DNA bar code for identifying pennisetum plants and application thereof

Abstract

The invention relates to the field of biological identification, and particularly discloses a DNA bar code for identifying pennisetum plants and application thereof.The DNA bar code comprises a section of specific nucleotide sequence of a chloroplast gene spacer psbA-trnH, screening finds that the section has enough variation sites between pennisetum species and is highly conservative in the species, and the section of specific nucleotide sequence can be used for identifying the pennisetum plants. Different species of pennisetum, such as pennisetum purpureum, Yu-grain and the like, can be effectively distinguished; the invention further comprises a specific primer pair for amplifying the bar code, a PCR kit containing the primer pair and application of the PCR kit in species identification, genetic relationship analysis, seed purity inspection and Chinese herbal medicine source tracing of the pennisetum plants. The method has the advantages of being high in identification speed, high in accuracy, good in repeatability, free of influences of plant development stages and the environment and the like, and the problems faced by a traditional morphological identification method are solved.

Description

Technical Field

[0001] This invention relates to the field of biological identification technology, and in particular to a DNA barcode for identifying plants of the genus *Pennisetum* and its application. Background Technology

[0002] *Pennisetum* is an important genus within the Poaceae family, comprising approximately 80-140 species widely distributed in tropical and subtropical regions worldwide. This genus includes important high-yielding forage grasses like elephant grass (*P. purpureum*), global food crops such as pearl millet (*P. glaucum*), and ornamental plants like pennisetum (*P. alopecuroides*). Therefore, plants in this genus have extremely high economic value in agriculture, animal husbandry, and landscaping.

[0003] Currently, the identification of plants in the genus *Pennisetum* mainly relies on traditional morphological methods, such as observing plant height, leaf morphology, inflorescence structure, spikelet and caryopsis characteristics. However, these methods have significant limitations: The morphological characteristics of plants are greatly affected by the growth environment (such as water, light, and soil fertility) and the developmental stage, resulting in unstable traits and making accurate identification difficult.

[0004] Highly subjective: Morphological identification relies heavily on the experience and expertise of the examiner, and different examiners may reach different conclusions, resulting in poor repeatability.

[0005] Identification of closely related species and hybrids is difficult: many species within the genus *Pennisetum* are morphologically similar, making them difficult to distinguish, especially during the seedling or vegetative growth stages. Furthermore, there is extensive hybridization within this genus (e.g., *Elephantgrass* is produced by crossing *Elephantgrass* with *Elephantgrass*), and the morphological characteristics of their hybrid offspring fall between those of their parents, making it difficult to accurately identify their parental origin and the authenticity of the hybridization based solely on morphology.

[0006] With the development of molecular biology, DNA barcoding technology has provided a new solution for species identification. This technology utilizes a standard, relatively short DNA fragment with sufficient variation in the genome to quickly and accurately identify species. Universal plant DNA barcodes such as matK, rbcL, ITS2, and psbA-trnH have been proposed. However, the identification efficiency of these universal barcodes varies significantly across different families and genera. For groups like *Pennisetum*, which have high species diversity and complex relationships, existing universal barcodes often suffer from insufficient interspecific variation or excessive intraspecific variation, resulting in low resolution and an inability to accurately distinguish all species within the genus.

[0007] Therefore, there is an urgent need in this field to find a high-resolution, specific DNA barcode fragment for plants of the genus *Pennisetum*, and to develop corresponding identification methods and kits to overcome the shortcomings of existing technologies. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of the existing technology and to propose a DNA barcode for identifying plants of the genus *Pennisetum* and its application.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: A DNA barcode for identifying plants of the genus *Pennisetum* comprises: a nucleotide sequence of the psbA-trnH intergenic spacer region of the chloroplast genome, the nucleotide sequence being 300-500 bp in length, the nucleotide sequence containing single nucleotide polymorphisms (SNPs) and insertion or deletion (InDel) sites that distinguish different species of *Pennisetum*.

[0010] As a further technical solution of the present invention, plants of the genus Pennisetum include, but are not limited to, elephant grass (Pennisetum purpureum), grass glaucum, alopecuroides, pasture grass (Pennisetum polystachion) and their closely related species.

