CN110511928B - Transcriptome SSR molecular marker system of moutan bark and application thereof - Google Patents

Abstract

The invention relates to a transcriptome SSR molecular marker system of moutan bark and application thereof, wherein 10 pairs of SSR molecular marker primers in moutan bark are screened, the primer system has high polymorphism and good universality, can reflect polymorphism in a plurality of peony samples, and can effectively identify the varieties and the quality of the moutan bark from different varieties.

Description

Transcriptome SSR molecular marker system of moutan bark and application thereof

Technical Field

The invention belongs to the field of biology, and relates to a transcriptome SSR molecular marker system of moutan bark and application thereof.

Background

The traditional Chinese medicine cortex moutan is dry root bark of Paeonia suffruticosa Andr of Ranunculaceae, and is mainly distributed in Chongqing, anhui, shaanxi, henan, shandong and other places in China. The moutan bark is a common bulk medicinal material, has wide clinical application, has the effects of clearing heat and cooling blood, and activating blood and dissolving stasis, and is also one of the raw materials of clinical large-variety preparations of traditional Chinese medicines, such as Liuwei Dihuang pills. Chongqing Zuijiang is the traditional genuine producing area of Chuan cortex moutan; the Anhui Paeonia ostii (P.ositi T.hong et J.X.zhang) is not in pharmacopoeia, but is one of the main sources of the moutan bark in the medicinal material market due to good medicinal material quality.

The existing research finds that nearly 1000 ornamental cultivated varieties of peony exist, and as the ornamental varieties and medicinal varieties have great difference in variety breeding and cultivation modes, the general medicinal varieties are single-petal single-color flowers, and the quality of tree peony bark is good; most ornamental varieties are flowers with multiple bright petals, and if the quality of the medicines for root bark is lower than that of medicinal varieties. However, as the demand of the market for moutan bark increases, the moutan bark from ornamental peony often fills the medicinal material market, which causes obvious quality difference of the moutan bark from different peony varieties, and further affects the clinical efficacy. Different varieties of moutan bark are very similar in appearance and shape, and are difficult to distinguish through external shape characteristics. Therefore, a reliable identification method is urgently needed to be established to distinguish the tree peony bark varieties from different sources and identify the tree peony bark from medicinal peony and ornamental peony so as to ensure the stability of the quality of tree peony bark medicinal materials.

The quality of the tree peony bark is mainly embodied in the content of effective components, the accumulation of the effective components depends on functional genes in a biosynthesis pathway in a plant body, and important references can be provided for the formation and evaluation of the quality of medicinal materials by analyzing the characteristic attributes of the functional genes. The characteristic mark of the functional gene can be used as the index for evaluating the quality of the medicinal materials and cultivating excellent varieties.

Molecular markers have become one of the most important tools to reveal genetic diversity and variety identification of species. The currently commonly used molecular marker types include Restriction Fragment Length Polymorphism (RFLP), random Amplified Polymorphic DNA (RAPD), restriction amplified polymorphic sequence (CAPS), single Nucleotide Polymorphism (SNP), simple repeat sequence (SSR), and the like. Among them, SSR (simple sequence repeat), also known as microsatellite sequence, is a DNA sequence consisting of 1 to 6 nucleotides repeated many times as a repeating motif, and their length is about 100 to 200 base pairs, and is widely present in eukaryotic genomes. The SSR sequence has the characteristics of wide distribution, co-dominant inheritance, multiple polymorphic sites, rich information content, good inter-species transferability, easy detection and good repeatability, and is widely applied to the research fields of variety identification, germplasm resource storage and utilization, genetic diversity analysis, molecular marker-assisted breeding and the like. Based on the SSR molecular marker in the transcriptome, the problems of long period, high cost and small data volume of EST-SSR (genomic-SSR) can be avoided, and the SSR molecular marker has the advantages of genomic-SSR; at the same time, this technique also makes full use of the results of transcriptome sequencing. The research has been carried out, and the molecular marker polymorphism and amplification effect developed based on the transcriptome SSR are found to be good, which indicates that the transcriptome SSR is suitable for developing the molecular marker. Peony as an important ornamental plant has the characteristics of 'multi-place, multi-variety and multi-element' origin and has extremely rich genetic diversity. In crop breeding, the deep understanding of molecular diversity is the basis for researching genetic diversity, variety identification and hybrid parent selection of crops, however, peony has few researches in this respect, the researches mainly focus on the identification, origin and the like of peony varieties, the researches focus on the ornamental value of peony, and the medicinal value of the plant is ignored. The peony varieties in partial regions are found to have rich genetic diversity through molecular marker research. Analyzing the peony varieties in Pengzhou Sichuan by adopting RAPD technology, and finding that most varieties with the same source represent relatively close relatives; in the research of constructing the original peony core germplasm resource library, a phenotype database and a molecular database of the original peony variety are established according to basic data, such as variety sources, classification systems and the like, and morphological and molecular marker characteristic data. And ISSR and AFLP marking are carried out by constructing the primary core germplasm resource of the original peony variety, so that the constructed core germplasm has rich genetic diversity. CDDP labeling has also been tried in genetic diversity analysis, variety specificity analysis and consistency check of peony, to screen core primers for differentiating peony germplasm resources and to discuss construction methods of peony molecular identity cards. The method is characterized in that the Spanish tree and the like take Paeonia ostii as experimental materials, SSR markers are developed by a magnetic bead enrichment method, the cross amplification capability of the Paeonia ostii in peony group plants is discussed, and the Paeonia purpurea is found to be one of main breeder species of the peony species in northwest and has a close relationship with the Paeonia suffruticosa in Sichuan; the paeonia ostii participates in the origin of the original peony variety; the line individuals and the original peony variety group individuals also have a certain genetic relationship.

