Primer pair for identifying ulmus pumila and ulmus lamellosa germplasm and application thereof

Abstract

The application discloses a primer pair for identifying Ulmus pumila and Ulmus lamellosa germplasm and application thereof, wherein the primer pair is Ulmus_p20, Ulmus_p61 or / and Ulmus_p165. Fresh tissue samples of the Ulmus pumila or Ulmus lamellosa to be detected are collected, and DNA of the tissue samples is extracted. The DNA is used as a template, and PCR amplification is carried out by using the primer pair. The amplification product is detected by electrophoresis. According to the detection result, it is determined that the sample to be detected is Ulmus pumila or Ulmus lamellosa. The application uses SSR molecular markers of transcriptome sequencing to identify Ulmus pumila and Ulmus lamellosa germplasm resources, is beneficial to promoting Ulmus pumila and Ulmus lamellosa breeding, and accelerates the utilization process of excellent germplasm. The specific primers of the application are used for amplifying Ulmus lamellosa and Ulmus pumila tissues respectively, and the length difference of the products of the two is significant, which indicates that the specific primers can effectively identify Ulmus pumila and Ulmus lamellosa germplasm.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and relates to a primer pair, in particular to a primer pair for identifying Ulmus pumila and Ulmus lamellosa germplasm. BACKGROUND

[0002] Ulmus pumila L. and Ulmus lamellosa are Ulmaceae Ulmus L. trees, and are native broad-leaved tree species widely distributed in China. There are more than 30 species of Ulmus in the Northern Hemisphere. There are 25 species and 6 varieties in China, which are distributed throughout the country, more in the north of the Yangtze River basin.

[0003] Both Ulmus pumila and Ulmus lamellosa are light-loving tree species with developed root systems and strong resistance, and are important soil-conserving and sand-fixing tree species in harsh ecological environments. The alar samara is edible (with an oil content of 20% to 40%) and the wood is of high quality, and is an important raw material tree species for medicine and light and chemical industries. The branches and leaves are resistant to pruning and have high ornamental value, and are widely used in landscape plant modeling. Therefore, Ulmus pumila and Ulmus lamellosa are multifunctional tree species integrating ecological, economic and ornamental values.

[0004] In the process of implementing the present application, the inventors found that at least one of the following technical problems existed in the prior art:

[0005] There are rich Ulmus pumila and Ulmus lamellosa germplasm resources in China. Ulmus pumila and Ulmus lamellosa are difficult to distinguish in early identification of germplasm because their phenotypic traits are similar except that there is a significant difference in flowering time (Ulmus pumila blooms in spring and Ulmus lamellosa blooms in autumn). SUMMARY

[0006] Therefore, the present application aims to provide a SSR molecular marker using transcriptome sequencing for rapidly identifying Ulmus pumila and Ulmus lamellosa germplasm.

[0007] Through long-term exploration and attempts, and multiple experiments and efforts, the inventors continuously reform and innovate to solve the above technical problems, and provide a technical solution, that is, a primer pair for identifying Ulmus pumila or Ulmus lamellosa germplasm, which is one or more of the following primer pairs:

[0008] Ulmus_p20:

[0009] upstream primer: 5'-TTCCCTCACCTCTCTGGCTT-3';

[0010] downstream primer: 5'-TGCCCAATTTTGCTTTATTGGC-3';

[0011] Ulmus_p61:

[0012] Upstream primer: 5'-CCACGAGTCGTCCATCTATCC-3';

[0013] Downstream primer: 5'-TTCCGAAGCCAACCCAGTTT-3';

[0014] Ulmus_p165:

[0015] Upstream primer: 5'-TTCGCGGAACACACTCTCAT-3';

[0016] Downstream primer: 5'-AGGAACGATACTGACGATCAAA-3'.

[0017] The application further provides an application of the primer pair for identifying Ulmus pumila or Ulmus lamellosa germplasm.

[0018] According to an embodiment of the method, the primer pair is Ulmus_p20:

[0019] The sample with the amplification product containing a fragment of 251 bp and not containing a fragment of 243 bp is Ulmus lamellosa;

[0020] The sample with the amplification product containing a fragment of 243 bp is Ulmus pumila.

