CN104195243B - Utilize the method for UBE3 gene constructed nucleic acid molecule formula plant identification kind - Google Patents

Utilize the method for UBE3 gene constructed nucleic acid molecule formula plant identification kind Download PDF

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CN104195243B
CN104195243B CN201410418156.1A CN201410418156A CN104195243B CN 104195243 B CN104195243 B CN 104195243B CN 201410418156 A CN201410418156 A CN 201410418156A CN 104195243 B CN104195243 B CN 104195243B
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crape myrtle
sequence
nucleic acid
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CN104195243A (en
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索志立
黄建民
黄磊
侯伯鑫
林道银
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HUNAN INSTITUTE OF LAGERSTROEMIA FLOWER AND TREE
Institute of Botany of CAS
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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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    • C12Q1/6888—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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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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    • C12Q2600/156—Polymorphic or mutational markers

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Abstract

The invention discloses a kind of method of plant identification kind.The invention provides the method for qualification crape myrtle kind, comprise the steps: 1) with the genomic dna of crape myrtle to be measured for template, carry out pcr amplification with the primer pair be made up of two single strand dnas shown in sequence 1 and sequence 2, obtain PCR primer; 2) described PCR primer is checked order, list the nucleic acid molecule formula of described crape myrtle to be measured according to sequencing result; 3) by step 2) the nucleic acid molecule formula of gained compares with the nucleic acid molecule formula of several corresponding crape myrtle kinds, thus determines the kind of described crape myrtle to be measured.Experiment proves, the method is accurate, simple to operation, can improve production efficiency and the quality monitoring level of related industries, effectively make up the deficiency of previous methods; In addition, the method achieves the qualification in non-florescence (as seedling stage) simultaneously.Present method is significant for the development and environment protection promoting the breeding of new variety of crape myrtle, crape myrtle flowers and treesmarket and urban landscaping cause.

Description

Utilize the method for UBE3 gene constructed nucleic acid molecule formula plant identification kind
Technical field
The invention belongs to biological technical field, relate to a kind of method of qualification or assistant identification plant variety, ubiquitin ligase gene (NuclearDNAE3ubiquitinligasegene, UBE3) is utilized to build the method for nucleic acid molecule formula plant identification kind in particular to one.
Background technology
Plant variety refers to that the kind formed through artificially breeding is basically identical, heredity is more stable, has some economic characters of human needs: as fancy points, medicinal proterties or other economic characters, as the cultivated plant colony of special producing data.As Varieties of Peony, Walnut Cultivars, apple variety, crape myrtle kind etc.
Plant variety general phenotypic character carries out identifying and identifying.But there is calendar variation in plant variety, can may not show discrepant proterties between kind in the season had in feature performance.Such as, crape myrtle kind is classified according to flower pattern and pattern usually, and the time beyond the florescence exists the problem of cultivar identification difficulty.
The precise Identification of kind and purity check are the important topics in economic plants and resource plant (as flowers) industry development always.In large-scale production field, with certain concrete plant variety for the production means and/or product carry out the enterprise etc. of scale operation, need to carry out accurate identification and analysis by the fractal detection technique of highly sensitive DNA molecular.
Crape myrtle (LagerstroemiaindicaL.) has another name called crapemyrtle, all round victory etc., is shrub or the arbor of Lythraceae (Lythraceae) Lagerstroemia, is Chinese famous ornamencal flower and tree in midsummer.Flue dust in the leaf energy absorbed air of crape myrtle, is excellent environmental protection seeds, has a extensive future in urban landscaping and green barren hill.
Whole world crape myrtle Cultivar is about more than 500, and maximum with the U.S., Sino-Japanese kind, wherein there be more than 200 kind in the U.S., in state-owned more than 100 kinds, there be more than 80 kind in Japan.Crape myrtle has the cultivation history of more than 1500 year in China, and the Tang Dynasty has just contained and planted crape myrtle as ornamencal flower and tree.At present, crape myrtle in Beijing, all there are growth or cultivation in Tianjin, Shanghai, Chongqing, Guangdong, Hainan, Guangxi, Fujian, Hunan, Hubei, Sichuan, Yunnan, Zhejiang, Shandong, Shaanxi, Jilin and Hong Kong, Macao, Taiwan.The accurate identification of crape myrtle germ plasm resource is carried out scientific research for various fields such as rearing new variety, physiology, ecology and is vital element task to the application of promotion crape myrtle in afforestation.
Summary of the invention
An object of the present invention is to provide a kind of method of qualification or assistant identification crape myrtle kind to be measured.
Provided by the present invention for the identification of or the method for assistant identification crape myrtle kind to be measured, be the method utilizing the variant sites in ubiquitin ligase gene sequence to build nucleic acid molecule formula and then qualification or assistant identification crape myrtle kind to be measured, specifically comprise the steps:
(1) with the genomic dna of crape myrtle to be measured for template, carry out pcr amplification with the primer pair be made up of two single strand dnas shown in sequence in sequence table 1 and sequence 2, obtain PCR primer;
(2) by front 76 Nucleotide of nucleotide sequence from 5 ' end that cut out described PCR primer after the order-checking of described PCR primer, (described 76 Nucleotide are the conserved sequence that PCR primer 5 ' is held; The conserved sequence that PCR primer 3 ' is held can certainly be cut out simultaneously), residue sequence is as sequence to be compared (non-conservative district);
(3) full sequence in described sequence to be compared and DNA sequence data storehouse is put together, utilize the softwares such as ClustalX or Mega to compare, automatically generate a DNA aligned sequences matrix;
Described DNA aligned sequences matrix is according to specific scoring rule, and the data set produced after two or more sequence opsition dependent being compared arrangement, reflects the similarity (as shown in Figure 1) between sequence to greatest extent.All on the books in the relevant general knowledge such as the specific scoring rule wherein related to and algorithm and the monograph of principle in " biological sequence analysis ".Whether described DNA aligned sequences matrix directly can be reflected on same nucleotide site and there are differences between each sequence, and there is what kind of difference.Find the discrepant position be listed in described DNA aligned sequences matrix of Nucleotide letter, check and approve this position and be positioned at the sequence of positions that described DNA aligned sequences matrix 5 ' hold and number, namely determine a polymorphic site.
Described DNA sequence dna storehouse is following (a1) or (a2):
(a1) be made up of sequence in sequence table 3 and/or sequence 4;
(a2) by least one in sequence in sequence table 3 and sequence 4, and at least one composition in sequence 5 to sequence 10;
Wherein, sequence 3 and sequence 4 are the sequence of length 620bp; Sequence 5 to sequence 10 is the sequence of length 611bp or 614bp.
