MNP marker site, primer composition and kit for coconut variety identification and application of MNP marker site, primer composition and kit

Abstract

The invention belongs to the field of biology, and particularly relates to an MNP marker site, a primer composition and a kit for coconut variety identification and application of the MNP marker site, the primer composition and the kit. According to the 240 MNP sites of the coconut and the corresponding primer composition, multiple PCR amplification can be performed, sequencing of amplification products is performed in combination with a next-generation sequencing platform, and the 240 MNP sites of the coconut and the corresponding primer composition have the advantages of being high in throughput, high in distinction degree, high in accuracy and the like in detection of coconut varieties. The 240 MNP marker loci, the primer composition and the kit provided by the invention can meet the requirements of coconut variety authenticity identification and coconut variety distinguishing, can be used for constructing a DNA fingerprint database of coconut varieties, can quickly and accurately determine a coconut variety to be detected by comparing the variety to be detected with coconut variety data in the database, and can be used for rapidly and accurately identifying the coconut variety to be detected. And the method also has a huge promotion effect on selection of cultivated varieties in the coconut cultivation process and breeding of coconut varieties.

Description

Technical Field

[0001] This invention belongs to the field of biology, specifically relating to MNP marker sites, primer compositions, and kits for coconut variety identification, and their applications. Background Technology

[0002] The differentiation of coconut varieties mainly relies on factors such as husk color, fruit shape, the taste of coconut juice and meat, and uses. For example, husk color: green for young coconuts, red for red coconuts, and golden for golden coconuts; fruit shape: red coconuts are long and narrow, while young and golden coconuts are mostly round; taste of coconut juice and meat: fragrant coconuts have a taro-like aroma, while sticky coconuts have a thick texture; uses: king coconuts are suitable for making coconut flakes, while young coconuts are suitable for drinking coconut juice directly; origin: Hainan coconuts are mainly tall varieties, while Thai fragrant coconuts are mainly dwarf varieties. However, it is difficult to accurately distinguish coconut varieties during other growth stages.

[0003] MNP stands for Multi-Nucleotide Polymorphism, a novel molecular marker technology that emerged with the development of molecular marker technology. Compared with other markers, MNP markers have the following advantages: (1) abundant alleles, high polymorphism, and 2 alleles per MNP locus. n (1) Strong variety differentiation ability: Due to the large number of alleles of MNP markers, the variety differentiation ability of the markers is guaranteed. Combining multiple MNP markers can achieve efficient variety differentiation. (2) High efficiency: Multiple sites can be amplified simultaneously in a single tube PCR reaction. Combined with high-throughput sequencing, hundreds or thousands of samples can be detected simultaneously. (3) High accuracy: Second-generation high-throughput sequencing can sequence the amplified marker products hundreds of times. The output result is a base sequence. There is no need for parallel experiments to verify the data. The data can be compared arbitrarily, and the data sharing is strong.

[0004] Based on the above advantages and characteristics, MNP marker technology has been widely used in crops such as rice, corn, melon, and kiwi. Currently, there are no research reports on MNP markers in coconuts. Therefore, MNP markers used for coconut variety identification are of great significance for the accurate identification of coconut varieties in the early stages. Summary of the Invention

[0005] This invention provides MNP marker sites, primer compositions, and kits for coconut variety identification, as well as their applications. These methods enable accurate and rapid identification of coconut varieties with strong discriminative power and high accuracy, achieving high-throughput identification of samples and playing a significant role in promoting the breeding of superior coconut varieties.

[0006] The technical solution of this invention is implemented as follows:

[0007] In a first aspect, the present invention provides an MNP marker site for coconut variety identification, wherein the MNP marker site is a genomic region with multiple nucleotide polymorphisms in a coconut population selected from the coconut genome, and the MNP marker site includes marker sites MNP-1 to MNP-240 on the coconut genome GCA_008124465.1.

[0008] The marker sites of MNP-1 to MNP-240 mentioned above are specifically shown in Table 1 of the specification. The start and end positions of the MNP markers marked in Table 1 are determined based on the GCA_008124465.1 sequence.

