CN109055370B - Straw WSC content gene marker based on Chinese rye 895 and application - Google Patents
Straw WSC content gene marker based on Chinese rye 895 and application Download PDFInfo
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Abstract
The invention discloses a stem WSC content gene marker based on Chinese rye 895 and application thereof. The invention provides a specific primer group, which consists of a primer A shown in a sequence 1, a primer B shown in a sequence 2 and a primer C shown in a sequence 3. The invention also discloses a method for identifying the WSC content traits of the stalks of the wheat to be detected. And detecting the genotype of the wheat to be detected based on the specific SNP locus. And (3) taking the genome DNA of the wheat to be detected as a template, adopting a specific primer group to carry out KASP, carrying out fluorescence scanning, and determining the genotype of the wheat to be detected based on the specific SNP locus. The WSC content of stems of TT genotype wheat is higher than that of CC genotype wheat. The specific SNP sites are: 36 th nucleotide of DNA molecule shown in sequence 4 of the sequence table in wheat genome; nucleotide 223 of DNA molecule shown in sequence 6 of the sequence table in wheat genome. The invention provides a good tool for breeding the QWSc-caas-1 RS utilizing the main QTL locus of the stem WSC content.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a stem WSC content gene marker based on Chinese rye 895 and application thereof.
Background
The grain filling stage is the key stage in determining wheat yield. The wheat main production area in China faces serious dry hot air harm in the middle and later grain filling period, and the yield is reduced by over 10 percent in serious cases. The carbon source in the wheat filling stage is mainly derived from photosynthetic products produced by flag leaves and Water-soluble carbohydrates (WSC) stored in stalks and leaf sheaths. Under normal water conditions, the contribution of the stem WSC to the yield is 10-20%, and the contribution to the yield under drought stress is up to 30-50%. In the evolution process of wheat varieties, the photosynthetic efficiency of leaves and the WSC storage amount of stalks are obviously improved, and the method has important contributions to the improvement of grain weight and yield of ears, the stress resistance of improved varieties and the increase of stable yield. However, the dry matter of the stalks of the current wheat variety still remains more in the middle and later stage of filling, and a larger genetic improvement space exists. Currently, the countries and organizations such as the American, Australia and International corn wheat improvement Center (CIMMYT) attach great importance to the physiological and genetic research related to the accumulation and transportation of WSC in the grain filling stage of wheat, and take high WSC content as an important target for future wheat breeding improvement so as to cope with the influence of global warming on wheat production. As an important grain crop in China, the wheat has important significance for high and stable yield breeding of the wheat in China by strengthening genetic research related to the carbohydrate of the stalks.
Research shows that the WSC content of the stems is in a stable accumulation state from heading to about 20 days after flowering, then metabolic attenuation occurs, and the WSC content is already at a low level about 30 days after flowering. The high-yield heat stress-resistant variety has high WSC storage amount at the early stage, high metabolic amount at the later stage and less WSC amount in the retained stalks. The analysis of the WSC content of the wheat straw 10-30 days after the flowering has important significance.
Single Nucleotide Polymorphisms (SNPs) are the most common genetic variation form among plant individuals, and the frequently occurring single nucleotide polymorphisms comprise base substitution and insertion and deletion, and are ideal molecular markers for genetic research of plant complex traits. With the development of biotechnology level, competitive Allele Specific PCR (KASP) technology is one of the methods for detecting SNP typing with high throughput, low cost and low error rate, and plays an important role in crop auxiliary breeding application.
Molecular markers can be classified into dominant markers and co-dominant markers according to their characteristics. For the KASP marker, the dominant marker type means that after the fluorescence scanning signal of F1 (first filial generation, the genotype of which is heterozygous genotype) obtained by hybridization of two homozygous genotype materials as parents is automatically typed by SNP typing software, the typing result is consistent with that of one of the parents (homozygous genotype), which is usually caused by that the gene sequence corresponding to one base in the heterozygous genotype cannot be effectively amplified; in addition, in the case of the dominant marker, since the gene sequence corresponding to one of the two polymorphic bases cannot be amplified efficiently, the fluorescence signal thereof is generally concentrated near the origin of the typing coordinate system and appears pink after being automatically typed by the Kluster Caller software. The co-dominant marker is that after the fluorescent scanning signal of F1 is automatically typed by typing software, the fluorescent scanning signal is gathered at the middle position of the diagonal line in the typing coordinate system and is displayed in green, because the gene sequences corresponding to two polymorphic bases in the heterozygous genotype can be amplified equally efficiently.