(4) the nucleic acid molecule formula of described crape myrtle to be measured is listed according to described DNA aligned sequences matrix;
The general formula of the nucleic acid molecule formula of described crape myrtle to be measured is:
B 152B 161B 173B 215B 221B 245B 294B 299B 426B 431B 440B 462B 527B 551B 557；
In described general formula, described B 152, described B 161, described B 173, described B 215, described B 221, described B 245, described B 294, described B 299, described B 426, described B 431, described B 440, described B 462, described B 527, described B 551with described B 557represent that (namely the sequence of positions numbering in polymorphic nucleotide site is determined based on aligned sequences for the 152nd, 161,173,215,221,245,294,299,426,431,440,462,527,551 and 557 site in described DNA aligned sequences matrix successively, just reasonable, meet science, otherwise do not have rule to follow);
In described general formula, each B is any one or two kinds of in A, T, C and G;
When described crape myrtle to be measured is homozygous, in described general formula, each B is any one in A, T, C and G, shows simple spike when checking order; When described crape myrtle to be measured is heterozygous, in described general formula, each B may be two kinds in A, T, C and G, can obtain significantly superposition bimodal when checking order.
(5) the nucleic acid molecule formula of crape myrtle to be measured described in step (4) gained and nucleic acid molecule formula group I are compared;
Described nucleic acid molecule formula group I is by following a1)-a8) totally 8 kinds of nucleic acid molecule formulas form:
a1)Y 152G 161C 173Y 215R 221G 245R 294C 299C 426C 431A 440A 462C 527A 551G 557；
a2)Y 152K 161Y 173Y 215G 221G 245G 294C 299C 426Y 431R 440A 462Y 527A 551G 557；
a3)T 152G 161C 173C 215A 221G 245G 294C 299C 426C 431A 440G 462C 527A 551A 557；
a4)Y 152K 161Y 173C 215R 221G 245G 294C 299C 426Y 431R 440R 462T 527A 551G 557；
a5)Y 152G 161C 173C 215R 221G 245G 294C 299C 426C 431R 440R 462C 527A 551R 557；
a6)Y 152K 161Y 173C 215R 221G 245G 294C 299C 426Y 431R 440R 462Y 527A 551R 557；
a7)C 152G 161C 173C 215G 221G 245G 294C 299C 426C 431G 440A 462C 527A 551G 557；
a8)C 152K 161Y 173C 215G 221G 245G 294C 299C 426Y 431G 440A 462Y 527A 551G 557；
At a1)-a8) in, M represents A and C; K represents G and T; Y represents T and C; R represents A and G; W represents A and T (namely the corresponding site of described crape myrtle to be measured is heterozygous);
(6) according to the comparison result of step (5), as follows cultivar identification is carried out to described crape myrtle to be measured:
If the nucleic acid molecule formula of described crape myrtle to be measured is a1) shown in nucleic acid molecule formula, then described crape myrtle to be measured is or candidate is crape myrtle kind ' emerald green dish rouge and powder ';
If the nucleic acid molecule formula of described crape myrtle to be measured is a2) shown in nucleic acid molecule formula, then described crape myrtle to be measured is or candidate is crape myrtle kind ' red ring straight branch purple ';
If the nucleic acid molecule formula of described crape myrtle to be measured is a3) shown in nucleic acid molecule formula, then described crape myrtle to be measured is or candidate is crape myrtle kind ' little Hua hybrid powder ';
If the nucleic acid molecule formula of described crape myrtle to be measured is a4) shown in nucleic acid molecule formula, then described crape myrtle to be measured is or candidate is crape myrtle kind ' Baoqing is pale purple ';
If the nucleic acid molecule formula of described crape myrtle to be measured is a5) shown in nucleic acid molecule formula, then described crape myrtle to be measured is or candidate is crape myrtle kind ' Big White Flower ';
If the nucleic acid molecule formula of described crape myrtle to be measured is a6) shown in nucleic acid molecule formula, then described crape myrtle to be measured is or candidate is crape myrtle kind ' lotus face ';
If the nucleic acid molecule formula of described crape myrtle to be measured is a7) shown in nucleic acid molecule formula, then described crape myrtle to be measured for or candidate be that crape myrtle kind ' builds people's powder ';
If the nucleic acid molecule formula of described crape myrtle to be measured is a8) shown in nucleic acid molecule formula, then described crape myrtle to be measured for or candidate be that crape myrtle kind ' spends more purple cloud '.
Certainly, in described method, the primer pair adopted in step (1) is not limited to the primer pair that in sequence table, sequence 1 and sequence 2 form, other the primer pair being positioned at conserved regions is also passable, as long as can increase the sequence described to be compared (non-conservative district) obtained in step (2).
Have again, in the process, the sequence described to be compared (non-conservative district) of comparing, the removal of its two ends conserved regions sequence is not limited to described in above step (2), as long as can contain the polymorphic site in the nucleic acid molecule formula identified for test sample (as crape myrtle kind); Accordingly, in step (4) formula of the numbering in the lower right corner of B also can because of 5 ' end conserved regions sequence accept or reject number and correspondingly change, but the relative position between polymorphic site is constant.
Further, the method for above qualification provided by the present invention or assistant identification crape myrtle kind to be measured, its operation steps can be converted into as follows:
(1) with the genomic dna of crape myrtle to be measured for template, carry out pcr amplification with the primer pair be made up of two single strand dnas shown in sequence in sequence table 1 and sequence 2, obtain PCR primer;
(2) described PCR primer is checked order, sequencing result and nucleotide sequence group I are compared;
Described nucleotide sequence group I by sequence 3-10 in sequence table totally 8 sequences form;
(3) according to the comparison result of step (2), as follows described crape myrtle to be measured is identified:
If containing sequence 3 in the nucleotide sequence of described PCR primer, then described crape myrtle to be measured is or candidate is crape myrtle kind ' Baoqing is pale purple ';
If containing sequence 4 in the nucleotide sequence of described PCR primer, then described crape myrtle to be measured is or candidate is crape myrtle kind ' lotus face ';
If containing sequence 5 in the nucleotide sequence of described PCR primer, then described crape myrtle to be measured be or candidate be crape myrtle kind ' kingfisher coils rouge and powder ';
If containing sequence 6 in the nucleotide sequence of described PCR primer, then described crape myrtle to be measured is or candidate is crape myrtle kind ' little Hua hybrid powder ';
If containing sequence 7 in the nucleotide sequence of described PCR primer, then described crape myrtle to be measured is or candidate is crape myrtle kind ' Big White Flower ';
If containing sequence 8 in the nucleotide sequence of described PCR primer, then described crape myrtle to be measured for or candidate be that crape myrtle kind ' spends more purple cloud ';
If containing sequence 9 in the nucleotide sequence of described PCR primer, then described crape myrtle to be measured be or candidate is crape myrtle kind ' the straight branch purple of red ring ';
If containing sequence 10 in the nucleotide sequence of described PCR primer, then described crape myrtle to be measured for or candidate be that crape myrtle kind ' builds people's powder '.
