CN112760402B - KASP primer group for detecting wheat spike number and biomass density and application thereof - Google Patents
KASP primer group for detecting wheat spike number and biomass density and application thereof Download PDFInfo
- Publication number
- CN112760402B CN112760402B CN202110129159.3A CN202110129159A CN112760402B CN 112760402 B CN112760402 B CN 112760402B CN 202110129159 A CN202110129159 A CN 202110129159A CN 112760402 B CN112760402 B CN 112760402B
- Authority
- CN
- China
- Prior art keywords
- primer
- wheat
- kasp
- seq
- nucleotide sequence
- 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.)
- Active
Links
- 241000209140 Triticum Species 0.000 title claims abstract description 96
- 235000021307 Triticum Nutrition 0.000 title claims abstract description 96
- 239000002028 Biomass Substances 0.000 title claims description 14
- 239000002773 nucleotide Substances 0.000 claims abstract description 27
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 27
- 108700028369 Alleles Proteins 0.000 claims abstract description 24
- 238000001514 detection method Methods 0.000 claims abstract description 21
- 238000012408 PCR amplification Methods 0.000 claims abstract description 13
- 238000003205 genotyping method Methods 0.000 claims abstract description 7
- 230000003321 amplification Effects 0.000 claims abstract description 4
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 4
- 239000012224 working solution Substances 0.000 claims description 12
- 238000006243 chemical reaction Methods 0.000 claims description 11
- 238000000034 method Methods 0.000 claims description 10
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 10
- 239000012498 ultrapure water Substances 0.000 claims description 10
- 241000495841 Oenanthe oenanthe Species 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 6
- 238000004925 denaturation Methods 0.000 claims description 4
- 230000036425 denaturation Effects 0.000 claims description 4
- 238000012257 pre-denaturation Methods 0.000 claims description 3
- 238000004153 renaturation Methods 0.000 claims description 3
- 108010047290 Multifunctional Enzymes Proteins 0.000 claims description 2
- 102000006833 Multifunctional Enzymes Human genes 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims 2
- 241000051135 Morella rubra Species 0.000 claims 1
- 235000014631 Myrica rubra Nutrition 0.000 claims 1
- 238000009395 breeding Methods 0.000 abstract description 16
- 230000001488 breeding effect Effects 0.000 abstract description 16
- 238000012216 screening Methods 0.000 abstract description 4
- 241001024327 Oenanthe <Aves> Species 0.000 abstract description 3
- 239000003550 marker Substances 0.000 description 25
- 108020004414 DNA Proteins 0.000 description 21
- 210000000349 chromosome Anatomy 0.000 description 10
- 108090000623 proteins and genes Proteins 0.000 description 10
- 241000196324 Embryophyta Species 0.000 description 9
- 239000003147 molecular marker Substances 0.000 description 9
- 238000012360 testing method Methods 0.000 description 9
- 230000002068 genetic effect Effects 0.000 description 8
- 239000000463 material Substances 0.000 description 7
- 230000000875 corresponding effect Effects 0.000 description 6
- 230000005284 excitation Effects 0.000 description 6
- 238000011144 upstream manufacturing Methods 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 5
- 235000013339 cereals Nutrition 0.000 description 4
- 238000013507 mapping Methods 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 238000007619 statistical method Methods 0.000 description 4
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 3
- 244000098338 Triticum aestivum Species 0.000 description 3
- 230000002596 correlated effect Effects 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 210000005069 ears Anatomy 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- 239000012807 PCR reagent Substances 0.000 description 2
- 238000007844 allele-specific PCR Methods 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000012252 genetic analysis Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000009394 selective breeding Methods 0.000 description 2
- 230000002195 synergetic effect Effects 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 108010068370 Glutens Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 238000012214 genetic breeding Methods 0.000 description 1
- 235000021312 gluten Nutrition 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000005065 mining Methods 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 230000000007 visual effect Effects 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
-
- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
-
- 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
-
- 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
- C12Q2600/156—Polymorphic or mutational 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
- C12Q2600/172—Haplotypes
Abstract
The invention provides a set of KASP primer group for detecting wheat spike number attachment density and application thereof, wherein the KASP primer group comprises the following components: a common primer 1 with a nucleotide sequence shown as SEQ ID NO.1, a primer 2 with a nucleotide sequence shown as SEQ ID NO.2, and a primer 3 with a nucleotide sequence shown as SEQ ID NO. 3; performing PCR amplification by taking the KASP primer group as a primer and taking a genome of the wheat to be detected as a template, wherein if an amplification result shows that the genotyping of the wheat to be detected is the same as that of the Yangmai 15, the wheat to be detected contains an allele T; otherwise, the wheat to be tested contains allele C; wheat containing allele T has a higher spike density than wheat containing allele C; the detection method can quickly and intuitively obtain the result of the number of wheat ears and the density of the wheat variety, and is suitable for large-scale breeding screening.