[0009] In a second aspect, the present invention provides a multiplex PCR primer composition for detecting the MNP marker site, the multiplex PCR primer composition comprising 240 pairs of primers, the nucleotide sequences of the 240 pairs of primers being shown in Table 1.

[0010] The primers for each MNP marker site mentioned above include an upper primer and a lower primer, as shown in Table 1 of the specification.

[0011] A third aspect of the present invention provides a detection kit for detecting the MNP marker site, the kit comprising the primer composition described above.

[0012] Furthermore, the kit also includes a multiplex PCR premix.

[0013] In a fourth aspect, the invention provides the application of the aforementioned multiplex PCR primer composition or the aforementioned detection kit in the identification of coconut variety authenticity.

[0014] A fifth aspect of the invention provides the application of the described multiplex PCR primer composition or the described detection kit in distinguishing coconut varieties.

[0015] In a sixth aspect, the present invention provides the application of the aforementioned multiplex PCR primer composition or the aforementioned detection kit in constructing a coconut variety DNA fingerprint database and / or in analyzing the genetic diversity of germplasm resources.

[0016] A seventh aspect of the present invention provides the application of the described multiplex PCR primer composition or the described detection kit in coconut breeding.

[0017] Furthermore, the KASP marker or primer composition can be used for variety selection, molecular marker-assisted breeding, detection of breeding materials, whole-genome selection breeding, or preparation of whole-genome breeding kits.

[0018] The eighth aspect of the present invention is to provide a method for constructing a coconut variety DNA fingerprint database. The specific application steps are as follows: extracting total DNA from all coconut varieties used to construct the coconut DNA fingerprint database, amplifying all coconut varieties using the above-mentioned multiplex PCR primer composition, and recording the genotype of 240 loci for each variety as the final MNP fingerprint data.

[0019] The specific operating method is as follows:

[0020] (1) Extract total DNA from coconut varieties for constructing a coconut DNA fingerprint database; perform a first round of multiplex PCR amplification on the total DNA of the coconut varieties using the primer composition or kit of the present invention, with 18 cycles; after purifying the amplification products, add sample tags and next-generation sequencing adapters based on a second round of PCR amplification; quantify the purified second-round amplification products.

[0021] (2) When testing coconut variety samples, high-throughput sequencing was performed by mixing equal amounts of the second-round amplification products;

[0022] (3) Perform data quality control and data analysis on the sequencing data of coconut variety samples, compare the sequencing results with the coconut reference sequence (GCA_008124465.1), and obtain the number of detection sites of the coconut variety at the MNP site, the number of sequencing sequences covering each MNP site and the genotype data of the MNP site, as the final DNA fingerprint database.

[0023] The ninth aspect of this invention is to provide a method for identifying the authenticity of coconut varieties, comprising the following steps:

[0024] Using accurately identified coconut varieties as control samples, gene data for the 240 MNP loci mentioned above were obtained for both the test sample and the control sample. Data comparisons were performed between varieties to obtain the percentage of differing loci. The percentage of differing loci p = (number of differing loci n / number of common loci N) × 100%. If the percentage of differing loci p between the test sample and the control sample is greater than 4%, the test sample and the control sample are not the same variety. If the percentage of differing loci p between the test sample and the control sample is less than or equal to 4%, the test sample and the control sample are the same variety or similar varieties.

[0025] The tenth aspect of the present invention is to provide an application in distinguishing coconut varieties, comprising the following steps:

[0026] Genotypic data of the above 240 MNP loci were obtained for each sample to be tested. The percentage of differential loci among the samples was obtained by comparing the genotypic data of the samples to be tested. The percentage of differential loci p = (number of differential loci n / number of common loci N) × 100%. If the percentage of differential loci p among the samples to be tested is greater than 4%, it is concluded that a significant difference was detected and the samples to be tested are not the same variety. If the percentage of differential loci p among the samples to be tested is less than or equal to 4%, it is concluded that no significant difference was detected and the samples to be tested are the same variety or similar varieties.