[0011] A DNA barcode for identifying plants of the genus *Pennisetum* further includes: a primer pair for amplifying the DNA barcode, wherein the nucleotide sequence of the primer pair is: SEQ ID NO:1 (forward primer F): 5'-GTT ATG CAT GAA CGT AAT GCT C-3'; SEQ ID NO:2 (reverse primer R): 5'-CGC GCA TGG TGG ATT CAC AAT CC-3'.

[0012] An application for identifying DNA barcodes of plants in the genus *Pennisetum* includes: a kit for identifying *Pennisetum* plants, said kit comprising primer pairs, PCR reaction buffer, a mixture of dNTPs, Taq DNA polymerase, and Mg... 2+ A database of standard DNA barcode sequences for ions, positive control templates, or different species of the genus *Pennisetum*.

[0013] An application of DNA barcoding for identifying plants of the genus *Pennisetum* also includes: identification of *Pennisetum* germplasm resources, evolutionary analysis of phylogenetic relationships, identification of the authenticity of hybrid offspring, seed purity testing, and traceability of Chinese medicinal materials and agricultural products.

[0014] A method for identifying plants of the genus *Pennisetum* based on DNA barcoding, comprising the following steps: S1: Extract genomic DNA from the plants of the genus *Pennisetum* to be tested; S2: Using the extracted DNA as a template, perform PCR amplification using the primer pair to obtain the PCR amplification product; S3: Sequencing the PCR amplification product (sent to professional institutions such as Sangon Biotech (Shanghai) Co., Ltd. for purification and bidirectional sequencing) to obtain the psbA-trnH spacer region sequence of the plant to be tested. S4: The obtained sequence is compared with the standard DNA barcode sequence database of different species in the genus *Pennisetum*, and the species identity of the plant to be tested is determined based on sequence similarity and specific variant sites.

[0015] As a further technical solution of the present invention, in S2, the PCR reaction program is as follows: pre-denaturation at 94℃ for 4 min; followed by 35 cycles: denaturation at 94℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 45 s; after the cycle, final extension at 72℃ for 7 min; and storage at 4℃.

[0016] As a further technical solution of the present invention, in step S2, 5 μL of PCR amplification product is taken and electrophoretically detected on a 1.5% agarose gel.

[0017] The beneficial effects of this invention are as follows: 1. High specificity and resolution: The excellent effect of the chloroplast psbA-trnH spacer region as a specific DNA barcode for the genus *Pennisetum* was discovered and verified. This fragment has accumulated sufficient single nucleotide polymorphisms (SNPs) and insertion / deletion sites among species of *Pennisetum*, which can clearly and stably distinguish different species of *Pennisetum* (such as elephant grass, yew, and *Pennisetum purpureum*), with a resolution far higher than other general barcodes.

[0018] 2. Accurate, reliable, and highly reproducible: DNA barcodes are the essential genetic characteristics of a species and are not affected by the plant's developmental stage, organ location, or environmental conditions. The identification results are objective, accurate, and highly reproducible, avoiding the subjectivity and instability of morphological identification.

[0019] 3. Fast and efficient: Sample identification can be completed in a short time (usually 1 to 2 working days), which is especially suitable for rapid detection of large batches of samples, such as seed purity testing and germplasm resource bank screening. The efficiency is much higher than that of field phenotypic identification, which requires long growth period observation.

[0020] 4. Wide range of applications: It can be used not only for the identification of mature plants of the genus Napier, but also for the identification of seedlings, seeds and even partially degraded samples (such as hay and Chinese medicinal materials). Attached Figure Description

[0021] Figure 1 This is a flowchart of a method for identifying plants of the genus *Pennisetum* based on DNA barcoding, as proposed in this invention. Detailed Implementation

[0022] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below in conjunction with specific embodiments.

[0023] A DNA barcode for identifying plants of the genus *Pennisetum* comprises: a nucleotide sequence of the psbA-trnH intergenic spacer region of the chloroplast genome, the nucleotide sequence being 300-500 bp in length, the nucleotide sequence containing single nucleotide polymorphisms (SNPs) and insertion or deletion (InDel) sites that distinguish different species of *Pennisetum*.

