CN102719543A - Method for identifying plant varieties by utilizing chemical molecular formulas of nucleotides - Google Patents

Method for identifying plant varieties by utilizing chemical molecular formulas of nucleotides Download PDF

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CN102719543A
CN102719543A CN2012102134887A CN201210213488A CN102719543A CN 102719543 A CN102719543 A CN 102719543A CN 2012102134887 A CN2012102134887 A CN 2012102134887A CN 201210213488 A CN201210213488 A CN 201210213488A CN 102719543 A CN102719543 A CN 102719543A
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sequence
nucleic acid
acid molecule
tree peony
measured
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CN102719543B (en
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索志立
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Institute of Botany of CAS
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Institute of Botany of CAS
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Abstract

The invention discloses a method for identifying plant varieties by utilizing the chemical molecular formulas of nucleotides. The method includes the following steps: (1) chloroplast genome DNA (deoxyribonucleic acid) sequences are utilized to design primers, and the primers are used for performing the PCR (Polymerase Chain Reaction) amplification on the genome DNA of plant varieties, so that PCR products are obtained; (2) the nucleotide sequences of the target DNA regions of the varieties are obtained by PCR direct sequencing, and the nucleotide sequences among the varieties are compared to find polymorphic sites and positions thereof and list the molecular formula corresponding to each variety; and (3) the molecular formulas of nucleotides are applied in the classifying identification and management of plant varieties. An experiment proves that the method which adopts the molecular formulas of nucleotides to identify the pedigrees of plant varieties is an accurate and simple method, and the defects of conventional analytical methods are remedied.

Description

Utilize the method for Nucleotide chemical molecular formula plant identification kind
Technical field
The present invention relates to a kind of biological technical field, relate in particular to a kind of method of utilizing Nucleotide chemical molecular formula plant identification kind.
Background technology
Plant variety is meant and forms some economic characters that kind of a property basically identical, heredity are more stable, have the human needs through artificially breeding: like fancy points, medicinal proterties or other economic characters, and the cultivated plant colony that uses as the special producing data.Like tree peony kind, walnut kind, apple variety etc.
Plant variety general using phenotypic character is identified and is discerned.But there is calendar variation in plant variety in the characteristic performance, possibly can not show discrepant proterties between kind in the season that has.For example, the tree peony kind is classified according to flower type and pattern usually, yet, have only the flowering period about one month every year, there is the difficult problem of cultivar identification in the time beyond the florescence, and the tree peony kind is through the grafting vegetative propagation; Some variety of watermelon had only to the ripe phase of melon could be identified according to speckle and the color and luster on melon crust surface, is difficult to differentiation other period.The accuracy of kind and the analysis of purity are the important topics in economic plants and resource plant (like the flowers) industry development always.In the large-scale production field; With certain concrete plant variety is that the production means and product carry out scale operation; Obtain the enterprise of profit through market sale; The identification of species title, analyze the plant variety purity of output in batches and solve the correlation technique problem, need accurately identify and analyze by the fractal detection technique of highly sensitive dna molecular.
Present DNA detection technology; Like DNA DNArandom amplified polymorphic DNA mark [random amplified polymorphic DNA (RAPD)], Tumor-necrosis factor glycoproteins transcribed spacer polymorphism mark [inters imple sequence repeat (ISSR)], AFLP mark [amplified fragment length polymorphism (AFLP)], the relevant amplification polymorphism mark [sequence-related amplified polymorphism (SRAP)] of sequence and little satellite or simple sequence repeat polymorphisms mark [microsatellites or polymorphic simple sequence repeats (SSRs)]; Can solve some problems in application facet; Like supposition of genetic affinity between kind etc., but all there be limitation and deficiency in various degree.
Summary of the invention
An object of the present invention is to provide the method for a kind of evaluation or assistant identification tree peony kind to be measured.
Method provided by the invention comprises the steps:
1) with primer to tree peony kind to be measured is carried out pcr amplification, obtain the PCR product in purpose zone; Said primer is to being made up of the dna molecular shown in the sequence 2 in sequence in the sequence table 1 and the sequence table;
2), obtain the nucleotide sequence in tree peony kind purpose to be measured zone with said PCR product order-checking;
3) nucleotide sequence and the nucleotide sequence cohort in said tree peony kind purpose to be measured zone are compared, find out mononucleotide polymorphism site, list tree peony kind nucleic acid molecule formula to be measured;
The general formula of the nucleic acid molecule formula of said tree peony kind to be measured is: B X1B X2B X3B Xn, wherein, B is the base kind on the mononucleotide polymorphism site, is in A, T, C and four kinds of bases of G any one; X1Be that the 1st mononucleotide polymorphism site numbered from 5 ' terminal sequence of positions in the nucleotide sequence in said tree peony kind purpose to be measured zone, X2Be that the 2nd mononucleotide polymorphism site numbered from 5 ' terminal sequence of positions in the nucleotide sequence in said tree peony kind purpose to be measured zone, analogize, XnBe that n mononucleotide polymorphism site numbered from 5 ' terminal sequence of positions in the nucleotide sequence in said tree peony kind purpose to be measured zone; Said B X1B X2B X3B XnFor all mononucleotide polymorphism sites are listed from 5 ' end in the nucleotide sequence in said tree peony kind purpose to be measured zone according to it successively;
Said nucleotide sequence cohort is made up of sequence in the sequence table 3, sequence 4, sequence 5 and sequence 6;
4) with said tree peony kind nucleic acid molecule formula to be measured and the comparison of nucleic acid molecule formula cohort, confirm tree peony kind to be measured;
Said nucleic acid molecule formula cohort is made up of following four kinds of nucleic acid molecule formulas: T 3C 301A 348G 416C 571G 712, G 3C 301G 348G 416A 571G 712, T 3A 301G 348G 416A 571T 712And T 3A 301G 348A 416A 571T 712
The corresponding tree peony kind cohort of each said nucleic acid molecule formula.
In the aforesaid method, in the step 1), the template of said pcr amplification is the genomic dna of said tree peony kind to be measured;
In the step 3); The said method of listing the nucleic acid molecule formula of tree peony kind to be measured comprises the steps: listing successively in order from 5 ' terminal the 3rd, 301,348,416,571 and 712 the Nucleotide letter of mononucleotide polymorphism site in the nucleotide sequence in said tree peony kind purpose to be measured zone; And with each mononucleotide polymorphism site in the nucleotide sequence in said tree peony kind purpose to be measured zone from 5 ' terminal sequence of positions number as subscript; Be marked on the lower right corner of the Nucleotide letter of said mononucleotide polymorphism site, obtain the nucleic acid molecule formula of tree peony kind to be measured;
The general formula of the nucleic acid molecule formula of said tree peony kind to be measured is B 3B 301B 348B 416B 571B 712
In the step 4), said T 3C 301A 348G 416C 571G 712Corresponding tree peony kind cohort is made up of following kind at least: ' Hua Erqiao ', ' acupuncture needle iron pink robe ', ' folded cloud ', ' Luoyang is red ', ' Corallium Japonicum Kishinouye platform ', ' hat generation carbuncle ', ' Venus snow wave ', ' piebald is white ' and ' scape is beautiful '; Said T 3C 301A 348G 416C 571G 712Corresponding tree peony kind cohort specifically is made up of following kind: ' Hua Erqiao ', ' acupuncture needle iron pink robe ', ' folded cloud ', ' Luoyang is red ', ' Corallium Japonicum Kishinouye platform ', ' hat generation carbuncle ', ' Venus snow wave ', ' piebald is white ' and ' scape is beautiful ';
When kind quantity to be tested increased, the kind number that is under the jurisdiction of this pedigree might increase; Below analogize.
