Linan bighead atractylodes rhizome chloroplast genome and application thereof

Abstract

The invention discloses a chloroplast genome of Linan atractylodes macrocephala koidz for the first time, the nucleotide sequence of the chloroplast genome is shown as SEQ ID NO.1, the chloroplast genome is applied to phylogenetic research of atractylodes macrocephala koidz, and the phylogenetic status of atractylodes macrocephala koidz in atractylodes is analyzed. According to the research, molecular systematics and phylogenomics methods are utilized, the phylogenetic relationship of rhizoma atractylodis species is established through a chloroplast genome of the Linan atractylodes macrocephala, on the basis, the phylogenetic relationship of the rhizoma atractylodis species is analyzed by applying the super DNA bar code, and suspected seedlings of the Linan atractylodes macrocephala are identified; a result obtained by adopting the molecular identification method provided by the invention is accurate and good in specificity; the method has the characteristics of simple operation, high detection efficiency, accurate detection and good repeatability.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to a Lin'an atractylodes rhizome leaf chloroplast genome and an application thereof. BACKGROUND

[0002] The core value of the DNA barcoding technology is to construct a molecular identity authentication system across morphological differences between species. The foundational work of this technology system was completed by the Hebert team, which innovatively used a 680bp-long fragment of the mitochondrial cytochrome oxidase subunit 1 (COI) gene to identify two hundred species of Lepidoptera insects, and first proposed that a DNA fragment can be used to distinguish species, similar to the DNA barcoding of commodities. By obtaining the corresponding gene sequence of a sample and comparing it with a well-constructed reference sequence, not only can species-level identification be achieved, but further genetic differences at the subspecies, population and even individual levels can be analyzed, thereby providing strong technical support for biodiversity protection, biological safety monitoring and sustainable utilization of resources.

[0003] In the evolution process of molecular species identification technology, the DNA barcoding technology has undergone a leap from a single marker sequence to the whole genome level. Traditional barcoding systems mainly rely on short fragment sequences such as rbcL and matK. However, due to the short length of conventional barcodes, the amount of information is limited, which leads to poor identification ability for recently diverged species. In recent years, super barcoding technology based on organelle genomes (especially chloroplast genomes) has emerged. Chloroplast, as a unique energy converter in plant cells, has a unique evolutionary feature: 1) a circular double-stranded DNA structure (about 120-160kb) contains more than 100 coding genes, which expands the information dimension by thousands of times compared to traditional barcodes; 2) the maternal inheritance property ensures the stability and traceability of the genome; 3) in the evolution process of about 150 million years, the chloroplast genome forms a precise co-evolution network with the nuclear genome and mitochondrial genome, and its structural conservation and sequence specificity together construct a unique molecular identity card for species. Using chloroplast whole genome sequences for species identification can significantly improve the identification efficiency, especially for closely related species, and there have been many beneficial attempts in this regard. This technological innovation not only meets the needs of modern molecular biology for high-precision species identification, but also provides revolutionary tools for biological safety monitoring, germplasm resource protection and other fields, and has significant technological progress and industrial application prospects.

[0004] The application scenarios of DNA barcoding technology have broken through the traditional taxonomy category and deeply penetrated into the following fields: 1) medicinal resource development field, such as accurate identification of endangered species such as ginseng and dendrobium; 2) ecosystem assessment, such as species composition analysis of vegetation community; 3) biological safety monitoring, such as early warning of invasive species; 4) conservation biology research, such as cryptic species diversity exploration. At present, DNA barcoding has been widely used in the fields of species classification, resource protection, system evolution and biodiversity research.

[0005] "Lian'an atractylodes", also known as "Yu atractylodes", is a cultivated variety of Atractylodes. Before the Wei, Jin and Southern and Northern Dynasties, atractylodes and atractylodes were collectively referred to as "Shu", and were not distinguished until the Wei and Jin Dynasties. In the Song Dynasty, they were officially divided into two drugs. Atractylodes was originally a wild product, mainly distributed in Zhejiang and southern Anhui, including "Shuzhou atractylodes", "Yu atractylodes", "Zhejiang atractylodes", "Qi atractylodes" and "Shexian atractylodes", and the quality of atractylodes produced in Zhejiang is the best. It has multiple effects, including invigorating the spleen and tonifying qi, drying dampness and promoting water, stopping sweating and calming the fetus. It is often used to treat loss of appetite, abdominal distension and diarrhea caused by spleen deficiency, dizziness caused by phlegm and fluid retention, self-sweating, edema and fetal movement instability.