Disclosure of Invention

In view of the above, the invention aims to provide a transcriptome SSR molecular marker system of moutan bark and an application thereof, which can effectively identify and distinguish varieties and qualities of moutan bark medicinal materials and decoction pieces from different sources through genetic polymorphism of functional genes.

In order to achieve the purpose, the invention provides the following technical scheme:

1. a transcriptome SSR molecular marker system of moutan bark comprises the following 10 primer pairs:

the nucleotide sequence of the primer pair P1 is shown as SEQ ID NO.1 and SEQ ID NO. 2;

the nucleotide sequence of the primer pair P2 is shown as SEQ ID NO.3 and SEQ ID NO. 4;

the nucleotide sequence of the primer pair P3 is shown as SEQ ID NO.5 and SEQ ID NO. 6;

the nucleotide sequence of the primer pair P4 is shown as SEQ ID NO.7 and SEQ ID NO. 8;

the nucleotide sequence of the primer pair P5 is shown as SEQ ID NO.9 and SEQ ID NO. 10;

the nucleotide sequence of the primer pair P6 is shown as SEQ ID NO.11 and SEQ ID NO. 12;

the nucleotide sequence of the primer pair P7 is shown as SEQ ID NO.13 and SEQ ID NO. 14;

the nucleotide sequence of the primer pair P8 is shown as SEQ ID NO.15 and SEQ ID NO. 16;

the nucleotide sequence of the primer pair P9 is shown as SEQ ID NO.17 and SEQ ID NO. 18;

the nucleotide sequence of the primer pair P10 is shown as SEQ ID NO.19 and SEQ ID NO. 20.

2. The transcriptome SSR molecular marker system of moutan bark is applied to variety identification and quality evaluation of moutan bark.

Preferably, the moutan bark includes but is not limited to single-petal red, double-petal red, paeonia ostii or luoyang ornamental peony varieties, and the luoyang ornamental peony varieties include but are not limited to Zhao powder, puju purple, clove purple, dazuo purple, pinna minor, haihuang, yaohuang, luoyang brocade, black peony and white peony.

3. The construction method of the transcriptome SSR molecular marker system of moutan bark comprises the following specific steps:

(1) Extracting fresh moutan bark sample RNA, constructing a moutan bark transcriptome library, performing sequencing analysis to obtain a moutan bark transcriptome sequence, and splicing transcripts by using Trinity software to obtain a Unigene;

(2) Unigene functional annotation the Unigene sequence is compared with databases of NR, swiss-Prot, GO, COG, KOG, eggNOG4.5 and KEGG by using BLAST software, a KEGGOrthology result of the Unigene in KEGG is obtained by using KOBAS2.0, the annotation information of the Unigene is obtained by comparing HMMER software with a Pfam database after the amino acid sequence of the Unigene is predicted, functional genes or fragments participating in biosynthesis pathways of effective components in moutan bark such as paeonol, paeoniflorin, gallic acid and the like are obtained, and a functional gene database is independently built;

(3) Screening and analyzing SSR loci of Unigene with the nucleotide length larger than 1000bp in a functional gene database by using MISA software; and (3) setting conditions in the obtained total SSR for screening, wherein the screening standard is as follows: the minimum number of times of repetition of the dinucleotides, the trinucleotides, the tetranucleotides, the pentanucleotides and the hexanucleotides is respectively 9, 6, 5, 4 and 4;

(4) Designing SSR primers by using Primer 5 software, wherein the Primer design parameters are that the length of the primers is 18-25 bp; the length of the PCR amplification product is 100-400 bp; the GC content is 40 to 60 percent;

(5) And (5) identifying the amplifiability and the universality of the primer designed in the step (4) in different moutan bark samples to obtain the SSR molecular marker system with the amplifiability and the universality in different moutan barks.

