CN108998561A - A method of exploitation elder SSR primer is sequenced based on transcript profile - Google Patents
A method of exploitation elder SSR primer is sequenced based on transcript profile Download PDFInfo
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Abstract
The present invention provides a kind of method that exploitation elder SSR primer is sequenced based on transcript profile, the described method includes: S1. constructs elder transcript profile library and is sequenced, sequencing result is subjected to splicing and is assembled into a complete transcript profile, longest transcript in every gene is taken to obtain the mixing library Unigenes by splicing as Unigene；S2. SSR detection is carried out to the library mixing Unigenes in step S1；S3. SSR design of primers is carried out, and identifies the polymorphism of SSR primer.The present invention designs 64148 pairs of SSR special primers altogether, has obtained the optimal system of golden elder fluorescence SSR-PCR by assay optimization scheme, in 100 pairs of fluorescent marker SSR primers of synthesis, has 25 pairs of primers to have good polymorphism.The method that exploitation elder SSR primer is sequenced the present invention is based on transcript profile is quick, accurate.
Description
Technical field
The invention belongs to molecular biology and bioinformatics technique fields, and in particular to one kind is opened based on transcript profile sequencing
The method for sending out elder SSR primer.
Background technique
Simple sequence repeats (Simple sequence repeat, SSR) are a kind of simple strings by 1-6 base composition
Join repetitive sequence, is distributed widely in eucaryote and prokaryotic gene group.EST-SSR and base can be divided into according to the source SSR
Because of a group SSR, traditional have that at high cost, test procedure is cumbersome, site is less etc. using genomic data developing SSR label and lack
Point, in recent years, as sequencing cost reduces, transcript profile sequencing has become the short, adaptable and fast think of of research molecular biology related content
The transcript profile data on one of road, different plants are exploited in succession, develop different plant SSR markers based on transcript profile sequencing
It studies also more and more.
Sambucus (Sambucus Linn) is Caprifoliaceae (Caprifoliaceae) plant, be deciduous tree or shrub,
About 20 kinds of this category, it is dispersed throughout temperate zone and subtropical zone.About 6 kinds of China, north and south produces.Elder is precious medicinal plant,
Many type fruit oil content are high (35%~44%), are one of the traditional oil trees that the Ministry of Agriculture announces.China's elder money
Source very abundant, with very strong adaptability, cold-resistant, drought resisting, disease resistance are strong, and it is not yet sharp in mountain forest to be sunk into sleep mostly
With, development and utilization to synthetism fogfruit, China is still in the junior stage, and the utilization of resources is insufficient, and it is only medicinal more, it is other
It comprehensively utilizes less, also has no marker development and the relevant reports such as molecular mark based on transcript profile sequencing, therefore
It is necessary to further develop microsatellite marker, to carry out elder Genetic Diversity of Germplasm, linkage map using SSR marker
Building and functional gene excavation lay the foundation.
Summary of the invention
In view of the above shortcomings of the prior art, inventor provides a kind of based on transcription through long-term technology and practical exploration
The method of group sequencing exploitation elder SSR primer.
One of the objects of the present invention is to provide a kind of elder SSR primer pairs.
The second object of the present invention is to provide a kind of elder SSR primer sets.
The third object of the present invention is to provide the application of a kind of above-mentioned elder SSR primer pair and/or primer sets.
The fourth object of the present invention is to provide a kind of method for obtaining elder SSR molecular marker.
The fifth object of the present invention is to provide a kind of method that exploitation elder SSR primer is sequenced based on transcript profile.
Specifically, the present invention adopts the following technical scheme:
The first aspect of the invention provides a kind of elder SSR primer pair, the sequence of the elder SSR primer pair
As shown in SEQ ID NO.1-2, as shown in SEQ ID NO.3-4, as shown in SEQ ID NO.5-6, such as SEQ ID NO.7-8 institute
Show, as shown in SEQ ID NO.9-10, as shown in SEQ ID NO.11-12, as shown in SEQ ID NO.13-14, such as SEQ
Shown in IDNO.15-16, as shown in SEQ ID NO.17-18, as shown in SEQ ID NO.19-20, such as SEQ ID NO.21-22
It is shown, as shown in SEQ ID NO.23-24, as shown in SEQ ID NO.25-26, as shown in SEQ ID NO.27-28, such as SEQ
Shown in IDNO.29-30, as shown in SEQ ID NO.31-32, as shown in SEQ ID NO.33-34, such as SEQ ID NO.35-36
It is shown, as shown in SEQ ID NO.37-38, as shown in SEQ ID NO.39-40, as shown in SEQ ID NO.41-42, such as SEQ
Shown in IDNO.43-44, as shown in SEQ ID NO.45-46, as shown in SEQ ID NO.47-48 or such as SEQ ID NO.49-
Shown in 50；
It should be noted that M is added in the end forward primer sequence 3' in each primer pair13Connector；M13Joint sequence are as follows:
5'-TGTAAAACGACGGCCAGT-3'；
The second aspect of the invention provides a kind of elder SSR primer sets, and the elder SSR primer sets are by following
Two or more composition in primer pair: as described in SEQ ID NO.1-2 primer pair, draw as described in SEQ ID NO.3-4
Object to, as described in SEQ ID NO.5-6 primer pair, as described in SEQ ID NO.7-8 primer pair, as described in SEQ ID NO.9-10
Primer pair, primer pair, primer pair, such as SEQ IDNO.15- as described in SEQ ID NO.13-14 as described in SEQ ID NO.11-12
