CN118256644A - Molecular marker for identifying germplasm of ulmus pumila and ulmus parvifolia and application thereof - Google Patents

Molecular marker for identifying germplasm of ulmus pumila and ulmus parvifolia and application thereof Download PDF

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CN118256644A
CN118256644A CN202410420213.3A CN202410420213A CN118256644A CN 118256644 A CN118256644 A CN 118256644A CN 202410420213 A CN202410420213 A CN 202410420213A CN 118256644 A CN118256644 A CN 118256644A
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ulmus
pumila
ulmus pumila
germplasm
molecular marker
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代剑峰
郑聪慧
王玉忠
徐振华
张苗苗
徐文凯
孙然
王桂鑫
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Hebei Academy Of Forestry And Grassland Sciences
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Abstract

The invention discloses a molecular marker for identifying the germplasm of Ulmus pumila and application thereof, wherein the molecular marker is Ulmus_p104 or/and Ulmus_p40 as the following primer pair, a fresh tissue sample of the Ulmus pumila or Ulmus pumila to be detected is collected, and a tissue sample DNA is extracted; using the DNA as a template and carrying out PCR amplification by using the molecular marker; detecting the amplified product by electrophoresis; and judging whether the sample to be detected is the ulmus pumila or the ulmus pumila according to the detection result. The SSR molecular markers for transcriptome sequencing are used for identifying the germplasm resources of the ulmus pumila and the ulmus pumila, which is beneficial to promoting the fine variety breeding of the ulmus pumila and accelerating the utilization process of the fine variety. The specific primers of the invention amplify the ulmus pumila and ulmus pumila tissues respectively, and the difference of the product lengths of the two is obvious, which indicates that the specific primers can effectively identify the germplasm of the ulmus pumila and the ulmus pumila.

