CN110511928A - A kind of the transcript profile SSR molecular marker system and its application of cortex moutan - Google Patents
A kind of the transcript profile SSR molecular marker system and its application of cortex moutan Download PDFInfo
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Abstract
The present invention relates to a kind of transcript profile SSR molecular marker system of cortex moutan and its applications, pass through 10 pairs of SSR molecular marker primers in screening cortex moutan, the primer system polymorphism is high, versatility is good, can embody polymorphism in multiple tree peony samples, and can cortex moutan kind to different cultivars source and quality effectively identified.
Description
Technical field
The invention belongs to biological fields, are related to the transcript profile SSR molecular marker system and its application of a kind of cortex moutan.
Background technique
Chinese medicine cortex moutan is the dry root skin of ranunculaceae peony Paeonia suffruticosa Andr., main point
It is distributed in the ground such as Chongqing, Anhui, Shaanxi, Henan, the Shandong in China.Cortex moutan is common bulk medicinal materials, and clinical application is extensive, tool
There are one of the raw material of the effect of clearing heat and cooling blood, activating microcirculation and removing stasis medicinal and the clinical big kind preparation such as Liuwei Dihuang Wan of Chinese medicine.Chongqing
Dianjiang is traditional Genuine producing area of the river root bark of tree peony；Anhui Feng Dan (P.ostii T.Hong et J.X.zhang) though do not take in pharmacopeia,
But because medical material quanlity is good, and one of main source as cortex moutan in medicinal material market.
It is existing the study found that tree peony have nearly 1000 it is ornamental cultivate kinds, since fancy breed and Plant's kind are in product
There is larger difference in kind breeding and cultivation mode, general Plant's kind is single-lobe monochrome flower, and cortex moutan quality is preferred；Fancy breed
The mostly bright-coloured flower of polyphyll, if root skin is used as medicine, quality is not so good as Plant's kind.But the increase in demand with market to cortex moutan, often
There is the cortex moutan in ornamental peonies source to be full of the cortex moutan that will lead to different cultivars tree peony source in medicinal material market to exist significantly
Quality difference, and then influence clinical efficacy.Since different cultivars cortex moutan is very much like on mode of appearance, it is difficult to by outer
Portion's morphological feature distinguishes.Therefore, it needs to establish the cortex moutan kind that reliable identification method carrys out distinguishing different, to medicine
It is identified with tree peony and the cortex moutan in ornamental peonies source, to guarantee the stability of cortex moutan quality of medicinal material.
The quality of cortex moutan is mainly reflected in the content of effective component, and the accumulation of effective component is dependent on raw in plant
Object synthesizes the functioning gene in access, by parsing the characteristic attribute of functioning gene, can be formed and be evaluated for medical material quanlity
Important references are provided.And to the signature of functional gene, it can be used as medical material quanlity evaluation and the index that excellent variety is cultivated.
Molecular labeling has become the genetic diversity and cultivar identification one of the most important instruments for disclosing species.It is common at present
Molecular labeling type include restriction fragment length polymorphism (RFLP), randomly amplified polymorphic DNA (RAPD), digestion amplification
Polymorphic sequence (CAPS), single nucleotide polymorphism (SNP), simple repeated sequence (SSR) etc..Wherein, SSR (simple
Sequence repeat) it is also known as microsatellite sequence, the DNA that forms is repeated as many times by 1~6 nucleotide complex radical member of attaching most importance to
Sequence, their length are about 100~200 base-pairs, are widely present in eukaryotic gene group.SSR sequence has
Widely distributed, codominant inheritance, polymorphic site is more, information content is abundant, metastatic is good between species, is easy to detect and repeat
The good feature of property, has been widely used in cultivar identification, Germ-plasma resources protection and utilization, analysis of genetic diversity, molecular labeling
The research fields such as assist-breeding.Genome SSR (genomic-SSR) week was both can avoid based on the SSR molecular marker in transcript profile
The problem that phase is long, data volume at high cost and EST-SSR is few, and have the advantages that genic-SSR；Meanwhile this technology
Take full advantage of the result of transcript profile sequencing.The molecular labeling developed from the research discovery carried out based on transcript profile SSR is more
State property and expanding effect are preferable, illustrate that transcript profile SSR is suitable for the exploitation for carrying out molecular labeling.Tree peony watches as important
Plant has the characteristics that " mostly, a variety of, polynary " origin, there is genetic diversity extremely abundant.In crop breeding, deep
Enter to understand molecular diversity, is to study crop genetic diversity, identification of species and the basis for selecting hybrid parent, but tree peony is at this
The research of aspect is on the low side, is concentrated mainly on to researchs such as Varieties of Peony identification, origins, research then biases toward the ornamental of peony
Property, and have ignored the medical value of the plant.The Varieties of Peony of some areas finds that it has by molecule marking research and enriches
Genetic diversity.It is analyzed using Varieties of Peony of the RAPD technology to Sichuan Pengzhou area, it is found that most source places are identical
Performance of cultivar be more close affiliation；Building Central Plains tree peony core authors library research in, according to kind come
The master datas such as source, classification system and form, molecular marker characteristic data, establish Central Plains tree peony cultivars phenotypic data library and
The molecular database of kind.And by the primary core collection resource of building Central Plains tree peony cultivars, ISSR and AFLP label is carried out
Prove that the Core Germplasms of building have genetic diversity abundant.CDDP label is also attempted for the genetic diversity point of tree peony
Analysis, varietY specificity analysis and consistency check, to screen the core primers of differentiation tree peony germ plasm resource and inquire into tree peony molecule
The construction method of identity card.Yu Haiping etc. is marked using magnesphere developing SSR using Feng Dan as experimental material, has inquired into phoenix
Intersection amplification ability of the pellet in Chinese herbaceous peony group plant, discovery Paeonia papaveracea are one of the main original seeds of northwest Varieties of Peony group, and
The two and four paeonia szechuanica Fangs have closer affiliation；Feng Dan takes part in the origin of Central Plains tree peony cultivars group；System's individual is male with Central Plains
Red Breeds individual also has certain affiliation.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of transcript profile SSR molecular marker system of cortex moutan and its answering
With effectively being reflected by the genetic polymorphism of functional gene to the cortex moutan medicinal material and medicine materical crude slice kind and quality of separate sources
Fixed and differentiation.