Description
Technical Field
The application relates to the field of wheat breeding, in particular to a KASP primer group for detecting the wheat spike density and application thereof.
Background
Wheat is one of the main grain crops in the world, the wheat yield is highly correlated with the wheat ear characteristics, the characteristics are intricate and complex, the wheat ear characteristics mainly comprise small ear number, ear length, small ear seed number, ear seed weight and the like, the ear length, the small ear seed number, the ear seed number and the ear seed weight are controlled by a main gene and a micro-effect polygene together, the wheat ear number is controlled by a polygene, and no main gene exists. Wheat ear attachment density refers to the number of attached ears per unit ear length (ear attachment density = total number of individual ears/ear length), which is closely related to wheat kernel yield, with higher ear attachment density, higher numbers of firm ears, and easier availability of high yield.
The field can not intuitively measure the small ear attachment density, the small ear attachment density can be determined only by measuring the ear length and the total small ear number, and the work load required for detecting the small ear attachment density among a large amount of materials in breeding is heavy.
Genetic studies on the earbud density at home and abroad are few, and the main gene genetic rate of the earbud density is up to 88.69% -92.14%, the influence of the environment is small, the early generation selection result in breeding is high in reliability (references: wei Yanli, wang Binlong, li Ruiguo, jiang Huili and Zhang Anjing. Genetic analysis of the earbud trait of large-ear wheat, wheat crop journal, 2015,35 (10): 1366-1371), and the genes are respectively located on chromosomes such as 1A, 3A, 2D, 4B, 4D, 6B and 6D (references: liu Kai, deng Zhiying, li Qingfang, zhang Ying, sun Cailing, tian Jichun and Chen Jiansheng. The high-density SNP genetic map is utilized to locate the earbud trait genes in China agricultural science, 2016, 42 (6): 820-831).
Molecular marker assisted selective breeding can select target characters at the DNA level, so that not only is the result stable, but also the selection can be performed at the seedling stage, the operation is simple and convenient, and the early generation selection of breeding is facilitated. The Single Nucleotide Polymorphism (SNP) marker has the characteristics of stable inheritance, large quantity, wide distribution, easy detection and the like, is suitable for detection and analysis with huge quantity, has a plurality of SNP chips suitable for different animals and plants in the market at present, and plays an important role in genetic breeding. After the linkage markers for important traits are mined by genetic analysis, they need to be converted into easy-to-use molecular markers. Kompetitive Allele Specific PCR (KASP) marking technology can distinguish different equivalent variations of SNP markers through fluorescent probes, has high flux, is rapid and stable, and is a molecular marker convenient for breeding and use.
The wheat 15 and 13 are wheat varieties bred by agricultural scientific research institute in Jiangsu-Shanghai river region, the wheat 15 has high grain weight, short plant height and high yield, compared with the wheat 13 with high grain weight which is equivalent to the wheat 15, and the wheat 15 and the wheat are all low quality weak gluten wheat, through years of character investigation, the wheat spike density of the wheat 15 is 1.85-1.95 per cm, the wheat spike density of the wheat 13 is 1.60-1.70 per cm, the wheat spike density of the common wheat is 1.65-1.75 per cm, the wheat spike density of the wheat 15 is obviously higher than that of the common wheat and the wheat spike density of the wheat 13, so that the mining of the wheat 15 is important for controlling the main effect QTL of the wheat spike density and developing the linkage molecular marker for breeding auxiliary selection of the wheat spike density. There is no report on the genetic basis of 15 spikelet of Yangmai at present.
Disclosure of Invention
In order to solve the problems, the invention acquires genotype data by utilizing an illuminea 90K wheat high-throughput gene chip, detects 1 major QTL locus YM15-SC-5A which comes from Yangmai 15 and is related to the earbud density, the close linkage mark of the locus is RAC875_c8690_446, and develops 1 KASP mark primer group Y15-SC-KASP according to the locus, so as to carry out high-efficiency screening on the earbud density, and the invention is realized by the following scheme:
the invention firstly provides a KASP primer group related to wheat spike density, which comprises a primer 1 (shared upstream primer) with a nucleotide sequence shown as SEQ ID NO.1, a primer 2 (downstream primer) with a nucleotide sequence shown as SEQ ID NO.2, and a primer 3 (downstream primer) with a nucleotide sequence shown as SEQ ID NO. 3; the KASP primer set described above can specifically detect the marker RAC875_c8690_446 on chromosome 5A.
