0017] The application provides a detection reagent comprising the InDel molecular marker primer set of Areca catechu and an acceptable adjuvant.

[0018] In some embodiments, the adjuvant comprises reagents required for PCR amplification and / or electrophoresis.

[0019] The application provides a detection kit comprising the InDel molecular marker primer set of Areca catechu and / or the detection reagent.

[0020] The application provides a gene chip comprising the InDel molecular marker primer set of Areca catechu and / or the detection reagent.

[0021] The application provides application of the InDel molecular marker primer set of Areca catechu, the detection reagent, the detection kit and / or the gene chip in construction of a genetic map of Areca catechu.

[0022] The application provides application of the InDel molecular marker primer set of Areca catechu, the detection reagent, the detection kit and / or the gene chip in genetic diversity analysis of Areca catechu.

[0023] The application provides application of the InDel molecular marker primer set of Areca catechu, the detection reagent, the detection kit and / or the gene chip in analysis of Areca catechu germplasm resources.

[0024] The application provides an analysis method for genetic diversity of Areca catechu, and is characterized by comprising the following steps:

[0025] Step 1, extracting genomic DNA of a to-be-tested Areca catechu sample;

[0026] Step 2, performing PCR amplification on the genomic DNA in step 1 by using the InDel molecular marker primer set of Areca catechu, the detection reagent, the detection kit and / or the gene chip;

[0027] Step 3, performing genetic diversity analysis according to the result of the PCR amplification in step 2 to obtain a genetic diversity analysis result of the to-be-tested Areca catechu sample.

[0028] The application is based on whole genome resequencing analysis results of one Areca catechu germplasm resource of known origin, and InDel sites are mined to develop molecular markers, 46 pairs of InDel molecular marker primer pairs with high polymorphism are obtained, the Shannon's diversity index changes in the range of 0.361-1.713, and the polymorphism information content changes in the range of 0.201-0.752. The clustering analysis result shows that 211 Areca catechu materials are divided into four groups at a genetic similarity coefficient of 0.36, and 31 samples of unknown origin are detected by using the 46 pairs of InDel markers, and clustering analysis is performed in combination with 211 samples of known origin, which proves that the InDel markers provided by the application can be used for genetic background detection and analysis of Areca catechu germplasm resources, and accurately identify the genetic relationship between the tested Areca catechu materials, and lay a foundation for genetic diversity analysis of Areca catechu germplasm resources and source detection of seeds, fruits and seedlings. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 An amplification spectrum of part of InDel primers of chromosome 15 is screened by using 8 samples;

[0030] Figure 2Genetic cluster analysis of 211 Areca germplasm resources based on 46 InDel markers

[0031] Figure 3 Genetic cluster analysis of 242 Areca germplasm resources based on 46 InDel markers

[0032] Figures 4 to 7 The results of PCR amplification and capillary electrophoresis of the Areca samples numbered 213, 214 and 215 in Table 1 by primers AC202, primer AC224, primer AC310 and primer AC422 in turn. DETAILED DESCRIPTION

[0033] The present application provides Areca InDel markers and their application in germplasm detection, and those skilled in the art can refer to the content herein to appropriately improve the process parameters. It is particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The methods and applications of the present application have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0034] The test materials used in the present application are all ordinary commercially available products and can be purchased in the market. The present application is further described below in combination with examples.

[0035] Example 1

[0036] 1. Materials and methods

[0037] 1.1 Experimental materials

[0038] The test materials include Areca germplasm resources collected at home and abroad and cultivated Areca in Hainan and various places, foreign materials mainly come from Vietnam, Indonesia, Sri Lanka, Fiji, Micronesia, etc., as well as Areca materials from Taiwan, Yunnan and cities and counties in Hainan Province of China; the materials from Hainan are called local species, and the materials from Yunnan, Taiwan of China and abroad are collectively called exotic species. The 242 Areca germplasm materials for testing are shown in Table 1. Among them, samples numbered 1-211 are known source samples, 212-226 are smuggled Areca fruits seized by Guangdong Customs, which are to be detected, and 227-242 are to be detected samples collected from Wenchang nursery.

