Carya illinoensis multiplex PCR (polymerase chain reaction) primer combination and application thereof

Abstract

The invention discloses a carya illinoensis multiplex PCR primer combination and application thereof, and belongs to the technical field of molecular markers. The multiplex PCR primer combination comprises eight primer combinations, each primer combination comprises three pairs of primers, and the sequences of the primers are as shown in SEQ ID NO. 1 to SEQ ID NO. 48. 24 polymorphic InDel markers are mined on the basis of apocarya transcriptome data, 24 pairs of primers are designed and synthesized, the 24 pairs of primers are optimized into 8 triple combinations, multi-site synchronous detection can be achieved, then apocarya germplasm resource diversity analysis and fingerprint spectrum construction work can be efficiently carried out, and the method has the advantages of high specificity, high specificity, high sensitivity and the like. And a reliable technical support is provided for genetic improvement, variety breeding, accurate propagation of improved varieties and the like of the carya illinoensis.

Description

Technical Field

[0001] This invention relates to the field of molecular marker technology, and in particular to a combination of multiplex PCR primers for thin-shelled pecans and their applications. Background Technology

[0002] Thin-shelled pecans ( Carya illinoinensis Pecans, also known as American hickory, long hickory, or pecan, are plants belonging to the genus *Caragana* in the family Juglandaceae. The kernels of thin-shelled pecans have an oil content as high as 70%, higher than that of camellia seeds, olives, and tung oil seeds, making them one of the woody tree species with the highest oil content.

[0003] Conventional methods for analyzing thin-shelled pecan germplasm resources include leaf morphology, female flower characteristics, and fruit properties. Phenotypic characteristics are easily affected by environmental factors such as plant developmental stage, seasonal changes, temperature fluctuations, and light intensity, leading to insufficient data stability. Furthermore, the judgment criteria are highly dependent on human experience, exhibiting strong subjectivity and easily introducing evaluation bias, ultimately reducing the reliability of the results. In contrast, DNA molecular marker technology demonstrates significant advantages: its detection results are unaffected by environmental conditions and show high consistency across different developmental stages and tissue parts (such as roots, stems, and leaves), making it a reliable tool for accurate plant variety identification, in-depth analysis of genetic diversity, and efficient fingerprinting. DNA molecular marker technology, with its advantages of being unaffected by environmental factors and consistent across different developmental stages and tissue parts, has become an effective means for plant variety identification, diversity analysis, and fingerprinting construction.

[0004] Conventional molecular markers such as microsatellites (Simple sequence repeats, SSRs) and insertions / deletions (InDels) can be used for multiplex detection, which involves placing two or more markers in a single polymerase chain reaction (PCR) for experimentation and subsequent detection. Compared to single-marker independent detection, this significantly shortens the experimental cycle, reduces reagent costs, and increases detection throughput. Unfortunately, a simple and stable multiplex detection technology system for thin-shelled pecans is still lacking, and further research and development are urgently needed. Summary of the Invention

[0005] The purpose of this invention is to provide a multiplex PCR primer combination for thin-shelled pecans and its application, thereby addressing the problems existing in the prior art. Based on the transcriptome data of thin-shelled pecans, this invention mines 24 polymorphic InDel markers and designs and synthesizes 24 primer pairs. These 24 primer pairs are then optimized into 8 triplet combinations, enabling simultaneous detection at multiple loci. This allows for efficient analysis of the diversity of thin-shelled pecan germplasm resources and the construction of fingerprint maps, providing reliable technical support for the genetic improvement, variety selection, and precise propagation of superior varieties of thin-shelled pecans.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a multiplex PCR primer set for thin-shelled pecans, comprising 8 primer sets, each containing 3 pairs of primers; Combination 1 contains primers as shown in SEQ ID NO.1-SEQ ID NO.6; Combination 2 contains primers as shown in SEQ ID NO.7-SEQ ID NO.12; Combination 3 contains primers as shown in SEQ ID NO.13-SEQ ID NO.18; Combination 4 contains primers as shown in SEQ ID NO.19-SEQ ID NO.24; Combination 5 contains primers as shown in SEQ ID NO.25-SEQ ID NO.30; Combination 6 contains primers as shown in SEQ ID NO.31-SEQ ID NO.36; Combination 7 contains primers as shown in SEQ ID NO.37-SEQ ID NO.42; Combination 8 contains primers as shown in SEQ ID NO.43-SEQ ID NO.48.

[0007] The present invention also provides a thin-shelled pecan multiplex PCR kit, wherein the kit contains the above-mentioned thin-shelled pecan multiplex PCR primer combination.

[0008] The present invention also provides the application of the above-mentioned thin-shelled pecan multiplex PCR primer combination or the above-mentioned kit in the analysis of genetic diversity of thin-shelled pecan germplasm, germplasm identification, assisted breeding or fingerprinting.