In the above-mentioned methods, described crape myrtle to be measured is any one in following 8 kinds: ' Baoqing is pale purple ', ' lotus face ', ' emerald green dish rouge and powder ', ' little Hua hybrid powder ', ' Big White Flower ', ' spending more purple cloud ', ' the straight branch of red ring is purple ', ' building people's powder '.
Another object of the present invention be to provide a kind of for the identification of or the test kit of assistant identification crape myrtle kind to be measured.
Provided by the present invention for the identification of or the test kit of assistant identification crape myrtle kind to be measured, specifically can comprise primer pair, record the contrast card of nucleic acid molecule formula group I;
Described primer pair is specifically made up of two single strand dnas shown in sequence in sequence table 1 and sequence 2;
Described nucleic acid molecule formula group I is specifically by following a1)-a8) totally 8 kinds of nucleic acid molecule formulas form:
a1)Y 152G 161C 173Y 215R 221G 245R 294C 299C 426C 431A 440A 462C 527A 551G 557；
a2)Y 152K 161Y 173Y 215G 221G 245G 294C 299C 426Y 431R 440A 462Y 527A 551G 557；
a3)T 152G 161C 173C 215A 221G 245G 294C 299C 426C 431A 440G 462C 527A 551A 557；
a4)Y 152K 161Y 173C 215R 221G 245G 294C 299C 426Y 431R 440R 462T 527A 551G 557；
a5)Y 152G 161C 173C 215R 221G 245G 294C 299C 426C 431R 440R 462C 527A 551R 557；
a6)Y 152K 161Y 173C 215R 221G 245G 294C 299C 426Y 431R 440R 462Y 527A 551R 557；
a7)C 152G 161C 173C 215G 221G 245G 294C 299C 426C 431G 440A 462C 527A 551G 557；
a8)C 152K 161Y 173C 215G 221G 245G 294C 299C 426Y 431G 440A 462Y 527A 551G 557；
At a1)-a8) in, M represents A and C; K represents G and T; Y represents T and C; R represents A and G; W represents A and T (namely the corresponding site of described crape myrtle to be measured is heterozygous);
Described crape myrtle to be measured can be any one in following 8 kinds: ' Baoqing is pale purple ', ' lotus face ', ' emerald green dish rouge and powder ', ' little Hua hybrid powder ', ' Big White Flower ', ' spending more purple cloud ', ' the straight branch of red ring is purple ', ' building people's powder '.
The application of described test kit in qualification or assistant identification crape myrtle kind to be measured also belongs to protection scope of the present invention.
Described crape myrtle to be measured is any one in following 8 kinds: ' Baoqing is pale purple ', ' lotus face ', ' emerald green dish rouge and powder ', ' little Hua hybrid powder ', ' Big White Flower ', ' spending more purple cloud ', ' the straight branch of red ring is purple ', ' building people's powder '.
Another object of the present invention is to provide a kind of method of qualification or assistant identification plant variety to be measured.
The method of qualification provided by the present invention or assistant identification plant variety to be measured, specifically can comprise the steps:
(1) belonged to the ubiquitin ligase gene sequence of multiple kinds of same plant by comparison, choose there is mononucleotide polymorphic site in the ubiquitin ligase gene sequence of described multiple kind region as target DNA region;
(2) utilize the sequence of both sides, described target DNA region conservative region, design the primer of the DNA fragmentation obtained containing described target DNA region that can increase;
(3) carry out pcr amplification respectively with the genomic dna of each kind in multiple kind described in described primer pair, from each kind, obtain the DNA fragmentation containing described target DNA region;
(4) DNA fragmentation containing described target DNA region that step (3) obtains from each kind is checked order, thus obtain the nucleotide sequence in the described target DNA region of each kind;
(5) nucleotide sequence in the described target DNA region of described multiple kind is put together compare, obtain DNA aligned sequences matrix, according to described DNA aligned sequences matrix determination mononucleotide polymorphic site, list the qualification nucleic acid molecule formula of each kind;
Described nucleic acid molecule formula by according to described DNA aligned sequences matrix from 5 ' end to 3 ' end or list the Nucleotide kind of all described mononucleotide polymorphic sites and sequence of positions described DNA aligned sequences matrix thereof from 3 ' end to 5 ' end successively and number and form; Described nucleic acid molecule formula general formula is B x1b x2b x3b xn, in general formula, B is the Nucleotide kind on mononucleotide polymorphic site, is any one or two kinds of in A, T, C, G tetra-kinds; x1be the sequence of positions numbering of the 1st mononucleotide polymorphic site in described DNA aligned sequences matrix from 5 ' end or 3 ' end, x2be the sequence of positions numbering of the 2nd mononucleotide polymorphic site in described DNA aligned sequences matrix from 5 ' end or 3 ' end, x3be the sequence of positions numbering of the 3rd mononucleotide polymorphic site in described DNA aligned sequences matrix from 5 ' end or 3 ' end, analogize, xnbe the sequence of positions numbering of the n-th mononucleotide polymorphic site in described DNA aligned sequences matrix from 5 ' end or 3 ' end;
Described B x1b x2b x3b xnfor all described mononucleotide polymorphic sites are listed from 5 ' end or 3 ' end successively according in described DNA aligned sequences matrix; Certainly in actually operating, distinguishing the site that effect is identical, only can retaining one, for building nucleic acid molecule formula;
(6) kind with identical described nucleic acid molecule formula is formed a pedigree, have the kind of different IPs thuja acid molecular formula as different pedigrees, all pedigrees form nucleic acid molecule formula pedigree chart;
In described nucleic acid molecule formula pedigree chart, the corresponding plant variety from a pedigree of each nucleic acid molecule formula;
(7) plant variety to be measured of same plant described in step (1) will be belonged to according to step (3)-(5) repetitive operation, by the described each kind in described plant variety alternative steps (3)-(5) to be measured, obtain the nucleic acid molecule formula of described plant variety to be measured; All nucleic acid molecule formulas in the nucleic acid molecule formula of described plant variety to be measured and described nucleic acid molecule formula pedigree chart are compared, the title determining described plant variety to be measured as follows and the pedigree be subordinate to thereof: if the nucleic acid molecule formula of described plant variety to be measured is identical with a nucleic acid molecule formula in described nucleic acid molecule formula pedigree chart, then described plant variety to be measured is or candidate is any one kind in described pedigree corresponding to a described nucleic acid molecule formula.