[0006] "Lian'an atractylodes" is a high-quality strain selected from Zhejiang's native medicinal material resources, with high content of effective ingredients. Its large-scale promotion can effectively solve the problems of germplasm degradation and quality fluctuation in the current atractylodes planting industry. Traditional medicinal plants are usually identified by destructive detection methods such as chemical composition analysis and microscopic identification, which have long detection periods, high costs and cannot cover the whole growth cycle monitoring.

[0007] Conducting fine assembly and phylogenetic analysis of the chloroplast genome of "Lian'an atractylodes" not only helps to elucidate the genetic evolution mechanism of atractylodes plants, but also provides key genetic information for developing scientific germplasm resource protection strategies, constructing core germplasm banks and targeted breeding. The molecular identification system developed based on the chloroplast genome sequence can realize the whole-process traceable management of "Lian'an atractylodes" seedlings. At present, the research on the chloroplast genome of "Lian'an atractylodes" is still blank. SUMMARY

[0008] In order to solve at least one of the above problems, the present application provides a kind of Lian'an atractylodes chloroplast genome and its application. The present application first proposes the chloroplast genome of Lian'an atractylodes, filling the blank of no chloroplast genome sequence of Lian'an atractylodes in the current genetic database.

[0009] The Lian'an atractylodes chloroplast genome proposed by the present application can be used to identify atractylodes varieties, providing technical support for atractylodes germplasm resource protection and species identification.

[0010] In order to achieve the above purpose, the present application adopts the following technical means:

[0011] The first aspect of the present application provides a Lin'an Atractylodes rhizome chloroplast genome, wherein the nucleotide sequence of the genome is shown as SEQ ID No. 1.

[0012] A method for obtaining a Lin'an Atractylodes rhizome chloroplast genome, comprising the following steps:

[0013] S1, using a modified CTAB method to extract total DNA of the Lin'an Atractylodes sample;

[0014] S2, performing double-end sequencing on the total DNA extracted in the step S1; wherein the sequencing depth is greater than or equal to 30x.

[0015] S3, splicing and assembling the chloroplast genome:

[0016] (1) using SPAdes 3.14.1 software to perform de novo assembly on the high-throughput sequencing data, and setting the k-mer parameter to 95;

[0017] (2) using the Atractylodes rhizome chloroplast genome sequence with the Gene Bank accession number OQ260036 as a reference sequence, performing blastn and Exonerate alignment respectively, selecting the scaffolds with gene matching, and sorting the splicing coverage;

[0018] (3) using PRICE and MITObim software to select the fragments belonging to the chloroplast genome from the assembled data, performing preliminary assembly, using bowtie2 to perform back-posting on the original sequencing reads, selecting the matched pairs of reads, using SPAdes to perform re-splicing, and obtaining the complete Lin'an Atractylodes sample chloroplast genome;

[0019] S4, annotating and correcting the chloroplast genome: using prodigal software to complete the annotation and correction of the chloroplast genome, thereby obtaining the complete Lin'an Atractylodes chloroplast genome, and using online software DOGMA to draw the chloroplast genome annotation diagram: the length of the Lin'an Atractylodes chloroplast genome is 153267 bp, the average sequencing depth is 1989.84X, the annotation reference is OQ260036, and 131 genes are encoded. Among them, there are 86 protein-coding genes, 8 rRNA genes, and 37 tRNAs; the atpF, ndhA, ndhB, petB, petD, rpl16, rpl2, rpoC1, rps16, trnA-UGC, trnG-UCC, trnI-GAU, trnK-UUU, trnL-UAA, and trnV-UAC genes each contain one intron, the clpP and ycf3 genes contain two introns, and the rps12 gene has a trans-splicing.

