InDel marker of areca and its application in provenance detection

Abstract

The present application relates to the field of molecular biology, in particular to a Areca catechu InDel marker and its application in provenance detection, which is based on the results of resequencing of one Areca catechu germplasm resource and reference genome sequence alignment analysis, to develop molecular markers by mining InDel sites, 46 pairs of InDel molecular marker primer pairs with high polymorphism are obtained, the Shannon's diversity index ranges from 0.361 to 1.713, and the polymorphism information content ranges from 0.201 to 0.752. The clustering analysis result shows that at the genetic similarity coefficient of 0.36, 211 Areca catechu materials are divided into 4 groups, the InDel marker pair provided by the present application can be effectively used for Areca catechu germplasm resource genetic background detection and analysis, and accurately identify the genetic relationship between the tested Areca catechu materials, which lays a foundation for Areca catechu germplasm resource genetic diversity analysis and provenance detection.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, specifically to the InDel marker in areca nut and its application in germplasm detection. Background Technology

[0002] Areca nut has a long history of cultivation in Hainan. It is a perennial palm tree that flowers and bears fruit 4-5 years after planting, resulting in a long non-production cycle. In recent years, with the development of the areca nut industry, the market demand for areca nut seedlings has been increasing. Due to the high overall quality and market price of fresh areca nut fruit grown locally in Hainan, there are fewer seed-producing fruits, leading to high prices for seed fruit. Areca nut seedling production is currently spontaneous, making it impossible to regulate the source of seedlings. Given the severe shortage of areca nut fruit and seedlings, seeds and seedlings from neighboring Southeast Asian countries such as Vietnam and Cambodia have entered the domestic market through various channels for cultivation and sale. These seeds and seedlings are inexpensive, and the fruit does not meet the characteristics of commercial fruit in Hainan, severely disrupting the Hainan areca nut seedling market. Different varieties of areca nut seedlings are difficult to distinguish at the early stages. After several years of planting, flowering, and fruiting, it is found that the fruit does not meet the requirements for commercial fruit and can only be sold as inferior fruit, or no one will buy it, seriously harming the interests of farmers. This study developed the InDel marker to conduct genotyping of areca nut germplasm resources and performed genetic cluster analysis on areca nut germplasm resources both domestically and internationally, providing technical support for early detection of the origin of areca nut seedlings.

[0003] InDel markers (Insertion-Deletion) were developed based on whole-genome resequencing and represent polymorphic variations resulting from the insertion or deletion of a short DNA sequence at allele loci. Two samples may differ in the number of nucleotide insertions or deletions in their entire genomes. Based on these insertion or deletion sites, PCR primers are designed to amplify these sites; these are InDel markers. Compared to SSR markers, InDel marker amplification products exhibit clearer and simpler banding patterns, and their stability and product separation are superior. InDel markers based on whole-genome resequencing are characterized by wide distribution within the genome, strong polymorphism, ease of detection, simple genotyping systems, and genetic stability. They have been successfully applied in areas such as plant and animal population genetic analysis, construction of molecular marker genetic maps, and genetic diversity analysis. They have been used in genetic research and variety identification of crops such as rice, maize, and cotton. However, there are relatively few reports on the application of InDel molecular markers to areca nut germplasm resources. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide InDel markers for areca nut and their application in seed source detection. The present invention provides InDel markers based on whole genome resequencing and constructs an InDel molecular marker primer set for obtaining areca nut.

[0005] This invention provides an InDel molecular marker primer set for areca nut, which 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) modified, substituted, deleted and / or added to one or more bases; and / or (3) A sequence having at least 80% homology with the nucleotide sequence shown in (1); and / or (4) Complementary sequences of sequences as shown in (1), (2) or (3).

[0006] In some embodiments, 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 substitution, deletion, or addition of 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 has at least 80% homology with the nucleotide sequence shown in (5) or (6); Where X is selected from any integer from 1 to 46.

[0007] In some specific embodiments, the InDel molecular marker primer set of the areca nut includes combinations 1 to 46.

[0008] The present invention provides a detection reagent comprising the InDel molecular marker primer set of areca nut and acceptable adjuvants.

