A DNA barcode for identifying Corylus species and its amplification primers and applications

Abstract

The present invention discloses a DNA barcode for identifying Corylus species and its amplification primers and applications, wherein the DNA barcode is a sequence formed by connecting the following four hypervariable regions of the chloroplast genome of the Corylus species: ndhF-rpl32, rpl32-trnL, ycf1 and trnS-trnG. The DNA barcode for identifying Corylus species provided by the present invention can achieve the same effectiveness as the identification of Corylus species by the chloroplast whole genome sequence, and can realize rapid and accurate identification of Corylus species.

Description

Technical Field

[0001] The invention belongs to the field of gene technology, and in particular relates to a DNA barcode for identifying Corylus species and an amplification primer and application thereof. Background Art

[0002] Hazelnut is a general term for plants of the genus Corylus L. in the family Betulaceae. There are about 20 species in the world, which are widely distributed in the northern temperate zone (Asia, Europe and North America). Hazelnuts are rich in oil and protein. They can be eaten fresh, fried or roasted, or pressed for oil or used as raw materials to make various foods. They are known as the "king of nuts".

[0003] Extensive natural hybridization among closely related species of Corylus, incomplete pedigree sorting and convergent evolution mechanisms have led to obvious irregularities in morphological characteristics, tissue structure and nut quality among different species of Corylus and even different populations of the same species. There are both interspecific differences and intraspecific variations. In addition, the phenotypic traits are also susceptible to unstable variations caused by environmental changes. It can be seen that the traditional morphological, anatomical and physicochemical property determination methods cannot scientifically and comprehensively evaluate and identify Corylus plants. Therefore, in order to achieve accurate utilization of Corylus germplasm at the species level, a faster and more effective method for species identification is needed.

[0004] Compared with traditional identification methods, DNA barcoding technology has the advantages of accuracy, high efficiency, strong repeatability, and no interference from the external environment. It has been widely used in plant classification and identification and systematic evolution research. In Corylus plants, although the internationally used barcodes ITS, matK, atpB-rbcL, psbA-trnH, etc. have a high identification rate at the genus or group level, the resolution at the species level is low, especially for closely related species, species and variants with close genetic distances. In view of this, in order to improve the development and utilization efficiency of germplasm resources, it is very necessary to develop specific DNA barcodes suitable for the identification of Corylus species. Summary of the invention

[0005] In response to one or more problems existing in the prior art, one aspect of the present invention provides a DNA barcode for identifying Corylus species, which is a sequence formed by connecting the following four hypervariable regions of the chloroplast genome of Corylus species: ndhF-rpl32, rpl32-trnL, ycf1 and trnS-trnG, wherein the ndhF-rpl32 represents the non-coding region between genes ndhF and rpl32, the rpl32-trnL represents the non-coding region between genes rpl32 and trnL, the ycf1 represents the coding region gene ycf1, and the trnS-trnG represents the non-coding region between genes trnS and trnG.

[0006] In some embodiments, the upstream and downstream primers used to amplify ndhF-rpl32 are shown as SEQ ID NOs: 3-4, respectively; the upstream and downstream primers used to amplify rpl32-trnL are shown as SEQ ID NOs: 5-6, respectively; the upstream and downstream primers used to amplify ycf1 are shown as SEQ ID NOs: 7-8, respectively; the upstream and downstream primers used to amplify trnS-trnG are shown as SEQ ID NOs: 9-10, respectively.

[0007] Another aspect of the present invention provides a primer combination for identifying Corylus species, comprising:

[0008] The upstream and downstream primers used to amplify ndhF-rpl32, whose nucleotide sequences are shown in SEQ ID NOs: 3-4, respectively;

[0009] The upstream and downstream primers used to amplify rpl32-trnL, whose nucleotide sequences are shown in SEQ ID NOs: 5-6, respectively;

[0010] The nucleotide sequences of the upstream and downstream primers used to amplify ycf1 are shown in SEQ ID NOs: 7-8, respectively;

[0011] The nucleotide sequences of the upstream and downstream primers used for amplifying trnS-trnG are shown in SEQ ID NOs: 9-10, respectively.

[0012] Another aspect of the present invention provides a kit for identifying Corylus species, comprising the above primer combination.

[0013] The use of the above-mentioned DNA barcode, primer combination, or kit in identifying Corylus species also belongs to the content of the present invention.