[0027] Beneficial effects:

[0028] This invention provides MNP marker sites, primer compositions, and kits for coconut variety identification, along with their applications. The provided 240 MNP sites for coconut and their corresponding primer compositions enable multiplex PCR amplification, followed by sequencing of the amplified products using a next-generation sequencing platform. This method offers high throughput, high discrimination, and high accuracy in coconut variety detection. Experiments have demonstrated that the 240 MNP marker sites, primer compositions, and kits provided by this invention can meet the requirements for identifying and differentiating 13 coconut varieties. Based on these detection results, this invention constructs a DNA fingerprint database for coconut varieties. By comparing the target variety with the coconut variety data in the database, the target coconut variety can be quickly and accurately identified.

[0029] The 240 MNP marker sites and primer compositions provided in this invention can also be applied to coconut breeding and related applications such as genetic resource diversity. They can be used for coconut variety selection, molecular marker-assisted breeding, detection of breeding materials, whole-genome selection breeding, or preparation of whole-genome breeding kits. They enable precise genetic diversity analysis of coconut germplasm resources, providing a wealth of genetic information and a basis for genetic improvement.

[0030] Therefore, the MNP marker sites and primer compositions provided by this invention provide important references for the selection of cultivars in the cultivation of coconuts, provide basic guarantees for the quality assurance of coconuts in the subsequent chocolate production process, and also provide an important foundation for research on coconut evolution, breeding and genetic engineering. Detailed Implementation

[0031] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0032] Example 1

[0033] Screening of MNP marker sites and design of primers for multiplex PCR amplification for coconut variety identification

[0034] S1. Screening of MNP marker sites for coconut variety identification: The genomes of 13 coconut varieties (Table 3) collected from the Wenchang Coconut Germplasm Resource Garden were sequenced. Sequencing data with more than 20-fold coverage were obtained for each sample. The publicly released coconut genome sequence GCA_000315295.1 was used as the reference genome. First, the sequence analysis was performed using Samtools (Version 1.2) and BCFtools (Version: 1.2) to obtain SNP sites on the coconut genome. MNP markers were screened according to the following principles: (1) The sequence should be a single copy in the genome as much as possible; (2) The marker sequence should have at least three non-contiguous SNPs; (3) The marker sequence length should be less than 250 bp; (4) The discrimination of the candidate MNP markers was further analyzed using the sequencing data of the 13 coconut varieties. Finally, 240 MNP marker sites were screened as shown in Table 1. On average, there were 10.9 marker sites per chromosome. The distribution of the number of marker sites on each chromosome is shown in Table 2.

[0035] Table 1. Locations of 240 coconut MNP marker sites and sequences of 240 detection primer pairs.

[0036]

[0037]

[0038]

[0039] Table 2 Distribution of marker sites on each chromosome

[0040]

[0041] S2. Design of primers for multiplex PCR amplification

[0042] Multiplex PCR amplification primers for the MNP sites were designed using primer design software. The primer design followed the principle of non-interference between primers, and all primers could be combined into a primer pool for multiplex PCR amplification. That is, all designed primers could be amplified normally in one amplification reaction. Finally, primer combinations for the 240 MNP sites described in Table 1 were screened.

[0043] Example 2

[0044] Evaluation of MNP markers, primer combinations, and kits for coconut variety identification

[0045] After synthesizing 240 primer pairs, 5 μL of each primer was mixed to form a primer mix, which is a 1:1 mixture of F-terminal and R-terminal primers. The developed MNP markers, primers, and kits were evaluated using 13 coconut varieties collected in Table 3 to test the detection rate, accuracy, and discrimination of the MNP marker sites.

[0046] The specific steps are as follows:

[0047] (1) Extract total DNA from the coconut variety to be tested; perform a first round of multiplex PCR amplification on the total DNA of the coconut variety using the 240 primer pairs or kit of the present invention, with 18 cycles; after purifying the amplification product, add sample tags and next-generation sequencing adapters based on a second round of PCR amplification; quantify the purified second-round amplification product.

[0048] (2) When testing coconut variety samples, high-throughput sequencing was performed by mixing equal amounts of the second-round amplification products;

[0049] (3) Perform data quality control and data analysis on the sequencing data of coconut variety samples, compare the sequencing results with the coconut reference sequence, and obtain the number of detection sites of the coconut variety at the MNP site, the number of sequencing sequences covering each MNP site, and the genotype data of the MNP site.

