CN104372003B - Molecular marker closely linked with major QTL of wheat height and uppermost internode length as well as acquisition method and application of molecular marker - Google Patents
Molecular marker closely linked with major QTL of wheat height and uppermost internode length as well as acquisition method and application of molecular marker Download PDFInfo
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Abstract
The invention discloses a molecular marker closely linked with the major QTL of wheat height and uppermost internode length as well as an acquisition method and application of the molecular marker. The acquisition method comprises the following steps: carrying out PCR amplification on DNA of a wheat variety Kenong9204 by adopting a marked primer Xcnl10; carrying out electrophoretic separation on the amplified product on 6.0% native polyacrylamide gel to obtain the amplified product with molecular weight of 700bp, namely the molecular marker of major QTL of wheat height and uppermost internode length. The molecular marker can be used for detecting whether a wheat variety or line contains QTL capable of reducing plant height and uppermost internode length, or not, so that the wheat variety or line containing QTL capable of reducing the plant height and uppermost internode length can be quickly screened for breeding, and the breeding progress for excellent wheat plant variety can be greatly accelerated.
Description
Technical field
The present invention relates to wheat molecular biotechnology and Breeding Application field, specifically a kind of and Plant Height in Wheat and fringe
The molecular labeling of lower internode length main effect QTL compact linkage and its acquisition methods and application.
Background technology
Plant Height in Wheat is closely related with lodging resistance, is influence improving yield of wheat, the important character of stable yields, reduces plant height and carries
Height lodging property is always the important goal of Yield Potential Breeding of Wheat.The plant height of wheat is by polygenes or a small number of key-genes plus a series of
Minor gene or modifier are controlled, and show as typical Inheritance of Quantitative Characters feature.Existing many domestic and foreign scholars are to wheat
The genetic mechanism of plant height is studied, and many plant height QTLs for being positioned are almost throughout 21 chromosomes of wheat.From 20th century
The sixties International Maize and Wheat Improvement Centers（CIMMYT）The first in the world time " green revolution " for initiating of Borlaug doctors NE
（Khush, Nature Reviews Genetics. 2: 815–822, 2001）, " agricultural 10 " dwarf gene is used for small
Since wheat breeding, the research of dwarf gene is paid attention to by increasing breeding expert.Although the dwarf gene named at present has 25
It is as many as individual, but up to more than 90% of short stem and Semi-dwarf cultivar is carried from agricultural 10 and duckbill wheat in Wheat Production
Dwarf gene, the hereditary basis that the unification in short source result in Bred Wheat Varieties is narrow.Therefore, constantly create and find new
Short source, enrich the diversity of dwarf gene, and by molecular marking technique, internode length under research and development Plant Height in Wheat and fringe
Gene, reduce internode length under Plant Height in Wheat and fringe, be extremely important in breeding work.
So far, the named Dwarfing gene of 4B chromosomes is positioned at for Rht-B1b, on 4B the short arm of a chromosome
（McVittie et al., Heredity. 40:67–70, 1978）；By quantitative character gene locus therefor（Quantitative
Trait locus, QTL）Analysis, using multiple mapping populations 4BS detect plant height QTL site (McCartney et al.,
Genome, 48: 870–883, 2005; Marza et al., Theoretical and Applied Genetics.
112: 688–698, 2006; Zhang et al., Journal of Genetics and Genomics. 35: 119–
127, 2008; Mao et al., Euphytica. 174: 343–356, 2010; Cui et al., Theoretical
and Applied Genetics. 122: 1517–1536, 2011).At present, not yet have named positioned at the short bars of 4BL
The relevant report of the molecular labeling of internode length close linkage under gene and fringe.
The content of the invention
It is an object of the invention to provide a kind of molecule mark with internode length main effect QTL compact linkage under Plant Height in Wheat and fringe
Note and its acquisition methods and application, by the molecule mark with internode length main effect QTL compact linkage under Plant Height in Wheat and fringe for obtaining
Remember and reduce the QTL of internode length under plant height and fringe whether having in detecting wheat breed or strain, to accelerate Distinctive Wheat plant type
The seed selection process of kind.
The purpose of the present invention is achieved through the following technical solutions：
A kind of molecular labeling of internode length main effect QTL compact linkage with Plant Height in Wheat and under fringe, the molecular labeling isXcnl10, its described molecular labelingXcnl10Upstream primer sequence：CTTACCTAGTACAATGTACTCTCTCT（Such as SEQ ID
NO:Described in 1）, downstream primer sequence：ATGATGGTCTGGATCTGG（Such as SEQ ID NO:Described in 2）.
