CN111363846A - Molecular marker for detecting wheat grain weight gene QTkw.saas-2D and application - Google Patents
Molecular marker for detecting wheat grain weight gene QTkw.saas-2D and application Download PDFInfo
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Abstract
The invention provides a molecular marker for detecting wheat grain weight gene QTkw.saas-2D and application thereof, belonging to the field of molecular genetic breeding. The invention provides an application of a substance for detecting polymorphism or genotype of an SNP marker XCRI-GJ043 in identification or auxiliary identification of wheat grain weight; the molecular marker XCRI-GJ043 is 526252853 th nucleotide on a wheat 2D chromosome, and the genotype of the molecular marker XCRI-GJ043 is GG, AA or GA. The invention can realize the rapid detection of the wheat grain weight gene QTkw.saas-2D and provides a molecular auxiliary selection means for the breeding of new high-yield wheat varieties.
Description
Technical Field
The invention belongs to the field of molecular genetic breeding, and particularly relates to a molecular marker for detecting wheat grain weight gene QTkw.saas-2D and application thereof.
Background
Wheat (Triticum aestivum L.) is one of the most important grain crops in the world, with the yield second only to corn and rice, the third largest grain crop. The worldwide wheat planting area accounts for 17.0 percent of the worldwide total grain planting area (2.17 hundred million hectares), the perennial wheat planting area in China is about 2500 million hectares, and the worldwide wheat planting area accounts for 21.8 percent of the national total grain planting area (1.13 hundred million hectares) (FAO, 2012; national State administration of statistics, 2014). Wheat is one of the most important food crops because it lives 40% of the world's population and provides 20% of the human energy and protein supply. It is reported that by 2050, the population of the world is more than 90 hundred million, and to meet the food demand of human beings, the food yield is at least improved by 70-100% based on the existing yield (680 ten thousand tons) (Paroda R, Dasguba S, Mal B, et al. proceedings of the same regional regulation on improving the yield in Asia; Bangkok (Thailand),26-27, Apr 2012), so that how to improve the yield is a huge problem in front of wheat breeders at present.
Wheat grain weight is a quantitative trait controlled by multiple genes, and multiple genes or QTLs associated with controlling wheat grain weight have been located (Kato K, Miura H, Sawada S. mapping QTLs controlling grain and Itscompositions on chromosom 5 Aofwhit. Theroreappl Gene, 2000,101: 1114;. KumarN, Kulwal PL, Gaura, TyagiAK, Khuran JP, Khuran P, Banyan HS, Gupta PK. QTLanalysis for grain weight in wheat grain and Euphyto, 2006,151: 135-. The subject group utilizes RIL group, combines genotype and phenotype analysis to position a new gene QTKW.saas-2D for controlling wheat grain weight, can explain 11.40% -23.62% of phenotype variation, and comparative analysis shows that the new gene is a new gene for controlling wheat grain weight and grain length, and is in urgent need of accelerating the excavation and utilization process. The development of molecular markers closely linked with the wheat and the genetic improvement of high-yield wheat are very necessary.
Disclosure of Invention
The invention aims to provide a molecular marker for detecting wheat grain weight gene QTkw.saas-2D and application thereof; the molecular marker is a co-dominant molecular marker closely linked with wheat grain weight gene QTkw.saas-2D; the molecular marker can realize the rapid detection of wheat grain weight gene QTkw.saas-2D, thereby being applied to high-yield wheat breeding.
In order to achieve the above purpose, the invention provides the following technical scheme:
a molecular marker for detecting wheat grain weight gene QTkw.saas-2D, which is named as XCRI-GJ 043; the molecular marker XCRI-GJ043 is located at 526252853 th nucleotide on the 2D chromosome of wheat, and the genotype of the molecular marker XCRI-GJ043 is GG, AA or GA.
Preferably, primer F1, primer F2 and primer R are included;
the nucleotide sequence of the primer F1 is shown as SEQ ID NO.1, and the 5' end G of the F1 is connected with a FAM group;
the nucleotide sequence of the primer F2 is shown as SEQ ID NO.2, and the 5' end of the F2 is connected with a HEX group;
the nucleotide sequence of the primer R is shown as SEQ ID NO. 3.
