A primer set for EST-SSR markers of Carya sylvestris and its kit and application

Abstract

The present invention provides a primer set for EST-SSR markers of thin-shelled pecans, a kit and an application thereof, belonging to the field of molecular marker technology. The primer set includes 16 pairs of primers, and the nucleotide sequence of each primer is shown in the sequence table SEQ ID No.1 to SEQ ID No.32. The primer set of the present invention can clarify the genetic diversity between germplasms, and at the same time can gather germplasms with pedigree relationships in one branch, and can be used for the analysis of genetic diversity and kinship of thin-shelled pecan germplasm resources. Compared with morphological identification, 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 innovation of thin-shelled pecan germplasm, so as to more effectively select suitable breeding parents.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular markers, and in particular relates to a primer set for EST-SSR markers of Carya serrata, a kit and an application thereof. Background Art

[0002] Thin-shelled pecan, also known as American pecan, longevity fruit, and pecan, is one of the world's famous dried fruit tree species. The kernel of the fruit contains up to 70% oil, which is higher than that of tea seeds, walnuts, olives, and tung seeds. It is one of the woody oil crops with the highest oil content. At present, the scarcity of good varieties is still an important factor restricting the improvement of the planting efficiency of thin-shelled pecans. In order to cultivate new germplasm with high yield, high quality, and multi-resistance, it is necessary to clarify the genetic diversity and kinship between the germplasm resources of thin-shelled pecans. In the long-term introduction and cultivation process, the same variety may have different names, resulting in very serious homonyms and homonyms, and the kinship between the varieties is unclear. This has a great impact on the selection of parents in the breeding process. Therefore, it is of great practical significance to establish a rapid and reliable technical system for the identification of kinship between germplasm resources of thin-shelled pecans.

[0003] Molecular markers are a useful tool for studying plant genetic diversity, with the advantages of high accuracy, good repeatability, and no interference from environmental conditions. Simple sequence repeats (SSR), also known as microsatellites, are co-dominant molecular markers with the characteristics of simple operation, high accuracy, and good stability. They are widely used in the analysis of plant genetic diversity and kinship. When the genetic similarity is 1 (the genetic distance is 0), it indicates that the plants come from the same asexual line. According to the source of SSR, it can be divided into expressed sequence tag microsatellites (EST-SSR) and genomic microsatellites (gSSR). Compared with gSSR, EST-SSR has higher interspecific universality and is therefore more instructive for interspecific hybridization. Therefore, it is necessary to develop EST-SSR markers that can be used for the diversity analysis and kinship identification of thin-shelled pecan germplasm resources. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a primer set for EST-SSR markers of Carya sylvestris and a kit and application thereof.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides a primer set for EST-SSR marker of Carya sylvestris. The primer set comprises 16 pairs of primers. The nucleotide sequence of each primer is shown in the sequence table SEQ ID No. 1 to SEQ ID No. 32.

[0007] The present invention also provides a kit for EST-SSR marking of Carya sylvestris, which comprises the above primer set.

[0008] Preferably, the kit also includes a PCR amplification system.

[0009] Preferably, the PCR amplification system is 12.5 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 9.5 μL of ddH2O.

[0010] The present invention also provides an application of the primer set or the kit in the analysis of genetic diversity and kinship of walnut germplasm resources.

[0011] The present invention also provides an application of the primer set or the kit in identifying the variety of Carya sylvestris.

[0012] Preferably, the above application comprises the following steps:

[0013] S1 takes the thin-shelled walnut sample to be identified and extracts genomic DNA;

[0014] S2 uses the genomic DNA obtained in step S1 as a template and performs PCR amplification using the primer set shown in SEQ ID No.1 to SEQ ID No.32;

[0015] S3 analyzed and identified the genetic diversity, phylogenetic relationship and variety of Carya sylvestris based on PCR amplification products.

[0016] Preferably, the thin-shelled pecan sample is thin-shelled pecan leaves or branches.

[0017] Preferably, the PCR amplification procedure is: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 35 s, annealing at 55-58°C for 35 s, extension at 72°C for 35 s, for a total of 32 cycles; and final extension at 72°C for 3 min.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention provides a primer set for EST-SSR markers of walnuts, a kit and an application thereof. The primer set of the present invention can clarify the genetic diversity among germplasms, and can gather germplasms with pedigree relationships in one branch, and can be used for the analysis of genetic diversity and kinship of walnut germplasm resources. Compared with morphological identification, the identification result obtained by the primer set of the present invention is not affected by the environment, and has the advantages of high efficiency, accuracy and stability. The identification result can provide basic data for walnut germplasm innovation, so as to more effectively select suitable breeding parents. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The polyacrylamide map of PCR products of 40 walnut germplasms amplified by 16 pairs of primers; wherein M is a marker; 1 to 40 correspond to the walnut germplasm resources described in Table 2;

[0021] Figure 2 This is the UPGMA cluster analysis diagram of SSR markers of 40 walnut germplasms. DETAILED DESCRIPTION

[0022] The invention provides a primer set for EST-SSR markers of Carya sylvestris. The primer set comprises 16 pairs of primers. The nucleotide sequence of each primer is shown in the sequence table SEQ ID No. 1 to SEQ ID No. 32. The specific primer set information is shown in Table 1.