Described method is highly sensitive, is applicable to the different varieties identifying a certain plant.
In the present invention, described plant is specially the different varieties of crape myrtle.
In the process, described target DNA region specifically can come from the ubiquitin ligase gene district in the genome of described plant.
In the present invention, in step (2), described primer is specially the primer pair be made up of two single strand dnas shown in sequence in sequence table 1 and sequence 2; Described target DNA region specifically comes from the PCR primer adopting described primer pair the genomic dna of described plant to be carried out to pcr amplification gained.
Further, multiple kind of described crape myrtle and the described target DNA region of correspondence thereof are as follows:
Multiple kinds of described crape myrtle are at least two kinds in following 8 kinds: ' Baoqing is pale purple ', ' lotus face ', ' emerald green dish rouge and powder ', ' little Hua hybrid powder ', ' Big White Flower ', ' spending more purple cloud ', ' the straight branch of red ring is purple ', ' building people's powder '.
The nucleotides sequence in the described region of DNA territory of described crape myrtle kind ' Baoqing is pale purple ' is classified as sequence 3 in sequence table;
The nucleotides sequence in the described region of DNA territory of described crape myrtle kind ' lotus face ' is classified as sequence 4 in sequence table;
The nucleotides sequence in the described region of DNA territory of described crape myrtle kind ' emerald green dish rouge and powder ' is classified as sequence 5 in sequence table;
The nucleotides sequence in the described region of DNA territory of described crape myrtle kind ' little Hua hybrid powder ' is classified as sequence 6 in sequence table;
The nucleotides sequence in the described region of DNA territory of described crape myrtle kind ' Big White Flower ' is classified as sequence 7 in sequence table;
The nucleotides sequence in the described region of DNA territory that described crape myrtle kind ' spends more purple cloud ' is classified as sequence 8 in sequence table;
The nucleotides sequence in the described region of DNA territory of described crape myrtle kind ' the straight branch of red ring is purple ' is classified as sequence 9 in sequence table;
The nucleotides sequence in the described region of DNA territory that described crape myrtle kind ' builds people's powder ' is classified as sequence 10 in sequence table.
More concrete, the nucleic acid molecule formula in the described region of DNA territory of described crape myrtle kind ' emerald green dish rouge and powder ' is Y 152g 161c 173y 215r 221g 245r 294c 299c 426c 431a 440a 462c 527a 551g 557;
The nucleic acid molecule formula in the described region of DNA territory of described crape myrtle kind ' the straight branch of red ring is purple ' is Y 152k 161y 173y 215g 221g 245g 294c 299c 426y 431r 440a 462y 527a 551g 557;
The nucleic acid molecule formula in the described region of DNA territory of described crape myrtle kind ' little Hua hybrid powder ' is T 152g 161c 173c 215a 221g 245g 294c 299c 426c 431a 440g 462c 527a 551a 557;
The nucleic acid molecule formula in the described region of DNA territory of described crape myrtle kind ' Baoqing is pale purple ' is Y 152k 161y 173c 215r 221g 245g 294c 299c 426y 431r 440r 462t 527a 551g 557;
The nucleic acid molecule formula in the described region of DNA territory of described crape myrtle kind ' Big White Flower ' is Y 152g 161c 173c 215r 221g 245g 294c 299c 426c 431r 440r 462c 527a 551r 557;
The nucleic acid molecule formula in the described region of DNA territory of described crape myrtle kind ' lotus face ' is Y 152k 161y 173c 215r 221g 245g 294c 299c 426y 431r 440r 462y 527a 551r 557;
The nucleic acid molecule formula in the described region of DNA territory that described crape myrtle kind ' builds people's powder ' is C 152g 161c 173c 215g 221g 245g 294c 299c 426c 431g 440a 462c 527a 551g 557;
The nucleic acid molecule formula in the described region of DNA territory that described crape myrtle kind ' spends more purple cloud ' is C 152k 161y 173c 215g 221g 245g 294c 299c 426y 431g 440a 462y 527a 551g 557;
Wherein, M represents A and C; K represents G and T; Y represents T and C; R represents A and G; W represents A and T (namely the corresponding site of described crape myrtle to be measured is heterozygous).
The present invention is for the qualification process of crape myrtle kind, experiment proves, the method of this molecular formula plant identification (as crape myrtle) kind that utilizes provided by the present invention is comparatively accurate, simple to operation, production efficiency and the quality monitoring level of related industries can be improved, effectively make up the deficiency of previous methods; In addition, the method achieves the accurate qualification at non-florescence (as seedling stage) simultaneously.Present method is significant for the development and environment protection promoting the breeding of new variety of crape myrtle, crape myrtle flowers and treesmarket and urban landscaping cause.
Accompanying drawing explanation
Fig. 1 is the partial schematic diagram of the DNA aligned sequences matrix obtained after the zw_UBE3 sequence of 8 crape myrtle kinds is carried out sequence alignment.Wherein, numeral " 397 " and " 502 " namely represents the sequence of positions numbering of corresponding nucleotide site in this DNA aligned sequences matrix; Namely the row having * to mark represent that 8 crape myrtle kinds are identical in the Nucleotide kind in this site;-represent corresponding site nucleotide deletion.Due to the DNA sequence dna length slightly difference obtained that checks order actual between kind, after comparison, in the sequence of some kinds, there will be nucleotide site disappearance.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
The spire of crape myrtle (LagerstroemiaindicaL.) kind ' emerald green dish rouge and powder ', ' the straight branch of red ring is purple ', ' little Hua hybrid powder ', ' Baoqing is pale purple ', ' lotus face ', ' Big White Flower ', ' building people's powder ', ' spending more purple cloud ' and ' red pawl is dark purple ' picks up from the crape myrtle plantation of cultivar resources of Shaoyang City crape myrtle flowers and trees institute of Hunan Province.
