CN109868330A - It is a kind of based on RNA-Seq exploitation lens EST-SSR label and application - Google Patents
It is a kind of based on RNA-Seq exploitation lens EST-SSR label and application Download PDFInfo
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Abstract
The invention discloses a kind of based on the lens EST-SSR label of RNA-Seq exploitation and application, 125 lens EST-SSR labels of package technique large-scale development are sequenced by transcript profile in the present invention, give the corresponding forward and reverse primer sequence of these labels, the screening process of these labels is provided, and elaborates that these EST-SSR mark the application in terms of Lentil Germplasm Resource analysis of genetic diversity.EST-SSR that the present invention develops label success rate is higher, rich polymorphism, it can amplify a plurality of types of bands in 94 parts of lens germplasm DNA, it shows good versatility, and the polymorphism very abundant of these EST-SSR primers, lens marker assisted selection is helped quite big.
Description
Technical field
The present invention relates to a kind of based on the lens EST-SSR label of RNA-Seq exploitation and application, is related to molecular labeling skill
Art field.
Background technique
Lens are annual self-pollination, long day property herbaceous plant, while being also that a kind of important cold season eats beans, two
Times body (2n=2x=14) plant, Genome Size is about 4G.Lens are the sixth-largest edible beans in the world.Lens are mankind's drinks
The important sources of protein, carbohydrate, vitamin and microelement in food, while being also livestock high value feed.Lens are because of it
Nitrogen fixing capacity and be beneficial to shift of crops.But since lens are often planted in dead soil, and by arid,
High temperature stress and various diseases, the shadow of the reasons such as wilt disease, anthracnose, rust, neck rot, root rot and bloom disease
It rings, the single rate of lens is still lower.According to FAO data in 2016, world's per unit area yield that is averaged only had 1,152kg/ha.Therefore,
Seem very urgent using the yield that suitable Breeding Strategies improve lens.
Successfully some qualitative trait genes are imported into lens mainstream kind by traditional breeding method, however
Conventional method is ineffective and time-consuming and laborious in importing quantity character gene, is usually influenced by environment or genotype-environment interaction
It is larger.And molecular labeling has a wide range of applications in Germplasm Resources Diversity research and marker assisted selection.Wherein simple weight
Complex sequences (Simple sequence repeat, SSR) is widely present in Eukaryotic genome, large number of, is repeated
Property it is high, be one of most common molecular labeling.EST-SSR is from EST library (EST) or transcript profile sequencing data
The SSR of excavation, compared with genome SSR, exploitation is relatively easy.
Currently, EST-SSR is widely used in the genetic research of barley, cherry and sugarcane, but it is in lens
Application study is less.
Summary of the invention
The present invention overcomes above-mentioned the deficiencies in the prior art, provide a kind of lens EST-SSR based on RNA-Seq exploitation
Label and application.The present invention provides 125 lens EST-SSR marks of package technique large-scale development are sequenced by transcript profile
Note, and elaborate that these EST-SSR mark the application in terms of Lentil Germplasm Resource analysis of genetic diversity.
One group of lens EST-SSR label, including forward primer and reverse primer corresponding to following 125 sites:
Note: * indicates that predicted value is unequal with actual value, lists actual value.
Further, application of the above-mentioned lens EST-SSR label in the analysis of lens germplasm genetic diversity.
Further, the primer of above-mentioned lens EST-SSR label obtains by the following method:
1) the different lens material in 6 parts of source places is chosen, RNA is extracted and establishes cDNA library, and in Illumina platform
Upper sequencing obtains original reads；
2) by above-mentioned steps 1) obtain original reads by data processing acquisition clean reads, by clean
Transcript and unigene is formed after reads assembling splicing；
3) SSR primer pair is designed using the unigene that step 2) obtains, and is chosen from 88 parts of other lens materials
4 parts of different lens material extraction DNA of source place carry out PCR amplification, are screened according to amplification situation as PCR amplification template
EST-SSR primer；
4) extraction step 1) described in 6 parts of lens materials and step 3) described in 88 parts of lens materials (total 94
Part) DNA profiling of the DNA as PCR amplification, as PCR expansion after the EST-SSR primer addition fluorescent marker screened in step 3)
The primer of increasing carries out PCR amplification, carries out fluorescent marker capillary electrophoresis using amplified production to verification step 3) in screening
EST-SSR primer out.