16 primer pairs, primer pair, primer pair, such as SEQ ID as described in SEQ ID NO.19-20 as described in SEQ ID NO.17-18
Primer pair described in NO.21-22, primer pair, the primer pair, such as described in SEQ ID NO.25-26 as described in SEQ ID NO.23-24
Primer pair described in SEQ ID NO.27-28, as described in SEQ IDNO.29-30 primer pair, draw as described in SEQ ID NO.31-32
Object to, primer pair, primer pair, such as SEQ ID NO.37- as described in SEQ ID NO.35-36 as described in SEQ ID NO.33-34
38 primer pairs, primer pair, primer pair, such as SEQ as described in SEQ ID NO.41-42 as described in SEQ ID NO.39-40
Primer pair described in IDNO.43-44, as described in SEQ ID NO.45-46 primer pair, as described in SEQ ID NO.47-48 primer pair,
With the primer pair as described in SEQ ID NO.49-50；
Preferably, the elder SSR primer sets are made of following primer pair: as described in SEQ ID NO.1-2 primer pair,
Primer pair, primer pair, the primer as described in SEQ ID NO.7-8 as described in SEQ ID NO.5-6 as described in SEQ ID NO.3-4
To, as described in SEQ ID NO.9-10 primer pair, as described in SEQ ID NO.11-12 primer pair, such as SEQ ID NO.13-14 institute
State primer pair, primer pair, primer pair, such as SEQ ID as described in SEQ ID NO.17-18 as described in SEQ IDNO.15-16
Primer pair described in NO.19-20, primer pair, the primer pair, such as described in SEQ ID NO.23-24 as described in SEQ ID NO.21-22
Primer pair described in SEQ ID NO.25-26, as described in SEQ ID NO.27-28 primer pair, draw as described in SEQ IDNO.29-30
Object to, primer pair, primer pair, such as SEQ ID NO.35- as described in SEQ ID NO.33-34 as described in SEQ ID NO.31-32
36 primer pairs, primer pair, primer pair, such as SEQ ID as described in SEQ ID NO.39-40 as described in SEQ ID NO.37-38
Primer pair described in NO.41-42, primer pair, the primer pair, such as described in SEQ ID NO.45-46 as described in SEQ IDNO.43-44
Primer pair described in SEQ ID NO.47-48 and the primer pair as described in SEQ ID NO.49-50.
The third aspect of the invention provides the application of above-mentioned elder SSR primer pair and/or primer sets；The application
Include:
1) application in elder analysis of genetic diversity；
2) application in building elder genetic map；
3) application in elder Germplasm Identification；
4) application in elder Genetic relationship；
5) application in the excavation of elder functional gene；
6) application in elder molecular mark.
The fourth aspect of the invention provides a kind of method for obtaining elder SSR molecular marker, comprising:
S1. using elder genomic DNA as template, with above-mentioned elder SSR primer pair or above-mentioned elder SSR primer sets
PCR amplification is carried out, pcr amplification product is obtained；
S2. pcr amplification product step S1 obtained carries out capillary electrophoresis detection, obtains elder SSR molecule mark
Note.
The fifth aspect of the invention provides the method that exploitation elder SSR primer is sequenced based on transcript profile, comprising:
S1. it constructs elder transcript profile library and is sequenced, sequencing result is subjected to splicing and is assembled into one complete turn
Record group takes longest transcript in every gene to obtain the mixing library Unigenes by splicing as Unigene；
S2. SSR detection is carried out to the library mixing Unigenes in step S1；
S3. SSR design of primers is carried out, and identifies the polymorphism of SSR primer.
Advantageous effects of the invention:
The present invention designs 64148 pairs of SSR special primers altogether.Elder fluorescence SSR- has been obtained by assay optimization scheme
The optimal system of PCR, in 100 pairs of fluorescent marker SSR primers of synthesis, wherein 80 pairs of primer amplifications have band, effectively amplification rate
It is 80%, wherein 25 pairs of primers (SEQ ID NO.1-50) have good polymorphism in 80 pairs of primers for having an amplified band.
The method that exploitation elder SSR primer is sequenced the present invention is based on transcript profile is quick, accurate, is elder SSR primer
Exploitation provides new approaches.The present invention is the building of high density elder genetic linkage maps, elder Germplasm Identification, elder something lost
Pass diversity analysis, elder Genetic relationship, elder important economical trait QTL positioning and elder molecular labeling auxiliary
Breeding etc. provides effective technical support.
Detailed description of the invention
Fig. 1 is the site golden elder SSR distribution map, in which: X-coordinate is SSR type, and Y coordinate numerical value is coordinate, specifically
Duplicate number should be corresponding with legend according to color, and Z coordinate is SSR number；
Fig. 2 is genotype of 8 elder samples in No. 5 sites；Wherein Fig. 2 (a)-(c) is three Canadian elder
Sample；Fig. 2 (d) is sambucus racemosa sample；Fig. 2 (e) is floral leaf elder sample；Fig. 2 (f) is golden plumage elder sample；Fig. 2
(g)-(h) is two elder samples.
Specific embodiment
It is noted that following detailed description is all illustrative, it is intended to provide further instruction to the application.Unless another
It indicates, all technical and scientific terms used herein has usual with the application person of an ordinary skill in the technical field
The identical meanings of understanding.
It should be noted that term used herein above is merely to describe specific embodiment, and be not intended to restricted root
According to the illustrative embodiments of the application.As used herein, unless the context clearly indicates otherwise, otherwise singular
Also it is intended to include plural form, additionally, it should be understood that, when in the present specification using term "comprising" and/or " packet
Include " when, indicate existing characteristics, step, operation, device, component and/or their combination.
