CN104818271B - The molecular labeling HRM5 of wheat widow Tillering gene Ltn3 a kind of and its application - Google Patents
The molecular labeling HRM5 of wheat widow Tillering gene Ltn3 a kind of and its application Download PDFInfo
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- CN104818271B CN104818271B CN201510222213.3A CN201510222213A CN104818271B CN 104818271 B CN104818271 B CN 104818271B CN 201510222213 A CN201510222213 A CN 201510222213A CN 104818271 B CN104818271 B CN 104818271B
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Abstract
The invention discloses the molecular labeling HRM5 with wheat widow's Tillering gene Ltn3 close linkages, for its nucleotide sequence as shown in SEQ ID No.1, the genetic distance of the molecular labeling and wheat widow's Tillering gene Ltn3 is 0.35cM.Detection and analysis show that the molecular labeling can accurately track the wheat widow Tillering gene, predict the shooting property of wheat, and then convenient progress Molecular design breeding.The invention also discloses a kind of method for the molecular labeling for identifying wheat widow's Tillering gene Ltn3, the accuracy of tiller prediction can be strengthened using method provided by the present invention, improve the success rate of special type breeding, speed up to the target of increase yield of wheat.
Description
Technical field
The present invention relates to Wheat Molecular Breeding field, and in particular to a kind of molecular labeling of wheat widow Tillering gene Ltn3
HRM5 and its application.
Background technology
Wheat is the second largest cereal crops that China is only second to rice, and cultivated area over the years accounts for the 22% of cultivated area respectively
~30%, the 22%~27% of the cereal crops gross area, it is mainly distributed on Henan, Hebei, Shandong, Shanxi, Shaanxi, Jiangsu, four
The provinces such as river, Anhui；Total output is more than 100,000,000 tons, accounts for the 22% of cereal crops yield, is the master of about half population of China
Food, therefore the selection and breeding of High-Yield Wheat Cultivar are always the emphasis of breeder's concern.
Yield Traits of Wheat is complicated quantitative character, by multiple quantitative character gene locus therefor (Quantitative
Trait locus, QTL) control, have that genetic force is low, characteristic big, selection difficulty is high affected by environment, so in selection and breeding
The problem of Cheng Zhong, traditional breeding method existence time is long, consumption is big, of high cost, achievement is small.Molecular mark is one
Kind carries out the effective ways of breeding using the molecular labeling associated with specific trait as supplementary means, has from environment bar
Part limitation, the whole growth period advantage such as detectable, efficiency of selection height in development of plants.
Wheat yield is made of three principal elements, i.e. yield=spike number × grain number per spike × grain weight.Tiller is to influence wheat
One of Main Agronomic Characters of spike number and then influence wheat yield, while be that a kind of monocotyledonous special branch shows again
As having important research significance.
Wheat tillering genetic research macro-progress is relatively slow, works outside Current Domestic mainly around Tillering gene QTL
Molecular mapping and its hereditary effect carry out.Part tiller mutant is by Dominant gene, thus some scholars are made
(Richards RA.A tiller inhibition gene in wheat and its affect are studied for qualitative character
on plant growth.Austr J Agric Res.1988,39:749-757；Duggan BL,Richards RA,
Tsuyuzaki H.Environmental effects on the expression of the tiller inhibition
(tin)gene in wheat.Funct Plant Biol,2002,29:45–53).Meanwhile also there are many scholars to make tiller
(Shaha MM, Gilla KS, Baenziger PS, Yen Y, Kaepplerc are studied for the quantitative character of controlled by multiple genes
SM,Ariyarathne HM.Molecular mapping of loci for agronomic traits on
chromosome 3A of bread wheat.Crop Sci.1999,39:1728-1732；Thank Yue, Longhai City, Hou Yongcui, Zheng You
The genetic analysis wheat crops journals .2006,26 of good Correlative Characters of A Oligoculm Wheat Line H 461 shape:21-23；Wang Yan wheats
Immortalized F2 is built and the QTL of plant height and shooting property positioning Shandong Agricultural University master thesis, and 2009；Warm star
The more tillers of Wangshuibai, the identification of Dwarf Mutants and correlation QTL positioning Agricultural University Of Nanjing master thesis, 2010；
Jinpeng Zhang,Jun Wu,Weihua Liu,Xiang Lu,Xinming Yang,Ainong Gao,Xiuquan Li,
Yuqing Lu,Lihui Li.Genetic mapping of a fertile tiller inhibition gene,ftin,
in wheat.Mol Breeding.2013,31:441-449).So far, tiller correlation major gene resistance QTL reported
In 1A, 2A, 3A, 6A chromosomes, minor gene is located at 5A, 3B, 7B, 1D, 5D chromosome, wherein the tin genes only on 1A and 3A
Research is more deep, completes finely positioning work.