In order to achieve the above objectives, the invention provides the following technical scheme:
1, the transcript profile SSR molecular marker system of a kind of cortex moutan, including following 10 primer pairs:
Primer pair P1, nucleotide sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2；
Primer pair P2, nucleotide sequence is as shown in SEQ ID NO.3 and SEQ ID NO.4；
Primer pair P3, nucleotide sequence is as shown in SEQ ID NO.5 and SEQ ID NO.6；
Primer pair P4, nucleotide sequence is as shown in SEQ ID NO.7 and SEQ ID NO.8；
Primer pair P5, nucleotide sequence is as shown in SEQ ID NO.9 and SEQ ID NO.10；
Primer pair P6, nucleotide sequence is as shown in SEQ ID NO.11 and SEQ ID NO.12；
Primer pair P7, nucleotide sequence is as shown in SEQ ID NO.13 and SEQ ID NO.14；
Primer pair P8, nucleotide sequence is as shown in SEQ ID NO.15 and SEQ ID NO.16；
Primer pair P9, nucleotide sequence is as shown in SEQ ID NO.17 and SEQ ID NO.18；
Primer pair P10, nucleotide sequence is as shown in SEQ ID NO.19 and SEQ ID NO.20.
2, a kind of transcript profile SSR molecular marker system of above-mentioned cortex moutan is in the cultivar identification and quality evaluation of cortex moutan
Application.
Preferably, the cortex moutan includes but is not limited to that single-lobe is red, polyphyll is red, Feng Dan or Luoyang ornamental peonies kind, institute
State Luoyang ornamental peonies kind include but is not limited to Zhao's powder, Pueraria lobota towel purple, lilac, big palm fibre is purple, duckweed is gorgeous in fact, it is extra large it is yellow, Yao is yellow, Luoyang
Bright and beautiful, black tree peony, white peony.
3, the construction method of the transcript profile SSR molecular marker system of above-mentioned a kind of cortex moutan, the specific steps are as follows:
(1) fresh cortex moutan sample RNA is extracted, cortex moutan transcript profile library is constructed and carries out sequencing analysis, obtains tree peony
Skin transcript profile sequence, and transcript is spliced to obtain Unigene with Trinity software；
(2) Unigene functional annotation using BLAST software by Unigene sequence and NR, Swiss-Prot, GO, COG,
KOG, eggNOG4.5, KEGG database compare, and obtain KEGGOrthology of the Unigene in KEGG using KOBAS2.0 and tie
Fruit has been predicted to be compared after the amino acid sequence of Unigene using HMMER software and Pfam database, has obtained the note of Unigene
Information is released, obtains participating in Cortex moutan such as Paeonol, Paeoniflorin, the function in gallic acid biosynthesis pathway
Gene or segment independently build up functional gene database；
(3) Unigene using MISA software to functional gene database nucleotide length greater than 1000bp carries out SSR
The screening and analysis in site；Condition is arranged again from obtained total SSR to be screened, screening criteria: dinucleotides, three nucleosides
Acid, tetranucleotide, pentanucleotide and the minimum number of repetition of Hexanucleotide are respectively 9,6,5,4,4；
(4) 5 software design SSR primer of Primer is used, design of primers parameter is that primer length is 18~25bp；PCR expands
Increasing product length is 100~400bp；G/C content is 40%~60%；
(5) amplifiable property and versatility identification are carried out in different cortex moutan samples to primer designed by step (4), is obtained
To the above-mentioned SSR molecular marker system in different cultivars cortex moutan with amplifiable property and versatility.