[0039] Table 1 Information of Areca germplasm resources materials for testing

[0040]

[0041]

[0042] The DNA of Areca catechu was extracted as follows: leaf or fruit samples were collected, about 1 g was weighed, and was ground into powder with liquid nitrogen in a mortar. The genomic DNA of the leaf was extracted by using a plant genomic DNA rapid extraction kit (Shanghai Genechem Co., Ltd.), and was dissolved in 100 μL TE buffer after being dried. 1 μL of the DNA stock solution was used to detect the mass and concentration of the extracted DNA by using a NanoDrop 2000 micro-ultraviolet spectrophotometer. The working solution was diluted to 20 ng / μL. The DNA stock solution and the working solution were stored in a -20 °C refrigerator for standby use.

[0043] 1.2 Experimental method

[0044] 1.2.1 InDel primer design and screening

[0045] According to the alignment analysis of the results of genome sequencing, there were a large number of InDel site differences between the long-elliptical fruit shape resources of Areca catechu and the reference genome. According to the InDel sites predicted in the re-sequencing data, the sites with an insertion / deletion base number of 10-50 bp were screened. According to the InDel sites discovered by sequence alignment, 420 InDel sites were selected for primer design according to their distribution on the genome. The primers designed for chromosome 15 are shown in Table 2 (as the primers of 420 pairs occupy a large space, only the primer information designed for chromosome 15 is listed). According to the genome sequence of Areca catechu, the selected sites were located on the genome, and 200 bp of base length on both sides of the InDel site was taken, a total of 400 bp length was used for primer design by using Primer 5.0. The upstream primer design range was 1-180 bp, the downstream primer design range was 220-400 bp, the product size was 100-400 bp, the GC content was 40%-60%, the annealing temperature was 55 °C-65 °C, and the primer length was set to 18-22 bp.

[0046] The test used a 15 μL reaction system, i.e. 15 μL reaction system containing dNTPs 0.15 mmol / L, Mg 2+ Buffer 1.5 mmol / L, Taq DNA polymerase 0.75 U, primer 0.3 μmol / L, and template DNA amount of 20 ng. The PCR reaction program was as follows: 94 °C pre-denaturation for 5 min; 94 °C denaturation for 30 s, 56 °C annealing and recombination for 30 s, 72 °C extension for 30 s, 33 cycles; 72 °C extension for 7 min, and storage at 4 °C. The amplification product was subjected to 8% non-denaturing polyacrylamide gel electrophoresis, silver staining, and color development. The gel was observed under a film observation lamp, and the band type was analyzed.

[0047] The synthesized primers were subjected to PCR detection by using 8 Areca catechu germplasm resource samples from different geographical sources. The PCR amplification gel running results of some primers for chromosome 15 are shown in Figure 1.Figure 1 The primers with clear band polymorphism were screened for genetic cluster analysis. As shown in Table 2, the primer pairs AC1515, AC1516 and AC1519 can all amplify specific bands with clear bands and can be used for subsequent cluster analysis. According to the above method, 420 pairs of primers were screened, and 46 pairs of primers with clear bands and good polymorphism were obtained (Table 3), which were used to detect the tested Areca germplasm resources. Figure 1

[0048] Table 2 InDel marker information of chromosome 15

[0049]

[0050]

[0051] Table 3 46 pairs of InDel marker information

[0052]

[0053]

[0054] 1.2.2 PCR amplification and capillary electrophoresis method

[0055] Using the extracted Areca genomic DNA as the template, PCR amplification was performed using the InDel fluorescence labeling detection technology. When the primers were used for PCR amplification, the 5' end of the upstream primer with a fluorescent group and the downstream primer directly amplified the DNA template to obtain PCR products with a fluorescent group.

[0056] The PCR amplification system was (10 μL): 20 ng of template DNA 1 μL, 0.5 μL of forward and reverse primers (10 umol / L) each, 5.0 μL of 2x Taq PCR Master Mix, and ddH2O to make up 10 μL. The PCR amplification program was set as: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 62-52°C gradient annealing for 30 s, 72°C extension for 30 s, running for 10 cycles; 95°C denaturation for 30 s, 52°C annealing for 30 s, 72°C extension for 30 s, running for 25 cycles; 72°C extension for 20 min, and finally 4°C storage; the reaction was performed on a Veriti384 PCR instrument.

