CN105925680A - Method for developing marker through tetraploid potato high-throughput sequencing and application of method - Google Patents
Method for developing marker through tetraploid potato high-throughput sequencing and application of method Download PDFInfo
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- 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
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- 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
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- C12Q2600/00—Oligonucleotides characterized by their use
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention discloses a method for developing a marker through tetraploid potato high-throughput sequencing and an application of the method. The method for developing the marker through the tetraploid potato high-throughput sequencing disclosed by the invention can greatly reduce the complexity of a potato genome and can facilitate operation; and by virtue of the method, high-density SNP (single nucleotide polymorphism) sites can be rapidly identified, a chromosome label difference genetic interval for controlling target character can be obtained, and finally, the molecular marker can be developed on the basis of the difference genetic interval. With the application of the method disclosed by the invention, the application of an 2b-RAD sequencing technology in potato tetraploid material marker development is successfully achieved; the difficulty on developing the potato tetraploid material marker is solved, and meanwhile, a development cycle of the character closely linked marker is greatly shortened. In addition, the method disclosed by the invention also provides a reference for genetic interval mining, marker development and genome research of other tetraploid organisms having complex genomes.
Description
Technical field
The invention belongs to biological technical field, be specifically related to the side of a kind of Tetraploid Potatoes high-flux sequence exploitation labelling
Method and application thereof.
Background technology
Tetraploid Potatoes (2n=4x=48) highly heterozygosis, genetic recombination frequency is high, and after selfing many generations, decline is serious,
But genetic background is narrow, gene bank is poor, molecular markers development difficulty.Therefore, the saturation of potato genetic collection of illustrative plates
Difference, molecular marker quantity are few, the most far apart in all many-sides compared with the crop such as Oryza sativa L., corn and soybean.At present
Genetic map use conventional tag, marker development and the genetic map construction such as SSR, AFLP to be also mainly two more
Carry out in times body level, in tetraploid level, directly develop labelling be rarely reported.Up to the present, it is available for Ma Ling
The direct applied molecular marker of potato breeding selection is the most limited, is badly in need of developing more and closely linked point of breeding character
Sub-labelling, the exploitation of related molecular marker is by the markup resources new for the offer of Rhizoma Solani tuber osi molecular mark.
It is on second filial generation order-checking basis that high flux simplifies gene order-checking (Reduced-representation sequencing)
On the one that grows up utilize enzyme incision technology, sequence capturing chip technology or other laboratory facilities to reduce species gene group
Complexity, checks order for specific genome area, and then the order-checking of reflection partial genome sequence structural information
Technology.Wherein using most commonly used is DNA (the Restriction-site associated that restriction enzyme site is relevant
DNA, RAD) sequencing technologies, i.e. RAD-seq.This technology utilizes restricted enzyme that genome is carried out enzyme action,
Produce a certain size fragment, build sequencing library, the RAD labelling produced after enzyme action is carried out high-flux sequence.
Owing to RAD labelling is the small pieces segment DNA label presented near specific cleavage site of full-length genome scope, represent
The sequence signature of whole genome, therefore by obtaining thousands of in most of biologies to the order-checking of RAD labelling
Single nucleotide polymorphism (Single nucleotide polymorphism, SNP) labelling up to ten thousand.The advantage of this technology
It is: (1) flux is high, is conventional molecular marker development technology by the quantity of once sequencing exploitation RAD labelling
10 times；(2) accuracy is high, and digitized signal and high coverage make its more traditional molecular marker accuracy be greatly promoted；
(3) data user rate is high, and cost performance is high, owing to the complexity of genome is greatly reduced, thus reduces and checks order into
This, thus be particularly suitable for studying at population level；(4) experimental period is short, owing to having high-throughout feature,
Ten hundreds of labellings can be produced through once sequencing, substantially reduce the construction cycle of conventional tag；(5) it is not subject to
Species without reference to genome can also be carried out extensive examination SNP site by the restriction of genome sequence.RAD-
Seq has been successfully applied to the exploitation of SNP marker, the structure of VHD genetic map, animals and plants important economical trait
QTL location, population genetic variations, phylogeny analysis and the auxiliary research field such as full-length genome de novo order-checking.
At present, RAD (IIB digest RAD, the 2b-RAD) technology of IIB type restricted enzyme is at single endonuclease digestion
RAD-seq technically grows up.This technology is to utilize IIB type restricted enzyme that genomic DNA is carried out enzyme action,
This fermentoid (such as Bsa Ⅺ and Alf I) can cut off in the upstream and downstream site in target site on genomic DNA
DNA, it is thus achieved that the DNA fragmentation that length is consistent.
Summary of the invention
First purpose of the present invention is to provide the development approach of a kind of polyploid crop molecular marker.