The invention has the beneficial effects that:

according to the invention, gene fragments with sequences of more than 1000bp are firstly screened according to the analysis of a cortex moutan transcriptome, SSR molecular marker primers related to effective component biosynthesis functional genes are searched based on the fragments, and 10 pairs of primer systems are obtained, the primer systems have high polymorphism and good universality, polymorphism can be embodied in a plurality of peony samples, the variety sources of cortex moutan of different variety sources can be effectively identified, and meanwhile, the primer systems can be used as an index for evaluating the quality of cortex moutan.

Drawings

In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:

FIG. 1 is a gel electrophoresis pattern of a primer pair P1 after amplification of a portion of a moutan bark sample, wherein N is a negative control; 1-Chongqing Taiping red single-petal tree peony bark sample; 2-Chongqing Paoyangping red petiole cortex moutan sample; 3-Chongqing Paeonia ostii white peony bark sample; 4-Anhui Paeonia ostii cortex moutan sample; 5-a sample of ornamental tree peony bark from luoyang; M-DNAmarker;

FIG. 2 is a gel electrophoresis pattern of the primer pair P2 after amplification of a portion of a moutan bark sample, wherein N is a negative control; 1-Chongqing Taiping red single-petal tree peony bark sample; 2-Chongqing Paoyangping red petiole cortex moutan sample; 3-Chongqing Paeonia ostii white peony bark sample; 4-Anhui Paeonia ostii cortex moutan sample; 5-a luoyang ornamental cortex moutan sample; M-DNAmarker;

FIG. 3 is a diagram showing a gel electrophoresis pattern of a portion of a moutan bark sample amplified by the primer pair P3, wherein N is a negative control; 1-Chongqing Taiping red single-petal tree peony bark sample; 2-Chongqing Paijing red petiolus moutan bark sample; 3-Chongqing Paeonia ostii white peony bark sample; 4-Anhui Paeonia ostii cortex moutan sample; 5-a luoyang ornamental cortex moutan sample; M-DNAmarker;

FIG. 4 is a gel electrophoresis pattern of the primer pair P4 after amplification of a portion of a moutan bark sample, wherein N is a negative control; 1-Chongqing Taiping red single-petal tree peony bark sample; 2-Chongqing Paijing red petiolus moutan bark sample; 3-Chongqing Paeonia ostii white peony bark sample; 4-Anhui Paeonia ostii cortex moutan sample; 5-a luoyang ornamental cortex moutan sample; M-DNAmarker;

FIG. 5 is a gel electrophoresis pattern of a primer pair P5 after amplification of a portion of a moutan bark sample, wherein 1-Chongqing Tejiangping red unipetal moutan bark sample; 2-Chongqing Paoyangping red petiole cortex moutan sample; 3-Chongqing Paeonia ostii white peony bark sample; 4-Anhui Paeonia ostii cortex moutan sample; 5-a luoyang ornamental cortex moutan sample; M-DNA marker;

FIG. 6 is a gel electrophoresis pattern of the primer pair P6 after amplification of a portion of a moutan bark sample, wherein N is a negative control; 1-Chongqing Taiping red single-petal tree peony bark sample; 2-Chongqing Paoyangping red petiole cortex moutan sample; 3-Chongqing Paeonia ostii white peony bark sample; 4-Anhui Paeonia ostii cortex moutan sample; 5-a luoyang ornamental cortex moutan sample; M-DNA marker;

FIG. 7 is a gel electrophoresis pattern of the primer pair P7 after amplification of a portion of a moutan bark sample, wherein N is a negative control; 1-Chongqing Taiping red single-petal tree peony bark sample; 2-Chongqing Paijing red petiolus moutan bark sample; 3-Chongqing Paeonia ostii white peony bark sample; 4-Anhui Paeonia ostii cortex moutan sample; 5-a luoyang ornamental cortex moutan sample; M-DNA marker;

FIG. 8 is a gel electrophoresis pattern of the primer pair P8 after amplification of a portion of a moutan bark sample, wherein N is a negative control; 1-Chongqing Taiping red single-petal tree peony bark sample; 2-Chongqing Paoyangping red petiole cortex moutan sample; 3-Chongqing Paeonia ostii white peony bark sample; 4-Anhui Paeonia ostii cortex moutan sample; 5-a sample of ornamental tree peony bark from luoyang; M-DNA marker;

FIG. 9 is a gel electrophoresis pattern of the primer pair P9 after amplification of a portion of a moutan bark sample, wherein N is a negative control; 1-Chongqing Taiping red single-petal tree peony bark sample; 2-Chongqing Paoyangping red petiole cortex moutan sample; 3-Chongqing Paeonia ostii white peony bark sample; 4-Anhui Paeonia ostii cortex moutan sample; 5-a luoyang ornamental cortex moutan sample; M-DNA marker;

FIG. 10 is a gel electrophoresis pattern of the primer pair P10 after amplification of a portion of a moutan bark sample, wherein N is a negative control; 1-Chongqing Taiping red single-petal tree peony bark sample; 2-Chongqing Paoyangping red petiole cortex moutan sample; 3-Chongqing Paeonia ostii white peony bark sample; 4-Anhui Paeonia ostii cortex moutan sample; 5-a luoyang ornamental cortex moutan sample; M-DNA marker;

FIG. 11 is a clustering analysis of moutan bark samples from different sources based on SSR polymorphism results.

