Barcodes prepared based on chloroplast genes of Apocynum venetum and their applications

Abstract

The invention relates to the technical field of identification of Apocynum, and in particular to a barcode prepared based on apocynum chloroplast genes and an application thereof. The DNA barcode label of the invention is designed based on the chloroplast genes of Apocynum, and can quickly distinguish and identify two varieties of Apocynum: Apocynum venetum and Apocynum pictum. Gene comparison shows that the ycf1 gene and rpoC2 gene of Apocynum venetum and Apocynum pictum are significantly different, and the specific sites are very different. The two varieties of Apocynum can be quickly distinguished by sequencing the two varieties. The method can provide a more accurate gene auxiliary tool for subsequent botanical classification of Apocynum.

Description

Technical field

[0001] The present invention relates to the technical field of Apocynum venetum identification, and in particular to a barcode prepared based on Apocynum venetum chloroplast genes and application thereof. [Background Technology]

[0002] Apocynum venetum, also known as tea tree and tea flower, belongs to the Apocynaceae family. It is a semi-shrubby perennial herb that reproduces by seeds and rhizomes. Apocynum venetum can grow in harsh natural environments, such as saline-alkali sandy wastelands. It has excellent ecological benefits, such as soil improvement and water conservation, and can also be cultivated as an ornamental plant. Its leaves can be used as medicine and can also be made into health tea. Apocynum venetum fiber, known as the "king of wild fibers," is a rare natural fiber material and therefore has great value for comprehensive utilization.

[0003] Although Apocynum venetum was named by Mr. Dong Zhengjun in 1952, its botanical classification remained unresolved. Local names were used, with the two species, red hemp and white hemp, being used instead. Subsequently, scholars such as Zhang Pengyun expressed differing opinions on the botanical classification of Apocynum venetum, primarily over whether to adopt Woodson's or Lin's classification system. The 1975 edition of the Flora of China records that there are three species commonly known as Apocynum venetum in my country, assigned to two genera. These include Apocynum venetum (A. venetum) and Poacynum pictum and P. hendersonii (P. hendersonii). Until recently, the botanical classification of Apocynum venetum remained unresolved, with ongoing debate among scholars, primarily focusing on the closeness of the relationship between species in the genus Poacynum and Apocynum venetum, and whether a new genus for Poacynum venetum was warranted. Based on observations of the stomatal morphology of the leaves, leaves, flowers, and seeds of Apocynum venetum and Apocynum venetum, some scholars believe that these characteristics are very similar between the two, and that there is no need to establish a new genus for Apocynum venetum. They suggest that the genus Apocynum venetum be abolished and Apocynum venetum be merged with Apocynum venetum. Other scholars believe that by comparing the ITS, trnL intron, and trnL-F non-coding region sequences of Apocynum venetum (Apocynum venetum), Canada hemp, and Apocynum venetum species, Apocynum venetum and Apocynum venetum, it was found that the ITS sequences of Apocynum venetum, Apocynum venetum, and Apocynum venetum are completely identical, but differ significantly from the ITS sequence of Canada hemp. In the trnL intron and trnL-F non-coding region, the sequences of Apocynum venetum and Apocynum venetum are completely identical, with only three sites of variation between them and Apocynum venetum. That is, the ITS, trnL, and trnL-F sequences of Apocynum venetum samples from different regions and populations, as well as those of Apocynum venetum and Apocynum pictum samples, are essentially unchanged. However, Apocynum venetum and these two plants have differences in these three DNA marker sequences, including more than a dozen SNPs and multiple base insertion / deletions. Therefore, it is believed that the relationship between Apocynum venetum and Apocynum pictum may be closer than the relationship between Apocynum venetum and other species within the genus Apocynum, and it is recommended that the genus Apocynum be withdrawn. Apocynum venetum and Apocynum pictum should be included in the genus Apocynum. Therefore, it is generally believed that Apocynum venetum plants in my country can be divided into one genus and two species, namely, Apocynum venetum, which includes two species: Apocynum venetum and Apocynum pictum.