Embodiment 1, the foundation utilizing the method for nucleic acid molecule formula qualification crape myrtle kind and application thereof
Can be used in effectively identifying the nucleic acid molecule formula of crape myrtle kind to build, the present inventor obtains one section of non-conservative district DNA sequence dna of ubiquitin ligase gene from following crape myrtle kind series, and has carried out statistical study to polymorphic site wherein.As the crape myrtle kind totally 8 kinds for examination material, specific as follows: ' emerald green dish rouge and powder ', ' the straight branch of red ring is purple ', ' little Hua hybrid powder ', ' Baoqing is pale purple ', ' lotus face ', ' Big White Flower ', ' building people's powder ' and ' spending more purple cloud '.
One, design of primers
According to the conserved regions at the DNA sequence dna two ends, one section of non-conservative district of ubiquitin ligase gene, design following primer:
Forward primer: 5 '-GTCCCCGTGATGTTTGA-3 ' (sequence 1);
Reverse primer: 5 '-GGTCCTTTGCCCGTAG-3 ' (sequence 2).
Two, the sequencing of pcr amplification and PCR primer
Using the young leaflet tablet as 8 crape myrtle kinds for examination material for experiment material, plant genome DNA extraction test kit (Chinese sky root biotechnology (Beijing) company limited product, its catalog number is DP305) is adopted to extract the genomic dna of each sample material.With gained genomic dna for template, the primer pair adopting step one to design carries out pcr amplification.Result shows, and it is the PCR primer of 771-780bp that each crape myrtle kind all can obtain a length.
Check order to each PCR primer after purifying, the length obtained containing some polymorphic sites is the DNA sequence dna (being designated as zw_UBE3) of 620bp.According to the sequencing result of 8 crape myrtle kinds for examination, be that the nucleotide sequence of described PCR primer is from the 77-696 position (620bp) that 5 ' end rises or for the nucleotide sequence of described PCR primer is from the 77-687 position (611bp) that 5 ' end rises or be 77-690 position (614bp) the nucleotide sequence of described PCR primer is held from 5 ' by zw_UBE3 sequence definition.77th Nucleotide of the nucleotide sequence of described PCR primer from 5 ' end is designated as the 1st, when these 21 Nucleotide of 468-488 position are 7 GGC repeating units, described zw_UBE3 sequence is the 77-696 position (be designated as zw_UBE3 sequence A) of nucleotide sequence from 5 ' end of described PCR primer; These 21 Nucleotide when 468-488 position are 4 GGC repeating units and (compared with above-mentioned zw_UBE3 sequence A disappearance 3 GGC repeating units) during 9 disappearance base position, and described zw_UBE3 sequence is the 77-687 position of nucleotide sequence from 5 ' end of described PCR primer; When these 21 Nucleotide of 468-488 position are 5 GGC repeating units and (compared with above-mentioned zw_UBE3 sequence A disappearance 2 GGC repeating units) during 6 disappearance base position, described zw_UBE3 sequence is the 77-690 position of nucleotide sequence from 5 ' end of described PCR primer.(the GGC repeating unit of this 468-488 position belongs to the high frequency generation area of variation, also longer or shorter sequence variation may be there is in the sequencing result of following crape myrtle kind to be measured, slightly may change because of the difference of testing sample, aligned sequences matrix can be obtained according to practical situation when the time comes, determine the sequence of positions numbering of polymorphic site below.)
Result shows, and the zw_UBE3 sequence of crape myrtle kind ' Baoqing is pale purple ' is sequence 3 in sequence table;
The zw_UBE3 sequence of crape myrtle kind ' lotus face ' is sequence 4 in sequence table;
The zw_UBE3 sequence of crape myrtle kind ' emerald green dish rouge and powder ' is sequence 5 in sequence table;
The zw_UBE3 sequence of crape myrtle kind ' little Hua hybrid powder ' is sequence 6 in sequence table;
The zw_UBE3 sequence of crape myrtle kind ' Big White Flower ' is sequence 7 in sequence table;
The zw_UBE3 sequence that crape myrtle kind ' spends more purple cloud ' is sequence 8 in sequence table;
The zw_UBE3 sequence of crape myrtle kind ' the straight branch of red ring is purple ' is sequence 9 in sequence table;
The zw_UBE3 sequence that crape myrtle kind ' builds people's powder ' is sequence 10 in sequence table.
Three, sequence alignment
The zw_UBE3 sequence of 8 of step 2 gained crape myrtle kinds is compared, obtains DNA aligned sequences matrix (alignedsequencematrix).And then obtain as 15 mononucleotide polymorphic sites of the crape myrtle cultivar identification in table 1.
Mononucleotide polymorphic site in the zw_UBE3 sequence of a table 18 crape myrtle kind
Note: M represents A and C; K represents G and T; Y represents T and C; R represents A and G; W represents A and T.
Namely the corresponding site of crape myrtle kind is heterozygous, and when checking order, corresponding heterozygous sites can detect that significantly superposition is bimodal." Position Number of polymorphic site " is the Position Number in described DNA aligned sequences matrix.For the sequence of 611bp, 3 GGC tumor-necrosis factor glycoproteinss are lacked afterwards at the 479th, so the physical location that the Position Number 527,551 and 557 in described DNA aligned sequences matrix corresponds to corresponding sequence is the 518th, the 542nd and the 548th due to the sequence relative to the longest 620bp.For the sequence of 614bp, 2 GGC tumor-necrosis factor glycoproteinss are lacked afterwards at the 482nd, so the physical location that the Position Number 527,551 and 557 in described DNA aligned sequences matrix corresponds to corresponding sequence is the 521st, the 545th and the 551st due to the sequence relative to the longest 620bp.
Only have and sorted by comparison, just can demonstrate the regularity of sample room sequence difference, this is the integral part of bioinformation theory and principle.Fig. 1 is the part sectional drawing of the zw_UBE3DNA aligned sequences matrix of 8 crape myrtle kinds.In Table 1, when comparing in the scope of these 8 kinds, site 245,299,426 and 551 look it is singlet site, but if the sequence of ' red pawl is dark purple ' in embodiment 2 is put into, these 4 sites just become polymorphic site, in order to look after accessibility and the integrity of content of the statement of this specification sheets, here still maintain this 4 sites, to echo with the formulaic representation of ' red pawl is dark purple ' in embodiment 2, be convenient to reading comprehension.