Detailed Description

Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

Unless otherwise specified, the methods in the examples of the present invention are conventional in the art, and the experimental materials, reagents and equipment in the following examples are commercially available.

Example 1 development of Paeonia suffruticosa bark transcriptome SSR polymorphic primer pairs

1 Total RNA extraction and purification

Digging fresh peony root, cleaning soil, quickly freezing in liquid nitrogen tank, and storing in refrigerator at-80 deg.C.

Using RNA extraction kit (Invitrogen) ^TM ，RiboMinus ^TM Plant Kit for RNA-Seq) to extract total RNA of a cortex moutan sample, and detecting the purity, concentration, integrity and the like of the RNA sample by using methods of Nanodrop, qubit2.0 and agent 2100 respectively so as to ensure that qualified samples are used for transcriptome sequencing.

2 library construction

After the sample is detected to be qualified, library construction is carried out according to

Ultra ^TM RNA Library Prep Kit for

The reagents and protocols provided were tested:

(1) Enriching eukaryotic mRNA by magnetic beads with Oligo (dT);

(2) Adding Fragmentation Buffer to randomly break mRNA;

(3) Using mRNA (5. Mu.L) as a template, mixing with hexabasic random primers (1. Mu.L), NEBNext First Strand and Synthesis Reaction Buffer (4. Mu.L), NEBNext First Strand and Synthesis Enzyme Mix (2. Mu.L) and the like, synthesizing a First cDNA Strand by gradient temperature rise, adding reagents such as NEBNext Second Strand and Synthesis Reaction Buffer (10X) (8. Mu.L), NEBNext Second Strand and Synthesis Enzyme Mix (4. Mu.L), water and the like, and synthesizing a Second cDNA Strand by incubation at 16 ℃ for 1 hour, and purifying magnetic bead cDNA using AMPure XP;

(4) Carrying out end repair on the purified double-stranded cDNA, adding A tail and connecting a sequencing joint, and then carrying out fragment size selection by using AMPure XP magnetic beads;

(5) And finally, obtaining a cDNA library through PCR enrichment.

3 quality control of library

After the library is constructed, the concentration and the Insert Size (Insert Size) of the library are detected by using the Qubit2.0 and Agilent 2100 respectively, and the effective concentration of the library is accurately quantified by using a Q-PCR method so as to ensure the quality of the library.

4 sequencing on machine

After the library was qualified, high throughput sequencing was performed using Illumina Hiseq.

And (4) performing Data filtering on Raw Data, removing the linker sequence and low-quality Reads in the Raw Data to obtain high-quality Clean Data. Clean Data was sequence assembled to obtain the Unigene library for this species.

5Unigene notes

The Unigene sequence was aligned to the NR, swiss-Prot, GO, COG, KOG, eggNOG4.5, KEGG databases using BLAST software and the KEGG orthopology results for Unigene in KEGG were obtained using KOBAS 2.0. Selecting BLAST parameters E-value not greater than 1E-5 and HMMER parameters E-value not greater than 1E-10 resulted in 46,398 Unigenes with annotated information.

The statistics of the gene annotation are shown in table 1.

Table 1.Unigene annotation statistics

Note: anotated databases: representing each function database; unknown _ Number: represents the Unigene number annotated to the database; 300 is less than or equal to length <1000: indicates the number of Unigenes annotated to the database with Unigene lengths of 300 or more and less than 1000 bases; length is more than or equal to 1000: indicating the number of unigenes annotated to the database that are greater than 1000 bases in length.

6 SSR molecules

The screened Unigene with more than 1kb is subjected to SSR analysis by using MISA software, and the result is shown in Table 2.

TABLE 2 SSR repeat motif types and distributions

From 6235 SSR loci identified from moutan bark transcriptome data, primer screening is firstly carried out, and the screening standard is as follows: the minimum number of times of repetition of the dinucleotides, the trinucleotides, the tetranucleotides, the pentanucleotides and the hexanucleotides is respectively 9, 6, 5, 4 and 4; the length of the primer is 18-25 bp; the length of the PCR amplification product is 150-300 bp; the GC content is 40 to 60 percent. The screened primer pairs are then randomly selected 132 for amplifiability and polymorphism and versatility validation.