[0057] ​After the PCR amplification products were diluted to the same concentration of fluorescent PCR products, capillary electrophoresis was used, and the following system was added to the detection reagent. Capillary electrophoresis fluorescence detection: after the PCR products were diluted, 1 μL of the diluted solution was added to 8.5 μL of deionized formamide, and 0.5 μL of LIZ-500 molecular weight marker. After the system was prepared, it was placed on the ABI 3730xL genetic analyzer for capillary electrophoresis detection.

[0058] The.fsa format raw data was exported from the ABI 3730xL genetic analyzer, classified and archived according to the detection site, and then imported into the GeneMarker analysis software for genotype data reading. The position of each peak was automatically compared with the LIZ-500 molecular weight marker in its lane to obtain the fragment size (bp) of the amplification product, and the Excel genotype raw data and PDF typing peak chart file were exported according to the site name.

[0059] 2. Experimental results

[0060] 2.1 Primer polymorphism analysis

[0061] After PCR amplification and capillary electrophoresis detection, the genetic parameters of each site were calculated using GenAlEx6.5 software, including the number of alleles, the number of effective alleles, the observed heterozygosity, the expected heterozygosity, and the Shannon's information index. The polymorphic information content of each site was calculated using PowerMaker V3.25 software, and the UPGMA (Unweighted pair group method with arithmetic mean) clustering analysis circular diagram was constructed using the online tool ITOL based on the genetic distance calculated by Powermarker.

[0062] The genotypes of 211 palm germplasm resources of known origin were detected using 46 pairs of InDel markers, and the genetic diversity of palm germplasm resources was analyzed (Table 4). The number of alleles (Na) ranged from 2 to 11, and the effective number of alleles (Ne) ranged from 1.261 to 4.648. The Shannon's diversity index ranged from 0.361 to 1.713, the polymorphic information content ranged from 0.201 to 0.752, and the genetic parameters of marker AC1516 were all the highest. Only 3 pairs of primers had a polymorphic information content less than 0.25, indicating that the InDel markers used in this study had high polymorphism in the tested palm germplasm resources.

[0063] Table 4 Polymorphism data statistics of 46 pairs of InDel primers

[0064]

[0065]

[0066] 2.2 Genetic and Evolutionary Analysis of Areca Nut Germplasm Resources

[0067] Genetic cluster analysis was performed on 211 germplasm resources from known sources using 46 pairs of InDel markers. Figure 2 ),Depend on Figure 2 It can be seen that at a genetic distance of 0.36, the tested population materials were divided into four major groups. Group I (light pink outer ring) included 9 materials, originating from Papua New Guinea and Taiwan, China, all of which were introduced species. Group II (light green outer ring) included 32 materials, originating from Fiji (6), Sri Lanka (12), Micronesia (3), Cambodia (1), Indonesia (3), Vietnam (6), and Yunnan (1), all of which were introduced species. Group III (light purple outer ring) included 24 materials from Yunnan, all of which were introduced species. Group IV (light blue outer ring) included 146 materials, originating from Hainan (144), Sri Lanka (1), and Yunnan (1). As shown in the figure, areca nut materials from the same countries and regions can be basically clustered together. Hainan native species are all clustered in Group IV, while introduced species are mainly clustered in Groups I, II, and III, showing a clear distinction from Hainan native species.