[0020] The second aspect of the present application provides an application of the chloroplast genome of the first aspect in identification of Atractylodes lancea germplasm.

[0021] In some embodiments of the present application, the identification of Atractylodes lancea germplasm comprises the following steps:

[0022] S1, total DNA of the Atractylodes lancea sample to be tested is extracted by using a modified CTAB method;

[0023] S2, double-end sequencing is performed on the total DNA extracted in step S1;

[0024] S3, splicing and assembly of the chloroplast genome;

[0025] S4, the nucleotide sequence of the assembled chloroplast genome of the Atractylodes lancea sample to be tested is compared with the nucleotide sequence of the chloroplast genome of Atractylodes lancea, SEQ ID No. 1, and when the similarity index reaches 99.9% or above, it is determined as Atractylodes lancea.

[0026] In some embodiments of the present application, the sequencing depth of step S2 is ≥30x.

[0027] In some embodiments of the present application, the method of assembly and annotation of the chloroplast genome in step S3 is specifically:

[0028] (1) the high-throughput sequencing data is de novo assembled by using SPAdes 3.14.1 software, and the k-mer parameter is set to 95;

[0029] (2) the chloroplast genome sequence of Atractylodes lancea with Gene Bank accession number OQ260036 is taken as a reference sequence, blastn and Exonerate are used for comparison respectively, the scaffold with gene matching is selected out, and the coverage of splicing is sorted;

[0030] (3) the fragments belonging to the chloroplast genome are selected out from the assembled data by using PRICE and MITObim software, and preliminary assembly is performed; the original sequencing reads are back-posted by using bowtie2, the paired reads matching are selected out, and SPAdes is used for re-splicing to obtain the complete chloroplast genome of the Atractylodes lancea sample to be tested;

[0031] (4) the annotation and correction of the chloroplast genome are completed by using prodigal software, so that the complete chloroplast genome of the Atractylodes lancea sample is obtained, and the online software DOGMA is used to draw the annotation diagram of the chloroplast genome.

[0032] The third aspect of the present application provides an application of the chloroplast genome of the first aspect in constructing a plant phylogenetic tree.

[0033] Advantages of the present application

[0034] Compared with the prior art, the present application has the following advantages:

[0035] The present application provides a Lin'an Atractylodes rhizome chloroplast genome, which is the first complete analysis and publication of the circular double-stranded structure of the genome, containing 131 functional genes with a total length of 153,267 bp. The technology has a milestone significance in ensuring the genetic purity of native medicinal materials, promoting the brand construction of Zhejiang-produced Atractylodes rhizome geographical indication products, etc. through the accurate identification of characteristic DNA sites; the research provides important technical support for the standardization and modernization transformation and upgrading of China's Atractylodes rhizome industry; by constructing the comparative genome framework of Atractylodes rhizome, the phylogenetic position of Lin'an Atractylodes rhizome in the Asteraceae plant is clarified, which provides core data support for the genetic background research and molecular breeding of Zhejiang-produced native medicinal materials.

[0036] The technology overcomes the technical bottlenecks of long detection period (mature period sampling is required), poor stability (environmental factors have a significant impact) caused by traditional identification relying on rootstock anatomical characteristics and chromatographic component analysis, eliminates the limitations of time and plant growth and development stage, realizes rapid and accurate identification of seedling sources, and the molecular identification method provided in the present application has the characteristics of simple operation, high detection efficiency, accurate detection and good repeatability.