[0024] In a preferred embodiment, the species of the genus Pennisetum include, but are not limited to, elephant grass (Pennisetum purpureum), yew grass (P. glaucum), prickly pear grass (P. alopecuroides), pasture prickly pear grass (P. polystachion), and their close relatives.

[0025] A DNA barcode for identifying plants of the genus *Pennisetum* further includes: a primer pair for amplifying the DNA barcode, wherein the nucleotide sequence of the primer pair is: SEQ ID NO:1 (forward primer F): 5'-GTT ATG CAT GAA CGT AAT GCT C-3'; SEQ ID NO:2 (reverse primer R): 5'-CGC GCA TGG TGG ATT CAC AAT CC-3'.

[0026] An application for identifying DNA barcodes of plants in the genus *Pennisetum* includes: a kit for identifying *Pennisetum* plants, the kit comprising primer pairs, PCR reaction buffer, a mixture of dNTPs, Taq DNA polymerase, and Mg... 2+ A database of standard DNA barcode sequences for ions, positive control templates, or different species of the genus *Pennisetum*.

[0027] An application of DNA barcoding for identifying plants of the genus *Pennisetum* also includes: identification of *Pennisetum* germplasm resources, evolutionary analysis of phylogenetic relationships, identification of the authenticity of hybrid offspring, seed purity testing, and traceability of Chinese medicinal materials and agricultural products.

[0028] A method for identifying plants of the genus *Pennisetum* based on DNA barcoding, comprising the following steps: S1: Extract genomic DNA from the plants of the genus *Pennisetum* to be tested; S2: Using the extracted DNA as a template, perform PCR amplification using the primer pair to obtain the PCR amplification product; S3: Sequencing the PCR amplification product (sent to professional institutions such as Sangon Biotech (Shanghai) Co., Ltd. for purification and bidirectional sequencing) to obtain the psbA-trnH spacer region sequence of the plant to be tested. S4: The obtained sequence is compared with the standard DNA barcode sequence database of different species in the genus *Pennisetum*, and the species identity of the plant to be tested is determined based on sequence similarity and specific variant sites.

[0029] In a preferred embodiment, in S2, the PCR reaction program is as follows: pre-denaturation at 94°C for 4 min; followed by 35 cycles: denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 45 s; final extension at 72°C for 7 min after the cycle; and storage at 4°C.

[0030] In a preferred embodiment, in S2, 5 μL of PCR amplification product is taken and electrophoretically detected on a 1.5% agarose gel.

[0031] Example 1 The DNA barcode and primer pairs provided by this invention are used to identify plants of the genus *Pennisetum*.

[0032] 1. Purpose: This embodiment aims to verify the effectiveness of the psbA-trnH gene spacer region provided by the present invention as a DNA barcode, as well as the amplification efficiency of the provided specific primer pair, and to demonstrate its process for accurately identifying different species of the genus Napier.

[0033] 2. Materials and Methods 2.1 Experimental Materials: Fresh leaf samples of *Pennisetum* species collected from germplasm banks or fields were selected, including: Elephant grass (Pennisetum purpureum Schum.) Pennisetum glaucum (L.) R. Br. Pennisetum alopecuroides (L.) Spreng. Hybrid (Elephant Grass × Miko) Closely related genus control: Setaria viridis (L.) P. Beauv. (Poaceae family, Setaria genus) For each species, 3 to 5 individuals from different geographical origins were selected as biological replicates.

[0034] 2.2 DNA Extraction: Total genomic DNA was extracted from each sample using a modified CTAB method or a commercial plant genomic DNA extraction kit (such as the TIANGEN Plant Genomic DNA Kit). The concentration and purity of the DNA (OD260 / 280 ratio between 1.8 and 2.0) were determined using a Nanodrop micro spectrophotometer, and the integrity of the DNA was assessed by 1% agarose gel electrophoresis.

