CN108660238A - Oat drought resistance related SNP molecular labeling based on GBS technologies and its application - Google Patents
Oat drought resistance related SNP molecular labeling based on GBS technologies and its application Download PDFInfo
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- CN108660238A CN108660238A CN201810296055.XA CN201810296055A CN108660238A CN 108660238 A CN108660238 A CN 108660238A CN 201810296055 A CN201810296055 A CN 201810296055A CN 108660238 A CN108660238 A CN 108660238A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention belongs to oat marker assisted selection technical field, the experience sex chromosome mosaicism of oat Identification of Drought is carried out to solve current oat dependent on biological character, provides a kind of oat drought resistance related SNP molecular labeling based on GBS technologies and its application.The SNP marker be nucleotide sequence shown in SEQ ID NO.1 from 5 ' ends rise the 126th, nucleotide sequence shown in SEQ ID NO.2 the 84th from 5 ' ends, nucleotide sequence shown in SEQ ID NO.3 is the 31st and 61 from holding 5 ', totally 4 SNP sites, loci polymorphism is respectively A/G, G/T, G/T, C/G.The marker assisted selection that the present invention is suitable for carrying out existing a large amount of oat germplasm early stage drought resistance screening works.
Description
Technical field
The invention belongs to oat marker assisted selection technical fields, disclose a kind of oat drought resistance based on GBS technologies
Related SNP molecular labeling and its application.The molecular labeling is the low anti-and highly resistance group that is stablized using oat drought resistance as material
Material, by simplifying genome-Genotyping（GBS-SNP）Base in technology and Fisher Test significance difference analysis and population
Because type frequency obtains.Using SNP marker disclosed in this invention, foundation is provided to the early stage drought resisting screening of oat germplasm, it can be big
It is big to shorten breeding cycle, improve oat Drought-resistant Breeding efficiency.
Background technology
Oat（Avena sativa L.）It is the seventh-largest crop culture in the world, plantation is in including northern China extensively
All over the world, due to rich in the beta glucan with hypoglycemic and blood fat needed for masses, and arid is always to restrict to make
An important factor for produce amount, drought-resistant breeding are also the important content of oat genetic breeding research.Shanxi Shanxi Academy of Agricultural Sciences
By conventional breeding for many years and phenotypic evaluation research, a variety of oat varieties for adapting to different damages caused by a drought of selection and breeding are used for Production of Large Fields.
The oat conventional breeding period is long, efficiency is low, and molecular labeling assists（MAS）Breeding is to accelerate the important hand of the accurate breeding of oat
Section.Therefore, carry out oat drought resisting（MAS）Breeding will effectively shorten oat breeding cycle, be the early stage drought resisting screening of oat germplasm
Foundation is provided.
The report about oat molecular labeling is concentrated mainly on SSR and AFLP labels at present, relative to above-mentioned technology, monokaryon
Nucleotide polymorphism（SNP）It is that most the molecular labeling of advantage, the GBS-SNP technologies based on sequencing are handled by digestion and removed at present
Genome repetitive sequence significantly reduces sequencing amount and sequencing cost, and can cover whole gene group, is not limited by reference gene group
System, thus it is widely applied to exploitation high density, in high-precision crops molecule marking research.
Invention content
The present invention carries out the experience sex chromosome mosaicism of oat Identification of Drought in order to solve current oat dependent on biological character,
Provide a kind of oat drought resistance related SNP molecular labeling based on GBS technologies and its application.
The present invention is realized by following technical solution：A kind of oat drought resistance related SNP molecule mark based on GBS technologies
Note, the SNP marker are nucleotide sequence shown in SEQ ID NO.1 the 126th from 5 ' ends, shown in SEQ ID NO.2
Nucleotide sequence is from 5 ' ends play the 84th, nucleotide sequence shown in SEQ ID NO.3 is the 31st and 61, totally 4 from holding 5 '
SNP site, loci polymorphism are respectively A/G, G/T, G/T, C/G.