The invention provides a kit for detecting wheat grain weight gene QTkw.saas-2D, which comprises the primer group.
Preferably, a PCR reaction buffer is also included.
The invention provides application of the molecular marker, the primer group or the kit in wheat grain weight detection.
Preferably, the method comprises the following steps: performing PCR amplification by using the wheat genome DNA to be detected as a template and the primer group as a primer to obtain an amplification product; when the fluorescence development of the amplification product is blue fluorescence, the wheat to be detected is high grain weight wheat; and when the fluorescence development of the amplification product is red fluorescence, the wheat to be detected is the wheat with low grain weight.
The invention provides application of the molecular marker, the primer group or the kit in comparison of different wheat grain weights.
Preferably, the method comprises the following steps: respectively taking wheat genome DNA to be compared as templates, and respectively taking the primer groups as primers to carry out PCR amplification to obtain amplification products; the grain weight of wheat in which the amplified product is developed into blue by fluorescence is larger than that of wheat in which the amplified product is developed into red by fluorescence.
The invention provides application of the molecular marker, the primer group or the kit in wheat breeding assistance.
Preferably, in the breeding process of wheat, the genotype of the molecular marker XCRI-GJ043 of wheat is detected, and the wheat with the genotype of GG is selected for subsequent breeding.
The invention has the beneficial effects that: the molecular marker for detecting the wheat grain weight gene QTkw.saas-2D provided by the invention is a co-dominant molecular marker closely linked with the wheat grain weight gene QTkw.saas-2D; the molecular marker can realize the rapid detection of wheat grain weight gene QTkw.saas-2D; the primer group or the kit for amplifying the molecular marker can effectively screen out a wheat strain line containing the QTkw.saas-2D gene in the seedling stage, so that the experiment cost is saved, the wheat strain line with high thousand grain weight can be quickly screened out, the selection efficiency is improved, and the molecular breeding process of high-yield wheat is accelerated.
Drawings
FIG. 1 is a genetic linkage diagram of a QTkw.saas-2D gene in example 1 of the present invention;
FIG. 2 shows the results of XCRI-GJ043 marker typing in example 2 of the present invention;
FIG. 3 is a relationship between QTkw.saas-2D allele and thousand kernel weight in the wheat RIL population of example 2 of the present invention.
Detailed Description
The invention provides a molecular marker for detecting wheat grain weight gene QTkw.saas-2D, which is named as XCRI-GJ 043. In the invention, the molecular marker XCRI-GJ043 is a co-dominant molecular marker closely linked with a wheat grain weight gene QTkw. In the invention, the molecular marker XCRI-GJ043 is located at 526252853 th nucleotide on the 2D chromosome of wheat, and the genotype of the molecular marker XCRI-GJ043 is GG, AA or GA.
The invention provides a primer group for amplifying the molecular marker, which comprises a primer F1, a primer F2 and a primer R; the nucleotide sequence of the primer F1 is shown as SEQ ID NO.1, and specifically comprises the following steps: 5'-GAAGGTGACCAAGTTCATGCTAATTTGATGGCAGCAACTGTAAGAG-3', respectively; the 5' end of the F1 is connected with a FAM group; the nucleotide sequence of the primer F2 is shown as SEQ ID NO.2, and specifically comprises the following steps: 5'-GAAGGTCGGAGTCAACGGATTCAATTTGATGGCAGCAACTGTAAGA-3', respectively; the 5' end of the F2 is connected with a HEX group; the nucleotide sequence of the primer R is shown as SEQ ID NO.3, and specifically comprises the following steps: 5' -GAGGAGCAAATGCATACCAACCCAA-3. In the present invention, the molar ratio of the primer F1, the primer F2 and the primer R is preferably 1:1: 1.
The invention provides a kit for detecting wheat grain weight gene QTkw.saas-2D, which comprises the primer group, wherein the kit preferably further comprises a PCR reaction buffer solution, the PCR reaction buffer solution is preferably 2 × Mastermix, and the kit preferably further comprises sterilized double distilled water.
The invention provides application of the molecular marker, the primer group or the kit in wheat grain weight detection.