The development approach of the polyploid crop molecular marker that the present invention provides comprises the steps:
1) polyploid crop filial generation is carried out character investigation, chooses the filial generation presenting a certain objective trait as filial generation first,
Choose and present the filial generation contrary with described a certain objective trait as filial generation second；
Described polyploid crop filial generation is polyploid crop parent's first and the hybrid generation of polyploid crop parent's second；
Described polyploid crop parent's first presents described objective trait；Described polyploid crop parent's second presents and described mesh
The character that mark shape is contrary；
2) described polyploid crop parent's first, described many crops polyploid parent's second, described filial generation first and institute are extracted respectively
State the genomic DNA of filial generation second, respectively obtain the genomic DNA of polyploid crop parent's first, polyploid crop parent
The genomic DNA of this second, filial generation first DNA mix pond and filial generation second DNA mixes pond；
It is mixed by obtain after the genomic DNA mixed in equal amounts of individuality each in filial generation first that described filial generation first DNA mixes pond
Close DNA sample；
It is mixed by obtain after the genomic DNA mixed in equal amounts of individuality each in filial generation second that described filial generation second DNA mixes pond
Close DNA sample；
Individual amount in described filial generation first and described filial generation second is no less than 30, described filial generation first and described filial generation second
In individual amount be specially 35；
3) genomic DNA, the gene of described polyploid crop parent's second to described polyploid crop parent's first respectively
Group DNA, described filial generation first DNA mix pond and described filial generation second DNA is mixed pond and carried out high flux simplification gene order-checking,
Respectively obtain the chromosome label densities scattergram of polyploid crop parent's first, the chromosome mark of polyploid crop parent's second
Sign density profile, filial generation first DNA and mix the chromosome label densities scattergram in pond and filial generation second DNA mixes the dyeing in pond
Body label densities scattergram；
4) comparison above-mentioned filial generation first DNA mixes the chromosome label densities scattergram in pond and filial generation second DNA mixes the dye in pond
Colour solid label densities scattergram 2 figure；
It is chosen at filial generation first DNA to mix special existence in the chromosome label densities scattergram in pond and mix in filial generation second DNA
The chromosome segment not having in the chromosome label densities scattergram in pond, divides as the filial generation special label densities of first chromosome
Butut；
It is chosen at filial generation second DNA to mix special existence in the chromosome label densities scattergram in pond and mix in filial generation first DNA
The chromosome segment not having in the chromosome label densities scattergram in pond, divides as the filial generation special label densities of second chromosome
Butut；
5) filial generation first chromosome special label densities scattergram described in comparison and the described filial generation special label densities of second chromosome
Scattergram, selection differences chromosome segment be for the purpose of label densities differential dyeing body section；
6) design synthetic primer according to described purpose label densities differential dyeing body section, develop molecular marker.
In said method, it is 2b-RAD order-checking that described high flux simplifies gene order-checking；
The restricted enzyme used in described 2b-RAD order-checking is IIB type restricted enzyme；
Described IIB type restricted enzyme is specially BsaXI.
In said method,
Described polyploid crop is Tetraploid Potatoes；Described filial generation is F1 generation；Specifically developing at molecular marker
Cheng Zhong, if other F1 generations do not occur the polyploid crop of trait segregation, should select F2 for segregating population as property
The colony of shape investigation.
In said method,
Described objective trait is Rhizoma Solani tuber osi Early mature apricot, and opposite to that character is the late-maturing character of Rhizoma Solani tuber osi；
Described Early mature apricot is that period of duration is less than 75 days；Described late-maturing character made a living the phase of educating more than 110 days；
Described molecular marker is the molecular marker relevant to described objective trait.
In said method,
Described polyploid crop parent's first is middle potato 3；
Described polyploid crop parent's second is middle potato 19.
In said method,
Described molecular marker is SCAR mark and/or CAPs labelling；
Described SCAR mark is made up of the single strand dna shown in sequence 1 and the single strand dna shown in sequence 2；
Described CAPs labelling is made up of the single strand dna shown in sequence 3 and the single strand dna shown in sequence 4.
Second object of the present invention is to provide the new application of said method.
The invention provides said method application in the exploitation of polyploid crop molecular marker.
Third object of the present invention is to provide the new use detecting the material whether Rhizoma Solani tuber osi to be measured contains DNA fragmentation first
On the way.
The invention provides and detect whether Rhizoma Solani tuber osi to be measured contains the material of DNA fragmentation first in following (1)-(6)
Application at least one:
(1) identify or assist qualification Rhizoma Solani tuber osi to be measured ripe property character；
(2) preparation is identified or the product of auxiliary qualification Rhizoma Solani tuber osi to be measured ripe property character；
(3) identify or assist and identify that Rhizoma Solani tuber osi to be measured is Early cropping potato or late-maturing Rhizoma Solani tuber osi；
(4) preparation is identified or is assisted and identifies that Rhizoma Solani tuber osi to be measured is Early cropping potato or the product of late-maturing Rhizoma Solani tuber osi；
(5) selection-breeding Early cropping potato；
(6) the late-maturing Rhizoma Solani tuber osi of selection-breeding.
In above-mentioned application, whether described detection Rhizoma Solani tuber osi to be measured contains the material of DNA fragmentation first is that PCR amplification contains
The primer of described DNA fragmentation first；
Described primer is following 1) or 2):
1) by the single stranded DNA shown in sequence 2 in the single strand dna shown in sequence in sequence table 1 and sequence table
Molecular primer is to A；
2) primer pair being made up of the single strand dna shown in sequence A and the single strand dna shown in sequence B
B；
Described sequence A is for deleting sequence 1 or increase or change one or several nucleotide, and has phase with sequence 1
The nucleotide of congenerous；
Described sequence B is for deleting sequence 2 or increase or change one or several nucleotide, and has phase with sequence 2
The nucleotide of congenerous.
Fourth object of the present invention is to provide a kind of qualification or the method for the auxiliary qualification ripe property of Rhizoma Solani tuber osi to be measured.
