Group of InDel markers for identifying areca-nut-shaped fruits and application of InDel markers
By using InDel-labeled primer sets and electrophoresis technology, the problem of fruit shape identification in areca nut seedlings has been solved, enabling efficient and accurate identification of jujube-shaped fruits, simplifying the operation process, and possessing significant application value.
Patent Information
- Application Number
- CN202511306981.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-12
- Publication Date
- 2025-12-12
AI Technical Summary
Current technology cannot effectively identify the fruit shape of areca seedlings, leading to the phenomenon of inferior products being passed off as superior ones and counterfeit products being sold in the market, which harms the interests of farmers. Furthermore, existing molecular marker technologies such as SSR markers are not very effective in the identification of areca varieties.
Using InDel-labeled primer sets, specific nucleotide sequences were designed for PCR amplification. Combined with electrophoresis, the size of the PCR amplification product fragments was used to distinguish between jujube-shaped, oval, and round fruits, providing an accurate method for variety identification.
It enables efficient and accurate identification of areca nut fruits with a date-like shape, simplifies the operation process, reduces instrument requirements, is suitable for large-scale identification, and has important application value.
Smart Images
Figure CN121109631A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of molecular biology, in particular to a set of InDel markers for identifying Bertholletia excelsa and application thereof. BACKGROUND
[0002] Bertholletia excelsa has a long history of cultivation in Hainan and has formed a unique local variety. Bertholletia excelsa is a perennial palm tree of the family Palmae, which flowers and bears fruit after 4-5 years of cultivation, and has a long non-production cycle. The commercially acceptable Bertholletia excelsa fruits in the local market of Hainan are elliptical, oval, and round fruits are not popular and belong to secondary fruits, and jujube-shaped fruits (Taiwan variety) are not willing to be purchased by enterprises. In recent years, with the development of the Bertholletia excelsa industry, the market demand for Bertholletia excelsa seedlings is increasing. Since the production of Bertholletia excelsa seedlings is in a spontaneous state and the source of seedlings cannot be monitored, there are situations such as using inferior goods as good ones and confusing true and false goods in the seedling market. Since jujube-shaped fruits (Taiwan variety) have no fresh fruit sales market, some unscrupulous traders use seed fruits to breed seedlings and sell them as local seedlings, it is difficult to identify different varieties of Bertholletia excelsa seedlings, and after several years of planting, it is found that the fruit shape cannot meet the requirements of commercial fruits, and there is no enterprise to purchase, which seriously damages the interests of farmers. The present application uses InDel molecular markers to carry out genotype detection analysis of different fruit shape varieties of Bertholletia excelsa, and provides technical support for early detection of Bertholletia excelsa seedlings.
[0003] InDel markers (Insertion-Deletion) are developed on the basis of whole genome resequencing, and are polymorphic variations produced by insertion or deletion of a short sequence at allelic sites. Two samples have a certain number of differences in the insertion or deletion of nucleotides in the whole genome, and according to the insertion or deletion site in the genome, a PCR primer amplifying these sites is designed, which is an InDel marker. 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 population genetic analysis, construction of molecular marker genetic map and genetic diversity analysis of animals and plants, and in the genetic research and variety identification of crops such as rice, corn and cotton. However, there is less research on the use of InDel molecular markers in Bertholletia excelsa variety identification. SUMMARY
[0004] Therefore, the present application aims to solve the technical problem by providing a set of InDel markers for identifying jujube-shaped Bertholletia excelsa and application thereof. The identification method provided by the present application can accurately identify jujube-shaped Bertholletia excelsa.
[0005] The present application provides a set of InDel marker primer groups for identifying jujube-shaped Bertholletia excelsa, which has the following characteristics:
[0006] (1) a nucleotide sequence as set forth in any one of SEQ ID NO: 1 to SEQ ID NO: 4; and / or
[0007] (2) a nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence as set forth in (1); and / or
[0008] (3) a sequence having at least 80% homology to the nucleotide sequence as set forth in (1); and / or
[0009] (4) a complementary sequence of the sequence as set forth in (1), (2) or (3).