The beneficial effects of the present invention are:
The present invention screens sequence according to cortex moutan transcriptome analysis in the genetic fragment of 1000bp or more first, and is based on this
A little segments find the SSR molecular marker primer with effective component biological synthetic functional gene-correlation again, and 10 pairs of primer bodies are obtained
System, the primer system polymorphism is high, versatility is good, and can embody polymorphism in multiple tree peony samples, not only can be to difference
The cortex moutan cultivar origin of cultivar origin is effectively identified, while can also be used as a finger of cortex moutan quality good or not evaluation
Mark.
Detailed description of the invention
In order to keep the purpose of the present invention, technical scheme and beneficial effects clearer, the present invention provides following attached drawing and carries out
Illustrate:
Fig. 1 is that primer pair P1 expands the gel electrophoresis spectrum after Paeonia suffruticosa skin sample, wherein N-negative control；1—
Chongqing Dianjiang peace clearance permit hemp nettle cortex moutan sample；The peaceful red hose-in-hose cortex moutan sample of 2-Chongqing Dianjiangs；3-Chongqing Dianjiangs
Phoenix pellet white peony skin sample；4-Anhui Feng Dan white peony skin samples；5-Luoyang are ornamental to use cortex moutan sample；M—
DNAmarker；
Fig. 2 is that primer pair P2 expands the gel electrophoresis spectrum after Paeonia suffruticosa skin sample, wherein N-negative control；1—
Chongqing Dianjiang peace clearance permit hemp nettle cortex moutan sample；The peaceful red hose-in-hose cortex moutan sample of 2-Chongqing Dianjiangs；3-Chongqing Dianjiangs
Phoenix pellet white peony skin sample；4-Anhui Feng Dan white peony skin samples；5-Luoyang are ornamental to use cortex moutan sample；M—
DNAmarker；
Fig. 3 is that primer pair P3 expands the gel electrophoresis spectrum after Paeonia suffruticosa skin sample, wherein N-negative control；1—
Chongqing Dianjiang peace clearance permit hemp nettle cortex moutan sample；The peaceful red hose-in-hose cortex moutan sample of 2-Chongqing Dianjiangs；3-Chongqing Dianjiangs
Phoenix pellet white peony skin sample；4-Anhui Feng Dan white peony skin samples；5-Luoyang are ornamental to use cortex moutan sample；M—
DNAmarker；
Fig. 4 is that primer pair P4 expands the gel electrophoresis spectrum after Paeonia suffruticosa skin sample, wherein N-negative control；1—
Chongqing Dianjiang peace clearance permit hemp nettle cortex moutan sample；The peaceful red hose-in-hose cortex moutan sample of 2-Chongqing Dianjiangs；3-Chongqing Dianjiangs
Phoenix pellet white peony skin sample；4-Anhui Feng Dan white peony skin samples；5-Luoyang are ornamental to use cortex moutan sample；M—
DNAmarker；
Fig. 5 is that primer pair P5 expands the gel electrophoresis spectrum after Paeonia suffruticosa skin sample, wherein 1-Chongqing Dianjiang is peaceful
Clearance permit hemp nettle cortex moutan sample；The peaceful red hose-in-hose cortex moutan sample of 2-Chongqing Dianjiangs；3-Chongqing Dianjiang phoenix pellet white peony skins
Sample；4-Anhui Feng Dan white peony skin samples；5-Luoyang are ornamental to use cortex moutan sample；M—DNA marker；
Fig. 6 is that primer pair P6 expands the gel electrophoresis spectrum after Paeonia suffruticosa skin sample, wherein N-negative control；1—
Chongqing Dianjiang peace clearance permit hemp nettle cortex moutan sample；The peaceful red hose-in-hose cortex moutan sample of 2-Chongqing Dianjiangs；3-Chongqing Dianjiangs
Phoenix pellet white peony skin sample；4-Anhui Feng Dan white peony skin samples；5-Luoyang are ornamental to use cortex moutan sample；M—DNA
marker；
Fig. 7 is that primer pair P7 expands the gel electrophoresis spectrum after Paeonia suffruticosa skin sample, wherein N-negative control；1—
Chongqing Dianjiang peace clearance permit hemp nettle cortex moutan sample；The peaceful red hose-in-hose cortex moutan sample of 2-Chongqing Dianjiangs；3-Chongqing Dianjiangs
Phoenix pellet white peony skin sample；4-Anhui Feng Dan white peony skin samples；5-Luoyang are ornamental to use cortex moutan sample；M—DNA
marker；
Fig. 8 is that primer pair P8 expands the gel electrophoresis spectrum after Paeonia suffruticosa skin sample, wherein N-negative control；1—
Chongqing Dianjiang peace clearance permit hemp nettle cortex moutan sample；The peaceful red hose-in-hose cortex moutan sample of 2-Chongqing Dianjiangs；3-Chongqing Dianjiangs
Phoenix pellet white peony skin sample；4-Anhui Feng Dan white peony skin samples；5-Luoyang are ornamental to use cortex moutan sample；M—DNA
marker；
Fig. 9 is that primer pair P9 expands the gel electrophoresis spectrum after Paeonia suffruticosa skin sample, wherein N-negative control；1—
Chongqing Dianjiang peace clearance permit hemp nettle cortex moutan sample；The peaceful red hose-in-hose cortex moutan sample of 2-Chongqing Dianjiangs；3-Chongqing Dianjiangs
Phoenix pellet white peony skin sample；4-Anhui Feng Dan white peony skin samples；5-Luoyang are ornamental to use cortex moutan sample；M—DNA
marker；
Figure 10 is that primer pair P10 expands the gel electrophoresis spectrum after Paeonia suffruticosa skin sample, wherein N-negative control；
1-Chongqing Dianjiang peace clearance permit hemp nettle cortex moutan sample；The peaceful red hose-in-hose cortex moutan sample of 2-Chongqing Dianjiangs；3-Chongqing pad
Jiang Fengdan white peony skin sample；4-Anhui Feng Dan white peony skin samples；5-Luoyang are ornamental to use cortex moutan sample；M—DNA
marker；
Figure 11 is the separate sources cortex moutan sample clustering analysis based on SSR polymorphism result.