What the present invention provided identifies or assist the method identifying the ripe property of Rhizoma Solani tuber osi to be measured is to detect whether Rhizoma Solani tuber osi to be measured contains
There is DNA fragmentation first,
If Rhizoma Solani tuber osi to be measured contains DNA fragmentation first, Rhizoma Solani tuber osi the most to be measured is or candidate is Early cropping potato；
If Rhizoma Solani tuber osi to be measured does not contains DNA fragmentation first, Rhizoma Solani tuber osi the most to be measured is or candidate is late-maturing Rhizoma Solani tuber osi.
In said method, whether described detection Rhizoma Solani tuber osi to be measured contains the method for DNA fragmentation first is following X1) or
X2):
X1) genomic DNA that direct Sequencing Rhizoma Solani tuber osi is individual；
X2) order-checking pcr amplification product containing DNA fragmentation first；
Primer used by described pcr amplification product is following 1) or 2):
1) by the single stranded DNA shown in sequence 2 in the single strand dna shown in sequence in sequence table 1 and sequence table
Molecular primer is to A；
2) primer pair being made up of the single strand dna shown in sequence A and the single strand dna shown in sequence B
B；
Described sequence A is for deleting sequence 1 or increase or change one or several nucleotide, and has phase with sequence 1
The nucleotide of congenerous；
Described sequence B is for deleting sequence 2 or increase or change one or several nucleotide, and has phase with sequence 2
The nucleotide of congenerous.
5th purpose of the present invention is to provide a kind of qualification or the product of auxiliary qualification Rhizoma Solani tuber osi to be measured ripe property character.
What the present invention provided identifies or assists the product identifying Rhizoma Solani tuber osi to be measured ripe property character for whether detecting Rhizoma Solani tuber osi to be measured
Material containing DNA fragmentation first；
Whether described detection Rhizoma Solani tuber osi to be measured contains the material of DNA fragmentation first is following Y1) or Y2) or Y3) or
Y4):
Y1) by the single stranded DNA shown in sequence 2 in the single strand dna shown in sequence in sequence table 1 and sequence table
Molecular primer is to A；
Y2) primer being made up of the single strand dna shown in sequence A and the single strand dna shown in sequence B
To B；
Described sequence A is for deleting sequence 1 or increase or change one or several nucleotide, and has phase with sequence 1
The nucleotide of congenerous；
Described sequence B is for deleting sequence 2 or increase or change one or several nucleotide, and has phase with sequence 2
The nucleotide of congenerous；
Y3) containing Y1) described primer is to A or Y2) the described primer PCR reagent to B；
Y4) containing Y1) described primer is to A or Y2) described primer is to B or Y3) reagent of described PCR reagent
Box.
In above-mentioned application or said method or the said goods,
The nucleotides sequence of described DNA fragmentation first is classified as the potato gene group (version of potato gene group reference sequences
Number be Potato (Solanum tuberosum group Phureja DM1-3) Genome Browser v4.03)
4132599-4133441 position, is also the DNA molecular shown in sequence 5.
Described Early cropping potato is the Rhizoma Solani tuber osi that period of duration is less than 75 days；Described late-maturing Rhizoma Solani tuber osi is that period of duration is more than 110
It Rhizoma Solani tuber osi；
Described Rhizoma Solani tuber osi is Tetraploid Potatoes.
The present invention utilizes 2b-RAD technology to provide a kind of the most quickly Tetraploid Potatoes high-flux sequence exploitation mark
The method of note, this method avoid the process that in other RAD technology, clip size selects, makes label in genome
Distribution more uniform, the most applicable particularly with the marker development of complex genome and the excavation of chromosome segment.With
Time the method without predicting genomic information, library construction simple and fast, label densities can easily be accommodated, with low cost,
It is also particularly well-suited for linkage map and the structure of mapping genetic variations in natural population.
It is experimentally confirmed: the method for the Tetraploid Potatoes high-flux sequence exploitation labelling of the present invention is greatly reduced Ma Ling
Potato genome complexity, easy and simple to handle, Rapid identification can go out highdensity SNP site, it is thus achieved that control objective trait
Chromosome label difference heredity is interval (section), is finally based on difference heredity section and can develop molecular marker.The present invention's
Method successfully achieves the application in Rhizoma Solani tuber osi tetraploid material marker development of the 2b-RAD sequencing technologies, not only solves
The problem of Rhizoma Solani tuber osi tetraploid material marker development difficulty of having determined, and greatly shorten and character close linkage labelling
Construction cycle.Additionally, the method for the present invention is also other polyploid complex genome, biological hereditary section excavates, marks
Note exploitation and genome research provide reference.
Accompanying drawing explanation
Fig. 1 is that Rhizoma Solani tuber osi parent mixes pond agarose quality measurements figure with extreme daughter DNA.Marker II is from upper
It is followed successively by 1200bp, 900bp, 700bp, 500bp, 300bp, 100bp under to；Parent 1: middle potato 3；Parent
2: middle potato 19；Mixed pond 1: extreme precocious filial generation mixes pond；Mixed pond 2: the most late-maturing filial generation mixes pond.
Fig. 2 is that the extreme filial generation of Rhizoma Solani tuber osi mixes pond chromosome special label densities scattergram.Vertical coordinate: be respectively Rhizoma Solani tuber osi
Article 12, chromosome, by Chr1 to Chr12；Abscissa: chromosome physical distance, unit (Mb)；Right side block diagram:
Different colours represents that different number of labels, numeral represent number of labels；In every chromosome, first (on) table
Show the special label in mixed pond 1, second (in) for the special label in mixed pond 2, the 3rd (under) it is label between two ponds
The absolute value of density contrast；Wherein, black region represents that difference label densities is big, and yellow area represents difference label densities
Taking second place, the depth of color represents the relative different of label densities.