[0010] In some embodiments, the InDel marker primer set comprises at least one of the following combinations:
[0011] Combination X:
[0012] (5) the forward primer has a nucleotide sequence as set forth in SEQ ID NO: (2X-1) and the reverse primer has a nucleotide sequence as set forth in SEQ ID NO: (2X); or
[0013] (6) a nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence as set forth in (5), and a nucleotide sequence having the same or similar function as the nucleotide sequence as set forth in (5); or
[0014] (7) a nucleotide sequence having at least 80% homology to the nucleotide sequence as set forth in (5) or (6);
[0015] wherein X is 1 or 2.
[0016] The present application provides detection reagents, including the InDel marker primer set for identifying P. jollyana and acceptable adjuvants.
[0017] In some embodiments, the adjuvants include reagents required for PCR amplification and / or electrophoresis.
[0018] The present application provides detection kits, including the InDel marker primer set for identifying P. jollyana and / or the detection reagents.
[0019] The present application provides gene chips, including the InDel marker primer set for identifying P. jollyana and / or the detection reagents.
[0020] The present application provides the use of the InDel marker primer set for identifying P. jollyana, the detection reagents, the detection kits and / or the gene chips in identifying P. jollyana.
[0021] The application provides a method for identifying Areca catechu L. of jujube-shaped fruits, comprising the following steps:
[0022] Step 1, extracting genomic DNA of an Areca catechu L. sample to be detected;
[0023] Step 2, performing PCR amplification on the genomic DNA in step 1 by using the InDel marker primer set for identifying Areca catechu L. of jujube-shaped fruits, the detection reagent, the detection kit and / or the gene chip;
[0024] When the InDel molecular marker primer set shown in combination 1 is used for PCR amplification, the obtained PCR product has a fragment size of 189 bp, indicating that the Areca catechu L. sample to be detected is of jujube-shaped fruits; the amplified PCR product has a fragment size of 198 bp and / or 217 bp, indicating that the Areca catechu L. sample to be detected is of oval-shaped fruits or round-shaped fruits;
[0025] When the InDel molecular marker primer set shown in combination 2 is used for PCR amplification, the obtained PCR product has a fragment size of 152 bp, indicating that the Areca catechu L. sample to be detected is of jujube-shaped fruits; the amplified PCR product has a fragment size of 132 bp, indicating that the Areca catechu L. sample to be detected is of oval-shaped fruits or round-shaped fruits.
[0026] In some embodiments, the PCR amplification system is as follows: 20 ng of template DNA 1 μL, 0.5 μL of forward and reverse primers each with a concentration of 10 umol / L, 5.0 μL of 2x Taq PCR Master Mix, and ddH2O is added to 10 μL.
[0027] In some embodiments, the PCR amplification reaction condition is as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 62-52℃ gradient annealing for 30 s, 72℃ extension for 30 s, running for 10 cycles; 95℃ denaturation for 30 s, 52℃ annealing for 30 s, 72℃ extension for 30 s, running for 25 cycles; 72℃ extension for 20 min.
[0028] The application provides a set of InDel markers for identifying Areca catechu L. of jujube-shaped fruits and application, the InDel molecular marker primer set provided by the application can accurately identify Areca catechu L. of jujube-shaped fruits, the method for identifying Areca catechu L. of jujube-shaped fruits by using the InDel molecular marker primer set is simple, accurate and efficient, has simple technical requirements, has low requirements on instrument operation, can identify Areca catechu L. of jujube-shaped fruits in a large batch, provides a basis for Areca catechu L. identification, and has important application value for Areca catechu L. variety identification evaluation, hybridization breeding and quality improvement. BRIEF DESCRIPTION OF DRAWINGS
[0029] Figure 1Electrophoretic pattern of a jujube-shaped areca nut variety amplified by InDel-labeled primer AC1605;
[0030] Figure 2 Electrophoretic pattern of oval-shaped areca nut amplified by InDel-labeled primer AC1605;
[0031] Figure 3 Electrophoretic pattern of amplified round-fruited areca nut variety using InDel-labeled primer AC1605;
[0032] Figure 4 Electrophoretic pattern of a jujube-shaped areca nut variety amplified by InDel-labeled primer AC1618;
[0033] Figure 5 Electrophoretic pattern of an oval-shaped areca nut variety amplified by InDel-labeled primer AC1618;
[0034] Figure 6 Electrophoretic pattern of amplified round-fruited areca nut variety using InDel-labeled primer AC1618;
[0035] Figure 7 Electrophoretic pattern of a jujube-shaped areca nut variety amplified by InDel-labeled primer AC617;
[0036] Figure 8 Electrophoretic pattern of an oval-shaped areca nut variety amplified by InDel-labeled primer AC617;
[0037] Figure 9 Electrophoretic pattern of a round-fruited areca nut amplified by InDel-labeled primer AC617. Detailed Implementation
[0038] This invention provides a set of InDel markers for identifying areca nuts with a date-shaped fruit and their applications. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. 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.