Specific embodiment
Below in conjunction with attached drawing, a preferred embodiment of the present invention will be described in detail.
If the method in the embodiment of the present invention without specified otherwise, is routinely method in such technology, the following example
In experimental material, reagent and instrument and equipment be obtained through commercial channels.
The exploitation of 1 cortex moutan transcript profile SSR polymorphism primer pair of embodiment
1 Total RNAs extraction and purifying
Take fresh tree peony root, cleans soil, it is quick-frozen in investment liquid nitrogen container rapidly, it is put into -80 DEG C of refrigerators and saves.
Using RNA extracts kit (InvitrogenTM, RiboMinusTMPlant Kit for RNA-Seq) extract it is male
The purity, dense of Nanodrop, Qubit 2.0,2100 method of Aglient detection RNA sample is respectively adopted in root bark of tree peony sample total serum IgE
Degree and integrality etc., to guarantee to carry out transcript profile sequencing using qualified sample.
2 library constructions
After sample detection is qualified, library construction is carried out, according to UltraTMRNA Library Prep Kit
for Provided reagent and operating instruction are tested:
(1) with the enrichment with magnetic bead eukaryote mRNA with Oligo (dT)；
(2) Fragmentation Buffer is added to be interrupted mRNA at random；
(3) with mRNA (5 μ L) for template, with hexabasic base random primer (random hexamers, 1 μ L), NEBNext
First Strand Synthesis Reaction Buffer(4μL)、NEBNext First Strand Synthesis
The mixing such as Enzyme Mix (2 μ L) synthesize first cDNA chain by gradient increased temperature, and NEBNext Second is added in reaction product
Strand Synthesis Reaction Buffer(10X)(8μL)、NEBNext Second Strand Synthesis
The reagents such as Enzyme Mix (4 μ L), water are pure using AMPure XP magnetic bead by 16 DEG C of incubations, 1 hour synthesis Article 2 cDNA chain
Change cDNA；
(4) the double-strand cDNA purified carries out end reparation plus A tail again and connects sequence measuring joints, then uses AMPure XP magnetic
Pearl carries out clip size selection；
(5) it is enriched with to obtain cDNA library finally by PCR.
3 library Quality Controls
It is big to the concentration and Insert Fragment in library using Qubit2.0 and Agilent 2100 respectively after the completion of library construction
Small (Insert Size) is detected, and accurate quantitative analysis is carried out using effective concentration of the Q-PCR method to library, to guarantee library
Quality.
Machine is sequenced on 4
After library inspection is qualified, high-flux sequence is carried out with Illumina Hiseq.
Data filtering is carried out to Raw Data, joint sequence therein is removed and low quality Reads obtains high quality
Clean Data.Clean Data is subjected to sequence assembling, obtains the library Unigene of the species.
5Unigene annotation
Using BLAST software by Unigene sequence and NR, Swiss-Prot, GO, COG, KOG, eggNOG4.5, KEGG number
It is compared according to library, obtains KEGG Orthology result of the Unigene in KEGG using KOBAS2.0.Select BLAST parameter E-
Value is not more than 1e-10 no more than 1e-5 and HMMER parameter E-value, and finally obtaining 46,398 has annotation information
Unigene。
The statistical result of gene annotation is shown in Table 1.