[0037] The super DNA barcode in the present application is applied to analyze the phylogenetic relationship of Atractylodes rhizome species, and to identify the seedling of suspected Lin'an Atractylodes rhizome, which shows that the super DNA barcode applied for protection in the present application can be used for phylogenetic research of Atractylodes rhizome plants, and can accurately identify the seedling of Lin'an Atractylodes rhizome. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 The chloroplast genome annotation map of Lin'an Atractylodes rhizome is shown;

[0039] Figure 2 The phylogenetic tree of Asteraceae plants based on the chloroplast genome sequence in Example 1 is shown;

[0040] Figure 3 The sample YZ and Lin'an Atractylodes rhizome chloroplast genome nucleotide sequence alignment chart in Experimental Example 2 is shown;

[0041] Figure 4 The sample YZ and Lin'an Atractylodes rhizome and other Atractylodes rhizome phylogenetic tree in Example 2 is shown. DETAILED DESCRIPTION

[0042] The following examples are put forth so as to provide those of ordinary skill in the art with the best procedures known to the inventors for the practice of the application. The examples are intended to be illustrative only and in no way limit the scope of the application. Those of skill in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are considered to be within the scope of this application. The examples are presented herein for purposes of illustration only and are not intended to limit the scope of the application.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. All publications and other references mentioned herein are incorporated by reference for the aspects to which they pertain. In case of conflict between the present specification and a document incorporated by reference, the present specification will control. In carrying out the methods of the present application, it will be within the ability of those skilled in the art to choose among available techniques for the practice of the application. Unless otherwise specified, the techniques employed in the examples are conventional techniques of the art that are well within the capabilities of those skilled in the art; unless otherwise specified, reagents used in the examples are commercially available. Those of skill in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are considered to be within the scope of this application.

[0044] The technical solutions of the present application are further described in detail below in conjunction with the specific embodiments.

[0045] Example 1 Construction of the complete chloroplast genome of Atractylodes lancea and phylogenetic tree analysis

[0046] (1) Extraction of total DNA from Atractylodes lancea chloroplast genome

[0047] Extraction of DNA from Atractylodes lancea tender leaves using the improved CTAB method: 0.2 g of Atractylodes lancea tender leaves were placed in 2 mL microcentrifuge tubes, rapidly frozen in liquid nitrogen, and each tube was added with an appropriate amount of quartz sand and steel balls. The leaves were ground into fine powder in a tissue grinder at a shaking speed of 1500 rpm per second for 28-29 times. 500 mL of DNA extraction solution (see Table 1 for the formula of the DNA extraction solution) was added, and the mixture was placed in a 60°C oven for about 45 min, with shaking every 15 min. An equal volume of chloroform:isopropyl alcohol (24:1) mixture was added, and the mixture was shaken on a shaker for about 30 min to mix until a clear layer was formed. The plant sample was balanced, and centrifuged at room temperature at 12000 rpm for 8 min. About 350 mL (depending on the amount of sample) of supernatant was transferred to another numbered 1.5 mL centrifuge tube, and an equal volume of pre-cooled ice alcohol was added. The mixture was shaken on a shaker for 15-20 min to precipitate the DNA. The mixture was balanced and centrifuged at room temperature at 12000 rpm for 3 min. The DNA was washed with 70% alcohol for 2-3 times. The extracted DNA was blown dry on a clean bench, and 30-50 uL of ddH2O was added to dissolve the DNA.

[0048] Table 1 Formula of DNA extraction solution

[0049]

[0050] (2) Total DNA paired-end sequencing

[0051] After the DNA sample is qualified, it is randomly broken by using a Covaris ultrasonic disrupter, and then end repair, A tailing, sequencing adapter addition, fragment screening, PCR amplification, and magnetic bead purification are performed by using an Illumina MiSeq Reagent Nano Kit v2 kit to complete the entire library preparation work.

[0052] Library detection mainly includes two methods: (1) AATI detects the integrity and insert size of the library DNA fragments. (2) QPCR detects the effective concentration of the library. After the library is qualified, Illumina NovaSeq 6000 platform is used for paired-end (PE) sequencing, the sequencing read length is 150, and the sequencing depth is not less than 30x (no less than 10 Gb data).

[0053] (3) Sequencing data quality control

[0054] The raw file obtained by the Illumina high-throughput sequencer after base calling and Bcl2Fastq conversion can obtain the original sequencing sequence (sequenced reads) in FASTQ (abbreviation fq) format, which is called raw data. The FASTQ format data contains sequence information and corresponding sequencing quality information of the sequencing sequence (reads), and each read is composed of four lines. The fastp (version 0.20.0, https: / / github.com / OpenGene / fastp) software is used to filter the raw data (Raw Data), including cutting off the sequencing adapter and primer sequence in the reads, filtering out the reads with an average quality value less than 0.5, and filtering out the reads with a continuous undetected base number greater than 5. After a series of quality controls, the high-quality reads obtained are called Clean Data.