The acquisition methods of the SNP marker are：Using paramagnetic particle method plant genome DNA extraction agent box, extraction is different
The genomic DNA of drought resisting degree oat natural population obtains oat gene group SNP information using GBS technologies, soft using Stacks
Part structure oat simplifies genome reference sequences and detects group SNP, and Fisher Test carry out significance difference analysis, determine
Significant difference, that is, FDR<0.001 SNP, according to genotype, distribution frequency threshold value is 1 between population, is obtained and oat drought resistance phase
The SNP marker of pass.
GBS technical limit spacing oat gene group SNP information specific methods are：The different drought resisting degree oat natural populations of extraction
Genomic DNA recycle the digestion piece of 220-450bp sizes with restriction enzyme PstI/MspI double digestion genomic DNAs
Section structure GBS libraries carry out double ends high-flux sequences using Illumina Hiseq microarray datasets, and sequencing pattern is 2 ×
150bp, lower machine data are preserved with both-end FASTQ formats, and software FastQC carries out data Quality Control, quality data acquisition methods
For：The double non-restriction enzyme site sequences in end of removal are polluted using the connector at the ends Adapter Removal removal 3'.
The Adapter Removal operating methods are：Mass filter, window size setting are carried out using slip window sampling
For 5bp, step-length is set as 1bp；It is moved along a base each time, takes the average Q value of 5 base calculation windows, if finally
Value≤2 Q of one base then only retain the base before the position；If average Q value≤20 of window only retain the window
Penultimate base and base before；Length filtration, if in double ends any one reads length≤50bp, go
Except double end reads.
Group's SNP detection method is：Quality data simplifies genome at oat using Stacks software combinations and refers to sequence
It is compared after row, populations filterings obtain oat group SNP site information, and clustering parameter is set as：-m4；
Populations parameters are set as：One site will at least occur in 1 group；Same site is detected in one group
The minimum percentage of individual 0.50；The minimum gene frequency 0.05 in one site.
The specific method that the relevant SNP marker of oat drought resistance determines is：The group SNP of acquisition uses GCTA softwares
Classified analysis on major constituents is carried out to all material, whether detection phenotype, that is, drought resistance and genotype, that is, group SNP groupings are consistent,
PCA shows that highly resistance and Di Kang groups are gathered for two clusters；It is aobvious that Fisher Test SNP genotypic differences are carried out to highly resistance and low anti-phenotype
The a certain genotype frequency of the analysis of work property, the site is 1 in low resist, and is 0 in highly resistance, obtains SNP relevant with drought resistance
Point.
The specific screening technique of oat Drought Resistance Germplasm is：The corresponding genotype of 4 SNP sites of the SNP marker
It is A/G, G/T, GG, when CC, then oat to be identified is the oat of low drought resisting；If 4 SNP sites of display lack（./.）,
Then oat to be identified is the oat of high drought resisting.
753,325 oats are assembled using the method for the invention and simplify genome reference sequences, annotate 74,657 altogether
A SNP site.It is finally obtained oat drought resistance related SNP molecular labeling.
The present invention is with oat natural population（The germplasm of Different Drought or improved variety）For research object, using GBS-
SNP genotyping techniques pass through Fisher Test significance difference analysis（FDR < 0.001）The genotype frequency between population
（Threshold value is set as 1）, molecular labeling of the exploitation for oat Drought Resistance Germplasm early stage assisting sifting.The SNP site is in low resist
Genotype be respectively AG, GT, GG, CC, in highly resistance the site be missing（./.）.These and oat drought resistance related SNP position
The invention of point solves and nowadays relies on the experience sex chromosome mosaicism that biological character carries out oat Identification of Drought, is based on the second generation
The GBS-SNP technologies of sequencing provide a SNP marker for oat Identification of Drought, to existing oat germplasm
Early stage drought resistance screening provides foundation.
