CN111676312A - Construction method and application of wheat KASP functional gene fingerprint - Google Patents
Construction method and application of wheat KASP functional gene fingerprint Download PDFInfo
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Abstract
The invention discloses a method for constructing a wheat KASP functional gene fingerprint, which comprises the following steps: (1) extracting DNA of a wheat variety to be tested; (2) carrying out PCR amplification on the DNA extracted in the step (1), wherein the used primers comprise 18 pairs of KASP fluorescence labeling primers, each pair of primers comprises two forward primers with label sequences, the sequences of the forward primers are shown as SEQ ID NO.1-36 of the sequence table, and the sequence of a universal reverse primer is shown as SEQ ID NO.37-54 of the sequence table; (3) and (3) based on the intensity of the fluorescence signal of the product in the amplification process, carrying out typing detection on the target product. The fingerprint of the present invention includes 18 pairs of KASP functional marker combinations comprising important wheat gene cloned successfully, PCR amplification technology, KASP genotyping technology and functional gene fingerprint construction of wheat variety based on the markers for controlling the adaptability and resistance of some important wheat functional genes.
Description
Technical Field
The invention relates to the technical field of wheat breeding and application, in particular to a construction method and application of a wheat KASP functional gene fingerprint.
Background
Wheat belongs to the wheat genus of the gramineae family, is one of the earliest cultivated crops in the world, and is one of the three major food crops in the world. In 43 countries around the world, wheat is the main food in 35-40% of the population. The wheat grains contain rich starch, more protein, a small amount of fat, various mineral elements and vitamin B, and are a commercial food with rich nutrition and higher economic value. Therefore, the method is particularly important for identifying commercial seed varieties.
The variety identification plays an important role in variety right protection and property dispute, and with the development of molecular biology, the appearance of the molecular marker technology provides a new means for variety identification. The molecular marker technology has the advantages of short period, no influence of environmental conditions, capability of performing high-throughput test analysis and the like, and is widely applied to the aspects of variety identification, seed purity identification and the like. However, the traditional fingerprint maps such as AFLP, SSR and the like have the defects of limited marker quantity, few detection sites, high site mutation rate and the like. The existing wheat fingerprint is constructed based on SSR markers, the SSR markers are randomly amplified, polymorphic sites are found, clear purpose is not achieved, accurate evaluation is not carried out on the interior of target genes, and the existing breeding requirements cannot be met.
In order to make up for the deficiency of SSR molecular markers, functional markers developed based on genes have been introduced. The functional marker is derived from the interior of a gene sequence for controlling phenotype, and after the phenotype function of the gene sequence is identified, polymorphic information in the sequence and the phenotype effect of a corresponding sequence are mined, so that a DNA marker, namely the functional marker, capable of distinguishing and predicting (multiple) alleles and relative characters is developed. Functional markers (functional markers) were developed based on polymorphisms in the gene sequence, and different allelic variations of these genes are directly related to phenotype. The invention constructs the fingerprint spectrum of the wheat variety based on the functional marker.
Disclosure of Invention
In order to overcome the defect that SSR markers do not aim at gene construction maps, the invention aims to provide a method for constructing a wheat KASP functional gene fingerprint map, which constructs the fingerprint map of the first functional gene by using the identified KASP functional markers for controlling important agronomic traits in wheat, makes up the one-sidedness of wheat identification aiming at single traits or genes, and provides scientific theoretical support for evaluation and utilization of wheat resources by breeders in the future.
The invention also aims to provide a wheat KASP functional gene fingerprint.
The invention also aims to provide application of the wheat KASP functional gene fingerprint.
One of the purposes of the invention is realized by adopting the following technical scheme:
a method for constructing a wheat KASP functional gene fingerprint comprises the following steps:
(1) extracting DNA of a wheat variety to be tested;
(2) carrying out PCR amplification on the DNA extracted in the step (1), wherein the used primers comprise 18 pairs of KASP fluorescence labeling primers, each pair of primers comprises two forward primers with label sequences, the sequences of the forward primers are shown as SEQ ID NO.1-36 of the sequence table, and the sequence of a universal reverse primer is shown as SEQ ID NO.37-54 of the sequence table;
(3) and (3) based on the intensity of the fluorescence signal of the product in the amplification process, carrying out typing detection on the target product.