[0068] Areca nut cultivation in Hainan has a history of over two thousand years. Over this long period, distinctive local varieties have developed, with the Hainan native variety being the most widely cultivated. The Hainan native variety produces oval or egg-shaped fruits with good processing suitability, and over 90% is processed into dried fruit for consumption. In my country, the edible part of processed areca nut is the pulp. The Hainan native variety, especially the oval-shaped fruit, has thick pulp that is chewy and durable. In other countries, areca nut is primarily consumed or used medicinally from the kernel, with a preference for larger kernels and thinner pulp. Due to different breeding objectives, there are genotypic differences between the Hainan native variety and cultivated areca nuts abroad. In international areca nut germplasm resource research, India began morphological evaluation of collected germplasm resources in 1957. One cultivar introduced from China exhibited excellent characteristics such as early maturity and high yield and was widely cultivated. Sri Lanka, geographically close to India, has a material from Sri Lanka in the fourth major Hainan native species cluster, which may have been introduced from India. Areca nut has a history of introduction and exchange, which may explain why some samples from Sri Lanka and Yunnan cluster together with native Hainan species. However, there is a clear distinction in genetic clustering between native Hainan species and introduced species.

[0069] 2.3 Analysis of genetic diversity of areca nut germplasm resources and estimation of seedling origin

[0070] Genotyping was performed on 31 samples of unknown origin using 46 pairs of InDel markers. The results were then combined with those from 211 germplasm resources of known origin for genetic cluster analysis. Figure 3 As shown in the figure, the 242 samples were divided into four major groups. Group I (light pink outer circle) included 9 samples, originating from Papua New Guinea and Taiwan, China. Group II (light green outer circle) included 24 samples, all originating from Yunnan, China. Group III (light purple outer circle) included 31 samples from known foreign sources and 1 sample from Yunnan, all of which were introduced species. It also included 15 samples from customs sources (red text), which clustered with samples from Vietnam and Cambodia, suggesting that these customs-originating samples were introduced species, most likely from Vietnam or Cambodia. Group IV (light blue outer circle) included 144 samples of Hainan native species and 16 samples from the Wenchang nursery, which clustered with Hainan native species, suggesting that these samples were all Hainan native species. After confirmation with the Wenchang nursery owner, the fruits of these samples all came from a Hainan native areca nut orchard, proving the effectiveness of our testing method. This study demonstrates that the InDel marker developed can be well applied to the detection of genetic background in areca germplasm resources, accurately identify the genetic relationships among the tested areca materials, and lay the foundation for the analysis of genetic diversity of areca germplasm resources and the detection of the origin of seeds and seedlings.

[0071] Using Powermarker software, genotype data from 242 samples were obtained through molecular marker detection based on 46 InDel marker pairs. Genetic distances between samples were calculated, and 17 representative materials from different countries and regions were selected for analysis (Table 5). Sample 1 from Taiwan, China, had the greatest genetic distance from samples 91 and 96, both of which originated from Hainan. Samples with relatively small genetic distances included 91 and 124, 96 and 129, and 91 and 150. These samples came from different regions of Hainan, indicating that the genetic distances between the tested materials from Hainan were relatively close.

[0072] Table 5. Genetic distances of 17 areca nut germplasm resources

[0073] 1 7 10 16 29 32 33 36 42 68 91 96 124 129 150 212 227 1 0 7 0.312 0 10 0.401 0.307 0 16 0.401 0.520 0.395 0 29 0.406 0.298 0.267 0.393 0 32 0.431 0.368 0.287 0.335 0.212 0 33 0.413 0.334 0.273 0.354 0.232 0.284 0 36 0.411 0.352 0.280 0.373 0.286 0.276 0.362 0 42 0.522 0.471 0.450 0.341 0.304 0.365 0.330 0.506 0 68 0.502 0.377 0.379 0.427 0.347 0.370 0.301 0.447 0.331 0 91 0.550 0.480 0.443 0.374 0.345 0.312 0.295 0.391 0.374 0.221 0 96 0.558 0.327 0.367 0.452 0.318 0.375 0.332 0.356 0.349 0.188 0.204 0 124 0.498 0.441 0.386 0.358 0.323 0.318 0.320 0.425 0.343 0.234 0.147 0.241 0 129 0.515 0.360 0.365 0.346 0.338 0.366 0.301 0.373 0.327 0.200 0.242 0.158 0.236 0 150 0.482 0.384 0.375 0.406 0.267 0.329 0.327 0.423 0.290 0.253 0.195 0.230 0.224 0.231 0 212 0.441 0.418 0.319 0.289 0.284 0.251 0.337 0.291 0.349 0.359 0.321 0.404 0.281 0.342 0.299 0 227 0.531 0.474 0.462 0.310 0.417 0.400 0.401 0.418 0.340 0.240 0.262 0.285 0.245 0.241 0.317 0.340 0

