AU2021106536A4 - Molecular Marker Closely Linked with Major QTL of Lateral Root Number in Wheat Seedling Stage and Application thereof - Google Patents
Molecular Marker Closely Linked with Major QTL of Lateral Root Number in Wheat Seedling Stage and Application thereof Download PDFInfo
- Publication number
- AU2021106536A4 AU2021106536A4 AU2021106536A AU2021106536A AU2021106536A4 AU 2021106536 A4 AU2021106536 A4 AU 2021106536A4 AU 2021106536 A AU2021106536 A AU 2021106536A AU 2021106536 A AU2021106536 A AU 2021106536A AU 2021106536 A4 AU2021106536 A4 AU 2021106536A4
- Authority
- AU
- Australia
- Prior art keywords
- wheat
- seedling stage
- identifying
- lateral roots
- primer pair
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Ceased
Links
- 241000209140 Triticum Species 0.000 title claims abstract description 69
- 235000021307 Triticum Nutrition 0.000 title claims abstract description 69
- 239000003147 molecular marker Substances 0.000 title claims abstract description 26
- 238000000034 method Methods 0.000 claims abstract description 18
- 238000009395 breeding Methods 0.000 claims abstract description 13
- 230000001488 breeding effect Effects 0.000 claims abstract description 13
- 108020004414 DNA Proteins 0.000 claims description 24
- LKVHMIDEEJWEAI-UHFFFAOYSA-N tetrakis-decylazanium Chemical compound CCCCCCCCCC[N+](CCCCCCCCCC)(CCCCCCCCCC)CCCCCCCCCC LKVHMIDEEJWEAI-UHFFFAOYSA-N 0.000 claims description 11
- 238000012408 PCR amplification Methods 0.000 claims description 10
- 239000000499 gel Substances 0.000 claims description 10
- 238000011144 upstream manufacturing Methods 0.000 claims description 9
- 238000006243 chemical reaction Methods 0.000 claims description 8
- 238000001962 electrophoresis Methods 0.000 claims description 6
- 238000000137 annealing Methods 0.000 claims description 4
- 238000004925 denaturation Methods 0.000 claims description 4
- 230000036425 denaturation Effects 0.000 claims description 4
- 229920002401 polyacrylamide Polymers 0.000 claims description 4
- 238000003752 polymerase chain reaction Methods 0.000 claims description 4
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 claims description 3
- 102000053602 DNA Human genes 0.000 claims description 3
- 108020004682 Single-Stranded DNA Proteins 0.000 claims description 3
- 244000098338 Triticum aestivum Species 0.000 claims description 3
- 239000012634 fragment Substances 0.000 claims description 3
- -1 methylene acrylamide Chemical compound 0.000 claims description 3
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 2
- 238000004806 packaging method and process Methods 0.000 claims description 2
- 239000003550 marker Substances 0.000 abstract description 10
- 230000003321 amplification Effects 0.000 abstract description 7
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 7
- 238000001514 detection method Methods 0.000 abstract description 3
- 230000008569 process Effects 0.000 abstract description 2
- 238000004458 analytical method Methods 0.000 description 10
- 239000000047 product Substances 0.000 description 9
- 108700028369 Alleles Proteins 0.000 description 5
- 230000002068 genetic effect Effects 0.000 description 5
- 230000000694 effects Effects 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 239000000654 additive Substances 0.000 description 3
- 230000000996 additive effect Effects 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 238000013507 mapping Methods 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- 235000021393 food security Nutrition 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 230000009418 agronomic effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000011536 extraction buffer Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000003517 fume Substances 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- 210000004209 hair Anatomy 0.000 description 1
- 239000003501 hydroponics Substances 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 125000001972 isopentyl group Chemical group [H]C([H])([H])C([H])(C([H])([H])[H])C([H])([H])C([H])([H])* 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000005065 mining Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 229910001961 silver nitrate Inorganic materials 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
- A01H1/045—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection using molecular markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
Abstract
The invention discloses a molecular marker closely linked with major QTL of lateral root
number in wheat seedling stage and application thereof, belonging to the technical field of crop
seed selection and cultivation. According to the invention, an InDel marker QA U5D-20 closely
linked with the major QTL for lateral root number in wheat seedling stage is developed, has
the advantages of co-dominance, stable amplification, quick detection and the like, can quickly
identify or assist in identifying the number of lateral roots in different wheat varieties in
seedling stage, and can predict lateral root number in wheat, thereby accelerating the molecular
breeding process of excellent root structure in wheat.