[0004] Therefore, in order to better distinguish the germplasm of the genus Apocynum, it is necessary to conduct in-depth research from a genetic perspective to find out the gene loci that can truly distinguish the germplasm of the genus Apocynum, and quickly distinguish different germplasms of the genus Apocynum at the molecular level, so as to achieve the purpose of simply, efficiently and accurately distinguishing different germplasms of the genus Apocynum, and provide more accurate genetic auxiliary tools for the subsequent botanical classification of Apocynum. [Summary of the invention]

[0005] In view of the above, it is necessary to conduct in-depth research on the genes of Apocynum venetum, find out the gene loci that can accurately and quickly distinguish the germplasm of Apocynum venetum varieties, and prepare the loci into gene barcodes. The barcodes will be able to quickly and accurately distinguish different Apocynum venetum germplasms, providing more accurate gene-assisted tools for subsequent Apocynum venetum botanical classification.

[0006] To achieve the above object, the technical solution adopted by the present invention is: a barcode is prepared based on the chloroplast gene of Apocynum venetum, wherein the barcode is a DNA barcode, and its nucleotide sequence is shown as SEQ ID NO.1-SEQ ID NO.4.

[0007] The present invention also includes the application of the barcode prepared based on the Apocynum venetum chloroplast gene in distinguishing different Apocynum venetum germplasm resources.

[0008] The present invention also includes a method for identifying different apocynum venetum germplasm resources using the barcode, the method comprising: extracting chloroplast DNA from apocynum venetum material to be identified, using the chloroplast DNA as a template, performing PCR amplification on its genomic DNA using rpoC2 gene primers: as shown in SEQ ID NO.5-SEQ ID NO.6 and ycf1 gene primers: as shown in SEQ ID NO.7-SEQ ID NO.8, and then sequencing the PCR products. If the sequenced rpoC2 gene sequence is as shown in SEQ ID NO.1, the gene sequence at 176-184 bp is: 5'-TAACCTCTC-3' and the gene sequence at 203-211 bp is: 5'-TAACCTCTC-3', then the sample is apocynum venetum (A. pictum); if the sequenced rpoC2 gene sequence is as shown in SEQ ID NO.2, then the sample is apocynum venetum (A. venetum); if the sequenced ycf1 gene sequence is as shown in SEQ ID NO. As shown in SEQ ID NO.3, the gene sequence at 153-158 bp is: 5'-AAAGAA-3', then the sample is A. venetum. If the sequenced ycf1 gene sequence is shown in SEQ ID NO.4, then the sample is A. pictum.

[0009] Furthermore, the total PCR amplification reaction system is 25 μl: 2×Reaction Mix 12.25 μl, GoldenDNA Polymerase 0.25 μl, 2.5 μl each of forward and reverse primers, 5 μl of ddH2O, and 2.5 μl of DNA template; the reaction conditions are: ① pre-denaturation at 94°C for 3 min; ② denaturation at 94°C for 30 s; ③ annealing at 58°C for 30 s; ④ extension at 72°C for 30 s; ⑤ steps ②-④ are cycled 35 times; ⑥ extension at 72°C for 7 min.

[0010] The present invention has the following beneficial effects: the DNA barcode of the present invention is designed based on the chloroplast gene of Apocynum venetum, and can quickly distinguish and identify two varieties of different Apocynum genus: Apocynum venetum (A. venetum) and Apocynum pictum (A. pictum). Gene comparison shows that the ycf1 gene and rpoC2 gene of Apocynum venetum (A. venetum) and Apocynum pictum are significantly different, and the specific sites are very different. By sequencing the above two genes, the two varieties of Apocynum venetum can be quickly distinguished. This method can provide a more accurate gene-assisted tool for subsequent botanical classification of Apocynum venetum.