In conjunction with specific example, the present invention is further illustrated, and following instance is not right merely to the explanation present invention
Its content is defined.If the actual conditions being not specified in embodiment, usually according to normal condition, or according to institute of sales company
The condition of recommendation；It is not particularly limited in the present invention, can be commercially available by commercial sources.
As previously mentioned, China's elder resource very abundant, but the development and utilization to synthetism fogfruit, China are still in
Primary stage, the utilization of resources is insufficient, only medicinal more, and other comprehensive utilizations are less, also has no the mark based on transcript profile sequencing
The relevant reports such as note exploitation and molecular mark.
In view of this, providing a kind of elder SSR primer pair, the synthetism in a kind of specific embodiment of the invention
The sequence of the wooden SSR primer pair as shown in SEQ ID NO.1-2, as shown in SEQ ID NO.3-4, as shown in SEQ ID NO.5-6,
As shown in SEQ ID NO.7-8, as shown in SEQ ID NO.9-10, as shown in SEQ ID NO.11-12, such as SEQ ID
Shown in NO.13-14, as shown in SEQ IDNO.15-16, as shown in SEQ ID NO.17-18, such as SEQ ID NO.19-20 institute
Show, as shown in SEQ ID NO.21-22, as shown in SEQ ID NO.23-24, as shown in SEQ ID NO.25-26, such as SEQ ID
Shown in NO.27-28, as shown in SEQ IDNO.29-30, as shown in SEQ ID NO.31-32, such as SEQ ID NO.33-34 institute
Show, as shown in SEQ ID NO.35-36, as shown in SEQ ID NO.37-38, as shown in SEQ ID NO.39-40, such as SEQ ID
Shown in NO.41-42, as shown in SEQ IDNO.43-44, as shown in SEQ ID NO.45-46, such as SEQ ID NO.47-48 institute
Show or as shown in SEQ ID NO.49-50；
It should be noted that M is added in the end forward primer sequence 3' in each primer pair13Connector；M13Joint sequence are as follows:
5'-TGTAAAACGACGGCCAGT-3'；
In still another embodiment of the invention, a kind of elder SSR primer sets, the elder SSR primer are provided
Group is made of two or more in following primer pair: primer pair, such as SEQ ID NO.3- as described in SEQ ID NO.1-2
4 primer pairs, primer pair, primer pair, such as SEQ ID as described in SEQ ID NO.7-8 as described in SEQ ID NO.5-6
Primer pair described in NO.9-10, primer pair, the primer pair, such as described in SEQ ID NO.13-14 as described in SEQ ID NO.11-12
Primer pair described in SEQ IDNO.15-16, as described in SEQ ID NO.17-18 primer pair, draw as described in SEQ ID NO.19-20
Object to, primer pair, primer pair, such as SEQ ID NO.25- as described in SEQ ID NO.23-24 as described in SEQ ID NO.21-22
26 primer pairs, primer pair, primer pair, such as SEQ ID as described in SEQ IDNO.29-30 as described in SEQ ID NO.27-28
Primer pair described in NO.31-32, primer pair, the primer pair, such as described in SEQ ID NO.35-36 as described in SEQ ID NO.33-34
Primer pair described in SEQ ID NO.37-38, as described in SEQ ID NO.39-40 primer pair, draw as described in SEQ ID NO.41-42
Object to, primer pair, primer pair, such as SEQ ID NO.47-48 as described in SEQ ID NO.45-46 as described in SEQ IDNO.43-44
The primer pair and the primer pair as described in SEQ ID NO.49-50；
In still another embodiment of the invention, the elder SSR primer sets are made of following primer pair: such as SEQ
Primer pair described in ID NO.1-2, primer pair, primer pair, such as SEQ as described in SEQ ID NO.5-6 as described in SEQ ID NO.3-4
Primer pair described in ID NO.7-8, primer pair, the primer pair, such as described in SEQ ID NO.11-12 as described in SEQ ID NO.9-10
Primer pair described in SEQ ID NO.13-14, as described in SEQ IDNO.15-16 primer pair, draw as described in SEQ ID NO.17-18
Object to, primer pair, primer pair, such as SEQ ID NO.23- as described in SEQ ID NO.21-22 as described in SEQ ID NO.19-20
24 primer pairs, primer pair, primer pair, such as SEQ as described in SEQ ID NO.27-28 as described in SEQ ID NO.25-26
Primer pair described in IDNO.29-30, as described in SEQ ID NO.31-32 primer pair, as described in SEQ ID NO.33-34 primer pair,
As described in SEQ ID NO.35-36 primer pair, as described in SEQ ID NO.37-38 primer pair, as described in SEQ ID NO.39-40
Primer pair, primer pair, primer pair, such as SEQ ID NO.45- as described in SEQ IDNO.43-44 as described in SEQ ID NO.41-42
46 primer pairs, primer pair and the primer pair as described in SEQ ID NO.49-50 as described in SEQ ID NO.47-48.
In still another embodiment of the invention, the application of above-mentioned elder SSR primer pair and/or primer sets is provided；
The application includes:
1) application in elder analysis of genetic diversity；
2) application in building elder genetic map；
3) application in elder Germplasm Identification；
4) application in elder Genetic relationship；
5) application in the excavation of elder functional gene；
6) application in elder molecular mark.
In still another embodiment of the invention, a kind of method for obtaining elder SSR molecular marker is provided, comprising:
S1. using elder genomic DNA as template, with above-mentioned elder SSR primer pair or above-mentioned elder SSR primer sets
PCR amplification is carried out, pcr amplification product is obtained；
S2. pcr amplification product step S1 obtained carries out capillary electrophoresis detection, obtains elder SSR molecule mark
Note.