Further, 6 parts of lens materials described in step 1) are the entire plant in lens seedling stage.
Further, data processing described in step 2) includes the following steps: to delete the reads comprising adapter, packet
Reads and low quality reads containing ploy-N.
Further, the reaction system of PCR amplification described in step 3): total volume is 10 μ l, contains 2.0 μ L of DNA
(10ng·μL-1), 1.0 μ L of primer pair (2.0pmol μ L-1)、2×Premix TaqTM5.0 μ L and ddH2O 2.0μL。
Further, the response procedures of PCR amplification described in step 3): 94 DEG C of 5min；94 DEG C of 30s, 52 DEG C of 45s, 72 DEG C
1min, 35 circulations；72 DEG C of 10min, 4 DEG C of preservations.
Further, the reaction system of PCR amplification described in step 4): 15 μ l of total volume contains 0.5 μ L of DNA
(10ng·μL-1), 1.5 μ L of primer pair (10pmol μ L-1), 1.5 μ L 10xBuffer, 0.2 μ L Taq enzyme (2.5U), 1.5 μ L
dNTP(2.5mmol·L-1)、0.75μL Mg2+With 10 μ L ddH2O。
Further, the response procedures of PCR amplification described in step 4): 94 DEG C of 5min；94 DEG C of 30s, difference annealing temperature
Degree annealing 30s, 72 DEG C of 30s, 30 circulations；72 DEG C of 5min, 60 DEG C of 40min, 4 DEG C of preservations.
The utility model has the advantages that
(1) RNA-Seq data volume based on the EST-SSR label that the present invention develops is big, has reliability.Root of the present invention
According to the lens RNA-Seq data of acquisition, 217,836 transcript and 161 are assembled, 095 unigene is closed respectively
Count 2.571 and 2.406 hundred million nucleotide.
(2) success rate is higher, rich polymorphism for the EST-SSR label that the present invention develops.Design 480 pairs of EST-SSR primers
Carry out primer screening.It meets the requirements the results show that sharing 276 pairs of primers.It is carried out using 94 parts of lens kinds 276 pairs of primers of confrontation
The verifying of SSR fluorescent marker Capillary Electrophoresis.The result shows that 125 pairs of primers be it is polymorphic, 43 pairs of primers are singlet, are accounted for respectively
The 26.04% and 8.96% of 480 pairs of primers.
(3) the EST-SSR versatility that the present invention develops is stronger.The present invention has collected the different lens germplasm in 94 parts of sources
It marks and tests and analyzes for EST-SSR, these 94 parts of germplasm origins are in 4 big geographic areas: 82 parts of China from East Asia
(wherein come from Xinjiang for 10 parts, 9 parts come from the Inner Mongol, and 7 parts come from Ningxia, and 10 parts come from Qinghai, and 11 parts come from Shanxi, and 8 parts come from
Shaanxi, 9 parts come from Gansu, and 9 parts come from Hubei, and 9 parts come from Yunnan), 1 part of Japan from East Asia；6 parts come from West Asia, (wherein
3 parts come from Turkey, and 3 parts come from Syria)；4 parts come from North America, (wherein coming from Canada for 3 parts, 1 part comes from the U.S.)；1 part
Morocco from north African.125 pairs of EST-SSR primers can amplify a plurality of types of in 94 parts of lens germplasm DNA
Band, it is shown that good versatility.
(4) the polymorphism very abundant of EST-SSR primer helps lens marker assisted selection quite big.
Detailed description of the invention
Fig. 1 EST-SSR fluorescent primer P233 is to part test material capillary electrophoresis result.
Three-dimensional PCA analysis chart of the Fig. 2 based on 125 EST-SSR, the 94 portions of lens marked.
The UPGMA dendrogram that Fig. 3 is marked based on EST-SSR.
The group structure analysis chart that Fig. 4 is marked based on EST-SSR.