In the present invention, the application preferably comprises the steps of: performing PCR amplification by using the wheat genome DNA to be detected as a template and the primer group as a primer to obtain an amplification product; when the fluorescence development of the amplification product is blue fluorescence, the wheat to be detected is high grain weight wheat; and when the fluorescence development of the amplification product is red fluorescence, the wheat to be detected is the wheat with low grain weight. In the present invention, the grain weight is thousand or hundred; in the specific implementation process of the invention, the thousand seed weight is adopted.
In the invention, the source of the wheat genome DNA to be detected is not particularly limited, and the wheat genome DNA obtained by extracting by adopting a conventional DNA extraction method in the field can be used. In the invention, the concentration of the wheat genome DNA to be detected is preferably 45-55 ng/muL, and more preferably 50 ng/muL. In the present invention, the concentration of the primer F1, the primer F2 and the primer R is independently 10 pmol/. mu.L.
In the present invention, the PCR amplification system comprises 2 × Master mix 1.25. mu.L, primer F1, primer F2 and primer R each 0.05. mu.L, sterilized double distilled water 2.35. mu.L, and 50ng DNA template 1.25. mu.L in terms of 5.0. mu.L. in the present invention, the PCR amplification procedure is as follows:
pre-denaturation at 94 ℃ for 15 min;
denaturation at 94 ℃ for 20s, annealing/extension at 61 ℃ for 60 s; denaturation at 94 ℃ for 20s, annealing/extension at 60.4 ℃ for 60 s; denaturation at 94 ℃ for 20s, annealing/extension at 59.8 ℃ for 60s … … for 10 cycles, with the annealing/extension temperature decreasing by 0.6 ℃ per cycle;
denaturation at 94 ℃ for 20s, annealing/extension at 55 ℃ for 1min, for a total of 26 cycles;
storing at 4 ℃.
After the PCR amplification is finished, the amplified PCR amplification product is scanned by a LuxScan-10K/D scanner to generate a tif file, the tif file is converted into a data signal value through software, and then the data signal value is typed through a typing software SNPTyper. When the fluorescence development of the amplification product is blue fluorescence, the gene QTkw.saas-2D of the wheat to be detected is GG homozygous type; the wheat to be detected is high-grain-weight wheat; when the fluorescence development of the amplification product is red fluorescence, the gene QTkw.saas-2D of the wheat to be detected is AA homozygous; the wheat to be detected is low grain weight wheat. And when the fluorescence development of the amplification product is green fluorescence, the gene QTkw.saas-2D of the wheat to be detected is AG heterozygosis.
The invention also provides application of the molecular marker, the primer group or the kit in comparison of different wheat grain weights.
In the present invention, the application preferably comprises the steps of: respectively taking wheat genome DNA to be compared as templates, and respectively taking the primer groups as primers to carry out PCR amplification to obtain amplification products; the thousand kernel weight of wheat of which the amplification product is in fluorescent coloration blue is greater than that of wheat of which the amplification product is in fluorescent coloration red. In the present invention, the amplification system, the amplification procedure and the method for detecting fluorescent signal of the PCR amplification are the same as the above-mentioned method for detecting the thousand-grain weight of wheat, and are not described herein again.
The invention provides application of the molecular marker, the primer group or the kit in wheat breeding assistance. In the invention, in the wheat breeding process, extracting the genome DNA of a wheat plant in the seedling stage of the wheat plant, and carrying out PCR amplification and fluorescence detection by using the genome DNA as a template according to the method; and screening the high grain weight wheat or the low grain weight wheat according to the breeding demand for subsequent breeding.
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The following example jimai 22 is described in the following documents: li Haosheng, Liu Jian Jun, Song Jian Min, etc. high-yield stable-yield disease-resistant eurytopic wheat new variety-Jimai 22[ J ] wheat crop academic newspaper, 2007, 27(4): 744. The public is available from the institute of agricultural sciences, Shandong province, to repeat the experiments of the present application, and is not available for other uses.
The following examples are purple wheat No. 2: the new wheat strain is obtained by separating a single plant from a dwarf-male-sterile alternate selection population (dwarf-male-sterile wheat/Shannon purple wheat No. 1) and breeding by a pedigree method, and is preserved by Guojun assistant researchers at the crop research institute of Shandong academy of agricultural sciences. The public is available from the institute of agricultural sciences, Shandong province, to repeat the experiments of the present application, and is not available for other uses.