The specific method comprises the following steps:

DNA extraction

Digging fresh root of Paeonia suffruticosa Andr, cleaning soil, removing core, and drying at 50 deg.C;

extracting moutan bark DNA by an improved CTAB method, wherein the operation steps are as follows:

(1) Accurately weighing 100mg of cortex moutan, placing in a mortar, and adding a small amount of PVP for co-grinding;

(2) Grinding the herbs into powder, transferring into 2.0mL centrifuge tube, adding 1.5mL TNE buffer (0.1 mol. L) pre-cooled at 4 deg.C ^-1 Tris，0.15mol·L ^-1 Sodium chloride, 0.02 mol. L ^-1 EDTA), leaching for 2 times at 4 ℃ for 30 minutes each time, centrifuging at 12000rpm for 10 minutes, and discarding the supernatant;

(3) 900 μ L of 2 × CTAB extraction buffer (0.1 mol. L) preheated at 65 deg.C was added to the tube ^-1 Tris-HCl，0.02mol·L ^-1 EDTA，1.4mol·L ^-1 Sodium chloride, 2% (W/V) CTAB and 1% (V/V) PVP), then adding 14 mu L beta-mercaptoethanol, placing in a water bath at 65 ℃ for heat preservation for 90 minutes, and turning over the sample for 2-3 times during the water bath;

(4) After the water bath is finished, when the sample is cooled to below 45 ℃, precisely sucking 900 mu L of chloroform-isoamylol (volume ratio 24;

(5) After standing and layering, sucking 700 mu L of supernatant into another clean 2.0mL centrifuge tube, adding equal volume of chloroform-isoamylol (volume ratio 24;

(6) Sucking 500 μ L of supernatant into another clean 1.5mL centrifuge tube, adding isopropanol (pre-cooling at-20 deg.C) in an amount of 2/3 volume of the supernatant, and standing at-20 deg.C for 30 min. Centrifuge at 12000rpm for 10 minutes. Discarding the supernatant;

(7) The centrifuge tube was centrifuged at 12000rpm for 5 minutes with 500. Mu.L of 70% ethanol, and the supernatant was discarded. Repeating the operation once;

(8) The centrifuge tube was filled with 500. Mu.L of absolute ethanol, centrifuged at 12000rpm for 5 minutes, and the supernatant was discarded. Repeating the operation once;

(9) Absorbing residual ethanol in the drying tube by using absorbent paper, horizontally placing the tube opening in a drying oven at 37 ℃ for 15-30 minutes until the ethanol is completely volatilized;

(10) Add 50. Mu.L of ddH2O to the tube and dissolve the DNA sufficiently. Storing in refrigerator at-20 deg.C.

24 parts of moutan bark (containing single-petal red and double-petal red) of Taiping river; 8 parts of Paeonia ostii white produced by Yaojiang river; 8 parts of paeonia ostii produced by Anhui; 10 parts of various ornamental varieties produced by Luoyang (Zhao powder, pujuan purple, clove purple, dazuo purple, nuanziyan, haihuang, yaohuang, luoyang brocade, black peony and white peony); the extraction of the genomic DNA of the sample was carried out according to the above-mentioned DNA extraction method.

DNA quality detection

DNA quality was checked by agarose gel electrophoresis using a DYCP-31DN model electrophoresis apparatus. mu.L of DNA sample was added to 1. Mu.L of 6 × loading buffer and gently mixed. After mixing uniformly, the mixed solution and 3 microliter DNA Marker are spotted into the sampling hole by a pipette gun; connecting electrophoresis conductor between power supply and electrophoresis apparatus, setting electrophoresis parameters, and setting voltage of 120V cm ^-1 Electrophoresis time is 40 minutes; and after the electrophoresis is finished, detecting the agarose gel by using a ZF-2 type ultraviolet analyzer, and marking and storing an electrophoresis pattern.

PCR amplification

In order to achieve good amplification effect, PCR enhancers (2% (W/V) BSA, 1% (W/V) PVP) were used in the PCR system. The PCR amplification system was prepared as shown in Table 3.

TABLE 3 PCR amplification System

The amplification procedure for the prepared PCR reaction solution was as follows: pre-denaturation at 94 ℃ for 4min; denaturation at 94 ℃ for 30s; annealing at 53 ℃ for 30s; extension at 72 ℃ for 30s; repeating the denaturation step for 30-35 cycles, extending at 72 deg.C for 5min, and taking out when the temperature drops to 25 deg.C for use. And detecting the amplification product by 2% agarose gel electrophoresis, wherein if a band of 100-400bp appears, the SSR primer has amplifiable property, and 83 pairs of amplifiable primers are screened out in total.

And then, the 83 pairs of amplifiable SSR primer pairs are expanded and applied to 4 different varieties of moutan bark samples, and a PCR amplification system and a PCR amplification program are shown as above. And (3) carrying out detection on the PCR amplification product by using non-denatured polyacrylamide gel electrophoresis with the mass concentration of 8% in combination with silver staining to screen out SSR primers with better polymorphism.