Specific embodiment
One, materials and methods
1, vegetable material
6 portions of lens (Lens culinaris Medikus) is chosen as test material, respectively A0000008 comes from
Shanxi Province, China, A0000094 come from Qinghai, and A0000170 comes from Shaanxi, China, and A0000370 comes from Morocco,
A0000669 is from Canada, and A0000677 is from Japan.Every part of material samples in triplicate, and is labeled as A0000008-1,
A0000008-2, A0000008-3, A0000094-1, A0000094-2, A0000094-3, A0000170-1, A0000170-2,
A0000170-3, A0000370-1, A0000370-2, A0000370-3, A0000669-1, A0000669-2, A0000669-3,
A0000677-1, A0000677-2 and A0000677-3, while RNA-Seq is carried out using Illumina HiSeq.
The 6 parts of lens germplasm and 88 parts of other lens germplasm for carrying out RNA-Seq are used for EST-SSR detection point simultaneously
Analysis.In addition 88 parts of germplasm origins are in 3 big geographic areas: 79 parts from East Asia China (wherein 10 parts come from Xinjiang, 9
Part come from the Inner Mongol, 7 parts come from Ningxia, 9 parts come from Qinghai, 10 parts come from Shanxi, 7 parts come from Shaanxi, 9 parts come from Gansu, 9 parts
From Hubei, 9 parts come from Yunnan)；6 parts come from West Asia, (wherein coming from Turkey for 3 parts, 3 parts come from Syria)；3 parts from north
Beauty, (wherein coming from Canada for 2 parts, 1 part comes from the U.S.).
Laboratory sample is from Shandong Crop Germplasm Resource Center, Jinan China.Details are given in Table 1.
1. 94 parts of lens germplasm origins of table ground
2, test method
(1) lens Illumina sequencing and transcript profile assembling
Using Trizol extraction method (Tiangen, BeiJing, China) to the whole strain (packet of 6 parts of lens seedling stages (after planting 4 weeks)
Including root, stem and leaf) RNA of material extracts respectively, and each sample takes 1.5 μ gRNA as RNA sample and prepares material.
The RNA sample of above-mentioned acquisition passes through NEBNext Ultra RNA Library Prep Kit for Illumina
(NEB, USA) prepares sequencing library, and index codes are added in the sequence of attributes of each sample.In short, utilizing
The magnetic bead of oligo-dT connection purifies mRNA from total serum IgE.In NEBNext the first chain synthesis reaction buffer under hot conditions
It is crushed in (5X) using bivalent cation, utilizes random hexamers and M-MuLV Reverse Transcriptase
(RNase H-) synthesizes the first chain cDNA, synthesizes the second chain cDNA followed by DNA polymerase i and RNA enzyme H, by nucleic acid outside
Remaining protruding terminus is converted flat end by enzyme cutting/polymerase activity, after the 3' terminal adenosine of DNA fragmentation is acidified, connection
NEBNext adapter with hairpin ring structure is to prepare to hybridize.Using AMPure XP system (Beckman Coulter,
Beverly, USA) purified library segment to selecting length for the cDNA segment of 150~200bp, uses the USER enzyme of 3 μ l
(NEB, USA) selects cDNA segment that is suitable, having connector to connect to be jointly processed by 15min at 37 DEG C with size, hereafter at 95 DEG C
Lower inactivation 5min.Then using treated DNA fragmentation as template, with Phusion High-Fidelity archaeal dna polymerase,
General PCR primer and Index (X) Primer carry out PCR.Finally, the purified pcr product in AMPure XP system, and
Library Quality is assessed in 2100 system of Agilent Bioanalyzer.Then, TruSeq PE Cluster Kit v3- is utilized
CBot-HS (Illumia) is indexed the cluster of coded samples in cBot Cluster Generation system.In fasciation
The library of Cheng Hou, preparation are sequenced on Illumina Hiseq platform, and generate double end reads (i.e. original reads).
Initial data stores reading in order and achieves (SRA), and label is in NCBI.
The initial data (original reads) of the FASTQ format of above-mentioned acquisition is handled by internal perl script first.
In this step, by deleting the reads comprising adapter, reads (the unknown base comprising ploy-N from initial data
10%) ratio is greater than to be obtained with low quality reads (reads that the base number of Q≤20 accounts for entire 50% or more read)
clean data.Q20, Q30, GC- content and sequence for calculating clean data simultaneously repeat horizontal.All downstream analysis
It is all based on the clean data of high quality.