Description of the drawings
Fig. 1 is oat GBS-SNP techniqueflow charts；Fig. 2 is oat GBS-SNP annotated sequences, and abscissa representative sample is indulged
Coordinate representation reads numbers；Fig. 3 is SNP site heterozygosity, and abscissa representative sample, ordinate indicates the pure of different SNP sites
Conjunction, heterozygosis, miss ratio.It can thus be appreciated that：The SNP site of homozygotic state in all sequencing samples（0/0 or 1/1）It occupies the minority, greatly
Different degrees of heterozygosis is presented in part（0/1）Or miss status（./.）；Fig. 4 is principal component analysis（PCA）, display sample is substantially
Gather for two clusters, all 11 parts of highly resistances, 4 parts of low anti-, 8 parts of moderate resistance germplasm and 6 non-identification of species gather for cluster；And other cluster is then
Including 14 parts of low anti-and 5 parts of moderate resistance germplasm；Remaining 6 unidentified oat varieties is then distributed in except two clusters.
Specific implementation mode
Implement below for illustrating the present invention, used oat sample standard deviation is by Shanxi Shanxi Academy of Agricultural Sciences variety of crops resource
Coarse cereals seminar of research institute collects, evaluates, and is stored in oat Germplasm Resources, paramagnetic particle method plant genome DNA extraction agent
Box extracts oat gene group DNA, carries out GBS library constructions and sequencing, and Stacks softwares simplify genome ginseng for building oat
It examines sequence and detects group SNP, R scripts（Fisher Test carry out significance difference analysis, genotype frequency threshold value between population
It is 1）It excavates and the relevant SNP marker of oat drought resistance.
A kind of oat drought resistance related SNP molecular labeling based on GBS technologies, the SNP marker are SEQ ID
Nucleotide sequence shown in NO.1 from 5 ' ends rise the 126th, nucleotide sequence shown in SEQ ID NO.2 the 84th from holding 5 ',
Nucleotide sequence shown in SEQ ID NO.3 is the 31st and 61 from 5 ' ends, and totally 4 SNP sites, loci polymorphism are respectively A/
G, G/T, G/T, C/G.
1. extracting genome DNA：This experiment is using the paramagnetic particle method plant base purchased from the Shanghai bio tech ltd Sheng Gong
Because of a group DNA extraction agent boxes, for extracting genomic DNA in oat young leaflet tablet tissue.With through the identification of drought-resistant character for many years
11 parts of highly resistances, 13 parts of moderate resistances, 11 parts of low anti-oat germplasm and 12 unidentified oat improved variety be test material, oat
Germination after two weeks, takes young leaflet tablet for extracting genomic DNA；
Plant sample is cracked using Buffer MPCB, releases plant genome DNA, protein, polysaccharide and cell fragment etc.
Impurity is by being centrifugation down.Supernatant is transferred in new centrifuge tube, MagicMag under the action of combining liquid
Genomic DNA in the absorption supernatant of Beeds selectivity.MagicMag Beeds are removed completely by the cleaning of cleaning solution
A small amount of impurity of absorption, the genomic DNA of MagicMag Beeds releases absorption, that is, obtain high-quality under the action of eluent
The genomic DNA of amount.It, can be with from 50mg plant tissues using this product extraction Plant Genome without phenol, chloroform
Obtain 15 μ g DNA, OD260/OD280Ratio is generally 1.7-1.9, and the DNA of extracting can be directly used for downstream tests.
2. GBS library constructions and sequencing：The step is completed by the bio tech ltd upper Shanghai's style Sen Nuo.With restricted
Restriction endonuclease PstI（CTGCAG）And MspI（CCGG）After carrying out digestion to the DNA of extraction, the digestion piece between 220-450bp is recycled
Section, carries out building library later according to dd-GBS methods.Double ends are carried out using llumina Hiseq microarray datasets（Paired-end,
PE）Sequencing is obtained lower machine data and is preserved with both-end FASTQ formats.Respectively to the lower machine data of each sample using FastQC into
Row quality control includes mainly base Mass Distribution（Per base sequence quality）, mass value（Per
sequence quality scores）, GC distribution（Per sequence GC content）, to the lower machine data matter of these reflections
The many index of amount is counted.