Fig. 3 is polymorphism mark SCAR5-5 the result figure in extreme precocious filial generation material.M:Marker II,
It is followed successively by 1200bp, 900bp, 700bp, 500bp, 300bp, 100bp from top to bottom；Parent 1: middle potato 3；
Parent 2: middle potato 19；55, it is that part is extreme precocious in 56,81,91,103,107,117,122 and 133
Filial generation is numbered.
Fig. 4 is polymorphism mark SCAR5-5 the result figure in the most late-maturing filial generation material.M:Marker II,
It is followed successively by 1200bp, 900bp, 700bp, 500bp, 300bp, 100bp from top to bottom；Parent 1: middle potato 3；
Parent 2: middle potato 19；2,4,5,10,23,34,52,54 and 60 is part the most late-maturing filial generation numbering.
Detailed description of the invention
Experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
The compound method of 2%CTAB in following embodiment: CTAB 20g+NaCl 81.81g+40mL EDTA
(0.5M pH8.0)+100mL Tris+HCl (1M PH8.0)+10g PVP, adds distilled water and is settled to 1L,
Autoclaving 40min, room temperature preserves, and adds the mercaptoethanol of 1%, and need to be placed in 65 DEG C of water preheating during use.
Solution chloroform in following embodiment: isoamyl alcohol (24:1), isopropanol are with needing first pre-cooling before.
In parent in following embodiment, potato 3 is Rhizoma Solani tuber osi early-maturing variety, and variety certification is numbered: state examines potato 2005005,
The public can obtain from research of agricultural science institute of China vegetable or flower institute.
In parent in following embodiment, potato 19 is Rhizoma Solani tuber osi late variety, and variety certification is numbered: state examines potato
2014002, the public can obtain from research of agricultural science institute of China vegetable or flower institute.
Embodiment 1, a kind of method of marker development of Tetraploid Potatoes material
One, material to be tested and character investigation thereof
1, material to be tested
The Rhizoma Solani tuber osi tetraploid F1 generation ripe property segregating population that the present invention is utilized, is by Tetraploid Potatoes early-maturing variety
Potato 3 (male parent, parent 1) obtains with potato 19 in Tetraploid Potatoes late variety (maternal, parent 2) hybridization
?.
2, character investigation
The ripe property character of F1 generation segregating population is investigated.Add up emerge (cotyledon is unearthed) and physiological maturity (plant
It is withered and yellow that blade reaches 50%) time, calculate period of duration (emerging to the natural law of physiological maturity).According to period of duration length,
Less than 75 days, period of duration is designated as precocious material, and period of duration was designated as late-maturing material more than 110 days, and period of duration is more than
75 days and less than 110 days be designated as middle ripe wood material.The final extreme precocious filial generation material of acquisition and the most late-maturing filial generation material
Each 35 parts.
Two, extreme daughter DNA mixes pond structure
1, parent and offspring individual extracting genome DNA
Gather parent 1, parent 2, extreme precocious filial generation material and the potato leaf of the most late-maturing filial generation material, use
The little mensuration extraction genomic DNA of CTAB of improvement.Specifically comprise the following steps that
(1) potato leaf 2 of lid size is taken in 1.5mL centrifuge tube.
(2) the 1.5mL centrifuge tube containing blade is put in liquid nitrogen after freezing, is ground into powder with grinding rod, add 65 DEG C
The CTAB 700uL (interpolation β mercaptoethanol, final concentration of 1%) of the 2% of preheating, mixing.
(3) put 65 DEG C of water-bath 1h, period every 10min to shake once.
(4) after taking-up is cooled to room temperature, the chloroform of addition equal-volume pre-cooling: isoamyl alcohol (24:1) solution 700uL, gently
The even 5min of jog.
(5) 14000rpm, 4 DEG C of centrifugal 10min, Aspirate supernatant about 500ul in new 1.5mL centrifuge tube,
Add the isopropanol of equal-volume pre-cooling, shake mixing the most up and down.
(6)-20 DEG C of refrigerators precipitate 30min (can be overnight), 14000rpm, 4 DEG C of centrifugal 10min；
(7) outwelling supernatant, add 1mL 70% washing with alcohol precipitation, turn upside down 6-8 time, 7500rpm is centrifuged
5min, abandons supernatant.
(8) repeated washing, takes 1mL 70% washing with alcohol precipitation, and 7500rpm is centrifuged 5min, abandons supernatant
(9) putting into fume hood, complete to ethanol volatilization, adding 100 μ L has the sterilized water of RNase to redissolve, 37 DEG C
Incubation 1h removes remaining RNA (RNase concentration: 0.1mg/mL).
(10) take 2 μ L DNA and carry out agarose gel electrophoresis detection.
2, extreme daughter DNA mixes pond structure
(1) genomic DNA of all extreme precocious filial generation material extracted is carried out mixed in equal amounts, build mixed pond 1.
And electrophoresis detection DNA mixes the quality in pond.Agarose gel electrophoresis testing result is as shown in Figure 1.
(2) genomic DNA of all the most late-maturing filial generation material extracted is carried out mixed in equal amounts, build mixed pond 2.
And electrophoresis detection DNA mixes the quality in pond.Agarose gel electrophoresis testing result is as shown in Figure 1.