[0021] According to an embodiment of the method, the primer pair is Ulmus_p61:

[0022] The sample with the amplification product containing a fragment of 330 bp is Ulmus lamellosa;

[0023] The sample with the amplification product not containing a fragment of 330 bp is Ulmus pumila.

[0024] According to an embodiment of the method, the primer pair is Ulmus_p165:

[0025] The sample with the amplification product containing a fragment of 293 bp or above is Ulmus lamellosa;

[0026] The sample with the amplification product containing a fragment of 265 bp or below is Ulmus pumila.

[0027] The application further discloses a method for identifying Ulmus pumila and Ulmus lamellosa germplasm by using the primer pair, and the identification steps are as follows:

[0028] Step S1: collecting fresh tissue samples of Ulmus pumila or Ulmus lamellosa to be tested, and extracting DNA of the tissue samples;

[0029] Step S2: using the primer pair to perform PCR amplification by taking the DNA extracted in step S1 as a template;

[0030] Step S3: electrophoretically detecting the amplification product;

[0031] Step S4: determining the sample to be Ulmus pumila or Ulmus lamellosa according to the detection result.

[0032] According to one embodiment of the method, the tissue sample is a young leaf.

[0033] According to one embodiment of the method, the electrophoresis is capillary electrophoresis or gel electrophoresis.

[0034] According to one embodiment of the method, the capillary electrophoresis is specifically as follows: 0.1 μL of the PCR product diluted to 1 μL is mixed with 15 μL of a mixture of formamide and a molecular weight marker at 100:1, and then added to a PCR plate, denatured at 95°C for 5 min, cooled at 4°C, and then centrifuged before being subjected to machine detection.

[0035] According to one embodiment of the method, in step S4, the original data obtained in step S3 are analyzed, and the positions of the molecular weight markers in each lane are compared with the positions of the sample peaks to obtain the fragment size.

[0036] According to one embodiment of the method, step S4 further comprises calculating a genetic distance matrix according to the original site data, and constructing a phylogenetic tree by using a similarity coefficient method according to the GS value matrix.

[0037] Compared with the prior art, one of the above technical solutions has the following advantages:

[0038] a) The present application uses SSR molecular markers of transcriptome sequencing to identify Ulmus pumila and Ulmus lamellosa germplasm resources, which is conducive to promoting the breeding of Ulmus pumila and Ulmus lamellosa varieties and accelerating the utilization process of excellent germplasm.

[0039] b) The specific primers of the present application amplify the tissues of Ulmus lamellosa and Ulmus pumila respectively, and the lengths of the products are significantly different, indicating that these specific primers can effectively identify Ulmus pumila and Ulmus lamellosa germplasm. BRIEF DESCRIPTION OF DRAWINGS

[0040] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be regarded as a limitation on the scope, and other related drawings can also be obtained by those skilled in the art without creative labor.

[0041] Figure 1 is an electrophoretogram of Ulmus pumila and Ulmus lamellosa germplasm after amplification by Ulmus_p20 primers.

[0042] Figure 2 is the electrophoretogram of white elm and peeling elm germplasm after amplification by Ulmus_p61 primer.

[0043] Figure 3 is the electrophoretogram of white elm and peeling elm germplasm after amplification by Ulmus_p165 primer.

[0044] Figure 4 is the phylogenetic tree constructed by the specific primer of the experimental group A.

[0045] Figure 5 is the phylogenetic tree constructed by the specific primer of the comparison group B. DETAILED DESCRIPTION

[0046] In order to make the objectives, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the present application. Therefore, the following detailed description of the embodiments of the present application provided in the drawings is not intended to limit the scope of the claimed present application, but only represents selected embodiments of the present application.

[0047] A large number of EST-SSR primer sequences are obtained by performing transcriptome sequencing on elm trees.

[0048] Among them, 200 pairs of primers are selected for stability, specificity and polymorphism test, 16 pairs of primers are screened out after test screening, and 3 pairs of specific primers are finally screened out through comparison analysis (Table 1) for the identification of white elm and peeling elm germplasm.

[0049] Table 1: Information of 3 pairs of specific EST-SSR primers screened

[0050]

[0051]

[0052] A cDNA library of elm is constructed according to the transcriptome data of white elm leaves. The site of primer Ulmus_p20 is a full-length sequence of 4465 bp, as shown in the sequence table SEQ ID NO. 1, the coding region is: 2170-4356 (-), and 728 amino acids are encoded, as shown in the sequence table SEQ ID NO. 16. The primer Ulmus_p165 is designed according to the sequence shown in SEQ ID NO. 1.