[0009] In some embodiments, the auxiliaries include reagents required for PCR amplification and / or electrophoresis.

[0010] The present invention provides a detection kit comprising the InDel molecular marker primer set of areca nut and / or the detection reagents described herein.

[0011] This invention provides a gene chip, including the InDel molecular marker primer set of areca nut and / or the detection reagents described herein.

[0012] This invention provides the application of the InDel molecular marker primer set of areca nut, the detection reagent, the detection kit, and / or the gene chip in constructing a genetic map of areca nut.

[0013] This invention provides the application of the InDel molecular marker primer set of areca nut, the detection reagent, the detection kit, and / or the gene chip in the analysis of genetic diversity of areca nut.

[0014] This invention provides the application of the InDel molecular marker primer set of areca nut, the detection reagent, the detection kit, and / or the gene chip in the analysis of areca nut germplasm resources.

[0015] This invention provides a method for analyzing the genetic diversity of areca nuts, characterized by comprising 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, the detection reagent, the detection kit and / or the gene chip described above; 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.

[0016] This invention, based on the whole-genome resequencing analysis of a known areca nut germplasm resource, identifies InDel sites and develops molecular markers, obtaining 46 highly polymorphic InDel molecular marker primer pairs. The Shannon's diversity index ranged from 0.361 to 1.713, and the polymorphism information content ranged from 0.201 to 0.752. Cluster analysis showed that at a genetic similarity coefficient of 0.36, 211 areca nut materials were divided into four groups. Furthermore, the 46 InDel marker pairs were used to perform genotyping on 31 samples from unknown sources, demonstrating that the InDel marker pairs provided by this invention can be used for genetic background detection and analysis of areca nut germplasm resources, accurately identifying the genetic relationships among the tested areca nut materials, and laying the foundation for genetic diversity analysis and origin detection of areca nut seeds and seedlings. Attached Figure Description

[0017] Figure 1 This shows the amplification patterns of InDel primers used to screen parts of chromosome 15 using 8 samples; Figure 2 Genetic clustering analysis of 211 areca germplasm resources based on 46 InDel markers is shown. Figure 3 Genetic clustering analysis of 242 areca germplasm resources based on 46 InDel markers is shown. Figures 4-7The results of PCR amplification and capillary electrophoresis of areca nut samples numbered 213, 214 and 215 in Table 1 are shown in order, using primers AC202, AC224, AC310 and AC422 respectively. Detailed Implementation

[0018] This invention provides InDel marking for areca nuts and its application in seed provenance detection. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0019] The test materials used in this invention are all commercially available products. The invention will be further illustrated below with reference to specific embodiments.

[0020] Example 1

[0021] 1. Materials and Methods

[0022] 1.1 Experimental Materials

[0023] The tested materials included areca germplasm resources collected both domestically and internationally, as well as cultivated areca nuts from Hainan and other regions. Foreign materials mainly came from Vietnam, Indonesia, Sri Lanka, Fiji, Micronesia, and other countries, as well as areca nuts from Taiwan, Yunnan, and various cities and counties in Hainan Province, my country. Those originating from Hainan are referred to as native species, while other materials from Yunnan, Taiwan, and other countries are collectively referred to as introduced species. Details of the 242 areca germplasm materials tested are shown in Table 1. Samples numbered 1-211 are from known sources; 212-226 are smuggled areca nuts seized by Guangdong Customs and are samples to be tested; and 227-242 are samples collected from the Wenchang Nursery and are also samples to be tested.

[0024] Table 1 Information on the tested areca germplasm resources

[0025]

[0026] DNA extraction from areca nut is performed as follows: Approximately 1g of leaf or fruit samples are collected from areca nut plants and rapidly ground into powder using liquid nitrogen in a mortar. Genomic DNA is extracted from the leaves using a plant genomic DNA rapid extraction kit (Sangon Biotech (Shanghai) Co., Ltd.). After air-drying, the extracted DNA is dissolved in 100 μL of TE buffer. 1 μL of the stock DNA solution is then extracted, and the quality and concentration of the extracted DNA are measured using a NanoDrop 2000 micro-UV spectrophotometer. The working solution is diluted to 20 ng / μL. Both the stock DNA solution and the working solution are stored at -20 ℃ for later use.