[0050] (1) MNP marker detection rate

[0051] Using the kit described in this invention, multiplex PCR amplification and sequencing library construction were performed on these 13 coconut DNA samples. Multiplex amplification, second-generation high-throughput sequencing, and data analysis were conducted, achieving the detection of 240×16=3696 markers in a single experiment. The average sequencing coverage of each sample was more than 300 times, demonstrating the high efficiency of MNP marker detection.

[0052] Table 3 shows the number of MNP markers detected in the sequencing data of these 13 samples. On average, 239.77 (99.90%) MNP markers were detected per variety, indicating that the developed coconut MNP markers have a high and stable detection rate. The national standard GB / T 38551-2020 requires a locus detection rate of no less than 95% for variety identification. A stable locus detection rate prevents significant variations in genetic similarity coefficients across different experiments due to large variations in detected loci, which could ultimately lead to significant biases in variety identification conclusions due to locus sampling.

[0053] Table 3. Statistics on the detection of MNP markers in coconut samples

[0054]

[0055] (2) Accuracy analysis of coconut MNP labeling method

[0056] The number of marker loci with both differences and identical genotypes among varieties is the basis for determining the conclusions in variety identification. Therefore, the accuracy of variety identification ultimately depends on the accuracy of marker locus genotyping. Since the true value is unknown, the absolute accuracy of any method cannot be calculated. In practice, either the reference value is assumed to be the true value when calculating the accuracy, or the precision is used to estimate the accuracy. Because the reference value for the genotype of the marker loci in a variety is also unknown, this experiment uses two reproducibility experiments to calculate the precision, and then calculates the genotyping accuracy: Accuracy = 1 - (1 - Precision) / 2. Here, precision refers to the proportion of marker loci whose genotyping results are consistent between the two experiments. A reproducibility experiment refers to two independent experiments conducted by different personnel, using different batches of reagents, and in different laboratories. The reproducibility experiment simulates identification from different batches; a high reproducibility rate means that the identification results from different laboratories can be accurately compared.

[0057] To verify the accuracy of the coconut MNP marker method, 12 varieties from Table 3 were randomly selected for a reproducibility experiment. After conducting two independent experiments, the MNP marker genotyping results were compared, the number of marker loci with different genotypes was counted, and the genotyping accuracy was calculated. Table 4 shows that a total of 2816 MNP markers were compared in the reproducibility experiment, with 2811 reproducible marker loci. The reproducibility rate of the MNP marker method for marker loci genotyping was 99.83%, and the genotyping accuracy was 99.91%. Therefore, in a single identification, the number of erroneous marker loci did not exceed 240 × (1 - 99.91%) = 0.216, and the deviation from the true value of the genetic similarity coefficient was less than 0.1%. Therefore, using the MNP molecular markers of this invention for variety identification yields highly consistent and reproducible results. It eliminates the need for parallel experiments to reduce experimental errors and allows for precise comparison between DNA fingerprint data from different sources, facilitating data sharing and greatly simplifying variety identification.

[0058] Table 4. Reproducibility of coconut marker genotyping results

[0059]

[0060] Example 3

[0061] Coconut MNP markers for coconut variety identification

[0062] The MNP marker method was used to detect the pairwise differentiation between 13 coconut samples. The proportion of MNP markers showing differences between each pair of coconut varieties was analyzed, resulting in 78 pairs of comparisons. The proportion of MNP markers showing differences between each pair of varieties directly reflects the ability of MNP markers to differentiate varieties. The results are shown in Table 5.

[0063] Based on the difference ratio, according to the current national standard for MNP markers, samples with a genetic similarity greater than 99% are considered "very similar or the same variety"; those with a genetic similarity between 96% and 99% are considered "similar varieties"; and those with a similarity below 96% are considered different varieties. As shown in Table 5, the genetic similarity of all 13 coconut samples was below 90%, indicating they were different varieties. Therefore, the MNP markers described in this invention can distinguish between the 13 coconut samples, enabling their identification.