The specific operation is as follows:

(1) Cleaning a glass plate, a sample grid, a measuring cylinder for glue filling and a beaker, and air-drying the glass plate;

(2) Placing the flat glass plate and the notched glass plate on a support of a desktop, dripping a small amount of 100% ethanol on the glass plate, uniformly scrubbing the glass plate with lens paper to prepare a rubber surface, and air-drying;

(3) Evenly coating proper vaseline on three edges of the flat glass plate and the notch glass plate, overlapping the flat glass plate and the notch glass plate, erecting the two glass plates on a table top by hands, and enabling the bottom surfaces of the two glass plates to be level to form a glue chamber;

(4) Placing the gel chamber into an electrophoresis apparatus main body, enabling one surface of the notched glass plate to face inwards, inserting the wedge-shaped inserting plate, placing the electrophoresis apparatus main body into an in-situ gel maker, rotating a handle of the gel maker to corresponding scales according to the thickness of gel required by an experiment, and enabling the wedge-shaped inserting plate to tightly press the gel chamber;

preparing non-denatured polyacrylamide gel:

(5) 8% polyacrylamide gel mixture (50 mL): 26.4mL deionized water, 13.3mL30% acrylamide, 10mL5 XTBE buffer, and rapidly adding 350. Mu.L 10% ammonium persulfate (catalyst), 33. Mu.L TEMED (accelerator) before pouring.

(6) And (5) preparing and filling glue. According to the size and thickness of the selected glue, a proper amount of 8% polyacrylamide gel mixed liquor is measured and poured into a clean beaker, the beaker is shaken up and filled with the glue, the sample grid with the thickness consistent with the glue is slowly inserted, and the glue is solidified for more than 30 minutes.

Performing non-denaturing polyacrylamide gel electrophoresis by using a DYCZ-24DN type electrophoresis apparatus:

(1) After gel polymerization, taking out the electrophoresis apparatus main body from the in-situ gel maker, and putting the electrophoresis apparatus main body into a lower groove of the electrophoresis apparatus;

(2) Pouring 140mL of 0.5 xTBE electrophoresis buffer solution into an upper groove formed by a glue chamber and an electrophoresis apparatus body, so that the buffer solution is over a notch glass plate;

(3) Pouring 200mL of 0.5 xTBE electrophoresis buffer solution into a lower groove of an electrophoresis apparatus;

(4) Slowly pulling out the sample grid vertically;

(5) Taking 3.5 mu L of PCR amplification sample, adding 0.7 mu L of 6 XDNA/RNAloading buffer, and mixing uniformly; after mixing evenly, a liquid transfer gun is used for dropping the mixed liquid and 3 mu L of 50bp Ladder DNAmarker into a sample loading hole;

(6) And (5) switching on an electrophoresis lead between the power supply of the electrophoresis apparatus and the electrophoresis apparatus, and setting electrophoresis parameters. The voltage is 200V cm ^-1 Electrophoresis time 45 minutes.

Silver staining for color development

(1) After electrophoresis, slowly taking down the gel, and rinsing the gel with deionized water for 2 times, 5 minutes each time;

(2) The gel was placed in silver stain (100 mL water, 1mL 20% silver nitrate), covered with a light shield to keep the gel dark, and stained on a low speed shaker for 13 minutes with gentle shaking.

(3) Slowly taking down the gel, and rinsing with deionized water for 2 times, 5 minutes each time;

(4) The gel was transferred to a developing solution (100mL 3% sodium hydroxide, 1mL formaldehyde, pre-cooled at 4 ℃), placed on a low speed shaker and shaken gently until DNA bands appeared,

(5) And (3) rapidly rinsing the gel with deionized water for 1 time, drying at room temperature, and then performing gel imaging and DNA fragment band number statistics.

Expanding the screened polymorphic primers into all the moutan bark DNAs for detection, and screening 10 pairs of polymorphic SSR primer pairs (shown in figures 1-10) based on the experimental results: the sequence information is shown in table 4.

TABLE 4 sequence information

And (3) polymorphism results of SSR primer pairs of tree peony bark from different sources in a system:

according to the DNAmarker and the size of a target fragment in the primer information, the artificially interpreted 10 pairs of polymorphic SSR primers are respectively named as A/B/C/D when 50 cortex moutan samples C are small to large, homozygotes are named as AA/BB/CC/DD, and heterozygotes are named as AB/BD/CD/BD.

