CN104531882A - Molecular marker primer pair, method and application for authenticating new dwarf main effect QTL of wheat - Google Patents
Molecular marker primer pair, method and application for authenticating new dwarf main effect QTL of wheat Download PDFInfo
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Abstract
The invention discloses a molecular marker primer pair, method and application for authenticating the new dwarf main effect QTL of wheat and belongs to the field of crop molecular breeding. The primer pair is one or more of a primer pair of a molecular marker WGGC6079, a primer pair of a molecular marker WGGC6880, a primer pair of a molecular marker WGGC8307 and a primer pair of a molecular marker WGGC6073. The primer pairs of the molecular markers are utilized for carrying out PCR augmentation on genome DNA of an authenticated wheat material, a PCR product is detected through polyacrylamide gel electrophoresis and silver staining, and if a DNA segment corresponding to wheat variety Hokuno number six in size can be augmented from the material to be detected, it is determined that the wheat material contains the new dwarf main effect QTL QPh-2A.2. By means of the molecular marker primer pair and the method, whether the wheat material contains the new dwarf main effect QTL QPh-2A.2 can be accurately authenticated.
Description
Technical field
The present invention relates to crop field of molecular breeding, particularly relate to a kind of identify the molecule marker of the new main effect QTL of short stem of wheat primer pair, method and application.
Background technology
Wheat is the third-largest food crop being only second to corn and paddy rice in China, the main grain ration of Ye Shi China population more than half simultaneously, ultimate production and consumption all occupy first place in the world, and therefore the Food Security of sustainable development to China of Wheat Production is significant.Plant height is the Main Agronomic Characters affecting wheat plant types and output.Reduce plant height and improve the important goal that lodging resistance is wheat breeding, dwarf gene or short source are then the basic substances of wheat breeding of short stem, and the heredity parsing of plant height, the excavation of dwarf gene and utilization have great importance to the cultivation of wheat high-yield variety of short stem.
Since 20th century agricultural sixties 10 dwarf gene is used to wheat breeding, the research of dwarf gene and to utilize by increasing geneticist and breeding expert pay attention to, the major objective proterties becoming improving yield of wheat in world wide, Breeding For Super High-yielding of short stem, the selection and popularization of of short stem, Semi-dwarf cultivar, world wheat output average year is made to increase progressively 3.4% (ten thousand equalitys, 1998).Widely used in current wheat breeding is the parent (Guo Baohong etc., 1997) carrying the Recessive dwarf genes such as Rhtl, Rht2, Rht8, Rht9.The genetic diversity in short source is low is unfavorable for that the yield and quality of wheat improves, and the excavation of dwarf gene and genetic research utilize more and more to be paid attention in breeding.Set about from the creation in new short source and two aspects that utilize in existing short source, widening the hereditary basis of wheat breed, note the transformation of background genotype of short stem simultaneously, is the critical path that wheat breeding work advances further.Up to now, in wheat, had been found that more than 40 major genes of short stem, be distributed on 20 sites, the wheat dwarf stem gene formally named has 22 (Rht1-Rht22) (McIntosh etc., 2010; Haque etc., 2011; Peng etc., 2011), the discovery of these dwarf genes has vital role for the improvement of yield and quality of wheat in world wide.But research work concentrates on Rht1, Rht2, Rht3, Rht8, Rht9, Rht10 and Rht12 mostly.The mode of inheritance of these dwarf genes or show as dominant, or show as recessiveness; Plant hormones regulators,gibberellins reacted or shows as sensitivity, or showing as insensitive, respectively having feature (Dong Yuchen, 2000), wherein, Rht1 and Rht2 recessive gene derives from the Daruma derived varietiess such as No. 10, agricultural, lay respectively on 4BS and 4DS karyomit(e), insensitive to GA3 reaction.It is short that Rht3 dominant gene derives from thumb, is positioned on 4BS karyomit(e), insensitive to GA3 reaction.The recessive Dwarfing gene of Rht8 and Rht9 derives from duckbill wheat, lays respectively on 2DL and 7BS karyomit(e), is responsive type to GA3 reaction.From No. 1, short change qualification out, be positioned on 4DS karyomit(e), be fall the strongest gene of stalk effect to Rht10 gene, insensitive to GA3 reaction.Rht12 derives from the radiomutant of Karcag522, on 5AL karyomit(e), with awns suppressor gene BI close linkage, insensitive to GA3 reaction.