[0064] Table 5 Comparison of the proportion of differences in coconut samples

[0065]

[0066] Example 4

[0067] The primer composition described in this invention does not conflict with each other and can be simultaneously and efficiently amplified by multiplex PCR. It has high identification accuracy and strong reproducibility. It can be used in detection kits for the MNP marker sites, meeting the requirements for DNA fingerprint database construction. It can be applied to coconut variety authenticity identification, differentiation of different coconut varieties, genetic diversity analysis of germplasm resources, and coconut breeding and other related fields.

[0068] Methods for constructing a coconut varietal DNA fingerprint database: Genomic DNA from all coconut varieties used to construct the database (13 varieties in Table 3) was extracted using the conventional CTAB method. Multiplex amplification, next-generation high-throughput sequencing, and data analysis were performed on different coconut varieties using 240 synthesized MNP marker primers shown in Table 1. The aligned genotype locus data were imported into Excel software, and the genotypes of 240 loci for each sample were recorded as the final DNA fingerprint database. When testing samples were required, genomic DNA was extracted from the samples, and the genotypes of the aforementioned 240 loci were determined and compared with the corresponding loci in the DNA fingerprint database. Referring to the varietal identification standards of other plants, varieties with a difference percentage greater than 4% were considered different varieties, and those less than or equal to 4% were considered the same variety. The percentage of differing loci, p, was calculated as (number of differing loci n / number of common loci N) × 100%.

[0069] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An MNP marker locus for coconut variety identification, characterized in that, The MNP marker sites include the marker sites MNP-1 to MNP-240 on the coconut genome GCA_000315295.1, and the marker sites MNP-1 to MNP-240 are shown in Table 1 of the specification.

2. A multiplex PCR primer composition for detecting the MNP marker site, characterized in that, The multiplex PCR primer composition comprises 240 primer pairs, the nucleotide sequences of which are shown below: 。 3. A detection kit for detecting the MNP marker site, characterized in that, The kit includes the primer composition described above.

4. The kit according to claim 3, characterized in that, The kit also includes a multiplex PCR premix.

5. The application of the multiplex PCR primer composition as described in claim 2 or the detection kit as described in claim 3 in the identification of coconut variety authenticity, differentiation of coconut varieties, construction of a coconut variety DNA fingerprint database and / or analysis of genetic diversity of coconut germplasm resources.

6. The application of the multiplex PCR primer composition as described in claim 2 or the detection kit as described in claim 3 in coconut breeding.

7. The application as described in claim 6, characterized in that, The multiplex PCR primer composition or the detection kit of claim 3 can be used for variety selection, molecular marker-assisted breeding, detection of breeding materials, whole-genome selection breeding, or preparation of whole-genome breeding kits.

8. A method for constructing a coconut variety DNA fingerprint database, characterized in that, The specific application steps are as follows: extract total DNA from all coconut varieties used to construct the coconut DNA fingerprint database, amplify all coconut varieties using the multiplex PCR primer composition described in claim 2, and record the genotype of 240 loci for each variety as the final MNP fingerprint data.

9. A method for identifying the authenticity of coconut varieties, characterized in that, Includes the following steps: Using accurately identified coconut varieties as control samples, gene data of the above 240 MNP loci in the test sample and control sample were obtained respectively, and the data between varieties were compared to obtain the percentage of differential loci. Percentage of differing loci p = (Number of differing loci n / Number of common loci N) × 100%; If the percentage of difference sites between the test sample and the control sample is greater than 4%, the conclusion is that a significant difference was detected, and the test sample and the control sample are not the same variety; if the percentage of difference sites between the test sample and the control sample is less than or equal to 4%, the conclusion is that no significant difference was detected, and the test sample and the control sample are the same variety or similar varieties.

10. An application for distinguishing coconut varieties, characterized in that, Includes the following steps: Genotype data of the above 240 MNP loci of the test samples were obtained respectively. The percentage of differential loci among the test samples was obtained by comparing the genotype data of the test samples. The percentage of differential loci p = (number of differential loci n / number of common loci N) × 100%. If the percentage of differential sites between the tested samples is greater than 4%, the conclusion is that a significant difference was detected, and the tested samples are not the same variety; if the percentage of differential sites between the tested samples is less than or equal to 4%, the conclusion is that no significant difference was detected, and the tested samples are the same variety or similar varieties.

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