[0055] (4) Chloroplast genome assembly

[0056] The clean data was preliminarily spliced, and the software used was SPAdes 3.14.1 software, and the k-mer parameter was set to 95; the publicly disclosed Atractylodes lancea chloroplast genome sequence (Gene Bank accession number: OQ260032.1) was used as a reference sequence, and blastn and Exonerate were used for alignment, and the alignment threshold was set to evalue 1e-10 and protein similarity threshold 70%, respectively. The scaffold with gene matching was selected, and the coverage of splicing was sorted, and the fragments obviously not belonging to the target genome were removed. The fragments belonging to the chloroplast genome were selected from the assembled data using PRICE and MITObim software, and preliminary assembly was performed; the original sequencing reads were back-posted using bowtie2, the matched pairs of reads were selected, and SPAdes was used for re-splicing, so as to obtain the complete Lin'an Atractylodes lancea chloroplast genome, and the online software DOGMA (Dual Organellar Genome Annotator) was used to draw the chloroplast genome annotation diagram, as shown in Figure 1

[0057] (5) Chloroplast genome annotation

[0058] The CDS of the chloroplast was annotated using prodigal v2.6.3 (https: / / www.github.com / hyattpd / Prodigal), the rRNA was predicted using hmmer v3.1b2 (http: / / www.hmmer.org / ), and the tRNA was predicted using aragorn v1.2.38 (http: / / 130.235.244.92 / ARAGORN / ).

[0059] Results: The length of the Lin'an Atractylodes lancea chloroplast genome is 153267 bp, the average sequencing depth is 1989.84X, the annotation reference is OQ260036, and 131 genes are encoded. Among them, there are 86 protein coding genes, 8 rRNA genes, and 37 tRNAs. The atpF, ndhA, ndhB, petB, petD, rpl16, rpl2, rpoC1, rps16, trnA-UGC, trnG-UCC, trnI-GAU, trnK-UUU, trnL-UAA, and trnV-UAC genes each contain one intron, the clpP and ycf3 genes contain two introns, and the rps12 gene has a trans-splicing, as shown in Table 2 below.

[0060] Table 2 Annotation gene information of the Lin'an Atractylodes lancea chloroplast genome

[0061] ​

[0062]

[0063] (6) Phylogenetic analysis method

[0064] The assembled Lin'an Atractylodes leaf chloroplast genome was combined with the published chloroplast genome sequences of 22 Asteraceae species (as shown in Table 3) on GenBank, and sequence alignment and manual adjustment were performed using MAFFT (v7) and BioEdit software, respectively.

[0065] Table 3 Sequence of chloroplast genomes of 22 species for phylogenetic analysis

[0066]

[0067]

[0068] The chloroplast whole genome was used for phylogenetic tree analysis, the same starting point was set for the circular sequence, and the sequences between species were subjected to multiple sequence alignment using MAFFT software (v7.427, --auto mode). The aligned data were subjected to phylogenetic tree construction using RAxML v8.2.10 (https: / / cme.h-its.org / exelixis / software.html) software, GTRGAMMA model was selected, maximum likelihood method (M) was used, and 1000 times of repetition was set for the bootstrap value. The results are shown in Figure 2

[0069] The results show that Lin'an Atractylodes and other Atractylodes species are clustered into one branch, and the closest relative of Atractylodes lancea, with a bootstrap support rate of 98 at the differentiation node, and high reliability.