Table 1.Unigene annotates statistical form
Note: Annotated databases: each functional database is indicated；Annotated_Number: it indicates that annotation arrives and is somebody's turn to do
The Unigene number of database；300≤length < 1000: indicate that annotation is more than or equal to 300 to the Unigene length of the database
And less than the Unigene number of 1000 bases；Length >=1000: indicate that annotation is greater than 1000 bases to the length of the database
Unigene number.
6 SSR molecules
Ssr analysis is done using Unigene of the MISA software to the 1kb or more that screening obtains, the results are shown in Table 2.
Table 2.SSR repeats motif type and distribution
From 6235 sites SSR that cortex moutan transcript profile data authentication goes out, primer screening is carried out first, screening criteria:
Dinucleotides, trinucleotide, tetranucleotide, pentanucleotide and the minimum number of repetition of Hexanucleotide are respectively 9,6,5,4,4；Primer
Length is 18~25bp；Pcr amplification product length is 150~300bp；G/C content is 40%~60%.Drawing after then screening
Object randomly chooses 132 pairs and carries out amplifiable property and polymorphism and versatility verifying.
Method particularly includes:
DNA is extracted
Take the new fresh root of Dianjiang tree peony, clean soil, removes core, 50 DEG C of drying for standby；
Cortex moutan DNA is extracted using the CTAB method of improvement, operating procedure is as follows:
(1) precision weighs 100mg cortex moutan medicinal material and is placed in mortar, and a small amount of PVP is added to be co-mulled and made into；
(2) medicinal material pulverize it is last be transferred in 2.0mL centrifuge tube, be added 1.5mL 4 DEG C be pre-chilled TNE buffer
(0.1mol·L-1Tris, 0.15molL-1Sodium chloride, 0.02molL-1EDTA), 4 DEG C extraction 2 times, 30 minutes every time,
12000rpm is centrifuged 10 minutes, discards supernatant；
(3) 2 × CTAB Extraction buffer (0.1molL that 900 μ L are preheated at 65 DEG C is added in pipe-1Tris-HCl,
0.02mol·L-1EDTA, 1.4molL-1Sodium chloride, 2% (W/V) CTAB, 1% (V/V) PVP), 14 μ L β-mercapto is then added
Base ethyl alcohol is placed in 65 DEG C of water-baths and keeps the temperature 90 minutes, stirs during water-bath sample 2~3 times；
(4) after water-bath terminates, 45 DEG C are cooled to hereinafter, accurate draw 900 μ L chloroform-isoamyl alcohol (volume ratios to sample
It 24:1) is added in centrifuge tube, mildly shaking makes into emulsion for 3~5 minutes, and 12000rpm is centrifuged 10 minutes；
(5) after stratification, 700 μ L supernatants is drawn into another clean 2.0mL centrifuge tube, isometric chlorine is added
Imitative-isoamyl alcohol (volume ratio 24:1) mildly shakes 2~3 minutes and is allowed to be sufficiently mixed, and 12000rpm is centrifuged 10 minutes；
(6) 500 μ L supernatants are drawn into another clean 1.5mL centrifuge tube, the isopropyl for accounting for 2/3 volume of supernatant is added
Alcohol (20 DEG C of ﹣ is pre-chilled in advance) is placed 30 minutes under the conditions of 20 DEG C of ﹣.12000rpm is centrifuged 10 minutes.Discard supernatant；
(7) 500 μ L, 70% ethyl alcohol is added in centrifuge tube, 12000rpm is centrifuged 5 minutes, discards supernatant.Repetitive operation one
It is secondary；
(8) 500 μ L dehydrated alcohols are added in centrifuge tube, 12000rpm is centrifuged 5 minutes, discards supernatant.Repetitive operation one
It is secondary；
(9) remaining ethyl alcohol in pipe is blotted with blotting paper, nozzle is placed horizontally at 37 DEG C of drying boxes 15~30 minutes, to second
Alcohol volatilizees completely；
(10) 50 μ L ddH2O, abundant dissolving DNA are added in centrifuge tube.20 DEG C of refrigerators of ﹣ are placed in save backup.
Dianjiang peace is red totally 24 parts of cortex moutan (red red with polyphyll containing single-lobe)；Dianjiang produces red white 8 parts of phoenix；Anhui produces Feng Dan
Totally 8 parts；Luoyang produces various fancy breeds, and (Zhao's powder, Pueraria lobota towel purple, lilac, big palm fibre purple, duckweed be gorgeous in fact, extra large Huang, Yao's Huang, Luo Yangjin, black
Tree peony, white peony) totally 10 parts；The extraction of sample gene group DNA is carried out by above-mentioned DNA extraction method.
DNA quality testing
Agarose gel electrophoresis detection is carried out to DNA mass using DYCP-31DN type electrophoresis apparatus.5 μ L DNA samples are taken, are added
Enter 1 μ L 6 × loading buffer, mixes gently.Mixed liquor and 3 μ L DNA Marker are clicked and entered with liquid-transfering gun after mixing
Sample hole；The electrophoresis conducting wire between electrophoresis apparatus power supply and electrophoresis apparatus is connected, electrophoretic parameters, voltage 120Vcm are set-1, when electrophoresis
Between 40 minutes；After electrophoresis, Ago-Gel is detected using ZF-2 type uv analyzer, and electrophorogram is marked,
It saves.