The DH population (doubled haploid) is a widely used material in crop genetic and breeding studies, formed by chromosome doubling of haploid material, and the genotype is homozygous. There are various methods for creating DH populations, and currently, the creation of DH populations is generally performed in wheat genetic and breeding studies by the wheat maize crossing method.
Disclosure of Invention
The invention aims to provide a stem WSC content gene marker based on Chinese rye 895 and application thereof.
The invention firstly protects a specific primer group which consists of a primer A, a primer B and a primer C;
primer A is (a1) or (a2) as follows:
(a1) a single-stranded DNA molecule shown in sequence 1 of the sequence table;
(a2) DNA molecules which are obtained by replacing and/or deleting and/or adding one or more nucleotides to the sequence 1 and have the same functions as the sequence 1;
primer B is (B1) or (B2) as follows:
(b1) a single-stranded DNA molecule shown in a sequence 2 of a sequence table;
(b2) DNA molecules obtained by replacing and/or deleting and/or adding one or more nucleotides to the sequence 2 and having the same functions as the sequence 2;
primer C is (C1) or (C2) as follows:
(c1) a single-stranded DNA molecule shown in sequence 3 of the sequence table;
(c2) and (b) a DNA molecule which is obtained by replacing and/or deleting and/or adding one or more nucleotides to the sequence 3 and has the same function as the sequence 3.
The invention also protects the application of the primer group, which is (d1), (d2), (d3), (d4), (d5), (d6), (d7) or (d 8):
(d1) identifying the genotype of the wheat based on the specific SNP locus;
(d2) the WSC content characters of the wheat stalks are identified in an auxiliary manner;
(d3) auxiliary screening or breeding of single wheat plants or wheat strains or wheat varieties with high stem WSC content;
(d4) auxiliary screening or breeding of single wheat plants or wheat strains or wheat varieties with low stem WSC content;
(d5) preparing a product for identifying the genotype of the wheat based on the specific SNP locus;
(d6) preparing a product for assisting in identifying the WSC content traits of the wheat stalks;
(d7) preparing a product for screening or breeding a single wheat plant or strain or variety with high stalk WSC content;
(d8) preparing a product for screening or breeding a single wheat plant or strain or variety with low stem WSC content;
the specific SNP sites are as follows (e1) or (e 2):
(e1) 36 th nucleotide of DNA molecule shown in sequence 4 of the sequence table in wheat genome;
(e2) nucleotide 223 of DNA molecule shown in sequence 6 of the sequence table in wheat genome.
The invention also provides a kit comprising the specific primer group.
The kit also includes a specific probe set. The specific probe set consists of a fluorescent probe A, a quenching probe A, a fluorescent probe B and a quenching probe B; the fluorescent probe A is shown as a sequence 8 in a sequence table, and the 5' end is connected with a fluorescent group; the fluorescent probe B is shown as a sequence 9 in a sequence table, and the 5' end is connected with a fluorescent group; the fluorescent groups in the fluorescent probe A and the fluorescent probe B are different; the quenching probe A is shown as a sequence 10 in a sequence table, and the 3' end is connected with a quenching group; the quenching probe B is shown as a sequence 11 in a sequence table, and the 3' terminal is connected with a quenching group. The fluorescent probe A is specifically connected with a FAM fluorescent group. The fluorescent probe B is specifically connected with a HEX fluorescent group. The quenching probe A is specifically connected with a quenching group BHQ. The quenching probe B is specifically connected with a quenching group BHQ.
The kit further comprises KASP 2 × Master Mix.
The invention also protects the application of the kit, which is (d1), (d2), (d3) or (d 4):
(d1) identifying the genotype of the wheat based on the specific SNP locus;
(d2) the WSC content characters of the wheat stalks are identified in an auxiliary manner;
(d3) auxiliary screening or breeding of single wheat plants or wheat strains or wheat varieties with high stem WSC content;
(d4) auxiliary screening or breeding of single wheat plants or wheat strains or wheat varieties with low stem WSC content;
the specific SNP sites are as follows (e1) or (e 2):
(e1) 36 th nucleotide of DNA molecule shown in sequence 4 of the sequence table in wheat genome;
(e2) nucleotide 223 of DNA molecule shown in sequence 6 of the sequence table in wheat genome.