Description
Molecular Marker Closely Linked with Major QTL of Lateral Root Number in
Wheat Seedling Stage and Application thereof
The invention relates to the technical field of crop seed selection and cultivation, in
particular to a molecular marker closely linked with major QTL for lateral root number in
wheat seedling stage and application thereof.
Wheat is an important cereal crop and the main food source, which plays an important role
in China's food security. Roots, as an important organ for absorbing water and nutrition in
wheat, play an important role in the growth and yield of aboveground parts. Wheat root
belongs to fibrous root, which is the general name of lateral roots and root hairs produced
on all primary roots, secondary roots and branches of wheat. Like most agronomic traits,
root-related traits are typical quantitative traits, which are controlled by multiple genes.
The isolation and identification of genes are facing great challenges, which limits the
development of molecular markers and the application of molecular breeding in wheat.
Therefore, analyzing the molecular genetic basis of root-related traits, mining alleles with
excellent number of lateral roots and using them in breeding will help to cultivate new
wheat varieties with strong root activity and ensure food security and sustainable
development of agriculture in China.
At present, scholars at home and abroad have used multiple genetic backgrounds and
different environmental conditions to locate QTL for lateral root number of wheat. The
QTL interval for lateral root number in wheat is large, and its linkage markers are far away from the target genes, so the molecular markers of lateral root number can't be effectively applied to molecular assisted breeding.
The purpose of the present invention is to provide a molecular marker closely linked with
the major QTL for lateral root number in wheat seedling stage, so as to detect whether
wheat varieties (lines) contain QTL loci for increasing lateral root number.
Another object of the present invention is to provide application of molecular marker
primer pair (SEQ ID NO:1 and SEQ ID:2) closely linked with major QTL for lateral root
number in wheat seedling stage in wheat root architecture breeding, including the
application of wheat sequences amplified by molecular marker QAU5D-20 in wheat
excellent root architecture breeding.
To achieve the purpose above, the present invention provides the following scheme:
The primer pair consists of two single-stranded DNAs shown in SEQ ID NO:1 and SEQ
ID NO:2.
The method for preparing the primer pair comprises the step of separately packaging the
two single-stranded NDAs of claim 1.
A product for identifying or assisting in identifying lateral root number in wheat seedling
stage, characterized in that the product contains the primer pair of claim 1.
The method for identifying or assisting in identifying lateral root number in wheat seedling
stage comprises the following steps:
Step (1) extracting genome DNA of wheat to be detected;
Step (2) taking the genome DNA of wheat to be detected as a template, and carrying out
PCR amplification by using the primer pair;
Step (3) carrying out agarose gel electrophoresis on the PCR products, and determining the
number of lateral roots of the wheat to be detected in the seedling stage according to the
electrophoresis result: If an amplified fragment with a molecular weight of 219 bp appears,
it is predicted that the wheat variety or strain has QTL loci that increase the number of
lateral roots in wheat seedling stage; otherwise, the wheat variety or strain does not have
QTL loci that increase the number of lateral roots in wheat seedling stage.
Preferably, in step (2), the PCR amplification reaction system is 10 L, and comprises:
1 L of the upstream primer shown in SEQ ID NO:1 with a concentration of 2 mol/L;
1 L of the downstream primer shown in SEQ ID NO:2 with a concentration of 2 mol/L;
1.5 L DNA template with concentration above 50 ng/[l;
pL 2x Taq PCR StarMix;
1.5 LddH20 is used to make up the reaction system.
Preferably, in step (2), the PCR amplification procedure is as follows:
Pre-denatured at 95C for 3 min; denaturation at 95C for 15 s, annealing at 60°C for 15
s, extension at 72C for 15 s, 34 cycles; extension at 72C for 5 min; store at 4°C.