[0023] Table 1 Primer information of 16 pairs of EST-SSR markers

[0024]

[0025]

[0026] In the present invention, the method for obtaining the primer set preferably comprises the following steps:

[0027] (1) Extract UTR and CDS sequences from the pecan genome database;

[0028] (2) Search for SSR loci using the MISA script;

[0029] (3) Use Primer3 software to design primers for the retrieved SSR loci to obtain the designed EST-SSR primers;

[0030] (4) Using the extracted genomic DNA of thin-shelled walnut as a template, PCR amplification was performed with the designed EST-SSR primers, and then polyacrylamide gel electrophoresis was performed. The primer set of EST-SSR markers of thin-shelled walnut was screened based on the electrophoresis result data.

[0031] In the present invention, UTR and CDS sequences are extracted from the pecan genome database. The present invention screens the pecan genome sequence and gene annotation file from the database, and then extracts the UTR and CDS sequences. Compared with the traditional development of SSR using transcriptome data, the assembly of raw data is not required, and the screening method is simpler and faster.

[0032] In the present invention, the MISA script is used to search for SSR sites. The search parameter setting is preferably that the minimum number of repetitions of mononucleotide, dinucleotide, trinucleotide, tetranucleotide, pentanucleotide, and hexanucleotide is 10, 6, 5, 5, 5, and 5 respectively; the search condition for composite SSR is preferably that the distance between two SSR fragments is ≤100bp.

[0033] In the present invention, Primer3 software is used to design primers for the retrieved SSR loci to obtain designed EST-SSR primers. The parameters of the primer design are preferably: primer size 18-27 bp, PCR product length 100-280 bp, GC content 40%-60%, and annealing temperature 57-63°C.

[0034] In the present invention, the genomic DNA of the extracted thin-shelled walnut is used as a template, PCR amplification is performed with the designed EST-SSR primers, and then polyacrylamide gel electrophoresis is performed, and the primer set of the thin-shelled walnut EST-SSR marker is screened according to the electrophoresis result data. The screening condition is preferably to select a primer pair containing the target band and with a clear background and stable results.

[0035] The present invention also provides a kit for EST-SSR marking of Carya sylvestris, which comprises the above primer set.

[0036] In the present invention, the kit also preferably includes a PCR amplification system, which preferably includes 12.5 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 9.5 μL of ddH2O.

[0037] The present invention also provides an application of the primer set or the kit in the analysis of genetic diversity and kinship of walnut germplasm resources.

[0038] The present invention also provides an application of the primer set or the kit in identifying the variety of Carya sylvestris.

[0039] In the present invention, the above application comprises the following steps:

[0040] S1 takes the thin-shelled walnut sample to be identified and extracts genomic DNA;

[0041] S2 uses the genomic DNA obtained in step S1 as a template and performs PCR amplification using the primer set shown in SEQ ID No.1 to SEQ ID No.32;

[0042] S3 analyzed and identified the genetic diversity, phylogenetic relationship and variety of thin-shelled pecan based on PCR amplification products.

[0043] In the present invention, the thin-shelled pecan sample is preferably a thin-shelled pecan leaf or branch. The PCR amplification procedure is preferably: 94°C pre-denaturation for 5 min; 94°C denaturation for 35 s, 55-58°C annealing and renaturation for 35 s, 72°C extension for 35 s, a total of 32 cycles; and finally 72°C extension for 3 min. Further preferred annealing temperatures: CiEST-SSR1, CiEST-SSR4 to CiEST-SSR8, CiEST-SSR10 to CiEST-SSR12, CiEST-SSR15 are all 55°C; CiEST-SSR2, CiEST-SSR3, CiEST-SSR13 are all 58°C; CiEST-SSR9, CiEST-SSR14 are all 57°C; CiEST-SSR16 is 56°C.