The molecular labelingXcnl10Acquisition methods, comprise the following steps：UsingXcnl10Labeled primer,
Upstream primer sequence：CTTACCTAGTACAATGTACTCTCTCT（Such as SEQ ID NO:Described in 1）、
Downstream primer sequence：ATGATGGTCTGGATCTGG（Such as SEQ ID NO:Described in 2）,
DNA to wheat breed section agriculture 9204 enters performing PCR amplification, and its PCR amplification system is 20 μ l, including：The DNA of 1 μ l
Template, the sense primer of 1 μ l, the anti-sense primer of 1 μ l, 2 × Taq PCR StarMix, the ddH of 7 μ l of 10 μ l2O；Using
Gradient touchdown PCR amplification program is expanded, and step is as follows：
（1）94 DEG C of 4 min of denaturation,
（2）15 renaturation drop of temperature programs of circulation, each circulation is denatured 45 s, 65 DEG C of s of renaturation 50 for 94 DEG C, often
It is individual circulate in 65 DEG C on the basis of reduce by 1 DEG C, 72 DEG C extension 55 s；
（3）30 regular-PCR programs of circulation：94oC is denatured 40 s, 50 DEG C of renaturation 40 s, 72 DEG C of 40 s of extension；
（4）72 DEG C of 5 min of extension；Terminate amplification, 15 DEG C of preservations；
The amplified production V of voltage stabilizing 120 electrophoretic separation on 6% non-denaturing polyacrylamide gel, argentation is corresponded to
Amplified production molecular weight be 700bp, as with the molecule mark of internode length main effect QTL compact linkage under Plant Height in Wheat and fringe
Note.
The DNA of wheat breed section of the present invention agriculture 9204 refers to acquisition after the plant leaf separation and Extraction of wheat section agriculture 9204
DNA.
Using above-mentioned molecular labelingXcnl10The method of detection wheat breed or strain, comprises the following steps：
A. useXcnl10Labeled primer enters performing PCR amplification, its PCR amplification bodies to the DNA of wheat breed to be measured or strain
It is 20 μ l to be, comprising：The DNA profiling of 1 μ l, the sense primer of 1 μ l, the anti-sense primer of 1 μ l, 2 × Taq PCR of 10 μ l
StarMix, and 7 μ l ddH2O；Expanded using gradient touchdown PCR amplification program：94 DEG C of 4 min of denaturation；15 circulation answer
Degree landing procedure warm in nature, each circulation is 94 DEG C of denaturation 45 s, 65 DEG C of s of renaturation 50, and each is reduced on the basis of circulating in 65 DEG C
1 DEG C, 72 DEG C of 55 s of extension；30 regular-PCR programs of circulation：94oC is denatured 40 s, 50 DEG C of s of renaturation 40,72 DEG C of extensions
40 s；72 DEG C of 5 min of extension；Terminate amplification, 15 DEG C of preservations；
B. by above-mentioned amplified production after the V of voltage stabilizing 120 electrophoretic separation on 6% non-denaturing polyacrylamide gel, if
The molecular weight for enough obtaining amplified production is the amplified fragments of 700bp, then the wheat breed or strain are have on the gene loci
There are the wheat breed or strain for reducing internode length gene under plant height and fringe；Otherwise, the wheat breed or strain belong in the site
It is wheat breed or strain without internode length gene under reduction plant height and fringe.
The wheat breed to be detected or strain can be parent for wheat section agriculture 9204, by using conventional hybridization simultaneously
Section agriculture 9204 wheat derived varieties or strain of the procreation seed selection to F2 more than generation, or other any wheat breeds or product
System.
The DNA of wheat breed described in detection method or strain refers to that will be obtained after wheat plant blade separation and Extraction
The DNA for obtaining.
Heretofore described 6% non-denaturing polyacrylamide gel refers to contain in 100ml polyacrylamide gel solution
5.85 g acrylamides and 0.15 g methene acrylamides.
In the present invention 2 × Taq PCR StarMix be premix containing optimization concentration high-purity Taq archaeal dna polymerases,
dNTPs、Mg2+, 2 times of concentration of instant of composition such as reaction buffer and stabilizer PCR solution, this product brand is
GenStar, by Beijing Kang Run Cheng Ye bio tech ltd commercial goods.
Wheat breed capital 411 of the present invention is Approved variety, and Beijing's variety certification was passed through in 1991；Lead within 1992
Cross Tianjin, Shanxi Province and national variety certification；Capital 411 can be asked for from national Germplasm Resources of Farm Crop storehouse, and the whole nation is unified to compile
Number be ZM020984；Section's agriculture 9204 is Approved variety, and Hebei province crop varietal approval committee was passed through in 2002；
By national variety certification, variety certification numbering is that state examines wheat 2003037 within 2003.