Preferably, in step (3), the gel is 8.0% non-denatured polyacrylamide gel, and every 100
mL of gel solution contains 7.8 g acrylamide and 0.2 g methylene acrylamide.
A molecular marker QAU5D-20 related to lateral root number in wheat seedling stage is a
DNA fragment obtained by PCR amplification with the primer pair of claim 1 using the
genomic DNA of common wheat TAA10 as a template.
The invention discloses the following technical effects:
(1) the primer pair provided by the invention consists of two single-stranded DNAs shown
in SEQ ID NO:1 and SEQ ID NO:2, and has the advantages of stable amplification, quick and efficient detection and the like, and can promote the application of QTL QLrn5D, a major QTL for lateral root number in wheat seedling stage, in molecular breeding of excellent root structure;
(2) the molecular marker QAU5D-20 provided by the invention is closely linked with
QLrn5D, which can promote the transfer and application of the locus in popularized
varieties, and is also beneficial to the polymerization breeding of the loci and other yield
related loci;
(3) The molecular marker QA U5D-20 provided by the invention is an InDel marker, which
has the advantages of co-dominance, stable amplification, quick and efficient detection,
etc. By using the molecular marker QA U5D-20, it is possible to quickly determine whether
QLrn5D exists in wheat resources and predict the characteristics of lateral root number in
wheat seedling stage, thereby effectively screening wheat varieties (lines) containing
QLrn5D loci and improving the root structure of wheat varieties;
(4) By using the molecular marker QA U5D-20 closely linked with QLrn5D, the phenotypic
identification can be greatly reduced, and it can be used in wheat seedling stage, thus
eliminating non-target plants, saving breeding cost and improving breeding efficiency.
In order to explain the embodiments of the present invention or the technical scheme in the
prior art more clearly, the figures needed in the embodiments will be briefly introduced
below. Obviously, the figures in the following description are only some embodiments of
the present invention, and for ordinary technicians in the field, other figures can be obtained
according to these figures without paying creative labor.
Figure 1 shows the amplified bands of molecular marker QA U5D-20 in some RIL families,
where M is 2000 bp DNA Ladder; AA is the amplified band of wheat variety TAA10, and
BB is the amplified band of wheat variety XX329.
Figure 2 is the result of QLrn5D location analysis using WinQTLCart 2.5;
Figure 3 is a single marker analysis result of the number of lateral roots in seedling stage
of 182 RIL families. AA indicates the allele of increasing the number of lateral roots in
seedling stage from TAA10; BB indicates the allelic progression of decreasing the number
of lateral roots in seedling stage from XX329; *** indicates significant difference (p <
0.001).
Various exemplary embodiments of the present invention will now be described in detail.
This part should not be considered as a limitation to the present invention, but as more
detailed information of certain aspects, characteristics, and embodiments of the present
invention.
It should be noted that the terms described in the present invention are only used to describe
specific embodiments and are not to limit the present invention. In addition, for the
numerical range in this invention, it should be understood that each intermediate value
between the upper limit and the lower limit is also specifically disclosed. Each smaller
range between any stated value or intermediate value within the stated range and any other
stated value or intermediate value within the stated range is also included in the present
invention. The upper and lower limits of these smaller ranges can be independently
included or excluded from the range.
Unless otherwise specified, all technical and scientific terms used herein have the same
meaning as commonly understood by those skilled in the art in the field of the present
invention. Although the present invention only describes preferred methods and materials,
any methods and materials similar or equivalent to those described herein can also be used
in the practice or testing of the present invention. All documents mentioned in this
specification are incorporated by reference to disclose and describe methods and/or
materials related to the documents. In the event of conflict with any incorporated document,
the content of this manual shall prevail.
Without departing from the scope or spirit of the present invention, various improvements
and changes can be made to the specific embodiments of the present specification, which
is apparent to those skilled in the art. Other embodiments derived from the description of
the present invention will be apparent to the skilled person. The specification and examples
of this application are only exemplary.
As used herein, "including", "composing", "having", "containing", etc., are all open terms,
which means including but not limited to.