Secondly, the application also provides application of the KASP primer group with the nucleotide sequences shown as SEQ ID NO. 1-SEQ ID NO.3 in identifying the earbud birth density.
Further, the application of the KASP primer group in identifying the tassel birth density specifically refers to performing PCR amplification by using the wheat genome DNA to be detected as a template and using the KASP primer group Y15-SC-KASP as a primer, adding the DNA of Yangma 15 as a control, detecting the PCR amplification product by using a multifunctional enzyme-labeled instrument, genotyping the marker RAC875_c8690_446 on the 5A chromosome at 144.40-145.70 CM by using Kmaster Caller software (Kbioscience), and determining the genotype of the SNP marker RAC875_c8690_446 linked to the tassel birth density related site (the site of the corresponding mutation is T/C) according to the analysis result; if the genotyping result is the same as that of Yangmai 15, the dominant allele T is carried, whereas the allele C is carried, and the wheat containing the allele T has a higher wheat spike biomass than the wheat containing the allele C. Thus, it was converted into a simple, high-throughput KASP (Kompetitive allele specific PCR, http:// www.lgcgroup.com/KASP) marker (Polymarker, http:// polymmarker. Tgac. Uk /) YM15-SC-KASP primer set, which facilitates discrimination between dominant and non-dominant alleles.
Further, the sample wheat is preferably wheat planted in the same ecological area.
Further, the PCR detection described in the above steps means:
preparing KASP marked primer working solution:
respectively taking 30 mu L (100 mu M) of an upstream primer (with a nucleotide sequence shown as SEQ ID NO. 1) and 12 mu L (100 mu M) of a downstream primer (with a nucleotide sequence shown as SEQ ID NO.2 and SEQ ID NO. 3), supplementing to 100 mu L with sterile ultrapure water, and fully and uniformly mixing to serve as a primer working solution marked by KASP for later use;
PCR amplification reaction system: 2. Mu.L (about 30 ng/. Mu.L) of the wheat DNA template to be tested, 0.08. Mu.L of primer working solution, 2.5. Mu.L of KASPMaster Mix (LGC Co., KBS-1016-002) and 5. Mu.L of sterile ultrapure water were used for replenishment;
PCR reaction procedure: firstly, pre-denaturation at 95 ℃ for 15min; secondly, denaturation at 95 ℃ for 20s, 65-57 ℃ (1 ℃ for each cycle down) for 60s, for 9 cycles; thirdly, denaturing for 20s at 95 ℃ and renaturation for 1min at 57 ℃ for 32 cycles; preserving at 10 ℃. Experiments were performed with a control (NTC) without template DNA added to the reaction system, with 1 or more controls per plate.
Wheat seedlings were taken and the genomic DNA of the wheat to be tested was extracted by CTAB (ref: stacey J, isaac P G. Isolation of DNAfrom plants. Methods mol. Biol.1994, 28:9-15.)
The specific detection method comprises the following steps: and (3) taking the wheat genome DNA to be detected as a template, and adopting the KASP primer group and the PCR reagent to carry out PCR amplification to obtain a PCR amplification product. The PCR reaction is performed at S1000 TM Thermal Cycler PCR (Bio-Rad Laboratories Inc.) and the PCR amplified products were scanned for fluorescence using a multifunctional microplate reader (PHERAstar Plus, BMG LABECH, germany). The FAM excitation wavelength is 485nm, and the emission wavelength is 520nm; the VIC excitation wavelength is 535nm, the emission wavelength is 556nm, the system reference fluorescence ROX excitation wavelength is 575nm, and the emission wavelength is 610nm. Genotyping was performed using Kmaster Caller software (Kbioscience), and the genotype of the SNP marker RAC875_c8690_446 linked to the tassel density-related site was determined based on the analysis results.
Thirdly, the application also provides a molecular marker RAC875_c8690_446 specifically amplified by the KASP primer group, the nucleotide sequence of the molecular marker is shown as SEQ ID NO.4, and the 51 st base of the nucleotide sequence from the 5' end is a SNP locus and a T/C allele exists.
Fourth, the application also provides a PCR detection kit for identifying the wheat spike biomass, which comprises a primer 1 with a nucleotide sequence shown as SEQ ID NO.1, a primer 2 with a nucleotide sequence shown as SEQ ID NO.2, and a primer 3 with a nucleotide sequence shown as SEQ ID NO. 3.