Molecular marker assisted selection directly selects genotype, has not by advantages such as envrionment conditions, Interaction among genes, genotype by environment interaction affect, greatly can improve the efficiency of selection of breeding work.Along with the development of biochip technology and the increasingly mature of DNA high throughput sequencing technologies, create third generation molecular marking technique-SNP marker.SNP refers to the DNA sequence polymorphism caused by the variation of single core thuja acid in genomic level, comprises the forms (Brookes etc., 2006) such as the conversion of single base, transversion, insertion and disappearance.SNP has very high frequency in nearly all species gene group, and in human genome, general every 1.3kb just has the existence of a SNP.The SNP site of a large amount of existence, makes people have an opportunity to find various genome mutation.SNP marker has diallele polymorphism usually, and its mutation rate is quite low, is a kind of stable sudden change.And SNP marker density is high, typical and easily Realization analysis automatization.The method detecting SNP has DNA chip technology, hybridization array analysis, homology hybrid method and direct sequencing etc.Wherein best with DNA chip technological method, obtain large-scale development and apply.SNP marker based on DNA chip technology is a kind of high-throughout molecular marking technique, and completing along with increasing farm crop genome sequencing, SNP marker will become the molecule marker (Fang Xuanjun etc., 2002) of plant genetics and breeding research middle ideal.At present, making a variation the wheat InfiniumiSelect 9k and the commercialization of Infinium iSelect 90k SNP chip that develop according to wheat est sequence SNP, providing important instrument for carrying out wheat snp analysis.
Summary of the invention
Based on the problem existing for above-mentioned prior art, the invention provides a kind of identify the molecule marker of the new main effect QTL of short stem of wheat primer pair, method and application, can the main effect QTL new of short stem of precise Identification wheat lines.
For solving the problems of the technologies described above, the invention provides a kind of primer pair identifying the molecule marker of the new main effect QTL of short stem of wheat, a pair or several that comprises in following primer pair is right:
WGGC6079: forward primer: 5 '-ATGGAGGTTTGGGATCAGCA-3 ' (SEQ ID NO.1), reverse primer: 5 '-ACTATGGTACACTCGTGCGT-3 ' (SEQ ID NO.2);
WGGC6880: forward primer: 5 '-CTTGCTGAGCTTGGTGGT-3 ' (SEQ ID NO.3), reverse primer: 5 '-TTGTTGCTGCACCATTACCC-3 ' (SEQ ID NO.4);
WGGC8307: forward primer: 5 '-CTTTGCCGTCAACGGCCA-3 ' (SEQ ID NO.5), reverse primer: 5 '-TACGTGGAGAGCAGCACTAC-3 ' (SEQ ID NO.6);
WGGC6073: forward primer: 5 '-CACAAGTACAAGGACCGCAG-3 ' (SEQ ID NO.7), reverse primer: 5 '-CAACAATCTCCATTTGCTTCTGA-3 ' (SEQ ID NO.8).
The present invention also provides a kind of method identifying the new main effect QTL molecule marker of short stem of wheat, comprises the following steps:
With the DNA of identified wheat lines for template, template described in primer pair of the present invention is utilized to carry out pcr amplification; Pcr amplification product detects through polyacrylamide gel electrophoresis and silver dye, if detected result is to amplify the DNA fragmentation with wheat breed north agriculture No. 6 DNA fragmentation formed objects, then determines that this institute identifies the QPh-2A.2 containing new main effect QTL of short stem in wheat lines.
In aforesaid method, the QPh-2A.2 of new main effect QTL of short stem derives from wheat breed No. 6, the agriculture in north, is positioned on wheat 2A chromosome long arm.
In aforesaid method, the primer pair of molecule marker WGGC6079, after carrying out pcr amplification to identified wheat lines DNA, electrophoresis can detect the DNA fragmentation with wheat breed north agriculture No. 6 formed objects, and between this DNA fragmentation and QPh-2A.2, genetic distance is 0cM;
The primer pair of molecule marker WGGC6880, after carrying out pcr amplification to identified wheat lines DNA, electrophoresis can detect the DNA fragmentation with wheat breed north agriculture No. 6 formed objects, and between this DNA fragmentation and QPh-2A.2, genetic distance is 0cM;
The primer pair of molecule marker WGGC8307, after carrying out pcr amplification to identified wheat lines DNA, electrophoresis can detect the DNA fragmentation with wheat breed north agriculture No. 6 formed objects, and between this DNA fragmentation and QPh-2A.2, genetic distance is 1.7cM;
The primer pair of molecule marker WGGC6073, after carrying out pcr amplification to identified wheat lines DNA, electrophoresis can detect the DNA fragmentation with wheat breed north agriculture No. 6 formed objects, and between this DNA fragmentation and QPh-2A.2, genetic distance is 3.0cM.