Embodiment 1
First, molecular markers closely linked with major QTL of lateral root number in wheat
seedling stage
Taking 182 RIL families derived from common wheat TAA1O and synthetic hexaploid
wheat XX329 as parents as test materials, the major QTL for lateral root number (QLrn5D)
was located in the interval of 5D chromosome by using QTL location analysis, and
according to the resequencing data of TAA1O and XX329, an InDel molecular marker
QAU5D-20 with polymorphism between parents was designed and developed near the
LOD prediction peak of QLrn5D segment. The upstream primer sequence of the molecular
marker QA U5D-20 is ttgataaaaagcccccttec (SEQ ID NO:1), and the downstream primer
sequence is gctaattagacccggcaaca (SEQ ID NO:2).
With the genome DNA of wheat variety TAA10 as template, PCR amplification was
carried out with the upstream primer shown in SEQ ID NO:1 and the downstream primer
shown in SEQ ID NO:2.
The PCR amplification reaction system is 10 L, and comprises:
(1) 1 L of the upstream primer shown in SEQ ID NO:1 with a concentration of 2 mol/L;
(2) 1 L of the downstream primer shown in SEQ ID NO:2 with a concentration of 2
pmol/L;
(3) 1.5 L DNA template with concentration 50 ng/[l;
(4) 5 L 2* Taq PCR StarMix;
(5) 1.5 pLddH20 is used to make up the reaction system.
The PCR amplification procedure is as follows:
Pre-denatured at 95C for 3 min; denaturation at 95C for 15 s, annealing at 60°C for 15
s, extension at 72C for 15 s, 34 cycles; extension at 72C for 5 min; store at 4°C.
Type of PCR instrument: S100TMThermal Cycler.
The amplified product was separated by electrophoresis on 8.0% non-denatured
polyacrylamide gel (containing 7.8 g acrylamide and 0.2 g methylene acrylamide in every
100 mL gel solution), and the molecular weight of the final amplified product was 219 bp.
Second, the acquisition method of molecular marker QA U5D-20
The method for obtaining the molecular marker QAU5D-20 specifically comprises the
following steps:
Step 1: Form the F8 RIL population of 182 families
A hybrid Fi is obtained by hybrid of TAA10 and XX329 as parents, and F2 is produced by
selfing of Fl. F2 performs selfing generation by generation to form an F8 RIL population
with 182 families.
Step 2: Extract the leaf DNA of wheat RIL population by CTAB method. The specific
method and procedure are as follows:
(1) Take 0.2 g of tender leaves of wheat, put them in a 1.5 ml Eppendorf tube, add liquid
nitrogen, and grind them into fine powder.
(2) Add 600 pl of1xCTAB extraction buffer preheated at 65C into the centrifuge tube,
and shake it gently and mix it evenly.
(3) Bath in a water bath at 65C for 60 min and shake the centrifuge tube carefully every
min.
(4) After 60 min, take out the centrifuge tube, add equal volume of chloroform: isoamyl
alcohol (24:1) into the fume hood, carefully and fully shake the centrifuge tube for 1 min,
and then let it stand until the organic phase changes from colorless to green to dark green.
(5) Centrifuge at 10000 rpm for 10 min at room temperature, and then suck 500 pl
supernatant into a new centrifuge tube.
(6) Add equal volume of pre-cooled isopropanol (-20°C) into the supernatant, carefully mix
it evenly, and then place it at -20°C for 20 min.
(7) Centrifuge at 10000 rpm for 10 min at room temperature, carefully pour out the
supernatant, rinse with 600 pl of 75% ethanol, and slightly shake the centrifuge tube to
suspend the DNA.
(8) Centrifuge at 8000 rpm for 2 min at room temperature, then carefully pour out the
supernatant and dry at room temperature.
(9) After the DNA is dried, add a proper amount of sterilized ddH20 to fully dissolve the
(10) Use ultraviolet spectrophotometer to detect the concentration and purity of DNA, and
store it in the refrigerator at -20°C for later use.
PCR amplification was carried out on all DNA extracted above with the upstream primer
shown in SEQ ID NO:1 and the downstream primer shown in SEQ ID NO:2.
