An amplification primer and a kit for identifying cyrtosperma macrocarpa

Abstract

This invention relates to the fields of molecular biology and genetic breeding technology, specifically to an amplification primer and a kit for identifying large-grain tiger nuts. This invention involves genome sequencing of representative tiger nut morphologies from germplasm resources, selecting black, medium-round, large, and medium-long nut morphologies as screening samples. A large number of tiger nut SSR sequence loci were discovered. After screening for single-copy loci, single-copy locus amplification primers were designed to screen and identify existing tiger nuts. Molecular markers specifically for distinguishing large-grain tiger nuts were discovered, and amplification primers targeting these markers were determined. These primers were then assembled to form a rapid molecular detection product capable of identifying and distinguishing large-grain tiger nut varieties. Verification through embodiments of this invention shows that the kit can accurately screen for large-grain tiger nut varieties from a diverse mix of varieties. The kit is low-cost, quick, highly reproducible, and provides accurate results.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology and genetic breeding technology, specifically to an amplification primer and a kit for identifying the large grain type of tiger nuts. Background Technology

[0002] Tiger nuts originated in Africa and are a newly emerging oilseed crop that has been vigorously promoted for cultivation in my country in recent years. They possess outstanding advantages such as high yield per acre, good health benefits, strong resistance to adverse conditions, and no competition for land with staple crops. Since 2019, in order to fully guarantee my country's self-sufficiency in vegetable edible oil and increase oilseed yields without occupying staple crop planting areas, tiger nuts have become a recommended emerging oil source crop by the Ministry of Science and Technology and the Ministry of Agriculture and Rural Affairs. In 2019, the Ministry of Science and Technology established my country's first national key research and development project on tiger nuts, conducting research on key technologies for the entire industrial chain of tiger nuts, laying the foundation for germplasm resources, breeding, and processing.

[0003] Among existing tiger nut germplasm resources, their grain shapes can be basically divided into medium-round, medium-long, and large. The stem bean color is commonly brownish-yellow, with black being relatively rare. Different grain shapes and stem bean colors are one of the main phenotypic bases for tiger nut quality classification, and also form the basis for industrialization processes such as specialized harvesting, storage, and processing. However, due to a very weak research foundation in tiger nut, and the lagging development of genetics and genomics, there is still no reference genome or highly reproducible primers. The identification of tiger nut germplasm resources with different grain shapes relies almost entirely on phenotypic and species identification. Phenotypic identification suffers from long time cycles and the results are easily affected by environmental and human factors. Molecular biology techniques, represented by DNA molecular markers, have been widely used in the trait detection of major food crops such as wheat, rice, and corn, while genetic research on tiger nut started late and has a weak foundation. Currently, only two oil synthesis-related genes for tiger nuts have been successfully identified through homologous cloning. The Jilin Academy of Agricultural Sciences has developed several RAPD markers for germplasm resource classification, and the Institute of Economic Crops of the Henan Academy of Agricultural Sciences has developed some KASP markers. However, these markers have not yet been fully validated in germplasm resources, nor can they be linked to the grain shape trait of tiger nuts. Therefore, specific sequences and detection markers related to large-grained tiger nuts are currently lacking worldwide. To better fulfill the national research and industrialization tasks, it is urgent to identify molecular detection markers for large-grained tiger nuts through screening and to develop molecular diagnostic products capable of distinguishing large-grained tiger nuts. Summary of the Invention

[0004] In order to overcome the shortcomings of the prior art, one of the objectives of the present invention is to provide an amplification primer that targets and amplifies the sequence shown in SEQ ID NO.1 as a molecular marker for large tiger nuts, for the identification and differentiation of large tiger nuts.

[0005] The second objective of this invention is to provide a kit for identifying large tiger nuts, including the amplification primers provided by this invention, which target and amplify the sequence shown in SEQ ID NO.1 to distinguish large tiger nuts.

[0006] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows: An amplification primer for targeted amplification of the sequence shown in SEQ ID NO.1.

[0007] Optionally, the above amplification primers consist of a forward primer and a reverse primer. The forward primer sequence is: 5'-AATCGAGCGAATCAGATTTCA-3'; the reverse primer sequence is: 5'-ATGTTGTCATCGCGCTTTC-3'.

[0008] A kit for identifying the large size of tiger nuts, comprising the above-mentioned amplification primers.