[0035] 2.3 PCR amplification and sequencing Primer sequences: PCR amplification was performed using the specific primer pairs provided in this invention.

[0036] Forward primer psbAF (SEQ ID NO: 1): 5'-GTT ATG CAT GAA CGT AAT GCT C-3' Reverse primer trnHR (SEQ ID NO: 2): 5'-CGC GCA TGG TGG ATT CAC AAT CC-3' The PCR reaction system (25 μL) is shown in Table 1 below: Table 1: PCR reaction system PCR reaction procedure: 94℃ pre-denaturation for 4 min; followed by 35 cycles: 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 45 s; after the cycles, final extension at 72℃ for 7 min; store at 4℃.

[0037] Product detection and sequencing: 5 μL of PCR product was electrophoresed on a 1.5% agarose gel. Successfully amplified samples were sent to professional institutions such as Sangon Biotech (Shanghai) Co., Ltd. for purification and bidirectional sequencing.

[0038] 2.4 Sequence Analysis and Alignment: The obtained forward and reverse sequences were assembled using SeqMan software to obtain the complete psbA-trnH spacer region sequence. Multiple alignments of all samples were performed using Clustal W or MEGA 11 software, and the sequences were manually proofread. Based on the alignment results, intraspecific homology and interspecific variation sites (including single nucleotide polymorphisms (SNPs) and insertions / deletions (InDels)) were analyzed.

[0039] 3. Results 3.1 Amplification effect: Using the primer pairs provided by this invention, all samples of the genus *Pennisetum* and the control *Setaria viridis* were successfully amplified with a clear band, ranging in size from 350 to 450 bp (slightly different depending on the species), and no non-specific bands were observed, indicating that the primers have high versatility and amplification efficiency.

[0040] 3.2 Sequence Characteristics and Variation Analysis Intraspecific consistency: Different individuals of the same species have completely identical psbA-trnH sequences or only a few base differences (intraspecific variation is much smaller than interspecific variation), indicating that the fragment is highly conserved within the species.

[0041] Interspecific variation: Multiple sequence alignment revealed abundant SNPs and InDel sites among different species of the genus *Pennisetum*. For example, in sequence comparisons between *Elephantgrass* and *Eriocheir sinensis*, more than 20 stable, specific variation sites were found, including several insertion / deletion mutations, which can serve as molecular markers to distinguish the two species.

[0042] Hybrid identification: The sequence of the hybrid (elephant grass × yak rice) was confirmed to be heterozygous from the sequences of the two parents. By comparing it with the standard sequences of the two parents, its hybrid origin can be clearly identified.

[0043] Intergeneric differentiation: The psbA-trnH sequence of the closely related species control foxtail grass differs significantly from the sequences of other species in the genus Napier, further demonstrating the potential of this barcode for differentiating Napier and its closely related genera.

[0044] 4. Conclusion: This embodiment fully verifies the effectiveness and reliability of the psbA-trnH gene spacer region described in this invention as a DNA barcode for *Phragmites australis* species. The primer pairs (SEQ ID NO: 1 and 2) can stably and efficiently amplify the target fragment. Through sequence alignment analysis, this barcode fragment can accurately and rapidly distinguish different species within the *Phragmites* genus through its unique SNP and InDel spectra, and can be applied to hybrid identification, fully achieving the intended purpose and effect of this invention.

[0045] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects: high specificity and resolution: the excellent effect of the chloroplast psbA-trnH spacer region as a specific DNA barcode for the genus *Pennisetum* was discovered and verified for the first time; this fragment has accumulated sufficient single nucleotide polymorphisms (SNPs) and insertion / deletion (InDel) sites among species of the genus *Pennisetum*, and can clearly and stably distinguish different species of the genus *Pennisetum* (such as elephant grass, yew, and *Pennisetum purpureum*), with a resolution far higher than other general barcodes.

[0046] Accurate, reliable, and highly reproducible: DNA barcodes are the essential genetic characteristics of a species and are not affected by the plant's developmental stage, organ location, or environmental conditions. The identification results are objective, accurate, and highly reproducible, avoiding the subjectivity and instability of morphological identification.