The molecular labeling of internode length close linkage under Plant Height in Wheat disclosed by the invention and fringeXcnl10Fully demonstrate small
Internode length main effect QTL under the plant height and fringe of wheat variety or strain, is expanded by molecular labeling to wheat breed or strain PCR, can
So that quickly to wheat breed or strain, whether the QTL with internode length under plant height and fringe judges, such that it is able to filter out
Wheat breed or strain with internode length QTL under reduction plant height and fringe, accelerate the seed selection process of wheat breed.
Educated what the present invention was provided for wheat with the molecule labelling method of internode length close linkage under Plant Height in Wheat and fringe
Kind, need to only detect the amplified band characteristic of these marks, it can be determined that the synergy variation presence or absence of plant height major gene loci,
To predict the plant height phenotype of wheat, for the breeding work for instructing Plant Height in Wheat to improve, fast accurate is not only screened, not by environment
Influence, selection target clearly, and has saved production cost, substantially increases the efficiency of selection and matter of wheat breed or strain
Amount, is directly realized identification of the target gene in Wheat Germplasm Resources and breeding progeny, is that wheat plant types breeding is opened up more
More new short bar genetic resources.
Brief description of the drawings
Fig. 1 is mapping area of the internode length main effect QTL on chromosome 4BL under the plant height and fringe of wheat breed section agriculture 9204
Between；In figure：Hollow rectangle represents chromosome, and its upper end dash area represents centromere position, and right side is the name of molecular labeling
Claim, left side of the digital is to mark position on chromosome, and unit is cM；Six kinds of molecular labelings are had in figure, Leaftype is shape
State is marked, and Xme is marked for SRAPs, and wPt is DArTs marks, and Xwmc and Xbarc is g-SSR marks, and Xcnl and Xcfe are
E-SSR is marked, and Xmag is STS marks, indicate the mark of black underscore forXcnl10；Chromosome lower right black and grey
QTL mapping of the internode length under each environment is interval under rectangle represents plant height and fringe respectively, and wherein Ph represents plant height, and Pl is represented
Internode length under fringe.
Fig. 2 is the labeled primer in section's agriculture 9204 with internode length main effect QTL compact linkage under Plant Height in Wheat and fringeXcnl10
And the labeled primer with the not close linkage of internode length main effect QTL under Plant Height in Wheat and fringeXmag2055、Xmag4087In 574 sections
The semi-dwarf mutant derived varieties of agriculture 9204（System）Heredity transmission result statistical chart.
Fig. 3 is that wheat breed section agriculture 9204 is the F of parent641 derivative strains for RIL colonies are usedXcnl10Mark draws
Thing enters the electrophoretic band figure of performing PCR amplification.
Specific embodiment
Example below is used to further describe the present invention, but the invention is not limited in any way.
Embodiment 1
With the molecular labeling of internode length main effect QTL compact linkage under Plant Height in Wheat and fringe,
Using labeled primerXcnl10,
Upstream primer sequence：CTTACCTAGTACAATGTACTCTCTCT（Such as SEQ ID NO:Described in 1）、
Downstream primer sequence：ATGATGGTCTGGATCTGG（Such as SEQ ID NO:Described in 2）；
DNA to wheat breed section agriculture 9204 enters performing PCR amplification, and its PCR amplification system is 20 μ l, including：The DNA of 1 μ l
Template, the sense primer of 1 μ l, the anti-sense primer of 1 μ l, 2 × Taq PCR StarMix, the ddH of 7 μ l of 10 μ l2O；Using
Gradient touchdown PCR amplification program, step is as follows：94 DEG C first 4 min of denaturation, next carries out 15 renaturation temperature drops of circulation
Fall program, and each circulation is 94 DEG C of denaturation 45 s, 65 DEG C of s of renaturation 50, and each reduces by 1 DEG C, 72 DEG C on the basis of circulating in 65 DEG C
Extend 55 s；30 regular-PCR programs of circulation are carried out again, i.e.,：94oC is denatured 40 s, 50 DEG C of s of renaturation 40, and 72 DEG C are prolonged
Stretch 40 s；5 min of last 72 DEG C of extensions；Terminate amplification, 15 DEG C of Refrigerator stores；Non denatured polypropylene by amplified production 6%
The V of voltage stabilizing 120 electrophoretic separation on acrylamide gel, argentation, the molecular weight for obtaining corresponding amplified production is 700bp.