[0009] Furthermore, the kit also includes a buffer solution for the amplification reaction.

[0010] Furthermore, the buffer solution used for the amplification reaction includes DNA polymerase, dATP, dCTP, dTTP, and dGTP.

[0011] The preparation method of the above-mentioned kit for identifying the large-sized tiger nuts includes first screening and determining specific molecular markers for the large-sized tiger nuts. The screening method includes the following steps: 1) Determine and obtain the tiger pea genome sequence information of the screening samples; 2) SSR site detection and primer design: Based on the tiger pea genome sequence information obtained in step 1), SSR sequences were screened and a batch of SSR primers were designed; 3) Screening and identifying molecular markers: Using the SSR primers designed in step 2), the samples were amplified and screened. Molecular markers associated with large-grained stem beans were screened based on the criteria of single copy, high reproducibility, and uniform distribution on the chromosome. 4) Using the amplification primers corresponding to the molecular markers identified in step 3), assemble the kit to obtain the kit; In step 3), the molecular markers identified are the sequences shown in SEQ ID NO.1.

[0012] Further, in step 2), use the MISA software to search the genome for SSRs using the following configuration parameters: Configuration parameter information: definition (unit_size,min_repeats):1-15 2-6 3-5 4-4 5-4 6-4 interruptions ( max_difference_between_2_SSRs ) : 100 Based on the SSR sequences found in the above steps, a batch of SSR primers were designed and developed using the software Primer 3.0. In step 3), the amplification primers corresponding to the molecular markers identified by screening consist of a forward primer and a reverse primer. The forward primer sequence is: 5'-AATCGAGCGAATCAGATTTCA-3'; the reverse primer sequence is: 5'-ATGTTGTCATCGCGCTTTC-3'.

[0013] Optionally, the stem bean traits of the screened samples include black, medium-round, large, and medium-long beans.

[0014] More preferably, the selected sample includes one black variety; three medium-round varieties; three large varieties; and one medium-long variety.

[0015] Optionally, the specific methods for determining and obtaining the tiger pea genome sequence information in step 1) include: (1) DNA extraction: Collect young leaves and extract high-quality, non-degradable, and high-purity genomic DNA; (2) Library construction: DNA containing biotin is captured by adsorption of avidin magnetic beads, and the DNA fragments are repaired at the ends, A is added, adapters are ligated, PCR amplification cycle number is evaluated, and the library is purified and discharged to complete the entire library preparation. (3) Library quality control: After the library is constructed, the library is first initially quantified using Qubit2.0 and diluted to 1 ng / μl. Then, the insert size of the library is detected using Agilent 2100. After the insert size meets the expectations, the effective concentration of the library is accurately quantified using Q-PCR to ensure the quality of the library. (4) Sequencing: After the library passes the test, the different libraries are pooled according to the effective concentration and the target amount of data to be sequenced and then BGI MGISEQ-2000 PE150 sequencing is performed. (5) Obtain sequence information: The raw image data file obtained in step (4) is converted into raw sequencing sequence through base calling analysis. After data quality control, filtering and splicing, tiger nut sequence information is obtained.

[0016] This invention utilizes genome sequencing of representative tiger nut morphologies from germplasm resources, selecting black, medium-round, large, and medium-long nut morphologies as screening samples. This process uncovered a large number of tiger nut SSR sequence loci. After screening for single-copy loci, primers for amplifying these loci were designed to screen and identify existing tiger nut varieties, families, and distinctive germplasm resources. Molecular markers specifically for distinguishing large-nut tiger nut morphologies were discovered, and primers targeting these markers were identified and assembled into a rapid molecular detection product capable of identifying and distinguishing large-nut tiger nut varieties. Verification through embodiments of this invention demonstrates that the kit can accurately screen for large-nut tiger nut varieties from a diverse mix of varieties.

