CN104818271A - Molecular marker HRM5 of wheat few-tillering gene Ltn3 and application thereof - Google Patents
Molecular marker HRM5 of wheat few-tillering gene Ltn3 and application thereof Download PDFInfo
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Abstract
The invention discloses a molecular marker HRM5 which is closely linked with wheat few-tillering gene Ltn3, wherein the nucleotide sequence of the molecular marker HRM5 is represented as the SEQ ID No.1. Genetic distance between the molecular marker and the wheat few-tillering gene Ltn3 is 0.35 cM. A test result proves that the molecular marker can accurately trace the wheat few-tillering gene and predict tillering characters of wheat, thereby further carrying molecular-designing breeding conveniently. The invention also discloses a method of identifying the molecular marker of the wheat few-tillering gene Ltn3. By means of the method, accuracy of tillering prediction can be increased, success rate of specific plant type breeding can be increased, and achievement of an object of increasing per unit area yield of wheat is accelerated.
Description
Technical field
The present invention relates to Wheat Molecular Breeding field, be specifically related to molecule marker HRM5 and the application thereof of the few Tillering gene Ltn3 of a grow wheat.
Background technology
Wheat is the second largest food crop that China is only second to paddy rice, cultivated area over the years accounts for 22% ~ 30% of cultivated area respectively, 22% ~ 27% of the food crop total area, is mainly distributed in the provinces such as Henan, Hebei, Shandong, Shanxi, Shaanxi, Jiangsu, Sichuan, Anhui; Ultimate production is more than 100,000,000 tons, accounts for 22% of food crop output, is the staple food that China is about half population, and therefore the seed selection of High-Yield Wheat Cultivar is the emphasis that breeding man pays close attention to always.
Yield Traits of Wheat is complicated quantitative character, by multiple quantitative character gene locus therefor (Quantitative trait locus, QTL) control, there is the characteristic that heritability is low, affected by environment greatly, selection difficulty is high, so in Breeding Process, the problem that traditional breeding method lifetime is long, consumption is large, cost is high, achievement is little.Molecular mark is a kind of effective ways adopting the molecule marker that is associated with specific trait to carry out breeding as supplementary means, have do not limit by envrionment conditions, all can detect at the whole growth period of development of plants, the advantage such as efficiency of selection height.
Wheat yield is made up of three principal elements, i.e. output=spike number × grain number per spike × grain weight.Tillering is affect one of wheat spike number and then the Main Agronomic Characters affecting wheat yield, is again monocotyledonous a kind of special branch phenomenon simultaneously, has important Research Significance.
Wheat tillering genetic research macro-progress is relatively slow, and Current Domestic works outward and mainly carries out around the Molecular mapping of Tillering gene QTL and hereditary effect thereof.Part tillers mutant by Dominant gene, thus some scholars it can be used as qualitative character to study (Richards RA.A tiller inhibition gene in wheat and its affect on plantgrowth.Austr J Agric Res.1988,39:749-757; Duggan BL, Richards RA, Tsuyuzaki H.Environmental effects on the expression of the tillerinhibition (tin) gene in wheat.Funct Plant Biol, 2002,29:45 – 53).Simultaneously, also there is a lot of scholar that the quantitative character of tillering as controlled by multiple genes is studied (ShahaMM, Gilla KS, Baenziger PS, Yen Y, Kaepplerc SM, Ariyarathne HM.Molecular mapping of loci for agronomic traits on chromosome 3A ofbread wheat.Crop Sci.1999,39:1728-1732; Thank to Yue, Longhai City, Hou Yongcui, Zheng Youliang. the genetic analysis of Correlative Characters of A Oligoculm Wheat Line H 461 shape. wheat crops journal .2006,26:21-23; Wang Yan. wheat immortalized F2 builds and the QTL of plant height and shooting property locates. Shandong Agricultural University's master thesis, 2009; Temperature star. Wangshuibai is tillered more, the qualification of Dwarf Mutants and relevant QTL locate. Agricultural University Of Nanjing's master thesis, 2010; Jinpeng Zhang, Jun Wu, Weihua Liu, Xiang Lu, XinmingYang, Ainong Gao, Xiuquan Li, Yuqing Lu, Lihui Li.Genetic mappingof a fertile tiller inhibition gene, ftin, in wheat.Mol Breeding.2013,31:441-449).Up to now, the relevant major gene QTL of tillering reported is positioned at 1A, 2A, 3A, 6A karyomit(e), and minor gene is positioned at 5A, 3B, 7B, 1D, 5D karyomit(e), and the tin gene studies wherein only on 1A and 3A is more deep, completes Fine Mapping work.