The model that PCR programs are used：TaKaRa PCR Thermal Cycler, all of PCR amplifications in the present invention
The model PCR instrument, the PCR instrument is used not to limit the content of the invention.
The screening side with the molecular labeling of internode length main effect QTL compact linkage under Plant Height in Wheat and fringe that the present invention is provided
Method, specifically includes following steps：
（i）With the shorter wheat breed section agriculture 9204 of internode length under semi-short-stalked, fringe for it is maternal, with internode length under bar high, fringe
Wheat breed capital 411 more long obtains Hybrids F1 for male parent hybridize, the F2 that F1 selfings are produced, and is contained using single seed descent
There are 188 F6 of family for RIL colonies；
（ii）With the CTAB methods of improvement, that is, the cetyl trimethylammonium bromide method for improveing（Vander Beek et al.,
1992）The DNA of above-mentioned each strain of RIL colonies is extracted, using diversity microarray technology（DArTs is marked）, simple sequence repeats
Mark（SSR marker）, the simple sequence repeat marker based on EST（EST-SSR is marked）, based on expressed sequence mark
Sign PCR amplification labels（STS is marked）, amplification label between simple repeated sequence（ISSR is marked）And SRAP
Mark（SRAP is marked）Genotyping is carried out to the family, the genotype value material of the RIL colonies is obtained；DArTs is marked
Analysis of scoring is the method that polymorphism between different genes group is distinguished by the technology of chip hybridization, by each strain genome
DNA recycles the relatively low restriction enzyme of cutting frequency to know by two kinds of digestion with restriction enzyme of different cutting frequencies
The joint of other sequence is connected with endonuclease bamhi, is then realized by the primer corresponding with the digestion joint to the special piece
The PCR amplifications of section.Amplicon is used to build clone library, and then passes it through fluorescence labeling, put on chip after denaturation
As probe.The target dna to be detected is also required to be expanded by same inscribe cleavage and PCR, by itself and process after allowing
The target sequence probe hybridization of fluorescence labeling, obtains the genotype value data of the RIL colonies；
The step of CTAB methods of improvement extract leaf DNA includes：The fresh blade for taking 0.2g or so is put into 2.0ml centrifuge tubes
In, 10 s or so in liquid nitrogen are rapidly joined, quickly wear into fine powder；Plus CTAB extract solution 0.6ml, shake up, 65 DEG C of water-bath 30min；
12000rpm is centrifuged 10min, takes supernatant, moves to another pipe；Plus isometric chloroform/isoamyl alcohol, mix 10min；12000rpm
Centrifugation 10min, takes supernatant（Do not inhale albumin layer）, move to another pipe；Plus isometric chloroform/isoamyl alcohol, mix 10min；
1200rpm is centrifuged 10min, takes supernatant（Do not inhale albumin layer）, another pipe 1200rpm centrifugations 10min is moved to, take supernatant（Do not inhale egg
White）, move to another pipe；Plus 1/10V 3M NaAc (Ph=5.2), mix, plus 2 times of absolute ethyl alcohols of volume, gently shake up,
White flock precipitate is produced, and puts the min of -20 DEG C of refrigerators about 20, increases DNA output；8000 rpm are centrifuged 6min, pour out supernatant；
Precipitation is used into 70% ethanol wash, 70% alcohol is poured out, air-dried；To alcohol-free taste, plus 100ul TE dissolvings, -20 DEG C of refrigerators are put long
Phase preserves；
（iii）Using the mapping softwares of MAPMAKER 3.0, the RIL colonies genotype data that will be obtained builds has 591
The wheat molecular marker genetic linkage mapses of marker site, with LOD >=2.5 as standard；287 DArTs are included in 591 marks
Mark, 178 g-SSR mark, 50 e-SSR marks, 17 STS marks, 45 SRAPs marks, 5 ISSRs marks, 7
Feature PCR is marked and 2 morphological markers；
（iv）RIL colonies 2 years field plantings and phenotypic evaluation of totally 8 experimental enviroments are carried out into, have been investigated respectively different
Internode length under the plant height and fringe of each strain under time, different location and Different nitrogen levels treatment conditions, wherein 8 environment
I.e.：2011-2012,2012-2013 continuous 2 years in Luancheng Agro-ecological System experiment station of the Chinese Academy of Sciences（T1 and T2：37°53’
N, 114 ° of 41 ' E）, 2012-2013 years be setting mansion farm in Inst. of Genetics and Development Biology, CAS Beijing（T3：
40 ° of 06 ' N, 116 ° of 24 ' E）And Xinxiang City, Henan Province industry research institute Huixian proving ground（T4：35 ° of 27 ' N, 113 ° of 48 ' E）
Plantation, point nitrogen high per experimental enviroment（High nitrogen, HN）With low nitrogen（Low nitrogen, LN）Two treatment.