Example 1
Establishment of wheat grain weight gene QTkw. saas-2D closely linked molecular marker development and detection method, identification of thousand grain weight phenotype of RIL population
Hybridizing a wheat variety Jimai 22 with a purple wheat variety No.2 to obtain F1, and selfing to obtain F2; using single seed descent, a RIL population comprising 93 lines was obtained. Thousand-grain weight shape of RIL group is identified in two environments of 2017, 2018 and 2018, 2019, field experiments are carried out in Lingcheng and Jinan test base of crop research institute of agricultural academy of sciences in Shandong province, and random block design is adopted in the experiments, the row spacing is 33cm, and the row length is 3 m. Two rows were planted per material, three replicates. And (3) harvesting grains in the mature period of the wheat, and measuring the thousand seed weight by using a seed test instrument, wherein each material has at least 500 grains and is repeated for 3 times.
Second, genotype scanning and genetic linkage map construction
The wheat 55K SNP chip is used for obtaining the genotypes of two parents and 93 strains, and the QTL analysis software IciMapping 4.1 is used for constructing the genetic linkage map of the RIL group.
(one) obtaining genotypes of two parents and 350 lines
93 RIL strains and 2 parents are extracted by CTAB method, which refers to Guo, J., Zhang, X, Hou, Y, Cai, J, Shen, X, Zhou, T, et al (2015), High-density mapping of the macro-order FHB7 derived from the thin nucleic acid and nucleic acid encoding with Fhb1 by marker-assisted selection, therapeutic and applied genes (128, 2301) 2316.
The integrity of the DNA samples was checked using agarose gels and the DNA concentration and quality was determined using Nanodrop (A260/280 and A260/230). After the detection is qualified (the total amount of DNA is more than 1 mu g, the DNA is complete, the RNA pollution is avoided, A260/280 and A260/230 meet the requirements), the DNA of a sample is hybridized with a wheat 55K SNP chip according to the requirements of the American African company operation manual AXIOMArray 2.0, and the obtained original data is subjected to SNP marking quality control to obtain the genotypes of two parents and 93 strains.
(II) constructing a genetic linkage map of the RIL population by using QTL analysis software IciMapping 4.1
And (3) screening the markers showing polymorphism among parents according to the genotype information obtained by the chip, and removing the markers with deletion rate of more than 10% and heterozygosity of more than 10%. All the remaining markers were clustered using the BIN function of QTL analysis software IcMapping 4.1 (Winfield, M.O., Allen, A.M., Burridge, A.J., Barker, G.L., Benbow, H.R., Wilkinson, P.A., Coghill, J., Waterfall, C., Davassi, A., Scopes, G, High-density SNPgenetic array for hexagonal and its second and third genes, Plant Biotechnology Journal, 2016, 14 (5): 1195) 1206. markers with the same separation type only retained one marker to represent the BIN marker. The significance of the separation of the markers was checked using the chi-square. By utilizing the function of MAP in IcMapping 4.1, BIN markers are firstly divided into different linkage groups (the LOD value is set to be 3.0), then sequencing is carried out by utilizing an nnTwoOpt algorithm, the sequencing of the markers is adjusted by taking SARF as a standard, the long and short arms of chromosomes are corrected according to the physical positions of the markers, and finally, a genetic linkage MAP is output.
(III) control of thousand kernel weight Gene analysis
Combining the genetic linkage map constructed in the second step with thousand-kernel-weight phenotype data under 4 environments in the first step, making an input file, utilizing a QTL positioning function (QTLmap in bi-partial locations, BIP) of an amphiphilic population in QTL analysis software IciMapping 4.1, carrying out additive effect QTL positioning by an Infinite Composite Interval Mapping (ICIM) based on stepwise regression, and selecting default values (the step length is set to be 1.0, the PIN is 0.001, the LOD value is set to be 2.5, and the ICIM-ADD is selected in the method).
As shown in figure 1, a control granule re-gene locus is located in the range of 521.5-527.4 Mb of 2D chromosome, can explain 14.10% -22.45% of phenotype variation, and is closely linked with AX-109875224(XCRI-GJ043), so that the molecular marker is developed into KASP molecular marker, and is conveniently applied to other materials.
