CN105821038A - Molecular marker for identifying wheat height and specific primers - Google Patents
Molecular marker for identifying wheat height and specific primers Download PDFInfo
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Abstract
The invention discloses a molecular marker for identifying the wheat height and specific primers .A primer set is composed of a specific primer pair A and a specific primer pair B; the specific primer A is composed of two primers, the primer F1 is shown in a sequence 1, and the primer R1 is shown in a sequence 2; the specific primer pair B is composed of two primers, and the prime F2 is shown in a sequence 3, and the primer R2 is shown in a sequence 4 .The molecular marker or the primer set is applied to molecular marker assistant selection, wheat varieties (materials) with a high wheat height phenotype or a low wheat height phenotype can be screened out rapidly, the breeding pace of new wheat varieties is accelerated, and the important theoretical significance and economic value are achieved.
Description
Technical field
The present invention relates to a kind of molecular marker for identifying Plant Height in Wheat and special primer.
Background technology
Semen Tritici aestivi is one of most important cereal crops, and its high yield and stable yields are the important leverages of world food safety.Along with population growth, cultivated land area and global climate deteriorate, improving yield of wheat and stable yields seem even more important.
Plant height is the key factor affecting wheat yield.Plant is too high the most easily causes lodging, but also can compete photosynthate with the growth of seed." Green revolution " that the application of Dwarf germplasm resource has caused, is greatly facilitated wheat increase yield and stable yields.Therefore, the research of plant height control gene is significant.
Li Xingmao etc. utilize the recombinant inbred lines (Recombinantinbredline of short anti-58 and Jing Dong No. 8, RIL) under 8 environment, two stable main effect QTLs expressed on chromosome 4D and 6A detected, wherein QPH.caas-6A is a novel site controlling plant height, soluble phenotypic variation 11.0%, therefore has important researching value.Develop and assisted selection offer good tool is not provided with the closely linked molecular marker of QPH.caas-6A, and the plant height control gene for this site of map based cloning creates conditions.
Summary of the invention
It is an object of the invention to provide a kind of molecular marker for identifying Plant Height in Wheat and special primer.
First the present invention protects a primer sets, is made up of first and special primer to second special primer；
First is made up of by described special primer two primers, and its target sequence is specific DNA molecular first, and described specific DNA molecular first includes primers F 1 and the target sequence of primer R1 in the genome of Semen Tritici aestivi；
Primers F 1 is following (a1) or (a2):
(a1) single strand dna shown in sequence 1 of sequence table；
(a2) sequence 1 through the replacement of one or several nucleotide and/or disappearance and/or interpolation and had the DNA molecular of identical function with sequence 1；
Primer R1 is following (a3) or (a4):
(a3) single strand dna shown in sequence 2 of sequence table；
(a4) sequence 2 through the replacement of one or several nucleotide and/or disappearance and/or interpolation and had the DNA molecular of identical function with sequence 2；
Second is made up of by described special primer two primers, and its target sequence is specific DNA molecular second, and described specific DNA molecular second includes primers F 2 and the target sequence of primer R2 in the genome of Semen Tritici aestivi；
Primers F 2 is following (a1) or (a2):
(a1) single strand dna shown in sequence 3 of sequence table；
(a2) sequence 3 through the replacement of one or several nucleotide and/or disappearance and/or interpolation and had the DNA molecular of identical function with sequence 3；
Primer R2 is following (a3) or (a4):
(a3) single strand dna shown in sequence 4 of sequence table；
(a4) sequence 4 through the replacement of one or several nucleotide and/or disappearance and/or interpolation and had the DNA molecular of identical function with sequence 4.
First specifically can be made up of by described special primer primers F 1 and primer R1.
Second specifically can be made up of by described special primer primers F 2 and primer R2.
Described specific DNA molecular first the most following molecular marker AP2。
Described specific DNA molecular second the most following molecular marker FAR.
The purposes of described primer sets is following (1) or (2) or (3) or (4): (1) auxiliary identifies the plant height of Semen Tritici aestivi to be measured；(2) assisting sifting has the wheat breed of low plant height；(3) assisting sifting has the wheat breed of high plant height；(4) plant height of more different wheat breeds to be measured is assisted.
The present invention also protects a kind of test kit, including primer sets, restricted enzyme MseI and restricted enzyme NlaIII described in any of the above.Described test kit may also include other reagent being conventionally used for PCR amplification and/or other are conventionally used for reagent that genome extracts and/or other are conventionally used for the reagent of polyacrylamide gel electrophoresis.