In still another embodiment of the invention, in the step S1,
PCR amplification actual conditions are as follows: expanded using two step fluorescent primer PCR amplification methods；
(1) add M13Connector PCR amplification system:
1 μ L, 2 × Mix 5 μ L, 10 μM of primers each 0.1 μ L, H of stoste containing DNA in 10 μ L PCR systems2O 3.8μL；PCR expands
Increase program and be shown in Table 1:
Table 1PCR amplification program
(2) fluorescent primer PCR amplification system: 2 μ L of the DNA of amplified production containing first step stoste in 20 μ L PCR systems, 2 ×
10 μ L of Mix, 10 μM of M13 connectors (adding fluorescent marker) and reverse primer each 0.15 μ L, H2O 7.7μL；Fluorescent primer PCR amplification
Program is shown in Table 2:
2 fluorescent primer PCR amplification program of table
In still another embodiment of the invention, elder can be Canadian elder in the above method
(Sambucus Canadensis), sambucus racemosa (Sambucus canadensis'Aurea'), floral leaf elder
(Sambucus nigra'Variegata'), golden plumage elder (Sambucus racemosa'Plumosa Aurea'), synthetism
Any one or more in wooden (Sambucus williamsii Hance)；
In still another embodiment of the invention, the method that exploitation elder SSR primer is sequenced based on transcript profile is provided,
Include:
S1. it constructs elder transcript profile library and is sequenced, sequencing result is subjected to splicing and is assembled into one complete turn
Record group takes longest transcript in every gene to obtain the mixing library Unigenes by splicing as Unigene；
S2. SSR detection is carried out to the library mixing Unigenes in step S1；
S3. SSR design of primers is carried out, and identifies the polymorphism of SSR primer.
In still another embodiment of the invention, in the step S1,
The elder is golden elder (Sambucus nigra)；
Spliced using Trinity software, splicing major parameter is SS_lib_type, min_kmer_cov, min_
glue；
In still another embodiment of the invention, in the step S2,
The search of the site SSR, search criterion are as follows: dinucleotides, trinucleotide, tetranucleotide, five are carried out using MISA software
Nucleotide and the minimum number of repetition of Hexanucleotide are respectively 6,5,5,5 and 5 times；
In still another embodiment of the invention, in the step S3,
SSR design of primers, the site SSR flanking sequence length >=50bp are carried out using 3.0 software of Primer.Design of primers
Major parameter:
(1) for annealing temperature (Tm) between 55-75 DEG C, the Tm value of upstream and downstream primer differs≤5 DEG C；
(2) pcr amplification product size is between 50-500bp, and primer length is between 15-30bp；
(3) G/C content is between 40%-60%, and primer free dimer and secondary structure go out between the primer of upstream and downstream
It is existing；
The Blast verifying of SSR primer is carried out in the library Unigenes to the SSR primer of design.
Embodiment
1 materials and methods
1.1 transcript profile data sources
Golden elder transcript profile data source carries out one plant of adult golden elder fruit in 2016 in this seminar
The result of 2000 high-flux sequence of Illumina HiSeq.The fruit of 3 developmental stages (Chinese olive, in ripe, late-maturing) is taken when sequencing
Real, commission Beijing Nuo Hezhi source genome company carries out the sequencing of RNA-Seq transcript profile after extracting RNA, and passes through De Novo method group
Dress obtains 257975 Unigene, as analysis background data.
1.2 sites transcript profile SSR identify
Golden elder transcript profile data are spliced using Trinity software, the transcript sequence information spliced
With the storage of FASTA format, splicing major parameter is SS_lib_type, min_kmer_cov, min_glue.It is obtained by splicing
The library Unigenes, and the search of the site SSR, search criterion are carried out with MISA program to mixed Unigenes Cooley are as follows: dinucleotide
Acid, trinucleotide, tetranucleotide, pentanucleotide and the minimum number of repetition of Hexanucleotide are respectively 6,5,5,5 and 5 times.
1.3 SSR design of primers
The sequence containing the site SSR is drawn using 3.0 design of primers program of Primer (2.3.5 editions, default parameters)
Object design, and the site SSR flanking sequence length >=50bp；The major parameter of design of primers: (1) annealing temperature (Tm) is in 55-
Between 75 DEG C, the Tm value of upstream and downstream primer differs≤5 DEG C；(2) pcr amplification product size is between 50-500bp, primer length
Between 15-30bp；(3) G/C content is between 40%-60%, and primer free dimer and second level knot between the primer of upstream and downstream
The appearance of structure.The blast verifying of SSR primer is carried out in the library Unigenes to the SSR primer of design.
1.4 vegetable materials and DNA are extracted
Choose 8 plants of elder mature leaf (3 plants of Canadian elder, 1 plant of sambucus racemosa, 1 plant of floral leaf elder,
1 plant of golden plumage elder, 2 plants of elder), DNA is all made of Ying Ruicheng biochemical technology (Shanghai) Co., Ltd. plant tissue genome
DNA extraction kit (paramagnetic particle method), -70 DEG C save backup.
1.5 fluorescent PCR amplification systems and program optimization
Primer screening is carried out with above-mentioned 8 elder mature leafs.Fluorescent primer PCR amplification system takes heminested PCR,
The optimization process such as decreased annealing temperature and progress grads PCR amplification obtain best fluorescent PCR amplification system and program.
Amplified production utilizes capillary electrophoresis detection: taking 0.3 μ L of PCR product, 0.5 μ L of molecular weight internal standard and deionization formyl
PCR plate is added in 9.5 μ L of amine mixing, and 95 DEG C of denaturation 5min are centrifuged, machine testing on 1 × Buffer buffer after 4 DEG C of coolings.