Further, the wheat KASP functional gene comprises wheat adaptability related genes: genes Rht-B1 and Rht-D1 for controlling plant height, genes Vrn-A1, Vrn-B1 and Vrn-D1 for influencing vernalization of wheat, and a photoperiod gene Ppd-D1 for influencing flowering of wheat; wheat resistance-related genes: 4 genes for resisting pre-harvest sprouting are respectively PHS1, Sdr-B1, VP-1B and MFT-A1, and the genes for resisting drought are 1-fehw3 and Dreb-B1; leaf rust-resistant genes Lr14a, Lr34 and Lr 68; the gene Yr15 for resisting stripe rust, the gene Fhb1 for resisting gibberellic disease and a 1BL/1RS translocation line.
In order to breed high-yield varieties with strong adaptability, breeders continuously improve and select crops, and the short-stalk wheat represented by the 'green revolution' promotes the great improvement of the yield after the 40 th generation of the 20 th century; vernalization and flowering are two important growth stages in the life cycle of wheat, and the improvement of the two traits leads the adaptability of wheat to be continuously enhanced. The invention relates to the three traits, wherein the genes for controlling the plant height are Rht-B1 and Rht-D1; genes influencing vernalization of wheat are Vrn-A1, Vrn-B1 and Vrn-D1; the gene influencing wheat flowering is a photoperiod gene Ppd-D1.
Biotic and abiotic stresses are major factors affecting crop yield stabilization. With global climate change and rapid variation of pathogenic bacteria, breeders never stop selecting resistance traits and related genes. The phenotypic characters related by the invention comprise ear sprouting, drought resistance, scab resistance, leaf rust resistance and stripe rust resistance, wherein 4 genes for ear sprouting resistance are respectively PHS1, Sdr-B1, VP-1B and MFT-A1; the drought-resistant genes are 1-fehw3 and Dreb-B1; leaf rust-resistant genes Lr14a, Lr34 and Lr 68; the gene Yr15 for resisting stripe rust, the gene Fhb1 for resisting gibberellic disease and a 1BL/1RS translocation line.
Further, the extraction process of the DNA in the step (1) is as follows:
a: preparing CTAB buffer solution, and preheating in a 65 ℃ water bath;
b: taking a proper amount of wheat tissue, cooling by liquid nitrogen, crushing, adding the CTAB buffer solution preheated in the step A, uniformly mixing, and heating in a water bath;
c: cooling the mixture obtained in the step B to room temperature, adding a mixed solvent of chloroform and isoamylol, uniformly mixing, centrifuging, taking supernate, adding RNaseA, uniformly mixing, and standing at room temperature for a period of time;
d: and D, adding precooled isopropanol into the mixture obtained in the step C, uniformly mixing, standing for precipitation, centrifuging, taking the precipitate, adding an ethanol solution for washing, and drying to obtain the DNA sample to be detected.
Further, the preparation process of the CTAB buffer solution in the step a is as follows: to each liter of CTAB buffer was added 20g CTAB, 200mL of 1.0M Tris-HCl buffer at pH 8.0, 81g NaCl, 40mL of 0.5M EDTA, 10g of 1% PVP.
Further, the volume ratio of chloroform to isoamyl alcohol in the step C is 24: 1; 0.7 times the volume of the mixture of isopropanol pre-cooled at-20 c was added in step D above.
Further, the PCR amplification reaction system comprises: the concentration of the DNA sample to be tested is 40 ng/. mu.L, 2.2. mu.L, 2.5. mu.L of KASP LowMixturee and Mg2+0.04. mu.L, primer solution 0.056. mu.L, dd H2O0.204 μ L; the KASP amplification program was: pre-denaturation at 94 ℃ for 15 min; then, denaturation is carried out for 20s at the temperature of 95 ℃; gradient annealing at 65 ℃ and extending for 25s for 10 cycles, each cycle decreasing by 1 ℃; finally, denaturation is carried out for 10s at 95 ℃; annealing at 57 deg.C and extending for 1min for 30 cycles; storing at 4 ℃.