Based on Left.fq and Right.fq, 2 are set by min_kmer_cov using Trinity, and every other parameter
Transcript profile assembling is carried out in the case where using as default.Elimination of Data Redundancy can by Corset (https: //
code.google.com/p/corset-project/).Corset can use the reads number for comparing upper transcript and
Expression pattern carries out hierarchical clustering to transcript and forms cluster, and a transcript longest in each cluster is retained, and claims
For unigene, and the analyses such as subsequent annotation are carried out with this.
For the comprehensive gene function information of acquisition, the function of unigene is annotated based on following seven large database concept:
NCBI non-redundant protein sequences (Nr, using diamond v0.8.22 with e-value=
1e-5)；NCBI nucleotide sequences (Nt, using NCBI blast 2.2.28+with e-value=1e-5)；Protein family (Pfam, using HMMER 3.0package and hmmscan with e-value=
0.01)；EuKaryotic Ortholog Groups (KOG, using diamond v0.8.22 with e-value=1e-3)；SwissProt (using diamond v0.8.22 with e-value=1e-5)；Kyoto Encyclopedia of
Genes and Genomes (KO, using KEGG Automatic Annotation Server with e-value=1e-10)；Gene Ontology (GO, using Blast2GO v2.5 and self-writing script with e-value
=1e-6)。
(2) lens DNA is extracted
Using Tiangeng biochemical technology (Beijing) company's plant genes group DNA extraction kit (DP320-03) to 94 parts
The seedling fresh leaf (after planting 4 weeks) of material (6 parts including RNA-Seq) carries out the extraction of genomic DNA, and in strict accordance with producer
Instruction carry out laboratory treatment.DNA mass and concentration are detected with NanoDrop One, and DNA is diluted to 25ng/ μ L storage
At -20 DEG C in case used in PCR.
(3) SSR detection and design of primers
Utilize MISA (MIcroSAtellite identification tool, http://pgrc.ipk-
Gatersleben.de/misa/misa.html transcript profile SSR detection) is carried out.Parameter setting are as follows: SSR repetitive unit length is most
Small value is mono- 10, two -6, three -5, four -5, five -5 and six -5.In a composite S SR, allow between two different SSR
It is 100bp that maximum, which interrupts size,.Utilize 3.0 software (https:/SourceMemi.net/Projects/primer of Primer
3/) and SSR primer pair is designed according to flank conserved sequence and filters out microsatellite locus.
(4) EST-SSR primer screening
Lens germplasm that EST-SSR primer screening is differed greatly using 4 source places (A0000036, A0000158,
A0000667, A0000376), every part of germplasm selects 3 single plants, totally 12 single plants, extracts DNA.Primer is by the prosperous biology in Beijing Chinese catalpa
Science and Technology Ltd.'s synthesis.Premix Taq used in PCR amplification and electrophoresisTM(TaKaRa TaqTMVersion 2.0) and
DL500DNA Marker is purchased from TaKaRa Biotechnology (Dalian) Co., Ltd.EST-SSR optimizing reaction system is
10 μ L, including 2.0 μ L of DNA (10ng μ L-1), 1.0 μ L of primer pair (2.0pmol μ L-1)、2×Premix TaqTM 5.0μL
And ddH2O 2.0μL.Pcr amplification reaction is enterprising in TAdvanced 96SG gradient gene-amplificative instrament (Biometra, Germany)
Row.PCR response procedures are 94 DEG C of 5min；94 DEG C of 30s, 52 DEG C of 45s, 72 DEG C of 1min, 35 circulations；72 DEG C of 10min, 4 DEG C of preservations.
Amplified production is in 8% non-denaturing polyacrylamide gel (acrylamide Acr ﹕ N'-N' methylene diacrylamide Bis=29 ﹕ 1,1
× tbe buffer liquid) on detected.Each 1.5 μ L, 50bp DNA Ladder of sample point sample is molecular weight standard, 260V electricity
Piezoelectricity swimming 1h30min, in 0.1% AgNO3Silver staining in solution, develops the color in NaOH solution.