Relative to wheat breed river agriculture 16 (state examines kind), wheat lines H461 has that widow is tillered, many grain number per spikes, many spikelet numbers, the characteristic (Hou Yongcui such as high thousand seed weight and high Ear weight, Zheng Youliang, Pu Zhien, Wei Yuming, Li Wei. spike number type New Wheat Variety Chuannong 16 and large spike widow are tillered 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, H461 and Chuanmai 107 is utilized to build genetic research colony, the few shooting property of further checking wheat H461, setting control Tillering gene, finds closely linked molecule marker, promote the map based cloning of Tillering gene, simultaneously tiller for wheat the is special initiative of material and Plant-type Breeding provides new gene resource, utilizes molecular marker assisted selection further, will strengthen the accuracy of prediction of tillering, improve Plant-type Breeding efficiency, accelerate the target realizing increasing yield of wheat.
Molecular marker assisted selection, does not rely on phenotype and selects, and namely not by the impact of the many factors such as envrionment conditions, Interaction among genes, genotype by environment interaction, but directly selects genotype, thus greatly can improve breeding efficiency.A kind of SNP that high-resolution fusion curve (HighResolution Melting, HRM) technology is risen in recent years in the world and mutation research instrument.This detection method have easy and simple to handle, specificity good, high-throughput, fast, the advantage such as testing cost is low, result is accurate, and achieve real stopped pipe operation and receive general concern.Therefore; filter out with Tillering gene closely linked; and be applicable to the molecule marker of quantitative fluorescent PCR platform HRM technology; can not only select wheat tillering gene; Effective Regulation wheat tillering occurs, and moulding reasonably tillers, and colony occurs, and improves choosing flux, speed and accuracy simultaneously; solve the technical bottleneck of large-scale promotion application, to mass-producing improvement wheat breeding colony quality and output significant.
Summary of the invention
The object of the present invention is to provide and the closely linked molecule marker of the few Tillering gene Ltn3 of wheat H461.
Another object of the present invention is to the fluorescence quantification PCR primer that described molecule marker is provided.
3rd object of the present invention is to provide the above-mentioned few Tillering gene Ltn3 application of closely linked molecule marker.
The object of the invention is to be achieved through the following technical solutions:
A woman who has recently been widowed's Tillering gene Ltn3 of the present invention is from wheat H461, and this gene is positioned at wheat 2D karyomit(e).Ltn3 significantly can reduce wheat tillering, and LOD value is greater than 5.31, explains the phenotypic variation of about 12%.
The nucleotide sequence of molecule marker HRM5 provided by the present invention is as shown in SEQ ID No.1.
The few Tillering gene Ltn3 of molecule marker of the present invention and wheat is positioned wheat 2D karyomit(e) altogether, and and genetic distance between Ltn3 be 0.35cM.
Present invention also offers a kind of method identifying the molecule marker of the few Tillering gene Ltn3 of wheat, using the genomic dna of Plant samples to be measured as template, fluorescent quantitative PCR is carried out with fluorescence quantification PCR primer, utilize amplification to carry out genotyping, be divided into the plant of same type to be accredited as plant containing the few Tillering gene Ltn3 of wheat by with H461.