Four, for the structure of the nucleic acid molecule formula of crape myrtle cultivar identification
According to the Nucleotide letter of each polymorphic site of crape myrtle kind listed in table 1 and the position in DNA aligned sequences matrix thereof, list the corresponding nucleic acid molecule formula of each crape myrtle kind, specific as follows:
The nucleic acid molecule formula of crape myrtle kind ' emerald green dish rouge and powder ' is: Y 152g 161c 173y 215r 221g 245r 294c 299c 426c 431a 440a 462c 527a 551g 557;
The nucleic acid molecule formula of crape myrtle kind ' the straight branch of red ring is purple ' is: Y 152k 161y 173y 215g 221g 245g 294c 299c 426y 431r 440a 462y 527a 551g 557;
The nucleic acid molecule formula of crape myrtle kind ' little Hua hybrid powder ' is: T 152g 161c 173c 215a 221g 245g 294c 299c 426c 431a 440g 462c 527a 551a 557;
The nucleic acid molecule formula of crape myrtle kind ' Baoqing is pale purple ' is: Y 152k 161y 173c 215r 221g 245g 294c 299c 426y 431r 440r 462t 527a 551g 557;
The nucleic acid molecule formula of crape myrtle kind ' Big White Flower ' is: Y 152g 161c 173c 215r 221g 245g 294c 299c 426c 431r 440r 462c 527a 551r 557;
The nucleic acid molecule formula of crape myrtle kind ' lotus face ' is: Y 152k 161y 173c 215r 221g 245g 294c 299c 426y 431r 440r 462y 527a 551r 557;
The nucleic acid molecule formula that crape myrtle kind ' builds people's powder ' is: C 152g 161c 173c 215g 221g 245g 294c 299c 426c 431g 440a 462c 527a 551g 557;
The nucleic acid molecule formula that crape myrtle kind ' spends more purple cloud ' is: C 152k 161y 173c 215g 221g 245g 294c 299c 426y 431g 440a 462y 527a 551g 557.
M above in each nucleic acid molecule formula all represents A and C; K all represents G and T; Y all represents T and C; R all represents A and G; W all represents A and T.Namely M, K, Y or R of containing in nucleic acid molecule formula are the heterozygous sites in crape myrtle kind genome, can obtain significantly superposition bimodal when checking order.
Five, by nucleic acid molecule formula qualification crape myrtle kind
(1) with the genomic dna of crape myrtle to be measured for template, carry out pcr amplification with the primer pair be made up of two single strand dnas shown in sequence in sequence table 1 and sequence 2, obtain PCR primer;
(2) by after the order-checking of described PCR primer, (described 76 Nucleotide are the conserved regions sequence that PCR primer 5 ' is held to front 76 Nucleotide of the nucleotide sequence cutting out described PCR primer from 5 ' end; The conserved regions sequence that PCR primer 3 ' is held can certainly be cut out simultaneously), residue sequence is as sequence to be compared (non-conservative district);
(3) full sequence in described sequence to be compared and DNA sequence dna storehouse is put together compare, obtain DNA aligned sequences matrix;
Described DNA sequence dna storehouse is following (a1) or (a2):
(a1) be made up of sequence in sequence table 3 and/or sequence 4;
(a2) by least one in sequence in sequence table 3 and sequence 4, and at least one composition in sequence 5 to sequence 10;
(4) the nucleic acid molecule formula of described crape myrtle to be measured is listed according to described DNA aligned sequences matrix;
The general formula of the nucleic acid molecule formula of described crape myrtle to be measured is:
B 152B 161B 173B 215B 221B 245B 294B 299B 426B 431B 440B 462B 527B 551B 557；
In described general formula, described B 152, described B 161, described B 173, described B 215, described B 221, described B 245, described B 294, described B 299, described B 426, described B 431, described B 440, described B 462, described B 527, described B 551with described B 557, represent the 152nd, 161,173,215,221,245,294,299,426,431,440,462,527,551 and 557 site (the 152nd, 161,173,215,221,245,294,299,426,431,440,462,527,551 and 557 nucleotide site of corresponding zw_UBE3 sequence matrix) in described DNA aligned sequences matrix successively;
In described general formula, each B is that any one or two kinds of in A, T, C and G are (when the concrete nucleotide site of described crape myrtle to be measured is homozygous site, in described general formula, each B is any one in A, T, C and G, shows simple spike when checking order; When the concrete nucleotide site of described crape myrtle to be measured is heterozygous site, in described general formula, each B is two kinds in A, T, C and G, can obtain significantly superposition bimodal when checking order).
(5) as follows cultivar identification is carried out to described crape myrtle to be measured:
If the nucleic acid molecule formula of described crape myrtle to be measured is Y 152g 161c 173y 215r 221g 245r 294c 299c 426c 431a 440a 462c 527a 551g 557, then described crape myrtle to be measured is or candidate is crape myrtle kind ' emerald green dish rouge and powder ';
If the nucleic acid molecule formula of described crape myrtle to be measured is Y 152k 161y 173y 215g 221g 245g 294c 299c 426y 431r 440a 462y 527a 551g 557, then described crape myrtle to be measured is or candidate is crape myrtle kind ' red ring straight branch purple ';
If the nucleic acid molecule formula of described crape myrtle to be measured is T 152g 161c 173c 215a 221g 245g 294c 299c 426c 431a 440g 462c 527a 551a 557, then described crape myrtle to be measured is or candidate is crape myrtle kind ' little Hua hybrid powder ';
If the nucleic acid molecule formula of described crape myrtle to be measured is Y 152k 161y 173c 215r 221g 245g 294c 299c 426y 431r 440r 462t 527a 551g 557, then described crape myrtle to be measured is or candidate is crape myrtle kind ' Baoqing is pale purple ';
If the nucleic acid molecule formula of described crape myrtle to be measured is Y 152g 161c 173c 215r 221g 245g 294c 299c 426c 431r 440r 462c 527a 551r 557, then described crape myrtle to be measured is or candidate is crape myrtle kind ' Big White Flower ';
If the nucleic acid molecule formula of described crape myrtle to be measured is Y 152k 161y 173c 215r 221g 245g 294c 299c 426y 431r 440r 462y 527a 551r 557, then described crape myrtle to be measured is or candidate is crape myrtle kind ' lotus face ';
If the nucleic acid molecule formula of described crape myrtle to be measured is C 152g 161c 173c 215g 221g 245g 294c 299c 426c 431g 440a 462c 527a 551g 557, then described crape myrtle to be measured for or candidate be that crape myrtle kind ' builds people's powder ';
If the nucleic acid molecule formula of described crape myrtle to be measured is C 152k 161y 173c 215g 221g 245g 294c 299c 426y 431g 440a 462y 527a 551g 557, then described crape myrtle to be measured for or candidate be that crape myrtle kind ' spends more purple cloud '.