The invention also provides a method for identifying the WSC content traits of the stalks of the wheat to be detected, which comprises the following steps: detecting the genotype of the wheat to be detected based on the specific SNP locus; the WSC content of stems of TT genotype wheat is higher than that of CC genotype wheat.
The invention also provides a wheat breeding method, which comprises the following steps: detecting the genotype of the wheat to be detected based on the specific SNP locus; TT genotype wheat is selected for breeding. The purpose of wheat breeding is to breed wheat with high stalk WSC content.
The invention also provides a method for identifying the WSC content traits of the stalks of the wheat to be detected, which comprises the following steps:
(1) using the genome DNA of wheat to be detected as a template, and adopting the specific primer group to perform KASP;
(2) after the step (1) is finished, performing fluorescence scanning to determine the genotype of the wheat to be detected based on the specific SNP locus;
(3) and (4) judging according to the genotype result: the WSC content of stems of TT genotype wheat is higher than that of CC genotype wheat.
The invention also provides a wheat breeding method, which comprises the following steps:
(1) using the genome DNA of wheat to be detected as a template, and adopting the specific primer group to perform KASP;
(2) after the step (1) is finished, performing fluorescence scanning to determine the genotype of the wheat to be detected based on the specific SNP locus;
(3) TT genotype wheat is selected for breeding.
The purpose of wheat breeding is to breed wheat with high stalk WSC content.
The invention also protects a specific DNA molecule, which is shown as a sequence 4 or a sequence 6 in the sequence table.
The invention also protects the application of the specific DNA molecule in identifying the WSC content traits of the wheat stalks. In the application, the specific DNA molecule is used as a detection target.
Any one of the specific SNP sites is as follows (e1) or (e 2):
(e1) 36 th nucleotide of DNA molecule shown in sequence 4 of the sequence table in wheat genome;
(e2) nucleotide 223 of DNA molecule shown in sequence 6 of the sequence table in wheat genome.
In any of the above methods, the method for determining the genotype of the wheat to be detected based on the specific SNP site comprises the following steps: and scanning by using a multifunctional microplate reader, automatically analyzing scanning data of the microplate reader by using Kluster Caller software, and if the data are displayed as pink (gathered near the origin of a coordinate axis in a fluorescence signal coordinate system of a typing result), determining that the genotype of the wheat to be detected based on the specific SNP is TT.
In any of the above methods, the method for determining the genotype of the wheat to be detected based on the specific SNP site comprises the following steps: scanning by using a multifunctional microplate reader, and then automatically analyzing scanning data of the microplate reader by using Kluster Caller software; if the wheat to be detected is pink (gathered near the origin of a coordinate axis in a fluorescence signal coordinate system of the typing result), the genotype of the wheat to be detected based on the specific SNP is TT; and if the wheat to be detected is displayed as blue (gathered near the right side close to the Y axis in the typing result fluorescent signal coordinate system), the genotype of the wheat to be detected based on the specific SNP is CC or CT.
Further, for a sample displayed as blue, selfing the corresponding wheat material, detecting the genotype of the individual plant of the offspring obtained by selfing based on the specific SNP according to the steps, and if the genotype of each individual plant of the detected offspring is non-TT homozygote, determining that the wheat material to be detected is CC homozygote; if the detected individual plants of the offspring are TT homozygotic and the other are not TT homozygotic, the wheat material to be detected is CT heterozygote.
In any of the above methods, the concentration of primer A, primer B and primer C in the reaction system of KASP is 0.192. mu.M, 0.192. mu.M and 0.48. mu.M, in that order.
In any of the above methods, the reaction sequence of KASP:
the first step is as follows: pre-denaturation at 95 ℃ for 15 min;
the second step is that: 9 cycles of 95 ℃ for 20s and 65 ℃ for 60 s;
the third step: denaturation at 95 ℃ for 20s, renaturation at 57 ℃ and extension for 60s, 32 cycles.
The WSC content of any one of the wheat stalks is the WSC content of the stalks of the wheat plants 20 days after the flowers are bloomed.