Optionally, said method comprising the steps of:
(1) using the genomic dna of Plant samples to be measured as template, fluorescent quantitative PCR is carried out with fluorescence quantification PCR primer:
(2) fluorescent quantitative PCR reaction system: SsoFast EvaGreen surpasses mixed solution 5 μ L, upstream primer and each 300ng of downstream primer, to add to total amount be 10 μ L to template DNA 100ng, Dnaes/RNase-free deionized water;
(3) quantitative fluorescent PCR program: 95 DEG C of denaturation 5min; 94 DEG C of sex change 15s, 53 DEG C of annealing 30s, totally 50 circulations; Melting curve scope is 65 ~ 95 DEG C, and read a number for every 0.2 DEG C, the time is 10s;
(4) result utilizing step (3) to obtain carries out gene type.
Wherein, the DNA of described plant to be identified takes from the blade in wheat samples tri-leaf period.
Present invention also offers the fluorescence quantification PCR primer of described molecule marker, wherein, upstream primer sequence is as shown in SEQ ID No.2, and downstream primer sequence is as shown in SEQ ID No.3.
Present invention also offers the application of described molecule marker HRM5 in wheat breeding.
Present invention also offers the application of described molecule marker HRM5 in preparation transgenic plant, wherein, described gene is few Tillering gene Ltn3.
Present invention also offers the application of the described method of one's duty invention in wheat breeding improvement.
We additionally provide described fluorescence quantification PCR primer and assist application in wheat plant types breeding at molecule marker.
In the present invention, the few Tillering gene Ltn3 and molecule marker HRM5 of wheat obtains by the following method:
(1) widow is utilized to tiller wheat H461 as female parent, with wheat river agriculture 16 for paternal hybrid, obtain Hybrids F1, F1 generation individual plant selfing obtains F2, adopt the F8 of single seed descent acquisition containing 223 strains for RIL colony, Stochastic choice 188 composition isolate genetic mapping colonies.
(2) with the DNA of each strain of the genetic mapping colony described in the extraction of CTAB method, choose the upper genomic 90K SNP chip of covering hexaploid wheat A, B, D announced of wheatgenomics (http://wheatgenomics.plantpath.ksu.edu/), with the DNA of parent H461 and river agriculture 16 for masterplate, carry out gene type, obtain the genotype data of described RIL colony.The banding pattern of parent H461 is designated as A, and the banding pattern of parent river agriculture 16 is designated as B.What F8 colony strain banding pattern derived from H461 is designated as A, and what derive from river agriculture 16 is designated as B, and heterozygous is H.
(3) tiller number of F8 colony plant described in wheat aging time field test.
(4) utilize JoinMap4.0 mapping software that the described RIL colony genotype data obtained is built wheat Molecular Markers Linkage Map, find optimum reference numerals and flag sequence, determine the linkage group of follow-up use.Utilize the Interval mapping model (IntervalMapping) of software MapQTL 6.0 and many QTL to make graph model (Multiple QTL Model), and in conjunction with F8 population tiller phenotypic data, few Tillering gene Ltn 3 is positioned in the section of a 3.49cM on 2DL karyomit(e).
(5) the SNP probe sequence in this section is obtained, by the gene order-checking data in IWGSC wheat China spring, obtain the more sequence information of target section, electronics extends sequence corresponding to SNP probe sequence two ends, and carry out sequential analysis, preliminary screening is used for the target area of follow-up fluorescence quantification PCR primer design.
(6) fluorescence quantification PCR primer is designed, for follow-up screening.Utilize BeaconDesigner 7.0 software design fluorescence quantification PCR primer 10 to (see table 1).Fluorescence quantification PCR primer standard: primer length 18 ~ 25bp, amplified production length 65 ~ 100bp, annealing temperature 60 DEG C, GC content is between 40% ~ 60%, and SNP difference locus products annealing temperature difference is greater than 0.2 DEG C.