In aforesaid method, pcr amplification product is detected as through polyacrylamide gel electrophoresis and silver dye:
(1) PCR amplification system is: reaction system is 10 μ L, comprises 10 × buffer 1 μ L, 15mmol L
-1mgCl
21 μ L, 2mmol L
-1dNTP 1 μ L, 20ng μ L
-1primer 1 μ L, 1U Taq enzyme, ddH
2o 4 μ L, 20ng μ L
-1genomic dna 2 μ L;
(2) pcr amplification program is: 94 DEG C of denaturation 5min; Then 94 DEG C of sex change 45s, 55 DEG C of annealing 45s, 72 DEG C extend 1.5min, 40 circulations; 10min is extended after last 72 DEG C;
(3) being detected as of amplified production: amplified production adds 3 μ L sample-loading buffers and formed and detect samples, and described sample-loading buffer is the methane amide of 98% containing mass concentration, 10mmol L
-1eDTA, pH are 8.0, mass concentration be 0.25% tetrabromophenol sulfonphthalein and mass concentration be 0.25% dimethylbenzene blue or green; Get described in 3 μ L and detect sample with constant voltage 100 ~ 150V on 8% non-denaturing polyacrylamide gel, electrophoresis 3.5 ~ 4.5 hours, after cma staining, carry out banding pattern statistics or scanning record.
The present invention further provides and a kind ofly identify the application of the primer pair of the molecule marker of the new main effect QTL of short stem of wheat in the new molecular mark of short stem of wheat.
The present invention further provides and a kind ofly identify that the primer pair of the molecule marker of the new main effect QTL of short stem of wheat is making the application in wheat new material of short stem.
Beneficial effect of the present invention is: by molecular marker primer pair of the present invention and method, whether important theory and practical significance can be had containing new main effect QTL QPh-2A.2 of short stem, this new main effect QTL QPh-2A.2 of short stem cultivation to the new high-yield variety of short stem of genetic diversity and wheat widening the short source of wheat in precise Identification wheat lines.And develop 11 with the molecule marker of this main effect QTL linkage, wherein, 4 can be used for this main effect QTL of molecular marker assisted selection with the molecule marker of main effect QTL compact linkage, and the deliberate genetic for this main effect QTL site is located and map based cloning has established solid basis.
Accompanying drawing explanation
In order to be illustrated more clearly in the technical scheme of the embodiment of the present invention, below the accompanying drawing used required in describing embodiment is briefly described, apparently, accompanying drawing in the following describes is only some embodiments of the present invention, for those of ordinary skill in the art, under the prerequisite not paying creative work, other accompanying drawings can also be obtained according to these accompanying drawings.
Fig. 1 is the position of wheat breed north No. 6, agriculture new main effect QTL Qph-2A.2 of short stem on 2A karyomit(e) and genetic linkage map spectrum information thereof;
Fig. 2 be with the molecule marker WGGC6073 of new main effect QTL compact linkage of short stem in parents and RILs population segment family electrophoresis detection result; Wherein M is that DNA Marker marks, and 1 and 2 are respectively parent swallow large 1817 and No. 6, northern agriculture, and 3,4,6,8,11 is new dwarf gene type family, and 5,7,9,10,12 is long-culm gene type family;
Fig. 3 be with the molecule marker WGGC6079 of new main effect QTL compact linkage of short stem in parents and RILs population segment family electrophoresis detection result; Wherein M is that DNA Marker marks, and 1 and 2 are respectively parent swallow large 1817 and No. 6, northern agriculture, and 3,4,6,8,11 is new dwarf gene type family, and 5,7,9,10,12 is long-culm gene type family;
Fig. 4 be with the molecule marker WGGC6880 of new main effect QTL compact linkage of short stem in parents and RILs population segment family electrophoresis detection result; Wherein M is that DNA Marker marks, and 1 and 2 are respectively parent swallow large 1817 and No. 6, northern agriculture, and 3,6,8,11 is new dwarf gene type family, and 4,5,7,9,10,12 is long-culm gene type family;
Fig. 5 be with the molecule marker WGGC8307 of new main effect QTL compact linkage of short stem in parents and RILs population segment family electrophoresis detection result; Wherein M is that DNA Marker marks, and 1 and 2 are respectively parent swallow large 1817 and No. 6, northern agriculture, and 7-10,12 is new dwarf gene type family, and 3-6,11 is long-culm gene type family.
Embodiment
Be clearly and completely described the technical scheme in the embodiment of the present invention below, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on embodiments of the invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to protection scope of the present invention.