Description

Molecular marker for identifying germplasm of ulmus pumila and ulmus parvifolia and application thereof
Technical Field
The invention belongs to the technical field of genetic engineering, and relates to a molecular marker, in particular to a molecular marker for identifying the germplasm of ulmus pumila and ulmus parvifolia.
Background
Ulmus pumila L. and Ulmus pumila Ulmus parvifolia are Ulmus (Ulmus L.) trees of Ulmus genus (Ulmaceae) of Ulmaceae family, which are broad-leaved tree species in rural area with relatively wide China distribution. The elm has more than 30 species and generates northern hemisphere. There are 25 varieties of 6 in our country, which are distributed throughout the country, more in the Yangtze river basin and more in the north.
Both the white elm and the hammer elm are happy tree species, have developed root systems and strong resistance, and are important excellent tree species for soil conservation and sand fixation in severe ecological environment areas; the samara is edible (oil content is 20% -40%), has excellent material quality, and is an important raw material tree species for medicine and light industry; the branches and leaves are resistant to pruning, have high ornamental value and are widely used for modeling garden plants. Therefore, the ulmus pumila and the ulmus pumila are multifunctional tree species which integrate ecological, economic and ornamental values.
In the process of implementing the present invention, the inventor finds that at least one of the following technical problems exists in the prior art:
The white elm and the hammer elm in China have abundant germplasm resources, have similar phenotype characters except obvious difference of flowering time (flowering in spring and autumn), are difficult to distinguish, and are difficult to identify in early germplasm.
Disclosure of Invention
In view of the above, the present invention aims to provide an SSR molecular marker using transcriptome sequencing to rapidly identify the germplasm of ulmus pumila and ulmus pumila.
The inventor continuously reforms and innovates through long-term exploration and trial and repeated experiments and efforts, and in order to solve the technical problems, the technical scheme provided by the invention is to provide a molecular marker for identifying the germplasm of ulmus pumila or ulmus parvifolia, wherein the molecular marker is one or more of the following primer pairs:
Ulmus_p104：
an upstream primer: 5'-CCCCATCTGGATTGGCAGTT-3';
a downstream primer: 5'-CGCCCTTTGACGGTTGGATA-3';
Ulmus_p40：
an upstream primer: 5'-GCCAGTTGGGTGGATCAAGA-3';
a downstream primer: 5'-GTGGGGATGCGAGAGAACAA-3'.
The invention also provides application of the molecular marker for identifying the germplasm of ulmus pumila or ulmus parvifolia.
The invention also discloses a method for identifying the germplasm of ulmus pumila and ulmus pumila by using the molecular marker, which comprises the following steps:
Step S1: collecting fresh tissue samples of the ulmus pumila or ulmus pumila to be detected, and extracting DNA of the tissue samples;
step S2: using the DNA extracted in the step S1 as a template, and carrying out PCR amplification by using the molecular marker;
Step S3: detecting the amplified product by electrophoresis;
Step S4: and judging whether the sample to be detected is the ulmus pumila or the ulmus pumila according to the detection result.
According to one embodiment of the method of the invention, the tissue sample is a young leaf.
According to one embodiment of the method of the invention, the electrophoresis is capillary electrophoresis or gel electrophoresis.
According to one embodiment of the method of the invention, the capillary electrophoresis is in particular: diluting 0.1 mu L of the PCR product in the step S2 to 1 mu L, and carrying out the PCR product with formamide and a molecular weight internal standard of 100:1, 15 mu L of the mixture is added into a PCR plate after being evenly mixed, denatured for 5min at 95 ℃, cooled at 4 ℃ and centrifuged, and then the mixture is detected by a machine.
According to one embodiment of the method of the invention, the primer pair is ulmus_p104:
the sample with the amplified product fragment size of 212bp is elm parvifolia;
The amplified product contains a sample with a fragment size of 209bp, which is Ulmus pumila.
According to one embodiment of the method of the invention, the primer pair is ulmus_p40:
The sample with 253bp of amplified product fragment size is Ulmus parvifolia;
The amplified product fragment size is 241-251 bp, and the sample is elm.
According to one embodiment of the method of the present invention, in the step S4: and (3) analyzing the original data obtained in the step (S3), and comparing and analyzing the positions of the internal molecular weight standards in each lane with the positions of the peak values of each sample to obtain the fragment size.
According to one embodiment of the method of the present invention, the step S4 further includes calculating a genetic distance matrix according to the original site data, and constructing a phylogenetic tree according to the GS value matrix by using a similarity coefficient method.
Compared with the prior art, one of the technical schemes has the following advantages:
a) The SSR molecular markers for transcriptome sequencing are used for identifying the germplasm resources of the ulmus pumila and the ulmus pumila, which is beneficial to promoting the fine variety breeding of the ulmus pumila and accelerating the utilization process of the fine variety.
B) The specific primers of the invention amplify the ulmus pumila and ulmus pumila tissues respectively, and the difference of the product lengths of the two is obvious, which indicates that the specific primers can effectively identify the germplasm of the ulmus pumila and the ulmus pumila.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some examples of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is an electrophoretogram of Ulmus pumila and Ulmus parvifolia germplasm after Ulmus_p104 primer amplification.
FIG. 2 is an electrophoretogram of Ulmus pumila and Ulmus parvifolia germplasm after Ulmus_p40 primer amplification.
FIG. 3 is a phylogenetic tree constructed from the group A specific primers of the experimental group.
FIG. 4 is a phylogenetic tree constructed with the group B specific primers of the control group.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, based on the embodiments of the invention, which are apparent to those of ordinary skill in the art without inventive faculty, are intended to be within the scope of the invention. Thus, the following detailed description of the embodiments of the invention, as presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention.