[0027] 1.2 Experimental Methods

[0028] 1.2.1 InDel primer design and screening

[0029] Based on the comparative analysis of genome sequencing results, there are numerous InDel site differences between the oblong-shaped areca nut resources and the reference genome. InDel sites predicted from resequencing data were selected, and sites with insertion / deletion bases ranging from 10 to 50 bp were identified. Based on the InDel sites discovered through sequence alignment and their distribution across the genome, primers were designed for 420 InDel sites. Primers designed for chromosome 15 are shown in Table 2 (due to the large amount of space required for 420 primer pairs, only primer information for chromosome 15 is listed). Based on the areca nut genome sequence, the selected sites were located on the genome. 200 bp were taken from each side of the InDel site, for a total length of 400 bp. Primers were designed using Primer 5.0, with upstream primers ranging from 1 to 180 bp and downstream primers from 220 to 400 bp. The product size was 100 to 400 bp, the GC content was 40% to 60%, the annealing temperature was 55℃ to 65℃, and the primer length was set to 18 to 22 bp.

[0030] The experiment used a 15 μL reaction system, meaning that the 15 μL reaction system contained 0.15 mmol / L dNTPs and Mg. 2+ The PCR reaction consisted of 1.5 mmol / L buffer, 0.75 U Taq DNA polymerase, 0.3 μmol / L primers, and 20 ng template DNA. The PCR program was as follows: 94 °C pre-denaturation for 5 min; 94 °C denaturation for 30 s, 56 °C annealing for 30 s, 72 °C extension for 30 s, for 33 cycles; 72 °C extension for 7 min, followed by storage at 4 °C. The amplified products were subjected to 8% non-denaturing polyacrylamide gel electrophoresis, silver staining, and photographed under a film viewing lamp. Band types were statistically analyzed.

[0031] PCR detection of synthesized primers was performed using eight areca nut germplasm samples from different geographical origins. The gel amplification results of partial primers for chromosome 15 are as follows: Figure 1 Primers with clear bands and good polymorphism are selected for use in genetic clustering analysis. For example... Figure 1 As shown, primer pairs AC1515, AC1516, and AC1519 all amplified clear and specific bands, which can be used for subsequent cluster analysis. Following the above method, 420 primer pairs were screened, resulting in 46 primer pairs with clear bands and good polymorphism (Table 3), which were then used to test the tested areca nut germplasm materials.

[0032] Table 2. InDel marker information for chromosome 15.

[0033] Table 3.46 InDel tag information table

[0034] 1.2.2 PCR amplification and capillary electrophoresis methods

[0035] Using extracted areca nut genomic DNA as a template, PCR amplification was performed using InDel fluorescent labeling detection technology. When using fluorescent primers for PCR amplification, the upstream primer with a fluorescent group at the 5' end and the downstream primer directly amplified the DNA template, resulting in PCR products with fluorescent groups.

[0036] The PCR amplification system was as follows (10 μL): 1 μL of 20 ng template DNA, 0.5 μL each of forward and reverse primers (10 μmol / L), 5.0 μL of 2×Taq PCR Master Mix, and ddH2O to make up to 10 μL. The PCR amplification program was set as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, gradient annealing at 62~52℃ for 30 s, extension at 72℃ for 30 s, for 10 cycles; 95℃ denaturation for 30 s, 52℃ annealing for 30 s, extension at 72℃ for 30 s, for 25 cycles; 72℃ extension for 20 min, and final storage at 4℃; the reaction was performed on a Veriti 384 PCR instrument.

[0037] After diluting the PCR amplification products to a uniform concentration, the fluorescent PCR products were subjected to capillary electrophoresis, and the detection reagents were added according to the following system. Capillary electrophoresis fluorescence detection: After appropriate dilution of the PCR products, 1 μL of the diluent was added to 8.5 μL of deionized formamide and 0.5 μL of LIZ-500 molecular weight internal standard. After preparing the system, it was placed on an ABI 3730xL genetic analyzer for capillary electrophoresis detection.