Said G 3C 301G 348G 416A 571G 712Corresponding tree peony kind cohort is made up of following kind at least: ' erythema is white ' and ' Feng Dan '; Said G 3C 301G 348G 416A 571G 712Corresponding tree peony kind cohort specifically is made up of following kind: ' erythema is white ' and ' Feng Dan ';
Said T 3A 301G 348G 416A 571T 712Corresponding tree peony kind cohort is made up of following kind at least: ' pea green ', ' Yao is yellow ', ' big Hu Hong ', ' sieve suede is red ' and ' SHOUANHONG '; Said T 3A 301G 348G 416A 571T 712Corresponding tree peony kind cohort specifically is made up of following kind: ' pea green ', ' Yao is yellow ', ' big Hu Hong ', ' sieve suede is red ' and ' SHOUANHONG ';
Said T 3A 301G 348A 416A 571T 712Corresponding tree peony kind cohort is made up of following kind at least: ' ROUFURONG ' and ' the red powder of phoenix '; Said T 3A 301G 348A 416A 571T 712Corresponding tree peony kind cohort specifically is made up of following kind: ' ROUFURONG ' and ' the red powder of phoenix '.
In the aforesaid method, in the step 4), the method for said definite tree peony kind to be measured comprises the steps:
If the nucleic acid molecule formula of said tree peony kind to be measured is T 3C 301A 348G 416C 571G 712, tree peony kind then to be measured is or the candidate is in the kind cohort of ' Hua Erqiao ', ' acupuncture needle iron pink robe ', ' folded cloud ', ' Luoyang is red ', ' Corallium Japonicum Kishinouye platform ', ' hat generation carbuncle ', ' Venus snow wave ', ' piebald is white ' and ' scape is beautiful ' composition any one;
If the nucleic acid molecule formula of said tree peony kind to be measured is G 3C 301G 348G 416A 571G 712, tree peony kind then to be measured be or the candidate for ' erythema is white ' and ' Feng Dan ' composition the kind cohort in any one;
If said nucleic acid molecule formula to be measured is T 3A 301G 348G 416A 571T 712, the name of tree peony kind then to be measured is called or the candidate is ' pea green ', ' Yao is yellow ', ' any one in the kind cohort of big Hu Hong ', ' sieve suede is red ' and ' SHOUANHONG ' composition;
If the nucleic acid molecule formula of said kind to be measured is T 3A 301G 348A 416A 571T 712, the name of tree peony kind then to be measured is called or the candidate is in the kind cohort of ' ROUFURONG ' and ' phoenix red powder ' composition any one.
In the aforesaid method; After step 4), also comprise the steps: the phenotypic characteristic of each kind in more said tree peony kind to be measured and the kind cohort under it; If tree peony kind to be measured with its under kind cohort in a kind of kind phenotypic characteristic (pattern or flower type) identical, then said tree peony kind to be measured be or the candidate is the kind of correspondence.
Another object of the present invention provides the test kit of a kind of evaluation or assistant identification tree peony kind to be measured.
Test kit provided by the invention, comprise primer to, record the comparison card of nucleotide sequence group and nucleic acid molecule formula group;
Said primer is to being that the primer be made up of the dna molecular shown in the sequence 2 in sequence in the sequence table 1 and the sequence table is right;
Said nucleotide sequence group is made up of sequence in the sequence table 3, sequence 4, sequence 5 and sequence 6;
Said nucleic acid molecule formula group is made up of following four kinds of nucleic acid molecule formulas: T 3C 301A 348G 416C 571G 712, G 3C 301G 348G 416A 571G 712, T 3A 301G 348G 416A 571T 712And T 3A 301G 348A 416A 571T 712
Said tree peony kind to be measured is specially ' Hua Erqiao ', ' acupuncture needle iron pink robe ', ' folded cloud ', ' Luoyang is red ', ' Corallium Japonicum Kishinouye platform ', ' hat generation carbuncle ', ' Venus snow wave ', ' piebald is white ', ' scape is beautiful ', ' erythema is white ', ' Feng Dan ', ' pea green ', ' Yao is yellow ', ' big Hu Hong ', ' sieve suede is red ', ' SHOUANHONG ', ' ROUFURONG ' or ' the red powder of phoenix '.
The application of above-mentioned test kit in evaluation or assistant identification tree peony kind to be measured also is the scope that the present invention protects; Said tree peony kind to be measured is specially ' Hua Erqiao ', ' acupuncture needle iron pink robe ', ' folded cloud ', ' Luoyang is red ', ' Corallium Japonicum Kishinouye platform ', ' hat generation carbuncle ', ' Venus snow wave ', ' piebald is white ', ' scape is beautiful ', ' erythema is white ', ' Feng Dan ', ' pea green ', ' Yao is yellow ', ' big Hu Hong ', ' sieve suede is red ', ' SHOUANHONG ', ' ROUFURONG ' or ' the red powder of phoenix '.
The 3rd purpose of the present invention provides the method for a kind of evaluation or assistant identification plant variety.
Method provided by the invention comprises the steps:
1) through the chloroplast gene group sequence of comparison plant a plurality of kinds, choose have mononucleotide polymorphism site difference in the chloroplast gene group sequence of said a plurality of kinds the zone as the target DNA zone;
2) utilize the sequence of both sides, said target DNA zone conservative region, can the increase primer in said target DNA zone of design;
3) with said primer each kind is carried out pcr amplification respectively respectively, obtain the target DNA zone of each said plant variety;
4) the check order target DNA zone of each said plant variety obtains the nucleotide sequence in the target DNA zone of each kind;
5) nucleotide sequence in the target DNA of all kinds zone relatively detects mononucleotide polymorphism site, lists the nucleic acid molecule formula of each kind; The nucleic acid molecule formula of said each kind is by forming to 3 ' all mononucleotide polymorphism sites terminal or that list successively from 3 ' end to 5 ' end and sequence of positions numbering thereof from 5 ' end in the nucleotide sequence in the target DNA zone of each kind; The nucleic acid molecule formula general formula of said each kind is B X1B X2B X3B Xn, wherein, B is the base kind on the mononucleotide polymorphism site, is in A, T, C and four kinds of bases of G any one; X1Be that the 1st mononucleotide polymorphism site numbered from 5 ' end or from 3 ' terminal sequence of positions at the nucleotide sequence in the target DNA zone of being detected, X2Be that the 2nd mononucleotide polymorphism site numbered from 5 ' end or from 3 ' terminal sequence of positions at the nucleotide sequence in the target DNA zone of being detected, analogize, XnBe that n mononucleotide polymorphism site numbered from 5 ' end or from 3 ' terminal sequence of positions at the nucleotide sequence in the target DNA zone of being detected;
Said B X1B X2B X3B XnFor all mononucleotide polymorphism sites are listed from 5 ' end or 3 ' end according to its nucleotide sequence in the target DNA zone of being detected successively;
The kind that 6) will have identical said nucleic acid molecule formula is formed same pedigree, and the kind with different IPs thuja acid molecular formula is formed different pedigrees; All pedigrees are formed nucleic acid molecule formula pedigree chart;
In the said nucleic acid molecule formula pedigree chart, each nucleic acid molecule formula correspondence is from the kind cohort of a pedigree;
7) will treat that measuring plants is according to step 3)-5) repetitive operation, with step 3)-5) in kind replace with and treat measuring plants, obtain the said nucleic acid molecule formula of treating the target DNA zone of measuring plants; With all nucleic acid molecule formulas comparisons in the nucleic acid molecule formula in said target DNA zone of treating measuring plants and the said nucleic acid molecule formula pedigree chart, confirm the said variety name of measuring plants and the pedigree that is subordinate to thereof treated.