The present invention also protects special primer described in any of the above to first.Described special primer is following (1) or (2) or (3) or (4) to the purposes of first: (1) auxiliary identifies the plant height of Semen Tritici aestivi to be measured；(2) assisting sifting has the wheat breed of low plant height；(3) assisting sifting has the wheat breed of high plant height；(4) plant height of more different wheat breeds to be measured is assisted.
The present invention also protects compositions first, is made up of first and restricted enzyme MseI special primer described in any of the above.The purposes of described compositions first is following (1) or (2) or (3) or (4): (1) auxiliary identifies the plant height of Semen Tritici aestivi to be measured；(2) assisting sifting has the wheat breed of low plant height；(3) assisting sifting has the wheat breed of high plant height；(4) plant height of more different wheat breeds to be measured is assisted.
The present invention also protects special primer described in any of the above to second.Described special primer is following (1) or (2) or (3) or (4) to the purposes of second: (1) auxiliary identifies the plant height of Semen Tritici aestivi to be measured；(2) assisting sifting has the wheat breed of low plant height；(3) assisting sifting has the wheat breed of high plant height；(4) plant height of more different wheat breeds to be measured is assisted.
The present invention also protects compositions second, is made up of second and restricted enzyme NlaIII special primer described in any of the above.The purposes of described primer sets second is following (1) or (2) or (3) or (4): (1) auxiliary identifies the plant height of Semen Tritici aestivi to be measured；(2) assisting sifting has the wheat breed of low plant height；(3) assisting sifting has the wheat breed of high plant height；(4) plant height of more different wheat breeds to be measured is assisted.
The present invention also protects the application of primer sets described in any of the above or test kit, for following (1) or (2) or (3) or (4): (1) auxiliary identifies the plant height of Semen Tritici aestivi to be measured；(2) assisting sifting has the wheat breed of low plant height；(3) assisting sifting has the wheat breed of high plant height；(4) plant height of more different wheat breeds to be measured is assisted.
In described application, identify that the method for the plant height of Semen Tritici aestivi to be measured is as follows:
1. with the genomic DNA of Semen Tritici aestivi to be measured as template, use the primer of primers F 1 and primer R1 composition to carrying out PCR amplification, then restricted enzyme MseI is used to carry out enzyme action, then polyacrylamide gel electrophoresis is carried out, if display does not have the band of 104bp, Semen Tritici aestivi to be measured for based on molecular marker AP2AP2-a genotype, if display has the band of 104bp, Semen Tritici aestivi to be measured for based on molecular marker AP2AP2-b gene type；
2. with the genomic DNA of Semen Tritici aestivi to be measured as template, use the primer of primers F 2 and primer R2 composition to carrying out PCR amplification, then restricted enzyme NlaIII is used to carry out enzyme action, then polyacrylamide gel electrophoresis is carried out, if display has the band of 278bp, Semen Tritici aestivi to be measured is FAR-a genotype based on molecular marker FAR, if display does not have the band of 278bp, Semen Tritici aestivi to be measured is FAR-b genotype based on molecular marker FAR；
If Semen Tritici aestivi to be measured is based on molecular marker AP2AP2-a genotype and FAR-a genotype based on molecular marker FAR, Semen Tritici aestivi to be measured is the low plant height Semen Tritici aestivi of candidate；If Semen Tritici aestivi to be measured is based on molecular marker AP2AP2-b gene type and FAR-b genotype based on molecular marker FAR, Semen Tritici aestivi to be measured is the high plant height Semen Tritici aestivi of candidate.
Step 1. with step 2. without sequencing.
3. described step the most also can be replaced following steps: with the genomic DNA of Semen Tritici aestivi to be measured as template, use the primer of primers F 2 and primer R2 composition to carrying out PCR amplification, then restricted enzyme NlaIII is used to carry out enzyme action, then polyacrylamide gel electrophoresis is carried out, if display does not have the band of 250bp, Semen Tritici aestivi to be measured is FAR-a genotype based on molecular marker FAR, if display has the band of 250bp, Semen Tritici aestivi to be measured is FAR-b genotype based on molecular marker FAR.