Wheat lines H461 has few tiller, more grain number per spikes, more small ears relative to wheat breed river agriculture 16 (state examines kind)
The characteristic such as several, high mass of 1000 kernel and high Ear weight (Hou Yongcui, Zheng Youliang, Pu Zhien, Wei Yuming, Li Wei spike number type new variety of wheat
River agriculture 16 and large spike widow tiller strain H461 hereditary difference First Report of Studies Sichuan Agricultural University journal .2003,21:94-9).
Meanwhile wheat breed Chuanmai 107 and tiller number are significantly higher than H461.Therefore, genetic research group is built using H461 and Chuanmai 107
Body, further verifies the few shooting property of wheat H461, location control Tillering gene, finds the molecular labeling of close linkage, promote
New gene resource is provided into the map based cloning of Tillering gene, while for the initiative and Plant-type Breeding of the special tiller material of wheat, into
One step utilizes molecular marker assisted selection, will strengthen the accuracy of tiller prediction, improves Plant-type Breeding efficiency, speed up to increase
The target of yield of wheat.
Molecular marker assisted selection, selects independent of phenotype, i.e., from environmental condition, Interaction among genes, genotype
With the influence of many factors such as environment interaction, but directly genotype is made choice, thus breeding efficiency can be greatly improved.It is high
A kind of SNP and mutation that melting curve (High Resolution Melting, the HRM) technology of resolution is risen in the world in recent years
Research tool.This detection method has easy to operate, specific good, high throughput, quick, testing cost is low, result is accurate etc.
Advantage, and realize real stopped pipe operation and receive universal concern.Therefore, filter out and Tillering gene close linkage
, and suitable for the molecular labeling of quantitative fluorescent PCR platform HRM technologies, wheat tillering gene can not only be made choice, effectively
Regulate and control wheat tillering, mould rational tiller and colony occurs, while improve choosing flux, speed and accuracy, solve
The technical bottleneck of large-scale promotion application, improves wheat breeding colony quality to scale and yield is of great significance.
The content of the invention
It is an object of the invention to provide the molecular labeling with wheat H461 widow's Tillering gene Ltn3 close linkages.
Another object of the present invention is to provide the fluorescence quantification PCR primer of the molecular labeling.
Third object of the present invention is the application for providing the molecular labeling of above-mentioned few Tillering gene Ltn3 close linkages.
The purpose of the present invention is what is be achieved through the following technical solutions：
A woman who has recently been widowed's Tillering gene Ltn3 of the present invention comes from wheat H461, which is located at wheat 2D chromosomes.Ltn3 can be shown
Writing reduces wheat tillering, and LOD value is more than 5.31, explains about 12% phenotypic variation.
The nucleotide sequence of molecular labeling HRM5 provided by the present invention is as shown in SEQ ID No.1.
Molecular labeling of the present invention and wheat widow Tillering gene Ltn3 common locations in wheat 2D chromosomes, and with Ltn3 it
Between genetic distance be 0.35cM.
Present invention also offers a kind of method for the molecular labeling for identifying wheat widow's Tillering gene Ltn3, with plant sample to be measured
The genomic DNA of product carries out fluorescent quantitative PCR with fluorescence quantification PCR primer, base is carried out using amplification as template
Because of type parting, the plant containing wheat widow's Tillering gene Ltn3 will be accredited as same type of plant with H461 points.