[0053] The site of primer Ulmus_p61 is a sequence with full length of 1414 bp, as shown in SEQ ID NO. 2, the coding region is 73-390 (+), and it encodes 105 amino acids, as shown in SEQ ID NO. 17. The primer Ulmus_p61 is designed according to the sequence shown in SEQ ID NO. 2.

[0054] The site of primer Ulmus_p165 is a sequence with full length of 2298 bp, as shown in SEQ ID NO. 3, the coding region is 1049-2074 (+), and it encodes 341 amino acids, as shown in SEQ ID NO. 18. The primer Ulmus_p165 is designed according to the sequence shown in SEQ ID NO. 3.

[0055] The steps of identifying Ulmus pumila and Ulmus lamellosa by using EST-SSR molecular markers obtained by transcriptome sequencing are as follows:

[0056] 1) Collect fresh and tender leaf samples of Ulmus pumila or Ulmus lamellosa to be tested, and extract DNA of the leaf samples.

[0057] The DNA of 26 samples of Ulmus pumila and Ulmus lamellosa germplasm (Table 2) is extracted by using Tian Gen high-efficiency plant genome DNA extraction kit.

[0058] Table 2 Ulmus pumila and Ulmus lamellosa germplasm

[0059]

[0060]

[0061] In this embodiment, two groups of EST-SSR specific primer controls are set. The experimental group A is three pairs of EST-SSR specific primers described in Table 1, which are Ulmus_p20, Ulmus_p61 and Ulmus_p165, respectively. The control group B is three other pairs of EST-SSR specific primers randomly selected from a large amount of experimental data obtained by the inventors during the completion of the present application, which are p81, p141 and p148 (the sequences are shown in Table 3), and the upstream and downstream sequences of the specific primers in the two groups are synthesized by Anhui General Biotechnology Co., Ltd.

[0062] Table 3 Specific EST-SSR primer information of group B

[0063]

[0064] 2) Amplification.

[0065] The DNA extracted in step 1) is used as a template, and the specific primers in the above A group and B group are used for PCR amplification, and the PCR instrument used is Hangzhou Jingge-K960 thermal cycler.

[0066] The PCR amplification uses a 20 μL reaction system, including: ddH2O 14.8 μL, dNTP 0.4 μL, PCR Buffer 2 μL, upstream primer 0.3 μL (20 μM), downstream primer 0.3 μL (μM), DNA template 2 μL, Taq 0.2 μL;

[0067] The PCR reaction program is: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 60℃ recombination for 45 s, 72℃ extension for 50 s, a total of 35 cycles; and finally 72℃ extension for 5 min.

[0068] 3) Capillary electrophoresis detection of amplification products.

[0069] 0.1 μL of the PCR amplification product in step 2) is diluted to 1 μL, mixed with 15 μL of a mixture of formamide and molecular weight marker 100:1, and then added to a PCR plate, denatured at 95℃ for 5 min, cooled at 4℃, and then centrifuged, and then detected by a gene analyzer (ABI 3730XL DNAanalyzer, capillary ABI96x50cm 4331246), and the specific parameters are shown in Table 4.

[0070] Table 4 Gene analyzer parameter settings

[0071]

[0072] After electrophoresis, the raw data obtained by the sequencer are analyzed by using Fragment (Plant) fragment analysis in Genemarker V2.2.0 software to obtain the fragment size. The electropherogram of Ulmus pumila and Ulmus lamellosa germplasm after amplification by Ulmus_p20 primer is as shown in Figure 1 The electropherogram of Ulmus pumila and Ulmus lamellosa germplasm after amplification by Ulmus_p61 primer is as shown in Figure 2 The electropherogram of Ulmus pumila and Ulmus lamellosa germplasm after amplification by Ulmus_p165 primer is as shown in Figure 3

[0073] Table 5 Length of A group primer amplification products of Ulmus pumila and Ulmus lamellosa germplasm

[0074]

[0075]

[0076] Table 6 Length of B group primer amplification products of Ulmus pumila and Ulmus lamellosa germplasm

[0077] ​

[0078] The results are shown in Table 5 and Table 6. In group A, in addition to the 251bp and 255bp heterozygote of tp1 and the 247bp and 251bp heterozygote of tp2, the rest are 251bp and 251bp, which are homozygotes, and the product length is obviously different from that of Ulmus pumila; under the amplification of P61 specific primer, the product is 330bp and 330bp homozygote or 330bp and 333bp heterozygote; under the amplification of P165 specific primer, the product length of the peeling elm germplasm is different, but it is obviously different from that of Ulmus pumila. In group B, the amplification products of the three pairs of SSR specific primers are different, and the differences between the germplasms are obvious, but it is difficult to effectively identify Ulmus pumila and peeling elm.