The inventor of the invention locates a main effect QTL site QWSc.caas-1RS on the short arm of the 1B chromosome, the synergistic allele comes from Mianmai 895 (Mianmai 895 is 1BL/1RS translocation line, and the source of the synergistic allele of QWSc.caas-1RS is 1RS), and the phenotypic variation is explained to be as high as 7.4% -24.3%. On the basis, the invention develops the KASP primer group of the SNP marker AX-111189530 closely linked with the QWSc.caas-1RS, and provides a good tool for breeding and utilizing the main effect QTL site QWSc.caas-1RS of the stem WSC content. The method can be used for molecular marker-assisted selective breeding of the WSC content major QTL QWSc. caas-1RS of the wheat straw. The method has low artificial error and high analysis flux, is suitable for detecting a large number of samples, and has important significance for accelerating genetic improvement by using excellent alleles of the QTL locus and improving the breeding efficiency.
Drawings
FIG. 1 is a genetic linkage diagram of the short-arm QTL QWSc. caas-1RS of chromosome 1B.
FIG. 2 shows the results of example 3.
FIG. 3 shows the results of example 4.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The test methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
Example 1 acquisition of closely-linked molecular marker AX-111189530 of wheat straw WSC content QTL site QWSc. caas-1RS and KASP primer set thereof
First, obtaining DH population
The Zhongmai 895 and Yangmai 16 are respectively one of the main varieties of Huang-Huai-mai areas and winter mai areas in the middle and lower reaches of Yangtze river in China, and have excellent comprehensive agronomic characters. Chinese wheat 895 is a semi-winter wheat variety bred by cooperation of the crop science research institute of Chinese academy of agricultural sciences and the cotton research institute of Chinese academy of agricultural sciences, and passes the national approval of Huang-Huai-Nanpian in 2012. The field performance of the Zhongmai 895 is fast in grouting speed, the WSC of the stem at the later stage is less in retention, and the yield is high.
A DH colony is constructed by taking Yangmai 16 as a female parent and Zhongmai 895 as a male parent, and contains 174 families.
II, obtaining SNP marker AX-111189530
1. Field test and measurement of WSC content of stalks
DH populations were planted in Henan New county and commercial dune test stations in 2016-2017. The method adopts a random block design, sets three times of repetition, and adopts a cell seeding mode to plant, wherein each cell has six rows, the row length is 3m, and the row spacing is 25 cm. The field management refers to the management of a local test station, and in addition, the control of diseases and insect pests such as powdery mildew, stripe rust, aphids and the like is emphasized.
Recording the flowering period of each test material, randomly shearing 20 main stems from each cell 20 days after flowering, removing leaves and ears, retaining stems, deactivating enzymes at 105 ℃ for 30min, and then drying at 80 ℃. The WSC content of the stalks is determined by near infrared spectroscopy with reference to the construction of Wang et al (2014). Each sample was subjected to 3 technical replicates and the average was taken for statistical analysis.
2. Genetic map construction
Before constructing the genetic map, firstly, the quality control is carried out on the obtained material genotype data, and markers which have no polymorphism, have a marker deletion rate of more than 10 percent and have a distortion separation of more than 30 percent are removed. And next, the BIN-Mapping function of IcMapping V4.0 software is used for optimizing the rest of the polymorphic markers for genetic map construction. The construction of genetic map was carried out using the JoinMap V4.0 and MSTmap Online.
3. QTL location and discovery of linked marker AX-111189530
QTL positioning is carried out on the WSC content of 174 families of a DH group in different environments by using QTL IciMapping V4.0 software and a complete composite interval mapping method (ICIM). The LOD value was chosen to be 3.0 as the threshold.
The genetic linkage diagram near the 1B chromosome short-arm QTL QWSc.caas-1RS constructed by using the wheat 660K SNP chip marker is shown in figure 1. The positioning result shows that a main QTL site for controlling the WSC content exists on the short arm of the 1B chromosome, the left marker and the right marker are AX-111189530 and AX-109507240 respectively, the site explains that the phenotypic variation reaches 7.4-24.3%, and the synergistic allele is from Michelia chinensis 895.
After a large number of sequence analyses, alignments and preliminary experiments, the flanking sequences of the SNP marker AX-111189530 were found to be specific on the 1B chromosome.