1.6 fluorescence SSR primer screenings
100 pairs of fluorescent primers are had from 3.0 software design of Primer gained by the farsighted Boxing section biotechnology in Beijing
The synthesis of limit company, way of purification PAGE.Primer screening principle is Tm value range: 55 DEG C -62 DEG C；The number of primer repetition base:
2bp, such as (AG), (AT) etc.；The number of repetition base: 8-11 times；PCR amplification system and program, which are selected from, tests resulting optimal knot
Fruit.
2 results and analysis
The distribution and design feature of SSR in 2.1 transcript profiles
(sequence overall length is scanned for by 257975 Unigene sequences to golden elder transcript profile
254175kb), it finds to contain 64148 sites SSR in wherein 51336 Unigene sequences, the site the SSR frequency of occurrences is
There is 1 SSR in 24.87% (ratio of SSR number of sites and total Unigene number), average every 3.96kb, and the type of SSR is abundant,
Wherein, mononucleotide repeats to be main Types, accounts for the 51.13% of total SSR.Followed by two, Trinucleotide repeats account for total respectively
The 38.63% of SSR and 9.11%.A/T and AG/CT is the advantage repetition primitive of mononucleotide and dinucleotides.Tetranucleotide, five cores
Thuja acid and Hexanucleotide repeat type negligible amounts account for 0.86%, 0.1% and 0.17% (table 3, Fig. 1) of sum respectively.
The distribution of 3 golden elder transcript profile difference SSR repetitive unit type of table
2.2 fluorescent PCR amplification systems and program optimization
Using golden elder leaf DNA as template, PCR system is optimized according to the primary condition of SSR-PCR and amplification program.
Fluorescent primer PCR amplification system and program are as follows:
Add M13Connector PCR amplification system: 1 μ L, 2 × Mix10 μ L, 10 μM of M of stoste containing DNA in 20 μ L PCR systems13It connects
Head and each 0.2 μ L of upstream and downstream primer, 0.1 μ L, 0.3 μ L, H2O 8.4μL；
Fluorescent primer PCR amplification program: 95 DEG C of initial denaturation 5min；Then 35 circulations are carried out, each circulation includes 95 DEG C
It is denaturalized 30s, 57 DEG C of annealing 30s (annealing temperature is different because of different primers), 72 DEG C of extension 30s；Last 72 DEG C of extensions 10min.
It is basic condition, the optimization such as decreased annealing temperature and progress grads PCR amplification with above-mentioned amplification system and program
Process obtains best fluorescent PCR amplification system and program is as follows:
Two step fluorescent primer PCR amplification methods:
(1) add M13Connector PCR amplification system: 15 μ L of μ L, 2 × Mix of stoste containing DNA, 10 μM of primers in 10 μ L PCR systems
Each 0.1 μ L, H2O 3.8μL；PCR amplification program is shown in Table 1.
(2) fluorescent primer PCR amplification system: 2 μ L of the DNA of amplified production containing first step stoste in 20 μ L PCR systems, 2 ×
10 μ L of Mix, 10 μM of M13 connectors (adding fluorescent marker) and reverse primer each 0.15 μ L, H2O 7.7μL；Fluorescent primer PCR amplification
Program is shown in Table 2.
The screening of 2.3 fluorescence SSR markers
51336 Unigene sequences in the site SSR carry out design of primers, devise 64148 pairs of SSR special primers altogether.For
The validity for verifying its primer selects 100 pairs of fluorescence SSR primers for having synthesized and having repeated primitive with dinucleotides at random, utilizes
The 100 pairs of primers (being shown in Table 4) screened carry out fluorescence SSR amplification to 8 parts of elder individuals not of the same race, the results showed that, 80 pairs are drawn
Object is augmented with band, i.e. amplification efficiency is 80%.There is 25 pairs of primers (SEQ ID NO.1- in expanding successful 80 pairs of primers
50) polymorphism is preferable, i.e., primer polymorphism efficiency is 31.25%.Fig. 2 is No. 5 primers in 8 parts of elder individuals not of the same race
Amplification situation.
Table 4
In the present invention, 257975 in transcript profile sequencing gained cDNA library are carried out to one plant of mature golden elder fruit
Unigene carries out SSR search, has obtained 64148 sites SSR, the frequency of occurrences 24.87%, two in most of species
Nucleotide and trinucleotide are the most common SSR repeat types, and from the point of view of golden elder SSR site structure, mononucleotide
(51.13%) frequency of occurrences highest, hence it is evident that be higher than dinucleotides (38.63%) and trinucleotide (9.11%)；Golden elder
It is AG/CT that dinucleotides advantage, which repeats primitive, in transcript profile.
Design of primers is carried out according to 257975 Unigene sequences, devises 64148 pairs of SSR special primers altogether.Pass through examination
It tests prioritization scheme and has obtained the optimal system of golden elder fluorescence SSR-PCR, in 100 pairs of fluorescent marker SSR primers of synthesis,
Wherein 80 pairs of primer amplifications have band, and effective amplification rate is 80%, and the failure of part primer amplification may be with primer and template quality
In relation to (20 pairs amplification failure primers be respectively in table 4 the 13rd, 14,18,20,23,29,34,36,50,52,57,58,59,
83,88,89,93,94,98 and 100 pairs of primers).25 pairs of primers have preferable polymorphism in 80 pairs of primers for having an amplified band, more
State property ratio is 31.25%, and polymorphism ratio size may be related with type, material to be tested and quantity.