4. described step the most also can be replaced following steps: with the genomic DNA of Semen Tritici aestivi to be measured as template, use the primer of primers F 2 and primer R2 composition to carrying out PCR amplification, then restricted enzyme NlaIII is used to carry out enzyme action, then polyacrylamide gel electrophoresis is carried out, if display have the band of 278bp and not there is the band of 250bp, Semen Tritici aestivi to be measured is FAR-a genotype based on molecular marker FAR, if display have the band of 250bp and not there is the band of 278bp, Semen Tritici aestivi to be measured is FAR-b genotype based on molecular marker FAR.
In described application, the method for the wheat breed that assisting sifting has low plant height is as follows: the plant height of Semen Tritici aestivi to be measured, screening have the wheat breed of low plant height to use said method to identify.
In described application, the method for the wheat breed that assisting sifting has high plant height is as follows: the plant height of Semen Tritici aestivi to be measured, screening have the wheat breed of high plant height to use said method to identify.
In described application, the relatively method of the plant height of different wheat breeds to be measured is as follows:
1. with the genomic DNA of Semen Tritici aestivi to be measured as template, use the primer of primers F 1 and primer R1 composition to carrying out PCR amplification, then restricted enzyme MseI is used to carry out enzyme action, then polyacrylamide gel electrophoresis is carried out, if display does not have the band of 104bp, Semen Tritici aestivi to be measured for based on molecular marker AP2AP2-a genotype, if display has the band of 104bp, Semen Tritici aestivi to be measured for based on molecular marker AP2AP2-b gene type；
2. with the genomic DNA of Semen Tritici aestivi to be measured as template, use the primer of primers F 2 and primer R2 composition to carrying out PCR amplification, then restricted enzyme NlaIII is used to carry out enzyme action, then polyacrylamide gel electrophoresis is carried out, if display has the band of 278bp, Semen Tritici aestivi to be measured is FAR-a genotype based on molecular marker FAR, if display does not have the band of 278bp, Semen Tritici aestivi to be measured is FAR-b genotype based on molecular marker FAR；
Based on molecular marker AP2For AP2-a genotype and plant height based on the Semen Tritici aestivi to be measured that molecular marker FAR is FAR-a genotype are less than based on molecular marker AP2For AP2-b gene type and based on the Semen Tritici aestivi to be measured that molecular marker FAR is FAR-b genotype.
Step 1. with step 2. without sequencing.
3. described step the most also can be replaced following steps: with the genomic DNA of Semen Tritici aestivi to be measured as template, use the primer of primers F 2 and primer R2 composition to carrying out PCR amplification, then restricted enzyme NlaIII is used to carry out enzyme action, then polyacrylamide gel electrophoresis is carried out, if display does not have the band of 250bp, Semen Tritici aestivi to be measured is FAR-a genotype based on molecular marker FAR, if display has the band of 250bp, Semen Tritici aestivi to be measured is FAR-b genotype based on molecular marker FAR.
4. described step the most also can be replaced following steps: with the genomic DNA of Semen Tritici aestivi to be measured as template, use the primer of primers F 2 and primer R2 composition to carrying out PCR amplification, then restricted enzyme NlaIII is used to carry out enzyme action, then polyacrylamide gel electrophoresis is carried out, if display have the band of 278bp and not there is the band of 250bp, Semen Tritici aestivi to be measured is FAR-a genotype based on molecular marker FAR, if display have the band of 250bp and not there is the band of 278bp, Semen Tritici aestivi to be measured is FAR-b genotype based on molecular marker FAR.
The present invention also protects special primer described in any of the above to first or compositions first or special primer to second or the application of compositions second, for following (1) or (2) or (3) or (4): (1) auxiliary identifies the plant height of Semen Tritici aestivi to be measured；(2) assisting sifting has the wheat breed of low plant height；(3) assisting sifting has the wheat breed of high plant height；(4) plant height of more different wheat breeds to be measured is assisted.
The present invention also protects molecular marker AP2, a kind of polymorphic form is (b1) or (b2), and another kind of polymorphic form is (b3) or (b4)；
(b1) DNA molecular shown in sequence 5 of sequence table；
(b2) sequence 5 of sequence table is from the DNA molecular shown in 5 ' end 47-305 position nucleotide；
(b3) DNA molecular shown in sequence 6 of sequence table；
(b4) sequence 6 of sequence table is from the DNA molecular shown in 5 ' end 47-305 position nucleotide.
Or (b2) is low plant height labelling (b1).Or (b4) is high plant height labelling (b3).