Further, the composition of the primer solution in the PCR amplification in the step (2) is: in a volume of 100. mu.L, each of 12. mu.L of two forward primers at a concentration of 100. mu.M, 30. mu.L of a universal reverse primer, dd H2O 46μL。
Further, after the PCR reaction is completed in the step (3), collecting fluorescence signals of the amplified products, carrying out typing detection on the products, displaying different allelic variation in the same locus in different colors, displaying materials of the same allelic variation type by using the same color point through a two-dimensional clustering chart, counting the allelic variation conditions of the wheat variety at 18 genetic loci, and calculating the genetic diversity of the locus; and further expressing two allelic variations of each gene by using black and white colors to construct a wheat fingerprint.
The second purpose of the invention is realized by adopting the following technical scheme:
a wheat KASP functional gene fingerprint is constructed by the above method.
The second purpose of the invention is realized by adopting the following technical scheme:
the application of the wheat KASP functional gene fingerprint in wheat variety identification is provided.
Compared with the prior art, the invention has the beneficial effects that: the method for constructing the wheat KASP functional gene fingerprint comprises 18 functional combinations formed by wheat genome KASP markers, utilizes a PCR amplification technology and a KASP genotyping technology to identify wheat varieties, can finish the identification of the wheat resource functional gene varieties and the evaluation of genetic diversity in a short time, and has the advantages of time saving, high throughput, rapidness, accuracy, convenient operation, difficult environmental influence on identification results and the like; compared with the traditional fingerprint constructed based on SSR markers, the method is used for constructing the functional gene fingerprint of the wheat variety based on the markers for controlling the development of important functional genes of the adaptability traits and the resistance traits in the wheat. The markers can be used for determining whether important genes are carried in the material, can be quickly applied to breeding practice, and have high application value. The method can effectively discriminate functional genes carried by wheat resources, reveal excellent genes of the varieties from the DNA level, provide technical support for reasonable utilization of excellent germplasm in the process of breeding the wheat varieties, and have good application prospect.
Drawings
FIG. 1 is the fingerprint of 50 wheat functional genes obtained by the present invention.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and the detailed description, and it should be noted that any combination of the embodiments or technical features described below can be used to form a new embodiment without conflict.
Example 1
In order to construct representative functional gene fingerprint spectra in China, 50 parts of wheat germplasm resources bred since new China is established in China are selected, and the materials are distributed in ten wheat ecological regions in China and have wide representativeness in different breeding years. Based on the materials, fingerprint maps of the materials on 18 key gene loci are constructed, 50 representative wheat varieties are shown in table 1, and the KASP functional genes and corresponding phenotypes of the wheat are shown in table 2.
TABLE 1
TABLE 2
(1) Extracting wheat gene DNA: extracting wheat leaf tissue genome DNA by a CTAB method, and comprising the following steps:
a: preparation of CTAB buffer per liter:
TABLE 3
| Composition (I) | Dosage of | |
| CTAB | 20g | |
| 1.0M Tris-HCl buffer (pH 8.0) | 200mL | |
| NaCl | 81g | |
| 0.5 | 40mL | |
| 1％PVP | 10g |
Placing the prepared CTAB buffer solution into a 65 ℃ water bath kettle for preheating in advance;
b: placing a proper amount of wheat leaf tissues into a 2.0mL centrifuge tube, adding small steel balls, cooling by liquid nitrogen, and quickly crushing by a sample grinding machine;
c: adding 800 μ L of preheated 2X CTAB buffer solution, mixing well, placing in 65 deg.C water bath for more than 30min, and mixing well by turning over every few minutes during water bath;
d: cooling to room temperature after the water bath is finished, adding 800 mu L chloroform/isoamylol (volume ratio is 24:1), reversing and mixing uniformly for several times until no obvious layering exists, and centrifuging at 12000rpm at 4 ℃ for 5-10 min;
e: carefully sucking the supernatant into a 1.5mL centrifuge tube, adding 5 μ L RNaseA, mixing uniformly, and standing at 37 ℃ for at least 15 min;
f: adding 0.7 volume times of isopropanol precooled at-20 ℃, slowly and uniformly inverting the mixture up and down, and standing the mixture for 30 to 60min to fully precipitate DNA; centrifuging at 12000rpm at 4 deg.C for 5 min;
g: discarding the supernatant or picking out DNA precipitate, adding 800 μ L70% ethanol, washing by turning upside down, centrifuging at 12000rpm for 2min, pouring out the supernatant, washing repeatedly for 2 times, and air drying at room temperature or in ventilation kitchen without alcohol smell;
h: the DNA was dissolved by adding 100. mu.L of 1 XTE (pH 8.0), the DNA was detected by electrophoresis on a 1% agarose gel, the concentration was measured, and the DNA was diluted to 40 ng/. mu.L. The remaining DNA sample was stored at-20 ℃ until use.