The embodiment of the present invention provides a kind of primer pair identifying the molecule marker of the new main effect QTL of short stem of wheat, the main effect QTL QPh-2A.2 new of short stem of wheat breed No. 6, the agriculture in north is derived from for molecule marker, this new main effect QTL of short stem is positioned on wheat 2A chromosome long arm (as shown in Figure 1), significantly can reduce Plant Height in Wheat, the soluble phenotypic variation of about 5.41% ~ 12.55%, should be as follows for the identification of the nucleotide sequence of the primer pair of the molecule marker of the new main effect QTL QPh-2A.2 of short stem of wheat breed No. 6, the agriculture in north: (as shown in SEQ ID NO.1-NO.8).
WGGC6079: forward primer: 5 '-ATGGAGGTTTGGGATCAGCA-3 ' (SEQ ID NO.1),
Reverse primer: 5 '-ACTATGGTACACTCGTGCGT-3 ' (SEQ ID NO.2);
WGGC6880: forward primer: 5 '-CTTGCTGAGCTTGGTGGT-3 ' (SEQ ID NO.3),
Reverse primer: 5 '-TTGTTGCTGCACCATTACCC-3 ' (SEQ ID NO.4);
WGGC8307: forward primer: 5 '-CTTTGCCGTCAACGGCCA-3 ' (SEQ ID NO.5),
Reverse primer: 5 '-TACGTGGAGAGCAGCACTAC-3 ' (SEQ ID NO.6);
WGGC6073: forward primer: 5 '-CACAAGTACAAGGACCGCAG-3 ' (SEQ ID NO.7),
Reverse primer: 5 '-CAACAATCTCCATTTGCTTCTGA-3 ' (SEQ ID NO.8).
Wherein, with the primer pair of above-mentioned molecule marker WGGC6079, after carrying out pcr amplification to wheat lines DNA to be identified, electrophoresis can detect the DNA fragmentation with wheat breed north agriculture No. 6 formed objects, and between this DNA fragmentation and QPh-2A.2, genetic distance is 0cM.
Wherein, with the primer pair of above-mentioned molecule marker WGGC6880, after carrying out pcr amplification to wheat lines DNA to be identified, electrophoresis can detect the DNA fragmentation with wheat breed north agriculture No. 6 formed objects, and between this DNA fragmentation and QPh-2A.2, genetic distance is 0cM.
Wherein, with the primer pair of above-mentioned molecule marker WGGC8307, after carrying out pcr amplification to wheat lines DNA to be identified, electrophoresis can detect the DNA fragmentation with wheat breed north agriculture No. 6 formed objects, and between this DNA fragmentation and QPh-2A.2, genetic distance is 1.7cM.
Wherein, with the primer pair of above-mentioned molecule marker WGGC6073, after carrying out pcr amplification to wheat lines DNA to be identified, electrophoresis can detect the DNA fragmentation with wheat breed north agriculture No. 6 formed objects, and between this DNA fragmentation and QPh-2A.2, genetic distance is 3.0cM.
Method of the present invention is: utilize the one or more pairs of primers in the primer pair of the primer pair of the primer pair of the primer pair of molecule marker WGGC6079, molecule marker WGGC6880, molecule marker WGGC8307, molecule marker WGGC6073, pcr amplification is carried out to the genomic dna of wheat lines to be identified, PCR primer detects through polyacrylamide gel electrophoresis and silver dye, if material to be detected can amplify the DNA fragmentation to wheat breed north agriculture No. 6 corresponding sizes, then illustrate in this material containing new main effect QTL QPh-2A.2 of short stem.
The invention provides a kind of molecule marking method identifying the new main effect QTL QPh-2A.2 of short stem of wheat, comprising: with the DNA of wheat lines to be identified for template, carry out pcr amplification with the primer pair of above-mentioned molecule marker; Pcr amplification product through 8% native polyacrylamide gel electrophoresis, then contaminates method soon with Silver Nitrate and detects, and can amplify the family be with the family of northern agriculture No. 6 wheat same clip containing new main effect QTL QPh-2A.2 of short stem.
The concrete steps that in said process, pcr amplification and product detect are:
(1) PCR amplification system: reaction system is 10 μ L, comprises 10 × buffer 1 μ L, 15mmol L
-1mgCl
21 μ L, 2mmol L
-1dNTP 1 μ L, 20ng μ L
-1primer 1 μ L, 1U Taq enzyme, ddH
2o 4 μ L, 20ng μ L
-1genomic dna 2 μ L.(2) pcr amplification program: 94 DEG C of denaturation 5min; Then 94 DEG C of sex change 45s, 55 DEG C of annealing 45s, 72 DEG C extend 1.5min, 40 circulations; 10min is extended after last 72 DEG C.(3) detection of amplified production: amplified production add 3 μ L sample-loading buffers (containing 98% methane amide, 10mmol L
-1eDTA, pH 8.0,0.25% tetrabromophenol sulfonphthalein, 0.25% dimethylbenzene is blue or green).Get 3 μ L samples with constant voltage 100 ~ 150V on 8% non-denaturing polyacrylamide gel, electrophoresis about 4h, after cma staining, carry out banding pattern statistics or scanning record.