[0079] The length of the amplification product of each germplasm is calculated by using Popgen32 software, and the phylogenetic tree is constructed by using the similarity coefficient method according to the GS value matrix. The analysis results are shown in Table 5 and Table 6. Figure 4 、 Figure 5 The phylogenetic tree of the three pairs of EST-SSR specific primers in group A Figure 4 ) divides the Ulmus pumila and peeling elm germplasms into two groups. The upper group contains Ul2, 4, 3, 6, 9, 5, 1, 11, 7, 13, 12, 14, 10, 8 and 15, which are all Ulmus pumila, and the lower group contains tp5, 6, 3, 2, 1, 7, 9, 4, 8, 10 and 11, which are all peeling elm. The three pairs of EST-SSR specific primers can completely distinguish the Ulmus pumila and peeling elm germplasms. The phylogenetic tree of the three pairs of EST-SSR specific primers in group B Figure 5 ) cannot effectively distinguish the Ulmus pumila and peeling elm germplasms, and the two types of germplasms are mixed together, which indicates that the three pairs of EST-SSR specific primers in group B cannot effectively distinguish the Ulmus pumila and peeling elm germplasms.

[0080] The above is only a preferred embodiment of the present application, and it should be pointed out that the above preferred embodiment should not be regarded as a limitation of the present application, and the protection scope of the present application should be limited by the scope defined in the claims. For ordinary skilled persons in the art, several improvements and refinements can be made without departing from the spirit and scope of the present application, and these improvements and refinements should also be regarded as the protection scope of the present application.

Claims

1. An application of a primer set, characterized in that, Used to identify germplasm of either white elm or peeled elm in the context of white elm and peeled elm; The primer set consists of the following primer pairs composition: Ulmus_p20: Upstream primer: 5'- TTCCCTCACCTCTCTGGCTT-3'; Downstream primer: 5'-TGCCCAATTTTGCTTTATTGGC-3'; Ulmus_p61: Upstream primer: 5'-CCACGAGTCGTCCATCTATCC-3'; Downstream primer: 5'-TTCGAAGCCAACCCAGTTT-3'; Ulmus_ p165: Upstream primer: 5'-TTCGCGGAACACACTCTCAT-3'; Downstream primer: 5'-AGGAACGATACTGACGATCAAA-3'.

2. A method for identifying white elm or peeled elm germplasm in white elm and peeled elm, characterized in that, The identification steps are as follows: Step S1: Collect fresh tissue samples of the white elm or peeled elm to be tested, and extract DNA from the tissue samples; Step S2: Using the DNA extracted in step S1 as a template, perform PCR amplification using the primer set described in claim 1; Step S3: Electrophoresis detection of amplification products; Step S4: Determine whether the sample to be tested is white elm or peeled elm based on the test results.

3. The method according to claim 2, characterized in that, The tissue sample was a young leaf.

4. The method according to claim 2, characterized in that, The electrophoresis is capillary electrophoresis or gel electrophoresis; the capillary electrophoresis is specifically performed as follows: take 0.1 μL of the PCR product from step S2, dilute it to 1 μL, mix it with 15 μL of a 100:1 mixture of formamide and molecular weight internal standard, add it to a PCR plate, denature at 95℃ for 5 min, cool at 4℃ and centrifuge, and then perform detection.

5. The method according to claim 2, characterized in that, In step S4: the raw data obtained in step S3 is analyzed, and the positions of the molecular weight internal standards in each lane are compared and analyzed with the positions of the peak values ​​of each sample to obtain the fragment size.

6. The method according to claim 5, characterized in that, Step S4 further includes calculating the genetic distance matrix based on the original locus data, and constructing a phylogenetic tree using the similarity coefficient method based on the GS value matrix.

Images