Thirdly, converting SNP marker AX-111189530 into KASP marker and designing primer set for detecting the marker
The SNP marker AX-111189530 is converted into a KASP marker for molecular marker-assisted selective breeding.
Sequence 4 in the sequence table is a flanking sequence of the SNP marker AX-111189530, wherein Y represents two polymorphic mononucleotides C or T of the SNP marker, namely the nucleotide at the position in the actual variety is C or T.
Searching in International wheat genome sequencing alliance (IWGSC) IWGSC (https:// heat-origin. versales. inra. fr /) by using sequence 4 in the sequence table to obtain 3 homologous sequences with the consistency of more than 90 percent with the sequence 4, wherein the homologous sequences are respectively shown as sequence 5, sequence 6 (the sequence 6 comprises the sequence 4, and Y is C) and sequence 7 in the sequence table, and are respectively positioned on wheat chromosomes 1A, 1B and 1D.
The specific SNP is located at the 36 th nucleotide of a DNA molecule shown in a sequence 4 of a sequence table in a wheat genome. Correspondingly, the specific SNP is located at the 223 rd nucleotide of the DNA molecule shown in the sequence 6 of the sequence table in the wheat genome.
Designing a primer group based on the KASP technology, and is called KASP primer group for short. The KASP primer set consists of two upstream primers (primer A and primer B) and one downstream primer (primer C).
The nucleotide sequence of the primer A is shown as a sequence 1 in a sequence table.
Sequence 1:
the nucleotide sequence of the primer B is shown as a sequence 2 in a sequence table.
Sequence 2:
the nucleotide sequence of the primer C is shown as a sequence 3 in the sequence table.
And (3) sequence: 5'-TGGGTACTACAGCTCGGTGCTA-3' are provided.
Example 2 establishment of the method
1. Extracting genome DNA of leaves of wheat to be detected, and diluting to obtain a template solution. The concentration of DNA in the template solution was about 30 ng/. mu.L.
2. KASP is performed.
Primer working solution: taking the primer A, the primer B and the primer C, supplementing to 100 mu L with sterile ultrapure water, and fully and uniformly mixing. In the primer working solution, the concentrations of the primer A, the primer B and the primer C are 12. mu.M, 12. mu.M and 30. mu.M in sequence.
KASP 2 XMASter Mix is a product of LGC company (KBS-1016-. The KASP 2 × Master Mix contains a fluorescent probe A, a fluorescent probe B, a quenching probe A, a quenching probe B, high fidelity Taq enzyme, dNTP and Mg2+And the like. Sequence of fluorescent Probe A5'-GAAGGTGACCAAGTTCATGCT-3', FAM fluorophore was attached to the 5 ' end. The sequence of the fluorescent probe B is 5'-GAAGGTCGGAGTCAACGGATT-3', and the 5 ' terminal is connected with HEX fluorescent group. The sequence of the quenching probe A is 5'-AGCATGAACTTGGTCACCTTC-3', and the 3 ' terminal is connected with a quenching group BHQ. The sequence of the quenching probe B is 5'-AATCCGTTGACTCCGACCTTC-3', and the 3 ' terminal is connected with a quenching group BHQ.
Reaction system: mu.L of the template solution prepared in the step 1, 0.08 mu L, KASP 2 mu x Master Mix2.5 mu L of the primer working solution, and supplementing to 5 mu L with sterile ultrapure water. In the reaction system, the concentrations of the primer A, the primer B and the primer C were 0.192. mu.M, 0.192. mu.M and 0.48. mu.M, respectively. The amount of the template DNA contained in the reaction system was about 60 ng.
Reaction procedure:
the first step is as follows: pre-denaturation at 95 ℃ for 15 min;
the second step is that: 9 cycles of 95 ℃ for 20s and 65 ℃ for 60 s;
the third step: denaturation at 95 ℃ for 20s, renaturation at 57 ℃ and extension for 60s, 32 cycles; storing at 10 deg.C.
3. A fluorescence scan is performed.
And (3) after the step 2 is finished, scanning by using a multifunctional microplate reader. The FAM excitation wavelength is 485nm, and the emission wavelength is 520 nm. The HEX excitation wavelength is 535nm and the emission wavelength is 556 nm. The excitation wavelength of the system reference fluorescence ROX is 575nm, and the emission wavelength is 610 nm.