Qualification result is: can amplify with the family of northern agriculture No. 6 wheat same DNA fragments is family containing new main effect QTL QPh-2A.2 of short stem.
The present invention further provides the application of the new main effect QTL of short stem of a kind of wheat identified in the new material initiative of short stem of wheat.
The present invention further provides the application of primer pair in the new material initiative of short stem of wheat of molecule marker.
The present invention further provides the new main effect QTL of short stem of a kind of wheat identified in the new Breeding Application of short stem of wheat.
The present invention further provides the application of primer pair in the new molecular mark of short stem of wheat of molecule marker.
The cultivation of new main effect QTL QPh-2A.2 of short stem provided by the present invention to the new high-yield variety of short stem of genetic diversity and wheat widening the short source of wheat has important theory and practical significance.And develop 11 with the molecule marker of this main effect QTL linkage, wherein, 4 can be used for this main effect QTL of molecular marker assisted selection with the molecule marker of main effect QTL compact linkage.Deliberate genetic for this main effect QTL site is located and map based cloning has established solid basis.
Below in conjunction with specific embodiment, the invention will be further described.
Embodiment
For examination material
The examination material that supplies of the present embodiment is: with the Landrace of Common Wheat swallow large 1817 of high stalk for maternal, utilizes little kind No. 6, the agriculture in north of short stem to be paternal hybrid, obtains F
1cross-fertilize seed, constructs " large No. 6, the 1817/ northern agriculture of swallow " the RILs F containing 269 familys with single seed descent
8, F
9, F
10and F
11colony.Local variety swallow large 1817 is one of wheat backbone parents, and be that former Yenching University crop is improved field and forms from the choosing of the little Bai Maizhong system in local variety Pingyao, Shanxi, this kind is famous with resistance, has cold-resistant, drought-enduring, resistance to lean, the feature such as ability for tillering is strong.The kind number be bred as has 53, and its offspring is distributed in northern Winter Wheat Area, particularly Northern Winter district (Zhuan Qiaosheng, 2003; Han Jun etc., 2009)." No. 6, northern agriculture " is the High-Yield Wheat Cultivar that Beijing Agricultural College is bred as the nineties in last century, shows the large grain of new anti-fall, cold-resistant, large fringe of short stem, precocity, the features such as Huang Hao that fall (Zhai Fenglin, 1995).The plant height Traits change of " swallow large 1817 " and " No. 6, northern agriculture " is very large, and the recombinant inbred lines genetic diversity derivative by it enriches, and is the ideal material of research plant height genetic mechanism.
The plant height trait phenotypes qualification of RILs colony
To " large No. 6, the 1817/ northern agriculture of swallow " the RILs colony of 269 familys and parent thereof be included in 2010-2011 (F
8), 2011-2012 (F
9), 2012-2013 (F
10) and 2013-2014 (F
11) plant in Beijing Agricultural College Ting Zi village testing station, Gaoyi County, Hebei province seed farm and testing station of Kaifeng academy of agricultural sciences.Test adopts randomized block design, duplicate rows district, row long 2.00m, line-spacing 20cm, repeats, manages according to a conventional method for 3 times.After Wheat in Grain Filling Stage plant height is stable, each family chooses the representative individual plant of 10 strains respectively, examines or check its plant height, using each mean value repeated as the raw data of statistical study.
The examination standard of plant height: the rhizome joint portion of wheat to the height (not containing awns) of main tip of the spike, unit: centimetre (cm).
Wheat leaf blade extracting genome DNA:
CTAB method is adopted to extract parent's No. 6, agriculture in north, swallow large 1817 and " large No. 6, the 1817/ northern agriculture of swallow " RILs F
8for the genomic dna of colony's plant leaf.
SSR molecular marker is analyzed and SNP chip analysis:
(1) screening of the SSR molecular marker of polymorphism between parents: the primer pair choosing 538 SSR, EST-SSR molecule markers announced in GrainGenes database (http://wheat.pw.usda.gov/cgi-bin/graingenes), pcr amplification is carried out to the genomic dna of parent's No. 6, agriculture in north and swallow large 1817, and detect through polyacrylamide gel electrophoresis and silver dye the molecule marker finding polymorphism, obtain SSR, EST-SSR molecule marker of 125 polymorphisms altogether.