Brief Description of the Drawings

[0011] Figure 1 This is a diagram of the differential fragments of the chloroplast gene rpoC2 of Apocynum venetum germplasm, from top to bottom they are Apocynum venetum, Apocynum venetum, Apocynum venetum, Apocynum venetum, Apocynum venetum, Apocynum venetum, Apocynum venetum, Apocynum venetum, Apocynum venetum, Apocynum venetum, Apocynum venetum, Apocynum venetum, Apocynum venetum, Apocynum venetum, Apocynum venetum and Apocynum venetum.

[0012] Figure 2-Figure 15 The rpoC2 gene sequencing peak diagram of the differential fragments of 14 different Apocynum venetum samples, among which, Figure 2 、 Figure 4 、 Figure 6 、 Figure 7 、 Figure 8 、 Figure 12 and Figure 13 The sample with the sequencing peak diagram is Apocynum sibiricum; Figure 3 、 Figure 5 、 Figure 9 、 Figure 10 、 Figure 11 、 Figure 14 and Figure 15 The sample is Lop Nivea.

[0013] Figure 16This is a diagram of the differential fragments of the chloroplast gene ycl1 of Apocynum venetum germplasm, from top to bottom they are Apocynum venetum, Apocynum venetum, Apocynum venetum, Apocynum venetum, Apocynum venetum, Apocynum venetum, Apocynum venetum, Apocynum venetum, Apocynum venetum, Apocynum venetum, Apocynum venetum, Apocynum venetum, Apocynum venetum, Apocynum venetum, Apocynum venetum and Apocynum venetum.

[0014] Figures 17-30 The ycl1 gene sequencing peak diagram of the differential fragments of 14 different Apocynum venetum samples, among which, Figure 17 、 Figure 19 、 Figure 21 、 Figure 22 、 Figure 23 、 Figure 27 and Figure 28 The sample with the sequencing peak diagram is Apocynum sibiricum; Figure 18 、 Figure 20 、 Figure 24 、 Figure 25 、 Figure 26 、 Figure 29 and Figure 30 The sample is Lop Nivea.

[0015] Figure 31 This is the electrophoresis diagram of gene rpoC2.

[0016] Figure 32 This is the electrophoresis diagram of gene ycf1. [Specific implementation method]

[0017] To make the above-mentioned objects, features, and advantages of the present invention more readily apparent, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings. The following description sets forth numerous specific details to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art may make similar modifications without departing from the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0018] Example 1

[0019] This example is about the screening of polymorphic regions in the intergenic regions of the chloroplasts of Apocynum venetum, and the specific method is as follows.

[0020] (1) The chloroplast genes of Apocynum kenaf and Apocynum scutellaria were downloaded from NCBI, and the coding sequences of these genes of Apocynum kenaf and Apocynum scutellaria were compared using DNAMAN. It was found that there were large fragment differences (InDel) between the rpoC2 and ycf1 genes of Apocynum kenaf and Apocynum scutellaria.

[0021] (2) Leaves of Apocynum kenaf (red stem, small flowers) and Apocynum sibiricum (red stem, medium flowers) were collected and the young leaves were thoroughly ground in a mortar. DNA was extracted using a DNA extraction kit (Tiangen DP320-03 DNAsecure New Plant Genomic DNA Extraction Kit) according to the instructions. The obtained DNA was stored at -20°C until use.

[0022] (3) Sequencing verification of the InDel between the ycf1 and rpoC2 genes of kenaf and white kenaf: The total reaction system was 25 μl: 2×Reaction Mix 12.25 μl, Golden DNA Polymerase 0.25 μl, 2.5 μl each of forward and reverse primers, 5 μl of ddH2O, and 2.5 μl of DNA template. The reaction conditions were: ① 94℃ pre-denaturation for 3 min; ② 94℃ denaturation for 30 s; ③ 58℃ annealing for 30 s; ④ 72℃ extension for 30 s; ⑤ 35 cycles of steps ②-④; ⑥ 72℃ extension for 7 min. PCR products of samples with good agarose gel electrophoresis results, bright colors, and no impurities were selected and sent for first-generation Sanger sequencing. The forward gene was used for DNA amplification. The primer pairs used for amplification are shown in Table 1.