Further, the PCR detection kit includes: 2 mu L of wheat DNA template to be detected with the concentration of 30 ng/mu L, 0.08 mu L of primer working solution, 2.5 mu L of KASP Master Mix and 5 mu L of sterile ultrapure water are supplemented; the primer working solution comprises: 130. Mu.L of 100. Mu.M primer, 212. Mu.L of 100. Mu.M primer and 312. Mu.L of 100. Mu.M primer were supplemented to 100. Mu.L with sterile ultra-pure water.
The application firstly finds a marker RAC875_c8690_446 on a wheat 5A chromosome, has the best group specificity and the most obvious correlation with the spike attaching density, and converts the marker into a KASP (kali-bacillus subtilis) marker primer group YM15-SC-KASP, so that the spike attaching density of the wheat can be rapidly screened by the marker through verification, convenience is provided for screening wheat materials with excellent allelic variation in the same ecological area, and the breeding efficiency is improved.
The KASP detection is suitable for breeding varieties (lines) in the middle and downstream regions of the Yangtze river.
Drawings
FIG. 1 is a schematic diagram of a partial genetic linkage map and spike density QTL mapping of FIG. 5A;
in the figure, a, b and c respectively represent the QTL of the spike birth density positioned in 2016, 2017 and average, and the corresponding LOD value on the right side of the QTL.
FIG. 2 is a schematic representation of alleles of the RAC875_c8690_446 marker sequence.
FIG. 3 is a schematic diagram showing the results of amplification detection of test markers of the RIL family of the KASP marker verification section in example 1.
FIG. 4 is a schematic diagram showing the amplification detection results of the test marker of KASP markers applied to a part of wheat lines in example 2.
Detailed Description
The wheat varieties and lines involved in the examples are provided by the institute of agricultural science in the region of the lower river in su
Example 1 screening for stable SNP loci that are significantly correlated with scion attachment density and validating
In this example, 198 parts of recombinant inbred line (F) derived from "Yangmai 15 XYangmai 13 9 ) As a material, the recombinant inbred line and the parents thereof are planted in the gulf head experimental base in Jiangsu Li-lower river region in 2015 and 2016 growing seasons continuously for 2 times. And adopting a random block design, wherein each line is planted in a single row, each row is 50 grains, the row length is 2.0m, the row spacing is 0.23m, the two times of repetition are carried out, and the conventional field management is carried out. The main stems, ear lengths, and spike numbers of 30 individual plants were randomly examined for each line in the wheat filling period of 2016 and 2017, and the average value of 30 individual plants was taken to calculate the average spike attachment density (total spike number/ear length of individual spikes) for each line.
Genomic DNA was extracted by CTAB method (ref: stacey J, isaac P G.isolation of DNA from plants. Methods mol. Biol.1994, 28:9-15.), genotype was obtained by using illuminea 90K chip, and genetic map was constructed. The genotype data was filtered and de-redundant using Icimapping v4.1 software (http:// www.isbreeding.net). The genetic map was constructed and corrected using JoinMap v4.0, compared to existing wheat 90K integration maps to determine the long and short arms of each chromosome, and mapped using MapChart2.3 (https:// www.wur.nl/en/show/Mapchart. Htm). QTL significantly correlated with spike density was detected using the complete interval mapping method (Inclusive composite interval mapping, ICIM) of IciMapping v4.1, with the LOD threshold set to 2.5.
The experiment shows that 1 relatively stable locus QYM15-SC-5A related to the tassel birth density is obtained, the synergistic gene is derived from Yangma 15, namely, the gene for increasing the tassel birth density is derived from Yangma 15, the peak position of the QTL is 144.40-145.70 CM on the 5A chromosome, the corresponding marking intervals are BS00068178 _51-RAC 875_c8690_446 and RAC875_c 8690_446-wsnp_Ex_c 5626_9897389 (shown in the accompanying figures 1 and table 1), the distance is 1.3 CM, and the phenotype contribution rate is 12.90% -25.31%.