(2) RILs F
8ssr analysis for colony: there is SSR, EST-SSR molecule marker of polymorphism for primer between obtain 125 parents,, No. 6, northern agriculture large 1817 to parent swallow simultaneously and 269 RILs F8 carry out pcr amplification for the genomic dna of colony, and according to the banding pattern of parents to the genotype identification of each family of RILs colony, obtain the molecular data of each SSR marker.
The genotype of large for parent swallow 1817 is designated as A, and the genotype of parent's No. 6, agriculture in north is designated as B.RILs F8 is designated as A for the large 1817 consistent familys of genotype in colony and parent swallow, and the family consistent with parent's No. 6, agriculture in north is designated as B.
(3) SNP chip detects and analyzes:
The wheat Infinium 9K iSelect SNP chip utilizing Illumina company to produce is large 1817 to parent swallow, No. 6, northern agriculture and RILs F8 carry out detection for the genomic dna of colony and analyze, and obtains the molecular data of SNP marker.
The structure of genetic linkage maps: the molecular data result integrating SSR, EST-SSR and SNP marker, utilizes mapping software Mapmaker/Exp version3.0 to build high-density genetic linkage maps.This genetic linkage maps comprises 2339 SNPs and 125 SSR, EST-SSR polymorphism mark, and total genetic distance is 2902cM.
The QTL positioning analysis of Plant Height in Wheat proterties
Utilize the ICIM-ADD program in QTLIciMapping v3.2 software, the plant height proterties of plant height phenotypic data to RIL colony of associating 4 years 8 environmental forms carries out complete Interval mapping and QTL site detects.Step=1.0cM, PSR=0.0010, getting LOD value is 2.50 to position for threshold value, calculates corresponding QTL site and to be correlated with the contribution rate that additive effect and this site make a variation to trait phenotypes.Result shows to navigate to the QTL site that 17 control plant height altogether, is distributed in 1B, 2A, 2D, 3A, 3B, 3D, 5A, 5B, 6A, on 11 karyomit(e)s such as 6B, 7A, wherein, the Qph-2A.2 effect value be positioned on 2A chromosome long arm is maximum, can detect in 8 environmental forms simultaneously.This site explains the maximum phenotypic variation of plant height proterties, the phenotype contribution rate of 8 environment is 5.41% ~ 12.55%, Qph-2A.2 site is the stable main effect QTL of this collective control plant height proterties, the LOD value in this site is respectively 2.80 ~ 7.97, additive effect is respectively 2.21 ~ 5.11 centimetres, the increase Plant height gene of this QTL site comes from parent " swallow large 1817 ", and reduces Plant height gene and come from parent's " No. 6, northern agriculture ".By comparing with the wheat dwarf stem gene reported and the chromosome position that controls plant height QTL, infer that QPh-2A.2 is the novel site controlling plant height proterties.
The molecule marker of exploitation and main effect QTL compact linkage:
For further Fine Mapping derives from the main effect QTL Qph-2A.2 new of short stem of No. 6, northern agriculture, according to the genome sequence of the SNP marker sequence information comparison two fringe false bromegrass with main effect QTL compact linkage the genome sequence of the two fringe false bromegrasses (can see http://www.plantgdb.org/BdGDB/cgi-bin/blastGDB.pl), specify the collinearity genomic segment of two fringe false bromegrasses corresponding to main effect QTL; Then, (the contigs sequence of Wheat volatiles can see https: //urgi.versailles.inra.fr/blast/blast.php) to utilize the contigs sequence of the gene order comparison Wheat volatiles in the collinearity genomic segment of two fringe false bromegrasses; According to the wheat contigs sequences Design Auele Specific Primer of homology, exploitation and the closely linked molecule marker of target QTL.
Develop and filter out 11 with molecule marker WGGC684, WGGC685, WGGC692, WGGC788, WGGC2137, WGGC2160, WGGC6073, WGGC6079, WGGC6880, WGGC8307, WGGC8308 (primer sequence is in table 1) of target main effect QTL compact linkage, in order to encrypt the molecule marker of this main effect QTL section.The plant height phenotypic data of RILs colony 4 years 8 environmental forms is utilized to locate further new main effect QTL Qph-2A.2 site of short stem, be positioned by this main effect QTL between molecule marker WGGC6079 and WGGC6880, the genetic distance between these two molecule markers is 2.3cM (Fig. 1).Utilize molecule marker WGGC6073, WGGC6079, WGGC6880, WGGC8307 can carry out molecular marker assisted selection, as shown in Fig. 2 to 5 to this new main effect QTL site of short stem.