4. Allelic typing was performed.
And (3) after the step 3 is completed, performing automatic analysis on the scanning data of the microplate reader by using Kluster Caller software (the specific method refers to the Kluster Caller software specification, and the public can obtain the scanning data from LGC company), and determining the genotype of the wheat to be detected based on the specific SNP according to the analysis result.
The marker is a dominant marker, if the fluorescence signal data of the amplification product of the wheat to be detected is automatically analyzed and displayed as pink through Kluster Caller software (gathered near the origin of a coordinate axis in a fluorescence signal coordinate system of a parting result), the genotype of the wheat to be detected based on the specific SNP is TT;
if the fluorescence signal data of the amplification product of the wheat to be detected is analyzed by Kluster Caller software and shows blue (gathered near the right side close to the Y axis in the parting result fluorescence signal coordinate system), the genotype of the wheat to be detected based on the specific SNP can be CC or CT;
further, for the sample showing blue, selfing the corresponding wheat material, detecting the genotype of the individual progeny obtained by selfing based on the specific SNP according to the steps, and if the genotype of each individual progeny detected is non-TT homozygote, determining that the wheat material to be detected is CC homozygote; if the detected individual plants of the offspring are TT homozygous (accounting for about 25% of the number of the detected samples) and non-TT homozygous, the wheat material to be detected is CT heterozygous.
Example 3 detection of specific SNP-based genotypes of Yangmai 16, Zhongmai 895 and part of DH population families Using KASP primer set
The wheat to be tested is: yangmai 16, Zhongmai 895, partial DH colony family obtained in step one of example 1. Yangmai 16 is a sample with CC genotype based on specific SNP which is verified by sequencing, and the genome DNA of Yangmai 16 is used as a CC genotype control sample. Zhongmai 895 is a sample with TT as the genotype based on specific SNP which has been sequenced and verified, and the genomic DNA of Zhongmai 895 is used as a control sample of TT genotype. The genomic DNA of Yangmai 16 and the genomic DNA of Mirabi 895 were mixed in equal amounts to obtain a control sample of heterozygous genotypes. Sterile ultrapure water was used as a blank control sample.
The method established in example 2 is used to detect the genotype of the wheat to be detected based on the specific SNP.
The results are shown in FIG. 2. The fluorescence signal data of Yangmai 16 was analyzed by Kluster Caller software to show blue color and the genotype was CC. The data of the fluorescence signal of Zhongmai 895 was analyzed by Kluster Caller software to show pink color and the genotype was TT. No heterozygous genotype exists in the DH family, the blue sample genotype analyzed by the Kluster Caller software in the DH family to be detected is CC, and the pink sample genotype analyzed by the Kluster Caller software in the fluorescence signal data is TT. The results of typing of the control sample of heterozygous genotypes aggregated near the right side of the X-axis due to the low efficiency of PCR amplification when the specific SNP in the template is T, resulting in a weak HEX fluorescence signal. In summary, the KASP marker is a dominant marker, and the KASP marker and/or the set of primers can distinguish between the "TT" and the "C _" genotypes at the site of the SNP marker AX-111189530, wherein "C _" is "CC" or "CT".
Example 4 assisted identification of WSC content of wheat straw by using KASP primer group
The wheat to be tested was 205 parts of the existing wheat variety (see table 1, national wheat improvement center).
1. Detection of WSC content of stem
The wheat to be tested was planted in Dezhou, Shandong in 2016 + 2017. The method adopts a completely random block design, sets three times of repeated and double-row blocks with the row length of 1.0m and the row width of 20cm, and carries out field management according to the management of a local test station. Recording the flowering period of each cell, randomly shearing 20 main stems from each cell 20 days after flowering, removing leaves and ears, retaining stems, deactivating enzyme at 105 ℃ for 30min, and drying at 80 ℃. The WSC content of the stalks is determined by near infrared spectroscopy with reference to the construction of Wang et al (2014). Each sample was subjected to 3 technical replicates and the average was taken for statistical analysis.
2. The method established in example 2 is used to detect the genotype of the wheat to be detected based on the specific SNP.