[0047] Fast and efficient: Sample identification can be completed in a short time (usually 1 to 2 working days), which is especially suitable for rapid detection of large batches of samples, such as seed purity testing and germplasm resource bank screening. The efficiency is much higher than that of field phenotypic identification, which requires long growth period observation.

[0048] Its applications are wide-ranging: it can be used not only for the identification of mature plants of the *Pennisetum* genus, but also for the identification of seedlings, seeds, and even partially degraded samples (such as hay and processed medicinal herbs). Its applications include, but are not limited to: Precise management and identification of germplasm resource banks.

[0049] Early identification of parental authenticity and hybrid offspring purity in hybridization breeding.

[0050] Testing the purity and authenticity of crop and forage seeds to combat counterfeit and substandard products.

[0051] In the field of traditional Chinese medicine, traceability and identification are carried out for medicinal plants of the genus *Phragmites* that are used interchangeably or are counterfeited.

[0052] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of the invention is limited to these examples; within the framework of the invention, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity.

[0053] This invention is intended to cover all such substitutions, modifications, and variations that fall within the broad scope of this specification. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. A DNA barcode for identifying plants of the genus *Pennisetum*, characterized in that, include: The nucleotide sequence of the psbA-trnH intergenic spacer region of the chloroplast genome, the nucleotide sequence being 300-500 bp in length, the nucleotide sequence containing single nucleotide polymorphisms and insertion or deletion sites that distinguish different species of the genus *Pennisetum*.

2. The DNA barcode for identifying plants of the genus *Pennisetum* according to claim 1, characterized in that, The species of the genus *Pennisetum* include, but are not limited to, elephant grass, *Gnaphalium affine*, *Pennisetum gracilis ... and their closely related species.

3. The DNA barcode for identifying plants of the genus *Pennisetum* according to claim 1, characterized in that, Also includes: Primer pairs used for amplifying DNA barcodes, the nucleotide sequences of which are: SEQ ID NO: 1: 5'-GTT ATG CAT GAA CGT AAT GCT C-3'; SEQ ID NO: 2: 5'-CGC GCA TGG TGG ATT CAC AAT CC-3'.

4. An application of DNA barcoding for identifying plants of the genus *Pennisetum*, characterized in that, include: A kit for identifying plants of the genus *Pennisetum*, the kit comprising primer pairs, PCR reaction buffer, a mixture of dNTPs, Taq DNA polymerase, and Mg... 2+ A database of standard DNA barcode sequences for ions, positive control templates, or different species of the genus *Pennisetum*.

5. The application of DNA barcoding for identifying plants of the genus *Pennisetum* according to claim 4, characterized in that, Also includes: Germplasm resource identification, phylogenetic analysis, authenticity identification of hybrid offspring, seed purity testing, and traceability of Chinese medicinal materials and agricultural products.

6. A method for identifying plants of the genus *Pennisetum* based on DNA barcoding, characterized in that, Identification using the DNA barcode for identifying plants of the genus *Pennisetum* as described in any one of claims 1-3 includes the following steps: S1: Extract genomic DNA from the plants of the genus *Pennisetum* to be tested; S2: Using the extracted DNA as a template, perform PCR amplification using the primer pair to obtain the PCR amplification product; S3: Sequencing the PCR amplification product to obtain the psbA-trnH spacer region sequence of the plant to be tested; S4: The obtained sequence is compared with the standard DNA barcode sequence database of different species in the genus *Pennisetum*, and the species identity of the plant to be tested is determined based on sequence similarity and specific variant sites.

7. The method for identifying plants of the genus *Pennisetum* based on DNA barcoding according to claim 6, characterized in that, In S2, the PCR reaction program is as follows: pre-denaturation at 94℃ for 4 min; followed by 35 cycles: denaturation at 94℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 45 s; final extension at 72℃ for 7 min after the cycle; and storage at 4℃.

8. The method for identifying plants of the genus *Pennisetum* based on DNA barcoding according to claim 7, characterized in that, In step S2, 5 μL of PCR amplification product is taken and electrophoresed on a 1.5% agarose gel for detection.

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