Wherein LN treatment, whole year does not apply fertilizer；And HN treatment, apply 300 kg hm-2 phosphoric acid diamines and 225 before annual sowing
Kg hm-2 urea used as base fertilizer, apply 150 kg hm-2 urea and topdress by the jointing stage；
Wherein wheat planting method：Each is 2 rows of plantation, and often row broadcasts 40；Row 3m, the cm of spacing in the rows 7.5, line-spacing 25 long
Cm, normal growth and results；Plant height assay method：Each strain randomly chooses 5, measurement ground end portion to fringe portion after results
The length on top（Do not include awn length）, take each family plant height of its mean value calculation；Internode length under fringe（Peduncle length,
PL, cm）：Each strain randomly chooses 5 after results, and the length of the first internode length of measurement fringe bottom takes its mean value calculation each
Internode length under family fringe；
（v）Using the softwares of MAPMAKER/EXP 3.0（Lander et al., 1987）Build linkage map.By 8 lists
Internode length phenotypic number under direct surveys are obtained in one environment plant height and fringe, according to the BIP block formats of IciMapping v3.3
It is required that after arranging, carrying out additivity QTL positioning.It is interval by stepping of 1cM, 1000 permutation tests are carried out, determine LOD threshold values；
（vi）Can be obtained by qtl analysis, detected respectively on chromosome 4BL under 8 environment the plant height QTL of stabilization expression and
Stablize internode length QTL under the fringe of expression under 5 environment, its confidential interval isXmag4087–wPt-1046, such as Fig. 1 and the institutes of Biao 1
Show；
The qtl analysis result of internode length under the plant height of table 1 and fringe
As shown in Table 1, this 13 QTL peak values existXcnl10Near, wherein Hebei（2011-2012 high and low nitrogen, 2012-
2013 high and low nitrogen）, Beijing（2012-2013 low nitrogen）, the high and low nitrogen in Henan 2012-2013）Plant height QTL under 7 environment altogether
Peak value away fromXcnl10Distance be only 0.20 cM；Beijing（2012-2013 nitrogen high）Plant height QTL peak value and Hebei under environment
（2012-2013 nitrogen high）Under fringe under environment internode length QTL peak values away fromXcnl10Distance be 0.80 cM；Hebei（2011–
2012 nitrogen high）, Hebei（2012-2013 low nitrogen）, Henan（2012-2013 nitrogen high）And Henan（2012-2013 low nitrogen）Under environment
Fringe under internode length QTL peak values away fromXcnl10Distance be respectively 9.20 cM, 6.20 cM, 6.20 cM and 8.20 cM.This 13
The contribution rate of individual QTL is up to 30.91%, and minimum is 9.33%, and all of QTL additive effects value is negative value.Xmag4087–wPt-1046In interval, plant height QTL is detected in 8 environment, and the average contribution rate of QTL is 23.10%；
Internode length QTL under fringe is detected under 5 environment, average contribution rate is 14.80%.IllustrateXmag4087–wPt-1046Area
It is interior that detect is QTL with the chain stabilization of internode length, main effect under Plant Height in Wheat and fringe, its labeled primerXcnl10In wheat
The amplified production molecular weight obtained in the DNA of kind section agriculture 9204 is 700bp, as with internode length QTL under Plant Height in Wheat and fringe
Chain molecular labeling.
Embodiment 2
The present invention provide with the molecular labeling of internode length main effect QTL compact linkage under Plant Height in Wheat and fringe in wheat line
Section's agriculture 9204 is 574 semi-dwarf mutant derived varieties of parent（System）In application.