[0039] The test materials used in this invention are all commercially available products. The invention will be further illustrated below with reference to specific embodiments.
[0040] Example 1
[0041] 1. Materials and Methods
[0042] 1.1 Experimental Materials
[0043] Six samples of date-shaped fruit (Taiwanese variety) (T1-T6), six samples of oval fruit (R1-R6), and six samples of round fruit (Y1-Y6) were selected. All of these samples were from the National Tropical Palm Germplasm Resource Nursery - Areca Nut Zone, Coconut Research Institute, Chinese Academy of Tropical Agricultural Sciences, Wenchang City, Hainan Province.
[0044] DNA extraction from areca nut leaves was performed as follows: Leaves were collected from mature areca nut plants, chopped, and approximately 1g of leaves were weighed and rapidly ground into powder using liquid nitrogen in a mortar. Genomic DNA was extracted from the leaves using a plant genomic DNA rapid extraction kit [Sangon Biotech (Shanghai) Co., Ltd.]. After air-drying, the extracted DNA was dissolved in 100μL of TE buffer. 1μL of the stock DNA solution was taken, and the quality and concentration of the extracted DNA were measured using a NanoDrop 2000 micro-UV spectrophotometer. The working solution was diluted to 20ng / μL. Both the stock DNA solution and the working solution were stored at -20℃ for later use.
[0045] 1.2 Experimental Methods
[0046] 1.2.1 InDel primer design and screening
[0047] Based on comparative analysis of genome sequencing results, a large number of InDel sites differed between the oblong-shaped areca nut resources and the reference genome. InDel sites predicted from resequencing data were selected, with insertion / deletion bases ranging from 10 to 50 bp. These selected sites were located on the genome according to the areca nut genome sequence. 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 expected to be 100 to 400 bp, with a GC content of 40% to 60%, an annealing temperature of 55℃ to 65℃, and primer lengths ranging from 18 to 22 bp. 420 primer pairs were designed and screened. PCR detection of the synthesized primers was performed using areca nut germplasm samples from different geographical origins. The experiment used a 15 μL reaction system, containing 0.15 mmol / L dNTPs and Mg2+. 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℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 30 s, 33 cycles; 72℃ extension for 7 min, and storage at 4℃. 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. Forty-six pairs of primers with clear bands and good polymorphism were obtained for testing the materials. Two primer pairs (AC1605 and AC1618) were selected for identification of jujube-shaped (Taiwanese) areca nuts, distinguishing them from the other two fruit shapes. Since too many primers could not distinguish between jujube-shaped and the other two fruit shapes, only one primer pair (AC617) is listed as an example. Primer information is shown in Table 1.
[0048] Table 1. InDel-labeled primers used for identifying areca nuts with jujube-shaped fruit.
[0049]
[0050] 1.2.2 PCR amplification and capillary electrophoresis methods
[0051] 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 to obtain PCR products with fluorescent groups.
[0052] 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.
[0053] 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.
[0054] 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.
[0055] 2. Experimental Results
[0056] 2.1 Amplification results of InDel-labeled primer AC1605
[0057] Eighteen areca nut samples were amplified using the molecular marker primer AC1605, followed by capillary electrophoresis to genotype the PCR products. The results are as follows: Figure 1 As shown, all jujube-shaped fruit (Taiwanese species) DNA templates amplified only one specific band of 189 bp. Figure 1 AC16.05 in the table corresponds to primer AC1605 in Table 1. The number before the "." in AC16.05 is the chromosome number, and the number after the "." is the primer number. To facilitate differentiation and statistical analysis during the experiment, the above format was used in the images. The following... Figures 2-9 Similarly, this indicates that the DNA of the jujube-shaped fruit (Taiwanese variety) is stably homozygous at this molecular marker site, and no other heterogeneous bands will appear; the oval-shaped fruit areca variety ( Figure 2 ) and round fruit areca varieties ( Figure 3 A band was amplified at 198bp or 217bp, or at both positions, but no band was observed at 189bp. This indicates that the InDel-labeled primer AC1605 can specifically identify areca nuts with a date-shaped fruit (Taiwanese variety).