Genomic DNA of Yangmai 16 was used as a control sample for CC genotype. Genomic DNA of Migmai 895 was used as a control sample for the TT genotype. Sterile ultrapure water was used as a blank control sample.
The results are shown in FIG. 3. The two blank samples were collected in the lower left corner of the fluorescence signal coordinate system and are shown in black. 895 parts of Chinese wheat and 107 parts of wheat varieties to be detected are gathered near the blank control position, the positions are shown as pink, and the genotype is TT. Yangmai 16 and another 98 wheat varieties to be tested are gathered near the right position near the X axis and are displayed as blue, and the genotypes of the 98 varieties are CC because the bred varieties are considered to be homozygous genotypes.
The results of 205 wheat varieties to be tested based on the genotype of the specific SNP and the WSC content of the stalks are shown in Table 1.
TABLE 1
The WSC content of 205 wheat varieties of the test group of different genotype materials was subjected to t-test by using a PROC TTEST model in the international general SAS9.2 statistical software, and the results are shown in Table 2. The WSC content of the wheat variety with the genotype TT is 7.0 percent higher than that of the wheat variety with the genotype CC, and the WSC content is obviously different at the level that p is less than 0.01. The results show that the KASP primer group can be used for molecular assisted selective breeding with the goal of improving the WSC content of wheat stalks.
TABLE 2
SEQUENCE LISTING
<110> institute of crop science of Chinese academy of agricultural sciences
<120> stem WSC content gene marker based on Chinese rye 895 and application
<130> GNCYX181752
<160> 11
<170> PatentIn version 3.5
<210> 1
<211> 48
<212> DNA
<213> Artificial sequence
<400> 1
gaaggtgacc aagttcatgc tgagtcccaa gtgcataata ccacttac 48
<210> 2
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<213> Artificial sequence
<400> 2
gaaggtcgga gtcaacggat tgagtcccaa gtgcataata ccacttat 48
<210> 3
<211> 22
<212> DNA
<213> Artificial sequence
<400> 3
tgggtactac agctcggtgc ta 22
<210> 4
<211> 71
<212> DNA
<213> Triticum aestivum
<220>
<221> misc_feature
<222> (36)
<223> y =t or c
<400> 4
catgtttacg agtcccaagt gcataatacc acttaygtta acccactatg gacagcctaa 60
ttagcaccga g 71
<210> 5
<211> 433
<212> DNA
<213> Triticum aestivum
<400> 5
gctggttgta gtagggagta taatatacta gtatcatgca tatgatacta catccatagt 60
gcatactatt ataggcggtc tcatttattg ccatgcatga cacatagtgg cataacattt 120
attatgatat tgtatctacc tgtgttacta taaccctctc tctcttcttt aattgtctcc 180
catataaaca tgtttacgag tcccaagtgc atgataccgc ttatgttacc ccactatggc 240
cagcctaaca taatcagtat catgcgcttg ggactcgcaa acatgcttat gcggcagaca 300
attaaagaag agagaaggcg ttatagtaac ataggtagat accgtatcat aataaatgtt 360
atgctactat gtgtcatgca tggcaataaa tgagaccacc tatgatacta ctaaggcctc 420
ttttggttca tag 433
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<220>
<221> misc_feature
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gagtatcata cactagtatc atgctacgtc tataatgcat agtatcatag atagattagt 60
atcatatatc gtatcattta ttgacataca agacacatag tagcataaaa ttaattatga 120
tatggtatct acctatatat gttatactat aaccctctct ctcttcttta attgtttgcc 180
atataagcat gtttacgagt cccaagtgca taataccact taygttaacc cactatggac 240
agcctaatta gcaccgagct gtagtaccca gttggggcgt cacttcatga gctgcacaag 300
gtcctgctaa ttaggctggt cataatggaa gtatcataag cggtattatg catgccaact 360
agattttttt gatgatgtag cacacaatta aatgaggaaa gagggtgtgt tgtaacatat 420
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<210> 7
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acatttatta tgttacggta tctacctatg ttactataac catctctctc ttctttaatt 180
gcctaccaca ttaccatgtt tgcgagtccc aagtgcatga tactacttat gttaccccca 240
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ggacggagga actagtctgc attgtgcatt taagttttgt ctgaagtcaa tgtacctcta 360
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gaaggtcgga gtcaacggat t 21
<210> 10
<211> 21
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agcatgaact tggtcacctt c 21
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Claims (7)
1. An application of a specific primer group is disclosed,
the specific primer group consists of a primer A, a primer B and a primer C;
the primer A is a single-stranded DNA molecule shown in a sequence 1 of a sequence table;
the primer B is a single-stranded DNA molecule shown in a sequence 2 of the sequence table;
the primer C is a single-stranded DNA molecule shown in a sequence 3 of the sequence table;
the application is (d1), (d2), (d3), (d4), (d5) or (d 6):
(d1) the WSC content characters of the wheat stalks are identified in an auxiliary manner;
(d2) auxiliary screening or breeding of single wheat plants or wheat strains or wheat varieties with high stem WSC content;
(d3) auxiliary screening or breeding of single wheat plants or wheat strains or wheat varieties with low stem WSC content;
(d4) preparing a product for assisting in identifying the WSC content traits of the wheat stalks;
(d5) preparing a product for screening or breeding a single wheat plant or strain or variety with high stalk WSC content;
(d6) preparing a product for screening or breeding a single wheat plant or strain or variety with low stem WSC content.