[0038] The raw data in .fsa format was exported from the ABI 3730xL genetic analyzer, classified and archived according to the detection sites, and then imported into GeneMarker analysis software to read the genotype data. The position of each peak was automatically compared with the LIZ-500 molecular weight internal standard in the lane to obtain the fragment size (bp) of the amplified product. The raw genotype data in Excel and PDF genotyping peak plot files were exported according to the site name.

[0039] 2. Experimental Results

[0040] 2.1 Primer Polymorphism Analysis

[0041] After PCR amplification and capillary electrophoresis, genetic parameters for each locus were calculated using GenAlEx 6.5 software, including the number of alleles, effective number of alleles, observed heterozygosity, expected heterozygosity, and Shannon's information index. The polymorphism information content for each locus was calculated using PowerMarker V3.25 software, and a UPGMA (Unweighted pair group method with arithmetic mean) clustering analysis circular plot was constructed using the online tool ITOL based on the genetic distances calculated by PowerMarker.

[0042] Genotyping of 211 areca nut germplasm resources from known sources was performed using 46 pairs of InDel markers, and genetic diversity analysis was conducted (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. Shannon's diversity index ranged from 0.361 to 1.713, and the polymorphism information content ranged from 0.201 to 0.752. Marker AC1516 showed the highest values ​​for all genetic parameters. Only three primer pairs had a polymorphism information content less than 0.25, indicating that the InDel markers used in this study exhibited high polymorphism in the tested areca nut germplasm resources.

[0043] Table 4. Statistical analysis of polymorphism data for 46 pairs of InDel primers

[0044] 2.2 Genetic and Evolutionary Analysis of Areca Nut Germplasm Resources

[0045] Genetic cluster analysis was performed on 211 germplasm resources from known sources using 46 pairs of InDel markers. Figure 2 ),Depend on Figure 2It 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 basically clustered in Groups I, II, and III, showing a clear distinction from Hainan native species.

[0046] 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 typically produces oval or egg-shaped fruits, which are well-suited for processing and are mostly processed from fresh fruit 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. In other countries, areca nut is primarily used for food or medicine, mainly the kernel, which is generally larger and has a thinner pulp. Due to different breeding objectives, there are genotypic differences between the Hainan native variety and foreign resources. 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 to Sri Lanka. 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.

[0047] 2.3 Analysis of genetic diversity of areca nut germplasm resources and estimation of seedling origin

[0048] 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 3As 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 species 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 in areca germplasm resources and the detection of the origin of seeds and seedlings.

[0049] 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.

[0050] Table 5. Genetic distances of 17 areca nut germplasm resources

[0051] 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 4-7 As shown ( Figure 4 AC2.02 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 5-7 Similarly.