PCR amplification
To reach good expanding effect, PCR reinforcing agent (2% (W/V) BSA, 1% (W/V) have been used in PCR system
PVP).PCR amplification system is prepared as shown in table 3.
Table 3.PCR amplification system
To above-mentioned configured PCR reaction solution, amplification program is as follows: 94 DEG C of initial denaturations, 4min；94 DEG C of denaturation, 30s；
53 DEG C of annealing, 30s；72 DEG C of extensions, 30s；It repeats denaturation and is fallen after rise to step 30-35 circulation, 72 DEG C of extension 5min, temperature is extended
It is taken out when to 25 DEG C spare.Amplified production is detected with 2% agarose gel electrophoresis, if there is the band of 100-400bp,
The SSR primer has amplifiable property, filters out 83 pairs of amplifiable property primers altogether.
Then this 83 pairs amplifiable SSR primer pair expansions are applied in 4 parts of different cultivars cortex moutan samples, PCR amplification
System and program are as noted above.Pcr amplification product 8% native polyacrylamide gel electrophoresis combination silver staining of mass concentration
Detection, filters out the preferable SSR primer of polymorphism.
Concrete operations are as follows:
(1) glass plate, sample lattice, encapsulating graduated cylinder and beaker are cleaned, glass plate gas is dry；
(2) flat mouth glass plate and recess glass plate being placed on the supporter of desktop, glass plate drips a small amount of 100% ethyl alcohol,
Glue face is uniformly cleaned with lens paper, gas is dry；
(3) appropriate vaseline is uniformly smeared on three sides of flat mouth glass plate and recess glass plate, and the overlapping of two plates will with hand
Two pieces of glass plates are holded up on the table, make two glass plate bottom surfaces concordantly to form glue room；
(4) glue room is put into electrophoresis apparatus main body, keeps the one of recess glass plate inward-facing, wedge-shaped plate is inserted into, by electrophoresis apparatus
Main body is put into adhesive-preparing device of normal position, according to the thickness of the glue of requirement of experiment, is rotated gel maker handle to corresponding scale, is inserted wedge shape
Plate compresses glue room；
Non-denaturing polyacrylamide gel is prepared:
(5) 8% polyacrylamide gel Compound mixed solutions (50mL): 26.4mL deionized water, 13.3mL30% acryloyl
Amine, 10mL5 × tbe buffer liquid are rapidly added 350 μ L, 10% ammonium persulfate (catalyst) before encapsulating, and 33 μ L TEMED (accelerate
Agent).
(6) match glue, encapsulating.For the size and thickness of selected glue, appropriate 8% polyacrylamide gel mixing is measured
Liquid pours into clean beaker, shakes up, encapsulating, slowly the sample lattice of insertion and glue consistency of thickness, 30 minutes or more solid wait be gelled.
Native polyacrylamide gel electrophoresis is carried out using DYCZ-24DN type electrophoresis apparatus:
(1) after gel polymerisation, electrophoresis apparatus main body is taken out from adhesive-preparing device of normal position, is put under electrophoresis apparatus in slot；
(2) 140mL 0.5 × TBE electrophoretic buffer is poured into the upper slot formed by glue room and electrophoresis apparatus main body, makes to delay
Fliud flushing there was not recess glass plate；
(3) 200mL 0.5 × TBE electrophoretic buffer is poured under electrophoresis apparatus in slot；
(4) slowly sample lattice are vertically extracted；
(5) 3.5 μ L PCR amplification samples are taken, 0.7 μ L 6 × DNA/RNAloading buffer is added, are mixed；After mixing
Mixed liquor and 3 μ L 50bp Ladder DNAMarker are clicked and entered into loading hole with liquid-transfering gun；
(6) the electrophoresis conducting wire between electrophoresis apparatus power supply and electrophoresis apparatus is connected, electrophoretic parameters are set.Voltage is 200Vcm-1,
Electrophoresis time 45 minutes.
Silver staining colour developing
(1) after electrophoresis, gel is slowly removed, is rinsed 2 times, every time 5 minutes with deionized water；
(2) gel is placed in silver staining liquid (100mL water, 20% silver nitrate of 1mL), covering barn door makes gel be in black
Dark state gently shakes 13 minutes on low speed shaking table and dyes.
(3) gel is slowly removed, is rinsed 2 times, every time 5 minutes with deionized water；
(4) gel is transferred in developer solution (3% sodium hydroxide of 100mL, 1mL formaldehyde, 4 DEG C of pre-coolings), is placed on low speed
It is gently shaken on shaking table up to there is DNA band,
(5) gel is rinsed rapidly 1 time with deionized water, gel imaging and DNA fragmentation band number is carried out after drying at room temperature
Mesh statistics.