[0009] This invention also provides a method for constructing a fingerprint spectrum of thin-shelled pecans, comprising the following steps: S1. Extract DNA from thin-shelled pecans; S2. Using the DNA from step S1 as a template, perform PCR amplification using each primer combination in the above-mentioned thin-shelled pecan multiplex PCR primer combination to obtain PCR products. S3. Perform electrophoresis detection on the PCR product, and arrange the detection results of each primer combination in series to obtain the fingerprint spectrum of the thin-shelled pecan.

[0010] Furthermore, the PCR amplification reaction system is as follows: 30 μL of 2×TSINGKE Master Mix, 1 μL of DNA, 1 μL each of 3 pairs of upstream and downstream primers, 13 μL of ddH2O, and a total volume of 50 μL.

[0011] Furthermore, the PCR amplification program is as follows: 94℃ pre-denaturation for 5 min; 95℃ denaturation for 10 s, 56℃ annealing for 1 min, 72℃ extension for 1 min, 35 cycles; final extension for 7 min.

[0012] This invention also provides an application of the thin-shelled pecan fingerprint constructed by the above-described method in the analysis of genetic diversity or identification of thin-shelled pecan germplasm.

[0013] The present invention discloses the following technical effects: (1) Low cost: Based on the transcriptome data of pecan, this invention mined 24 polymorphic InDel markers and designed and synthesized 24 primer pairs. The 24 primer pairs were optimized into 8 triple combinations, reducing the required reaction volume from the conventional 75 μL to 50 μL, and the amount of reagents used was reduced by about 33.33%. At the same time, the different markers within the same combination have different amplified fragment lengths, which can be clearly distinguished by conventional agarose gel electrophoresis, resulting in low detection costs.

[0014] (2) Time-saving and efficient: Conventional PCR only amplifies one target site. The triple PCR designed in this invention achieves simultaneous amplification of three target sites in the same reaction, reducing repeated operations, reducing the overall process time, and significantly improving experimental efficiency.

[0015] (3) Simple operation: Each primer pair in this invention contains 6 primers, and the amount of each primer added is uniformly 1 μL, which significantly reduces the need for frequent adjustment of sampler parameters during sample addition. In addition, the PCR amplification program of all primer pairs is consistent, and there is no need to adjust the annealing temperature or extension time, further simplifying the experimental procedure. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 Agarose gel electrophoresis images of 20 thin-shelled pecan germplasms amplified using primer combinations 1-8 of the present invention; M: marker; 1-20: corresponding to thin-shelled pecan germplasms S1-S20 in Table 2; Figure 2 This is a UPGMA cluster diagram of 20 thin-shelled pecan germplasm accessions constructed based on primer combinations 1-8 of this invention. Detailed Implementation

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] Example 1: Obtaining multiplex PCR primer combinations for thin-shelled pecans Transcriptome sequencing data from three pecan varieties ('Pawnee', 'Wichita', and 'Stuart') obtained previously by our research group were used to identify insertion / deletion (InDel) markers. Raw sequencing data was filtered to obtain Clean data, which was then aligned to the reference genome (Carya illinoinensis Pawnee v1.1) using the BWA program to generate SAM files. The SAM files were then converted to BAM files using the SAMtools program, and PCR repeat data were labeled using Picard. Finally, InDel sites were identified using the GATK program.

[0024] A total of 135,622 InDel loci were identified on the reference genome. InDels were filtered based on alignment quality >400, bias ≥15, and variant length ≥15, leaving 740 loci. After removing InDels containing SSRs, 255 InDel loci remained. Primers were designed for these 255 InDel loci, resulting in 242 primer pairs. These 242 InDel primer pairs were screened using three pecan varieties ('Pawnee', 'Wichita', and 'Stuart'), revealing 135 pairs exhibiting polymorphism. InDel markers with 2-3 alleles and clear amplification bands were selected for multiplex PCR experiments.

[0025] Primers were redesigned based on the InDel site length. For InDel markers with lengths of 15-20 bp, 21-25 bp, and 26-30 bp, primers were designed to amplify products with lengths of approximately 160, 250, and 350 bp, respectively. Three pairs of primers with different target product lengths were grouped together, and PCR amplification was performed using genomic DNA from 'Pawnee', 'Wichita', and 'Stuart' as templates. After repeated experiments with different combinations, eight triplet PCR primer combinations with 24 InDel markers were finally obtained. Specific information is shown in Table 1.

[0026] Table 1 Primer information for triple primer combinations Example 2: Application of multiplex PCR primer combinations for thin-shelled pecans In this embodiment, the genetic diversity analysis and fingerprinting of thin-shelled pecans were performed using the eight triple PCR primer combinations from Example 1. The steps are as follows: (1) Extraction of genomic DNA from thin-shelled pecans Twenty leaf samples of thin-shelled pecan germplasm (details provided in Table 2, from the Jiangsu Provincial Germplasm Resource Bank of the Institute of Botany, Chinese Academy of Sciences) stored at -20℃ were used to extract DNA using the BioTeke nucleic acid extraction kit. The quality and concentration of DNA were determined using 1% agarose gel electrophoresis and a NanoDrop spectrophotometer. Qualified DNA samples were diluted to 10 ng / µL and stored at -20℃ for later use.