Using 574 semi-dwarf mutant derived varieties of section's agriculture 9204（System）Internode length main effect QTL is tight under carrying out Plant Height in Wheat and fringe
Close chain molecular labeling application verification research.It is comprised the following steps that：
（1）By 574 semi-dwarf mutant derived varieties of section's agriculture 9204（System）Field planting is carried out, the blade of the plant of each strain is taken
The DNA of separation and Extraction acquisition is carried out using the CTAB methods of the improvement；
The described semi-dwarf mutant derived varieties of 574 section's agricultures 9204（System）It is parent by with section's agriculture 9204 and its pedigree is shown in Table 2
This, is multiplied to F by using conventional hybridization2More than generation, obtained by being choosing then in conjunction with plant height and yield traits general performance
The new variety of wheat or strain for obtaining；
The semi-dwarf mutant derived varieties of 2 574 section's agricultures of table 9204（System）And its genealogical table
（2）Using the labeled primer with internode length main effect QTL compact linkage under Plant Height in Wheat and fringeXcnl10And and wheat
The labeled primer of internode length main effect QTL not close linkage under plant height and fringeXmag2055、Xmag4087The DNA of acquisition is carried out
PCR is expanded, and its PCR amplification system is：The DNA profiling of 1 μ l, the forward primer of 1 μ l, the reverse primer of 1 μ l, the 2 of 10 μ l
×TaqPCR StarMix, the ddH of 7 μ l2O；Amplification condition is gradient touchdown PCR：94 DEG C of 4 min of denaturation, then 15 are followed
The renaturation drop of temperature program of ring, 94 DEG C of each circulation is denatured 45 s, and 65 DEG C of s of renaturation 50 (- 1 DEG C/circulation) and 72 DEG C prolong
Stretch 55 s；It is last 30 circulation regular-PCR programs be：94 DEG C of denaturation 40 s, 50 DEG C of renaturation 40 s, 72 DEG C of 40 s of extension；
5 min of last 72 DEG C of extensions；15 DEG C preserve after amplification terminates；Amplified production is steady on 6% non-denaturing polyacrylamide gel
120 V are pressed to be separated by electrophoresis, argentation detects its amplified fragments size；
（3）With the labeled primer of the not close linkage of internode length main effect QTL under Plant Height in Wheat and fringeXmag2055、Xmag4087
Sequence be shown in Table 3, its DNA fragmentation for amplifying 195bp and 530bp respectively in section's agriculture 9204；
The labeled primer of internode length main effect QTL not close linkage under table 3 and Plant Height in Wheat and fringeXmag2055、Xmag4087DNA sequence dna and extension fragments molecules amount
（4）Testing result is analyzed,Xcnl10Amplified production molecular weight for 700bp molecular labeling occur, i.e.,
The amplified production kind consistent with the amplified production of section agriculture 9204 or strain are the derivative with internode length under semi-dwarf mutant, shorter fringe
Strain；Xmag2055、Xmag4087Amplified production molecular weight be respectively 195bp and 530bp molecular labeling occur, show section
Agriculture 9204 derives the descendant inheritting inhereditary material of section's agriculture 9204.
Concrete outcome is as follows：Using the labeled primer with internode length main effect QTL compact linkage under Plant Height in Wheat and fringeXcnl10And the labeled primer with the not close linkage of internode length main effect QTL under Plant Height in Wheat and fringeXmag2055、Xmag4087It is right
574 semi-dwarf mutant derived varieties of section's agriculture 9204（System）DNA enter performing PCR amplification, the corresponding amplified fragments of section's agriculture 9204 spread out at it
Transmission result in raw offspring is as shown in Figure 2.
Figure it is seen that the molecular labeling with internode length main effect QTL compact linkage under Plant Height in Wheat and fringeXcnl10
574 semi-dwarf mutant derived varieties of section's agriculture 9204（System）Amplification banding pattern it is identical with section agriculture 9204, corresponding plant height half is short
Stalk proterties is corresponded to completely；With the labeled primer of the not close linkage of internode length main effect QTL under Plant Height in Wheat and fringeXmag2055、Xmag4087ShowingXcnl10In 574 semi-dwarf mutant derived varieties of section's agriculture 9204（System）Amplification banding pattern have respectively 52.8% and
55.9% is identical with section agriculture 9204, and corresponding plant height semi-dwarf mutant proterties is not exclusively corresponded to；The above results prove molecular labelingXcnl10It is the molecular labeling with internode length close linkage under Plant Height in Wheat and fringe, the molecular labeling can very effectively be used for small
Among the wheat plant type molecular marker assisted selection procedure of breeding.
Embodiment 3
The molecular labeling with the major gene loci close linkage of internode length under Plant Height in Wheat and fringe that the present invention is providedXcnl10It is the F of parent in wheat breed section agriculture 92046For in 41 derivative strains of RIL colonies and section's agriculture 9204 and capital 411
Application.
Male parent is made for maternal, capital 411 with section's agriculture 9204 and prepares combination, by single seed descent（SSD methods）Construct F7 restructuring
Inbred line population（RIL colonies）.Use labeled primerXcnl10The derivative strain of capital 411, section's agriculture 9204 and 41 is analyzed.