Optionally, the described method comprises the following steps：
(1) using the genomic DNA of Plant samples to be measured as template, quantitative fluorescent PCR is carried out with fluorescence quantification PCR primer
Amplification：
(2) fluorescent quantitative PCR reaction system：SsoFast EvaGreen surpass 5 μ L of mixed liquor, sense primer and downstream
It is 10 μ L that each 300ng of primer, template DNA 100ng, Dnaes/RNase-free deionized water, which add to total amount,；
(3) quantitative fluorescent PCR program：95 DEG C of pre-degeneration 5min；94 DEG C of denaturation 15s, 53 DEG C of annealing 30s, totally 50 circulations；
Melting curve scope is 65~95 DEG C, number of every 0.2 DEG C of reading, time 10s；
(4) result obtained using step (3) carries out Genotyping.
Wherein, the DNA of the plant to be identified is derived from the blade in wheat samples tri-leaf period.
Present invention also offers the fluorescence quantification PCR primer of the molecular labeling, wherein, upstream primer sequence such as SEQ ID
Shown in No.2, downstream primer sequence is as shown in SEQ ID No.3.
Present invention also offers applications of the molecular labeling HRM5 in wheat breeding.
Present invention also offers applications of the molecular labeling HRM5 in prepare transgenosis plant, wherein, the gene
For few Tillering gene Ltn3.
Present invention also offers application of the well-behaved invention the method in wheat breeding improvement.
We additionally provide application of the fluorescence quantification PCR primer in molecular labeling aids in wheat plant types breeding.
In the present invention, wheat widow Tillering gene Ltn3 and molecular labeling HRM5 are prepared by the following：
(1) by the use of widow tiller wheat H461 as female parent, it is paternal hybrid with wheat river agriculture 16, obtains Hybrids F1, F1 generation
Individual plant selfing obtains F2, obtains the F8 containing 223 strains for RIL colonies using single seed descent, randomly chooses 188 strain structures
Into genetic mapping colony.
(2) DNA of each strain of the genetic mapping colony is extracted with CTAB methods, chooses wheatgenomics
(http://wheatgenomics.plantpath.ksu.edu/) on covering hexaploid wheat A, B, D genome announced
90K SNP chips, using parent H461 and the DNA of river agriculture 16 as masterplate, carry out Genotyping, obtain the gene of the RIL colonies
Type data.The banding pattern of parent H461 is denoted as A, and the banding pattern of parent river agriculture 16 is denoted as B.F8 colonies strain banding pattern derives from the note of H461
For A, from the B that is denoted as of river agriculture 16, heterozygous H.
(3) tiller number of F8 colonies plant described in wheat aging time field test.
(4) the RIL colonies genotype data structure wheat molecule of acquisition is connected using JoinMap4.0 mapping softwares
Collection of illustrative plates is locked, finds optimal reference numerals and flag sequence, determines the linkage group subsequently used.Utilize the area of software MapQTL 6.0
Between make graph model (Interval Mapping) and more QTL make graph model (Multiple QTL Model), and combine F8 colonies
Few Tillering gene Ltn 3 is positioned on 2DL chromosomes in the section of a 3.49cM by tiller phenotypic data.
(5) the SNP probe sequences in the section are obtained, by the gene order-checking data in IWGSC wheat China springs, are obtained
The more sequence informations of target zone are obtained, electronics extends the corresponding sequence in SNP probe sequences both ends, and carries out sequence analysis, just
Step screening is used for the target area of follow-up fluorescence quantification PCR primer design.
(6) fluorescence quantification PCR primer is designed, for subsequently screening.It is glimmering using 7.0 Software for Design of Beacon Designer
Fluorescent Quantitative PCR primer 10 is to (being shown in Table 1).Fluorescence quantification PCR primer design standard：18~25bp of primer length, amplified production length
65~100bp is spent, 60 DEG C of annealing temperature, for G/C content between 40%~60%, SNP difference locus products annealing temperature differences are big
In 0.2 DEG C.