[0027] Table 2. Thin-shelled pecan germplasm tested (2) Multiplex PCR DNA from 20 germplasm accessions was amplified using eight triple PCR primer combinations. The PCR system consisted of 30 μL of 2×TSINGKEMaster Mix (green), 1 μL of DNA, 1 μL each of three pairs of forward and reverse primers (10 μM each), and 13 μL of ddH2O, for a total of 50 μL. The PCR amplification program was as follows: 94℃ pre-denaturation for 5 min; 95℃ denaturation for 10 s, 56℃ annealing for 1 min, 72℃ extension for 1 min, for 35 cycles; final extension for 7 min.

[0028] (3) Detection of PCR products The PCR products were detected using 3% agarose gel electrophoresis, and the results are shown below. Figure 1 Based on the electrophoretic separation of PCR products, the polymorphism of the bands was statistically analyzed: among the 24 markers, MP-7 produced 3 polymorphic bands across 20 germplasms, while the other markers produced only 2 bands each. For each marker, a band at the same position was recorded as "1", and no band was recorded as "0", resulting in a total of 49 0-1 numbers. The first two characters correspond to the detection result of the first marker, and subsequent codes are arranged sequentially according to the marker order.

[0029] (4) Genetic diversity analysis The genetic similarity coefficient (GS) between samples was calculated using the SimQual program in Ntsys 2.1 software, and a genetic similarity coefficient matrix was obtained. The genetic diversity among germplasms was then determined using the GS matrix. UPGMA clustering was performed using the SHAN method, and a clustering tree was constructed. The results are shown below. Figure 2 .

[0030] Depend on Figure 2 It can be seen that the genetic similarity values ​​among the 20 germplasms are all around 0.8, with 'ZY1' and 'Sioux' having the highest similarity; 'Zhongshan12' has the lowest genetic similarity with the other 19 germplasms, suggesting that its genome may have significant differences from other germplasms.

[0031] (5) Fingerprint mapping By integrating the 0-1 feature data of 24 InDel markers, fingerprint maps of 20 germplasm resources were successfully constructed. Except for 'ZY1' and 'Sioux', which share the same map features, the other germplasm resources all have unique fingerprint identifiers (see Table 3).

[0032] Table 3. Fingerprint patterns of 20 thin-shelled pecan germplasm accessions The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A multiplex PCR primer combination for thin-shelled pecans, characterized in that, It contains 8 primer combinations, each containing 3 pairs of primers; Combination 1 contains primers as shown in SEQ ID NO.1-SEQ ID NO.6; Combination 2 contains primers as shown in SEQ ID NO.7-SEQ ID NO.12; Combination 3 contains primers as shown in SEQ ID NO.13-SEQ ID NO.18; Combination 4 contains primers as shown in SEQ ID NO.19-SEQ ID NO.24; Combination 5 contains primers as shown in SEQ ID NO.25-SEQ ID NO.30; Combination 6 contains primers as shown in SEQ ID NO.31-SEQ ID NO.36; Combination 7 contains primers as shown in SEQ ID NO.37-SEQ ID NO.42; Combination 8 contains primers as shown in SEQ ID NO.43-SEQ ID NO.

48.

2. A multiplex PCR kit for thin-shelled pecans, characterized in that, The kit contains the thin-shelled pecan multiplex PCR primer combination as described in claim 1.

3. The application of the thin-shelled pecan multiplex PCR primer combination of claim 1 or the kit of claim 2 in the analysis of genetic diversity of thin-shelled pecan germplasm, germplasm identification, assisted breeding or fingerprinting.

4. A method for constructing a fingerprint spectrum of thin-shelled pecans, characterized in that, Includes the following steps: S1. Extract DNA from thin-shelled pecans; S2. Using the DNA from step S1 as a template, perform PCR amplification using each primer combination in the thin-shelled pecan multiplex PCR primer combination described in claim 1 to obtain PCR products. S3. Perform electrophoresis detection on the PCR product, and arrange the detection results of each primer combination in series to obtain the fingerprint spectrum of the thin-shelled pecan.

5. The construction method according to claim 4, characterized in that, The PCR amplification reaction system was as follows: 30 μL of 2×TSINGKE Master Mix, 1 μL of DNA, 1 μL each of 3 pairs of upstream and downstream primers, 13 μL of ddH2O, and a total volume of 50 μL.

6. The construction method according to claim 4, characterized in that, The PCR amplification program was as follows: 94℃ pre-denaturation for 5 min; 95℃ denaturation for 10 s, 56℃ annealing for 1 min, 72℃ extension for 1 min, 35 cycles; final extension for 7 min.

7. The application of a thin-shelled pecan fingerprint constructed by the method described in any one of claims 4-6 in the analysis of genetic diversity or germplasm identification of thin-shelled pecan germplasm.

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