It is comprised the following steps that：
（1）Wheat breed section agriculture 9204, capital 411 and 41 derivative strains are carried out into field planting, the plant of each strain is taken
Blade carries out separation and Extraction DNA using the SDS methods；The derivative strain of wherein section's agriculture 9204 refers to：Female parent is made with section's agriculture 9204,
Male parent is made in capital 411, the strain obtained by single seed descent；
（2）UsingXcnl10Sense primer（Such as SEQ ID NO:Described in 1）And anti-sense primer（Such as SEQ ID NO:1 institute
State）The DNA of acquisition is entered into performing PCR amplification, the DNA to wheat breed section agriculture 9204 enters performing PCR amplification, and its PCR amplification system is
20 μ l, including：The DNA profiling of 1 μ l, the sense primer of 1 μ l, the anti-sense primer of 1 μ l, 2 × Taq PCR of 10 μ l
StarMix, the ddH of 7 μ l2O；Expanded using gradient touchdown PCR amplification program：94 DEG C first 4 min of denaturation, next carries out 15
The renaturation drop of temperature program of individual circulation, each circulation is 94 DEG C of denaturation 45 s, 65 DEG C of s of renaturation 50, and each circulates in 65 DEG C
On the basis of reduce by 1 DEG C, 72 DEG C extension 55 s；30 regular-PCR programs of circulation are carried out again, i.e.,：94oC is denatured 40 s, 50
DEG C renaturation 40 s, 72 DEG C of 40 s of extension；5 min of last 72 DEG C of extensions；Terminate amplification, 15 DEG C of Refrigerator stores；By amplified production
The V of voltage stabilizing 120 electrophoretic separation on 6% non-denaturing polyacrylamide gel, argentation detection；
（3）Testing result is analyzed, such asXcnl10There is molecular size range in the amplified production that labeled primer is obtained
It is that the band of 700 bp, i.e. amplified production strain consistent with the amplified production of section agriculture 9204 are with reducing under plant height and fringe
The strain of internode length gene loci.
Result is as shown in figure 3, swimming lane 1-44 is respectively capital 411 in Fig. 3（J）, section's agriculture 9204（KN）、DNA Marker（M）
And 41 derivative strain 1-41 of section's agriculture 9204.Wherein, banding pattern and the banding pattern identical of section's agriculture 9204 are the carrying reduction plant height
With the derivative strain of internode length gene loci under fringe, that is, derive strain 1,6,10,11,12,13,14,18,22,27,30,32,
34th, 37,38 and 41, it is the strain that can be used for the excellent strain type high-yield breeding of the short bar of next step that these derive strain；And capital 411 and spread out
Health product is 2,3,4,5,7,8,9,15,16,17,19,20,21,23,24,25,26,28,29,31,33,35,36,39 and 40 expansions
The electrophoretic separation banding pattern for increasing production thing is different with section 9204 banding patterns of agriculture, i.e., these strains do not have described reduces internode under plant height and fringe
Gene loci.
Thus prove：UtilizeXcnl10Carry out molecular marker assisted selection, can Effective selection have and reduce plant height, save under fringe
Between major gene loci long kind or strain, the mark has been greatly improved High-Yield Wheat Cultivar for wheat assistant breeding
Or the efficiency of selection and quality of strain.
Claims (8)
1. with the molecular labeling of internode length main effect QTL compact linkage under Plant Height in Wheat and fringe, it is characterised in that the molecular labeling isXcnl10, its molecular labelingXcnl10Upstream primer sequence such as SEQ ID NO:Described in 1, downstream primer sequence such as SEQ ID
NO:Described in 2.
2. with the acquisition methods of the molecular labeling of internode length main effect QTL compact linkage under Plant Height in Wheat and fringe, it is characterised in that bag
Include following steps：
Enter performing PCR to the DNA of wheat breed section agriculture 9204 with Xcnl10 labeled primers to expand, its PCR amplification system is 20 μ l, bag
Include：The DNA profiling of 1 μ l, the sense primer of 1 μ l, the anti-sense primer of 1 μ l, 2 × Taq PCR StarMix, 7 μ l of 10 μ l
DdH2O；Expanded using gradient touchdown PCR amplification program, step is as follows：
（1）94 DEG C of 4 min of denaturation,
（2）15 renaturation drop of temperature programs of circulation, each circulation is 94 DEG C of denaturation 45 s, 65 DEG C of s of renaturation 50, and each is followed
Ring reduces by 1 DEG C, 72 DEG C of 55 s of extension on the basis of 65 DEG C；
（3）30 regular-PCR programs of circulation：94 DEG C of denaturation 40 s, 50 DEG C of renaturation 40 s, 72 DEG C of 40 s of extension；
（4）72 DEG C of 5 min of extension；Terminate amplification, 15 DEG C of preservations；
The amplified production V of voltage stabilizing 120 electrophoretic separation on 6% non-denaturing polyacrylamide gel, obtains corresponding amplified production
Molecular weight be 700bp, as with the molecular labeling of internode length main effect QTL compact linkage under Plant Height in Wheat and fringe；Wherein
The upstream primer sequence such as SEQ ID NO of Xcnl10 marks:Described in 1, downstream primer sequence such as SEQ ID NO:Described in 2.