TABLE 1 QY15-SC-5A genetic Effect and flanking markers therefor
As shown in fig. 1, 1 synergistic gene is detected by QTL mapping from locus QYM15-SC-5A of Yangmai 15 related to the tassel bearing density, the QTL peak position is 144.40-145.70 CM on the 5A chromosome, the corresponding marker interval in 2015-2016 is BS00068178 _51-RAC 875_c8690_446, the average corresponding marker interval in 2016-2017 is RAC875_c 8690_446-wsnp_ex_c5626_ 9897389, the locus is found to be at the tail end of the long arm of the wheat 5A chromosome by comparison with a wheat reference genome, and is completely inconsistent with the previously reported positions Qsc-5a.1, qsc-5a.2, qsc-5a.3, qsc-5a.4 of the short arm of the 5A chromosome (reference literature: sun Zhongpei, liu Tianxiang, left-hand Xiya, zhaochen, wang Zhonghua, li Chunlian. Analysis of the trait QTL of ordinary wheat ear, wheat theory, 2017,37 (4): 452-457, relating to the markers Xbarc316, IWA825, IWA3530, IWA6166, IWA6412, xbarc319, IWA 3355). The applicant further preliminarily screens SNP markers from the QTL interval according to marker homology, selects the SNP marker with high specificity and highest correlation with the tassel landing density in the interval to carry out KASP marker conversion, determines that the genome of the RAC875_c8690_446 marker has the best specificity and the most obvious correlation with the tassel landing density, and designs KASP primers by using a polymmarker (http:// polymmarker. Tgac. Uk /), wherein the primers are synthesized by the International Biotechnology research institute of Huameric (North) Co. Finally, the RAC875_c8690_446 marker is successfully converted into a KASP marker Y15-SC-KASP, wherein the corresponding mutation site is T/C, namely, the site of the nucleotide sequence SEQ ID NO.4, from the 5' end, of which 51 th base exists a T/C allele (SNP), is shown in FIG. 2; for wheat breeding, the dominant allelic variation is high in the earbud, the Yangmai 15 carries the dominant allelic variation T, and the earbud of the wheat carrying the allele T has higher earbud density than the wheat containing the allele C.
This example designed YM15-SC-KASP primer set for this SNP locus, comprising primer 1 having the nucleotide sequence shown as SEQ ID NO.1, primer 2 having the nucleotide sequence shown as SEQ ID NO.2, and primer 3 having the nucleotide sequence shown as SEQ ID NO. 3. Wherein primer 1 is used as a common primer, and is an upstream primer, and primer 2 and primer 3 are downstream primers. The upstream primer (primer 1) ensures the specificity of the PCR amplified 5A chromosome, and the 3' -end of the downstream primer (primer 2 and primer 3) is the complementary base A/G of the allelic variation base T/C of the marker RAC875_c8690_446.
Preparing KASP marked primer working solution:
30 mu L (100 mu M) of an upstream primer (the nucleotide sequence is shown as SEQ ID NO. 1) and 12 mu L (100 mu M) of a downstream primer (the nucleotide sequence is shown as SEQ ID NO.2 and SEQ ID NO. 3) are respectively taken, and the upstream primer and the downstream primer are supplemented to 100 mu L by sterile ultrapure water and are fully and uniformly mixed to be used as a primer working solution marked by KASP for standby.
PCR amplification reaction system: 2. Mu.L (about 30 ng/. Mu.L) of the wheat DNA template to be tested, 0.08. Mu.L of primer working solution, 2.5. Mu.L of KASPMaster Mix (LGC Co., KBS-1016-002) and 5. Mu.L of sterile ultrapure water were used for replenishment;
PCR reaction procedure: firstly, pre-denaturation at 95 ℃ for 15min; secondly, denaturation at 95 ℃ for 20s, 65-57 ℃ (1 ℃ for each cycle down) for 60s, for 9 cycles; thirdly, denaturing for 20s at 95 ℃ and renaturation for 1min at 57 ℃ for 32 cycles; preserving at 10 ℃. Experiments were performed with a control (NTC) without template DNA added to the reaction system, with 1 or more controls per plate.
Wheat seedlings were taken and the genomic DNA of the wheat to be tested was extracted by CTAB (ref: stacey J, isaac P G. Isolation of DNA from plants. Methods mol. Biol.1994, 28:9-15.)
And (3) taking the wheat genome DNA to be detected as a template, and adopting the KASP primer group and the PCR reagent to carry out PCR amplification to obtain a PCR amplification product. The PCR reaction is performed at S1000 TM Thermal Cycler PCR (Bio-Rad Laboratories Inc.) and the PCR amplified products were scanned for fluorescence using a multifunctional microplate reader (PHERAstar Plus, BMG LABECH, germany). The FAM excitation wavelength is 485nm, and the emission wavelength is 520nm; the VIC excitation wavelength is 535nm, the emission wavelength is 556nm, the system reference fluorescence ROX excitation wavelength is 575nm, and the emission wavelength is 610nm. Genotyping was performed using Kmaster Caller software (Kbioscience), and the genotype of the SNP marker RAC875_c8690_446 linked to the tassel density-related site was determined based on the analysis results.