The subscript of above-mentioned nucleic acid molecule formula also can be the figure place that begins to count from any comparable starting point.
In the aforesaid method, in the step 1), said target DNA zone is a part psbf-petL intergenic region;
In the step 3), the template of said pcr amplification is the genomic dna of each said plant variety;
In the step 5), the nucleic acid molecule formula of said each kind is by forming from 5 ' terminal Nucleotide letter and sequence of positions numbering thereof to 3 ' terminal all mononucleotide polymorphism sites of listing successively in the nucleotide sequence in the target DNA zone of each kind; The nucleic acid molecule formula general formula of said each kind is B X1B X2B X3B Xn, wherein, B is the base kind on the mononucleotide polymorphism site, is in A, T, C and four bases of G any one; X1Be that the 1st mononucleotide polymorphism site numbered from 5 ' terminal sequence of positions in the nucleotide sequence in the target DNA zone of being detected, X2Be that the 2nd mononucleotide polymorphism site numbered from 5 ' terminal sequence of positions in the nucleotide sequence in the target DNA zone of being detected, analogize, XnBe that n mononucleotide polymorphism site numbered from 5 ' terminal sequence of positions in the nucleotide sequence in the target DNA zone of being detected;
Said B X1B X2B X3B XnFor all mononucleotide polymorphism sites are listed from 5 ' end in the nucleotide sequence in the target DNA zone of being detected according to it successively;
In the step 7); The said method of confirming the said pedigree of treating the variety name of measuring plants and being subordinate to comprises the steps: if the nucleic acid molecule formula in said target DNA zone of treating measuring plants is identical with a nucleic acid molecule formula in the said nucleic acid molecule formula pedigree chart, and then said plant variety to be measured be or the candidate is any one kind in the said pedigree of said nucleic acid molecule formula correspondence.
In the aforesaid method; In the step 5); The said method of listing the nucleic acid molecule formula of each kind comprises the steps: all mononucleotide polymorphism sites in the nucleotide sequence in the target DNA of each kind zone are listed from 5 ' terminal order in the nucleotide sequence in said target DNA zone according to it successively, and will said each mononucleotide polymorphism site in the nucleotide sequence in said target DNA zone from the sequence of positions of 5 ' end number as the alphabetical subscript of the Nucleotide of said mononucleotide polymorphism site; Obtain the nucleic acid molecule formula of each kind.
In the aforesaid method, in the step 1), said plant is a tree peony, and the nucleotide sequence of the said part psbE-petL intergenic region of a plurality of kinds of said plant and correspondence thereof is following:
Said plant variety is ' Hua Erqiao ', ' acupuncture needle iron pink robe ', ' folded cloud ', ' Luoyang is red ', ' Corallium Japonicum Kishinouye platform ', ' hat generation carbuncle ', ' Venus snow wave ', ' piebald is white ', ' scape is beautiful ', ' erythema is white ', ' Feng Dan ', ' pea green ', ' Yao is yellow ', ' big Hu Hong ', ' sieve suede is red ', ' SHOUANHONG ', ' ROUFURONG ' and ' the red powder of phoenix ';
The nucleotides sequence of the part psbE-petL intergenic region of said ' Hua Erqiao ', ' acupuncture needle iron pink robe ', ' folded cloud ', ' Luoyang is red ', ' Corallium Japonicum Kishinouye platform ', ' hat generation carbuncle ', ' Venus snow wave ', ' piebald is white ', ' scape is beautiful ' is classified the sequence 3 in the sequence table as;
The nucleotides sequence of the part psbE-petL intergenic region of said ' erythema is white ', ' Feng Dan ' is classified the sequence 4 in the sequence table as;
Said ' pea green ', ' Yao yellow ', ' nucleotides sequence of the part psbE-petL intergenic region of big Hu Hong ', ' sieve suede is red ', ' SHOUANHONG ' is classified the sequence 5 in the sequence table as;
The nucleotides sequence of the part psbE-petL intergenic region of said ' ROUFURONG ', ' the red powder of phoenix ' is classified the sequence 6 in the sequence table as;
Step 2) in, said primer is to be made up of the dna molecular shown in the sequence 2 in sequence in the sequence table 1 and the sequence table;
In the step 4), the nucleotide sequence in the target DNA zone of said ' Hua Erqiao ', ' acupuncture needle iron pink robe ', ' folded cloud ', ' Luoyang is red ', ' Corallium Japonicum Kishinouye platform ', ' hat generation carbuncle ', ' Venus snow wave ', ' piebald is white ', ' scape is beautiful ' is the sequence 3 in the sequence table;
The nucleotide sequence in the target DNA zone of said ' erythema is white ', ' Feng Dan ' is the sequence 4 in the sequence table;
Said ' pea green ', ' Yao yellow ', ' nucleotide sequence in the target DNA zone of big Hu Hong ', ' sieve suede is red ', ' SHOUANHONG ' is the sequence 5 in the sequence table;
The nucleotide sequence in the target DNA zone of said ' ROUFURONG ' and ' the red powder of phoenix ' is the sequence 6 in the sequence table;
Step 2) in, said primer is to be made up of the dna molecular shown in the sequence 2 in sequence in the sequence table 1 and the sequence table;
In the step 4), the nucleotide sequence in the target DNA zone of said ' Hua Erqiao ', ' acupuncture needle iron pink robe ', ' folded cloud ', ' Luoyang is red ', ' Corallium Japonicum Kishinouye platform ', ' hat generation carbuncle ', ' Venus snow wave ', ' piebald is white ', ' scape is beautiful ' is the sequence 3 in the sequence table;
The nucleotide sequence in the target DNA zone of said ' erythema is white ', ' Feng Dan ' is the sequence 4 in the sequence table;
Said ' pea green ', ' Yao yellow ', ' nucleotide sequence in the target DNA zone of big Hu Hong ', ' sieve suede is red ', ' SHOUANHONG ' is the sequence 5 in the sequence table;
The nucleotide sequence in the target DNA zone of said ' ROUFURONG ' and ' the red powder of phoenix ' is the sequence 6 in the sequence table;
In the step 5), the nucleic acid molecule formula of said ' Hua Erqiao ', ' acupuncture needle iron pink robe ', ' folded cloud ', ' Luoyang is red ', ' Corallium Japonicum Kishinouye platform ', ' hat generation carbuncle ', ' Venus snow wave ', ' piebald is white ' and ' scape is beautiful ' is T 3C 301A 348G 416C 571G 712
The nucleic acid molecule formula of said ' erythema is white ' and ' Feng Dan ' is G 3C 301G 348G 416A 571G 712
Said ' pea green ', ' Yao yellow ', ' the nucleic acid molecule formula of big Hu Hong ', ' sieve suede is red ' and ' SHOUANHONG ' is T 3A 301G 348G 416A 571T 712
The nucleic acid molecule formula of said ' ROUFURONG ' and ' the red powder of phoenix ' is T 3A 301G 348A 416A 571T 712
The method of describing in this patent makes up the nucleic acid molecule formula and is applicable to enterprise's formula earning activities such as plant variety classification, evaluation, investigation and management; In the research of public and social interest sexology was not included in, the nucleic acid molecule formula that this paper uses was meant the structure of genotype type polymorphic mononucleotide letter sequence of positions number structure residing with it in the variable region sequences that utilizes the plant chloroplast genomic dna, that be used to distinguish kind.