[0014] In another aspect, the present invention also provides a method for identifying Corylus species, comprising the following steps:

[0015] S1) using chloroplast DNA of different samples to be tested as templates, and using the above primer combinations to perform PCR amplification respectively, to obtain PCR amplification products of the following four hypervariable regions: ndhF-rpl32, rpl32-trnL, ycf1, and trnS-trnG;

[0016] S2) sequencing the PCR amplification products of the four hypervariable regions obtained in step S1) to obtain four hypervariable region sequences of chloroplast DNA of different samples to be tested, and splicing and proofreading the four hypervariable region sequences of different samples to be tested in the same order to obtain DNA barcodes corresponding to different samples to be tested;

[0017] S3) performing cluster analysis based on the DNA barcodes of different samples to be tested obtained in step S2) to identify Corylus species.

[0018] In some embodiments, the reaction procedure of the PCR amplification in step S1) is: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 45 s, gradient annealing at 60-50°C for 1 min, extension at 72°C for 1 min, 30 cycles; and final extension at 72°C for 10 min.

[0019] In some embodiments, the cluster analysis in step S3) is to construct an ML phylogenetic tree using DNA barcodes of different samples to be tested, and if the Corylus species are distributed in different branches with a support rate of not less than 75%, they are determined to be different species.

[0020] The DNA barcode for identifying Corylus species provided by the above technical solution is a sequence formed by connecting the following four hypervariable regions of the chloroplast genome of Corylus species: ndhF-rpl32, rpl32-trnL, ycf1 and trnS-trnG. When using the DNA barcode to identify Corylus species, the same effect as using the chloroplast whole genome sequence to identify Corylus species can be achieved, and rapid and accurate identification of Corylus species can be achieved. The kit and method provided by the present invention can be applied to the identification of germplasm resources in agricultural scientific research and production, and provide an important basis for the classification management and application of Corylus germplasm resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 mVISTA global alignment of chloroplast genomes of 17 species of the genus Corylus;

[0022] Figure 2 This is a variation analysis diagram of nucleotide polymorphism (Pi) in the chloroplast genome of 17 species of the genus Corylus;

[0023] Figure 3 This is the ML phylogenetic tree of Corylus species constructed based on the complete chloroplast genome sequences;

[0024] Figure 4 This is the ML phylogenetic tree of Corylus species constructed based on the triple combination of ndhF-rpl32+rpl32-trnL+ycf1 sequences;

[0025] Figure 5 This is the ML phylogenetic tree of Corylus species constructed based on the four-combination sequence of ndhF-rpl32+rpl32-trnL+ycf1+atpH-atpI;

[0026] Figure 6 This is the ML phylogenetic tree of Corylus species constructed based on the four-combination sequence of ndhF-rpl32+rpl32-trnL+ycf1+trnS-trnG. DETAILED DESCRIPTION

[0027] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0028] The methods used in the following examples are conventional methods unless otherwise specified. The methods for obtaining various biological materials described in the examples are only to provide an experimental method for obtaining them to achieve the specific disclosed purpose, and should not be a limitation on the source of the biological materials of the present invention. In fact, the sources of the biological materials used are extensive, and any biological materials that can be obtained without violating laws and moral ethics can be replaced and used according to the instructions in the examples.

[0029] The embodiments are implemented on the premise of the technical solution of the present invention, and provide detailed implementation methods and specific operation processes. The embodiments will help to understand the present invention, but should not be used as a limitation to the contents of the present invention.

[0030] The primers involved in the following examples can all be synthesized using existing techniques.

[0031] Example 1: Screening and determination of highly variable polymorphic regions in the chloroplast genome of the genus Corylus