[0052] 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 germplasm resource analysis, characterized in that, The InDel molecular marker primer set consists of combinations 1 to 46; The nucleotide sequence of the upstream primer of combination 1 is shown in SEQ ID NO:1, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:2; The nucleotide sequence of the upstream primer of combination 2 is shown in SEQ ID NO:3, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:4; The nucleotide sequence of the upstream primer of combination 3 is shown in SEQ ID NO:5, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:6; The nucleotide sequence of the upstream primer of combination 4 is shown in SEQ ID NO:7, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:8; The nucleotide sequence of the upstream primer of combination 5 is shown in SEQ ID NO:9, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:10; The nucleotide sequence of the upstream primer of combination 6 is shown in SEQ ID NO:11, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:12; The nucleotide sequence of the upstream primer of combination 7 is shown in SEQ ID NO:13, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:14; The nucleotide sequence of the upstream primer of combination 8 is shown in SEQ ID NO:15, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:16; The nucleotide sequence of the upstream primer of combination 9 is shown in SEQ ID NO:17, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:18; The nucleotide sequence of the upstream primer of combination 10 is shown in SEQ ID NO:19, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:20; The nucleotide sequence of the upstream primer of combination 11 is shown in SEQ ID NO:21, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:22; The nucleotide sequence of the upstream primer of combination 12 is shown in SEQ ID NO:23, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:24; The nucleotide sequence of the upstream primer of combination 13 is shown in SEQ ID NO:25, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:26; The nucleotide sequence of the upstream primer of combination 14 is shown in SEQ ID NO:27, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:28; The nucleotide sequence of the upstream primer of combination 15 is shown in SEQ ID NO:29, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:30; The nucleotide sequence of the upstream primer of combination 16 is shown in SEQ ID NO:31, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:32; The nucleotide sequence of the upstream primer of combination 17 is shown in SEQ ID NO:33, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:34; The nucleotide sequence of the upstream primer of combination 18 is shown in SEQ ID NO:35, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:36; The nucleotide sequence of the upstream primer of combination 19 is shown in SEQ ID NO:37, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:38; The nucleotide sequence of the upstream primer of combination 20 is shown in SEQ ID NO:39, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:40; The nucleotide sequence of the upstream primer of combination 21 is shown in SEQ ID NO:41, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:42; The nucleotide sequence of the upstream primer of combination 22 is shown in SEQ ID NO:43, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:44; The nucleotide sequence of the upstream primer of combination 23 is shown in SEQ ID NO:45, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:46; The nucleotide sequence of the upstream primer of combination 24 is shown in SEQ ID NO:47, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:48; The nucleotide sequence of the upstream primer of combination 25 is shown in SEQ ID NO:49, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:50; The nucleotide sequence of the upstream primer of combination 26 is shown in SEQ ID NO:51, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:52; The nucleotide sequence of the upstream primer of combination 27 is shown in SEQ ID NO:53, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:54; The nucleotide sequence of the upstream primer of combination 28 is shown in SEQ ID NO:55, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:56; The nucleotide sequence of the upstream primer of combination 29 is shown in SEQ ID NO:57, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:58; The nucleotide sequence of the upstream primer of the combination 30 is shown in SEQ ID NO:59, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:60; The nucleotide sequence of the upstream primer of combination 31 is shown in SEQ ID NO:61, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:62; The nucleotide sequence of the upstream primer of combination 32 is shown in SEQ ID NO:63, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:64; The nucleotide sequence of the upstream primer of combination 33 is shown in SEQ ID NO:65, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:66; The nucleotide sequence of the upstream primer of combination 34 is shown in SEQ ID NO:67, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:68; The nucleotide sequence of the upstream primer of combination 35 is shown in SEQ ID NO:69, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:70; The nucleotide sequence of the upstream primer of the combination 36 is shown in SEQ ID NO:71, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:72; The nucleotide sequence of the upstream primer of combination 37 is shown in SEQ ID NO:73, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:74; The nucleotide sequence of the upstream primer of the combination 38 is shown in SEQ ID NO:75, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:76; The nucleotide sequence of the upstream primer of combination 39 is shown in SEQ ID NO:77, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:78; The nucleotide sequence of the upstream primer of combination 40 is shown in SEQ ID NO:79, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:80; The nucleotide sequence of the upstream primer of combination 41 is shown in SEQ ID NO:81, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:82; The nucleotide sequence of the upstream primer of combination 42 is shown in SEQ ID NO:83, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:84; The nucleotide sequence of the upstream primer of combination 43 is shown in SEQ ID NO:85, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:86; The nucleotide sequence of the upstream primer of combination 44 is shown in SEQ ID NO:87, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:88; The nucleotide sequence of the upstream primer of combination 45 is shown in SEQ ID NO:89, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:90; The nucleotide sequence of the upstream primer of combination 46 is shown in SEQ ID NO:91, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:

92.

2. A detection reagent, characterized in that, Includes the InDel molecular marker primer set as described in claim 1 and acceptable auxiliaries.

3. The detection reagent according to claim 2, characterized in that, The auxiliaries include reagents required for PCR amplification and / or electrophoresis.

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

5. A gene chip, characterized in that, Includes the InDel molecular marker primer set as described in claim 1 or the detection reagent as described in claim 2 or 3.

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

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

8. The application of the InDel molecular marker primer set of claim 1, the detection reagent of claim 2 or 3, the detection kit of claim 4, or the gene chip of claim 5 in the analysis of areca germplasm resources.

9. 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 on the genomic DNA described in Step 1 using the InDel molecular marker primer set described in claim 1, the detection reagent described in claim 2 or 3, the detection kit described in claim 4, or the gene chip described in claim 5; 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.

Images