2. The application of the kit is disclosed in the specification,
the kit comprises a specific primer group as described in claim 1;
the application is (d1), (d2) or (d 3):
(d1) the WSC content characters of the wheat stalks are identified in an auxiliary manner;
(d2) auxiliary screening or breeding of single wheat plants or wheat strains or wheat varieties with high stem WSC content;
(d3) and (3) auxiliary screening or breeding of a single wheat plant or strain or variety with low stem WSC content.
3. A method for identifying WSC content traits of stalks of wheat to be detected comprises the following steps: detecting the genotype of the wheat to be detected based on the specific SNP locus; the WSC content of stems of TT genotype wheat is higher than that of CC genotype wheat;
the specific SNP sites are as follows (e1) or (e 2):
(e1) 36 th nucleotide of DNA molecule shown in sequence 4 of the sequence table in wheat genome;
(e2) nucleotide 223 of DNA molecule shown in sequence 6 of the sequence table in wheat genome.
4. A breeding method for breeding wheat with high stem WSC content comprises the following steps: detecting the genotype of the wheat to be detected based on the specific SNP locus; selecting TT genotype wheat for breeding;
the specific SNP sites are as follows (e1) or (e 2):
(e1) 36 th nucleotide of DNA molecule shown in sequence 4 of the sequence table in wheat genome;
(e2) nucleotide 223 of DNA molecule shown in sequence 6 of the sequence table in wheat genome.
5. A method for identifying WSC content traits of stalks of wheat to be detected comprises the following steps:
(1) using the genome DNA of wheat to be detected as a template, and adopting the specific primer group described in claim 1 to carry out KASP;
(2) after the step (1) is finished, performing fluorescence scanning to determine the genotype of the wheat to be detected based on the specific SNP locus;
(3) and (4) judging according to the genotype result: the WSC content of stems of TT genotype wheat is higher than that of CC genotype wheat;
the specific SNP sites are as follows (e1) or (e 2):
(e1) 36 th nucleotide of DNA molecule shown in sequence 4 of the sequence table in wheat genome;
(e2) nucleotide 223 of DNA molecule shown in sequence 6 of the sequence table in wheat genome.
6. A breeding method for breeding wheat with high stem WSC content comprises the following steps:
(1) using the genome DNA of wheat to be detected as a template, and adopting the specific primer group described in claim 1 to carry out KASP;
(2) after the step (1) is finished, performing fluorescence scanning to determine the genotype of the wheat to be detected based on the specific SNP locus;
(3) selecting TT genotype wheat for breeding;
the specific SNP sites are as follows (e1) or (e 2):
(e1) 36 th nucleotide of DNA molecule shown in sequence 4 of the sequence table in wheat genome;
(e2) nucleotide 223 of DNA molecule shown in sequence 6 of the sequence table in wheat genome.
7. The application of a specific DNA molecule in identifying the content character of the wheat straw WSC is shown as a sequence 4 or a sequence 6 in a sequence table.
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| Title |
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| Development and validation of KASP assays for genes underpinning key economic traits in bread wheat;Awais Rasheed, 等;《Theor Appl Genet》;20160615;第129卷;第1843-1860页 * |
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