[0023] (4) The sequencing sequence was compared with the sequence downloaded from NCBI to confirm that the InDel sequence between the rpoC2 and ycf1 genes of Apocynum kenaf and Apocynum sibiricum did exist.

[0024] Therefore, after manual verification and screening, it was concluded that the chloroplast gene rpoC2 gene and the chloroplast gene ycf1 can be used as specific identification DNA barcodes to effectively distinguish the core germplasm of Lop Nivea and Lop Nivea.

[0025] Example 2

[0026] (1) DNA of 14 Apocynum kenaf or Apocynum scutellaria germplasms was obtained according to the method of Example 1 (Table 2).

[0027] (2) Amplification and sequencing of DNA barcode gene rpoC2 and ycf1 fragments: The total reaction system is 25 μl: 2×Reaction Mix 12.25 μl, Golden DNA Polymerase 0.25 μl, forward and reverse primers 2.5 μl each, ddH2O 5 μl, DNA template 2.5 μl. Reaction conditions are: ①94℃ pre-denaturation for 3 min; ②94℃ denaturation for 30 s; ③58℃ annealing for 30 s; ④72℃ extension for 30 s; ⑤steps ②-④ are cycled 35 times; ⑥72℃ extension for 7 min. Reference Figure 31 Gene rpoC2 and Figure 32Electrophoresis diagram of gene ycf1. Select PCR products of samples with good agarose electrophoresis effect, bright color, and no mixed bands, and send them for sequencing. DNA amplification was performed using the forward gene. The primer pairs used for amplification are shown in Table 1.

[0028] Table 1 Amplification primers for genes rpoC2 and ycf1

[0029]

[0030] (3) Sequence alignment and quality check: DNAMAN was used to check the quality of the sequences. The sequencing results were checked by multiple sequence alignment to identify the fragments with differences between the chloroplast gene rpoC2 and the chloroplast gene yc11.

[0031] The sequencing results of the rpoC2 gene of Lop Nivea kenaf are shown in SEQ ID NO: 1 in the sequence listing; the sequencing results of the rpoC2 gene of Lop Nivea kenaf are shown in SEQ ID NO: 2 in the sequence listing.

[0032] The sequencing results of the ycf1 gene of Lop Neff (Apocynum kenaf) are shown in SEQ ID NO: 3 in the sequence listing; the sequencing results of the ycf1 gene of Lop Neff (Apocynum kenaf) are shown in SEQ ID NO: 4 in the sequence listing.

[0033] Sequence comparison of chloroplast rpoC2 genes in different germplasms is as follows Figure 1 As shown, Figure 1 The samples in the figure are the samples in Table 1 from top to bottom. It can be seen from the figure that the gene sequence of the Apocynum kenaf sample at 176-184bp is: 5'-TAACCTCTC-3' and the gene sequence at 203-211bp is: 5'-TAACCTCTC-3', while the genes at 176-184bp and 203-211bp of the Apocynum kenaf sample are both deleted genes.

[0034] Sequence comparison of chloroplast ycl1 genes in different germplasms Figure 16 As shown, Figure 16 The samples in the figure are the samples in Table 1 from top to bottom. It can be seen from the figure that the gene at 153-158bp of the Lop Necklace sample is a deleted gene; while the gene sequence at 153-158bp of the Lop Necklace sample is: 5'-AAAGAA-3'.

[0035] The specific sequencing peak diagram is as follows Figure 2-Figure 15 and Figures 17-30 For details, see Table 2.