1 10 pairs of SSR primer sequences of table and expanding fragment length
(7) high-resolution fusion curve (HRM) is analyzed
A) between parent polymorphic molecular marker screening：Choose 10 pairs of primers of above-mentioned design, with parent H461 and
The DNA of CN16 is masterplate, carries out PCR amplification, obtains 1 pair of quantitative fluorescent PCR molecular labeling primer to work well altogether, is named as
HRM5-F/R (nucleotide sequence is as shown in SEQ ID No.2 and 3).Amplified production be with polymorphism molecular labeling HRM5,
Nucleotide sequence is as shown in SEQ ID No.1
B) the HRM analyses of F8 colonies：The PCR primer of the molecular labeling HRM5 with polymorphism obtained with above-mentioned steps,
The DNA of parent H461, CN16 and F8 colonies plant is expanded, carries out genotype identification, obtains molecular marker data.Parent H461's
Type is denoted as A, the long 72bp of amplified fragments size, and single base difference site is C.The type of parent CN16 is denoted as B, amplified fragments length
72bp, single base difference site are T.F8 colonies strain type origin is denoted as A in H461, and B is denoted as from CN16.
C) densification of linkage map：According to the appraising datum of molecular labeling HRM5, with reference to the Genotyping of 90KSNP chips
Data, mapping software JoinMap 4.0 build hereditary high density collection of illustrative plates.Utilize the Interval mapping model of software MapQTL 6.0
(Interval Mapping) and more QTL make graph model (Multiple QTL Model), and combine F8 population tiller Phenotype Numbers
According to the few Tillering gene Ltn3 of positioning, the genetic distance between the position of few Tillering gene Ltn3 and molecular labeling, discovery point are calculated
Son mark HRM5 and wheat widow Tillering gene Ltn3 common locations are in wheat 2D chromosomes, and the genetic distance between Ltn3 is
0.35cM, LOD value are more than 5.31, explain phenotypic variation about 12%.
Beneficial effect：
Present invention firstly discloses the few Tillering gene Ltn3 from wheat H461, positioned at wheat 2D chromosomes, significantly drop
Low wheat tillering.The gene has higher utility value in wheat plant types (regulation and control tiller) breeding.
Present invention firstly discloses accurately detect wheat H461 a woman who has recently been widowed's Tillering gene Ltn3's based on quantitative fluorescent PCR platform
Molecular labeling HRM5, and be codominant marker, detection precise and high efficiency, the convenient stabilization of amplification.
Molecular labeling HRM5 disclosed by the invention and few Tillering gene Ltn3 are extremely significantly correlated, and it is special that presentation isolates mark
Sign, the accuracy for molecular marker assisted selection is high, improves the selection mirror for the specific tiller kind of wheat for adapting to varying environment
Determine efficiency, and success rate is high.
Brief description of the drawings
Fig. 1 be positions of the wheat H461 widow's Tillering gene Ltn3 on 2D chromosomes and with molecular labeling HRM5 of the present invention it
Between Genetic linkage map.
Fig. 2 is that river agriculture 16, H461, Chuanmai 107 and the leaf DNA in continuous 37 tri-leaf period of wheat are made of fluorescence quantification PCR primer
The result of genotyping.
Fig. 3 is that the F7RIL colonies offspring of H461 × Chuanmai 107 makes the result of genotyping of fluorescence quantification PCR primer.
Embodiment
Below will the present invention is described in detail by embodiment.It will be appreciated that following embodiments are given
Go out merely to play the purpose of explanation, be not used to limit the scope of the present invention.Those skilled in the art exists
In the case of without departing substantially from spirit of the invention and spirit, various modifications and replacement can be carried out to the present invention.