Further filtering data include some belt lacings, and double end reads1 5 ' hold the non-restriction enzyme site sequences of 6 bp
CTGCAG or reads2 5 ' holds the non-restriction enzyme site sequence C CGG of 4bp.Connector using 3 ' end of Adapter Removal removals is dirty
Dye.Mass filter is carried out using sliding window, window size is set as 5bp, and step-length is set as 1bp, is moved along one each time
A base takes the average Q value of 5 base calculation windows, if value≤2 Q of the last one base, before only retaining the position
Base only retain the window penultimate base and base before if average Q value≤20 of window.It filters out and is less than
The reads of 50bp.
3. group SNP is detected：The reads of each sample is clustered using the ustacks in Stacks software packages, together
One stack represents a restriction enzyme site（loci）, clustering parameter is set as-m4, to the loci and loci of each sample
Sequencing depth is counted.The loci of all samples is merged with cstacks in next step, is at most allowed between different sample loci
2 mispairing obtain the catalog consensus sequences of each loci.Using sstacks by the loci sequences of each sample with
Populations filterings obtain group SNP after catalog consensus sequence alignments.Major parameter includes：One site is most
To occur in 1 group less；The individual minimum percentage 0.50 in same site is detected in one group（When this in group
The miss rate in site is more than 50%, then removes the site）；The minimum gene frequency 0.05 in one site.
4. the determination of drought resistance related SNP label：For acquisition group SNP using GCTA softwares to all material into
Row classified analysis on major constituents, PCA show that highly resistance and Di Kang groups are gathered for two clusters；Using Fisher Test（Make R scripts, FDR by oneself
< 0.001）More high and low anti-material, determines the SNP of significant difference；The frequency being distributed between population according to genotype, unites respectively
Count four kinds of genotype in highly resistance and low anti-population（The genotype of 0/0 homozygous ref types, the gene of the ref/alt of 0/1 heterozygosis
Type, the genotype of 1/1 homozygous alt, the sites /are missing）The frequency that individual occurs sets population because Population is few
The threshold value of genotype frequency is 1, i.e., it is low it is anti-in the frequency that occurs of certain genotype individuals be 1, the genotype individuals occur in highly resistance
Frequency be 0, further identification with the relevant SNP site of oat drought resistance.Oat drought resistance associated SNP positions application is shown in Table 1.
Table 1
Note：* indicate that drought resistance degree, * more multilists show that drought resistance is stronger.
Sequence table
<110>Agricultural Biotechnology Research Center of Shanxi Province；Shanxi Shanxi Academy of Agricultural Sciences variety of crops resource is ground
Study carefully institute
<120>Oat drought resistance related SNP molecular labeling based on GBS technologies and its application
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 139
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
gtcgggttcg gtgtccgcta ggggtctttt ttggtccgaa accgaaaaca agcggttatt 60
ttatagtttt ggtgttttta aaatatttgc tagagatgct cttaggagtt accaattaag 120
gataactcta ataaaaaga 139
<210> 2
<211> 139
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
catctgaaat ctggagcaat agcactacac aaaaggaaag ctccagtgac aatgcatggg 60
gtaaaccagc tggaggaggt agtggattgg atgctggtgg cagctcttgg ggtggagcag 120
cagtcaacaa ggacagtga 139
<210> 3
<211> 148
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
gtgttgacat atatttggtc actgaaacca ggaaacataa caaagctgta cagaactatt 60
ctagcaactg aactcagcgg gcagtgacct cactaagtta caattttcat gattatggaa 120
cttctaaaag atagatgaaa taaagtag 148
Claims (7)
1. a kind of oat drought resistance related SNP molecular labeling based on GBS technologies, it is characterised in that：The SNP marker is
Nucleotide sequence shown in SEQ ID NO.1 is from 5 ' ends play the 126th, nucleotide sequence shown in SEQ ID NO.2 is from holding 5 '
84th, nucleotide sequence shown in SEQ ID NO.3 play the 31st and 61 from 5 ' ends, totally 4 SNP sites, loci polymorphism divide
It Wei not A/G, G/T, G/T, C/G.