A large number of EST-SSR primer sequences were obtained by transcriptome sequencing elms.
200 Pairs of primers are selected for stability, specificity and polymorphism inspection, 16 pairs of primers are screened through inspection, and 2 pairs of specific primers (table 1) are screened finally through comparative analysis for germplasm identification of ulmus pumila and ulmus pumila.
Table 1 screening 2 pairs of specific EST-SSR primer information
Constructing an elm cDNA library according to transcriptome data of the elm leaves, wherein the site of the primer Ulmus_p40 is a segment of full-length 939bp coding sequence, as shown in a sequence table SEQ ID NO.1, and totally encoding 312 amino acids. The primer Ulmus_p40 was designed according to the sequence shown in SEQ ID NO. 1.
Constructing an elm cDNA library according to transcriptome data of the Ulmus pumila leaves, wherein the site of the primer Ulmus_p104 is a segment of full-length 990bp coding sequence, which is shown as a sequence table SEQ ID NO.2, and totally codes 329 amino acids. Primer Ulmus_p104 was set according to the sequence shown in SEQ ID NO. 2.
The EST-SSR molecular markers obtained by transcriptome sequencing are used for identifying the ulmus pumila and the ulmus pumila, and the steps are as follows:
1) Collecting fresh and tender leaf samples of the ulmus pumila or ulmus pumila to be detected, and extracting DNA of the leaf samples.
26 Parts of DNA of tender leaf samples of the ulmus pumila and ulmus pumila germplasm (Table 2) are extracted by using a high-efficiency plant genome DNA extraction kit of the root of the heaven.
TABLE 2 Ulmus pumila and Ulmus pumila germplasm
In this example, two sets of EST-SSR specific primer controls were set, and experimental set A was set up to 2 pairs of EST-SSR specific primers described in Table 1, ulmus_p104 and Ulmus_p40, respectively. The control group B is other 4 pairs of EST-SSR specific primers randomly selected by the inventor in a large amount of experimental data in the process of completing the invention, wherein the other 4 pairs of EST-SSR specific primers are p20, p61, p141 and p148 (the sequences are shown in Table 3), and the upstream and downstream sequences of the two groups of specific primers are synthesized by Anhui general biotechnology Co.
TABLE 3 group-specific EST-SSR primer information
2) And (5) amplifying.
And (3) taking the DNA extracted in the step (1) as a template, and respectively carrying out PCR amplification by using the group A and group B specific primers, wherein the model of a PCR instrument is Hangzhou lattice-K960 thermal cycler.
The PCR amplification adopts a 20 mu L reaction system, and comprises the following steps: ddH2O 14.8. Mu.L, dNTP 0.4. Mu.L, PCR Buffer 2. Mu.L, upstream primer 0.3. Mu.L (20. Mu.M), downstream primer 0.3. Mu.L (. Mu.M), DNA template 2. Mu.L, taq 0.2. Mu.L;
the PCR reaction procedure was: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, renaturation at 60℃for 45s, extension at 72℃for 50s for 35 cycles; finally, the mixture is extended for 5min at 72 ℃.
3) And detecting the amplified product by capillary electrophoresis.
Diluting 0.1 mu L of the PCR amplification product obtained in the step 2) to 1 mu L, and carrying out PCR amplification reaction on the diluted product with formamide and a molecular weight internal standard of 100:1 mixing well 15. Mu.L of the mixture, adding into a PCR plate, denaturing at 95℃for 5min, cooling at 4℃and centrifuging, and then detecting by a machine (a gene analyzer ABI 3730XL DNA analyzer, a capillary ABI 96X 50cm 4331246), wherein the specific parameters are shown in Table 4.
Table 4 gene analyzer parameter settings
After electrophoresis, the original data obtained by the sequencer is analyzed by Fragment (Plant) fragment analysis in GENEMARKER V2.2.0 software to obtain fragment size. The electrophoresis patterns of the Ulmus pumila and Ulmus pumila germplasm after the Ulmus_p104 primer amplification are shown in figure 1; the electrophoretogram of Ulmus pumila and Ulmus pumila germplasm after Ulmus_p40 primer amplification is shown in FIG. 2.
TABLE 5 amplification product Length of primers for Ulmus pumila and Ulmus pumila germplasm A group
TABLE 6 primer amplification product Length of Ulmus pumila and Ulmus pumila germplasm B group
The results are shown in Table 5 and Table 6, in the A group, the products of the Ulmus parvifolia germplasm LY1-6 amplified by Ulmus_P40 specific primers are 253bp and 253bp, and are homozygotes; under the amplification of Ulmus_P104 specific primers, 212bp and 212bp are homozygotes, and the length difference between the homozygotes and the Ulmus pumila product is obvious. In the B group, 4 pairs of SSR specific primer amplification products are different, the difference between germplasm is obvious, but the Ulmus pumila and Ulmus pumila cannot be effectively identified. The products of the Ulmus parvifolia germplasm LY1-6 amplified by the P20 specific primer are 243bp and 243bp, and are homozygotes; however, ul7 and Ul10 detected homozygotes of 243bp and 243bp in the same manner.
And calculating genetic distances of various mass amplification product lengths by Popgen software, and constructing a phylogenetic tree by using a similarity coefficient method according to the GS value matrix. As shown in FIG. 3 and FIG. 4, the phylogenetic tree of EST-SSR specific primers of group A (FIG. 3) divides the Ulmus pumila and Ulmus pumila germplasm into two groups, the upper group contains Ul3, 11, 6, 12, 13, 14, 15, 9, 1, 2, 4, 8, 10, 5 and 7, and the lower group contains LY4, 1, 2, 3, 5 and 6, and the Ulmus pumila and Ulmus pumila germplasm can be completely distinguished by using 2 pairs of EST-SSR specific primers. The EST-SSR specific primer phylogenetic tree of group B (figure 4) cannot effectively distinguish and identify the germplasm of ulmus pumila and ulmus parvifolia, and the two germplasm types are mixed together, which indicates that the EST-SSR specific primer of group B cannot effectively identify the germplasm of ulmus pumila and ulmus parvifolia.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that the above-mentioned preferred embodiment should not be construed as limiting the invention, and the scope of the invention should be defined by the appended claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and such modifications and adaptations are intended to be comprehended within the scope of the invention.