The polymorphism primer filtered out is expanded in all cortex moutan DNA of the present invention and is detected, based on
Upper experimental result, filter out 10 pairs of polymorphism SSR primer pairs (Fig. 1~10) altogether: its sequence information is as shown in table 4.
4. sequence information of table
Separate sources cortex moutan SSR primer pair system polymorphism result:
According to target fragment size in DNAMarker and primer information, artificial interpretation 10 is to polymorphism SSR primer at 50 parts
Cortex moutan sample C is small to A/B/C/D is respectively designated as greatly, and homozygote is named as AA/BB/CC/DD, and heterozygote is named as AB/BD/
CD/BD。
5. cortex moutan sample situation of table
Using 32 software of POPGENE to 10 pairs of polymorphism SSR primers in 50 parts of cortex moutan sample DNAs of different sources
Amplification situation analyzed.The variation range of number of alleles (Na) is 2~4, average 2.8000, and wherein primer P10 is most
Height, primer P2, P4 and P9's is minimum.Average each site SSR effective number of allele (Ne) is 2.0045.According to Nei ' s
1973 method calculating observation heterozygosity (Ho) ranges are 0.0816~0.6800, average 0.3947.It is expected that heterozygosity (He) range is
0.3232~0.6455, average 0.4863.Nei ' s it is expected that heterozygosity (H) range is 0.3200~0.6390, average 0.4814.
Polymorphism information content (PIC) is from 0.2688 to 0.5711, average value 0.4069.Primer P8 and P10 are high polymorphic locus
(PIC > 0.5), remaining 8 are moderate polymorphic site (0.25 < PIC < 0.5).Shannon information index (I) range is
0.5004~1.0944, average 0.7741.The above result shows that the genetic diversity of Peony Resource is relatively abundanter.(table 6)
The analysis of genetic diversity of table 6.SSR primer
50 parts of cortex moutan samples are divided into analysis of genetic diversity between 5 groups of carry out groups according to geographic origin and kind.
" peaceful red " the single-lobe cortex moutan sample (number S1~S12) acquired using Chongqing Dianjiang as first group, (number by polyphyll sample
S13~S24) it is used as second group, " Feng Dan " sample (number S25~S32) is used as third group, " Feng Dan " sample of Anhui acquisition
(number S33~S40) is used as the 4th group, and the cortex moutan sample (number S41~S50) of Luoyang, henan acquisition is used as the 5th group.
The analysis of POPGENE 32 is the results show that observation heterozygosity (Ho) range is 0.2750~0.4917.It is expected that heterozygosity
(He) range is 0.2692~0.4263.Nei ' s expectation heterozygosity (H) range is that 0.2580~0.3993, H is that reflection group loses
Multifarious common counter is passed, is sorted according to genetic diversity to 5 tree peony groups: the 4th group > the 5th group > third group > the first
Group > the second group.The range of number of alleles (Na) be 1.700~2.400, the 4th group and the 5th group of quantity it is more, second group of number
Amount is minimum.Shannon information index (I) range be 0.3639~0.6587, up to the 4th group, minimum second group.It is comprehensive
Result above, it can be seen that genetic diversity highest (H=0.3993, the I=of Anhui " Feng Dan " in 5 tree peony groups
0.6587), the genetic diversity of Chongqing Dianjiang " peaceful red " polyphyll tree peony is minimum (H=0.2580, I=0.3639).
First group and second group of observation heterozygosity (Ho) is above desired heterozygosity (He), and the coefficient of inbreeding (Fis) is
Negative value (first group: Fis=﹣ 0.8656, second group: Fis=﹣ 0.9704) shows that intragroup heterozygote is superfluous, and homozygote lacks
It is weary.Third group, the 4th group and the 5th group observation heterozygosity (Ho) be below desired heterozygosity (He), the coefficient of inbreeding is positive value
(Fis=0.0356~0.2129), shows intragroup heterozygote missing, and homozygote is superfluous.The coefficient of inbreeding of 5 groups of tree peonies is average
Value is negative value (Fis=﹣ 0.2794), also indicates that group's heterozygote is superfluous.(table 7)
The genetic diversity relevant parameter of 7. separate sources cortex moutan of table
According to Nei ' s genetic distance corresponding between 50 parts of cortex moutan samples, used on PowerMarker V3.25 software
UPGMA method constructs dendrogram.As shown in figure 11,50 parts of samples are integrally divided into three apparent branch Cluster1, Cluster2
And Cluster3.Wherein Cluster1 includes A and B Liang Ge branch, shares 24 parts of samples, is all from Chongqing Dianjiang.A is by all
" peaceful red " polyphyll tree peony (number S13~S24) composition, B are made of all single-lobe tree peonies (number S1~S12).