The present invention also protects molecular marker FAR, and a kind of polymorphic form is (c1) or (c2) or (c3), and another kind of polymorphic form is (c4) or (c5) or (c6)；
(c1) DNA molecular shown in sequence 7 of sequence table；
(c2) sequence 7 of sequence table is from the DNA molecular shown in 5 ' end 1-548 position nucleotide；
(c3) sequence 7 of sequence table is from the DNA molecular shown in 5 ' end 271-548 position nucleotide；
(c4) DNA molecular shown in sequence 8 of sequence table；
(c5) sequence 8 of sequence table is from the DNA molecular shown in 5 ' end 1-548 position nucleotide；
(c6) sequence 8 of sequence table is from the DNA molecular shown in 5 ' end 271-548 position nucleotide.
Or (c2) or (c3) is low plant height labelling (c1).Or (c5) or (c6) is high plant height labelling (c4).
The present invention also protects described molecular marker AP2And/or the application of described molecular marker FAR, for following (1) or (2) or (3) or (4): (1) auxiliary identifies the plant height of Semen Tritici aestivi to be measured；(2) assisting sifting has the wheat breed of low plant height；(3) assisting sifting has the wheat breed of high plant height；(4) plant height of more different wheat breeds to be measured is assisted.
In described application, identify that the method for the plant height of Semen Tritici aestivi to be measured is as follows:
If Semen Tritici aestivi the most to be measured carries (b1) or (b2), Semen Tritici aestivi to be measured for based on molecular marker AP2AP2-a genotype, if Semen Tritici aestivi to be measured carries (b3) or (b4), Semen Tritici aestivi to be measured for based on molecular marker AP2AP2-b gene type；
If Semen Tritici aestivi the most to be measured carries (c1) or (c2) or (c3), Semen Tritici aestivi to be measured is FAR-a genotype based on molecular marker FAR, if Semen Tritici aestivi to be measured carries (c4) or (c5) or (c6), Semen Tritici aestivi to be measured is FAR-b genotype based on molecular marker FAR；
If Semen Tritici aestivi to be measured is based on molecular marker AP2AP2-a genotype and FAR-a genotype based on molecular marker FAR, Semen Tritici aestivi to be measured is the low plant height Semen Tritici aestivi of candidate；If Semen Tritici aestivi to be measured is based on molecular marker AP2AP2-b gene type and FAR-b genotype based on molecular marker FAR, Semen Tritici aestivi to be measured is the high plant height Semen Tritici aestivi of candidate.
Step 1. with step 2. without sequencing.
In described application, the method for the wheat breed that assisting sifting has low plant height is as follows: the plant height of Semen Tritici aestivi to be measured, screening have the wheat breed of low plant height to use said method to identify.
In described application, the method for the wheat breed that assisting sifting has high plant height is as follows: the plant height of Semen Tritici aestivi to be measured, screening have the wheat breed of high plant height to use said method to identify.
In described application, the relatively method of the plant height of different wheat breeds to be measured is as follows:
If Semen Tritici aestivi the most to be measured carries (b1) or (b2), Semen Tritici aestivi to be measured for based on molecular marker AP2AP2-a genotype, if Semen Tritici aestivi to be measured carries (b3) or (b4), Semen Tritici aestivi to be measured for based on molecular marker AP2AP2-b gene type；
If Semen Tritici aestivi the most to be measured carries (c1) or (c2) or (c3), Semen Tritici aestivi to be measured is FAR-a genotype based on molecular marker FAR, if Semen Tritici aestivi to be measured carries (c4) or (c5) or (c6), Semen Tritici aestivi to be measured is FAR-b genotype based on molecular marker FAR；
Based on molecular marker AP2For AP2-a genotype and plant height based on the Semen Tritici aestivi to be measured that molecular marker FAR is FAR-a genotype are less than based on molecular marker AP2For AP2-b gene type and based on the Semen Tritici aestivi to be measured that molecular marker FAR is FAR-b genotype.
Step 1. with step 2. without sequencing.
Semen Tritici aestivi to be measured concretely wheat cultivation kind described in any of the above.
Low plant height described in any of the above refers to plant height less than 82cm.
High plant height described in any of the above refers to plant height more than 82cm.
Molecular marker in the present invention or primer sets are applied to molecular marker assisted selection, allow rapid screening out the wheat breed (material) with high strain high or low plant height phenotype, thus accelerate the incubation paces of new variety of wheat, there is important theory significance and economic worth.