From 198 parts of "recombinant inbred line of Yangmai 15×Yangmai 13", 74 families were randomly selected and amplified together with two parents according to the method described above, and the detection result is shown in fig. 3. The fluorescence signal data of the amplified products are analyzed by Kmaster Caller software and gathered at a position (blue) close to an X axis in a parting result fluorescence signal coordinate system, and the position is the same as Yangma 15, namely, the genotype of 51 th base (SNP locus) of the 51 st base (SNP locus) of a nucleotide sequence (such as SEQ ID NO. 4) of a molecular marker RAC875_c8690_446 of the wheat is proved to be T; the fluorescence signal data of the amplified products are analyzed by Kmaster Caller software to be gathered at a position (red) close to a Y axis in a coordinate system, and the genotype of the wheat at the SNP locus is proved to be C when the position is different from the type of Yangmai 15; the lower left hand corner of fig. 3 shows a black sample as a blank. The KASP test results and field test results for 74 families along with the two parents are shown in table 2, table 3 and fig. 3.
TABLE 2 average values of the spike-to-shoot densities and KASP typing results for 74 families and parents
As can be seen from Table 2, wheat containing allele T had a higher ear biomass than wheat containing allele C.
TABLE 3 results of the average value T of the earbud birth densities of partial RIL lines carrying the different genotypes of RAC875_c8690_446
The genotype and phenotype of 74 RIL families were tested using a double sample T using Excel 2019, shown in table 3, and the results indicated: the genotype of Yangmai 15 is T, the genotype of Yangmai 13 is C, the average value of the biomass density of the wheat ears of the genotype T in 74 families is 12.0 percent higher than that of the genotype C, and the significant difference exists at the p <0.01 level, which indicates that the primer group of the KASP marker Y16-GF-KASP and the genotype detection system can be applied to molecular marker assisted breeding of the wheat ears biomass density (the statistical method of the table 3 is a conventional method in the field, and the statistical method of the table 3 can be concretely disclosed in the literature of ' GaJun Liu, experimental statistical method ' and Chinese agricultural publishing, 9 month 2000 '). Figure 3 shows that the material typing results are good, the material typing is completely consistent with the chip detection data, which indicates that the KASP mark is successfully developed and can be further used for detecting breeding materials.
EXAMPLE 2 KASP primer set breeding applications
And (3) field test: in this example, the wheat breeding identification nursery grown in the middle and downstream regions of the gulf head in 2018 and 2019 of the agricultural sciences in Jiangsu and lower river region was used as a material, the main stem spike length of the wheat breeding identification nursery grown in the middle and downstream regions of the gulf head in 2019 and 2020 was investigated, the main stem spike length of 30 individual plants was randomly investigated for each line per spike number, the average value of 30 individual plants was taken per spike number, the average spike number per line was calculated, the average value of 2 years was taken, and the KASP primer set obtained in example 1 was used to genotype 80 high-generation lines grown in the wheat breeding identification nursery grown in the agricultural sciences in the first experimental region in the gulf head in 2018-2020 (see fig. 4 and table 4 for detection results). The fluorescence signal data of the amplified products are analyzed and gathered on the parting result by Kmaster Caller software, and the fluorescence signal data is analyzed and gathered on the parting result by Kmaster Caller software to be the same as that of Yangmai 15, namely, the genotype of the wheat strains in a molecular marker RAC875_c8690_446 is proved to be T; if fluorescence signal data of amplified products of wheat strains are different from that of Yangmai 15 in type through Kluster Caller software analysis, the genotype of the wheat strains at the SNP locus is proved to be C, and the wheat spike density of wheat containing allele T is higher than that of wheat containing allele C.
TABLE 4 average value of the spike density and genotype detection results for 80 high-generation lines
TABLE 5 average value T of the tassel birth densities of test lines carrying different genotypes
Table 5 reference: "cover Jun Yuan", test statistical method, chinese agriculture Press, 9 months of 2000). Table 5 the dual sample T test results of Excel 2019 show that: the average value of the earbud biomass of the variety with genotype T is 14.74% higher than that of the variety with genotype C, and the T test result t=8.15 shows that the earbud biomass density of the wheat containing the allele T is higher than that of the wheat containing the allele C at the p <0.01 level. Meanwhile, the primer group and the genotype detection system of the KASP marker YM15-SC-KASP can be applied to molecular marker assisted selective breeding of wheat spike biomass.
From the above experimental results, it can be derived that: the PCR amplification is carried out on the wheat genome DNA by the primer 1, the primer 2 and the primer 3, whether the wheat genome DNA carries the gene with high spike attaching density of the Yangmai 15 can be directly judged by KASP typing, the detection method is simple to operate, the detection result is very visual, and the detection effect is obvious and effective.