2. a kind of oat drought resistance related SNP molecular labeling based on GBS technologies according to claim 1, feature exist
In：The acquisition methods of the SNP marker are：Using paramagnetic particle method plant genome DNA extraction agent box, different drought resisting journeys are extracted
The genomic DNA for spending oat natural population obtains oat gene group SNP information, using Stacks software buildings using GBS technologies
Oat simplifies genome reference sequences and detects group SNP, and Fisher Test carry out significance difference analysis, determines that difference is aobvious
It is FDR to write<0.001 SNP, according to genotype, distribution frequency threshold value is 1 between population, is obtained relevant with oat drought resistance
SNP marker.
3. a kind of oat drought resistance related SNP molecular labeling based on GBS technologies according to claim 2, feature exist
In：GBS technical limit spacing oat gene group SNP information specific methods are：The base of the different drought resisting degree oat natural populations of extraction
Because a group DNA recycles the endonuclease bamhi structure of 220-450bp sizes with restriction enzyme PstI/MspI double digestion genomic DNAs
The libraries GBS to be built, double ends high-flux sequence is carried out using Illumina Hiseq microarray datasets, sequencing pattern is 2 × 150bp,
Lower machine data are preserved with both-end FASTQ formats, and software FastQC carries out data Quality Control, and quality data acquisition methods are：Removal
Double non-restriction enzyme site sequences in end are polluted using the connector at the ends Adapter Removal removal 3'.
4. a kind of oat drought resistance related SNP molecular labeling based on GBS technologies according to claim 3, feature exist
In：The Adapter Removal operating methods are：Mass filter is carried out using slip window sampling, window size is set as 5
Bp, step-length are set as 1 bp；It is moved along a base each time, the average Q values of 5 base calculation windows are taken, if finally
Value≤2 Q of one base then only retain the base before the position；If average value≤20 Q of window, only retain
The window penultimate base and base before；Length filtration, if in double ends any one reads length≤
50 bp then remove double end reads.
5. a kind of oat drought resistance related SNP molecular labeling based on GBS technologies according to claim 2, feature exist
In：Group's SNP detection method is：Quality data compares after simplifying genome reference sequences at oat using Stacks software combinations
Right, populations filterings obtain oat group SNP site information, and clustering parameter is set as：-m4；Populations parameters
It is set as：One site will at least occur in 1 group；The minimum percentage of individual in same site is detected in one group
0.50；The minimum gene frequency 0.05 in one site.
6. a kind of oat drought resistance related SNP molecular labeling based on GBS technologies according to claim 2, feature exist
In：The specific method that the relevant SNP marker of oat drought resistance determines is：The group SNP of acquisition is using GCTA softwares to institute
There is material to carry out classified analysis on major constituents, whether detection phenotype, that is, drought resistance and genotype, that is, group SNP groupings are consistent, PCA is aobvious
Show that highly resistance and Di Kang groups are gathered for two clusters；Fisher Test SNP genotypic difference conspicuousnesses are carried out to highly resistance and low anti-phenotype
The a certain genotype frequency of analysis, the site is 1 in low resist, and is 0 in highly resistance, obtains and the relevant SNP site of drought resistance.
7. a kind of oat drought resistance related SNP molecular labeling based on GBS technologies as claimed in claim 1 or 2 is in oat drought resisting
Application in germplasm screening, it is characterised in that：The specific screening technique of oat Drought Resistance Germplasm is：4 of the SNP marker
The corresponding genotype of SNP site is A/G, G/T, GG, and when CC, then oat to be identified is the oat of low drought resisting；If display 4
SNP site lacks（./.）, then oat to be identified is the oat of high drought resisting.
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Application publication date: 20181016 |