TABLE 5 cortex moutan sample Condition

And (3) analyzing the amplification conditions of 10 pairs of polymorphic SSR primers in 50 parts of moutan bark sample DNA of different production places by utilizing POPGENE 32 software. The allele number (Na) varied from 2 to 4, with an average of 2.8000, with the highest primer P10 and the lowest primers P2, P4 and P9. The average effective allelic factor (Ne) per SSR locus was 2.0045. The observed heterozygosity (Ho) was calculated according to the method of Nei's 1973 to be in the range of 0.0816 to 0.6800, on average 0.3947. The desired heterozygosity (He) is in the range of 0.3232 to 0.6455, on average 0.4863.Nei's expected heterozygosity (H) ranges from 0.3200 to 0.6390, with an average of 0.4814. The Polymorphic Information Content (PIC) was from 0.2688 to 0.5711, with an average value of 0.4069. The primers P8 and P10 are highly polymorphic sites (PIC > 0.5), and the other 8 are moderately polymorphic sites (0.25 < PIC < 0.5). The Shannon information index (I) ranges from 0.5004 to 1.0944, with an average of 0.7741. The results show that the genetic diversity of the peony resources is relatively rich. (Table 6)

TABLE 6 genetic diversity analysis of SSR primers

Dividing 50 moutan bark samples into 5 groups according to geographical sources and varieties for analyzing the genetic diversity among groups. The method comprises the steps of taking 'Taiping red' single-petal moutan bark samples (serial numbers S1-S12) collected from Chongqing Teojiang as a first group, taking 'Paodan' samples (serial numbers S13-S24) as a second group, taking 'Paodan' samples (serial numbers S25-S32) as a third group, taking 'Paodan' samples (serial numbers S33-S40) collected from Anhui as a fourth group, and taking moutan bark samples (serial numbers S41-S50) collected from Henan Luoyang as a fifth group.

The POPGENE 32 analysis result shows that the observed heterozygosity (Ho) range is 0.2750-0.4917. The heterozygosity (He) is desirably in the range of 0.2692 to 0.4263.Nei's expected heterozygosity (H) ranges from 0.2580 to 0.3993, H is a common index reflecting population genetic diversity, and 5 peony populations are sorted according to genetic diversity: fourth group > fifth group > third group > first group > second group. The allelic factor (Na) ranges from 1.700 to 2.400, with the fourth and fifth groups being more abundant and the second group being the least abundant. The Shannon information index (I) ranges from 0.3639 to 0.6587, the highest index is the fourth group, and the lowest index is the second group. Combining the above results, it can be seen that the genetic diversity of "paeonia ostii" in anhui is the highest among 5 peony populations (H =0.3993, i = 0.6587), and the genetic diversity of "taiping red" chongqing zuojiang heavy peony is the lowest (H =0.2580, i = 0.3639).

The observed heterozygosity (Ho) of the first and second groups is higher than the expected heterozygosity (He), and the inbreeding coefficients (Fis) are negative (Fis = -0.8656 of the first group and Fis = -0.9704 of the second group), indicating that heterozygosity is excessive and homozygosity is deficient in the group. The observed heterozygosity (Ho) of the third, fourth and fifth groups was lower than the desired heterozygosity (He), and the coefficients of inbreeding were all positive (Fis = 0.0356-0.2129), indicating a loss of heterozygotes and an excess of homozygotes within the population. The mean value of the inbreeding coefficients of 5 groups of peonies was negative (Fis = -0.2794), also indicating a population heterozygote surplus. (Table 7)

TABLE 7 genetic diversity-related parameters of moutan bark from different sources

And constructing a dendrogram by using a UPGMA method on PowerMarker V3.25 software according to the corresponding Nei's genetic distance among 50 moutan bark samples. As shown in FIG. 11, the 50 samples were divided into three distinct branches Cluster1, cluster2 and Cluster3 as a whole. Wherein Cluster1 comprises two branches A and B, and 24 samples are obtained in total and are from Chongqing Teojiang. A is composed of all 'Taiping red' double-petal peonies (numbered S13 to S24), and B is composed of all single-petal peonies (numbered S1 to S12). The clustering condition of Cluster1 shows that peony varieties with the same producing area and the same flower color have closer relationship. There are 16 moutan bark samples in Cluster2, including 8 "Paeonia ostii" samples from Chongqing Yaojiang (Nos. S25 to S32) and 8 "Paeonia ostii" samples from Anhui (Nos. S33 to S40)). Moutan bark samples (numbers S41-S50) produced by 10 ornamental peonies collected from Henan Luoyang are gathered in Cluster3. Chongqing underlay river is "red and flat". Therefore, the cortex moutan sample polymorphism analysis based on the SSR molecular marker system can effectively distinguish and identify the cortex moutan samples from different sources through cluster analysis.

Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.