[0017] The kit of this invention has the following advantages for rapid identification of tiger nut varieties: (1) low detection cost, with a single sample detection cost of about 0.5 yuan when using conventional PAGE detection technology; (2) simple and easy-to-operate detection technology, convenient and flexible detection throughput, which can be flexibly adjusted according to detection needs; (3) low requirements for detection equipment, only a PCR instrument and electrophoresis equipment are needed; (4) short time, high repeatability and accurate results. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the population validation results of the molecular markers for the large grain type trait of tiger nuts identified in Example 1. Figure 2 This is a schematic diagram of the detection results for experimental group 1; Figure 3 This is a schematic diagram of the detection results for experimental group 2. Detailed Implementation

[0019] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the description of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0020] The method for determining the tiger nut gene sequence information in the following examples is as follows: 1. Genome sequencing (1) DNA extraction: Collect young leaves and extract high-quality, non-degradable, and high-purity genomic DNA.

[0021] (2) Library construction: DNA containing biotin is captured by adsorption of avidin magnetic beads, and the DNA fragments are repaired at the ends, A is added, adapters are ligated, PCR amplification cycle number is evaluated, and the library is purified and discharged to complete the entire library preparation.

[0022] (3) Library quality control: After the library is constructed, the library is first initially quantified using Qubit2.0 and diluted to 1ng / μl. Then, the insert size of the library is detected using Agilent 2100. After the insert size meets the expectations, the effective concentration of the library is accurately quantified using Q-PCR (effective concentration of library >2nM) to ensure the quality of the library.

[0023] (4) Sequencing: After the library passes the test, different libraries are pooled according to the effective concentration and the target amount of data to be sequenced and then BGI MGISEQ-2000 PE150 sequencing is performed.

[0024] 2. Data Processing (1) Raw sequencing data The raw image data files obtained from the next-generation sequencing platform are converted into raw sequencing reads (Raw Data or Raw Reads) through base calling analysis. The results are stored in FASTQ (fq) file format, which contains the sequence information of the sequencing reads and their corresponding sequencing quality information.

[0025] (2) Clean Data Raw sequencing data contains adapter information, low-quality bases, and undetected bases (represented by N). This information can cause significant interference to subsequent data analysis. By using a fine filtering method to remove this interference, the resulting data is called clean data or clean reads. The data format of this file is exactly the same as that of raw data.

[0026] (3) Data quality control and filtering ① Reads containing adapter sequences need to be filtered out; ② Remove consecutive bases with a mass less than 15 at both ends of the sequencing read; ③ Remove reads when the final length of the sequencing read is less than 50 bp; ④ Only retain paired reads.

[0027] Example 1 In this embodiment, molecular markers for the large-grain trait of tiger nuts were screened, and the specific method was as follows: 1. Selected screening samples: 8HS-2-9 (black), 2022-Y (medium round grain), DL-3-Y (large grain), 48 (medium round grain), DL-17-Y (large grain), 224-5 (medium round grain), DL-17-C (large grain), 383-5 (medium long grain); samples were provided by the Tiger Pea Research Laboratory of the Institute of Economic Crops, Henan Academy of Agricultural Sciences.

[0028] 2. Determine and obtain gene sequence information of the screening samples; 3. Marker site discovery and primer design (1) SSR site detection and primer design ① Use the MISA software (http: / / pgrc.ipk-gatersleben.de / misa / ) with the following configuration parameters to search the genome for SSRs: Configuration parameter information: definition (unit_size,min_repeats):1-15 2-6 3-5 4-4 5-4 6-4 interruptions ( max_difference_between_2_SSRs ) : 100 ② SSR primer design Based on the SSR sequences found in the above steps, a batch of SSR primers were designed and developed using Primer 3.0 software: ③ Screening criteria Single-copy, highly reproducible, and evenly distributed on chromosomes, the synthesized material is used to scan the population and screen for molecular markers associated with large-grained stem beans.

[0029] 4. Screening results: The molecular marker for large tiger nuts obtained by screening is the sequence shown in SEQ ID NO.1; the corresponding forward primer sequence is: 5'-AATCGAGCGAATCAGATTTCA-3'; the reverse primer sequence is: 5'-ATGTTGTCATCGCGCTTTC-3'.

[0030] 5. Group validation: The selected amplification primers from step 4 were used to amplify the selected samples, whereby... PCR system: 5 μL 2xEs Taq Master Mix, 3 μL sterile water, 0.5 μL forward primer, 0.5 μL reverse primer, 1 μL tiger pea leaf DNA (15 μg / μL).

[0031] PCR program: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 30 s, 65℃ annealing for 30 s, 72℃ extension for 30 s, 15 cycles, with the annealing temperature decreasing by 1℃ per cycle, 95℃ denaturation for 30 s, 50℃ annealing for 30 s, 72℃ extension for 30 s, 25 cycles, 72℃ extension for 5 min.