Consider from SSR loci polymorphism potential, 64148 pairs of SSR primers of this researching and designing also availability with higher,
Be for elder Research on Genetic Variation it is feasible, this result of study, which also indicates that, utilizes golden elder transcript profile data mining
SSR marker is the analysis of elder Genetic Diversity of Germplasm, molecular mark, genetic map construction and functional gene
Excavation etc. lay a good foundation.Further work will be enlarged by primer screening range, and a large amount of SSR primers that transcript profile sequencing obtains will
It can be provided for researchs such as the resource classification of elder and other caprifoliaceae plants, the assignment of genes gene mapping and comparative genomics more, more
Effective molecular labeling.
The foregoing is merely preferred embodiment of the present application, are not intended to limit this application, for the skill of this field
For art personnel, various changes and changes are possible in this application.Within the spirit and principles of this application, made any to repair
Change, equivalent replacement, improvement etc., should be included within the scope of protection of this application.
SEQUENCE LISTING
<110>Shandong Forest Science Academy
<120>a kind of method that exploitation elder SSR primer is sequenced based on transcript profile
<130>
<160> 50
<170> PatentIn version 3.3
<210> 1
<211> 38
<212> DNA
<213>artificial sequence
<400> 1
tgtaaaacga cggccagtaa aagggtttgg cttgggga 38
<210> 2
<211> 20
<212> DNA
<213>artificial sequence
<400> 2
gtcaccccca caaaccctag 20
<210> 3
<211> 38
<212> DNA
<213>artificial sequence
<400> 3
tgtaaaacga cggccagtaa aatcgtctcg gaggctcc 38
<210> 4
<211> 20
<212> DNA
<213>artificial sequence
<400> 4
agcgcccttt atcagaaccc 20
<210> 5
<211> 38
<212> DNA
<213>artificial sequence
<400> 5
tgtaaaacga cggccagtaa acgatgggtt gctgttgc 38
<210> 6
<211> 21
<212> DNA
<213>artificial sequence
<400> 6
gatctgcttg taaggcactg c 21
<210> 7
<211> 38
<212> DNA
<213>artificial sequence
<400> 7
tgtaaaacga cggccagtaa agggcttgac cgacttgt 38
<210> 8
<211> 20
<212> DNA
<213>artificial sequence
<400> 8
tcatctttgc tccctcggaa 20
<210> 9
<211> 38
<212> DNA
<213>artificial sequence
<400> 9
tgtaaaacga cggccagtaa caaccccact cagagtcg 38
<210> 10
<211> 20
<212> DNA
<213>artificial sequence
<400> 10
taggcggtta gggtccacat 20
<210> 11
<211> 38
<212> DNA
<213>artificial sequence
<400> 11
tgtaaaacga cggccagtaa cctggtgtcc acgaagtc 38
<210> 12
<211> 20
<212> DNA
<213>artificial sequence
<400> 12
ttgttctagg ccccactcca 20
<210> 13
<211> 38
<212> DNA
<213>artificial sequence
<400> 13
tgtaaaacga cggccagtaa cgtgagcatc aaccccaa 38
<210> 14
<211> 21
<212> DNA
<213>artificial sequence
<400> 14
tcaattgtcg gccttcactg t 21
<210> 15
<211> 38
<212> DNA
<213>artificial sequence
<400> 15
tgtaaaacga cggccagtaa ctcactgcca ccactagc 38
<210> 16
<211> 20
<212> DNA
<213>artificial sequence
<400> 16
attcagccgc ctccaatctc 20
<210> 17
<211> 38
<212> DNA
<213>artificial sequence
<400> 17
tgtaaaacga cggccagtaa ctgtagcgtg aggccaaa 38
<210> 18
<211> 20
<212> DNA
<213>artificial sequence
<400> 18
tatacccgtg ctctggggaa 20
<210> 19
<211> 38
<212> DNA
<213>artificial sequence
<400> 19
tgtaaaacga cggccagtaa cttcaaacag catcgccg 38
<210> 20
<211> 20
<212> DNA
<213>artificial sequence
<400> 20
agcgtgattg cagggaaaga 20
<210> 21
<211> 38
<212> DNA
<213>artificial sequence
<400> 21
tgtaaaacga cggccagtaa gaactgtagc gtgaggcc 38
<210> 22
<211> 20
<212> DNA
<213>artificial sequence
<400> 22
tatacccgtg ctctggggaa 20
<210> 23
<211> 38
<212> DNA
<213>artificial sequence
<400> 23
tgtaaaacga cggccagtaa gaccctgttt ggttgggg 38
<210> 24
<211> 20
<212> DNA
<213>artificial sequence
<400> 24
cctgttctcg tggagccttt 20
<210> 25
<211> 38
<212> DNA
<213>artificial sequence
<400> 25
tgtaaaacga cggccagtaa gatcccatcg ccggaaag 38
<210> 26
<211> 20
<212> DNA
<213>artificial sequence
<400> 26
tgggcaagga tgactttggg 20
<210> 27
<211> 38
<212> DNA
<213>artificial sequence
<400> 27
tgtaaaacga cggccagtaa gcgcgtcact ttcccata 38
<210> 28
<211> 20
<212> DNA
<213>artificial sequence
<400> 28
agacactggg cacaagttgt 20
<210> 29
<211> 38
<212> DNA
<213>artificial sequence
<400> 29
tgtaaaacga cggccagtaa gcgcttgggc atttgttt 38
<210> 30
<211> 20
<212> DNA
<213>artificial sequence
<400> 30