Sequence listing
<110> institute of agricultural science in Jiangsu Li-Shanghai region
<120> KASP primer set for detecting wheat spike number and biomass and use thereof
<141> 2021-01-29
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
tcatctcgtg tgatctcata ccgaa 25
<210> 2
<211> 43
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
gaaggtgacc aagttcatgc tgctttgctt tatgcccata cca 43
<210> 3
<211> 43
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
gaaggtcgga gtcaacggat tgctttgctt tatgcccata ccg 43
<210> 4
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<220>
<221> mutation
<222> (51)..(51)
<223> t is c
<400> 4
ggcttgatat ctgcatgagg cgtcatctcg tgtgatctca taccgaatag tggtatgggc 60
ataaagcaaa gctatacgac tatagaaatg ttcaatgact g 101
Claims (6)
1. A KASP primer set for detecting wheat ear biomass, wherein the KASP primer set comprises: primer 1 with nucleotide sequence shown as SEQ ID NO.1, primer 2 with nucleotide sequence shown as SEQ ID NO.2, and primer 3 with nucleotide sequence shown as SEQ ID NO. 3.
2. The use of a set of KASP primers according to claim 1 for detecting wheat ear biomass.
3. The use according to claim 2, wherein the use is that the KASP primer set is used as a primer, the genomic DNA of the wheat to be tested is used as a template for PCR amplification, and then the PCR amplification product is detected and genotyping is performed by using a multifunctional enzyme-labeled instrument, and if the amplification result shows that the genotyping of the wheat to be tested is the same as that of yangmei 15, the wheat to be tested contains an allele T; otherwise, the wheat to be tested contains allele C; wheat containing allele T had a higher ear biomass than wheat containing allele C.
4. The use according to claim 3, wherein the PCR amplification is:
PCR reaction system: 2 mu L of wheat DNA template to be detected with the concentration of 30 ng/mu L, 0.08 mu L of primer working solution and 2.5 mu L of KASP Master Mix are supplemented to 5 mu L by sterile ultrapure water;
the preparation method of the primer working solution comprises the following steps: 30. Mu.L of primer 1 at a concentration of 100. Mu.M, 12. Mu.L of primer 2 at a concentration of 100. Mu.M, 12. Mu.L of primer 3 at a concentration of 100. Mu.M, and 100. Mu.L of the mixture were supplemented with sterile ultrapure water;
a PCR reaction procedure; firstly, pre-denaturation at 95 ℃ for 15min; second, denaturation at 95℃of 20s, 60s at 65-57℃with 1℃drop per cycle for 9 cycles; thirdly, denaturation at 95 ℃ for 20s, renaturation at 57 ℃ for 1min and 32 cycles.
5. A PCR detection kit for detecting the earpick density comprises a common primer 1 with a nucleotide sequence shown as SEQ ID NO.1, a primer 2 with a nucleotide sequence shown as SEQ ID NO.2 and a primer 3 with a nucleotide sequence shown as SEQ ID NO. 3.
6. The PCR detection kit of claim 5, wherein the PCR detection kit comprises: 2 mu L of wheat DNA template to be detected with the concentration of 30 ng/mu L, 0.08 mu L of primer working solution, 2.5 mu L of KASP Master Mix and 5 mu L of sterile ultrapure water are supplemented;
the preparation method of the primer working solution comprises the following steps: 30. Mu.L of primer 1 at a concentration of 100. Mu.M, 12. Mu.L of primer 2 at a concentration of 100. Mu.M, 12. Mu.L of primer 3 at a concentration of 100. Mu.M were supplemented to 100. Mu.L with sterile ultrapure water.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110129159.3A CN112760402B (en) | 2021-01-29 | 2021-01-29 | KASP primer group for detecting wheat spike number and biomass density and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110129159.3A CN112760402B (en) | 2021-01-29 | 2021-01-29 | KASP primer group for detecting wheat spike number and biomass density and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112760402A CN112760402A (en) | 2021-05-07 |
| CN112760402B true CN112760402B (en) | 2024-01-26 |
Family
ID=75704115
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110129159.3A Active CN112760402B (en) | 2021-01-29 | 2021-01-29 | KASP primer group for detecting wheat spike number and biomass density and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112760402B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115786565B (en) * | 2022-09-20 | 2023-07-18 | 四川农业大学 | SNP molecular marker linked with wheat spike number QTL and application thereof |
| CN116769961B (en) * | 2023-07-28 | 2024-01-26 | 四川农业大学 | Wheat spike number per spike QTL linkage molecular marker developed by multi-sieve-mixing-determining four-step method and application |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109182586A (en) * | 2018-10-25 | 2019-01-11 | 江苏省农业科学院 | KASP primer sets and its application for wheat scab resistance detection |