Experiment of the present invention proves; Method provided by the invention is for utilizing the plant chloroplast genomic dna sequence; Form with the nucleic acid molecule formula is identified and pedigree analysis plant variety, is a kind of comparatively accurate, simple and direct method, can improve the production efficiency and the quality monitoring level of related industries; Remedied the deficiency of previous methods, can make analysis precision reach the level of a nucleic acid molecule.Specifically be to increase, check order, set up the nucleic acid molecule formula, set up different pedigrees according to different nucleic acid molecule formulas through 18 ornamental tree peony chloroplast(id) psbE-petL intergenic regions; Should can be used for detecting by pedigree crowd's table that different IPs thuja acid molecular formula is set up and treat whether measuring plants is 18 ornamental tree peonies, and method is easy and easy to operate; Combine with morphological specificity, further accurately identify the kind of treating measuring plants.
Embodiment
Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.
View and admire the tree peony kind with 18 kinds among the following embodiment: ' Hua Erqiao ', ' acupuncture needle iron pink robe ', ' folded cloud ', ' Luoyang is red ', ' Corallium Japonicum Kishinouye platform ', ' hat generation carbuncle ', ' Venus snow wave ', ' piebald is white ', ' scape is beautiful ', ' erythema is white ', ' Feng Dan ', ' pea green ', ' Yao is yellow ', ' big Hu Hong ', ' sieve suede is red ', ' SHOUANHONG ', ' ROUFURONG ' and ' the red powder of phoenix '.The tree peony kind is through the grafting vegetative propagation.
The fresh blade that is used to analyze was gathered spring, utilized discolour silica gel (siccative) rapid drying immediately to preserve.
Embodiment 1, evaluation tree peony kind
We's ratio juris: plant materials contains 3 genomes, is respectively chloroplast gene group, Mitochondrial Genome Overview and cell nucleus gene group.Present method is to utilize conventional Protocols in Molecular Biology from the plant chloroplast genome, to gather one of innovative approach of bioinformation.The DNA of plant (thymus nucleic acid) sequence is made up of A, G, C, four kinds of bases of T (Nucleotide).According to the initial of their English names, be referred to as A (Adenine VITAMIN B4), T (Thymine thymus pyrimidine), G (Guanine guanine), C (Cytosine cytosine(Cyt)) respectively.
Present method utilizes ordinary method (extracting test kit like CTAB method or commercially available plant genome DNA) from the vegetable material of a plurality of purpose kinds, to extract DNA respectively earlier; From the chloroplast gene group, select 1 again to several DNA zones; Utilize the conserved regions sequences Design primer at its two ends; Utilize polymerase chain reaction (Polymerase Chain Reaction is called for short PCR) to amplify corresponding D NA fragment, this regional dna sequence dna in the genome of each kind of order-checking acquisition; Compare the difference between it; Utilize on the same site discrepant nucleic acid molecule letter and count (or confirming a comparable start position arbitrarily) from certain comparable start position of 5 ' end; Calculate the sequential bits of mutating alkali yl in homologous sequence time numeral; In the lower right corner of mutating alkali yl letter, enclose the position digital in this mutational site, such mutating alkali yl is described coupling together in order; Can construct the nucleic acid molecule formula; This nucleic acid molecule formula can be used for distinguishing or plant identification kind (pedigree or strain), has a same nucleic acid molecule formula (in the time of promptly need identifying a plurality of kind that belongs to same pedigree or strain) when one to raise variety, and can combine phenotypic character to carry out the deep evaluation of plant variety (pedigree or strain).Phenotypic character refers to the proterties that plant materials shows, and comprises morphological characters, physiological characteristic, characteristic etc.
1, primer design
To the DNA VA, can design more than a pair of primer, all can be used for analysis, plant identification kind.But, select one of the highest zone of its intermediate-resolution to be used usually, can satisfy the demand.
The psbE-petL intergenic region of choosing the tree peony chloroplast genomic dna has designed a pair of new primer; Forward primer psbE-petL-356F (primer sequence: 5 '-CCTTCTTCTGACACAGCAATG-3 '; Sequence 1) and reverse primer psbE-petL-1219R (primer sequence: 5 '-TTACCATTATAGACAGCACTAACAA-3 ', sequence 2).
2, the amplification of psbE-petL intergenic region
The Plant Genome extraction test kit (DP305) of utilization China day root Bioisystech Co., Ltd extracts 18 kinds respectively and views and admires the tree peony kind: ' Hua Erqiao '; ' acupuncture needle iron pink robe '; ' folded cloud '; ' Luoyang is red '; ' Corallium Japonicum Kishinouye platform '; ' hat generation carbuncle '; ' Venus snow wave '; ' piebald is white '; ' scape is beautiful '; ' erythema is white '; ' Feng Dan '; ' pea green '; ' Yao is yellow '; ' big Hu Hong '; ' sieve suede is red '; ' SHOUANHONG '; ' ROUFURONG '; The genomic dna of ' the red powder of phoenix '.The DNA dilution is the working fluid concentration (25ng/ul) of every microlitre 25 nanograms, carries out pcr amplification, the sequence in the target DNA zone that it is 728bp that each kind all can obtain a length with above-mentioned primer psbE-petL-356F and psbE-petL-1219R.
Taq archaeal dna polymerase that adopts in the pcr amplification process and PCR damping fluid are conventional, common commercially available biochemical reagents; What buy in this experiment implementation process is the product of precious biotechnology (Dalian) ltd; PIN is TaKaRa Code:DR100B, and the operational guidance of enclosing according to product carries out pcr amplification.Pcr amplification is at Applied Biosystems Verit TMCarry out on 96-Well Thermal Cycler (Model#:9902, made in Singapore) the PCR appearance.
The program of pcr amplification is: 94 degree kept 4 minutes earlier; Carry out following 34 temperature cycle again, being set to of each temperature cycle: 94 degree 1 minute, 53 degree (annealing temperature) 40 seconds, 72 degree 1.5 minutes; At last, 72 degree extended 8 minutes.