[0032] In this example, the inventors used the chloroplast genome annotation of Corylus heterophylla as a reference. The chloroplast genome of Corylus heterophylla (GenBank accession number: KX822769) was used as the reference sequence, and the Shuffle-LAGAN model of the online mVISTA software was used to compare the variations between the chloroplast genomes of the 17 Corylus species in Table 1 below. Figure 1As shown. It can be seen that the sequence similarity of the chloroplast genome region of most Corylus species is above 90%, the protein coding region is relatively conservative, and the sequence differentiation and variation are mainly distributed in the non-coding region, especially the intergenic region, such as trnK-rps16, trnQ-psbK, trnS-trnG, trnG-trnR, atpH-atpI, trnF-ndhJ, ndhC-trnV, trnP-rpl33, rps3-rpl22, ndhF-rpl32, rpl32-trnL, ndhD-psaC, ndhF-rpl32, rpl32-trnL, ndhD-psaC, etc., as well as ycf1 (ycf1a and ycf1b) located in the coding region. The inventors believe that these hypervariable regions can be used as potential molecular markers for the identification of Corylus species, and used MAFFT 7.0 (Katoh and Standley, 2013) to perform a global alignment of the chloroplast genomes of the 17 Corylus species shown in Table 1 below, and then used Se-Al software to manually adjust the alignment results, and open the inversion alignments that appeared in the sequence to avoid erroneous data polymorphism. Then DnaSP 5.0 was used for sliding window analysis, and the hotspots of variation were screened out by counting the variation sites and sequence polymorphism (Pi) in the chloroplast genome. The window length was set to 800bp and the step length was set to 100bp during the operation. The results are as follows: Figure 2 As shown in Figure 2, it can be seen that the Pi value of the intergenic region is significantly greater than the Pi value of the coding gene, which is consistent with Figure 1 The results are consistent with those shown in Figure 1, that is, sequence differentiation and variation are mainly distributed in the intergenic region. Figure 2 The positions of the hypervariable regions trnS-trnG, trnG-trnR, atpH-atpI, trnP-rpl33, rps3-rpl22, ndhF-rpl32, rpl32-trnL, ndhD-psaC, and ycf1 (ycf1a and ycf1b) are exemplarily shown.

[0033] Based on the above Pi values ​​and mVISTA result analysis, the inventors used DNA barcodes spliced ​​from the sequences of several molecular markers among the above potential molecular markers (such as several of trnK-rps16, trnQ-psbK, trnS-trnG, trnG-trnR, atpH-atpI, trnF-ndhJ, ndhC-trnV, trnP-rpl33, rps3-rpl22, ndhF-rpl32, rpl32-trnL, ndhD-psaC, ndhF-rpl32, rpl32-trnL, ndhD-psaC and ycf1, etc.) to construct an ML phylogenetic tree for the 17 Corylus species in Table 1 for cluster analysis, and compared the ML phylogenetic tree constructed using the chloroplast whole genome sequences of the 17 Corylus species in Table 1 to determine the DNA barcode that can quickly and accurately identify Corylus species. This embodiment takes the hypervariable regions atpH-atpI, ndhF-rpl32, rpl32-trnL, ycf1 and trnS-trnG as examples to determine a DNA barcode that can quickly and accurately identify Corylus species.

[0034] Table 1: Chloroplast genome sequences of 17 Corylus species

[0035]

[0036] 1.1. According to the sequence of the chloroplast genome of Corylus species, primer sequences for amplifying the hypervariable regions atpH-atpI, ndhF-rpl32, rpl32-trnL, ycf1 and trnS-trnG were designed. The primer sequence information is shown in Table 2 below.

[0037] Table 2: Corresponding primers and PCR amplification information of the five hypervariable regions

[0038]

[0039] 1.2. Using the selected 5 hypervariable regions and the primer sequences designed in step 1.1 above to identify the 17 Corylus species listed in Table 1, specifically comprising the following steps:

[0040] (1) Extraction of genomic DNA from Corylus species

[0041] 100 mg of leaf samples of the 17 Corylus species listed in Table 1 were taken respectively into 2.0 mL centrifuge tubes, and appropriate amount of PVP powder was added and ground with liquid nitrogen. The remaining steps were carried out according to the standard operating procedures of the plant genomic DNA extraction kit to obtain the respective chloroplast genomic DNA.

[0042] (2) PCR amplification

[0043] Using the chloroplast genomic DNA extracted in step (1) as a template, PCR amplification was performed on the five hypervariable regions. The total amount of the amplification reaction system was 40 μL, including 20 μL of 2x Es Taq MasterMix enzyme; 0.5 μL of forward primer (20 μM); 0.5 μL of reverse primer (20 μM); 1 μL of template DNA; ddH 2 O 18μL. The PCR amplification program was: 94℃ pre-denaturation for 5min; 94℃ denaturation for 45s, 60-50℃ gradient renaturation for 1min, 72℃ extension for 1min, 30 cycles; final extension at 72℃ for 10min. The PCR amplification products were detected by 1.2% agarose gel electrophoresis, and PCR products of the five hypervariable regions of each Corylus species were obtained.