The PCR amplification reaction system is 10 L, and comprises:
(1) 1 L of the upstream primer shown in SEQ ID NO:1 with a concentration of 2 mol/L;
(2) 1 L of the downstream primer shown in SEQ ID NO:2 with a concentration of 2
pmol/L;
(3) 1.5 L DNA template with concentration 50 ng/[l;
(4) 5 L 2* Taq PCR StarMix;
(5) 1.5 pL ddH20 is used to make up the reaction system.
The PCR amplification procedure is as follows:
Pre-denatured at 95C for 3 min; denaturation at 95C for 15 s, annealing at 60°C for 15
s, extension at 72C for 15 s, 34 cycles; extension at 72C for 5 min; store at 4°C.
Type of PCR instrument: SIO00TThermal Cycler.
The amplified products were separated by electrophoresis on 8.0% non-denatured
polyacrylamide gel, and the electrophoresis buffer was 1*TBE, and the constant pressure
was 180 V for 3 hours.
Silver nitrate staining step: after electrophoresis, the stripped rubber block was dyed in the
staining solution for 15 min, washed with deionized water for 3 times, then transferred to
a developer for development until the amplification band was clearly visible, and washed
with water for 2 times.
Put the developed rubber block on the film viewing lamp, record the tape type, and take
pictures to get gel pictures, as shown in Figure 1.
The result of band pattern reading: the size of TAA10 amplification product is 219 bp, and
that of XX329 amplification product is 196 bp. among the 182 RIL populations, 83 have
the same band pattern as TAA10 and 91 have the same band pattern as XX329.
Step 3: construct a wheat molecular marker genetic linkage map
Using JoinMap 4.0 mapping software, the genetic linkage map of wheat molecular markers
with 44 marker loci was constructed from the obtained RIL population genotype data, with
LOD 32.5 as the standard.
Step 4: investigate the number of lateral roots of 182 lines in RIL population
182 families of RIL population were cultured under the condition of hydroponics, and the
number of lateral roots at seedling stage on the 8th day after germination was investigated.
Each strain has 3 biological replicates, and each biological replicate has at least 8 seedlings.
Measuring method of lateral root number at seedling stage: use root scanner to measure
root uniformly, scan and record the primary root of wheat seedlings, and then use Adobe
Photoshop software to count and record the number of lateral roots on the primary root at
seedling stage.
Step 5: carry out additive QTL mapping analysis
Using the above-mentioned 182 RIL genotype and phenotype values, WinQTLCart 2.5
was used for additive QTL mapping analysis, and the analysis results are shown in Table
1 and Figure 2.
Table 1 Mapping results of major QTL(QLrn5D) of lateral root number in seedling stage
Nearest LOD Additive QTL location R(2 marker value effect R (%)
QLrn5D QAU5D-20 55.4 3.98 1.36 8.4
The results of QTL analysis showed that the interval was located at the nearest marker
QAU5D-20 on 5D chromosome, and the LOD value was 3.98. The QTL for lateral root
number located could explain 8.4% of the phenotypic variation, and the allele from TAA10
increased the number of lateral roots by 1.36.
Step 6: significant analysis of lateral root number difference based on single marker
QAU5D-20
The 182 RIL families were Step 6: significant analysis of lateral root number difference
based on single marker QA U5D-20
Based on genotype results of the 182 RIL families by marker QAU5D-20, and significant
difference of lateral root number of different genotypes in seedling stage was analyzed.
The results are shown in Figure 3.
The result showed that the allele from TAA10 could increase lateral root number in
seedling stage compared with the effect of reducing the number of lateral roots from
XX329 allele. Therefore, the amplification product with molecular weight of 219 bp
obtained by the upstream primer shown in SEQ ID NO:1 and the downstream primer shown
in SEQ ID NO:2 in the DNA of wheat variety TAA10 is a molecular marker closely linked with QTL of lateral root number in wheat seedling stage (i.e., molecular marker QAU5D
Third, application of molecular marker QAU5D-20
Based on the previous test and analysis, it can be known that:
(1) The primer pair consisting of the upstream primer shown in SEQ ID NO:1 and the
downstream primer shown in SEQ ID NO:2 can be used for identifying or assisting in
identifying the number of lateral roots in wheat seedling stage, and can also be applied in
obtaining molecular markers related to lateral root number in wheat seedling stage;
(2) The molecular marker QAU5D-20 can be applied in identifying or assisting in
identifying the number of lateral roots in wheat seedling stage, and can also be used in
breeding wheat genotypes with the number of lateral roots.