The dna fragmentation that above-mentioned 18 kinds amplification is obtained send the big gene ltd of Beijing China (Beijing Genome Institute is called for short BGI), utilizes above-mentioned primer; At 3730xl DNA analyzer (Applied Biosystems; Foster City, California, USA) directly PCR order-checking on the sequenator; Each kind sampling has been checked order individual more than 3 or 3, and the order-checking experimental result in the kind between individuality is consistent.The result is specific as follows:
The length that ' Hua Erqiao ', ' acupuncture needle iron pink robe ', ' folded cloud ', ' Luoyang is red ', ' Corallium Japonicum Kishinouye platform ', ' hat generation carbuncle ', ' Venus snow wave ', ' piebald is white ', the amplification of ' scape is beautiful ' kind, order-checking obtain is that the nucleotides sequence in 728bp (psbE-petL district) is classified the sequence 3 in the sequence table as;
The length that the amplification of ' erythema is white ', ' Feng Dan ' kind, order-checking obtain is that the nucleotides sequence in 728bp (psbE-petL district) is classified the sequence 4 in the sequence table as;
' pea green ', ' Yao yellow ', ' length that big Hu Hong ', ' sieve suede is red ', the amplification of ' SHOUANHONG ' kind, order-checking obtain is that the nucleotides sequence in 728bp (psbE-petL district) is classified the sequence 5 in the sequence table as;
The length that ' ROUFURONG ', ' the red powder of phoenix ' kind amplification, order-checking obtain is that the nucleotides sequence in 728bp (psbE-petL district) is classified the sequence 6 in the sequence table as.
3, sequence alignment
With the length of above-mentioned 18 kinds is that the sequence (sequence 3-sequence 6) of 728bp is compared result such as table 1:
Table 1 is 18 positions, variable base site between 4 genetic pedigrees viewing and admiring the tree peony kind
In sequence 3-sequence 6; From 5 ' terminal the 3rd, 301,348,416,571,712 Nucleotide is polymorphic site between 18 kinds; The Nucleotide in all the other sites is all identical, lists corresponding nucleic acid molecule formula according to the sequence of each kind, and is specific as follows:
Be respectively T, C, A, G, C, G from 5 ' terminal the 3rd, 301,348,416,571,712 Nucleotide in the nucleotide sequence in ' Hua Erqiao ', ' acupuncture needle iron pink robe ', ' folded cloud ', ' Luoyang is red ', ' Corallium Japonicum Kishinouye platform ', ' hat generation carbuncle ', ' Venus snow wave ', ' piebald is white ', ' scape is beautiful ' psbE-petL district; Utilize these several Nucleotide letters and residing order-checking position thereof (from 5 ' end meter), listing the nucleic acid molecule formula is T 3C 301A 348G 416C 571G 712, the Nucleotide in this nucleic acid molecule formula is interracial polymorphic site, this Nucleotide of numeral in each Nucleotide letter lower right corner in the sequence of order-checking from 5 ' terminal position;
Be respectively G, C, G, G, A, G from 5 ' terminal the 3rd, 301,348,416,571,712 Nucleotide in the nucleotide sequence in the chloroplast DNA psbE-petL district of ' erythema is white ' and ' Feng Dan ' kind; Utilize these several Nucleotide and residing order-checking position thereof (from 5 ' end meter), listing the nucleic acid molecule formula is G 3C 301G 348G 416A 571G 712, the Nucleotide in this nucleic acid molecule formula is interracial polymorphic site, and this Nucleotide of numeral in each Nucleotide letter lower right corner in the sequence of order-checking from 5 ' terminal position;
' pea green ', ' Yao yellow ', ' be respectively T, A, G, G, A, T from the 3rd, 249,301,416,571,712 Nucleotide of 5 ' end in the nucleotide sequence in the chloroplast DNA psbE-petL district of big Hu Hong ', ' sieve suede is red ', ' SHOUANHONG ' kind; Utilize these several Nucleotide and residing order-checking position thereof (from 5 ' end meter), listing the nucleic acid molecule formula is T 3A 301G 348G 416A 571T 712, the Nucleotide in this nucleic acid molecule formula is from discrepant Nucleotide between 5 ' end kind, and this Nucleotide of numeral in each Nucleotide letter lower right corner in the sequence of order-checking from the position of 5 ' end;
Be respectively T, A, G, A, A, T from 5 ' terminal the 3rd, 249,301,416,571,712 Nucleotide in the nucleotide sequence in the chloroplast DNA psbE-petL district of ' ROUFURONG ', ' the red powder of phoenix ' kind; Utilize these several Nucleotide letters and residing order-checking position thereof (from 5 ' end meter), listing the nucleic acid molecule formula is T 3A 301G 348A 416A 571T 712, the Nucleotide in this nucleic acid molecule formula is interracial polymorphic site, and this Nucleotide of numeral in each Nucleotide letter lower right corner in the sequence of order-checking from 5 ' terminal position;
Nucleic acid molecule formula according to above-mentioned 18 kinds obtains 18 kinds of pedigrees of viewing and admiring the tree peony kind, and the corresponding kind of different nucleic acid molecule formulas is under the jurisdiction of different genetic pedigrees, and the corresponding kind of identical nucleic acid molecule formula is under the jurisdiction of same pedigree;
Therefore, the tree peony of above-mentioned 18 kinds is under the jurisdiction of four different pedigrees respectively, the nucleic acid molecule formula pedigree chart below 4 pedigrees are formed, and the corresponding one group of kind of each nucleic acid molecule formula:
Table 2 is a nucleic acid molecule formula pedigree chart
Pedigree I: the nucleic acid molecule formula is T 3C 301A 348G 416C 571G 712' Hua Erqiao ', ' acupuncture needle iron pink robe ', ' folded cloud ', ' Luoyang is red ', ' Corallium Japonicum Kishinouye platform ', ' hat generation carbuncle ', ' Venus snow wave ', ' piebald is white ', ' scape is beautiful '
Pedigree II: the nucleic acid molecule formula is G 3C 301G 348G 416A 571G 712' erythema is white ', ' Feng Dan '
Pedigree III: the nucleic acid molecule formula is T 3A 301G 348G 416A 571T 712' pea green ', ' Yao is yellow ', ' big Hu Hong ', ' sieve suede is red ', ' SHOUANHONG '
Pedigree IV: the nucleic acid molecule formula is T 3A 301G 348A 416A 571T 712' ROUFURONG ', ' the red powder of phoenix '.
Owing to contain 1 to raise variety in each pedigree, further the combining form characterized is to concrete kind; With Macro or mass analysis such as pedigree, nucleic acid molecule formula, morphological specificitys, work out out 18 kinds of systematic searching tables of viewing and admiring the tree peony kind, see table 3:
Table 3 is 18 systematic searching tables of viewing and admiring the tree peony kind
Figure BDA00001803764900101
Figure BDA00001803764900111
Annotate: wherein, the psbE-petL intergenic region of the green body genomic dna that " psbE-petL district " refers to be adopted; " 728bp " representes that the ordering length of this intergenic region dna sequence dna is 728 nucleic acid molecules; The nucleic acid molecule formula is like " T 3C 301A 348G 416C 571G 712" being illustrated in the molecular formula that contains 6 polymorphum mononucleotides [single nucleotide polymorphism (SNP)] in the ordering aligned sequences in this zone; the numeral in Nucleotide letter (T; C, G or A) lower right corner is from the position of the corresponding SNP of sequence 5 ' end.Utilize the nucleic acid molecule formula at first to view and admire the tree peony variety plot and be divided into 4 pedigrees, then, in each pedigree, utilize phenotypic character to distinguish kind 18.