Embodiment 1
The present embodiment is used for the preparation method for illustrating wheat widow's Tillering gene Ltn3 and molecular labeling HRM5：
(1) by the use of widow tiller wheat H461 as female parent, it is paternal hybrid with wheat river agriculture 16, obtains Hybrids F1, F1 generation
Individual plant selfing obtains F2, obtains the F8 containing 223 strains for RIL colonies using single seed descent, randomly chooses 188 strain structures
Into genetic mapping colony.
(2) DNA of each strain of the genetic mapping colony is extracted with CTAB methods, chooses wheatgenomics
(http://wheatgenomics.plantpath.ksu.edu/) on covering hexaploid wheat A, B, D genome announced
90K SNP chips, using parent H461 and the DNA of river agriculture 16 as masterplate, carry out Genotyping, obtain the gene of the RIL colonies
Type data.The banding pattern of parent H461 is denoted as A, and the banding pattern of parent river agriculture 16 is denoted as B.F8 colonies strain banding pattern derives from the note of H461
For A, from the B that is denoted as of river agriculture 16, heterozygous H.
(3) tiller number of F8 colonies plant described in wheat aging time field test.
(4) the RIL colonies genotype data structure wheat molecule of acquisition is connected using JoinMap4.0 mapping softwares
Collection of illustrative plates is locked, finds optimal reference numerals and flag sequence, determines the linkage group subsequently used.Utilize the area of software MapQTL 6.0
Between make graph model (Interval Mapping) and more QTL make graph model (Multiple QTL Model), and combine F8 colonies
Few Tillering gene Ltn3 is positioned on 2DL chromosomes in the section of a 3.49cM by tiller phenotypic data.
(5) the SNP probe sequences in the section are obtained, by the gene order-checking data in IWGSC wheat China springs, are obtained
The more sequence informations of target zone are obtained, electronics extends the corresponding sequence in SNP probe sequences both ends, and carries out sequence analysis, just
Step screening is used for the target area of follow-up fluorescence quantification PCR primer design.
(6) fluorescence quantification PCR primer is designed, for subsequently screening.It is glimmering using 7.0 Software for Design of Beacon Designer
Fluorescent Quantitative PCR primer 10 is to (as shown in table 1).Fluorescence quantification PCR primer design standard：18~25bp of primer length, amplification production
Thing 65~100bp of length, 60 DEG C of annealing temperature, for G/C content between 40%~60%, SNP difference locus products annealing temperatures are poor
It is different to be more than 0.2 DEG C.
(7) high-resolution fusion curve (HRM) is analyzed
A) between parent polymorphic molecular marker screening：Choose 10 pairs of primers of above-mentioned design, with parent H461 and
The DNA of CN16 is masterplate, carries out PCR amplification, obtains 1 pair of quantitative fluorescent PCR molecular labeling to work well altogether.
B) the HRM analyses of F8 colonies：The molecular labeling HRM5 with polymorphism obtained with above-mentioned steps, expands parent
The DNA of H461, CN16 and F8 colony plant, carries out genotype identification, obtains molecular marker data.The type of parent H461 is denoted as
A, the long 72bp of amplified fragments size, single base difference site are ' C '.The type of parent CN16 is denoted as B, the long 72bp of amplified fragments,
Single base difference site is ' T '.F8 colonies strain type origin is denoted as A in H461, and B is denoted as from CN16.
C) densification of linkage map：According to the appraising datum of molecular labeling HRM5, with reference to the Genotyping of 90KSNP chips
Data, mapping software JoinMap 4.0 build hereditary high density collection of illustrative plates.Utilize the Interval mapping model of software MapQTL 6.0
(Interval Mapping) and more QTL make graph model (Multiple QTL Model), and combine F8 population tiller Phenotype Numbers
According to the few Tillering gene Ltn3 of positioning, the genetic distance between the position of few Tillering gene Ltn3 and molecular labeling, discovery point are calculated
Son mark HRM5 and wheat widow Tillering gene Ltn3 common locations are in wheat 2D chromosomes, and the genetic distance between Ltn3 is
0.35cM, LOD value are more than 5.31, and explanation phenotypic variation about 12%, Fig. 1 is wheat H461 widow's Tillering gene Ltn3 in 2D chromosomes
On position and the Genetic linkage map between molecular labeling HRM5 of the present invention.