Third, design of specific primer and establishment of method
Aiming at two alleles of a target SNP marker AX-109875224, designing a set of KASP primers, wherein the set comprises 2 upstream primers and 1 universal downstream primer;
the primer sequence of the SNP marker XCRI-GJ043 is as follows:
XCRI-GJ043-F1:5′-AATTTGATGGCAGCAACTGTAAGAG-3′(SEQ ID NO.4);
XCRI-GJ043-F2:5′-CAATTTGATGGCAGCAACTGTAAGA-3′(SEQ IDNO.5);
XCRI-GJ043-R:5′-GAGGAGCAAATGCATACCAACCCAA-3′(SEQ ID NO.3);
the fluorescent label FAM is 5'-GAAGGTGACCAAGTTCATGCT-3' (SEQ ID NO. 6);
the fluorescent tag HEX is 5'-GAAGGTCGGAGTCAACGGATT-3' (SEQ ID NO. 7).
A single-stranded DNA molecule primer F1 obtained by adding a fluorescent label FAM to the 5' end of the single-stranded DNA molecule shown in SEQ ID NO. 4; amplifying a fragment with the genotype of GG (namely the genotype of the SNP marker XCRI-GJ043 is homozygous for G) by using the primer F1 and the single-stranded DNA molecule shown by SEQ ID NO.3, and irradiating a product carrying the FAM sequence after PCR amplification by using fluorescence to show blue;
the single-stranded DNA molecule primer F2 obtained by adding a fluorescent label HEX at the 5' end of the single-stranded DNA molecule shown in SEQ ID NO.5 and the single-stranded DNA molecule shown in SEQ ID NO.3 amplify the segment of which the genotype of the SNP marker XCRI-GJ043 is AA (namely the SNP marker XCRI-GJ043 is homozygous for A), and the product carrying the HEX sequence after PCR amplification shows red color by fluorescent irradiation.
Fourth, SNP typing
Extracting genome DNA of young wheat leaves according to the method, and performing PCR amplification by using the genome DNA as a template and adopting a primer XCRI-GJ043-F1, a primer XCRI-GJ043-F2 and a primer XCRI-GJ 043-R.
The volume of the PCR reaction system is 5.0 mu L, including 2 × Master mix1.25 mu L, primers XCRI-GJ043-F1, XCRI-GJ043-F2 and XCRI-GJ043-R each 0.05 mu L, sterilized double distilled water 2.35 mu L, and 50ng DNA template 1.25 mu L.
The reaction procedure of the PCR amplification can be specifically as follows:
pre-denaturation at 94 ℃ for 15 min;
denaturation at 94 ℃ for 20s, annealing/extension at 61 ℃ for 60 s; denaturation at 94 ℃ for 20s, annealing/extension at 60.4 ℃ for 60 s; denaturation at 94 ℃ for 20s, annealing/extension at 59.8 ℃ for 60s … … for 10 cycles, with the annealing/extension temperature decreasing by 0.6 ℃ per cycle;
denaturation at 94 ℃ for 20s, annealing/extension at 55 ℃ for 1min, for a total of 26 cycles;
storing at 4 ℃.
Scanning the reaction product by a LuxScan-10K/D scanner to generate a tif file, converting the tif file into a data signal value through software, and typing through a typing software SNPTyper. The genotype showing blue fluorescence was GG, and the genotype showing red fluorescence was AA. The results are shown in FIG. 2, wherein the upper left of the graph is the strain with the SNP marker XCRI-GJ043 genotype of AA, and the lower right is the strain with the SNP marker XCRI-GJ043 genotype of GG.
Example 2
Molecular marker XCRI-GJ 043-based analysis of relationship between QTkw.saas-2D allele and thousand kernel weight
93 RIL DNAs were extracted by the two CTAB method in example 1, and SNP typing was performed on 93 DNA samples by the four steps in example 1.
The RIL group allele analysis shows that the average thousand-grain weight of the material with the genotype of QTkw.saas-2Da is higher than that of the material with the genotype of QTkw.saas-2Db (the difference is 5.16g, and P is 4.08 × e-7), which indicates that the QTkw.saas-2D gene controls the height of the wheat grain weight and can be tracked and detected by using an XCRI-GJ043 molecular marker (shown in figure 3).