[0032] The results are as follows Figure 1As shown, the amplification product band distribution of different tiger nuts shows that the large-grain tiger nuts have a characteristic electrophoretic band in the 300bp-350bp range. Compared with other tiger nut varieties that have characteristic bands in the 300bp-350bp range, the characteristic band of the large-grain tiger nuts is longer than 20bp.

[0033] Example 2 This embodiment provides a kit that can distinguish between large tiger nuts, including the amplification primers screened and determined in Example 1.

[0034] The kit provided in Example 2 was used to verify the effectiveness of distinguishing large tiger nuts. 1. Experimental group: Group 1: 24 tiger nut trial materials from Henan Province in 2022; Group 2: Family data of tiger nuts from the Institute of Economic Crops, Henan Academy of Agricultural Sciences, totaling 52 families, coded as follows: 252, 273, 306, 295, 322, 296, 449, 330, 297, 4, 315, 275, 251, 307, 541, Yu You Sha No. 1, 462, 574, 548, 363, 353, 319, 468, Yu You Sha No. 4, 323, 411, 549, 543, 426, 407, 538, 518, 369, 539, 625, 586, 555, 294, 405, 26, 394, 276, 485, 544, 317, 597, 8HS-3, DL-17-C, 8HS-2-2, PDL-15-5; 2. Experimental methods: DNA was extracted from the leaves of all experimental tiger pea plants, and the DNA was amplified using the kit provided in Example 2. The amplification products were then analyzed by electrophoresis. The PCR system consisted of 5 μL of 2xEs Taq Master Mix, 3 μL of sterile water, 0.5 μL of forward primer, 0.5 μL of reverse primer, and 1 μL of tiger pea leaf DNA (15 μg / μL).

[0035] PCR program: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 30 s, 65℃ annealing for 30 s, 72℃ extension for 30 s, 15 cycles, with the annealing temperature decreasing by 1℃ per cycle, 95℃ denaturation for 30 s, 50℃ annealing for 30 s, 72℃ extension for 30 s, 25 cycles, 72℃ extension for 5 min.

[0036] Results for Group 1 are as follows Figure 2As shown: Sample 8 can be clearly distinguished by the presence of a characteristic electrophoretic band in the 300bp~350bp range. Compared with other tiger nuts with characteristic bands in the 300bp~350bp range, the characteristic band of sample 8 is longer than 20bp, thus identifying sample 8 as a large tiger nut, which is consistent with the actual results. Results for Group 2 are as follows Figure 3 As shown, samples 50 and 52 can be clearly distinguished by the presence of characteristic electrophoretic bands in the 300bp~350bp range. Compared to other tiger nuts with characteristic bands in the 300bp~350bp range, the characteristic bands of samples 50 (DL-17-C) and 52 (PDL-15-5) are longer than 20bp. DL-17-C and PDL-15-5 were identified as large tiger nuts, which is consistent with the actual results.

[0037] In actual testing, the kit of this invention typically includes a positive control (i.e., large-particle material) as needed. The specific process is as follows: using the amplification primers provided by this invention, as shown in SEQ ID NO.1, PCR amplification and electrophoresis are performed on the unknown sample and the positive control material. The electrophoretic band amplified in the 300-350bp range of the positive control is used as the standard. If the band pattern of the unknown sample is completely identical to that of the positive control, it is determined to be a large-particle material; otherwise, it is determined to be other particle types.

[0038] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An amplification primer, characterized in that, The targeted amplification is performed on the sequence shown in SEQ ID NO.1; it consists of a forward primer and a reverse primer, the forward primer sequence being: 5'-AATCGAGCGAATCAGATTTCA-3'; the reverse primer sequence being: 5'-ATGTTGTCATCGCGCTTTC-3'.

2. A kit for identifying the large-sized tiger nuts, characterized in that, Includes the amplification primers as described in claim 1.

3. The kit for identifying the large size of tiger nuts as described in claim 2, characterized in that, It also includes buffer solutions for amplification reactions.

4. The kit for identifying the large size of tiger nuts as described in claim 3, characterized in that, The buffer solution used for the amplification reaction includes DNA polymerase, dATP, dCTP, dTTP, and dGTP.

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