The PCR reaction system of the present embodiment is: 10 × buffer 1 μ L, 15mmol L
-1mgCl
21 μ L, 2mmol L
-1dNTP 1 μ L, 20ng μ L
-1primer 1 μ L, 1U Taq enzyme, ddH
2o 4 μ L, 20ng μ L
-1genomic dna 2 μ L.
The pcr amplification condition of the present embodiment is: 94 DEG C of denaturation 5min; Then 94 DEG C of sex change 45s, 55 DEG C of annealing 45s, 72 DEG C extend 1.5min, 40 circulations; 10min is extended after last 72 DEG C.
Table 1. and the closely linked molecule marker of new main effect QTL Qph-2A.2 of short stem
| Molecule marker title | Forward primer sequence | Reverse primer sequences |
| WGGC684 | TTGCTGATAAGAAGAAGTTGC | CATGTGTACTCCTTGAAGAGC |
| WGGC685 | TAGGGAAGAGGAAGAGAAGAA | GAAGGAACTAAGGATTGTGGT |
| WGGC692 | ACTGATGTATAGACGCTCTGG | TATAGGGCATGGACTGATAAA |
| WGGC788 | CATACACCTGTGTTCCATTCT | GAACAGCTTGAGGTTAGACAA |
| WGGC2137 | GTAGAGCCTTGGCATTTTT | CCGAAAGTCCTCTCCTTATT |
| WGGC2160 | GGCCATGGAAGAGAGTCAAA | TCAACTGGCCGTCGTCAGTC |
| WGGC6073 | CACAAGTACAAGGACCGCAG | CAACAATCTCCATTTGCTTCTGA |
| WGGC6079 | ATGGAGGTTTGGGATCAGCA | ACTATGGTACACTCGTGCGT |
| WGGC6880 | CTTGCTGAGCTTGGTGGT | TTGTTGCTGCACCATTACCC |
| WGGC8307 | CTTTGCCGTCAACGGCCA | TACGTGGAGAGCAGCACTAC |
| WGGC8308 | CTTTGCCGTCAACGGCCG | TACGTGGAGAGCAGCACTAC |
The above; be only the present invention's preferably embodiment, but protection scope of the present invention is not limited thereto, is anyly familiar with those skilled in the art in the technical scope that the present invention discloses; the change that can expect easily or replacement, all should be encompassed within protection scope of the present invention.Therefore, protection scope of the present invention should be as the criterion with the protection domain of claims.
Claims (7)
1. identify a primer pair for the molecule marker of the new main effect QTL of short stem of wheat, it is characterized in that, a pair or several that comprises in following primer pair is right:
WGGC6079: forward primer: 5 '-ATGGAGGTTTGGGATCAGCA-3 ' (SEQ ID NO.1), reverse primer: 5 '-ACTATGGTACACTCGTGCGT-3 ' (SEQ ID NO.2);
WGGC6880: forward primer: 5 '-CTTGCTGAGCTTGGTGGT-3 ' (SEQ ID NO.3), reverse primer: 5 '-TTGTTGCTGCACCATTACCC-3 ' (SEQ ID NO.4);
WGGC8307: forward primer: 5 '-CTTTGCCGTCAACGGCCA-3 ' (SEQ ID NO.5), reverse primer: 5 '-TACGTGGAGAGCAGCACTAC-3 ' (SEQ ID NO.6);
WGGC6073: forward primer: 5 '-CACAAGTACAAGGACCGCAG-3 ' (SEQ ID NO.7), reverse primer: 5 '-CAACAATCTCCATTTGCTTCTGA-3 ' (SEQ ID NO.8).
2. identify a method for the new main effect QTL molecule marker of short stem of wheat, it is characterized in that, comprise the following steps:
With the DNA of identified wheat lines for template, template described in the primer pair described in claim 1 is utilized to carry out pcr amplification; Pcr amplification product detects through polyacrylamide gel electrophoresis and silver dye, if detected result is to amplify the DNA fragmentation with wheat breed north agriculture No. 6 DNA fragmentation formed objects, then determines the QPh-2A.2 containing new main effect QTL of short stem in identified wheat lines.
3. the method for the molecule marker of the new main effect QTL of short stem of qualification wheat according to claim 2, is characterized in that, the QPh-2A.2 of described new main effect QTL of short stem derives from wheat breed No. 6, the agriculture in north, is positioned on wheat 2A chromosome long arm.