The cluster situation of Cluster1 illustrates that the place of production is identical, the identical Varieties of Peony of pattern has closer affiliation.In Cluster2
16 parts of cortex moutan samples are shared, Chongqing Dianjiang " Feng Dan " sample (number S25~S32) is picked up from including 8 parts and 8 parts is picked up from Anhui
" Feng Dan " sample (number S33~S40)).Produced cortex moutan sample (the number S41 of 10 ornamental peonies acquired from Luoyang, henan
~S50) all it is gathered in Cluster3.Chongqing Dianjiang " peaceful red ".Thus, the tree peony shown based on SSR molecular marker system
Skin sample polymorphism analysis can effectively distinguish the cortex moutan sample with identification separate sources by clustering.
Finally, it is stated that preferred embodiment above is only used to illustrate the technical scheme of the present invention and not to limit it, although logical
It crosses above preferred embodiment the present invention is described in detail, however, those skilled in the art should understand that, can be
Various changes are made to it in form and in details, without departing from claims of the present invention limited range.
Sequence table
<110>Southwest University
<120>the transcript profile SSR molecular marker system and its application of a kind of cortex moutan
<160> 20
<170> SIPOSequenceListing 1.0
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ggtcctatct cttcacgaaa atc 23
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<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
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<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
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<211> 22
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<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 (4)
1. a kind of transcript profile SSR molecular marker system of cortex moutan, which is characterized in that including following 10 primer pairs:
Primer pair P1, nucleotide sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2；
Primer pair P2, nucleotide sequence is as shown in SEQ ID NO.3 and SEQ ID NO.4；
Primer pair P3, nucleotide sequence is as shown in SEQ ID NO.5 and SEQ ID NO.6；
Primer pair P4, nucleotide sequence is as shown in SEQ ID NO.7 and SEQ ID NO.8；
Primer pair P5, nucleotide sequence is as shown in SEQ ID NO.9 and SEQ ID NO.10；
Primer pair P6, nucleotide sequence is as shown in SEQ ID NO.11 and SEQ ID NO.12；
Primer pair P7, nucleotide sequence is as shown in SEQ ID NO.13 and SEQ ID NO.14；
Primer pair P8, nucleotide sequence is as shown in SEQ ID NO.15 and SEQ ID NO.16；
Primer pair P9, nucleotide sequence is as shown in SEQ ID NO.17 and SEQ ID NO.18；
Primer pair P10, nucleotide sequence is as shown in SEQ ID NO.19 and SEQ ID NO.20.
2. a kind of transcript profile SSR molecular marker system of cortex moutan described in claim 1 is in the cultivar identification and quality of cortex moutan
Application in evaluation.
3. application according to claim 2, which is characterized in that the cortex moutan include but is not limited to single-lobe is red, polyphyll is red,
Feng Dan or Luoyang ornamental peonies kind, the Luoyang ornamental peonies kind includes but is not limited to Zhao's powder, Pueraria lobota towel purple, lilac, big
Palm fibre is purple, duckweed is gorgeous in fact, extra large Huang, Yao's Huang, Luo Yangjin, black tree peony, white peony.
4. a kind of construction method of the transcript profile SSR molecular marker system of cortex moutan described in claim 1, which is characterized in that tool
Steps are as follows for body:
(1) fresh cortex moutan sample RNA is extracted, cortex moutan transcript profile library is constructed and carries out sequencing analysis, cortex moutan is obtained and turns
Record group sequence, and transcript is spliced to obtain Unigene with Trinity software；
(2) Unigene functional annotation using BLAST software by Unigene sequence and NR, Swiss-Prot, GO, COG, KOG,
EggNOG4.5, KEGG database compare, obtain KEGG Orthology of the Unigene in KEGG using KOBAS2.0 as a result,
It has predicted to compare after the amino acid sequence of Unigene using HMMER software and Pfam database, has obtained the annotation of Unigene
Information obtains participating in Cortex moutan such as Paeonol, Paeoniflorin, the function base in gallic acid biosynthesis pathway
Cause or segment, independently build up functional gene database；
(3) Unigene using MISA software to functional gene database nucleotide length greater than 1000bp carries out the site SSR
Screening and analysis；Condition is arranged again from obtained total SSR to be screened, screening criteria: dinucleotides, trinucleotide, four
Nucleotide, pentanucleotide and the minimum number of repetition of Hexanucleotide are respectively 9,6,5,4,4；
(4) 5 software design SSR primer of Primer is used, design of primers parameter is that primer length is 18~25bp；PCR amplification produces
Object length is 100~400bp；G/C content is 40%~60%；
(5) amplifiable property and versatility identification are carried out in different cortex moutan samples to primer designed by step (4), is obtained
With the above-mentioned SSR molecular marker system of amplifiable property and versatility in different cultivars cortex moutan.
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| CN111733279A (en) * | 2020-07-27 | 2020-10-02 | 洛阳师范学院 | Peony EST-SSR primer group and application thereof |
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