[0044] (3) Sequencing and sequence assembly

[0045] The size of the PCR product detected by electrophoresis was consistent with the amplification length of the target fragment designed by the specific primers, and the PCR product with a single band was subjected to Sanger bidirectional sequencing on the ABI3730XL sequencer to obtain the sequences of the five hypervariable regions of each Corylus species. The sequencing results were manually corrected using Mega software, and then the hypervariable region sequences were spliced ​​using PhyloSuite software. The following three combined splicing sequences were obtained for each Corylus species: ndhF-rpl32+rpl32-trnL+ycf1, ndhF-rpl32+rpl32-trnL+ycf1+atpH-atpI, and ndhF-rpl32+rpl32-trnL+ycf1+trnS-trnG, which were used as candidate DNA barcodes for identifying Corylus species.

[0046] (4) Construction of ML phylogenetic tree

[0047] (4.1) Using the other five closely related genera and species in the Betulaceae family (as shown in Table 3) as the outgroup, the spliced ​​sequences of three combinations of these five closely related genera and species were obtained according to the above steps (1)-(3).

[0048] Table 3: Chloroplast genome information of outgroups used to construct phylogenetic trees

[0049]

[0050] (4.2) The chloroplast genome sequences of the five closely related species shown in Table 3 above were used as outgroups, and a phylogenetic tree was constructed based on the chloroplast genome sequences of the 17 Corylus species shown in Table 1 above. The best model was selected using the ModelFinder software according to the BIC (Bayesian information criterion) standard, and the Software, using maximum likelihood (ML) method to construct phylogenetic tree, the results are as follows Figure 3 As shown, the ML phylogenetic tree constructed based on the chloroplast whole genome sequences of 17 Corylus species is shown. It can be seen that the phylogenetic tree constructed based on the chloroplast whole genome sequences of 17 Corylus species has a high support (all above 76%), indicating that the chloroplast whole genome sequence as a super DNA barcode can well identify Corylus species, and analyze the phylogenetic relationship between Corylus species and between Corylus and closely related genera of Betulaceae (Corylus, Carpinus, Ironwood, Alnus, Betula).

[0051] (4.3) The spliced ​​sequences of the three combinations of the five closely related genera shown in Table 3 were used as outgroups, and ML phylogenetic trees were constructed based on the spliced ​​sequences of the three combinations of 17 Corylus species (i.e., candidate DNA barcodes). The best model was selected according to the BIC standard using the ModelFinder software, and the Software, using the maximum likelihood method to construct a phylogenetic tree, the results are as follows Figure 4-6 As shown, Figure 4 represents the ML phylogenetic tree constructed based on the candidate DNA barcodes ndhF-rpl32+rpl32-trnL+ycf1 of 17 Corylus species, Figure 5 represents the ML phylogenetic tree constructed based on the candidate DNA barcodes ndhF-rpl32+rpl32-trnL+ycf1+atpH-atpI of 17 Corylus species, Figure 6 This shows the ML phylogenetic tree constructed based on the candidate DNA barcodes ndhF-rpl32+rpl32-trnL+ycf1+trnS-trnG of 17 Corylus species. Figure 4 and Figure 5 Although the ML phylogenetic tree shown has good identification ability for Corylus species, its support for some interspecific relationships is low (less than 75%), especially in analyzing the evolutionary relationship between Corylus and its closely related genera. In contrast, the candidate DNA barcode ndhF-rpl32+rpl32-trnL+ycf1+trnS-trnG has a similar topological structure to the chloroplast whole genome phylogenetic tree, and the support of each branch is not less than 75%, which can accurately identify Corylus species and reflect the evolutionary relationship between Corylus and its closely related genera. The above results show that the identification of undetermined Corylus species does not need to rely on the chloroplast whole genome sequence, and can be achieved quickly and accurately using only the candidate DNA barcode ndhF-rpl32+rpl32-trnL+ycf1+trnS-trnG.

[0052] In this embodiment, the inventors also verified the effect of candidate DNA barcodes obtained by different splicing orders of the following hypervariable regions in identifying 17 species of Corylus: ndhF-rpl32, rpl32-trnL, ycf1, trnS-trnG, all of which can achieve the effect of rapid and accurate identification of Corylus species, and all have a topological structure similar to the phylogenetic tree of the chloroplast genome. Therefore, the present invention determines that the sequential splicing sequence of the hypervariable regions ndhF-rpl32, rpl32-trnL, ycf1, trnS-trnG on the chloroplast genome of Corylus species is used as the DNA barcode for identifying Corylus species.