acgggagagt tgcatgacag 20
<210> 31
<211> 38
<212> DNA
<213>artificial sequence
<400> 31
tgtaaaacga cggccagtaa ggcggcaata ccccaaat 38
<210> 32
<211> 22
<212> DNA
<213>artificial sequence
<400> 32
accatgtatg caaggtacaa ca 22
<210> 33
<211> 38
<212> DNA
<213>artificial sequence
<400> 33
tgtaaaacga cggccagtaa ggctatccaa gtgagcgg 38
<210> 34
<211> 20
<212> DNA
<213>artificial sequence
<400> 34
ttccgagacg cacgtattcc 20
<210> 35
<211> 38
<212> DNA
<213>artificial sequence
<400> 35
tgtaaaacga cggccagtaa gggttcgggt tcctaagg 38
<210> 36
<211> 22
<212> DNA
<213>artificial sequence
<400> 36
gaggttggag gtctttcaca ga 22
<210> 37
<211> 38
<212> DNA
<213>artificial sequence
<400> 37
tgtaaaacga cggccagtaa gtgggcagaa gcaagtga 38
<210> 38
<211> 20
<212> DNA
<213>artificial sequence
<400> 38
gagctgaatc tggtcgaggg 20
<210> 39
<211> 38
<212> DNA
<213>artificial sequence
<400> 39
tgtaaaacga cggccagtaa tcgccgccat ctcttcat 38
<210> 40
<211> 20
<212> DNA
<213>artificial sequence
<400> 40
tcctctaagc ctcctccgtt 20
<210> 41
<211> 38
<212> DNA
<213>artificial sequence
<400> 41
tgtaaaacga cggccagtaa tgtcctaggc gaagtggg 38
<210> 42
<211> 20
<212> DNA
<213>artificial sequence
<400> 42
cccactgaaa tgagggcagt 20
<210> 43
<211> 38
<212> DNA
<213>artificial sequence
<400> 43
tgtaaaacga cggccagtaa ttgcgtggcc tcgaaaac 38
<210> 44
<211> 20
<212> DNA
<213>artificial sequence
<400> 44
tgtttcaggg acgcaaagga 20
<210> 45
<211> 38
<212> DNA
<213>artificial sequence
<400> 45
tgtaaaacga cggccagtac aaaaacctca gggctcgt 38
<210> 46
<211> 20
<212> DNA
<213>artificial sequence
<400> 46
ccagtgatgc ttatgtgcgc 20
<210> 47
<211> 42
<212> DNA
<213>artificial sequence
<400> 47
tgtaaaacga cggccagtac aaagggttac atgcactttt ct 42
<210> 48
<211> 21
<212> DNA
<213>artificial sequence
<400> 48
tgcaacacct caaactgtcc t 21
<210> 49
<211> 38
<212> DNA
<213>artificial sequence
<400> 49
tgtaaaacga cggccagtac aactaggttg gggagtgg 38
<210> 50
<211> 21
<212> DNA
<213>artificial sequence
<400> 50
acaaccacaa atgccgttag c 21
Claims (10)
1. a kind of elder SSR primer pair, which is characterized in that the sequence of the elder SSR primer pair such as SEQ ID NO.1-2
It is shown, as shown in SEQ ID NO.3-4, as shown in SEQ ID NO.5-6, as shown in SEQ ID NO.7-8, such as SEQ ID
Shown in NO.9-10, as shown in SEQ ID NO.11-12, as shown in SEQ ID NO.13-14, as shown in SEQ IDNO.15-16,
As shown in SEQ ID NO.17-18, as shown in SEQ ID NO.19-20, as shown in SEQ ID NO.21-22, such as SEQ ID
Shown in NO.23-24, as shown in SEQ ID NO.25-26, as shown in SEQ ID NO.27-28, such as SEQ IDNO.29-30 institute
Show, as shown in SEQ ID NO.31-32, as shown in SEQ ID NO.33-34, as shown in SEQ ID NO.35-36, such as SEQ ID
Shown in NO.37-38, as shown in SEQ ID NO.39-40, as shown in SEQ ID NO.41-42, such as SEQ IDNO.43-44 institute
Show, as shown in SEQ ID NO.45-46, as shown in SEQ ID NO.47-48 or as shown in SEQ ID NO.49-50；
M is added in the end forward primer sequence 3' in each primer pair13Connector；M13Joint sequence are as follows: 5'-
TGTAAAACGACGGCCAGT-3'。
2. a kind of elder SSR primer sets, which is characterized in that the elder SSR primer sets are by two kinds in following primer pair
Or two or more compositions: primer pair, primer pair, such as SEQ ID as described in SEQ ID NO.3-4 as described in SEQ ID NO.1-2
Primer pair described in NO.5-6, primer pair, primer pair, such as SEQ as described in SEQ ID NO.9-10 as described in SEQ ID NO.7-8
Primer pair described in ID NO.11-12, as described in SEQ ID NO.13-14 primer pair, as described in SEQ IDNO.15-16 primer pair,
As described in SEQ ID NO.17-18 primer pair, as described in SEQ ID NO.19-20 primer pair, as described in SEQ ID NO.21-22
Primer pair, primer pair, primer pair, such as SEQ ID as described in SEQ ID NO.25-26 as described in SEQ ID NO.23-24
Primer pair described in NO.27-28, primer pair, the primer pair, such as described in SEQ ID NO.31-32 as described in SEQ IDNO.29-30
Primer pair described in SEQ ID NO.33-34, as described in SEQ ID NO.35-36 primer pair, draw as described in SEQ ID NO.37-38
Object to, primer pair, primer pair, such as SEQ IDNO.43-44 as described in SEQ ID NO.41-42 as described in SEQ ID NO.39-40
The primer pair, primer pair, primer pair and such as SEQ ID as described in SEQ ID NO.47-48 as described in SEQ ID NO.45-46
Primer pair described in NO.49-50.