Three, high flux simplification gene order-checking filters out differential dyeing body section
Parent 1 qualified for quality testing, parent 2, mixed pond 1 and mixed pond 2 are carried out 2b-RAD high flux and simplifies gene
Group order-checking, respectively obtain the chromosome label densities scattergram of parent 1, the chromosome label densities scattergram of parent 2,
The chromosome label densities scattergram in mixed pond 1 and the chromosome label densities scattergram in mixed pond 2, and to each chromosome
Label densities figure is compared, and finds out special signature, then draws the chromosome in mixed pond 1 further according to special signature
The chromosome special label densities figure in special label densities figure and mixed pond 2, close finally according to 2 special labels of chromosome
Degree figure filters out the differential dyeing body section in mixed pond 1 and mixed pond 2.Specifically comprise the following steps that
1, Jian Ku
Utilize 2b-RAD technology, by restricted enzyme Bsa XI, complete genome DNA is carried out enzyme action, use
Standard type NNN joint, builds above-mentioned 4 potato samples (parent 1, parent 2, mixed pond 1 and mixed pond 2)
Label sequencing library, 4 samples carry out single end sequencing at Hiseq2500v2 platform, obtain the dye of parent 1 respectively
Colour solid label densities scattergram, the chromosome label densities scattergram of parent 2, the chromosome label densities in mixed pond 1 divide
The chromosome label densities scattergram in Butut and mixed pond 2.
2, mass filter
Original reads step 1 obtained carries out mass filter according to following condition:
(1) rejecting does not contains the sequence of BsaXI enzyme action recognition site；
(2) low quality sequence (low quality sequence definition: be less than 20 more than the mass fraction of 10 bases) is rejected；
(3) more than 10 sequences having consecutive identical base are rejected.
3, individual order-checking label statistics
Individual high-quality reads step 2 obtained utilizes SOAP software mapping to single times of Rhizoma Solani tuber osi of checking order
On the reference sequences of type DM, it is thus achieved that can be used for unique number of tags and the degree of depth of typing.
4, the examination of full-length genome scope SNP and phenotypic analysis
(1) reference sequences is built
Extract from Rhizoma Solani tuber osi is with reference to genome sequence and comprise the label of Bsa XI restriction enzyme site as reference sequences, Ma Ling
Potato is as follows with reference to genome network address:
http://www.ncbi.nlm.nih.gov/genome/？Term=Solanum%20tuberosum.
(2) individual SNP marker typing
Utilize SOAP software (parameter is set to :-M4 v2 r0) mapping to ginseng individual high-quality reads
Examine in sequence.Method of maximum likelihood ML is utilized to carry out the typing in site.In order to ensure SNP site typing accuracy and
Preciseness, carries out the filtration of following condition:
A () is up to the label site of 3 SNP in only leaving and taking label；
B () does not carries out typing in the individual interior label degree of depth more than 500.
5, according to above genotyping result, to personality analysis such as the chromosome label densities scattergrams that 4 samples build,
Comparison mixes the chromosome label densities scattergram in pond 1 and the chromosome label densities scattergram in mixed pond 2；It is chosen at mixed pond
Special existence in 1 chromosome label densities scattergram and the chromosome that do not has at mixed pond 2 chromosome label densities scattergram
Section, as the chromosome special label densities scattergram in mixed pond 1；It is chosen at the chromosome label densities distribution of mixed pond 2
Special existence in figure and the chromosome segment that do not has in the chromosome label densities scattergram in mixed pond 1, as mixed pond 2
Chromosome special label densities scattergram；Mixed pond 1 and the chromosome special label densities scattergram such as Fig. 2 in mixed pond 2
Shown in.
6, difference interval screening
Comparison mixes the chromosome special label densities scattergram in pond 1 and the chromosome special label densities scattergram in mixed pond 2,
Filter out the chromosome segment that label densities differs greatly, be differential dyeing body section.That the present invention filters out and horse
The differential dyeing body section that the ripe property of bell potato is relevant is positioned at the position of No. 5 chromosome about 4Mb.
Four, marker development and checking
1, design of primers
Transfer different regions label information, including reference sequences and sequence location etc..According to the position of difference label, from
Potato gene group sequence website (http://solanaceae.plantbiology.msu.edu/pgsc_download.shtml)
The sequence of each 700bp in this difference label left and right of middle download.Mega5.2 is utilized the sequence of sequence label with download to be carried out
Comparison, determines the accuracy of this label.Premier5 primer-design software is utilized to carry out design of primers, by primer sequence
Length is set as that 22bp, forward primer are arranged at 1-550bp, and downstream primer is arranged at 850-1400bp, primer
Amplified fragments size is set as between 400-900bp, and Tm value is set as between 55-63 DEG C, and design 3 is to primer altogether,
And deliver the synthesis of Shanghai Sheng Gong biological engineering company limited.Primer is as shown in table 1:
Table 1, the design of primer
2, the detection of molecular marker
(1) respectively with the genomic DNA of parent 1 and parent 2 as template, the primer of step 1 design is used to carry out
PCR expands, and amplified production utilizes quality and the specificity of agarose gel electrophoresis detection primer.
Above-mentioned PCR amplification system (10 μ L) is as shown in table 2:
Table 2, PCR amplification system
Above-mentioned PCR amplification program is as shown in table 3:
Table 3, PCR amplification program
Electrophoresis: applied sample amount is 10 μ L, agarose gel is 1.2%, voltage 120V, electrophoresis 25min.
According to the amplification of 3 pairs of primers, SCAR5-5 primer is amplified band in parent 1, and in parent 2 not
Amplifying band, therefore SCAR5-5 primer can be developed as SCAR mark for identifying the ripe property character of Rhizoma Solani tuber osi.