Sequence listing

<110> university of southwest

<120> transcriptome SSR molecular marker system of moutan bark and application thereof

<160> 20

<170> SIPOSequenceListing 1.0

<210> 1

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

ggtcctatct cttcacgaaa atc 23

<210> 2

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

catgaggtta acgcaaagga 20

<210> 3

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

cgtccgacgt acaactttga 20

<210> 4

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gtgctcacct cttcctcgtc 20

<210> 5

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atgtagccga ccccatgtag 20

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

tttgcacaca aaacatgcac 20

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

tcctctaaat tatccgcccc 20

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

cggtgttgca gttgtagtcg 20

<210> 9

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

agggagggat ggacaaaact 20

<210> 10

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tcactagaag gagtgggagc at 22

<210> 11

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ggggagatca gaaacacaca ac 22

<210> 12

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

tcaaagaaac cccagttctc ac 22

<210> 13

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

gagaaccatc attccattca ag 22

<210> 14

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

ctcaatctct tcttcctttg cc 22

<210> 15

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

ggcaaacaat ttggcgtagt 20

<210> 16

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

cgcacccttc cacaaataat 20

<210> 17

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

tgaagaaggt ggtgaggagg 20

<210> 18

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

tggacaatga gagcaaaacg 20

<210> 19

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

tgtagctgaa cctgtcgtgc 20

<210> 20

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

ctccccatca gtaactcgga 20

Claims (3)

1. A transcriptome SSR molecular marker primer pair combination of moutan bark is characterized by comprising the following 10 primer pairs:

the nucleotide sequence of the primer pair P1 is shown as SEQ ID NO.1 and SEQ ID NO. 2;

the nucleotide sequence of the primer pair P2 is shown as SEQ ID NO.3 and SEQ ID NO. 4;

the nucleotide sequence of the primer pair P3 is shown as SEQ ID NO.5 and SEQ ID NO. 6;

the nucleotide sequence of the primer pair P4 is shown as SEQ ID NO.7 and SEQ ID NO. 8;

the nucleotide sequence of the primer pair P5 is shown as SEQ ID NO.9 and SEQ ID NO. 10;

the nucleotide sequence of the primer pair P6 is shown as SEQ ID NO.11 and SEQ ID NO. 12;

the nucleotide sequence of the primer pair P7 is shown as SEQ ID NO.13 and SEQ ID NO. 14;

the nucleotide sequence of the primer pair P8 is shown as SEQ ID NO.15 and SEQ ID NO. 16;

the nucleotide sequence of the primer pair P9 is shown as SEQ ID NO.17 and SEQ ID NO. 18;

the nucleotide sequence of the primer pair P10 is shown as SEQ ID NO.19 and SEQ ID NO. 20;

the cortex moutan is selected from single-petal red, multiple-petal red, paeonia ostii, zhao powder, radix Puerariae Lobatae, flos Caryophylli purple, DAZONGZI, LIANZI, HAIHUAN, YAOHUANG, LUOYANGJIN, black peony, and white peony.

2. The application of the transcriptome SSR molecular marker primer pair combination of moutan bark in variety identification and quality evaluation of moutan bark in claim 1; the variety of the cortex moutan is single-petal red, double-petal red, paeonia ostii, zhao powder, pueraria lobata purple, clove purple, dazuo purple, pinna minor flamboyant, haihuang, yaohuang, luoyang brocade, black peony and white peony.

3. The screening method of the transcriptome SSR molecular marker primer pair combination of moutan bark as claimed in claim 1, is characterized in that the specific steps are as follows:

(1) Extracting RNA of a fresh cortex moutan sample, constructing a cortex moutan transcriptome library, performing sequencing analysis to obtain a cortex moutan transcriptome sequence, and splicing transcripts by using Trinity software to obtain a Unigene;

(2) Unigene functional annotation the Unigene sequence is compared with databases of NR, swiss-Prot, GO, COG, KOG, eggNOG4.5 and KEGG by using BLAST software, a KEGG ontology result of the Unigene in KEGG is obtained by using KOBAS2.0, the annotation information of the Unigene is obtained by comparing HMMER software with a Pfam database after the amino acid sequence of the Unigene is predicted, and a functional gene or a fragment participating in a biosynthesis pathway of paeonol, paeoniflorin and gallic acid in moutan bark is obtained to independently build a functional gene database;

(3) Using MISA software to screen and analyze the SSR locus of the Unigene with the nucleotide length of more than 1000bp in the functional gene database, and then setting conditions in the obtained total SSR for screening, wherein the screening standard is as follows: the minimum number of times of repetition of the dinucleotides, the trinucleotides, the tetranucleotides, the pentanucleotides and the hexanucleotides is respectively 9, 6, 5, 4 and 4;

(4) Designing SSR primers by using Primer 5 software, wherein the Primer design parameters are that the length of the primers is 18-25 bp; the length of the PCR amplification product is 100-400 bp; the GC content is 40 to 60 percent;

(5) And (3) identifying the amplifiable property and the universality of the primer designed in the step (4) in different moutan bark samples to obtain the SSR molecular marker primer pair combination with the amplifiable property and the universality in different moutan bark samples.

Images