[0044] In the present invention, the specific operations for analyzing and identifying the genetic diversity, kinship and variety of thin-shelled pecans are as follows: data conversion is performed on the PCR amplification product, and when the SSR marker is amplified, it is represented by 1, and when the SSR marker is not amplified, it is represented by 0. The results of each pair of SSR marker amplification are converted into a binary matrix, and the genetic diversity parameters, including the observed allele number, effective allele number, Shannon information index, observed heterozygosity and expected heterozygosity of each site, are calculated using PopGene software; the polymorphic information amount of each primer is calculated using PowerMarker software; the genetic similarity between each sample to be tested is calculated using Ntsys software, and a phylogenetic tree is constructed based on the unweighted arithmetic mean clustering method (UPGMA).

[0045] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0046] Example 1

[0047] A method for obtaining a primer set for EST-SSR markers of Carya sylvestris, comprising the following steps:

[0048] (1) Download the genome sequence and gene annotation file of Carya sylvestris from the public database (ftp: / / parrot.genomics.cn / gigadb / pub / 10.5524 / 100001_101000 / 100571 / ), and extract UTR and CDS sequences based on the downloaded data;

[0049] (2) The MISA script was used to search for SSR loci, and the search parameters were set to a minimum repeat number of 10 for mononucleotide, dinucleotide, trinucleotide, tetranucleotide, pentanucleotide, and hexanucleotide, respectively, and the number of nucleotides between two SSRs of the composite SSR was ≤100 bp.

[0050] (3) Primer3 software was used to design primers for the retrieved SSR loci, and the primer design parameters were as follows: primer size 18-27 bp, PCR product length 100-280 bp, GC content 40%-60%, and annealing temperature 57-63° C. 250 pairs of primers were randomly selected and synthesized by Nanjing Qingke Biotechnology Co., Ltd.

[0051] (4) DNA of six pecan varieties ('Wichita', 'Stewart', 'Cado', 'Colby', 'Shaoxing', 'Pony') was used as a template for PCR amplification of the 250 primer pairs; wherein the DNA was extracted using the Biotech Plant DNA Extraction Kit according to the instructions, and then the DNA quality was detected by 1% mass volume fraction agarose gel electrophoresis, and the 260 / 280 value was measured by NanoDrop spectrophotometer. Qualified DNA samples were diluted to 10 ng / μL and stored at -20°C for later use.

[0052] (5) After the PCR amplification product of step (4) is separated by 10% PAGE gel, silver stained, and developed, primers containing the target band with clear background and stable results are selected.

[0053] Through the electrophoresis results, 16 pairs of primers with target bands, clear background and stable results were finally obtained. The specific information of the primer sets of the 16 pairs of EST-SSR markers of Hickory nut are shown in Table 1.

[0054] Example 2

[0055] A kit for EST-SSR markers of thin-shelled walnuts, the kit comprising the primer sets of 16 pairs of EST-SSR markers of thin-shelled walnuts described in Example 1 and a PCR amplification system, the PCR amplification system comprising 12.5 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 9.5 μL of ddH2O.

[0056] Example 3

[0057] The primer set of 16 pairs of EST-SSR markers of Carya vesiculata described in Example 1 was used for genetic diversity and kinship analysis of Carya vesiculata germplasm resources, and the steps were as follows:

[0058] (1) Taking healthy leaves of samples to be identified: In this embodiment, a total of 40 germplasm resources to be identified were taken and their healthy leaves were taken back to the laboratory and stored at -20°C for future use. The information of the 40 germplasm resources to be identified is shown in Table 2.

[0059] Table 2. The tested walnut germplasms

[0060]

[0061]

[0062] DNA was extracted from the healthy leaves of the 40 samples to be identified. The DNA was extracted using the Biotech Plant DNA Extraction Kit, and the extraction method was carried out according to the instructions. The DNA quality was detected by 1% mass volume fraction agarose gel electrophoresis, and the 260 / 280 value was measured by NanoDrop spectrophotometer. The qualified DNA samples were diluted to 10 ng / μL and stored at -20°C for use.

[0063] (2) Using the 16 pairs of EST-SSR marker primers described in Example 1, PCR amplification was performed on the 40 EST-SSR markers of step (1):

[0064] The PCR amplification system (25 μL in total) was: 12.5 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 9.5 μL of ddH2O.

[0065] The PCR amplification program was as follows: 94℃ pre-denaturation for 5min; 94℃ denaturation for 35s, 55-58℃ annealing for 35s, 72℃ extension for 35s, for a total of 32 cycles; and finally 72℃ extension for 3min. Specific annealing temperatures: CiEST-SSR1, CiEST-SSR4 to CiEST-SSR8, CiEST-SSR10 to CiEST-SSR12, and CiEST-SSR15 were all 55℃; CiEST-SSR2, CiEST-SSR3, and CiEST-SSR13 were all 58℃; CiEST-SSR9 and CiEST-SSR14 were all 57℃; and CiEST-SSR16 was 56℃.