(2) CAPS5-24 primer all can amplify clear and zero difference band in parent 1 and parent 2, will
The pcr amplification product that CAPS5-24 primer amplification obtains carries out enzyme action, and utilizes 2.0% agarose gel to detect
(voltage 100v, electrophoresis 40min).
Above-mentioned enzyme action system (15 μ L): PCR primer 8 μ L, Cutsmart 1.5 μ L, BsaXI restriction endonuclease (0.5 μ L/U)
0.2μL、ddH2O 5.3μL。
Above-mentioned endonuclease reaction condition: 37 DEG C of reaction 3h.
CAPS5-24 primer pcr amplification product in parent 1 can be by restricted enzyme BsaXI enzyme action, and parent
Pcr amplification product in basis 2 can not be by restricted enzyme BsaXI enzyme action, and therefore CAPS5-24 primer can be developed
As CAPs labelling for identifying the ripe property character of Rhizoma Solani tuber osi.
3, the checking of molecular marker SCAR5-5
Choosing molecular marker SCAR5-5 utilizes extreme precocious filial generation to verify with the most late-maturing material.Concrete steps are such as
Under: respectively with the genomic DNA of parent 1, parent 2, extreme precocious filial generation and the most late-maturing material as template, adopt
Carry out PCR amplification with molecular marker SCAR5-5, respectively obtain pcr amplification product, by pcr amplification product fine jade
Sepharose electrophoresis detection.Above-mentioned PCR amplification and product detection method are with step 3.
Molecular marker SCAR5-5 checking knot in parent 1, parent 2, extreme precocious filial generation and the most late-maturing material
Fruit is as shown in Figure 3 and Figure 4.As can be seen from Figure 3: except material 103, molecular marker SCAR5-5 is extremely
Precocious filial generation obtains, with all amplifications in parent 1, the band that size is 843bp, and above-mentioned size is the core of the band of 843bp
Nucleotide sequence is that (version number of potato gene group reference sequences is Potato (Solanum to potato gene group
Tuberosum group Phureja DM1-3) Genome Browser v4.03) 4132599-4133441 position,
Also the DNA molecular shown in sequence 5 it is；Figure 4, it is seen that molecular marker SCAR5-5 is the most late-maturing
Filial generation and parent 2 all do not amplify band.These results suggest that: the molecular marker SCAR5-5 of present invention exploitation
Property ripe with the Tetraploid Potatoes linkage of characters, can be used for the qualification of Tetraploid Potatoes ripe property character.
Claims (10)
1. a development approach for polyploid crop molecular marker, comprises the steps:
1) polyploid crop filial generation is carried out character investigation, chooses the filial generation presenting a certain objective trait as filial generation first,
Choose and present the filial generation contrary with described a certain objective trait as filial generation second；
Described polyploid crop filial generation is polyploid crop parent's first and the hybrid generation of polyploid crop parent's second；
Described polyploid crop parent's first presents described objective trait；Described polyploid crop parent's second presents and described mesh
The character that mark shape is contrary；
2) described polyploid crop parent's first, described many crops polyploid parent's second, described filial generation first and institute are extracted respectively
State the genomic DNA of filial generation second, respectively obtain the genomic DNA of polyploid crop parent's first, polyploid crop parent
The genomic DNA of this second, filial generation first DNA mix pond and filial generation second DNA mixes pond；
3) genomic DNA, the gene of described polyploid crop parent's second to described polyploid crop parent's first respectively
Group DNA, described filial generation first DNA mix pond and described filial generation second DNA is mixed pond and carried out high flux simplification gene order-checking,
Respectively obtain the chromosome label densities scattergram of polyploid crop parent's first, the chromosome mark of polyploid crop parent's second
Sign density profile, filial generation first DNA and mix the chromosome label densities scattergram in pond and filial generation second DNA mixes the dyeing in pond
Body label densities scattergram；
4) comparison above-mentioned filial generation first DNA mixes the chromosome label densities scattergram in pond and filial generation second DNA mixes the dye in pond
Colour solid label densities scattergram 2 figure；
It is chosen at filial generation first DNA to mix special existence in the chromosome label densities scattergram in pond and mix in filial generation second DNA
The chromosome segment not having in the chromosome label densities scattergram in pond, divides as the filial generation special label densities of first chromosome
Butut；
It is chosen at filial generation second DNA to mix special existence in the chromosome label densities scattergram in pond and mix in filial generation first DNA
The chromosome segment not having in the chromosome label densities scattergram in pond, divides as the filial generation special label densities of second chromosome
Butut；
5) filial generation first chromosome special label densities scattergram described in comparison and the described filial generation special label densities of second chromosome
Scattergram, selection differences chromosome segment be for the purpose of label densities differential dyeing body section；
6) design synthetic primer according to described purpose label densities differential dyeing body section, develop molecular marker.
Method the most according to claim 1, it is characterised in that: described high flux simplifies gene order-checking and is
2b-RAD checks order；
The restricted enzyme used in described 2b-RAD order-checking is IIB type restricted enzyme；
Described IIB type restricted enzyme is specially BsaXI.
Method the most according to claim 1 and 2, it is characterised in that:
Described polyploid crop is Tetraploid Potatoes；Described filial generation is F1 generation；
Described objective trait is Rhizoma Solani tuber osi Early mature apricot, and opposite to that character is the late-maturing character of Rhizoma Solani tuber osi；
Described Early mature apricot is that period of duration is less than 75 days；Described late-maturing character made a living the phase of educating more than 110 days；
Described molecular marker is the molecular marker relevant to described objective trait；
Described polyploid crop parent's first is middle potato 3；
Described polyploid crop parent's second is middle potato 19.