Claims (10)

1. A molecular marker for identifying the germplasm of ulmus pumila or ulmus parvifolia, characterized in that the molecular marker is one or more of the following primer pairs:
Ulmus_p104：
an upstream primer: 5'-CCCCATCTGGATTGGCAGTT-3';
a downstream primer: 5'-CGCCCTTTGACGGTTGGATA-3';
Ulmus_p40：
an upstream primer: 5'-GCCAGTTGGGTGGATCAAGA-3';
a downstream primer: 5'-GTGGGGATGCGAGAGAACAA-3'.
2. Use of a molecular marker according to claim 1 for the identification of ulmus pumila or ulmus parvifolia germplasm.
3. A method for identifying the germplasm of ulmus pumila and ulmus pumila by using the molecular marker of claim 1, which is characterized by comprising the following identification steps:
Step S1: collecting fresh tissue samples of the ulmus pumila or ulmus pumila to be detected, and extracting DNA of the tissue samples;
step S2: using the DNA extracted in the step S1 as a template, and carrying out PCR amplification by using the molecular marker;
Step S3: detecting the amplified product by electrophoresis;
Step S4: and judging whether the sample to be detected is the ulmus pumila or the ulmus pumila according to the detection result.
4. A method according to claim 3, wherein the tissue sample is young leaves.
5. A method according to claim 3, wherein the electrophoresis is capillary electrophoresis or gel electrophoresis.
6. The method according to claim 5, wherein the capillary electrophoresis is specifically: diluting 0.1 mu L of the PCR product in the step S2 to 1 mu L, and carrying out the PCR product with formamide and a molecular weight internal standard of 100:1, 15 mu L of the mixture is added into a PCR plate after being evenly mixed, denatured for 5min at 95 ℃, cooled at 4 ℃ and centrifuged, and then the mixture is detected by a machine.
7. A method according to claim 3, wherein the primer pair is ullmus_p 104: the sample with the amplified product fragment size of 212bp is elm parvifolia;
The amplified product contains a sample with a fragment size of 209bp, which is Ulmus pumila.
8. A method according to claim 3, wherein the primer pair is ullmus_p40:
The sample with 253bp of amplified product fragment size is Ulmus parvifolia;
The amplified product fragment size is 241-251 bp, and the sample is elm.
9. A method according to claim 3, wherein in step S4: and (3) analyzing the original data obtained in the step (S3), and comparing and analyzing the positions of the internal molecular weight standards in each lane with the positions of the peak values of each sample to obtain the fragment size.
10. The method of claim 8, wherein step S4 further comprises calculating a genetic distance matrix from the original site data, and constructing a phylogenetic tree from the GS value matrix using a similarity coefficient method.
CN202410420213.3A 2024-04-09 2024-04-09 Molecular marker for identifying germplasm of ulmus pumila and ulmus parvifolia and application thereof Pending CN118256644A (en)

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