[0053] Example 2: Kit for identifying Corylus species

[0054] This embodiment provides a kit for identifying Corylus species, which includes primers for amplifying the following four hypervariable regions of the chloroplast genome of Corylus species: ndhF-rpl32, rpl32-trnL, ycf1 and trnS-trnG, specifically including:

[0055] The upstream and downstream primers used to amplify ndhF-rpl32, whose nucleotide sequences are shown in SEQ ID NOs: 3-4, respectively;

[0056] The upstream and downstream primers used to amplify rpl32-trnL, whose nucleotide sequences are shown in SEQ ID NOs: 5-6, respectively;

[0057] The nucleotide sequences of the upstream and downstream primers used to amplify ycf1 are shown in SEQ ID NOs: 7-8, respectively;

[0058] The nucleotide sequences of the upstream and downstream primers used for amplifying trnS-trnG are shown in SEQ ID NOs: 9-10, respectively.

[0059] For the convenience of identification and detection, the kit also includes a reaction system and PCR reaction program for PCR amplification, wherein a 40 μL reaction system includes: 15-25 μL of 2x Es Taq MasterMix enzyme; 0.5-1 μL of forward primer (20 μM); 0.5-1 μL of reverse primer (20 μM); ddH 2 The PCR reaction program included: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 45 s, 60-50℃ gradient annealing for 1 min, 72℃ extension for 1 min, 30 cycles; and final extension at 72℃ for 10 min.

[0060] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A DNA barcode for identifying Corylus species, characterized in that: The DNA barcode is a sequence formed by connecting the following four hypervariable regions of the chloroplast genome of Corylus species: ndhF-rpl32, rpl32-trnL, ycf1 and trnS-trnG, wherein the ndhF-rpl32 represents the non-coding region between the genes ndhF and rpl32, the rpl32-trnL represents the non-coding region between the genes rpl32 and trnL, the ycf1 represents the coding region gene ycf1, and the trnS-trnG represents the non-coding region between the genes trnS and trnG; The upstream and downstream primers used to amplify ndhF-rpl32 are shown in SEQ ID NOs: 3-4, respectively; The upstream and downstream primers used to amplify rpl32-trnL are shown in SEQ ID NOs: 5-6, respectively; The upstream and downstream primers used to amplify ycf1 are shown in SEQ ID NOs: 7-8, respectively; The upstream and downstream primers used for amplifying trnS-trnG are shown in SEQ ID NOs: 9-10, respectively.

2. A primer combination for identifying species of the genus Corylus, characterized in that: The primer combination comprises: The upstream and downstream primers used to amplify ndhF-rpl32, whose nucleotide sequences are shown in SEQ ID NOs: 3-4, respectively; The upstream and downstream primers used to amplify rpl32-trnL, whose nucleotide sequences are shown in SEQ ID NOs: 5-6, respectively; The nucleotide sequences of the upstream and downstream primers used to amplify ycf1 are shown in SEQ ID NOs: 7-8, respectively; The nucleotide sequences of the upstream and downstream primers used for amplifying trnS-trnG are shown in SEQ ID NOs: 9-10, respectively.

3. A kit for identifying Corylus species, characterized in that: The kit comprises the primer combination according to claim 2.

4. Use of the DNA barcode according to claim 1, the primer combination according to claim 2 or the kit according to claim 3 in identifying Corylus species.

5. A method for identifying Corylus species, characterized in that: The method comprises the following steps: S1) using chloroplast DNA of different samples to be tested as templates, and using the primer combination of claim 2 to perform PCR amplification respectively, to obtain PCR amplification products of the following four hypervariable regions: ndhF-rpl32, rpl32-trnL, ycf1 and trnS-trnG; S2) sequencing the PCR amplification products of the four hypervariable regions obtained in step S1) to obtain four hypervariable region sequences of chloroplast DNA of different samples to be tested, and splicing and proofreading the four hypervariable region sequences of different samples to be tested in the same order to obtain DNA barcodes corresponding to different samples to be tested; S3) performing cluster analysis based on the DNA barcodes of different samples to be tested obtained in step S2) to identify Corylus species.

6. The method according to claim 5, characterized in that The reaction procedure of the PCR amplification in step S1) is: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 45 s, gradient annealing at 60-50°C for 1 min, extension at 72°C for 1 min, 30 cycles; and final extension at 72°C for 10 min.

7. The method according to claim 5 or 6, characterized in that: The cluster analysis in step S3) is to construct an ML phylogenetic tree using DNA barcodes of different samples to be tested. If the Corylus species are distributed in different branches with a support rate of not less than 75%, they are determined to be different species.

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