| CN109811074A (en) * | 2018-10-26 | 2019-05-28 | 中国科学院成都生物研究所 | A kind of label and its application for assisting detection wheat Gao Dansui spikelet number character |
| CN109825621A (en) * | 2019-02-22 | 2019-05-31 | 四川农业大学 | Wheat spikelet number QTL chain SNP marker and its application |
| CN112080582A (en) * | 2020-10-26 | 2020-12-15 | 中国科学院遗传与发育生物学研究所农业资源研究中心 | KASP molecular marker closely linked with spike length major QTL locus and application thereof |
-
2021
- 2021-01-29 CN CN202110129159.3A patent/CN112760402B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109182586A (en) * | 2018-10-25 | 2019-01-11 | 江苏省农业科学院 | KASP primer sets and its application for wheat scab resistance detection |
| CN109811074A (en) * | 2018-10-26 | 2019-05-28 | 中国科学院成都生物研究所 | A kind of label and its application for assisting detection wheat Gao Dansui spikelet number character |
| CN109825621A (en) * | 2019-02-22 | 2019-05-31 | 四川农业大学 | Wheat spikelet number QTL chain SNP marker and its application |
| CN112080582A (en) * | 2020-10-26 | 2020-12-15 | 中国科学院遗传与发育生物学研究所农业资源研究中心 | KASP molecular marker closely linked with spike length major QTL locus and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| Transcript-specific, single-nucleotide polymorphism discovery and linkage analysis in hexaploid bread wheat (Triticum aestivum L.);Alexandra M. Allen 等;Plant Biotechnology Journal(第9期);1086–1099 * |
| 小麦粒重主效QTL近等基因系的构建和效应评价;孔忠新 等;麦类作物学报;第37卷(第3期);312-318 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112760402A (en) | 2021-05-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110295251B (en) | SNP molecular marker linked with wheat effective tillering number QTL and application thereof | |
| CN110656198B (en) | Wheat plant height related KASP primer group and application thereof | |
| CN112981001B (en) | Molecular marker BnC04Y2255 for identifying dwarf cabbage type rape and application thereof | |
| CN109055395B (en) | Photoperiod insensitive Hd1 allele and molecular marker and application thereof | |
| CN112760402B (en) | KASP primer group for detecting wheat spike number and biomass density and application thereof | |
| CN112080582A (en) | KASP molecular marker closely linked with spike length major QTL locus and application thereof | |
| CN115786565B (en) | SNP molecular marker linked with wheat spike number QTL and application thereof | |
| CN109762812A (en) | Wheat growth gesture related SNP and its as target spot identification wheat growth gesture character in application | |
| CN111778352A (en) | KASP primer group related to wheat grain weight and application thereof | |
| CN112941232B (en) | Wheat scab resistance related molecular marker and application thereof | |
| CN113273489B (en) | Molecular marker-assisted breeding method for high-yield wheat with resistance to gibberellic disease | |
| CN113736910A (en) | Linkage molecular marker of peanut single plant pod number main effect QTL site qPN7 and application thereof | |
| CN109811077B (en) | KASP marker closely linked with wheat dwarf gene and application thereof | |
| CN116590466A (en) | KASP primer group related to wheat grain weight and application | |
| CN114480698B (en) | Molecular marker related to wheat set spike number and/or grain hardness characteristics and application | |
| CN115927718A (en) | KASP molecular marker for identifying wheat grain weight and grain width and application thereof | |
| CN115852021A (en) | SNP molecular marker for identifying wheat grain weight and grain length and application thereof | |
| CN109439788A (en) | KASP molecular labeling and its application with Plant Height in Wheat major gene loci close linkage | |
| CN112779348B (en) | Major QTL site of ear number per unit area of wheat, KASP primer closely linked with major QTL site and application of KASP primer | |
| CN115029465A (en) | KASP and dCAPS markers cosegregating with rape seed secondary dormancy major QTL and application thereof | |
| CN109913580B (en) | Application of molecular marker closely linked with rice blast resistance gene | |
| CN108728576B (en) | Marker of wheat chlorophyll content related gene and application thereof | |
| Sanghvi et al. | Molecular markers in plant biotechnology | |
| CN111004857A (en) | Molecular marker primer of major QTL (quantitative trait locus) site of soybean branch number and application of molecular marker primer | |
| CN108841994B (en) | Green-keeping related gene marker in winter wheat under genetic background of wheat 895 and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |