An InDel molecular marker for identifying walnut germplasm

Abstract

This invention belongs to the field of molecular marker technology and specifically discloses an InDel molecular marker for identifying slender-shelled pecan germplasm. The primers comprise 34 pairs, the nucleotide sequences of which are shown in SEQ ID NO. 1 to SEQ ID NO. 68. The 34 pairs of primers described herein can clearly identify genetic differences between germplasms and cluster related germplasms into a single branch, making them suitable for evaluating genetic diversity, analyzing kinship, and identifying cultivars of slender-shelled pecan. The identification results obtained using the primer set are unaffected by the environment and possess the advantages of high efficiency, accuracy, and stability. These identification results can provide essential data for slender-shelled pecan variety planting, germplasm development, and cultivar identification.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular markers and specifically discloses an InDel molecular marker for identifying walnut germplasm. Background Art

[0002] Pecan, also known as the American pecan, longevity fruit, and pecan, is one of the world's most famous dried fruit tree species. The kernel of its fruit contains approximately 70% oil, a higher content than camellia seeds, walnuts, olives, and tung seeds, making it one of the highest-oil-rich woody oil crops. In Jiangsu, Anhui, Zhejiang, and other regions, pecans have become a crucial strategic resource for ensuring national grain and oil security. Currently, the heterogeneity of varieties and the scarcity of high-quality seeds remain major constraints on increasing the profitability of pecan cultivation. To ensure the purity of cultivated varieties and to create high-quality new germplasm, it is necessary to clarify the genetic differences and phylogenetic relationships among pecan germplasm resources. Pecans are highly heterozygous, making morphological distinctions between varieties difficult, and morphological identification is susceptible to environmental influences. Establishing a rapid and reliable identification system for pecan germplasm resources is of great practical significance.

[0003] Insertion / deletion (InDel) refers to the insertion or deletion of nucleotide fragments of different sizes at the same site in the genome between closely related species or different individuals of the same species. Specific primers can be designed based on the sequences on both sides of the InDel site for PCR amplification. Traditional molecular marker development is generally based on only one sequence, while InDel markers are based on sequence differences between different individuals. InDel markers are widely distributed in the genome, have high density, and are stable in variation. They have the advantages of low development cost, good reproducibility, accurate results, simple typing, and easy operation. The development of InDel markers that can be used for the analysis of thin-shelled pecan germplasm is of great significance. Summary of the Invention

[0004] The purpose of the present invention is to provide an InDel molecular marker for identifying walnut germplasm, so as to solve the problem that walnut varieties are scarce and difficult to distinguish.

[0005] To achieve the above-mentioned object, the technical solution of the present invention is: an InDel molecular marker for identifying walnut germplasm, comprising 34 pairs of primers, the nucleotide sequence of each primer is shown in the sequence listing SEQ ID NO: 1 to SEQ ID NO: 68:

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[0010] The beneficial effects of this technical solution are:

[0011] (1) The identification results obtained by the primer set of the present invention are not affected by the environment and have the advantages of high efficiency, accuracy and stability. The identification results can provide basic data for the planting of thin-shelled walnut varieties, germplasm creation, and variety identification.

[0012] (2) InDel markers are widely distributed in the genome, have high density, and stable mutations. They have the advantages of low development cost, good reproducibility, accurate results, simple typing, and easy operation.

[0013] Furthermore, one or more of the 34 primer pairs were used in the genetic diversity evaluation, kinship analysis and variety identification of thin-shelled pecan germplasm resources.

[0014] Furthermore, the identification process includes the following steps:

[0015] S1 takes the genomic DNA of the thin-shelled pecan sample to be identified;

[0016] S2 uses the DNA in step S1 as a template and constructs an amplification system using InDel molecular markers to perform PCR amplification;

[0017] S3 used polyacrylamide gel electrophoresis system to detect PCR products.

[0018] Furthermore, the InDel molecules are labeled as one or more pairs of the 34 primer pairs.

[0019] Furthermore, the amplification system was 20 μL, including 10 μL of 2×Tsingke MasterMix, 1 μL of 10 μM upstream primer, 1 μL of 10 μM downstream primer, 1 μL of 10 ng / μL DNA, and 7 μL of ddH2O.

[0020] Furthermore, the PCR amplification program was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 35 s, annealing at 55°C for 35 s, and extension at 72°C for 35 s, for a total of 32 cycles; and final extension at 72°C for 3 min. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is an InDel molecular marker for identifying walnut germplasm of the present invention. Example 15 shows the PCR pattern of 34 walnut germplasms amplified by primer pairs;

[0022] Figure 2This is a UPGMA cluster analysis diagram of InDel markers of 34 pieces of walnut germplasm according to an embodiment of the present invention for identifying walnut germplasm. DETAILED DESCRIPTION

[0023] The following is further described in detail through specific implementation methods:

[0024] The present invention provides a walnut InDel molecular marker primer set, which includes 34 pairs of primers. The nucleotide sequence of each primer is shown in the sequence table SEQ ID No. 1 to SEQ ID No. 68. Specific primer set information is shown in Table 1.

[0025] Table 1 Information of 34 pairs of InDel-labeled primers

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[0029] Example 1

[0030] The genetic diversity and phylogenetic relationship of Carya elata germplasm resources were analyzed using 34 pairs of InDel primers (SEQ ID No. 1 to SEQ ID No. 68) in the following steps:

[0031] (1) Taking healthy leaves of samples to be identified: In the embodiment, a total of 34 germplasm resources to be identified were taken, and their healthy leaves were taken back to the laboratory and stored at -20°C for use. The information of the 34 germplasm resources to be identified is shown in Table 2.

[0032] Table 2. Tested Carya serrata germplasms

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[0034]

[0035] DNA was extracted from healthy leaves of 34 samples to be identified using the Biotech Plant DNA Extraction Kit according to the manufacturer's instructions. DNA quality was assessed by electrophoresis on a 1% (w / v) agarose gel and the 260 / 280 value was measured using a NanoDrop spectrophotometer. Qualified DNA samples were diluted to 10 ng / μL and stored at -20°C until use.

[0036] (2) PCR amplification of the 34 walnut germplasms in step (1) was performed using the 34 pairs of walnut InDel marker primer sets described above:

[0037] The PCR amplification system (20 μL in total) was as follows: 10 μL of 2×Tsingke Master Mix, 1 μL of 10 μM upstream primer, 1 μL of 10 μM downstream primer, 1 μL of 10 ng / μL DNA, and 7 μL of ddH2O.

[0038] The PCR amplification program was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 35 s, annealing at 55°C for 35 s, and extension at 72°C for 35 s, for a total of 32 cycles; and final extension at 72°C for 3 min.

[0039] (3) Detect PCR products using a polyacrylamide gel electrophoresis system. The PCR amplification products obtained in step (2) were separated using 10% PAGE, silver stained, and developed to obtain the genotype of the amplified germplasm. According to the electrophoretic separation of the PCR products, the polymorphism of the bands was statistically analyzed: bands with the same electrophoretic mobility in the products amplified by the same primers were considered to have homology, and products at the same position with bands were recorded as "1" and without bands as "0". The results of each pair of InDel marker amplification were converted into a binary 0-1 matrix, and the genetic diversity parameters, including the number of observed alleles, the number of effective alleles, and the Shannon information index at each site, were calculated using PopGene software; the polymorphic information amount of each primer was calculated using PowerMarker software. The results are shown in Table 3.

[0040] Table 3 Genetic diversity of 34 InDel-labeled primers in 34 Carya elata accessions

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[0043] The results in Table 3 show that 34 primer pairs amplified a total of 78 polymorphic loci in 34 individuals of Hickory nut. Each primer pair amplified an average of 2.29 polymorphic loci. The minimum number of effective alleles ranged from 1.035 (InDel-20) to 2.352 (InDel-1), with an average of 1.62. The average Shannon Information Index (SI) was 0.56, and the average Polymorphic Information Content (PIC) was 0.35. Six primer pairs exhibited high PIC values (PIC > 0.5), 20 pairs had intermediate PIC values (0.25 < PIC < 0.5), and the remaining eight pairs had low polymorphism (0 < PIC < 0.25).

[0044] in, Figure 1 Shown are the amplification results of 15 pairs of InDel-labeled primers.

[0045] Depend on Figure 1The results showed that the InDel marker of the present invention can effectively amplify the DNA of different thin-shelled pecan varieties, the target bands are clear, the results are stable, and the genetic differences of different thin-shelled pecan varieties can be well reflected.

[0046] The SM (Similarity coefficient) matrix between samples was calculated using the SimQual program of Ntsys 2.1 software. Cluster analysis using the unweighted pair group method with arithmetic mean (UPGMA) was performed based on the genetic similarity matrix using the SHAN program. The kinship clustering tree diagram between individuals was constructed using the Treeplot module. Figure 2 shown.

[0047] Combine Figure 2 It can be seen that the varieties 'P1', 'P2', 'P3', 'P4', and 'P5' with clear pedigree relationships and their parents 'Pawnee' and 'Shaoxing' are clustered in the same branch. This result proves that the 34 pairs of walnut InDel markers can be used to determine the genetic relationship between walnut germplasm resources.

[0048] The above description is merely an embodiment of the present invention. Common knowledge regarding the specific structure and characteristics of the solution is not described in detail herein. It should be noted that those skilled in the art may make various modifications and improvements without departing from the structure of the present invention. These modifications and improvements should also be considered within the scope of protection of the present invention and will not affect the effectiveness of the implementation of the present invention or the practical application of the patent.

Claims

1. An InDel molecular marker for identifying walnut germplasm, characterized in that: A total of 34 pairs of primers are included, and the nucleotide sequences of the primers are shown in the sequence listing SEQ ID NO: 1 to SEQ ID NO: 68:

2. An InDel molecular marker for identifying walnut germplasm according to claim 1, and application of the 34 pairs of primer combinations in genetic diversity evaluation, kinship analysis and variety identification of walnut germplasm resources.

3. The identification process of an InDel molecular marker for identifying walnut germplasm according to claim 1, characterized in that: The following steps are involved: S1 takes the genomic DNA of the thin-shelled pecan sample to be identified; S2 uses the DNA in step S1 as a template and constructs an amplification system using the 34 pairs of InDel molecular marker primers described in claim 1 to perform PCR amplification; S3 used polyacrylamide gel electrophoresis system to detect PCR products.

4. The identification process of an InDel molecular marker for identifying vegetative walnut germplasm according to claim 3, characterized in that: The amplification system is 20 μL, comprising 10 μL of 2×Tsingke MasterMix, 1 μL of 10 μM upstream primer, 1 μL of 10 μM downstream primer, 1 μL of 10 ng / μL DNA, and 7 μL of ddH2O.

5. The identification process of an InDel molecular marker for identifying walnut germplasm according to claim 3, characterized in that: The PCR amplification program was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 35 s, annealing at 55°C for 35 s, and extension at 72°C for 35 s, for a total of 32 cycles; and final extension at 72°C for 3 min.

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