Six, the new taxa key of crape myrtle kind
According to the nucleic acid molecule formula of each kind of step 5 gained, develop the new taxa key of 8 crape myrtle kinds, in table 2.
The new taxa key of a table 28 crape myrtle kind
1a.Y 215type
2a.G 161c 527type: molecular formula is: Y 152g 161c 173y 215r 221g 245r 294c 299c 426c 431a 440a 462c 527a 551g 557.' emerald green dish rouge and powder '
2b.K 161y 527type: molecular formula is: Y 152k 161y 173y 215g 221g 245g 294c 299c 426y 431r 440a 462y 527a 551g 557.' the straight branch of red ring is purple '
1b.C 215type
3a.A 440type: molecular formula is: T 152g 161c 173c 215a 221g 245g 294c 299c 426c 431a 440g 462c 527a 551a 557.' little Hua hybrid powder '
3b.R 440type
4a.T 527type: molecular formula is: Y 152k 161y 173c 215r 221g 245g 294c 299c 426y 431r 440r 462t 527a 551g 557.' Baoqing is pale purple '
4b.C 527type: molecular formula is: Y 152g 161c 173c 215r 221g 245g 294c 299c 426c 431r 440r 462c 527a 551r 557.' Arabescato '
4c.Y 527type: molecular formula is: Y 152k 161y 173c 215r 221g 245g 294c 299c 426y 431r 440r 462y 527a 551r 557.' lotus face '
3c.G 440type
5a.C 527type: molecular formula is: C 152g 161c 173c 215g 221g 245g 294c 299c 426c 431g 440a 462c 527a 551g 557.' building people's powder '
5b.Y 527type: molecular formula is: C 152k 161y 173c 215g 221g 245g 294c 299c 426y 431g 440a 462y 527a 551g 557.' spending more purple cloud '
Separately, the morphologic description of the crape myrtle kind in this key (table 2) is delivered in such as Publication about Document:
1. Huang builds the people, and Hou Baixin, Suo Zhi stand .2013a. Shaoyang City crape myrtle Varietal Investigation research I. agricultural journal 3 (3): 47-53.
2. Huang builds the people, and Hou Baixin, Suo Zhi stand .2013b. Shaoyang City crape myrtle Varietal Investigation research II. agricultural journal 3 (4): 35-41.
3. Huang builds the people, and Hou Baixin, Suo Zhi stand .2013c. Shaoyang City crape myrtle Varietal Investigation research III. agricultural journal 3 (5): 34-41.
Embodiment 2, identify other crape myrtle kinds
In order to verify the validity that nucleic acid molecule formula that embodiment 1 sets up is identified for plant of Lagerstroemia and specificity, the blade of this crape myrtle kind that the present inventor also acquires ' red pawl is dark purple ', and according to the method for embodiment 1, extract its genomic dna, carry out pcr amplification and order-checking with the primer pair be made up of two single strand dnas shown in sequence 1 and sequence 2, obtain the zw_UBE3 sequence of this crape myrtle kind.Wherein, the zw_UBE3 sequence of crape myrtle kind ' red pawl is dark purple ' is for shown in sequence in sequence table 11.
The sequence of the zw_UBE3 sequence of this crape myrtle kind of sequence 11 and aforementioned 8 crape myrtle kinds is put together, software (as ClustalX or Mega software) is utilized to compare, obtain DNA aligned sequences matrix, be further listed in the nucleic acid molecule formula of ' the light pawl powder crystal of emerald green dish ', for: C 152k 161t 173c 215r 221k 245g 294y 299m 426y 431g 440a 462y 527w 551g 557.Visible, the nucleic acid molecule formula of ' red pawl is dark purple ' this crape myrtle kind contains K 245type site, the 8 kind nucleic acid molecule formula (Gs corresponding with the crape myrtle kind of 8 in embodiment 1 245type) all not identical.
Thus confirm validity and the specificity of the inventive method.
In addition, the blade material of the two crape myrtle (Lagerstroemiavenusta) in wild species west is also acquired from Meng Lun town, Xishuangbanna Prefecture, Yunnan Province Mengla County Chinese Academy of Sciences Xishuangbanna tropical botanical garden, method similar to the above is utilized to obtain zw_UBE3 sequence, as shown in sequence in sequence table 12.Utilize method similar to the above to carry out DNA sequence dna comparison, find the two crape myrtle wild species in west and the sequence difference of totally 9 crape myrtle kinds is very large above, be very easy to distinguish.The sequence of the front 466bp of DNA aligned sequences matrix has had very large difference, utilizes the 13rd nucleotide site just these wild species and aforementioned 9 kinds can be distinguished completely.The sequence of the two crape myrtle in west is very large in the variation of GGC repeat region, and create a large amount of insertion/deletion site, for brevity, the present inventor is front 466 Nucleotide of comparison only.
Result shows: in the zw_UBE3 sequence of the two crape myrtle in west, be T 13type, and other sample is C 13type.
Visible, the inventive method is also effective to detection crape myrtle wild species.According to the literature and this patent contriver investigation, current crape myrtle cultivar origin is in the cultivation of wild species and cross-breeding approach, and the heredity that many crape myrtle wild species such as the two crape myrtle wild species in known west still do not participate in existing crape myrtle kind as hybrid strain is formed.At nature, now and/or in the future, there is the natural and artificial hybridization kind between crape myrtle wild species and between crape myrtle kind and crape myrtle wild species, these cross-fertilize seed are important sources of breeding of new variety, imply that present method still has huge value and potentiality in following classification application.