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
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Claims (10)
1. A molecular marker for detecting wheat grain weight gene QTkw.saas-2D, which is characterized in that the molecular marker is named XCRI-GJ 043; the molecular marker XCRI-GJ043 is located at 526252853 th nucleotide on the 2D chromosome of wheat, and the genotype of the molecular marker XCRI-GJ043 is GG, AA or GA.
2. The primer group for amplifying the molecular marker of claim 1, which comprises a primer F1, a primer F2 and a primer R;
the nucleotide sequence of the primer F1 is shown as SEQ ID NO.1, and the 5' end of the F1 is connected with a FAM group;
the nucleotide sequence of the primer F2 is shown as SEQ ID NO.2, and the 5' end of the F2 is connected with a HEX group;
the nucleotide sequence of the primer R is shown as SEQ ID NO. 3.
3. A kit for detecting wheat grain weight gene qtkw.saas-2D, comprising the primer set of claim 2.
4. The kit of claim 3, further comprising a PCR reaction buffer.
5. Use of the molecular marker of claim 1, the primer set of claim 2, or the kit of claim 3 for detecting wheat grain weight.
6. Use according to claim 5, characterized in that it comprises the following steps: performing PCR amplification by using the wheat genome DNA to be detected as a template and the primer group as a primer to obtain an amplification product; when the fluorescence development of the amplification product is blue fluorescence, the wheat to be detected is high grain weight wheat; and when the fluorescence development of the amplification product is red fluorescence, the wheat to be detected is the wheat with low grain weight.
7. Use of the molecular marker of claim 1, the primer set of claim 2, or the kit of claim 3 or 4 for comparison of different wheat grain weights.
8. Use according to claim 7, characterized in that it comprises the following steps: respectively taking wheat genome DNA to be compared as templates, and respectively taking the primer groups as primers to carry out PCR amplification to obtain amplification products; the grain weight of wheat in which the amplified product is developed into blue by fluorescence is larger than that of wheat in which the amplified product is developed into red by fluorescence.
9. Use of the molecular marker of claim 1, the primer set of claim 2, or the kit of claim 3 or 4 for assisting in wheat breeding.
10. The use according to claim 9, wherein during the wheat breeding process, the genotype of the molecular marker XCRI-GJ043 of wheat is detected, and wheat with the genotype of GG is selected for subsequent breeding.
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CN111893209A (en) * | 2020-08-27 | 2020-11-06 | 中国科学院遗传与发育生物学研究所农业资源研究中心 | Detection marker for insertion deletion site related to thousand grain weight of wheat and application of detection marker |
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CN109402284A (en) * | 2018-10-26 | 2019-03-01 | 山东省农业科学院作物研究所 | One main effect QTL for influencing wheat spike length and its application |
CN110241248A (en) * | 2019-07-09 | 2019-09-17 | 中国科学院遗传与发育生物学研究所 | KASP label relevant to Grain Weight in Common Wheat under condition of salt stress and its application |
CN110527742A (en) * | 2019-09-19 | 2019-12-03 | 中国科学院遗传与发育生物学研究所 | To the long relevant KASP label of wheatear under low-salt conditions and its application |
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CN109402284A (en) * | 2018-10-26 | 2019-03-01 | 山东省农业科学院作物研究所 | One main effect QTL for influencing wheat spike length and its application |
CN110241248A (en) * | 2019-07-09 | 2019-09-17 | 中国科学院遗传与发育生物学研究所 | KASP label relevant to Grain Weight in Common Wheat under condition of salt stress and its application |
CN110527742A (en) * | 2019-09-19 | 2019-12-03 | 中国科学院遗传与发育生物学研究所 | To the long relevant KASP label of wheatear under low-salt conditions and its application |
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CN111893209A (en) * | 2020-08-27 | 2020-11-06 | 中国科学院遗传与发育生物学研究所农业资源研究中心 | Detection marker for insertion deletion site related to thousand grain weight of wheat and application of detection marker |
CN111893209B (en) * | 2020-08-27 | 2023-06-23 | 中国科学院遗传与发育生物学研究所农业资源研究中心 | Indel site detection marker related to thousand grain weight of wheat and application thereof |
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