CN110616276B - Method for assisting in identifying surface area characters of wheat seedling-stage roots and special primer group thereof - Google Patents
Method for assisting in identifying surface area characters of wheat seedling-stage roots and special primer group thereof Download PDFInfo
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Abstract
The invention discloses a method for assisting in identifying the surface area character of a wheat seedling root and a special primer group thereof. The invention provides application of a substance for detecting the genotype of an SNP AX95025477 locus on a wheat chromosome 7BL in identification or auxiliary identification of wheat root system characters; the SNP AX95025477 site is the 36 th site of the nucleotide sequence shown in the sequence 4. The invention discloses an SNP locus AX95025477 related to root system characters in a wheat seedling stage. The SNP locus can be used for screening wheat with excellent related root system characters, and plays an important role in cultivating water-fertilizer efficient wheat varieties.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for assisting in identifying the surface area character of a wheat seedling-stage root and a special primer set thereof.
Background
The root system, one of the plant three major organs, is the first site of mineral nutrient absorption and water conduction, and plays a role in fixing and supporting the growth of plants (guo heiyu, 2013). The first green revolution improves the yield of grains by improving the traits of the overground parts of crops (such as dwarf breeding) and sufficiently supplying rich water, but the variation of the underground parts is not taken into consideration, and the improvement of the underground root system plays an important role in improving the eurytopic property of wheat and the utilization efficiency of the rich water (Li jin, 2009; Gewin, 2010; ZHao, 2011; Zhang, 2014). Den Herder et al (2010) indicated that it is difficult to guarantee the planting yield by continuously increasing the amount of fertilizer and water used to increase the yield of wheat, and it is necessary to improve the nutrient absorption and nutrient utilization of crops and improve the fertilizer and water utilization efficiency, and the core content of the improvement is to improve the root system. The genetic analysis is carried out on the wheat root system characteristics under the specific environmental conditions, the genetic regulation and control mechanism of the root system growth and development in the seedling stage is excavated, the scientific basis can be provided for the molecular marker-assisted screening and the genetic engineering means to improve the root system characteristics, and the method has important significance for cultivating new wheat varieties with suitable root system configurations under specific climates and soil environments such as nitrogen and phosphorus reduction, drought and the like.
The supply of nitrogen and phosphorus can significantly affect the development and the morphogenesis of the wheat root system. Under the condition of low nitrogen, the root system development is seriously hindered, the dry weight is reduced, and further, the transportation of nutrients to the overground part is influenced, so that the tillering is reduced, and the yield is reduced. Narayanan et al (2014) studies showed a significant positive correlation of dry root weight and total root surface area with dry stem leaf weight, and a significant positive correlation of root number and total root length with grain yield (Xie et al, 2017). The ideal root system configuration is different under different soil environment conditions, and the 'deep root system' is beneficial to the crop to adapt to drought (Ludlow and Muchow, 1990; Wasson et al.,2012) and low nitrogen environment (Garnett et al., 2009); and the 'shallow root system' is beneficial to the adaptation of crops to low-phosphorus environment (Lynch, 2011).
The discovery of genes for controlling root system characters and gene markers thereof is a precondition for developing molecular marker-assisted breeding, and the genetic analysis research on the root system characters of wheat is relatively lagged behind relative to the overground agronomic characters. Wheat root development is controlled by multiple genes, and more than 300 QTL (quantitative trait loci) loci (QTL) of wheat roots are reported at present and distributed on each chromosome except 1D (Christopher et al, 2013; Kabir et al, 2015; Maccaferri et al, 2016; Ren et al, 2017). CAO and the like (2014) find that qTaLRO-B1 is QTL influencing the root length and related characters of common wheat by researching root systems of filial generations F1 and F2 of wheat varieties with two different root systems of 178A (short root system) and 178B (long root system). In addition, the QTL which is carried on the rice chromosome 9 and is related to the control of the root system depth is beneficial to improving the drought resistance of the plant and enhancing the absorption of the plant to the root system nutrient elements, the markers corresponding to the genetic sections RM242-RM201 of the gene are applied to molecular assisted breeding, and a plant line which carries the gene and has excellent root system properties is obtained (Steele et al, 2006).
The Zhongmai 895 is a semi-winter multi-spike middle and late maturing variety bred by the combination of Zhongmai 16 as female parent and litchi reclamation No.4 as male parent in the institute of crop science and technology of Chinese academy of agricultural sciences and the institute of Cotton research of Chinese academy of agricultural sciences. The characteristics of high yield, wide adaptability, high temperature resistance in the later grouting stage and the like are shown in a species comparison test and a field demonstration in 2013-2015. Yangmai 16 is the variety with the largest planting area in the middle and lower Yangtze river wheat areas and has the characteristics of high grouting speed, high grain weight and the like.
Disclosure of Invention
An object of the present invention is to provide an application of a substance for detecting the genotype of SNP AX95025477 site on wheat chromosome 7 BL.
The application of the substance for detecting the genotype of the SNP AX95025477 locus on the wheat chromosome 7BL in identification or auxiliary identification of the wheat root system character is provided;
the SNP AX95025477 site is the 36 th site of the nucleotide sequence shown in the sequence 4.
In the application, the genotype of the SNP AX95025477 site is GG, CC or CG;
or, the root system property is root system surface area, and in the embodiment, the root system surface area specifically refers to total root surface area.
Or, the invention also provides the application of the substance for detecting the genotype of the SNP AX95025477 locus on the wheat chromosome 7BL in breeding wheat with excellent root system characters.
The SNP AX95025477 site is the 36 th site of the nucleotide sequence shown in the sequence 4.
In the above-mentioned application, the first and second substrates,
the genotype of the SNP AX95025477 site is GG or CC or CG;
or the wheat with the excellent root system character is the wheat with large root system surface area, in particular the wheat with large total root system surface area.
In the application, the substance for detecting the genotype of the SNP AX95025477 site on wheat chromosome 7BL is 1) or 2) as follows:
1) the primer set is a primer set, and the primer set is a or b as follows:
the primer set shown in a consists of a single-stranded DNA molecule shown in a sequence 1 in a sequence table, a single-stranded DNA molecule shown in a sequence 2 in the sequence table and a single-stranded DNA molecule shown in a sequence 3 in the sequence table;
the primer set shown in b consists of a single-stranded DNA molecule or a derivative thereof shown in 22 th-39 th sites of a sequence 1 in a sequence table, a single-stranded DNA molecule or a derivative thereof shown in 22 th-39 th sites of a sequence 2 in the sequence table and a single-stranded DNA molecule shown in a sequence 3 in the sequence table;
2) a PCR reagent or a kit containing the primer set.
In the application, the concentrations of the single-stranded DNA molecule or the derivative thereof shown in the sequence 1 in the sequence table, the single-stranded DNA molecule or the derivative thereof shown in the sequence 2 in the sequence table and the single-stranded DNA molecule shown in the sequence 3 in the sequence table in the PCR reagent containing the primer set are all 0.25 uM.
The single-stranded DNA molecule shown in the sequence 1 in the sequence table is formed by connecting a fluorescent sequence (in an embodiment, FAM fluorescent sequence F5 ' -GCCGGCCTTCCATCTACTCCATGA-3 ') to the 5' end of the single-stranded DNA molecule shown in the 22 th-39 th site of the sequence 1;
the single-stranded DNA molecule shown in the sequence 2 in the sequence table is formed by connecting another fluorescent sequence (in the embodiment, HEX fluorescent sequence F5 ' -GCCGGCCTTCCATCTACTCCATGG-3 ') to the 5' end of the single-stranded DNA molecule shown in the 22 th-39 th site of the sequence 2;
or the fluorescent group sequence is a fluorescent sequence FAM or a fluorescent sequence HEX.
The 2 nd object of the present invention is to provide the following method:
the invention provides a method for identifying or assisting in identifying wheat root system characters, which comprises the following steps: and detecting whether the genotype of the SNP AX95025477 site on the wheat chromosome 7BL is GG or CC or CG, wherein the genotype of the SNP AX95025477 site is CC, and the root surface area of the wheat to be detected is larger than that of the wheat to be detected of which the genotype of the SNP AX95025477 site is GG or CG.
The invention also provides a method for breeding wheat with large root surface area, which is used for detecting whether the genotype of the SNP AX95025477 locus on the 7BL of the wheat chromosome is GG, CC or CG and breeding the wheat to be detected with the genotype of the SNP AX95025477 locus being CC to obtain the wheat with large root surface area;
the SNP AX95025477 site is the 36 th site of the nucleotide sequence shown in the sequence 4.
In the method, the method for detecting whether the genotype of the SNP AX95025477 site on the wheat chromosome 7BL is GG, CC or CG is A) or B) as follows:
A) direct sequencing;
B) and carrying out PCR amplification products on the genome DNA of the wheat to be detected by using the primer set, and carrying out genotyping on the PCR amplification products.
In the above-mentioned method, the first step of the method,
the genotyping method adopts a fluorescence enzyme labeling instrument (specifically, PHERAstar)plusA fluorescence microplate reader), if the PCR product only shows the color (FAM fluorescence, blue) of the 5' end connecting fluorescence sequence in the DNA molecule shown in the sequence 1, the genotype of the SNP AX95025477 locus on the wheat chromosome 7BL to be detected is GG; if the PCR product only shows the color (HEX fluorescence, red) of the 5' end connecting fluorescence sequence in the DNA molecule shown in the sequence 2, the genotype of the SNP AX95025477 locus on the wheat chromosome 7BL to be detected is CC;
and if the PCR product shows the color of the 5 'end connected fluorescent sequence in the DNA molecule shown in the sequence 1 and the color (green) of the 5' end connected fluorescent sequence in the DNA molecule shown in the sequence 2, the genotype of the SNP AX95025477 locus on the wheat chromosome 7BL to be detected is CG.
The invention also provides a substance for identifying or assisting in identifying the root system characteristics of wheat, which is the substance for detecting the genotype of the SNP AX95025477 locus on the wheat chromosome 7BL in the application.
In the above, the root system character of the wheat is the root system character of the wheat in the seedling stage, the root system character of the wheat in the seedling stage is embodied in the root surface area, and in the embodiment of the invention, the root surface area is the total root surface area.
In the above, the wheat to be detected is specifically any one or more of the following varieties: afu, CA1055, CA1133, alternate 987, jindong No. 8, qinong 731, wan wheat 52, jining 16, jimai 21, lumai 15, lumai 21, mianyang 26, ningmai No. 9, and zhen mai No. 6.
Experiments prove that the SNP locus AX95025477 of the related locus of the root system character in the seedling stage of the wheat is found, the wheat with excellent related root system characters can be screened by using the SNP locus, and the important role is played in cultivating water-fertilizer efficient wheat varieties.
Drawings
FIG. 1 is a diagram of seedling root systems of Zhongmai 895 (right) and Yangma 16 (left).
FIG. 2 is a diagram of the linkage of a SNP marker and QROSA. caas-7BL gene.
FIG. 3 shows the KASP marker detection results of the root surface area genes of the tested wheat varieties.
Detailed Description
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.
All primer syntheses were performed by Oakco Biotechnology, Inc. of Beijing. All wheat material used in the examples below was from the national center for crop germplasm preservation, national academy of agricultural sciences, china.
Example 1 acquisition of SNP site of Gene related to root System at seedling stage, marker AX95025477 thereof, and Special primer
Yangmai 16 (China agriculture development group, Inc., variety number: CNA 20030436.4)/Zhongmai 895 (Henan New big agriculture development Co., Ltd., national examined wheat 2012010) DH groups are selected, and the groups comprise 200 families in total.
FIG. 1 is a diagram of seedling root systems of Zhongmai 895 (right) and Yangma 16 (left).
1. Root system character investigation
The root test adopts water culture method, that is, 30 seeds of each variety are treated with 10% of water for 20-30 min, and washed with sterile water for 5-6 times. Selecting H2O2The treated seeds are full and consistent in size, placed in a culture dish paved with filter paper, and subjected to germination acceleration in a dark room of an incubator. Seeds with consistent germination are selected for each material, the seeds are germinated and cultured, the culture tray is placed in nutrient solution to be cultured in a controllable greenhouse, and the nutrient solution is prepared according to the reference (Ren et al, 2012). The root characteristics at seedling stage were investigated after 10 days of continuous culture. The investigational traits include longest root length, lateral root length, main root length, total root length, lateral root surface area, main root surface area, total root tip count, and root dry weight.
2. Primer acquisition and labeling analysis
SNP (Single nucleotide polymorphism) markers in Boo biological Limited (CapitalBio Corporation, Beijing, China; http:// bioservices. CapitalBio. com) Using Illumina SNP genotyping assays, the major steps were as follows: 1) carrying out whole genome amplification on the genome DNA of the wheat variety to be detected; 2) cutting the amplified product by using random endonuclease; 3) hybridizing the DNA fragment with a chip, connecting a 50-mers length specific capture probe on the microbead of the chip, and combining the product of the enzyme digestion of gDNA with a probe complementary sequence; 4) washing to remove DNA fragments which are not hybridized or mismatched and hybridized; 5) carrying out single-base extension on dinitrophenol (dinitrophenol) and biotin (biotin) -labeled nucleotide substrates (A/T and C/G) on a capture probe, wherein only a probe which is complementarily combined with gDNA can be extended; by staining, A/T and C/G will mark different fluorochromes respectively; 6) and scanning the chip, interpreting and outputting a typing result according to the two types of fluorescence by using software.
660k SNP chip typing is carried out on Yangmai 16/Zhongmai 895DH groups by utilizing an Illumina SNP genotyping research platform, wherein the 660k SNP chip typing comprises series markers such as BS, Bobwhite, CAP, D _ contig and the like, the total number is 630518, and 626276 SNP markers have difference in the Yangmai 16/Zhongmai 895DH groups.
Second, the discovery of the associated Gene mapping and the linkage marker AX95025477
Basic statistics and multiple comparison analysis are carried out by using SAS9.2 software (SAS institute.2000), stepwise regression is carried out on SNP data and root system characters by combining a Glmselect program of SAS, and the associated sites are judged according to a P value (P < 0.01). AX95025477 was located in association with the site qrosa. caas-7BL (P < 0.001).
Allele-specific marker identification of site III, AX95025477
Extracting the whole genome DNA of 16 Yangmai 16/Zhongmai 895DH groups respectively, using each genome DNA as a template, and using allele specific marker KASP of SNP locus AX94573388TMGenotyping test, the results are shown in table 1, and C: C typing fragments appear; the SNP locus AX95025477 is shown to be capable of effectively identifying the root surface area gene of the wheat variety.
TABLE 1 isolation of allelic variants at the AX95025477 locus in the Yangmai 16/Miyami 895DH population
The result shows that the genotype of the AX95025477 locus is only C: C, and the genotype conforms to the phenotype that the wheat variety has larger root surface area, and the AX95025477 locus can effectively identify the root surface area gene and the genotype of the wheat variety. The surface area of the wheat variety root with the genotype of the AX95025477 site being C: C is larger than that of the wheat variety root with the genotype of the AX95025477 site being C: G or G: G.
The position AX95025477 was integrated into the Wheat genetic map according to the Wheat molecular marker map published by Wheat DArT maps Version 1.2(http:// www.triticarte.com.au) and Allen et al (2011), and the 700.83Mb position of QROSA. caas-7BL on chromosome 7BL was determined as shown in FIG. 2.
The SNP AX95025477 site corresponding to marker AX95025477 is the 36 th position in sequence 4, and the nucleotide is C or G; the genotype of the SNP locus is CC, CG or GG; the DNA molecule shown in the sequence 4 is located at the 700.83Mb position on the wheat chromosome 7 BL.
Development of primers special for KASP markers
Based on the nucleotide sequence (sequence 4) of the SNP AX95025477 site at the 700.83Mb position on wheat chromosome 7B, a primer set for detecting the AX95025477 site was designed as follows:
an upstream primer: f5 '-GAAGGTGACCAAGTTCATGCTCTCGTGGTGTTCCTCGCG-3' (SEQ ID NO: 1),
an upstream primer: f5 '-GAAGGTCGGAGTCAACGGATTCTCGTGGTGTTCCTCGCC-3' (SEQ ID NO: 2),
a downstream primer: r5 '-CTCAGCGAGTCGATCTTCTCC-3' (SEQ ID NO: 3).
In the sequence 1, the 1 st to 21 st positions are specific fluorescent sequences FAM added at the 5' end of the upstream specific primer: 5'-GAAGGTGACCAAGTTCATGCT-3' (SEQ ID NO: 5, blue in color when irradiated with fluorescence) and upstream specific primers at positions 22 to 39;
in the sequence 2, the 1 st to 21 st sites are the sites for adding a specific fluorescent sequence HEX at the 5' end of an upstream primer: f5 '-GAAGGTCGGAGTCAACGGATT-3' (SEQ ID NO: 6, showing red color by fluorescent irradiation), and the upstream specific primers at positions 22-39.
Amplifying a fragment (sequence 7, and G at 18 th site) with the genotype G at the SNP AX95025477 site by adding the single-stranded DNA molecule shown as the sequence 1 of the specific fluorescent sequence FAM and the single-stranded DNA molecule shown as the sequence 3 at the 5' end, and irradiating a product obtained after PCR amplification of the sequence carrying the FAM by fluorescence to show blue;
the single-stranded DNA molecule shown in the sequence 2 with the specific fluorescent sequence HEX added at the 5' end and the single-stranded DNA molecule shown in the sequence 3 amplify a fragment with the AX95025477SNP site genotype of C: C (the sequence 7 and the 18 th position are C), and a product obtained after PCR amplification of the sequence carrying the HEX shows red color by fluorescent irradiation.
The single-stranded DNA molecule shown in the sequence 1 with the specific fluorescent sequence FAM added at the 5 'end, the single-stranded DNA molecule shown in the sequence 2 with the specific fluorescent sequence HEX added at the 5' end and the single-stranded DNA molecule shown in the sequence 3 amplify the fragment with the SNP AX95025477 site genotype of C: G, and the product after PCR amplification shows green through fluorescent irradiation.
Establishment of method for detecting wheat root system character by SNP AX95025477 locus
Extracting the genome DNA of the wheat to be detected as a template, and performing PCR amplification by using a primer shown as a sequence 1 with a specific fluorescent sequence FAM added at the 5 'end, a primer shown as a sequence 2 with a specific fluorescent sequence HEX added at the 5' end and a primer shown as a sequence 3.
The PCR reaction system of 12 μ L for PCR amplification comprises: a primer shown as a sequence 1 of a specific fluorescent sequence FAM is added at the 5 'end, a primer shown as a sequence 2 of a specific fluorescent sequence HEX and a primer shown as a sequence 3 are added at the 5' end, and the final concentration of each primer is 0.25uM and 1 mmol.L-1Tris-HCl (pH 9.0) of (b) to a final concentration of 5 mmol. L-1KCl to a final concentration of 2.5 mmol. multidot.L-1MgCl of2(Promega Co.) to a final concentration of 200. mu. mol. L-1dNTPs (TaKaRa Co.), Taq DNA polymerase 1.5U (Tiangen Co.), template DNA 15ng, and the balance water.
The PCR amplification reaction is carried out on a PTC-200PCR amplification instrument, and the Touch down PCR amplification program is as follows:
pre-denaturation at 94 ℃ for 15 min; (Touch down program) denaturation at 94 ℃ for 30s, annealing at 61 ℃ for 60s, extension at 72 ℃ for 30s, and 11 cycles, wherein the annealing temperature is reduced by 0.6 ℃ in each cycle; (amplification procedure) denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 60s, extension at 72 ℃ for 30s, 26 cycles; extending for 5min at 72 ℃; storing at 10 deg.C.
The obtained PCR amplification product is put in PHERAStarplusGenotyping with fluorescent irradiation on a fluorescent microplate reader, followed by KlustercallerTMAnd reading the typed data by software.
If the PCR amplification product only displays a blue image (the fluorescent color connected with the 5' end in the DNA molecule shown in the sequence 1), the nucleotide of the SNP AX95025477 site of the wheat genome to be detected is G, and the genotype is G; if the PCR amplification product only displays a red image (the fluorescent color of the 5' end connection in the DNA molecule shown in the sequence 2), the nucleotide at the SNP AX95025477 site is C, and the genotype is C: C; if the PCR amplification product shows a green image, the nucleotide at the site SNPAX95025477 is C/G, and the genotype is C: G.
As can be seen from the results of combining the surface areas of the three, the root surface area of wheat showing only red color (SNP AX95025477 site genotype C: C) as the PCR amplification product was larger than that of wheat showing only blue color (SNP AX95025477 site genotype G: G) or green color (SNPAX95025477 site genotype C: G) as the PCR amplification product.
Example 2 application of SNP AX95025477 site in detection of wheat root system traits
Of the 14 varieties: zhongmai 895 and Yangmai 16 were used as control groups, and others were used as detection groups: afu, CA1055, CA1133, alternate 987, jindong No. 8, qinong 731, wan wheat 52, jining 16, jimai 21, lumai 15, lumai 21, mianyang 26, ningmai No. 9, and zhen mai No. 6.
One, conventionally detecting the root system characters of different wheat varieties to be detected in seedling stage
The root system characters of the wheat variety in the seedling stage are identified in 2015 winter in a greenhouse of the institute of crop science of the academy of agricultural sciences. Selecting 30 full and consistent-sized seeds from each wheat variety to be detected, and using 10% of H2O2Treating for 20-30 min, and washing with sterile water for 5-6 times; then placing the seeds in a culture dish paved with filter paper, and accelerating germination in a dark room in an incubator for 18-24 h; then 25 seeds with consistent germination are selected and placed on a seedling net, and after 6 days of growth, big seeds are selectedTransferring 10 uniform wheat seedlings into culture plates, and repeating for 3 times on each culture plate; then the culture dish is placed in nutrient solution to be cultured in a controllable greenhouse. Nutrient solution formulation references (Ren Y, He X, Liu D, Li J, ZHao X, Li B, Tong Y, Zhang A, Li Z. major qualitative trail loci for a minor root morphology of wheat seeds 148, molecular Breeding,2012,30: 139) 148, publicly available from the institute of crop science, academy of agriculture sciences, China. The seedling culture conditions are 22 +/-1 ℃ and 50-60% of relative humidity. The nutrient solution is changed every 3 days, and the roots in the seedling stage are harvested after continuous culture for 10 days. The root systems of the harvested varieties are scanned by a scanner, then root properties including the longest root length, the lateral root length, the main root length, the total root length, the lateral root surface area, the main root surface area, the total root tip number and the dry weight of the root system are analyzed by image analysis software Win RHIO (Regent Instruments, Canada), and finally the total root surface area is counted.
Second, application of SNP AX95025477 locus in detection of wheat root system characters
1. Extracting genome DNA of wheat to be detected
And respectively extracting the genome DNA of each wheat to be detected.
2. PCR amplification
PCR amplification was carried out using the genomic DNA as a template, and the primer indicated by sequence 1, to which the specific fluorescent sequence FAM was added at the 5 'end, the primer indicated by sequence 2, to which the specific fluorescent sequence HEX was added at the 5' end, and the primer indicated by sequence 3, obtained in example 1.
The PCR reaction system of 12 μ L for PCR amplification comprises: a primer shown as a sequence 1 of a specific fluorescent sequence FAM is added at the 5 'end, a primer shown as a sequence 2 of a specific fluorescent sequence HEX and a primer shown as a sequence 3 are added at the 5' end, and the final concentration of each primer is 0.25uM and 1 mmol.L-1Tris-HCl (pH 9.0) at a final concentration of 5 mmol. multidot.L-1KCl to a final concentration of 2.5 mmol. multidot.L-1MgCl of2(Promega Co.) to a final concentration of 200. mu. mol. L-1dNTPs (TaKaRa), Taq DNA polymerase 1.5U (Tiangen), template DNA 15ng, and water as the rest.
The PCR amplification reaction is carried out on a PTC-200PCR amplification instrument, and the Touch down PCR amplification program is as follows:
pre-denaturation at 94 ℃ for 15 min; (Touch down program) denaturation at 94 ℃ for 30s, annealing at 61 ℃ for 60s, extension at 72 ℃ for 30s, and 11 cycles, wherein the annealing temperature is reduced by 0.6 ℃ in each cycle; (amplification procedure) denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 60s, extension at 72 ℃ for 30s, 26 cycles; extending for 5min at 72 ℃; storing at 10 deg.C.
The obtained PCR amplification product (sequence 4) is put in PHERAStarplusGenotyping with fluorescent irradiation on a fluorescent microplate reader, followed by KlustercallerTMAnd reading the typed data by software.
If the PCR amplification product only displays a blue image (the fluorescence color connected with the 5' end in the DNA molecule shown in the sequence 1), the genotype of the wheat SNP AX95025477 to be detected is G; if the PCR amplification product only displays a red image (the fluorescence color of the 5' end connection in the DNA molecule shown in the sequence 2), the genotype of the SNP AX95025477 locus is C: C; if the PCR amplification product shows a green image, the genotype of the SNP AX95025477 site is C: G.
The surface area of wheat roots of which the PCR amplification product only shows red color (the genotype of the SNP AX95025477 site is C: C) is larger than that of wheat of which the PCR amplification product only shows blue color or green color (the genotype of the SNP AX95025477 site is G: G or the genotype of the SNP AX95025477 site is C: G).
The genotype (FIG. 3) of each of the above-mentioned wheat and the root system characteristics (root surface area) of the wheat are shown in Table 2.
TABLE 214 wheat variety SNP site genotype and root surface area results
Note: chinese rye 895 is CC; yangmai 16 is GG.
The above results show that: afu, Roche 987, Jingdong No. 8, Lumai No. 15, Ningmai No. 9 and Zhenmai No. 6 have C: C genotypes at SNP sites, and the wheat has large root surface area. From the above results, it can also be seen that the root surface area of wheat having SNP site with genotype CC is larger than that of wheat having SNP site with genotype GG.
The result shows that the SNP locus can rapidly and accurately identify whether the wheat variety has larger root surface area.
SEQUENCE LISTING
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<400> 3
ctcagcgagt cgatcttctc c 21
<210> 4
<211> 71
<212> DNA
<213> Artificial sequence
<220>
<221> misc_feature
<222> (36)..(36)
<223> n is c or g
<400> 4
ctcagcgagt cgatcttctc cagcgcgcac agcggngcga ggaacaccac gaggacgaac 60
aggatgagca g 71
<210> 5
<211> 21
<212> DNA
<213> Artificial sequence
<400> 5
gaaggtgacc aagttcatgc t 21
<210> 6
<211> 21
<212> DNA
<213> Artificial sequence
<400> 6
gaaggtcgga gtcaacggat t 21
<210> 7
<211> 53
<212> DNA
<213> Artificial sequence
<220>
<221> misc_feature
<222> (18)..(18)
<223> n is c or g
<400> 7
ctcgtggtgt tcctcgcncc gctgtgcgcg ctggagaaga tcgactcgct gag 53
Claims (5)
1. Detection of SNP on wheat chromosome 7BL AX95025477The application of the substance of the genotype of the locus in the identification or the auxiliary identification of the surface area of the root system of the wheat in the seedling stage;
the SNP AX95025477 site is the 36 th site of a nucleotide sequence shown in SEQ ID NO. 4;
the genotype of the SNP AX95025477 site is GG or CC or CG;
SNP AX95025477the root surface area of the wheat to be detected with the locus genotype CC is larger than SNP AX95025477The genotype of the locus is GG or CG.
2. Detection of SNP on wheat chromosome 7BL AX95025477The application of the substance of the genotype of the locus in the breeding of wheat with excellent seedling-stage root surface area;
the SNP AX95025477 site is the 36 th site of a nucleotide sequence shown in SEQ ID NO. 4;
the genotype of the SNP AX95025477 site is GG or CC or CG;
the wheat with the excellent seedling stage root system surface area is the wheat with the large root system surface area;
SNP AX95025477the root surface area of the wheat to be detected with the locus genotype CC is larger than that of SNP AX95025477The genotype of the locus is GG or CG.
3. Use according to any one of claims 1-2, characterized in that:
the detection of the SNP on the wheat chromosome 7BL AX95025477The substance of the genotype of the locus is 1) or 2):
1) a set of primers;
2) a PCR reagent or kit comprising the set of primers;
the primer set is the following a or b:
the primer set shown in a consists of a single-stranded DNA molecule shown in SEQ ID NO.1, a single-stranded DNA molecule shown in SEQ ID NO.2 and a single-stranded DNA molecule shown in SEQ ID NO. 3;
in SEQ ID NO.1, positions 1 to 21 are the specific fluorescent sequence FAM added at the 5' end of the upstream specific primer: 5'-GAAGGTGACCAAGTTCATGCT-3', positions 22-39 are upstream specific primers;
in SEQ ID NO.2, positions 1-21 are positions obtained by adding a specific fluorescent sequence HEX to the 5' end of an upstream primer: f5 '-GAAGGTCGGAGTCAACGGATT-3', the 22 th-39 th site is upstream specific primer;
the primer set shown in b consists of single-stranded DNA molecules shown in 22 th to 39 th positions of SEQ ID NO.1, single-stranded DNA molecules shown in 22 th to 39 th positions of SEQ ID NO.2 and single-stranded DNA molecules shown in SEQ ID NO. 3.
4. Method for identifying or assisting in identifying surface area of root system of wheat in seedling stageThe method comprises the following steps: detection of SNP on wheat chromosome 7BL AX95025477The genotype of the locus is GG, CC or CG, SNP AX95025477The root surface area of the wheat to be detected with the locus genotype CC is larger than SNP AX95025477The genotype of the locus is GG or CG;
the SNP AX95025477 site is the 36 th site of a nucleotide sequence shown in SEQ ID NO. 4;
the detection of SNP on wheat chromosome 7BL AX95025477The method for determining whether the genotype of the site is GG, CC or CG is A) or B) as follows:
A) direct sequencing;
B) carrying out PCR amplification on the wheat genome DNA to be detected by using the primer set shown in a in claim 3, and carrying out genotyping on PCR amplification products;
the genotyping method comprises the steps of irradiating by adopting a fluorescent microplate reader, and if the PCR amplification product only shows the color of a 5' end connecting fluorescent sequence in a DNA molecule shown as SEQ ID NO.1, carrying out SNP on the wheat chromosome 7BL to be detectedAX95025477The genotype of the locus is GG; if the PCR amplification product only shows the color of the 5' end connecting fluorescence sequence in the DNA molecule shown in SEQ ID NO.2, the SNP on the wheat chromosome 7BL to be detected AX95025477The genotype of the locus is CC; if the PCR amplification product shows the color of the 5 'end connecting fluorescent sequence in the DNA molecule shown in SEQ ID NO.1 and the color of the 5' end connecting fluorescent sequence in the DNA molecule shown in SEQ ID NO.2, the SNP on the wheat chromosome 7BL to be detectedAX95025477The genotype of the site is GC.
5. A method for selectively breeding wheat with large root surface area by detecting SNP on 7BL of wheat chromosome AX95025477The genotype of the locus is GG, CC or CG, and SNP is selected and bred AX95025477Obtaining wheat with large root surface area by using the wheat to be detected with CC locus genotype;
the SNP AX95025477 site is 36 th site of a nucleotide sequence shown in SEQ ID NO. 4;
the detection of SNP on wheat chromosome 7BL AX95025477The method for determining whether the genotype of the site is GG, CC or CG is as follows A)Or B):
A) direct sequencing;
B) carrying out PCR amplification on the wheat genome DNA to be detected by using the primer set shown in a in claim 3, and carrying out genotyping on PCR amplification products;
the genotyping method is characterized in that after the genotyping method adopts a fluorescence microplate reader for irradiation, if the PCR amplification product only shows the color of a 5' end connecting fluorescence sequence in a DNA molecule shown as SEQ ID NO.1, the SNP on the wheat chromosome 7BL to be detectedAX95025477The genotype of the locus is GG; if the PCR amplification product only shows the color of the 5' end connecting fluorescence sequence in the DNA molecule shown in SEQ ID NO.2, the SNP on the wheat chromosome 7BL to be detected AX95025477The genotype of the locus is CC; if the PCR amplification product shows the color of the 5 'end connecting fluorescence sequence in the DNA molecule shown in SEQ ID NO.1 and the color of the 5' end connecting fluorescence sequence in the DNA molecule shown in SEQ ID NO.2, the SNP on the wheat chromosome 7BL to be detectedAX95025477The genotype of the locus was GC.
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| CN109234443A (en) * | 2018-11-15 | 2019-01-18 | 中国农业科学院作物科学研究所 | The relevant KASP label of wheat 6B chromosome root system depth and its application |
| CN109762812A (en) * | 2019-03-05 | 2019-05-17 | 中国农业科学院作物科学研究所 | Wheat growth gesture related SNP and its as target spot identification wheat growth gesture character in application |
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| WO2003027243A2 (en) * | 2001-09-27 | 2003-04-03 | Pioneer Hi-Bred International, Inc. | Phytate polynucleotides and methods of use |
| JP2003259898A (en) * | 2002-03-12 | 2003-09-16 | National Agricultural Research Organization | Method for identifying wheat variety |
| CN103952402B (en) * | 2014-04-18 | 2016-03-09 | 中国农业科学院作物科学研究所 | A kind of SNP site relevant to root system of plant proterties and application thereof |
| CN107805673B (en) * | 2017-11-20 | 2020-06-05 | 中国农业科学院作物科学研究所 | SNP (Single nucleotide polymorphism) marker related to root system characters of wheat in seedling stage and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108977439A (en) * | 2018-09-03 | 2018-12-11 | 中国农业科学院作物科学研究所 | A kind of method and its primer special group of auxiliary identification wheat canopy temperature character |
| CN109234443A (en) * | 2018-11-15 | 2019-01-18 | 中国农业科学院作物科学研究所 | The relevant KASP label of wheat 6B chromosome root system depth and its application |
| CN109762812A (en) * | 2019-03-05 | 2019-05-17 | 中国农业科学院作物科学研究所 | Wheat growth gesture related SNP and its as target spot identification wheat growth gesture character in application |
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