Embodiment 2
The extraction of 1.1DNA
Test material chooses H461, river agriculture 16 (CN16), Chuanmai 107 (CM107) and continuous wheat 37 (MM37), wherein river agriculture
16th, Chuanmai 107 and continuous wheat 37 are more tiller kinds, and H461 is few tiller kind.Wheat samples tri-leaf period is extracted using CTAB methods
Leaf DNA.
The screening of the primer of 1.2 detection wheat widow's Tillering gene Ltn3
1.2.1 design of primers
(1) the SNP probe sequences in the section are obtained, by the gene order-checking data in IWGS wheat China springs, are obtained
The more sequence informations of target zone are obtained, electronics extends the corresponding sequence in SNP probe sequences both ends, and carries out sequence analysis, just
Step screening is used for the target area of follow-up fluorescence quantification PCR primer design.
(2) fluorescence quantification PCR primer is designed, for subsequently screening.Design fluorescence quantification PCR primer.Quantitative fluorescent PCR draws
Thing design standard：18~25bp of primer length, amplified production 65~100bp of length, 60 DEG C of annealing temperature, G/C content 40%~
Between 60%, SNP difference locus products annealing temperatures difference is more than 0.2 DEG C.
1.2.2 the otherness between quantitative fluorescent PCR platform test primer and its parent
(1) river agriculture 16, H461, Chuanmai 107 and the leaf DNA in continuous 37 tri-leaf period of wheat are extracted.
(2) using the DNA obtained by step (1) as template, HRM5-F (sequence shown in SEQ ID No.2) of the present invention
With HRM5-R (sequence shown in SEQ ID No.3) fluorescent quantitative PCR is carried out for primer.
(3) fluorescent quantitative PCR reaction system：It is each that SsoFast EvaGreen surpass 5 μ L of mixed liquor, upstream and downstream primer
It is 10 μ L that 300ng, template DNA 100ng, Dnaes/RNase-free deionized water, which add to total amount,.
(4) quantitative fluorescent PCR program：95 DEG C of pre-degeneration 5min；94 DEG C of denaturation 15s, 53 DEG C of annealing 30s, totally 50 circulations；
Melting curve scope is 65~95 DEG C, number of every 0.2 DEG C of reading, time 10s.
(5) with step (4) acquired results file, BioRadPrecisionMelt softwares is imported and carry out Genotyping, as a result
Such as Fig. 2.During melting curve is drawn, according to base-pair A=T, the temperature that C ≡ G unwind is different,
Sample is divided into two types by BioRadPrecisionMelt.H461 is type A, and continuous 37/ Chuanmai 107 of wheat of river agriculture 16/ is type
B。
The applicability of primer sequence HRM5-F/R during 1.3 crowd surveillances
(1) using H461 as female parent, Chuanmai 107 obtains F1 for paternal hybrid, and F1 selfings obtain F2, pass through single seed descent plus generation
To F7 colony is verified for RIL.Extract the leaf DNA in each strain tri-leaf period in colony.
(2) using the DNA obtained by step (1) as template, quantitative fluorescent PCR expansion is carried out using primer provided by the present invention
Increase, fluorescent dye is SsoFast EvaGreen.
(3) fluorescent quantitative PCR reaction system：It is each that 5 μ L SsoFast EvaGreen surpass mixed liquor, upstream and downstream primer
It is 10 μ L that 300ng, 100ng template DNA, Dnaes/RNase-free deionized waters, which add to total amount,.
(4) quantitative fluorescent PCR program：95 DEG C of pre-degeneration 5min；94 DEG C of denaturation 15s, 53 DEG C of annealing 30s, totally 50 circulations；
Melting curve scope is 65~95 DEG C, number of every 0.2 DEG C of reading, time 10s.