Accompanying drawing explanation
Fig. 1 Plant Height in Wheat main effect site QPH.caas-6A mapping on 6A chromosome is interval.
Fig. 2 uses the method for embodiment 2 to detect based on molecular marker AP2Genotype, the result of part Experiment material.
Fig. 3 uses the method for embodiment 2 to detect genotype based on molecular marker FAR, the result of part Experiment material.
Detailed description of the invention
Below example facilitates a better understanding of the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is conventional method.Test material used in following embodiment, if no special instructions, is and is commercially available from routine biochemistry reagent shop.Quantitative test in following example, is respectively provided with three times and repeats experiment, results averaged.
The discovery of embodiment 1, molecular marker and primer pair
Carry out a large amount of sequence analysis, comparison and preliminary experiment, it was found that two molecular marker (molecular marker AP of Plant Height in Wheat main effect site QPH.caas-6A2With molecular marker FAR), and for identifying the primer pair of molecular marker.
Molecular marker AP2Two kinds of polymorphic form respectively as shown in the sequence 5 of sequence table and the sequence 6 of sequence table.Two kinds of polymorphic form of molecular marker FAR are respectively as shown in the sequence 7 of sequence table and the sequence 8 of sequence table.
Plant Height in Wheat main effect site QPH.caas-6A is shown in Fig. 1 in the mapping interval on 6A chromosome.Right side is the title of molecular marker, and left side numeral is the genetic distance between labelling, and unit is cM.The position of black triangles display QPH.caas-6A.Molecular marker AP2With molecular marker FAR, target QTL is positioned in 1.8cM interval.
For identifying molecular marker AP2Primer to as follows:
F1 (sequence 1 of sequence table): 5 '-GGTTAGAACATTATATTATTAATTACTGGTTGATTTCTTATATTG-3 '；
R1 (sequence 2 of sequence table): 5 '-CTCGTATGAACAAGTAATTCAATTCAATTATAAATAAGTC-3 '.
The target sequence of F1 and R1 is molecular marker AP2。
Molecular marker AP2Target sequence such as
For identifying that the primer of molecular marker FAR is to as follows:
F2 (sequence 3 of sequence table): 5 '-CCCATACTACTCTGGAACTTGCCCATCTC-3 '；
R2 (sequence 4 of sequence table): 5 '-ATATTTACCAACTCCTGGGATGTCCCAACA-3’。
The target sequence of F2 and R2 is the 1st to 548 nucleotide of molecular marker FAR.
Embodiment 2, the foundation of method
1, extract the genomic DNA of Semen Tritici aestivi to be measured, obtain the template solution that DNA concentration is 100ng/ μ l.
2, with step 1 extract genomic DNA as template, use F1 and R1 composition primer to carrying out PCR amplification, obtain pcr amplification product.
3, take the pcr amplification product that step 2 obtains, use restricted enzyme MseI to carry out enzyme action, then carry out polyacrylamide gel electrophoresis, if display has the band of 259bp, Semen Tritici aestivi to be measured for based on molecular marker AP2AP2-a genotype (dwarf gene type), if display has the band of 104bp and 155bp, Semen Tritici aestivi to be measured for based on molecular marker AP2AP2-b gene type (long-culm gene type).Above criterion is based on enzyme action situation completely, but in practical operation, the incomplete phenomenon of enzyme action, AP often occurs2The Semen Tritici aestivi to be measured of-b gene type is likely to show the band with 259bp, therefore it is not recommended that carry out genotype judgement by the band of 259bp.In practical operation, owing to single double-stranded DNA mobility speed is different, there is interference stripes near 155bp band, therefore it is not recommended that carry out genotype judgement by the band of 155bp.
Therefore, actual application is advised being judged by following standard: take the pcr amplification product that step 2 obtains, use restricted enzyme MseI to carry out enzyme action, then carry out polyacrylamide gel electrophoresis, if display does not have the band of 104bp, Semen Tritici aestivi to be measured for based on molecular marker AP2AP2-a genotype (dwarf gene type), if display has the band of 104bp, Semen Tritici aestivi to be measured for based on molecular marker AP2AP2-b gene type (long-culm gene type).