(5) SSR fluorescent marker Capillary Electrophoresis
In each pair of end addition of primer 5 ' fluorescent marker FAM (6-carboxy-fluorescein) of the EST-SSR that filters out or
HEX(5-hexachloro-fluorescein).Fluorescent primer used is closed by Beijing Zi Xi Biotechnology Co., Ltd in test
At.The ddH of primer sterilizing2O dissolves and is diluted to 10 μm of ol μ L-1It is spare.DNA used in test is the 94 parts small of extraction
The DNA of hyacinth bean material.Other reagents are purchased from TaKaRa Biotechnology (Dalian) Co., Ltd.The PCR of Capillary Electrophoresis
Amplification uses the reaction system of 15 μ L, comprising: 0.5 μ L DNA (10ng μ L-1)、2μmol·L-1, 1.5 μ L primer pairs
(10pmol·μL-1), 1.5 μ L 10xBuffer, 0.2 μ L Taq enzyme (2.5U), 1.5 μ L dNTP (2.5mmolL-1)、0.75
μL Mg2+With 10 μ L ddH2O.PCR reaction system is 94 DEG C of 5min；94 DEG C of 30s, different annealing temperature annealing 30s, 72 DEG C of 30s,
30 circulations；72 DEG C of 5min, 60 DEG C of 40min, 4 DEG C of preservations.PCR product is diluted 50 times, is added respectively in each hole of 96 orifice plates
Enter 9.5 μ L diluted internal standard (GS500LIZ:HIDI=1:120) and 0.5 μ L dilution after PCR product, 95 DEG C of denaturation 5min, in
Cooled on ice 2min, then be placed on ABI 3730XL DNA analysis instrument (Applied Biosystems, Foster City, USA)
Carry out automatic fluorescence detection.Capillary Electrophoresis program is prerunning 15kV, 2min；2kV voltage sample introduction 10s；Electrophoresis 15kV,
20min.Finally setting endogenous control (Size Standard) and parameter (Parameters), using Data Collection with
Genemapper V4.0 software carries out data collection and image analysis.
Two, data statistics and analysis
According to Genemapper V4.0 analysis as a result, reading the numerical value of each allele of each fluorescent marker, it is lower than
500 be considered as invalid peak, gives up and does not have to.The size of each allele is recorded, is stored at Excel table, further according to not
Call format with software makees corresponding conversion.PCA analysis is carried out using NTSYS-pc V2.10e.Utilize PowerMarker
V3.25 calculates the genetic diversity parameter of each EST-SSR label, comprising: major allele frequency, number of alleles, base
Because of diversity, heterozygosity and polymorphism information content (PIC) etc..Material of participating in the experiment is based in conjunction with MEGA V6.0 analysis software
The phylogenetic tree of UPGMA method constructs.Group structure analysis is carried out to material of participating in the experiment using Structure V2.3.4, parameter is set
It is fixed as follows: Length of Burnin Period=10000, Number of MCMC Reps after Burnin=
10000, subgroup quantity K (Number of population)=1-20, recurring number (Number of Iterations)=20.
According to Delta K (Δ K) value determine best group structure and subgroup number (network address of on-line analysis is http: //
taylor0.biology.ucla.edu/struct_harvest/)。
Three, result
(1) lens Illumina sequencing and transcript profile assembling
18 lens sample A0000008-1, A0000008-2, A0000008-3, A0000094-1, A0000094-2,
A0000094-3, A0000170-1, A0000170-2, A0000170-3, A0000370-1, A0000370-2, A0000370-3,
A0000669-1, A0000669-2, A0000669-3, A0000677-1, A0000677-2 and A0000677-3 pass through
The sequencing of IlluminaHiSeq system produces 0.677 respectively, 0.713,0.746,0.684,0.816,0.755,0.888,
0.890,0.749,0.810,0.756,0.690,0.704,0.701,0.737,0.884,0.991 and 0.961 hundred million raw
reads.Be respectively 0.657 by filtered sequencing data, 0.684,0.731,0.657,0.802,0.742,0.873,
0.876,0.738,0.798,0.744,0.681,0.675,0.673,0.710,0.852,0.954 and 0.927 hundred million clean
reads.These clean reads' is spliced into 217,836 Transcript and 161, and 095 unigene adds up to respectively
2.571 and 2.406 hundred million nucleotide.The average length of Transcript be 1,180bp, N50 and N90 be respectively 2,075bp and
479bp；The average length of Unigene is that Isosorbide-5-Nitrae 94bp, N50 and N90 are respectively 2,203bp and 714bp (table 2).