(5) with step (4) acquired results file, BioRadPrecisionMelt softwares is imported and carry out Genotyping, as a result
Such as Fig. 3.96 plants of strains of sampling observation at random, 38 plants of fragments that can be amplified with H461 same types, to contain few Tillering gene Ltn3
Plant, prediction strain plant tiller number after maturation it is relatively low.58 plants can amplify the Type B piece identical with river agriculture 16, Chuanmai 107
Section, not contain the plant of few Tillering gene Ltin3, predicts that these plant tiller number after maturation is higher.
(6) between wheat aging time field test 96 F7 plant tiller number, the results are shown in Table 2, few Tillering gene Ltn3
Molecular labeling HRM5 prediction genetic group tillering ability result.The plant the mean tillering number identical with H461 types is 2.47
It is a, substantially less than with river agriculture 16, the plant tillering number (average 9.64) of Chuanmai 107 type.Actual result is consistent with expected results,
Illustrate the few Tillering gene Ltn3 of the present invention significantly reduces tiller number really；The molecular labeling HRM5 of the present invention at the same time
Few Tillering gene Ltn3 can be identified with tracking.
Table 2
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (6)
- A kind of 1. wheat widow Tillering geneLtn3Molecular labeling HRM5, it is characterised in that the nucleosides of the molecular labeling HRM5 Acid sequence is as shown in SEQ ID No.1；The molecular labeling and wheat widow's Tillering geneLtn3Common location in wheat 2D chromosomes, and withLtn3Between heredity away from From for 0.35cM；Wheat widow's Tillering geneLtn3Wheat tillering is significantly reduced, LOD value is more than 5.31.
- 2. wheat widow Tillering gene described in claim 1Ltn3Molecular labeling HRM5 identification method, it is characterised in that to treat The genomic DNA of Plant samples is surveyed as template, fluorescent quantitative PCR is carried out with fluorescence quantification PCR primer, is tied using amplification Fruit carries out genotyping, will divide with wheat H461 and is accredited as same type of plant containing wheat widow's Tillering geneLtn3's The plant of molecular labeling HRM5；The upstream primer sequence of the fluorescence quantification PCR primer is as shown in SEQ ID No.2, downstream primer sequence such as SEQ ID Shown in No.3.
- 3. according to the method described in claim 2, it is characterised in that it includes following steps：（1）Using the genomic DNA of Plant samples to be measured as template, quantitative fluorescent PCR expansion is carried out with fluorescence quantification PCR primer Increase；（2）Fluorescent quantitative PCR reaction system：SsoFast EvaGreen surpass 5 μ L of mixed liquor, sense primer and anti-sense primer It is 10 μ L that each 300ng, template DNA 100ng, deionized water, which add to total amount,；Wherein, the upstream primer sequence such as SEQ ID Shown in No.2, the downstream primer sequence is as shown in SEQ ID No.3；（3）Quantitative fluorescent PCR program：95 DEG C of pre-degenerations 5 minutes；94 DEG C of denaturation are annealed 30 seconds for 15 seconds, 53 DEG C, totally 50 circulations； Melting curve scope is 65 ~ 95 DEG C, and number of every 0.2 DEG C of reading, the time is 10 seconds；（4）Utilize step（3）The result of acquisition carries out Genotyping.
- 4. the fluorescence quantification PCR primer of molecular labeling HRM5 described in claim 1, it is characterised in that upstream primer sequence such as SEQ Shown in ID No.2, downstream primer sequence is as shown in SEQ ID No.3.
- 5. applications of the molecular labeling HRM5 in wheat breeding described in claim 1.
- 6. application of the fluorescence quantification PCR primer in molecular labeling aids in wheat plant types breeding described in claim 4.
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| CN107177667B (en) * | 2017-05-18 | 2020-09-01 | 四川农业大学 | Wheat head density QTL (quantitative trait locus) linked HRM (high resolution melting) molecular marker and application thereof |
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| CN115786565B (en) * | 2022-09-20 | 2023-07-18 | 四川农业大学 | SNP molecular marker linked with wheat spike number QTL and application thereof |
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