To sum up, the primer pair and the molecular marker QA U5D-20 provided by the invention
can greatly accelerate the molecular marker-assisted breeding process of the number of
lateral roots in wheat seedling stage, and have important application value for genetic
improvement of wheat roots and cultivation of high-yield and high-quality wheat varieties.
The embodiments above only describe the preferred mode of the invention, but do not limit
the scope of the invention. On the premise of not departing from the design spirit of the
invention, various modifications and improvements made by ordinary technicians in the
field to the technical scheme of the invention shall be subject to the protection scope
determined by the claims of the invention.
Claims (10)
1. A primer pair for identifying or assisting in identifying the number of lateral roots in
wheat seedling stage, characterized in that the primer pair consists of two single-stranded
DNAs shown in SEQ ID NO:1 and SEQ ID NO:2.
2. The method for preparing the primer pair of claim 1, which is characterized by
comprising the step of separately packaging the two single-stranded NDAs of claim 1.
3. A product for identifying or assisting in identifying the number of lateral roots in wheat
seedling stage, characterized in that the product contains the primer pair of claim 1.
4. The method for identifying or assisting in identifying the number of lateral roots in wheat
seedling stage, which is characterized by comprising the following steps:
(1) extracting genome DNA of wheat to be detected;
(2) taking the genome DNA of wheat to be detected as a template, and carrying out PCR
amplification by using the primer pair of claim 1;
(3) carrying out agarose gel electrophoresis on the PCR products, and determining the
number of lateral roots of the wheat to be detected in the seedling stage according to the
electrophoresis result.
5. The method according to claim 4, wherein in step (2), the PCR amplification reaction
system is 10 L, and comprises:
1 L of the upstream primer shown in SEQ ID NO:1 with a concentration of 2 mol/L;
1 L of the downstream primer shown in SEQ ID NO:2 with a concentration of 2 mol/L;
1.5 L DNA template with concentration above 50 ng/L;
pL 2x Taq PCR StarMix;
1.5 LddH20 is used to make up the reaction system.
6. The method for identifying or assisting in identifying the number of lateral roots in wheat
seedling stage according to claim 4, characterized in that in step (2), the PCR amplification
procedure is as follows:
Pre-denatured at 95C for 3 min; denaturation at 95C for 15 s, annealing at 60°C for 15
s, extension at 72C for 15 s, 34 cycles; extension at 72C for 5 min; store at 4C.
7. The method according to claim 4, characterized in that in step (3), the gel is 8.0% non
denatured polyacrylamide gel, and every 100 mL of gel solution contains 7.8 g acrylamide
and 0.2 g methylene acrylamide.
8. A molecular marker QA U5D-20 related to the number of lateral roots in wheat seedling
stage, characterized in that the molecular marker QAU5D-20 is a DNA fragment obtained
by PCR amplification with the primer pair of claim 1 using the genomic DNA of common
wheat TAA10 as a template.
9. The primer pair according to claim 1, which has any one of the following applications:
(a) its application in identifying or assisting in identifying the number of lateral roots in
wheat seedling stage;
(b) Application in obtaining molecular markers related to the number of lateral roots in
wheat seedling stage.