2. lens transcript profile of table sequencing assembling result
(2) polymorphism analysis of EST-SSR label
According to RNA-Seq as a result, devising 26,449 pairs of EST-SSR primers in total, 480 pairs of primers have therefrom been randomly choosed
It is verified.EST-SSR primer screening is carried out using the material that 4 parts of source places differ greatly.The results show that in 480 pairs of primers
There are 276 pairs of primer amplifications to go out clearly band, remaining primer is without amplified production or amplifies complicated product.Whole is utilized later
94 parts of lens materials carry out the verifying of EST-SSR fluorescent marker Capillary Electrophoresis to 276 pairs of primers.The result shows that 125 pairs of primers
It is polymorphic (table 3), 43 pairs of primers are singlet, account for the 26.04% and 8.96% of 480 pairs of primers respectively, remaining primer capillary
Electrophoresis tube is ineffective, is referred to no amplified production or amplifies in the primer of complicated product, the forward directions of 125 labels of acquisition
Primer and reverse primer.Fig. 1 is fluorescent primer P233 to part test material capillary electrophoresis result, allele clear reading
Accurately and reliably.Table 3 the results show that major allele frequency range from 0.1882 to 0.9946, average value 0.7143；Equipotential
Gene number range is from 2 to 17, average value 5.1520；From 0.0107 to 0.8836, average value is gene diversity range
0.3800；Heterozygosity range is from 0 to 1, average value 0.2541；From 0.0106 to 0.8727, average value is PIC range
0.3407.The above result shows that the polymorphism very abundant of this 125 pairs of EST-SSR primers, helps lens marker assisted selection
It helps quite big..
3. 125 pairs of the table EST-SSR primers with polymorphism
(3) analysis of genetic diversity of EST-SSR label
PCA analysis
Using NTSYS-pc V2.10e the 94 parts of lens germplasm all participated in the experiment are carried out with the PCA marked based on EST-SSR
Analysis, obtains three-dimensional space dendrogram (Fig. 2).Preceding three-dimensional contribution rate is 98.45%.The results show that (black is ellipse for Chinese Resources
Circle) it is clearly separated with the distribution of foreign resources (grey oval) spatially, show affiliation between the two farther out,
It is significant related to geographic origin.Chinese Resources significantly build up the right side for being mainly distributed on dendrogram together, only 2 parts of resources
Except, this shows that the hereditary basis of Chinese Resources is totally consistent and more narrow, dramatically different with foreign resources；And foreign resources
Broad, equally distributed space characteristics are presented, shows their genetic background extensively and Distribution center is equal compared with Chinese Resources
It is even.Only have 1 part of Japanese resource distribution in Chinese Resources in foreign resources, shows the genetic affinity of this part of germplasm and Chinese Resources
It is relatively close.
UPGMA clustering
It is calculated using PowerMarker V3.25 and obtains genetic distance (Nei ' s, 1972), analyzed in conjunction with MEGA V6.0 soft
Part is drawn 94 parts of lens germplasm (including 6 parts of RNA-Seq germplasm and remaining 88 parts verifying germplasm) and is marked based on EST-SSR
The UPGMA dendrogram (Fig. 3) of note, open circle represent 6 parts of lens resources of RNA-Seq；Filling circle represents remaining 88 parts
Lens resource；There is no the circle of box to represent Chinese lens resource；The circle come is outlined by box represents foreign lens resource.
The results show that 94 parts of lens germplasm are divided into 2 big groups.2 subgroups are divided into again in first group, the first subgroup only has
3 parts, it all is from the Syria and Turkey of West Asia.Second subgroup is nearly all Chinese Resources, only 1 part Japanese resource.In
Two major features are presented in state's resource, first is that the genetic affinity between each resource-saving is close, second is that the in one's duty resource of each province is often gathered
It gathers together, such as Xinjiang, Shanxi, Yunnan, Hubei, but each province resource also has intersection and infiltration.This shows Chinese Resources
Generally hereditary basis is narrow, but the genetic affinity of resource has certain difference between each province.Second group is divided into 2 Asias again
Group, most of is foreign resources.3 parts of America & Canadas from North America in first subgroup, 1 part of Morocco from north African,
Second subgroup source place is more, 3 parts of Syria and Turkey from West Asia, and 2 parts come from Xinjiang, China and Shaanxi, and 1 part comes from
The Canada of North America.Genetic affinity between foreign resources farther out, shows that its hereditary basis is more wide in range.