[0070] Example 2 Identification of suspected Lin'an Atractylodes samples

[0071] The sample YZ of suspected "Lin'an Atractylodes" seedlings collected in the wild was identified, and the specific steps are as follows:

[0072] (1) Total DNA extraction of the sample YZ to be tested: the improved CTAB method was used, which was the same as step (1) of Example 1;

[0073] (2) Genomic second-generation sequencing: I11umina Hiseq PE150 was used for double-end sequencing, which was the same as step (2) of Example 1;

[0074] (3) Quality control of sequencing data: the same as step (3) of Example 1;

[0075] (4) Chloroplast genome splicing and assembly: the same as step (4) of Example 1; ​

[0076] (5) The assembled chloroplast genome of the sample to be identified YZ was aligned with the nucleotide sequence of Atractylodes macrocephala-LA assembled in Example 1 (as shown in SEQ ID NO. 1). The results are shown in Table 2. Figure 3

[0077] The results show that the length of the chloroplast genome of sample YZ is 153165 bp, and the sequence identity with the sequence SEQ ID NO. 1 of Atractylodes macrocephala-LA is 99.93%. It can be judged that the sample A is Atractylodes macrocephala-LA.

[0078] The chloroplast genome of sample YZ was combined with the nucleotide sequence of the super DNA barcode and the chloroplast genome sequences of another 5 Atractylodes chinensis, and the multiple sequence alignment was performed by using MAFFT software (v7.427, --auto mode). The data of the aligned good data were used to construct a phylogenetic tree by using RAxML v8.2.10 (https: / / cme.h-its.org / exelixis / software.html) software, selecting GTRGAMMA model, and using maximum likelihood method (M). The results are shown in Table 3. Figure 4

[0079] The results show that the test sample YZ is clustered with Atractylodes macrocephala-LA, and the bootstrap support of the differentiation node is 100, which has high reliability. The phylogenetic analysis also proves that the test sample YZ is Atractylodes macrocephala-LA.

[0080] All the documents mentioned in the present application are cited as references in the present application, just as each document is cited as a reference. In addition, it should be understood that those skilled in the art can make various modifications or changes to the present application after reading the above teaching of the present application, and these equivalent forms also fall within the scope defined by the present application.​​

Claims

1. A chloroplast genome of Atractylodes macrocephala from Lin'an, characterized in that: The nucleotide sequence of the genome is shown in SEQ ID No.

1.

2. The application of the Lin'an Atractylodes macrocephala chloroplast genome as described in claim 1 in the identification of Atractylodes macrocephala germplasm.

3. The application according to claim 2, characterized in that: Includes the following steps: S1. Total DNA was extracted from the Atractylodes macrocephala samples using a modified CTAB method. S2. Perform paired-end sequencing on the total DNA extracted in step S1; S3, splicing and assembly of chloroplast genomes; S4. The chloroplast genome nucleotide sequence of the assembled Atractylodes macrocephala sample to be tested is compared with the chloroplast genome nucleotide sequence of Atractylodes macrocephala from Lin'an. When the similarity index reaches 99.9% or higher, it is identified as Atractylodes macrocephala from Lin'an.

4. The application according to claim 3, characterized in that, The sequencing depth in step S2 is ≥30×.

5. The application according to claim 3, characterized in that, The specific method for assembling and annotating the chloroplast genome in step S3 is as follows: (1) De novo assembly of high-throughput sequencing data was performed using SPAdes 3.14.1 software, with the k-mer parameter set to 95; (2) The genome sequence of Atractylodes macrocephala chloroplasts with Gene Bank accession number OQ260036 was used as a reference sequence and compared with blastn and Exonerate respectively. The scaffolds with matching genes were selected and the coverage of the splicing was sorted. (3) Use PRICE and MITObim software to select fragments belonging to the chloroplast genome from the assembled data and perform preliminary assembly; use bowtie2 to reassemble the original sequencing reads, pick out the matching pairs of reads, and use SPAdes to reassemble them to obtain the complete chloroplast genome of the Atractylodes macrocephala sample to be tested. (4) The chloroplast genome was annotated and proofread using the prodigal software, thus obtaining the complete chloroplast genome of the Atractylodes macrocephala sample. The chloroplast genome annotation map was drawn using the online software DOGMA.

6. The application of the Atractylodes macrocephala chloroplast genome as described in claim 1 in constructing a plant phylogenetic tree.

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