4. according to described method arbitrary in claim 1-3, it is characterised in that:
Described molecular marker is SCAR mark and/or CAPs labelling；
Described SCAR mark is made up of the single strand dna shown in sequence 1 and the single strand dna shown in sequence 2；
Described CAPs labelling is made up of the single strand dna shown in sequence 3 and the single strand dna shown in sequence 4.
5. arbitrary described method application in the exploitation of polyploid crop molecular marker in claim 1-4.
6. detect Rhizoma Solani tuber osi to be measured whether contain the material of DNA fragmentation first in (1)-(6) as follows at least one
In application:
(1) identify or assist qualification Rhizoma Solani tuber osi to be measured ripe property character；
(2) preparation is identified or the product of auxiliary qualification Rhizoma Solani tuber osi to be measured ripe property character；
(3) identify or assist and identify that Rhizoma Solani tuber osi to be measured is Early cropping potato or late-maturing Rhizoma Solani tuber osi；
(4) preparation is identified or is assisted and identifies that Rhizoma Solani tuber osi to be measured is Early cropping potato or the product of late-maturing Rhizoma Solani tuber osi；
(5) selection-breeding Early cropping potato；
(6) the late-maturing Rhizoma Solani tuber osi of selection-breeding.
Application the most according to claim 6, it is characterised in that: whether described detection Rhizoma Solani tuber osi to be measured contains DNA
The material of fragment first is the PCR amplification primer containing described DNA fragmentation first；
Described primer is following 1) or 2):
1) by the single stranded DNA shown in sequence 2 in the single strand dna shown in sequence in sequence table 1 and sequence table
Molecular primer is to A；
2) primer pair being made up of the single strand dna shown in sequence A and the single strand dna shown in sequence B
B；
Described sequence A is for deleting sequence 1 or increase or change one or several nucleotide, and has phase with sequence 1
The nucleotide of congenerous；
Described sequence B is for deleting sequence 2 or increase or change one or several nucleotide, and has phase with sequence 2
The nucleotide of congenerous.
8. identify or a method for the auxiliary qualification ripe property of Rhizoma Solani tuber osi to be measured, be to detect whether Rhizoma Solani tuber osi to be measured contains DNA
Fragment first,
If Rhizoma Solani tuber osi to be measured contains DNA fragmentation first, Rhizoma Solani tuber osi the most to be measured is or candidate is Early cropping potato；
If Rhizoma Solani tuber osi to be measured does not contains DNA fragmentation first, Rhizoma Solani tuber osi the most to be measured is or candidate is late-maturing Rhizoma Solani tuber osi；
Whether described detection Rhizoma Solani tuber osi to be measured contains the method for DNA fragmentation first is following X1) or X2):
X1) genomic DNA that direct Sequencing Rhizoma Solani tuber osi is individual；
X2) order-checking pcr amplification product containing DNA fragmentation first；
Primer used by described pcr amplification product is following 1) or 2):
1) by the single stranded DNA shown in sequence 2 in the single strand dna shown in sequence in sequence table 1 and sequence table
Molecular primer is to A；
2) primer pair being made up of the single strand dna shown in sequence A and the single strand dna shown in sequence B
B；
Described sequence A is for deleting sequence 1 or increase or change one or several nucleotide, and has phase with sequence 1
The nucleotide of congenerous；
Described sequence B is for deleting sequence 2 or increase or change one or several nucleotide, and has phase with sequence 2
The nucleotide of congenerous.
9. identify or a product for auxiliary qualification Rhizoma Solani tuber osi to be measured ripe property character, for detecting whether Rhizoma Solani tuber osi to be measured contains
The material of DNA fragmentation first；
Whether described detection Rhizoma Solani tuber osi to be measured contains the material of DNA fragmentation first is following Y1) or Y2) or Y3) or
Y4):
Y1) by the single stranded DNA shown in sequence 2 in the single strand dna shown in sequence in sequence table 1 and sequence table
Molecular primer is to A；
Y2) primer being made up of the single strand dna shown in sequence A and the single strand dna shown in sequence B
To B；
Described sequence A is for deleting sequence 1 or increase or change one or several nucleotide, and has phase with sequence 1
The nucleotide of congenerous；
Described sequence B is for deleting sequence 2 or increase or change one or several nucleotide, and has phase with sequence 2
The nucleotide of congenerous；
Y3) containing Y1) described primer is to A or Y2) the described primer PCR reagent to B；
Y4) containing Y1) described primer is to A or Y2) described primer is to B or Y3) reagent of described PCR reagent
Box.