Group structure analysis
Analysis (the figure of the group structure based on EST-SSR is carried out to 94 parts of lens germplasm using STRUCTURE V2.3.4
4).The result shows that Delta K (Δ K) value numerical value highest in K=2, material of participating in the experiment are divided into 2 subgroups.It is wherein dark to indicate
First subgroup, the overwhelming majority are Chinese Resources, only 1 part of Japan material (A0000677)；Light color indicates the second subgroup, most of
For foreign resources, only 2 parts of Chinese materials (A0000170 and A0000491), respectively from the Shaanxi and Xinjiang of China.This knot
Fruit analyzes with PCA and UPGMA clustering is consistent.
Claims (9)
1. one group of lens EST-SSR label, which is characterized in that including forward primer corresponding to following 125 sites and instead
To primer:
2. application of the lens EST-SSR label as described in claim 1 in the analysis of lens germplasm genetic diversity.
3. a kind of method for obtaining lens EST-SSR label as described in claim 1, which is characterized in that including walking as follows
It is rapid:
1) the different lens material in 6 parts of source places is chosen, RNA is extracted and establishes cDNA library, and surveyed on Illumina platform
Sequence obtains original reads；
2) by above-mentioned steps 1) obtain original reads by data processing acquisition clean reads, by clean reads group
Transcript and unigene is formed after dress splicing；
3) SSR primer pair is designed using the unigene that step 2) obtains, and chooses source from 88 parts of other lens materials
Ground 4 lens material extraction DNA of difference carry out PCR amplification as PCR amplification template, screen EST-SSR according to amplification situation
Primer；
4) extraction step 1) described in 6 parts of lens materials and step 3) described in 88 parts of lens materials (94 parts total)
DNA profiling of the DNA as PCR amplification, as PCR amplification after the EST-SSR primer addition fluorescent marker screened in step 3)
Primer carries out PCR amplification, carry out fluorescent marker capillary electrophoresis using amplified production to verification step 3) in filter out
EST-SSR primer.
4. method as claimed in claim 3, which is characterized in that 6 parts of lens materials described in step 1) are lens seedling
The entire plant of phase.
5. method as claimed in claim 3, which is characterized in that data processing described in step 2) includes the following steps: to delete
Except the reads comprising adapter, the reads comprising ploy-N and low quality reads.
6. method as claimed in claim 3, which is characterized in that the reaction system of PCR amplification described in step 3): total volume is
10 μ l contain 2.0 μ L of DNA (10ng μ L-1), 1.0 μ L of primer pair (2.0pmol μ L-1)、2×Premix TaqTM5.0μL
And ddH2O 2.0μL。
7. method as claimed in claim 3, which is characterized in that the response procedures of PCR amplification described in step 3): 94 DEG C
5min；94 DEG C of 30s, 52 DEG C of 45s, 72 DEG C of 1min, 35 circulations；72 DEG C of 10min, 4 DEG C of preservations.
8. such as the described in any item methods of claim 3-7, which is characterized in that the reactant of PCR amplification described in step 4)
System: 15 μ l of total volume contains 0.5 μ L of DNA (10ng μ L-1), 1.5 μ L of primer pair (10pmol μ L-1)、1.5μL
10xBuffer, 0.2 μ L Taq enzyme (2.5U), 1.5 μ L dNTP (2.5mmolL-1)、0.75μL Mg2+With 10 μ L ddH2O。
9. such as the described in any item methods of claim 3-7, which is characterized in that the reaction interval of PCR amplification described in step 4)
Sequence: 94 DEG C of 5min；94 DEG C of 30s, different annealing temperature annealing 30s, 72 DEG C of 30s, 30 circulations；72 DEG C of 5min, 60 DEG C of 40min, 4
DEG C save.
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| CN114574627A (en) * | 2022-04-25 | 2022-06-03 | 山东省农业科学院 | Pea neutral SNaPshot marker and application thereof in population genetic diversity analysis |
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| CN114574627B (en) * | 2022-04-25 | 2023-08-22 | 山东省农业科学院 | Pea neutral SNaPshot marker and application thereof in population genetic diversity analysis |
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