The psbE-petL intergenic region sequence (sequence 3-sequence 6) of the primer of above-mentioned amplification psbE-petL intergenic region, 18 kinds and the nucleic acid molecule formula of 18 kinds can be formed test kit, can utilize this test kit and aforesaid method to detect tree peony kind to be measured: if the nucleic acid molecule formula of said kind to be measured is T 3C 301A 348G 416C 571G 712, then said tree peony to be measured is or the candidate is in the kind cohort formed of ' Hua Erqiao ', ' acupuncture needle iron pink robe ', ' folded cloud ', ' Luoyang is red ', ' Corallium Japonicum Kishinouye platform ', ' hat generation carbuncle ', ' Venus snow wave ', ' piebald is white ', ' scape is beautiful ' any one;
If the nucleic acid molecule formula of said kind to be measured is G 3C 301G 348G 416A 571G 712, then said tree peony to be measured is or the candidate is in the kind cohort formed of ' erythema is white ', ' Feng Dan ' any one;
If the nucleic acid molecule formula of said kind to be measured is T 3A 301G 348G 416A 571T 712, then said tree peony to be measured is or the candidate is ' pea green ', ' Yao is yellow ', ' any one in the kind cohort of big Hu Hong ', ' sieve suede is red ', ' SHOUANHONG ' composition;
If the nucleic acid molecule formula of said kind to be measured is T 3A 301G 348A 416A 571T 712, then said tree peony to be measured is or the candidate is in the kind cohort formed of ' ROUFURONG ', ' phoenix red powder ' any one.
Compare through the nucleic acid molecule formula; After confirming the affiliated kind cohort of said tree peony to be measured; Phenotypic characteristic through comparing each kind in said tree peony to be measured and the kind cohort under it carries out cultivar identification; If said tree peony to be measured with its under kind group in a kind of kind phenotypic characteristic identical, then said tree peony to be measured be or the candidate is the kind of correspondence.
Can find out from above-mentioned, can utilize nucleic acid molecule that kind to be measured is carried out hierarchical classification earlier, combine phenotypic characteristic to confirm tree peony kind to be measured again.
Embodiment 2, evaluation tree peony kind
With 18 kinds of tree peony kinds picking up from production base, the tree peony gardener village, Caozhou: ' Hua Erqiao ', ' acupuncture needle iron pink robe ', ' folded cloud ', ' Luoyang is red ', ' Corallium Japonicum Kishinouye platform ', ' hat generation carbuncle ', ' Venus snow wave ', ' piebald is white ', ' scape is beautiful ', ' erythema is white ', ' Feng Dan ', ' pea green ', ' Yao is yellow ', ' big Hu Hong ', ' sieve suede is red ', ' SHOUANHONG ', ' ROUFURONG ' identify that according to the method (step 2-3) of embodiment 1 each kind is got 5 strains with ' phoenix pellet powder '; The result is all correct.
The south of the River tree peony kind of picking up from Hunan Province's Shaoyang City ' fragrant red ' is identified that according to the method (step 2-3) of embodiment a kind is got 5 strains; The nucleotides sequence in its chloroplast DNA psbE-petL district is classified the sequence 7 in the sequence table as as a result, and its nucleic acid molecule formula is T 3T 249C 301G 348G 416A 571G 712, and the Nucleotide of the 249th base position of the nucleotide sequence in the chloroplast DNA psbE-petL district of above-mentioned 18 kinds of tree peony kinds is C.Explain that ' fragrant red ' is not any one in 18 kinds of tree peony kinds, thereby prove that method specificity of the present invention is good.
Figure IDA00001803765600011
Figure IDA00001803765600021
Figure IDA00001803765600041
Figure IDA00001803765600051
Figure IDA00001803765600061

Claims (10)

1. identify or the method for assistant identification tree peony kind to be measured for one kind, comprise the steps:
1) with primer to tree peony kind to be measured is carried out pcr amplification, obtain the PCR product in purpose zone; Said primer is to being made up of the dna molecular shown in the sequence 2 in sequence in the sequence table 1 and the sequence table;
2), obtain the nucleotide sequence in tree peony kind purpose to be measured zone with said PCR product order-checking;
3) nucleotide sequence and the nucleotide sequence cohort in said tree peony kind purpose to be measured zone are compared, find out mononucleotide polymorphism site, list tree peony kind nucleic acid molecule formula to be measured;
The general formula of the nucleic acid molecule formula of said tree peony kind to be measured is: B X1B X2B X3B Xn, wherein, B is the base kind on the mononucleotide polymorphism site, is in A, T, C and four kinds of bases of G any one; X1Be that the 1st mononucleotide polymorphism site numbered from 5 ' terminal sequence of positions in the nucleotide sequence in said tree peony kind purpose to be measured zone, X2Be that the 2nd mononucleotide polymorphism site numbered from 5 ' terminal sequence of positions in the nucleotide sequence in said tree peony kind purpose to be measured zone, analogize, XnBe that n mononucleotide polymorphism site numbered from 5 ' terminal sequence of positions in the nucleotide sequence in said tree peony kind purpose to be measured zone; Said B X1B X2B X3B XnFor all mononucleotide polymorphism sites are listed from 5 ' end in the nucleotide sequence in said tree peony kind purpose to be measured zone according to it successively;
Said nucleotide sequence cohort is made up of sequence in the sequence table 3, sequence 4, sequence 5 and sequence 6;
4) with said tree peony kind nucleic acid molecule formula to be measured and the comparison of nucleic acid molecule formula cohort, confirm tree peony kind to be measured;
Said nucleic acid molecule formula cohort is made up of following four kinds of nucleic acid molecule formulas: T 3C 301A 348G 416C 571G 712, G 3C 301G 348G 416A 571G 712, T 3A 301G 348G 416A 571T 712And T 3A 301G 348A 416A 571T 712
The corresponding tree peony kind cohort of each said nucleic acid molecule formula.
2. method according to claim 1 is characterized in that:
In the step 1), the template of said pcr amplification is the genomic dna of said tree peony kind to be measured;
In the step 3); The said method of listing the nucleic acid molecule formula of tree peony kind to be measured comprises the steps: listing successively in order from 5 ' terminal the 3rd, 301,348,416,571 and 712 the Nucleotide letter of mononucleotide polymorphism site in the nucleotide sequence in said tree peony kind purpose to be measured zone; And with each mononucleotide polymorphism site in the nucleotide sequence in said tree peony kind purpose to be measured zone from 5 ' terminal sequence of positions number as subscript; Be marked on the lower right corner of the Nucleotide letter of said mononucleotide polymorphism site, obtain the nucleic acid molecule formula of tree peony kind to be measured;
The general formula of the nucleic acid molecule formula of said tree peony kind to be measured is B 3B 301B 348B 416B 571B 712
In the step 4), said T 3C 301A 348G 416C 571G 712Corresponding tree peony kind cohort is made up of following kind at least: ' Hua Erqiao ', ' acupuncture needle iron pink robe ', ' folded cloud ', ' Luoyang is red ', ' Corallium Japonicum Kishinouye platform ', ' hat generation carbuncle ', ' Venus snow wave ', ' piebald is white ' and ' scape is beautiful ';
Said G 3C 301G 348G 416A 571G 712Corresponding tree peony kind cohort is made up of following kind at least: ' erythema is white ' and ' Feng Dan ';
Said T 3A 301G 348G 416A 571T 712Corresponding tree peony kind cohort is made up of following kind at least: ' pea green ', ' Yao is yellow ', ' big Hu Hong ', ' sieve suede is red ' and ' SHOUANHONG ';
Said T 3A 301G 348A 416A 571T 712Corresponding tree peony kind cohort is made up of following kind at least: ' ROUFURONG ' and ' the red powder of phoenix '.