[0074] PCR amplification and capillary electrophoresis were performed on the test samples using 46 primer pairs. The test samples included the 31 samples listed in Table 1, and the primers were all those listed in Table 3. Due to space limitations, this invention only lists the results of PCR amplification and capillary electrophoresis of areca nut samples numbered 213, 214, and 215 in Table 1 using primers AC202, AC224, AC310, and AC422, as follows: Figures 4 to 7 As shown ( Figure 4AC2.02 in the table corresponds to primer AC202 in Table 3. The number before the "." in AC2.02 is the chromosome number, and the number after the "." is the primer number. This format was used in the images for easy identification and statistical analysis during the experiment. Figures 5 to 7 Similarly.

[0075] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An InDel molecular marker primer set for areca nut, characterized in that, It has the following characteristics: (1) Any nucleotide sequence as shown in SEQ ID NO:1 to SEQ ID NO:92; and / or (2) A nucleotide sequence having the nucleotide sequence shown in (1) obtained by deleting and / or adding one or more bases; and / or (3) A sequence having at least 80% homology to the nucleotide sequence shown in (1); and / or (4) Complementary sequences to sequences shown in (1), (2) or (3).

2. The InDel molecular marker primer set for areca nut according to claim 1, characterized in that, The amplification primer set includes at least one of the following combinations: Combination X: (5) The upstream primer has the nucleotide sequence shown in SEQ ID NO:(2X-1), and the downstream primer has the nucleotide sequence shown in SEQ ID NO:(2X); or (6) A nucleotide sequence obtained by substituting, deleting, or adding one or more bases to the nucleotide sequence shown in (5), and which has the same or similar function as the nucleotide sequence shown in (5); or (7) A nucleotide sequence that is at least 80% homologous to the nucleotide sequence shown in (5) or (6); Where X is selected from any integer from 1 to 46.

3. A detection reagent, characterized in that, Includes the InDel molecular marker primer set for areca as described in claim 1 or 2, and acceptable adjuvants.

4. The detection reagent according to claim 3, characterized in that, The auxiliaries include reagents required for PCR amplification and / or electrophoresis.

5. A test kit, characterized in that, Includes the InDel molecular marker primer set for areca nut as described in claim 1 or 2 and / or the detection reagent as described in claim 3 or 4.

6. A gene chip, characterized in that, Includes the InDel molecular marker primer set for areca nut as described in claim 1 or 2 and / or the detection reagent as described in claim 3 or 4.

7. The application of the InDel molecular marker primer set of areca nut as described in claim 1 or 2, the detection reagent as described in claim 3 or 4, the detection kit as described in claim 5, and / or the gene chip as described in claim 6 in constructing a genetic map of areca nut.

8. The application of the InDel molecular marker primer set of areca nut as described in claim 1 or 2, the detection reagent as described in claim 3 or 4, the detection kit as described in claim 5, and / or the gene chip as described in claim 6 in the analysis of genetic diversity of areca nut.

9. The application of the InDel molecular marker primer set of areca nut as described in claim 1 or 2, the detection reagent as described in claim 3 or 4, the detection kit as described in claim 5, and / or the gene chip as described in claim 6 in the analysis of areca nut germplasm resources.

10. A method for analyzing the genetic diversity of areca nut, characterized in that, Includes the following steps: Step 1: Extract genomic DNA from the areca nut sample to be tested; Step 2: Perform PCR amplification of the genomic DNA described in Step 1 using the InDel molecular marker primer set of areca nut as described in claim 1 or 2, the detection reagent as described in claim 3 or 4, the detection kit as described in claim 5, and / or the gene chip as described in claim 6; Step 3: Perform genetic diversity analysis based on the PCR amplification results described in Step 2 to obtain the genetic diversity analysis results of the areca nut sample to be tested.

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