3. a kind of elder SSR primer sets as claimed in claim 2, which is characterized in that the elder SSR primer sets by with
Lower primer pair composition: primer pair, primer pair, such as SEQ ID as described in SEQ ID NO.3-4 as described in SEQ ID NO.1-2
Primer pair described in NO.5-6, primer pair, primer pair, such as SEQ as described in SEQ ID NO.9-10 as described in SEQ ID NO.7-8
Primer pair described in ID NO.11-12, as described in SEQ ID NO.13-14 primer pair, as described in SEQ IDNO.15-16 primer pair,
As described in SEQ ID NO.17-18 primer pair, as described in SEQ ID NO.19-20 primer pair, as described in SEQ ID NO.21-22
Primer pair, primer pair, primer pair, such as SEQ ID as described in SEQ ID NO.25-26 as described in SEQ ID NO.23-24
Primer pair described in NO.27-28, primer pair, the primer pair, such as described in SEQ ID NO.31-32 as described in SEQ IDNO.29-30
Primer pair described in SEQ ID NO.33-34, as described in SEQ ID NO.35-36 primer pair, draw as described in SEQ ID NO.37-38
Object to, primer pair, primer pair, such as SEQ IDNO.43-44 as described in SEQ ID NO.41-42 as described in SEQ ID NO.39-40
The primer pair, primer pair, primer pair and such as SEQ ID as described in SEQ ID NO.47-48 as described in SEQ ID NO.45-46
Primer pair described in NO.49-50.
4. elder SSR primer pair as described in claim 1 and/or elder SSR primer sets described in claim 2 or 3
Application；The application includes:
1) application in elder analysis of genetic diversity；
2) application in building elder genetic map；
3) application in elder Germplasm Identification；
4) application in elder Genetic relationship；
5) application in the excavation of elder functional gene；
6) application in elder molecular mark.
5. a kind of method for obtaining elder SSR molecular marker, which is characterized in that method includes:
S1. using elder genomic DNA as template, elder SSR primer pair or claim 2 described in claim 1 or power
Benefit requires the 3 elder SSR primer sets to carry out PCR amplification, obtains pcr amplification product；
S2. pcr amplification product step S1 obtained carries out capillary electrophoresis detection, obtains elder SSR molecular marker.
6. obtaining the method for elder SSR molecular marker as claimed in claim 5, which is characterized in that synthetism in the method
Wood be Canadian elder (Sambucus Canadensis), sambucus racemosa (Sambucus canadensis'Aurea'),
Floral leaf elder (Sambucus nigra'Variegata'), golden plumage elder (Sambucus racemosa'Plumosa
Aurea'), any one or more in elder (Sambucus williamsii Hance).
7. a kind of method that exploitation elder SSR primer is sequenced based on transcript profile, which is characterized in that method includes:
S1. it constructs elder transcript profile library and is sequenced, sequencing result is subjected to splicing and is assembled into a complete transcript profile,
Longest transcript in every gene is taken to obtain the mixing library Unigenes by splicing as Unigene；
S2. SSR detection is carried out to the library mixing Unigenes in step S1；
S3. SSR design of primers is carried out, and identifies the polymorphism of SSR primer.
8. the method that exploitation elder SSR primer is sequenced based on transcript profile as claimed in claim 7, which is characterized in that described
In step S1,
The elder is golden elder (Sambucus nigra)；
Spliced using Trinity software, splicing major parameter is SS_lib_type, min_kmer_cov, min_glue.
9. the method that exploitation elder SSR primer is sequenced based on transcript profile as claimed in claim 7, which is characterized in that described
In step S2,
The search of the site SSR, search criterion are as follows: dinucleotides, trinucleotide, tetranucleotide, five nucleosides are carried out using MISA software
Acid and the minimum number of repetition of Hexanucleotide are respectively 6,5,5,5 and 5 times.
10. the method that exploitation elder SSR primer is sequenced based on transcript profile as claimed in claim 7, which is characterized in that described
In step S3,
SSR design of primers, the site SSR flanking sequence length >=50bp are carried out using 3.0 software of Primer；The ginseng of design of primers
Number includes:
(1) for annealing temperature (Tm) between 55-75 DEG C, the Tm value of upstream and downstream primer differs≤5 DEG C；
(2) pcr amplification product size is between 50-500bp, and primer length is between 15-30bp；
(3) G/C content is between 40%-60%, and between the primer of upstream and downstream primer free dimer and secondary structure appearance；
The Blast verifying of SSR primer is carried out in the library Unigenes to the SSR primer of design.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103642912A (en) * | 2013-11-29 | 2014-03-19 | 中国农业科学院作物科学研究所 | Method for developing mung bean simple sequence repeat (SSR) primer based on transcriptome sequencing |
| CN106834510A (en) * | 2017-03-20 | 2017-06-13 | 西南林业大学 | A kind of method that pseudo-ginseng SSR primers are developed based on transcript profile sequence |
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2018
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103642912A (en) * | 2013-11-29 | 2014-03-19 | 中国农业科学院作物科学研究所 | Method for developing mung bean simple sequence repeat (SSR) primer based on transcriptome sequencing |
| CN106834510A (en) * | 2017-03-20 | 2017-06-13 | 西南林业大学 | A kind of method that pseudo-ginseng SSR primers are developed based on transcript profile sequence |
Non-Patent Citations (2)
| Title |
|---|
| 孙华等: "珍贵观赏树种接骨木的研究现状与展望", 《湖北农业科学》 * |
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