3. method according to claim 1 and 2 is characterized in that:
In the step 4), the method for said definite tree peony kind to be measured comprises the steps:
If the nucleic acid molecule formula of said tree peony kind to be measured is T 3C 301A 348G 416C 571G 712, tree peony kind then to be measured is or the candidate is in the kind cohort of ' Hua Erqiao ', ' acupuncture needle iron pink robe ', ' folded cloud ', ' Luoyang is red ', ' Corallium Japonicum Kishinouye platform ', ' hat generation carbuncle ', ' Venus snow wave ', ' piebald is white ' and ' scape is beautiful ' composition any one;
If the nucleic acid molecule formula of said tree peony kind to be measured is G 3C 301G 348G 416A 571G 712, tree peony kind then to be measured be or the candidate for ' erythema is white ' and ' Feng Dan ' composition the kind cohort in any one;
If said nucleic acid molecule formula to be measured is T 3A 301G 348G 416A 571T 712, the name of tree peony kind then to be measured is called or the candidate is ' pea green ', ' Yao is yellow ', ' any one in the kind cohort of big Hu Hong ', ' sieve suede is red ' and ' SHOUANHONG ' composition;
If the nucleic acid molecule formula of said kind to be measured is T 3A 301G 348A 416A 571T 712, the name of tree peony kind then to be measured is called or the candidate is in the kind cohort of ' ROUFURONG ' and ' phoenix red powder ' composition any one.
4. according to arbitrary described method among the claim 1-3; It is characterized in that: the phenotypic characteristic that after step 4), also comprises the steps: each kind in more said tree peony kind to be measured and the kind cohort under it; If tree peony kind to be measured with its under kind cohort in a kind of kind phenotypic characteristic identical, then said tree peony kind to be measured be or the candidate is the kind of correspondence.
5. identify or the test kit of assistant identification tree peony kind to be measured for one kind, comprise primer to, record the data comparison card of nucleotide sequence group and nucleic acid molecule formula group;
Said primer is to being that the primer be made up of the dna molecular shown in the sequence 2 in sequence in the sequence table 1 and the sequence table is right;
Said nucleotide sequence group is made up of sequence in the sequence table 3, sequence 4, sequence 5 and sequence 6;
Said nucleic acid molecule formula group is made up of following four kinds of nucleic acid molecule formulas: T 3C 301A 348G 416C 571G 712, G 3C 301G 348G 416A 571G 712, T 3A 301G 348G 416A 571T 712And T 3A 301G 348A 416A 571T 712
Said tree peony kind to be measured is specially ' Hua Erqiao ', ' acupuncture needle iron pink robe ', ' folded cloud ', ' Luoyang is red ', ' Corallium Japonicum Kishinouye platform ', ' hat generation carbuncle ', ' Venus snow wave ', ' piebald is white ', ' scape is beautiful ', ' erythema is white ', ' Feng Dan ', ' pea green ', ' Yao is yellow ', ' big Hu Hong ', ' sieve suede is red ', ' SHOUANHONG ', ' ROUFURONG ' or ' the red powder of phoenix '.
6. the application of the described test kit of claim 5 in evaluation or assistant identification tree peony kind to be measured; Said tree peony kind to be measured is specially ' Hua Erqiao ', ' acupuncture needle iron pink robe ', ' folded cloud ', ' Luoyang is red ', ' Corallium Japonicum Kishinouye platform ', ' hat generation carbuncle ', ' Venus snow wave ', ' piebald is white ', ' scape is beautiful ', ' erythema is white ', ' Feng Dan ', ' pea green ', ' Yao is yellow ', ' big Hu Hong ', ' sieve suede is red ', ' SHOUANHONG ', ' ROUFURONG ' or ' the red powder of phoenix '.
7. identify or the method for assistant identification plant variety for one kind, comprise the steps:
1) through the chloroplast gene group sequence of comparison plant a plurality of kinds, choose have mononucleotide polymorphism site difference in the chloroplast gene group sequence of said a plurality of kinds the zone as the target DNA zone;
2) utilize the sequence of both sides, said target DNA zone conservative region, can the increase primer in said target DNA zone of design;
3) with said primer each kind is carried out pcr amplification respectively, obtain the target DNA zone of each said plant variety;
4) the check order target DNA zone of each said plant variety obtains the nucleotide sequence in the target DNA zone of each kind;
5) nucleotide sequence in the target DNA of all kinds zone relatively detects mononucleotide polymorphism site, lists the nucleic acid molecule formula of each kind; The nucleic acid molecule formula of said each kind is by forming from 5 ' terminal Nucleotide letter and sequence of positions numbering thereof to 3 ' all mononucleotide polymorphism sites terminal or that list successively from 3 ' end to 5 ' end in the nucleotide sequence in the target DNA zone of each kind; The nucleic acid molecule formula general formula of said each kind is B X1B X2B X3B Xn, wherein, B is the base kind on the mononucleotide polymorphism site, is in A, T, C and four bases of G any one; X1Be that the 1st mononucleotide polymorphism site numbered from 5 ' end or from 3 ' terminal sequence of positions in the nucleotide sequence in the target DNA zone of being detected, X2Be that the 2nd mononucleotide polymorphism site numbered from 5 ' end or from 3 ' terminal sequence of positions in the nucleotide sequence in the target DNA zone of being detected, analogize, XnBe that n mononucleotide polymorphism site numbered from 5 ' end or from 3 ' terminal sequence of positions in the nucleotide sequence in the target DNA zone of being detected;
Said B X1B X2B X3B XnFor all mononucleotide polymorphism sites are listed from 5 ' end or 3 ' end in the nucleotide sequence in the target DNA zone of being detected according to it successively;
The kind that 6) will have identical said nucleic acid molecule formula is formed same pedigree, and the kind with different IPs thuja acid molecular formula is formed different pedigrees; All pedigrees are formed nucleic acid molecule formula pedigree chart;
In the said nucleic acid molecule formula pedigree chart, each nucleic acid molecule formula correspondence is from the kind cohort of a pedigree;
7) will treat that measuring plants is according to step 3)-5) repetitive operation, with step 3)-5) in kind replace with and treat measuring plants, obtain the said nucleic acid molecule formula of treating the target DNA zone of measuring plants; With all nucleic acid molecule formulas comparisons in the nucleic acid molecule formula in said target DNA zone of treating measuring plants and the said nucleic acid molecule formula pedigree chart, confirm the said variety name of measuring plants and the pedigree that is subordinate to thereof treated.