The reaction system of PCR amplification: 2 × TaqPCRMix7.5 μ l, F1 (10mM) 1 μ l, R1 (10mM) 1 μ l, template solution 1.5 μ l, aseptic H2O4μl。
The response procedures of PCR amplification: 94 DEG C of denaturations 3min；94 DEG C of degeneration 15s, 56 DEG C of annealing 20s, 72 DEG C of extension 30s, 35 circulations；Last 72 DEG C extend 5min.
Enzyme action system (20 μ l): restricted enzyme 0.5 μ l, pcr amplification product 10 μ l, 10 × NEBbuffer2 μ l, ddH2O7.5μl。
Enzyme action condition: 37 DEG C, 3h.
4, the pcr amplification product that step 2 obtains is taken, restricted enzyme NlaIII is used to carry out enzyme action, then polyacrylamide gel electrophoresis is carried out, if display has the band of 278bp, Semen Tritici aestivi to be measured is FAR-a genotype (dwarf gene type) based on molecular marker FAR, if display has the band of 28bp and 250bp, Semen Tritici aestivi to be measured is FAR-b genotype (long-culm gene type) based on molecular marker FAR.In practical operation, owing to 28bp band is the least, it is not easy to the observation after electrophoresis, therefore it is not recommended that carry out genotype judgement by the band of 28bp.
Therefore, actual application is advised being judged by following standard: take the pcr amplification product that step 2 obtains, restricted enzyme NlaIII is used to carry out enzyme action, then polyacrylamide gel electrophoresis is carried out, if display has the band of 278bp, Semen Tritici aestivi to be measured is FAR-a genotype (dwarf gene type) based on molecular marker FAR, if display does not have the band of 278bp, Semen Tritici aestivi to be measured is FAR-b genotype (long-culm gene type) based on molecular marker FAR.
Therefore, actual application is advised being judged by following standard: take the pcr amplification product that step 2 obtains, restricted enzyme NlaIII is used to carry out enzyme action, then polyacrylamide gel electrophoresis is carried out, if display does not have the band of 250bp, Semen Tritici aestivi to be measured is FAR-a genotype (dwarf gene type) based on molecular marker FAR, if display has the band of 250bp, Semen Tritici aestivi to be measured is FAR-b genotype (long-culm gene type) based on molecular marker FAR.
Therefore, actual application is advised being judged by following standard: take the pcr amplification product that step 2 obtains, restricted enzyme NlaIII is used to carry out enzyme action, then polyacrylamide gel electrophoresis is carried out, if display have the band of 278bp and not there is the band of 250bp, Semen Tritici aestivi to be measured is FAR-a genotype (dwarf gene type) based on molecular marker FAR, if display have the band of 250bp and not there is the band of 278bp, Semen Tritici aestivi to be measured is FAR-b genotype (long-culm gene type) based on molecular marker FAR.
The reaction system of PCR amplification: 2 × TaqPCRMix7.5 μ l, F2 (10mM) 1 μ l, R2 (10mM) 1 μ l, template solution 1.5 μ l, aseptic H2O4μl。
The response procedures of PCR amplification: 94 DEG C of denaturations 3min；94 DEG C of degeneration 15s, 63 DEG C of annealing 20s, 72 DEG C extend 30s, 35 circulations；Last 72 DEG C extend 5min.
Enzyme action system (20 μ l): restricted enzyme 0.5 μ l, pcr amplification product 10 μ l, 10 × NEBbuffer2 μ l, ddH2O7.5μl。
Enzyme action condition: 37 DEG C, 3h.
Embodiment 3, the application of primer pair
Experiment material is 166 wheat breeds (are China Yellow River-Huai River region wheat and promote mainly kind), is specifically shown in Table 1.
1, each experiment material being planted respectively and be placed in Anyang and Liang Ge place, Suixi, each place arranges three and repeats to process (each practice ground repeating to process is 350 square meters), Routine Management, obtains the plant height meansigma methods of each test material.
2, the method using embodiment 2 to provide detects each experiment material based on molecular marker AP2Genotype and genotype based on molecular marker FAR.
The method using embodiment 2 detects based on molecular marker AP2Genotype, the result of part Experiment material is shown in Fig. 2.In Fig. 2, swimming lane 1 to 17 be followed successively by Shijiazhuang 15, No.6 Xiaoyan, Huaihe River wheat 18, Jimai 20, in educate No. 9, Lankao 2, Gaoyou 503, flower training 5, interior township 5, good star 99, Anhui wheat 29, Shandong wheat 21, Luohan No.2, tobacco grower 18, Shan 715, short anti-58, Lankao 24.