10. The molecular marker QAU5D-20 according to claim 8, which has any one of the
following applications:
(a) its application in identifying or assisting in identifying the number of lateral roots in
wheat seedling stage;
(b) Application in breeding wheat with genotype of lateral roots number in seedling stage.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU2021106536A AU2021106536A4 (en) | 2021-08-23 | 2021-08-23 | Molecular Marker Closely Linked with Major QTL of Lateral Root Number in Wheat Seedling Stage and Application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU2021106536A AU2021106536A4 (en) | 2021-08-23 | 2021-08-23 | Molecular Marker Closely Linked with Major QTL of Lateral Root Number in Wheat Seedling Stage and Application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| AU2021106536A4 true AU2021106536A4 (en) | 2021-11-11 |
Family
ID=78488534
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU2021106536A Ceased AU2021106536A4 (en) | 2021-08-23 | 2021-08-23 | Molecular Marker Closely Linked with Major QTL of Lateral Root Number in Wheat Seedling Stage and Application thereof |
Country Status (1)
| Country | Link |
|---|---|
| AU (1) | AU2021106536A4 (en) |
-
2021
- 2021-08-23 AU AU2021106536A patent/AU2021106536A4/en not_active Ceased
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110117673B (en) | Molecular marker of brassica napus dwarf trait locus and application thereof | |
| Liu et al. | Genetic analysis and mapping of a striped rind gene (st3) in melon (Cucumis melo L.) | |
| CN105695478B (en) | Gene for regulating plant type and yield of plant and application thereof | |
| CN109929945B (en) | Molecular marker BrSF2604 primer of main effect QTL sites in flowering phase and mature phase of brassica napus and application thereof | |
| Nantawan et al. | Linkage mapping and quantitative trait loci analysis of sweetness and other fruit quality traits in papaya | |
| CN111961746B (en) | SNP molecular marker related to blight and disease resistance of cotton on land and application thereof | |
| Li et al. | Gynoecy instability in cucumber (Cucumis sativus L.) is due to unequal crossover at the copy number variation-dependent Femaleness (F) locus | |
| Ma et al. | Construction of chromosome segment substitution lines of Dongxiang common wild rice (Oryza rufipogon Griff.) in the background of the japonica rice cultivar Nipponbare (Oryza sativa L.) | |
| CN105543222B (en) | The molecular labeling InDeL_33 of soybean 100-grain weight main effect QTL and its application | |
| CN114107537B (en) | Molecular marker closely linked with main effect QTL of lateral root number in wheat seedling stage, detection primer and application thereof | |
| González et al. | Genetic structure based on EST–SSR: a putative tool for fruit color selection in Japanese plum (Prunus salicina L.) breeding programs | |
| CN106868182B (en) | Specific primer pair and application thereof in detection of rice resistance to bacterial blight | |
| Luan et al. | Genetic mapping reveals a candidate gene for egusi seed in watermelon | |
| Zhang et al. | Genome-wide association study of the candidate genes for grape berry shape-related traits | |
| CN113046467B (en) | SNP locus obviously associated with wheat stripe rust resistance and application thereof in genetic breeding | |
| CN107058532B (en) | Molecular marker and specific primer pair for detecting bacterial leaf blight resistance of rice | |
| CN113151553A (en) | Molecular marker coseparated with gene Clbl of watermelon plant few lateral branches and application | |
| CN116121431B (en) | Molecular marker closely linked with lateral root number gene locus in wheat seedling stage and application thereof | |
| CN110004242B (en) | Molecular marker BrSF0239 primer of main QTL sites in flowering phase and mature phase of brassica napus and application thereof | |
| AU2021106536A4 (en) | Molecular Marker Closely Linked with Major QTL of Lateral Root Number in Wheat Seedling Stage and Application thereof | |
| Osae et al. | CAPS marker-base genetic linkage mapping and QTL analysis for watermelon ovary, fruit and seed-related traits | |
| CN110468229A (en) | Rice wide spectrum high resistance to hoja blanca gene Xa45's (t) isolates molecular labeling Hxjy-1 | |
| CN110423838A (en) | The molecular labeling of main effect QTL section close linkage related to corn seed keeping quality is located at and its application | |
| CN113881799B (en) | Functional molecular marker for screening/detecting tobacco root black rot main effect resistance locus and application thereof | |
| CN114032323B (en) | Co-dominant SSR marker closely linked with cigar black shank resistance gene and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FGI | Letters patent sealed or granted (innovation patent) | ||
| MK22 | Patent ceased section 143a(d), or expired - non payment of renewal fee or expiry |