Table 1 10 pairs of SSR primer sequences and expanding fragment length
(7) high-resolution fusion curve (HRM) is analyzed
A) screening of polymorphic molecular marker between parent: the 10 pairs of primers choosing above-mentioned design, with the DNA of parent H461 and CN16 for masterplate, carry out pcr amplification, obtain 1 altogether to respond well quantitative fluorescent PCR molecule marker primer, called after HRM5-F/R (nucleotide sequence is as Suo Shi SEQ ID No.2 and 3).Amplified production is have polymorphism molecule marker HRM5, and nucleotide sequence is as shown in SEQ ID No.1
B) HRM of F8 colony analyzes: the PCR primer with the molecule marker HRM5 of polymorphism obtained by above-mentioned steps, and the DNA of amplification parent H461, CN16 and F8 colony plant, carries out genotype identification, obtain molecular marker data.The type of parent H461 is designated as A, the long 72bp of amplified fragments size, and single base difference site is C.The type of parent CN16 is designated as B, the long 72bp of amplified fragments, and single base difference site is T.F8 colony strain type origin is designated as A in H461, and what derive from CN16 is designated as B.
C) densification of linkage map: according to the appraising datum of molecule marker HRM5, in conjunction with the genotype data of 90KSNP chip, mapping software JoinMap 4.0 builds hereditary high-density collection of illustrative plates.The Interval mapping model of software MapQTL 6.0 (Interval Mapping) and many QTL is utilized to make graph model (Multiple QTL Model), and locate few Tillering gene Ltn3 in conjunction with F8 population tiller phenotypic data, calculate the genetic distance between the position of few Tillering gene Ltn3 and molecule marker, find that molecule marker HRM5 and the few Tillering gene Ltn3 of wheat is positioned wheat 2D karyomit(e) altogether, and and genetic distance between Ltn3 be 0.35cM, LOD value is greater than 5.31, interpret table form variation about 12%.
Beneficial effect:
The present invention makes public for the first time the few Tillering gene Ltn3 from wheat H461, is positioned at wheat 2D karyomit(e), significantly reduces wheat tillering.This gene has higher utility value in wheat plant types (regulation and control are tillered) breeding.
The present invention makes public for the first time the molecule marker HRM5 accurately detecting wheat H461 a woman who has recently been widowed Tillering gene Ltn3 based on quantitative fluorescent PCR platform, and is codominant marker, detects precise and high efficiency, amplification is convenient stable.
Molecule marker HRM5 disclosed by the invention and few Tillering gene Ltn3 pole significant correlation, present and be divided into from marker characteristic, accuracy for molecular marker assisted selection is high, improve the selection determination rates of the specific kind of tillering of wheat adapting to varying environment, and success ratio is high.
Accompanying drawing explanation
Fig. 1 be the position of the few Tillering gene Ltn3 of wheat H461 on 2D karyomit(e) and and molecule marker HRM5 of the present invention between Genetic linkage map.
Fig. 2 is the result of fluorescence quantification PCR primer, river agriculture 16, H461, Chuanmai 107 and the leaf DNA in continuous wheat 37 tri-leaf period being made to genotyping.
Fig. 3 is the result that the F7RIL colony offspring fluorescence quantification PCR primer of H461 × Chuanmai 107 does genotyping.
Embodiment
Below will the present invention is described in detail by embodiment.It will be appreciated that providing of following examples is only object in order to play explanation, being not used to limit scope of the present invention.Those skilled in the art, when not deviating from aim of the present invention and spirit, can carry out various amendment and replacement to the present invention.
Embodiment 1
The present embodiment is for illustration of the preparation method of the few Tillering gene Ltn3 and molecule marker HRM5 of wheat:
(1) widow is utilized to tiller wheat H461 as female parent, with wheat river agriculture 16 for paternal hybrid, obtain Hybrids F1, F1 generation individual plant selfing obtains F2, adopt the F8 of single seed descent acquisition containing 223 strains for RIL colony, Stochastic choice 188 composition isolate genetic mapping colonies.