4. the method for the molecule marker of the new main effect QTL of short stem of the qualification wheat according to Claims 2 or 3, is characterized in that,
The primer pair of described molecule marker WGGC6079, after carrying out pcr amplification to identified wheat lines DNA, electrophoresis can detect the DNA fragmentation with wheat breed north agriculture No. 6 formed objects, and between this DNA fragmentation and QPh-2A.2, genetic distance is 0cM;
The described primer pair stating molecule marker WGGC6880, after carrying out pcr amplification to identified wheat lines DNA, electrophoresis can detect the DNA fragmentation with wheat breed north agriculture No. 6 formed objects, and between this DNA fragmentation and QPh-2A.2, genetic distance is 0cM;
The primer pair of described molecule marker WGGC8307, after carrying out pcr amplification to identified wheat lines DNA, electrophoresis can detect the DNA fragmentation with wheat breed north agriculture No. 6 formed objects, and between this DNA fragmentation and QPh-2A.2, genetic distance is 1.7cM;
The primer pair of described molecule marker WGGC6073, after carrying out pcr amplification to identified wheat lines DNA, electrophoresis can detect the DNA fragmentation with wheat breed north agriculture No. 6 formed objects, and between this DNA fragmentation and QPh-2A.2, genetic distance is 3.0cM.
5. the method for the molecule marker of the new main effect QTL of short stem of the qualification wheat according to Claims 2 or 3, is characterized in that, described pcr amplification product is detected as through polyacrylamide gel electrophoresis and silver dye:
(1) PCR amplification system is: reaction system is 10 μ L, comprises 10 × buffer 1 μ L, 15mmol L
-1mgCl
21 μ L, 2mmol L
-1dNTP 1 μ L, 20ng μ L
-1primer 1 μ L, 1U Taq enzyme, ddH
2o 4 μ L, 20ng μ L
-1genomic dna 2 μ L;
(2) pcr amplification program is: 94 DEG C of denaturation 5min; Then 94 DEG C of sex change 45s, 55 DEG C of annealing 45s, 72 DEG C extend 1.5min, 40 circulations; 10min is extended after last 72 DEG C;
(3) being detected as of amplified production: amplified production adds 3 μ L sample-loading buffers and formed and detect samples, and described sample-loading buffer is the methane amide of 98% containing mass concentration, 10mmol L
-1eDTA, pH are 8.0, mass concentration be 0.25% tetrabromophenol sulfonphthalein and mass concentration be 0.25% dimethylbenzene blue or green; Get described in 3 μ L and detect sample with constant voltage 100 ~ 150V on 8% non-denaturing polyacrylamide gel, electrophoresis 3.5 ~ 4.5 hours, after cma staining, carry out banding pattern statistics or scanning record.
6. the application of primer pair in the new molecular mark of short stem of wheat of the molecule marker of the new main effect QTL of short stem of qualification wheat according to claim 1.
7. the primer pair of the molecule marker of the new main effect QTL of short stem of qualification wheat according to claim 1 is making the application in the new material of short stem of wheat.
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| CN105821038A (en) * | 2016-05-24 | 2016-08-03 | 中国农业科学院作物科学研究所 | Molecular marker for identifying wheat height and specific primers |
| CN108179220A (en) * | 2018-02-27 | 2018-06-19 | 西北农林科技大学 | The KASP labels of wheat dwarf stem gene Rht12 close linkages and its application |
| CN109811077A (en) * | 2019-03-25 | 2019-05-28 | 中国农业科学院作物科学研究所 | With the KASP label of wheat dwarf stem gene close linkage and its application |
| CN115873976A (en) * | 2022-09-23 | 2023-03-31 | 甘肃省农业科学院作物研究所 | Molecular marker for identifying height character of broom corn millet and application thereof |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105821038A (en) * | 2016-05-24 | 2016-08-03 | 中国农业科学院作物科学研究所 | Molecular marker for identifying wheat height and specific primers |
| CN105821038B (en) * | 2016-05-24 | 2018-07-20 | 中国农业科学院作物科学研究所 | Molecular labeling and special primer for identifying Plant Height in Wheat |
| CN108179220A (en) * | 2018-02-27 | 2018-06-19 | 西北农林科技大学 | The KASP labels of wheat dwarf stem gene Rht12 close linkages and its application |
| CN108179220B (en) * | 2018-02-27 | 2021-04-23 | 西北农林科技大学 | KASP marker tightly linked with wheat dwarf gene Rht12 and application thereof |
| CN109811077A (en) * | 2019-03-25 | 2019-05-28 | 中国农业科学院作物科学研究所 | With the KASP label of wheat dwarf stem gene close linkage and its application |
| CN109811077B (en) * | 2019-03-25 | 2022-04-12 | 中国农业科学院作物科学研究所 | KASP marker closely linked with wheat dwarf gene and application thereof |
| CN115873976A (en) * | 2022-09-23 | 2023-03-31 | 甘肃省农业科学院作物研究所 | Molecular marker for identifying height character of broom corn millet and application thereof |
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