[0036] Table 2 Sequencing results of 14 Apocynum venetum samples

[0037] Sample name Germplasm source rpoC2 gene sequencing peak diagram yc11 gene sequencing peak map Extra fine leaves and medium flowers <![CDATA[A. pictum )]]> Figure 2 Figure 17 BianFH-48 1 <![CDATA[A. venetum )]]> Figure 3 Figure 18 Green stem and white flowers <![CDATA[A. Poacynum pictum pictum )]]> Figure 4 Figure 19 BianFH-0224 <![CDATA[A. venetum )]]> Figure 5 Figure 20 TD-02078 <![CDATA[A. pictum )]]> Figure 6 Figure 21 purple spot flower <![CDATA[A. pictum )]]> Figure 7 Figure 22 Red stem medium flower <![CDATA[A. pictum )]]> Figure 8 Figure 23 Lilan117 <![CDATA[Apocynum venetum (A. venetum )]]> Figure 9 Figure 24 GXA2017 <![CDATA[Apocynum venetum (A. venetum )]]> Figure 10 Figure 25 2-868710031041 <![CDATA[Apocynum venetum (A. venetum )]]> Figure 11 Figure 26 Inner Mongolia white hemp <![CDATA[Apocynum venetum (A. pictum )]]> Figure 12 Figure 27 white hemp <![CDATA[Apocynum venetum Linn. pictum )]]> Figure 13 Figure 28 Red stem small flower <![CDATA[Apocynum venetum (A. vetnetum )]]> Figure 14 Figure 29 LiZJ0643 <![CDATA[Apocynum venetum (A. venetum )]]> Figure 15 Figure 30

[0038] Through sequencing results, it can be concluded that the mutation sites in rpoC2 gene and ycl1 gene of Lop Necklace samples are very obvious. Therefore, rpoC2 gene and ycl1 gene fragments can be used as specific DNA barcodes to identify and distinguish the core germplasm of Lop Necklace and Lop Necklace.

[0039] In summary, the DNA barcode prepared according to the rpoC2 gene and ycl1 gene of the chloroplast can quickly distinguish between Apocynum sibiricum and Apocynum kenaf, which is an accurate and efficient method for distinguishing Apocynum sibiricum and Apocynum kenaf germplasm.

[0040] The above-described embodiments merely illustrate several embodiments of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make various modifications and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A method for identifying different Apocynum venetum germplasm resources using DNA barcodes, characterized in that: The method comprises the following steps: extracting chloroplast DNA of a sample of apocynum venetum to be identified, using the chloroplast DNA as a template, using primers with sequences of SEQ ID NO.5 and SEQ ID NO.6 to amplify the rpoc2 gene sequence by PCR, and using primers with sequences of SEQ ID NO.7 and SEQ ID NO.8 to amplify the ycf1 gene sequence by PCR; and then sequencing the PCR products. If the sequenced rpoC2 gene sequence is shown in SEQ ID NO.1, the gene sequence at 176-184 bp is: 5'-TAACCTCTC-3' and the gene sequence at 203-211 bp is: 5'-TAACCTCTC-3', then the sample is apocynum venetum ( A. pictum ), if the sequenced rpoC2 gene sequence is shown as SEQ ID NO.2, then the sample is Lop Nivea ( A. venetum ); if the sequenced ycf1 gene sequence is shown in SEQ ID NO.3, the gene sequence at 153-158bp is: 5'-AAAGAA-3', then the sample is Lop Nivea ( A. venetum ), if the sequenced ycf1 gene sequence is shown as SEQ ID NO.4, then the sample is Lophatherum vulgare ( A. pictum ).

2. The method according to claim 1, characterized in that The total reaction system of the PCR amplification is 25 µl: 2×Reaction Mix 12.25 µl, Golden DNA Polymerase 0.25 µl, forward and reverse primers 2.5 µl each, ddH2O5 µl, DNA template 2.5 µl; The reaction conditions were as follows: ① pre-denaturation at 94 °C for 3 min; ② denaturation at 94 °C for 30 s; ③ annealing at 58 °C for 30 s; ④ extension at 72 °C for 30 s; ⑤ steps ②-④ were cycled 35 times; ⑥ extension at 72 °C for 7 min.

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