The method using embodiment 2 detects genotype based on molecular marker FAR, and the result of part Experiment material is shown in Fig. 3.In Fig. 3, swimming lane 1 to 14 is followed successively by Shijiazhuang 15, short rich No. 3, No.6 Xiaoyan, short anti-58, Huaihe River wheat 18, Jimai 20, Jinan 17, Lankao 2, good star 99, faces wheat No. 4, Shandong wheat 23, Luohan No.2, interior township 188, mountain agriculture 20.
The results are shown in Table 1.In 166 wheat breeds: 138 kinds are shown as AP2-a genotype and FAR-a genotype, plant height meansigma methods is 80.7cm；20 kinds are shown as AP2-b gene type and FAR-b genotype, plant height meansigma methods is 84.1cm；8 kinds are shown as AP2-a genotype and FAR-b genotype or AP2-b gene type and FAR-a genotype, plant height meansigma methods is 82.4cm；The genetic effect of statistical test display QPH.caas-6A has reached significant difference (P < 0.05).Using 82cm as threshold value, if Semen Tritici aestivi to be measured based on molecular marker AP2Genotype be AP2-a genotype and genotype based on molecular marker FAR are FAR-a genotype, can be judged as the Semen Tritici aestivi (i.e. the plant height Semen Tritici aestivi less than 82cm) with low plant height phenotype of candidate, and accuracy is 71.3%.Using 82cm as threshold value, if Semen Tritici aestivi to be measured based on molecular marker AP2Genotype be AP2-b gene type and genotype based on molecular marker FAR are FAR-b genotype, can be judged as the Semen Tritici aestivi (i.e. the plant height Semen Tritici aestivi more than 82cm) with high plant height phenotype of candidate, and accuracy is 55%.Through order-checking, AP2When-a genotypic variety uses the primer pair of F1 with R1 composition the amplified production that obtains all with the phase in the sequence 5 of sequence table it is contemplated that target region is consistent, AP2When-b gene type kind uses the primer pair of F1 with R1 composition the amplified production that obtains all with the phase in the sequence 6 of sequence table it is contemplated that target region is consistent.Through order-checking, when FAR-a genotypic variety uses the primer pair of F2 with R2 composition the amplified production that obtains all with the phase in the sequence 7 of sequence table it is contemplated that target region is consistent, the amplified production obtained during the primer pair that FAR-b genotypic variety employing F2 with R2 forms all with the phase in the sequence 8 of sequence table it is contemplated that target region is consistent.
The genotype call results of each wheat breed of table 1 and plant height measurement result
Claims (10)
1. primer sets, is made up of second first and special primer special primer；
First is made up of by described special primer two primers, and its target sequence is specific DNA molecular first, and described specific DNA molecular first includes primers F 1 and the target sequence of primer R1 in the genome of Semen Tritici aestivi；
Primers F 1 is following (a1) or (a2):
(a1) single strand dna shown in sequence 1 of sequence table；
(a2) sequence 1 through the replacement of one or several nucleotide and/or disappearance and/or interpolation and had the DNA molecular of identical function with sequence 1；
Primer R1 is following (a3) or (a4):
(a3) single strand dna shown in sequence 2 of sequence table；
(a4) sequence 2 through the replacement of one or several nucleotide and/or disappearance and/or interpolation and had the DNA molecular of identical function with sequence 2；
Second is made up of by described special primer two primers, and its target sequence is specific DNA molecular second, and described specific DNA molecular second includes primers F 2 and the target sequence of primer R2 in the genome of Semen Tritici aestivi；
Primers F 2 is following (a1) or (a2):
(a1) single strand dna shown in sequence 3 of sequence table；
(a2) sequence 3 through the replacement of one or several nucleotide and/or disappearance and/or interpolation and had the DNA molecular of identical function with sequence 3；
Primer R2 is following (a3) or (a4):
(a3) single strand dna shown in sequence 4 of sequence table；
(a4) sequence 4 through the replacement of one or several nucleotide and/or disappearance and/or interpolation and had the DNA molecular of identical function with sequence 4.
2. primer sets as claimed in claim 1, it is characterised in that: first is made up of by described special primer primers F 1 and primer R1, and second is made up of by described special primer primers F 2 and primer R2.
3. a test kit, including special primer group, restricted enzyme MseI and restricted enzyme NlaIII；Described special primer group is the primer sets described in claim 1 or 2.