(2) with the DNA of each strain of the genetic mapping colony described in the extraction of CTAB method, choose the upper genomic 90K SNP chip of covering hexaploid wheat A, B, D announced of wheatgenomics (http://wheatgenomics.plantpath.ksu.edu/), with the DNA of parent H461 and river agriculture 16 for masterplate, carry out gene type, obtain the genotype data of described RIL colony.The banding pattern of parent H461 is designated as A, and the banding pattern of parent river agriculture 16 is designated as B.What F8 colony strain banding pattern derived from H461 is designated as A, and what derive from river agriculture 16 is designated as B, and heterozygous is H.
(3) tiller number of F8 colony plant described in wheat aging time field test.
(4) utilize JoinMap4.0 mapping software that the described RIL colony genotype data obtained is built wheat Molecular Markers Linkage Map, find optimum reference numerals and flag sequence, determine the linkage group of follow-up use.Utilize the Interval mapping model (IntervalMapping) of software MapQTL 6.0 and many QTL to make graph model (Multiple QTL Model), and in conjunction with F8 population tiller phenotypic data, few Tillering gene Ltn3 is positioned in the section of a 3.49cM on 2DL karyomit(e).
(5) the SNP probe sequence in this section is obtained, by the gene order-checking data in IWGSC wheat China spring, obtain the more sequence information of target section, electronics extends sequence corresponding to SNP probe sequence two ends, and carry out sequential analysis, preliminary screening is used for the target area of follow-up fluorescence quantification PCR primer design.
(6) fluorescence quantification PCR primer is designed, for follow-up screening.Utilize BeaconDesigner 7.0 software design fluorescence quantification PCR primer 10 to (as shown in table 1).Fluorescence quantification PCR primer standard: primer length 18 ~ 25bp, amplified production length 65 ~ 100bp, annealing temperature 60 DEG C, GC content is between 40% ~ 60%, and SNP difference locus products annealing temperature difference is greater than 0.2 DEG C.
(7) high-resolution fusion curve (HRM) is analyzed
A) screening of polymorphic molecular marker between parent: the 10 pairs of primers choosing above-mentioned design, with the DNA of parent H461 and CN16 for masterplate, carries out pcr amplification, obtains 1 altogether to respond well quantitative fluorescent PCR molecule marker.
B) HRM of F8 colony analyzes: the molecule marker HRM5 with polymorphism obtained with above-mentioned steps, and the DNA of amplification parent H461, CN16 and F8 colony plant, carries out genotype identification, obtain molecular marker data.The type of parent H461 is designated as A, the long 72bp of amplified fragments size, and single base difference site is ' C '.The type of parent CN16 is designated as B, the long 72bp of amplified fragments, and single base difference site is ' T '.F8 colony strain type origin is designated as A in H461, and what derive from CN16 is designated as B.
C) densification of linkage map: according to the appraising datum of molecule marker HRM5, in conjunction with the genotype data of 90KSNP chip, mapping software JoinMap 4.0 builds hereditary high-density collection of illustrative plates.The Interval mapping model of software MapQTL 6.0 (Interval Mapping) and many QTL is utilized to make graph model (Multiple QTL Model), and locate few Tillering gene Ltn3 in conjunction with F8 population tiller phenotypic data, calculate the genetic distance between the position of few Tillering gene Ltn3 and molecule marker, find that molecule marker HRM5 and the few Tillering gene Ltn3 of wheat is positioned wheat 2D karyomit(e) altogether, and and genetic distance between Ltn3 be 0.35cM, LOD value is greater than 5.31, interpret table form variation about 12%, Fig. 1 be the position of the few Tillering gene Ltn3 of wheat H461 on 2D karyomit(e) and and molecule marker HRM5 of the present invention between Genetic linkage map.