[0066] (3) Analysis of PCR amplification products

[0067] After the PCR amplification products obtained in step (2) were separated by 10% PAGE, silver-stained, and developed, the genotypes of the amplified germplasms were obtained. According to the electrophoresis separation of the PCR products, the polymorphism of the bands was counted: Bands with the same electrophoretic mobility in the amplification products of the same primer were considered homologous. For the products at the same position, if there was a band, it was recorded as "1", and if there was no band, it was recorded as "0". The results of the amplification of each pair of SSR markers were converted into a binary 0-1 matrix, and PopGene software was used to calculate the genetic diversity parameters, including the number of observed alleles, effective number of alleles, Shannon information index, observed heterozygosity, and expected heterozygosity at each locus; PowerMarker software was used to calculate the polymorphism information content of each primer, and the results are shown in Table 3.

[0068] Table 3 Genetic diversity of primers of 16 EST-SSR markers in 40 pecan germplasms

[0069]

[0070]

[0071] The results in Table 3 show that the primer sets of 16 pairs of EST-SSR markers amplified a total of 116 polymorphic loci in 40 pecan individuals, with an average of 7.25 polymorphic loci amplified by each pair of primers. The minimum effective number of alleles was 2.27 (CiEST-SSR11), the maximum was 8.60 (CiEST-SSR7), and the average was 4.64. The average observed heterozygosity was 0.74, and the average expected heterozygosity was 0.75, indicating a high heterozygosity among pecan germplasms. The average Shannon information index was 1.62, and the average polymorphism information content (PIC) was 0.72. Among them, 15 pairs of primers showed high PIC values (PIC>0.5), and the PIC value of the other 1 pair of primers was medium (0.25<PIC<0.5), indicating that the selected SSR loci had a relatively high degree of polymorphism.

[0072] Among them, Figure 1 are the amplification results of the primers of 16 pairs of EST-SSR markers.

[0073] From Figure 1 the results show that the primer sets of 16 pairs of pecan EST-SSR markers can effectively amplify the DNA of different pecan varieties, with clear target bands, stable results, and good polymorphism among different pecan varieties, and can well distinguish different pecan varieties.

[0074] The SM (Similarity coefficient) matrix between samples was calculated using the SimQual program of Ntsys2.1 software. Unweighted pair group method with arithmetic mean (UPGMA) cluster analysis was performed based on the genetic similarity matrix using the SHAN program. The kinship clustering tree between individuals was constructed using the Treeplot module (see Figure 2 ).

[0075] according to Figure 2 It can be seen that varieties with clear pedigree relationships such as 'Mahan' and 'Wichita', 'Mandan' and 'Osage' are clustered in one branch, and morphologically similar varieties such as Shaoxing and Stewart, 'Huangbo No. 1' and 'Huangbo No. 2', Jiande No. 100 and 'Mohank' are also clustered in one branch. This result proves that the use of 16 pairs of thin-shell pecan EST-SSR markers can successfully determine the genetic relationship between thin-shell pecan germplasm resources.

[0076] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A primer set for EST-SSR markers of Carya sylvestris, characterized in that: The primer set consists of 16 pairs of primers, and the nucleotide sequence of each primer is shown in the sequence table SEQ ID No. 1 to SEQ ID No.

32.

2. A kit for EST-SSR markers of Carya sylvestris, characterized in that: The kit comprises the primer set according to claim 1.

3. The kit according to claim 2, characterized in that The kit also includes a PCR amplification system.

4. The kit according to claim 3, characterized in that The PCR amplification system is 12.5 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 9.5 μL of ddH2O.

5. Use of the primer set according to claim 1 or the kit according to any one of claims 2 to 4 in the analysis of genetic diversity and kinship of Carya elata germplasm resources.

6. Use of the primer set according to claim 1 or the kit according to any one of claims 2 to 4 in identifying a variety of Carya sylvestris.

7. The use according to claim 5, characterized in that: The following steps are involved: S1 takes the thin-shelled walnut sample to be identified and extracts genomic DNA; S2 uses the genomic DNA obtained in step S1 as a template and performs PCR amplification using the primer set shown in SEQ ID No.1 to SEQ ID No.32; S3 analyzed and identified the genetic diversity, phylogenetic relationship and variety of Carya sylvestris based on PCR amplification products.

8. The use according to claim 7, characterized in that: The thin-shelled pecan sample is thin-shelled pecan leaves or branches.

9. The use according to claim 7, characterized in that: The PCR amplification procedure is: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 35 s, annealing at 55-58°C for 35 s, 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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