8. method according to claim 7 is characterized in that:
In the step 1), said target DNA zone is a part psbE-petL intergenic region;
In the step 3), the template of said pcr amplification is the genomic dna of each said plant variety;
In the step 5), the nucleic acid molecule formula of said each kind is by forming from 5 ' terminal Nucleotide letter and sequence of positions numbering thereof to 3 ' terminal all mononucleotide polymorphism sites of listing successively in the nucleotide sequence in the target DNA zone of each kind; The nucleic acid molecule formula general formula of said each kind is B X1B X2B X3B Xn, wherein, B is the base kind on the mononucleotide polymorphism site, is in A, T, C and four bases of G any one; X1Be that the 1st mononucleotide polymorphism site numbered from 5 ' terminal sequence of positions in the nucleotide sequence in the target DNA zone of being detected, X2Be that the 2nd mononucleotide polymorphism site numbered from 5 ' terminal sequence of positions in the nucleotide sequence in the target DNA zone of being detected, analogize, XnBe that n mononucleotide polymorphism site numbered from 5 ' terminal sequence of positions in the nucleotide sequence in the target DNA zone of being detected;
Said B X1B X2B X3B XnFor all mononucleotide polymorphism sites are listed from 5 ' end in the nucleotide sequence in the target DNA zone of being detected according to it successively;
In the step 7); The said method of confirming the said pedigree of treating the variety name of measuring plants and being subordinate to comprises the steps: if the nucleic acid molecule formula in said target DNA zone of treating measuring plants is identical with a nucleic acid molecule formula in the said nucleic acid molecule formula pedigree chart, and then said plant variety to be measured be or the candidate is any one kind in the said pedigree of said nucleic acid molecule formula correspondence.
9. according to claim 7 or 8 described methods, it is characterized in that:
In the step 5); The said method of listing the nucleic acid molecule formula of each kind comprises the steps: all mononucleotide polymorphism sites in the nucleotide sequence in the target DNA of each kind zone are listed from 5 ' terminal order in the nucleotide sequence in said target DNA zone according to it successively, and will said each mononucleotide polymorphism site in the nucleotide sequence in said target DNA zone from the sequence of positions of 5 ' end number as the alphabetical subscript of the Nucleotide of said mononucleotide polymorphism site; Obtain the nucleic acid molecule formula of each kind.
10. according to arbitrary described method among the claim 7-9, it is characterized in that:
In the step 1), said plant is a tree peony, and the nucleotide sequence of the said part psbE-petL intergenic region of a plurality of kinds of said plant and correspondence thereof is following:
Said plant variety is ' Hua Erqiao ', ' acupuncture needle iron pink robe ', ' folded cloud ', ' Luoyang is red ', ' Corallium Japonicum Kishinouye platform ', ' hat generation carbuncle ', ' Venus snow wave ', ' piebald is white ', ' scape is beautiful ', ' erythema is white ', ' Feng Dan ', ' pea green ', ' Yao is yellow ', ' big Hu Hong ', ' sieve suede is red ', ' SHOUANHONG ', ' ROUFURONG ' and ' the red powder of phoenix ';
The nucleotides sequence of the part psbE-petL intergenic region of said ' Hua Erqiao ', ' acupuncture needle iron pink robe ', ' folded cloud ', ' Luoyang is red ', ' Corallium Japonicum Kishinouye platform ', ' hat generation carbuncle ', ' Venus snow wave ', ' piebald is white ', ' scape is beautiful ' is classified the sequence 3 in the sequence table as;
The nucleotides sequence of the part psbE-petL intergenic region of said ' erythema is white ', ' Feng Dan ' is classified the sequence 4 in the sequence table as;
Said ' pea green ', ' Yao yellow ', ' nucleotides sequence of the part psbE-petL intergenic region of big Hu Hong ', ' sieve suede is red ', ' SHOUANHONG ' is classified the sequence 5 in the sequence table as;
The nucleotides sequence of the part psbE-petL intergenic region of said ' ROUFURONG ', ' the red powder of phoenix ' is classified the sequence 6 in the sequence table as;
Step 2) in, said primer is to be made up of the dna molecular shown in the sequence 2 in sequence in the sequence table 1 and the sequence table;
In the step 4), the nucleotide sequence in the target DNA zone of said ' Hua Erqiao ', ' acupuncture needle iron pink robe ', ' folded cloud ', ' Luoyang is red ', ' Corallium Japonicum Kishinouye platform ', ' hat generation carbuncle ', ' Venus snow wave ', ' piebald is white ', ' scape is beautiful ' is the sequence 3 in the sequence table;
The nucleotide sequence in the target DNA zone of said ' erythema is white ', ' Feng Dan ' is the sequence 4 in the sequence table;
Said ' pea green ', ' Yao yellow ', ' nucleotide sequence in the target DNA zone of big Hu Hong ', ' sieve suede is red ', ' SHOUANHONG ' is the sequence 5 in the sequence table;
The nucleotide sequence in the target DNA zone of said ' ROUFURONG ' and ' the red powder of phoenix ' is the sequence 6 in the sequence table;
In the step 5), the nucleic acid molecule formula of said ' Hua Erqiao ', ' acupuncture needle iron pink robe ', ' folded cloud ', ' Luoyang is red ', ' Corallium Japonicum Kishinouye platform ', ' hat generation carbuncle ', ' Venus snow wave ', ' piebald is white ' and ' scape is beautiful ' is T 3C 301A 348G 416C 571G 712
The nucleic acid molecule formula of said ' erythema is white ' and ' Feng Dan ' is G 3C 301G 348G 416A 571G 712
Said ' pea green ', ' Yao yellow ', ' the nucleic acid molecule formula of big Hu Hong ', ' sieve suede is red ' and ' SHOUANHONG ' is T 3A 301G 348G 416A 571T 712
The nucleic acid molecule formula of said ' ROUFURONG ' and ' the red powder of phoenix ' is T 3A 301G 348A 416A 571T 712
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CN103509869A (en) * 2013-09-29 2014-01-15 中国科学院植物研究所 Method for identifying juglans plant by using UBE3 gene-based nucleotide formula
CN104195243A (en) * 2014-07-02 2014-12-10 中国科学院植物研究所 Method for identifying plant variety by constructing nucleotide molecular formula by virtue of UBE3 gene
CN104195243B (en) * 2014-07-02 2016-03-30 中国科学院植物研究所 Utilize the method for UBE3 gene constructed nucleic acid molecule formula plant identification kind
CN107699615A (en) * 2017-10-27 2018-02-16 山东省林木种质资源中心 A kind of method that nucleic acid molecule formula identification Malus spectabilis kind is built using DNA sequence dna
CN107699615B (en) * 2017-10-27 2018-11-06 山东省林木种质资源中心 A method of identifying Malus spectabilis kind using DNA sequence dna structure nucleic acid molecule formula
CN109554445A (en) * 2019-01-23 2019-04-02 山东省花生研究所（山东省农业科学院花生工程技术研究中心） A kind of effectively easy method of parsing Arachis inter-species genetic affinity

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