4. special primer is to first or compositions first；
First is made up of by described special primer two primers, and its target sequence is specific DNA molecular first, and described specific DNA molecular first includes primers F 1 and the target sequence of primer R1 in the genome of Semen Tritici aestivi；
Primers F 1 is following (a1) or (a2):
(a1) single strand dna shown in sequence 1 of sequence table；
(a2) sequence 1 through the replacement of one or several nucleotide and/or disappearance and/or interpolation and had the DNA molecular of identical function with sequence 1；
Primer R1 is following (a3) or (a4):
(a3) single strand dna shown in sequence 2 of sequence table；
(a4) sequence 2 through the replacement of one or several nucleotide and/or disappearance and/or interpolation and had the DNA molecular of identical function with sequence 2；
First and restricted enzyme MseI are made up of by described compositions first described special primer.
5. special primer is to second or compositions second；
Second is made up of by described special primer two primers, and its target sequence is specific DNA molecular second, and described specific DNA molecular second includes primers F 2 and the target sequence of primer R2 in the genome of Semen Tritici aestivi；
Primers F 2 is following (a1) or (a2):
(a1) single strand dna shown in sequence 3 of sequence table；
(a2) sequence 3 through the replacement of one or several nucleotide and/or disappearance and/or interpolation and had the DNA molecular of identical function with sequence 3；
Primer R2 is following (a3) or (a4):
(a3) single strand dna shown in sequence 4 of sequence table；
(a4) sequence 4 through the replacement of one or several nucleotide and/or disappearance and/or interpolation and had the DNA molecular of identical function with sequence 4；
Second and restricted enzyme NlaIII are made up of by described compositions second described special primer.
6. the application of test kit described in special primer group described in special primer group described in claim 1 or claim 2 or claim 3, for following (1) or (2) or (3) or (4):
(1) auxiliary identifies the plant height of Semen Tritici aestivi to be measured；
(2) assisting sifting has the wheat breed of low plant height；
(3) assisting sifting has the wheat breed of high plant height；
(4) plant height of more different wheat breeds to be measured is assisted.
7. special primer described in claim 4 to special primer described in compositions first described in first or claim 4 or claim 5 to the application of compositions second described in second or claim 5, for following (1) or (2) or (3) or (4):
(1) auxiliary identifies the plant height of Semen Tritici aestivi to be measured；
(2) assisting sifting has the wheat breed of low plant height；
(3) assisting sifting has the wheat breed of high plant height；
(4) plant height of more different wheat breeds to be measured is assisted.
8. molecular marker AP2, a kind of polymorphic form is (b1) or (b2), and another kind of polymorphic form is (b3) or (b4)；
(b1) DNA molecular shown in sequence 5 of sequence table；
(b2) sequence 5 of sequence table is from the DNA molecular shown in 5 ' end 47-305 position nucleotide；
(b3) DNA molecular shown in sequence 6 of sequence table；
(b4) sequence 6 of sequence table is from the DNA molecular shown in 5 ' end 47-305 position nucleotide.
9. molecular marker FAR, a kind of polymorphic form is (c1) or (c2) or (c3), and another kind of polymorphic form is (c4) or (c5) or (c6)；
(c1) DNA molecular shown in sequence 7 of sequence table；
(c2) sequence 7 of sequence table is from the DNA molecular shown in 5 ' end 1-548 position nucleotide；
(c3) sequence 7 of sequence table is from the DNA molecular shown in 5 ' end 271-548 position nucleotide；
(c4) DNA molecular shown in sequence 8 of sequence table；
(c5) sequence 8 of sequence table is from the DNA molecular shown in 5 ' end 1-548 position nucleotide；
(c6) sequence 8 of sequence table is from the DNA molecular shown in 5 ' end 271-548 position nucleotide.
10. molecular marker AP described in claim 82And/or the application of molecular marker FAR described in claim 9, for as follows (1) or (2) or (3) or (4):
(1) auxiliary identifies the plant height of Semen Tritici aestivi to be measured；
(2) assisting sifting has the wheat breed of low plant height；
(3) assisting sifting has the wheat breed of high plant height；
(4) plant height of more different wheat breeds to be measured is assisted.
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| CN109762812B (en) * | 2019-03-05 | 2022-05-31 | 中国农业科学院作物科学研究所 | Wheat growth potential related SNP and application thereof as target point in identification of wheat growth potential traits |
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