[0058] 2.2 Amplification results of InDel-labeled primer AC1618
[0059] Eighteen areca nut samples were amplified using molecular marker primer AC1618, followed by capillary electrophoresis to genotype the PCR products. The results are shown in the figure. All jujube-shaped fruit (Taiwanese species) DNA templates showed a single 152 bp specific band that could be amplified. Figure 4 This indicates that the DNA of the jujube-shaped fruit (Taiwanese variety) is stably homozygous at this molecular marker site, and no other heterogeneous bands will appear; the oval-shaped fruit areca variety ( Figure 5 ) and round fruit areca varieties ( Figure 6A band was amplified at 132 bp, but no band was observed at 152 bp. This indicates that the InDel-labeled primer AC1618 can specifically identify areca nuts with jujube-shaped fruit (Taiwanese variety).
[0060] 2.3 Amplification results of InDel-labeled primer AC617
[0061] Eighteen areca nut samples were amplified using the molecular marker primer AC617, followed by capillary electrophoresis to genotype the PCR products. The results are as follows: Figure 7 As shown, all jujube-shaped fruit (Taiwanese variety) DNA templates amplified only one specific 200bp band, without any other banding patterns; oval-shaped fruit areca varieties ( Figure 8 ) and round fruit areca varieties ( Figure 9 The bands can be amplified at 200bp or 228bp, or at both positions. The band patterns of round fruit Y4 and oval fruit R1, R2, R3 and R4 are the same as those of jujube-shaped fruit, indicating that the InDel marker primer AC617 cannot distinguish jujube-shaped fruit (Taiwanese species) areca nut.
[0062] 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. A set of InDel-labeled primers for identifying areca nuts with a date-shaped fruit, characterized in that, It has the following characteristics: (1) Any nucleotide sequence as shown in SEQ ID NO:1 to SEQ ID NO:4; 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 to the nucleotide sequence shown in (1); and / or (4) Complementary sequences to sequences shown in (1), (2) or (3).
2. The InDel-labeled primer set for identifying areca nuts of the jujube shape according to claim 1, characterized in that, The InDel-labeled primer set includes at least one of the following combinations: Combination X: (5) The forward primer has the nucleotide sequence shown in SEQ ID NO:(2X-1), and the reverse 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 1 or 2.
3. A detection reagent, characterized in that, Includes the InDel-labeled primer set for identifying areca nut of the jujube shape 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-labeled primer set for identifying areca nut of the jujube shape 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-labeled primer set for identifying areca nut of the jujube shape 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-labeled primer set for identifying jujube-shaped areca nuts 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 identifying jujube-shaped areca nuts.
8. A method for identifying areca nut fruits with a date-like shape, 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-labeled primer set for identifying jujube-shaped areca nuts 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; When PCR amplification is performed using the InDel molecular marker primer set shown in combination 1, if the size of the obtained PCR product fragment is 189bp, it indicates that the detected areca nut sample is date-shaped; if the size of the obtained PCR product fragment is 198bp and / or 217bp, it indicates that the detected areca nut sample is oval or round. When PCR amplification is performed using the InDel molecular marker primer set shown in combination 2, if the size of the obtained PCR product fragment is 152bp, it indicates that the detected areca nut sample is date-shaped; if the size of the obtained PCR product fragment is 132bp, it indicates that the detected areca nut sample is oval or round.
9. The method according to claim 8, characterized in that, The PCR amplification system consisted of: 1 μL of 20 ng template DNA, 0.5 μL each of forward and reverse primers at a concentration of 10 μmol / L, 5.0 μL of 2×Taq PCR Master Mix, and ddH2O added to bring the total volume to 10 μL.
10. The method according to claim 8 or 9, characterized in that, The PCR amplification reaction conditions were as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 62-52℃ gradient annealing for 30 s, 72℃ extension for 30 s, for 10 cycles; 95℃ denaturation for 30 s, 52℃ annealing for 30 s, 72℃ extension for 30 s, for 25 cycles; 72℃ extension for 20 min.