3. it is according to claim 2 to be obtained with the molecular labeling of internode length main effect QTL compact linkage under Plant Height in Wheat and fringe
Take method, it is characterised in that after the DNA of wheat breed section agriculture 9204 refers to the plant leaf separation and Extraction of wheat section agriculture 9204
The DNA of acquisition.
4. it is according to claim 2 to be obtained with the molecular labeling of internode length main effect QTL compact linkage under Plant Height in Wheat and fringe
Take method, it is characterised in that described 6% non-denaturing polyacrylamide gel refers to contain in 100ml polyacrylamide gel solution
There are 5.85 g acrylamides and 0.15 g methene acrylamides.
5. it is a kind of using the Markers for Detection wheat breed or the method for strain that such as claim 2 is obtained, it is characterised in that bag
Include following steps：
A. enter performing PCR to the DNA of wheat breed to be measured or strain with the labeled primer of Xcnl10 to expand, its PCR amplification system
It is 20 μ l, comprising：The DNA profiling of 1 μ l, the sense primer of 1 μ l, the anti-sense primer of 1 μ l, 2 × Taq PCR of 10 μ l
StarMix, and 7 μ l ddH2O；Expanded using gradient touchdown PCR amplification program：94 DEG C of 4 min of denaturation；15 circulation answer
Degree landing procedure warm in nature, each circulation is 94 DEG C of denaturation 45 s, 65 DEG C of s of renaturation 50, and each is reduced on the basis of circulating in 65 DEG C
1 DEG C, 72 DEG C of 55 s of extension；30 regular-PCR programs of circulation：94oC is denatured 40 s, 50 DEG C of s of renaturation 40,72 DEG C of extensions
40 s；72 DEG C of 5 min of extension；Terminate amplification, 15 DEG C of preservations；The wherein upstream primer sequence of the labeled primer of Xcnl10 such as SEQ
ID NO:Described in 1, downstream primer sequence such as SEQ ID NO:Described in 2；
B. by above-mentioned amplified production after the V of voltage stabilizing 120 electrophoretic separation on 6% non-denaturing polyacrylamide gel, if obtaining
The molecular weight for obtaining amplified production is the amplified fragments of 700bp, then the wheat breed or strain are have drop on the gene loci
The wheat breed or strain of internode length gene under low plant height and fringe；Otherwise, the wheat breed or strain belong in the site for not
Wheat breed or strain with internode length gene under reduction plant height and fringe.
6. it is according to claim 5 detection wheat breed or strain method, it is characterised in that the wheat breed or product
System is, as parent, to be spread out by using conventional hybridization and multiplying section agriculture 9204 wheat of the seed selection to F2 more than generation with the agriculture 9204 of wheat section
Health product kind or strain.
7. detection wheat breed according to claim 5 or 6 or the method for strain, it is characterised in that the wheat breed
Or the DNA of strain refers to the DNA that will be obtained after wheat plant blade separation and Extraction.
8. the method for detection wheat breed according to claim 7 or strain, it is characterised in that described 6% non denatured is gathered
Acrylamide gel refers to containing 5.85 g acrylamides and 0.15 g methene acryloyls in 100ml polyacrylamide gel solution
Amine.
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| CN109913573B (en) * | 2019-04-08 | 2022-06-14 | 鲁东大学 | Closely linked molecular marker of wheat ear grain number major QTL and application thereof |
| CN110982933B (en) * | 2020-03-02 | 2020-06-30 | 鲁东大学 | Molecular marker closely linked with major QTL (quantitative trait locus) of wheat grain length and application thereof |
| CN110982932B (en) * | 2020-03-02 | 2020-06-30 | 鲁东大学 | Molecular marker closely linked with major QTL of wheat plant height and application thereof |
| CN111269966A (en) * | 2020-03-18 | 2020-06-12 | 中国科学院遗传与发育生物学研究所农业资源研究中心 | Method for obtaining specific molecular marker of aegilops tauschii and application thereof |
| CN113151576B (en) * | 2021-06-07 | 2022-10-21 | 鲁东大学 | Molecular marker closely linked with major QTL of wheat plant height as well as acquisition method and application thereof |
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