Embodiment 2
The extraction of 1.1DNA
Test materials chooses H461, river agriculture 16 (CN16), Chuanmai 107 (CM107) and continuous wheat 37 (MM37), and wherein river agriculture 16, Chuanmai 107 and continuous wheat 37 for tillering kind more, and H461 to be tillered kind for widow.CTAB method is adopted to extract the leaf DNA in wheat samples tri-leaf period.
The screening of the primer of the few Tillering gene Ltn3 of 1.2 detection wheat
1.2.1 design of primers
(1) the SNP probe sequence in this section is obtained, by the gene order-checking data in IWGS wheat China spring, obtain the more sequence information of target section, electronics extends sequence corresponding to SNP probe sequence two ends, and carry out sequential analysis, preliminary screening is used for the target area of follow-up fluorescence quantification PCR primer design.
(2) fluorescence quantification PCR primer is designed, for follow-up screening.Design fluorescence quantification PCR primer.Fluorescence quantification PCR primer standard: primer length 18 ~ 25bp, amplified production length 65 ~ 100bp, annealing temperature 60 DEG C, GC content is between 40% ~ 60%, and SNP difference locus products annealing temperature difference is greater than 0.2 DEG C.
1.2.2 the otherness between quantitative fluorescent PCR platform test primer and parent thereof
(1) river agriculture 16, H461, Chuanmai 107 and the leaf DNA in continuous wheat 37 tri-leaf period is extracted.
(2) using the DNA of step (1) gained as template, HRM5-F of the present invention (sequence shown in SEQ ID No.2) and HRM5-R (sequence shown in SEQ ID No.3) carries out fluorescent quantitative PCR for primer.
(3) fluorescent quantitative PCR reaction system: SsoFast EvaGreen surpasses mixed solution 5 μ L, each 300ng of upstream and downstream primer, to add to total amount be 10 μ L to template DNA 100ng, Dnaes/RNase-free deionized water.
(4) quantitative fluorescent PCR program: 95 DEG C of denaturation 5min; 94 DEG C of sex change 15s, 53 DEG C of annealing 30s, totally 50 circulations; Melting curve scope is 65 ~ 95 DEG C, and read a number for every 0.2 DEG C, the time is 10s.
(5) with step (4) acquired results file, import BioRadPrecisionMelt software and carry out gene type, result is as Fig. 2.In drafting melting curve process, different according to the temperature that base pair A=T, C ≡ G unwinds, sample is divided into two types by BioRadPrecisionMelt.H461 is type A, and river agriculture 16/ continuous wheat 37/ Chuanmai 107 is type B.
The suitability of primer sequence HRM5-F/R in 1.3 crowd surveillance processes
(1) take H461 as female parent, Chuanmai 107 is that paternal hybrid obtains F1, and F1 selfing obtains F2, is added verify colony for F7 for RIL by single seed descent.Extract the leaf DNA in the tri-leaf period of each strain in colony.
(2) using the DNA of step (1) gained as template, utilize primer provided by the present invention to carry out fluorescent quantitative PCR, fluorescence dye is SsoFast EvaGreen.
(3) fluorescent quantitative PCR reaction system: 5 μ L SsoFast EvaGreen surpass mixed solution, to add to total amount be 10 μ L for each 300ng, 100ng template DNA of upstream and downstream primer, Dnaes/RNase-free deionized water.
(4) quantitative fluorescent PCR program: 95 DEG C of denaturation 5min; 94 DEG C of sex change 15s, 53 DEG C of annealing 30s, totally 50 circulations; Melting curve scope is 65 ~ 95 DEG C, and read a number for every 0.2 DEG C, the time is 10s.
(5) with step (4) acquired results file, import BioRadPrecisionMelt software and carry out gene type, result is as Fig. 3.Random sampling observation 96 strain strain, 38 strains can amplify the fragment of type identical with H461, are the plant containing few Tillering gene Ltn3, and prediction strain plant tiller number after maturation is lower.58 strains can amplify and river agriculture 16, Type B fragment that Chuanmai 107 is identical, are the plant not containing few Tillering gene Ltin3, predict that these plant tiller number after maturation is higher.