Claims (4)

1. a method for qualification or assistant identification crape myrtle kind to be measured, comprises the steps:
(1) with the genomic dna of crape myrtle to be measured for template, carry out pcr amplification with the primer pair be made up of two single strand dnas shown in sequence in sequence table 1 and sequence 2, obtain PCR primer;
(2) by after described PCR primer order-checking, cut out front 76 Nucleotide of nucleotide sequence from 5 ' end of described PCR primer, residue sequence is as sequence to be compared;
(3) full sequence in described sequence to be compared and DNA sequence dna storehouse is put together compare, obtain DNA aligned sequences matrix;
Described DNA sequence dna storehouse is following (a1) or (a2):
(a1) be made up of sequence in sequence table 3 and/or sequence 4;
(a2) by least one in sequence in sequence table 3 and sequence 4, and at least one composition in sequence 5 to sequence 10;
(4) the nucleic acid molecule formula of described crape myrtle to be measured is listed according to described DNA aligned sequences matrix;
The general formula of the nucleic acid molecule formula of described crape myrtle to be measured is:
B 152B 161B 173B 215B 221B 245B 294B 299B 426B 431B 440B 462B 527B 551B 557；
In described general formula, described B 152, described B 161, described B 173, described B 215, described B 221, described B 245, described B 294, described B 299, described B 426, described B 431, described B 440, described B 462, described B 527, described B 551with described B 557, represent the 152nd, 161,173,215,221,245,294,299,426,431,440,462,527,551 and 557 site in described DNA aligned sequences matrix successively;
In described general formula, each B is any one or two kinds of in A, T, C and G;
(5) the nucleic acid molecule formula of crape myrtle to be measured described in step (4) gained and nucleic acid molecule formula group I are compared;
Described nucleic acid molecule formula group I is by following a1)-a8) totally 8 kinds of nucleic acid molecule formulas form:
a1)Y 152G 161C 173Y 215R 221G 245R 294C 299C 426C 431A 440A 462C 527A 551G 557；
a2)Y 152K 161Y 173Y 215G 221G 245G 294C 299C 426Y 431R 440A 462Y 527A 551G 557；
a3)T 152G 161C 173C 215A 221G 245G 294C 299C 426C 431A 440G 462C 527A 551A 557；
a4)Y 152K 161Y 173C 215R 221G 245G 294C 299C 426Y 431R 440R 462T 527A 551G 557；
a5)Y 152G 161C 173C 215R 221G 245G 294C 299C 426C 431R 440R 462C 527A 551R 557；
a6)Y 152K 161Y 173C 215R 221G 245G 294C 299C 426Y 431R 440R 462Y 527A 551R 557；
a7)C 152G 161C 173C 215G 221G 245G 294C 299C 426C 431G 440A 462C 527A 551G 557；
a8)C 152K 161Y 173C 215G 221G 245G 294C 299C 426Y 431G 440A 462Y 527A 551G 557；
At a1)-a8) in, K represents G and T; Y represents T and C; R represents A and G;
(6) according to the comparison result of step (5), as follows cultivar identification is carried out to described crape myrtle to be measured:
If the nucleic acid molecule formula of described crape myrtle to be measured is a1) shown in nucleic acid molecule formula, then described crape myrtle to be measured is or candidate is crape myrtle kind ' emerald green dish rouge and powder ';
If the nucleic acid molecule formula of described crape myrtle to be measured is a2) shown in nucleic acid molecule formula, then described crape myrtle to be measured is or candidate is crape myrtle kind ' red ring straight branch purple ';
If the nucleic acid molecule formula of described crape myrtle to be measured is a3) shown in nucleic acid molecule formula, then described crape myrtle to be measured is or candidate is crape myrtle kind ' little Hua hybrid powder ';
If the nucleic acid molecule formula of described crape myrtle to be measured is a4) shown in nucleic acid molecule formula, then described crape myrtle to be measured is or candidate is crape myrtle kind ' Baoqing is pale purple ';
If the nucleic acid molecule formula of described crape myrtle to be measured is a5) shown in nucleic acid molecule formula, then described crape myrtle to be measured is or candidate is crape myrtle kind ' Big White Flower ';
If the nucleic acid molecule formula of described crape myrtle to be measured is a6) shown in nucleic acid molecule formula, then described crape myrtle to be measured is or candidate is crape myrtle kind ' lotus face ';
If the nucleic acid molecule formula of described crape myrtle to be measured is a7) shown in nucleic acid molecule formula, then described crape myrtle to be measured for or candidate be that crape myrtle kind ' builds people's powder ';
If the nucleic acid molecule formula of described crape myrtle to be measured is a8) shown in nucleic acid molecule formula, then described crape myrtle to be measured for or candidate be that crape myrtle kind ' spends more purple cloud '.
2. method according to claim 1, is characterized in that: described crape myrtle to be measured is any one in following 8 kinds: ' Baoqing is pale purple ', ' lotus face ', ' emerald green dish rouge and powder ', ' little Hua hybrid powder ', ' Big White Flower ', ' spending more purple cloud ', ' the straight branch of red ring is purple ', ' building people's powder '.
3. for the identification of or the test kit of assistant identification crape myrtle kind to be measured, comprise primer pair, record the contrast card of nucleic acid molecule formula group I;
Described primer pair is made up of two single strand dnas shown in sequence in sequence table 1 and sequence 2;
Described nucleic acid molecule formula group I is by following a1)-a8) totally 8 kinds of nucleic acid molecule formulas form:
a1)Y 152G 161C 173Y 215R 221G 245R 294C 299C 426C 431A 440A 462C 527A 551G 557；
a2)Y 152K 161Y 173Y 215G 221G 245G 294C 299C 426Y 431R 440A 462Y 527A 551G 557；
a3)T 152G 161C 173C 215A 221G 245G 294C 299C 426C 431A 440G 462C 527A 551A 557；
a4)Y 152K 161Y 173C 215R 221G 245G 294C 299C 426Y 431R 440R 462T 527A 551G 557；
a5)Y 152G 161C 173C 215R 221G 245G 294C 299C 426C 431R 440R 462C 527A 551R 557；
a6)Y 152K 161Y 173C 215R 221G 245G 294C 299C 426Y 431R 440R 462Y 527A 551R 557；
a7)C 152G 161C 173C 215G 221G 245G 294C 299C 426C 431G 440A 462C 527A 551G 557；
a8)C 152K 161Y 173C 215G 221G 245G 294C 299C 426Y 431G 440A 462Y 527A 551G 557；
At a1)-a8) in, K represents G and T; Y represents T and C; R represents A and G;
Described crape myrtle to be measured is any one in following 8 kinds: ' Baoqing is pale purple ', ' lotus face ', ' emerald green dish rouge and powder ', ' little Hua hybrid powder ', ' Big White Flower ', ' spending more purple cloud ', ' the straight branch of red ring is purple ', ' building people's powder '.
4. the application of test kit described in claim 3 in qualification or assistant identification crape myrtle kind to be measured;
Described crape myrtle to be measured is any one in following 8 kinds: ' Baoqing is pale purple ', ' lotus face ', ' emerald green dish rouge and powder ', ' little Hua hybrid powder ', ' Big White Flower ', ' spending more purple cloud ', ' the straight branch of red ring is purple ', ' building people's powder '.
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