10. according to described in the application described in claim 6 or 7 or the method described in claim 8 or claim 9
Product, it is characterised in that:
The nucleotides sequence of described DNA fragmentation first is classified as the 4132599-4133441 position of potato gene group；
Described Early cropping potato is the Rhizoma Solani tuber osi that period of duration is less than 75 days；Described late-maturing Rhizoma Solani tuber osi is that period of duration is more than 110
It Rhizoma Solani tuber osi；
Described Rhizoma Solani tuber osi is Tetraploid Potatoes.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107153777A (en) * | 2017-05-03 | 2017-09-12 | 武汉菲沙基因信息有限公司 | A kind of method for the diplodization degree for estimating tetraploid species gene group |
| CN107937591A (en) * | 2017-12-12 | 2018-04-20 | 华中农业大学 | The SNP marker of the Ⅺ relevant QTL site of end low-temperature resistance saccharification of tuberosum chromosomes and its application |
| CN109182505A (en) * | 2018-09-29 | 2019-01-11 | 南京农业大学 | Mastadenitis of cow key SNPs site rs75762330 and 2b-RAD Genotyping and analysis method |
| CN114223453A (en) * | 2021-12-20 | 2022-03-25 | 山东农业大学 | Method for creating apple non-fusion allopolyploid rootstock based on whole genome mutagenesis |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103555717A (en) * | 2013-11-18 | 2014-02-05 | 新疆农业科学院哈密瓜研究中心 | Functional molecular markers of related genes of sweetness and sourness characters of muskmelon and application of markers |
| CN104232627A (en) * | 2013-06-13 | 2014-12-24 | 深圳华大基因科技有限公司 | 2b-RAD pooling technology |
| CN104726450A (en) * | 2015-03-25 | 2015-06-24 | 广东省农业科学院蔬菜研究所 | Molecular markers in close linkage with specific resistance gene for capsicum phytophthora blight of root rot and application thereof |
| CN105063217A (en) * | 2015-08-19 | 2015-11-18 | 中国农业科学院蔬菜花卉研究所 | Method for identifying or assisting in identifying shapes of potatoes and special primer pair for method |
| CN105506075A (en) * | 2015-12-10 | 2016-04-20 | 湖北省农业科学院果树茶叶研究所 | SNP (Single Nucleotide Polymorphism) marker related to resistance to alternaria kikuchiana and application |
| CN105525000A (en) * | 2016-01-20 | 2016-04-27 | 江西师范大学 | QTL-seq based method for mining cold-tolerant gene of Dongxiang wild rice |
| CN105803094A (en) * | 2016-05-06 | 2016-07-27 | 中国农业科学院蔬菜花卉研究所 | SCAR mark for appraisal or auxiliary appraisal of maturity of potatoes and application of SCAR mark |
-
2016
- 2016-05-06 CN CN201610298485.6A patent/CN105925680B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104232627A (en) * | 2013-06-13 | 2014-12-24 | 深圳华大基因科技有限公司 | 2b-RAD pooling technology |
| CN103555717A (en) * | 2013-11-18 | 2014-02-05 | 新疆农业科学院哈密瓜研究中心 | Functional molecular markers of related genes of sweetness and sourness characters of muskmelon and application of markers |
| CN104726450A (en) * | 2015-03-25 | 2015-06-24 | 广东省农业科学院蔬菜研究所 | Molecular markers in close linkage with specific resistance gene for capsicum phytophthora blight of root rot and application thereof |
| CN105063217A (en) * | 2015-08-19 | 2015-11-18 | 中国农业科学院蔬菜花卉研究所 | Method for identifying or assisting in identifying shapes of potatoes and special primer pair for method |
| CN105506075A (en) * | 2015-12-10 | 2016-04-20 | 湖北省农业科学院果树茶叶研究所 | SNP (Single Nucleotide Polymorphism) marker related to resistance to alternaria kikuchiana and application |
| CN105525000A (en) * | 2016-01-20 | 2016-04-27 | 江西师范大学 | QTL-seq based method for mining cold-tolerant gene of Dongxiang wild rice |
| CN105803094A (en) * | 2016-05-06 | 2016-07-27 | 中国农业科学院蔬菜花卉研究所 | SCAR mark for appraisal or auxiliary appraisal of maturity of potatoes and application of SCAR mark |
Non-Patent Citations (4)
| Title |
|---|
| PENG ZHANG 等: "Mining candidate genes associated with powdery mildew resistance in cucumber via super-BSA by specific length amplified fragment (SLAF) sequencing", 《BMC GENOMICS》 * |
| YONGLI ZHAO 等: "QTL mapping for bacterial wilt resistance in peanut (Arachis hypogaea L.)", 《MOL BREEDING》 * |
| YU GUO 等: "An improved 2b-RAD approach (I2b-RAD) offering genotyping tested by a rice (Oryza sativa L.) F2 population", 《BMC GENOMICS》 * |
| 王洋坤 等: "RAD-seq 技术在基因组研究中的现状及展望", 《遗传》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107153777A (en) * | 2017-05-03 | 2017-09-12 | 武汉菲沙基因信息有限公司 | A kind of method for the diplodization degree for estimating tetraploid species gene group |
| CN107153777B (en) * | 2017-05-03 | 2020-01-21 | 武汉菲沙基因信息有限公司 | Method for estimating doubling degree of tetraploid species genome |
| CN107937591A (en) * | 2017-12-12 | 2018-04-20 | 华中农业大学 | The SNP marker of the Ⅺ relevant QTL site of end low-temperature resistance saccharification of tuberosum chromosomes and its application |
| CN109182505A (en) * | 2018-09-29 | 2019-01-11 | 南京农业大学 | Mastadenitis of cow key SNPs site rs75762330 and 2b-RAD Genotyping and analysis method |
| CN114223453A (en) * | 2021-12-20 | 2022-03-25 | 山东农业大学 | Method for creating apple non-fusion allopolyploid rootstock based on whole genome mutagenesis |
| CN114223453B (en) * | 2021-12-20 | 2022-12-27 | 山东农业大学 | Method for creating apple non-fusion allopolyploid rootstock based on whole genome mutagenesis |
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