(6) tiller number of these 96 F7 plant of field test between wheat aging time, the results are shown in Table 2, and the molecule marker HRM5 of few Tillering gene Ltn3 predicts the result of genetic group tillering ability.The plant the mean tillering number identical with H461 type is 2.47, significantly lower than the plant tillering number (average 9.64) with river agriculture 16, Chuanmai 107 type.Actual result is consistent with expected results, illustrates that few Tillering gene Ltn3 of the present invention has the effect significantly reducing tiller number really; Molecule marker HRM5 of the present invention can identify few Tillering gene Ltn3 with tracking simultaneously.
Table 2
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (10)
1. the molecule marker HRM5 of the few Tillering gene Ltn3 of a grow wheat, it is characterized in that, nucleotide sequence is as shown in SEQ ID No.1.
2. molecule marker according to claim 1, is characterized in that, the few Tillering gene Ltn3 of described molecule marker and wheat is positioned wheat 2D karyomit(e) altogether, and and genetic distance between Ltn3 be 0.35cM.
3. molecule marker according to claim 1 and 2, is characterized in that, the few Tillering gene Ltn3 of wheat significantly reduces wheat tillering, and LOD value is greater than 5.31.
4. identify the method for the molecule marker HRM5 of the few Tillering gene Ltn3 of wheat for one kind, it is characterized in that, using the genomic dna of Plant samples to be measured as template, fluorescent quantitative PCR is carried out with fluorescence quantification PCR primer, utilize amplification to carry out genotyping, be divided into the plant of same type to be accredited as plant containing the few Tillering gene Ltn3 of wheat by with H461.
5. method according to claim 4, is characterized in that, comprises the following steps:
(1) using the genomic dna of Plant samples to be measured as template, fluorescent quantitative PCR is carried out with fluorescence quantification PCR primer;
(2) fluorescent quantitative PCR reaction system: SsoFast EvaGreen surpasses mixed solution 5 μ L, upstream primer and each 300ng of downstream primer, to add to total amount be 10 μ L to template DNA 100ng, Dnaes/RNase-free deionized water;
(3) quantitative fluorescent PCR program: 95 DEG C of denaturation 5min; 94 DEG C of sex change 15s, 53 DEG C of annealing 30s, totally 50 circulations; Melting curve scope is 65 ~ 95 DEG C, and read a number for every 0.2 DEG C, the time is 10s;
(4) result utilizing step (3) to obtain carries out gene type.
6. the fluorescence quantification PCR primer of molecule marker described in any one in claim 1-3, it is characterized in that, upstream primer sequence is as shown in SEQ ID No.2, and downstream primer sequence is as shown in SEQ ID No.3.
7. the application of molecule marker HRM5 in wheat breeding.
8. the application of molecule marker HRM5 in preparation transgenic plant, wherein, described gene is few Tillering gene Ltn3.
9. the application of the method described in claim 4 or 5 in wheat breeding improvement.
10. fluorescence quantification PCR primer according to claim 6 assists the application in wheat plant types breeding at molecule marker.
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| CN106636406A (en) * | 2016-12-26 | 2017-05-10 | 四川农业大学 | Molecular marker R207 coseparated with wheat few-tillering gene Ltn3 and application of molecular marker R207 |
| CN107177667A (en) * | 2017-05-18 | 2017-09-19 | 四川农业大学 | HRM molecular labelings chain wheat spike density QTL and its application |
| 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 |
| CN110106274A (en) * | 2019-05-06 | 2019-08-09 | 四川农业大学 | Molecular labeling and its application with wheatear extraction degree QTL QPel.HN.6D close linkage |
| CN115786565A (en) * | 2022-09-20 | 2023-03-14 | 四川农业大学 | SNP molecular marker linked with wheat spikelet number QTL and application thereof |
| 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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