(2) Carrying out PCR amplification on the DNA extracted in the step (1), wherein the used primers comprise 18 pairs of KASP fluorescent labeled primers, each pair of primers comprises two forward primers with tag sequences shown in SEQ ID NO.1-36 of the sequence table, the tag sequences are FAM (5 'AAGGTGACCAAAGTTCATGCT 3') and HEX (5 'AAGGTCGGAGTCAACGGATT 3'), and a universal reverse primer with the sequence shown in SEQ ID NO.37-54 of the sequence table is used for ensuring the total amplification length<120bp, detailed in Table 4, and the amplification process is as follows: the reaction was performed in 384-well fluorescent quantitative plates, and the total volume of the reaction mixture was 5.0. mu.L per well. The preparation process of the mixed reaction system comprises the following steps: comprises 2.2 muL of DNA sample to be detected with the concentration of 40 ng/muL, 2.5 muL of KASP Low Mixturee and Mg2+0.04. mu.L, primer solution 0.056. mu.L, dd H2O0.204 μ L; the primer solution had the composition: in a volume of 100. mu.L, each of 12. mu.L of two forward primers at a concentration of 100. mu.M, 30. mu.L of a universal reverse primer, dd H2O 46μL。
The KASP amplification program was: pre-denaturation at 94 ℃ for 15 min; then, denaturation is carried out for 20s at the temperature of 95 ℃; gradient annealing at 65 ℃ and extending for 25s for 10 cycles, each cycle decreasing by 1 ℃; finally, denaturation is carried out for 10s at 95 ℃; annealing at 57 deg.C and extending for 1min for 30 cycles; storing at 4 deg.C;
(3) after the reaction is finished, collecting the fluorescence signal of the amplified product by using a QuantStaudio TM 7Flex fluorescence quantitative instrument (Applied biosystemsbby Life Technologies), and detecting the product by typing; visualization of data and interpretation of results was achieved by QuantStaudio (TM) real-time PCR Software v1.3(Applied Biosystems by Life Technologies). The variation types of all the identified materials in each gene are displayed by a two-dimensional clustering chart, a negative control (ddH2O) is used as a judgment base point, different materials present points with different colors on corresponding coordinates according to the carried allelic variation types, and the genetic diversity of the 18 gene loci of the 50 wheat varieties is counted. The results are shown in table 5, the genetic diversity variation range of all gene loci is between 0.1 and 0.5, and the genetic diversity variation ranges are classified into two categories according to the agronomic traits controlled by the genetic diversity variation ranges, wherein the two categories are respectively as follows: the average diversity of the adversity stress response gene locus is 0.33, the average diversity of the adaptive gene locus is 0.4, and the genetic diversity of the two genes is 0.53 by comprehensively considering. On the basis of the above 18 gene loci, 50 wheat fingerprints were constructed, in which two allelic variations of each gene are represented by black and white in two colors, and the results are shown in Table 5 and FIG. 1.
TABLE 4
TABLE 5
The method for constructing the wheat KASP functional gene fingerprint comprises 18 functional combinations formed by wheat genome KASP markers, utilizes a PCR amplification technology and a KASP genotyping technology to identify wheat varieties, can finish the identification of the wheat resource functional gene varieties and the evaluation of genetic diversity in a short time, and has the advantages of time saving, high throughput, rapidness, accuracy, convenient operation, difficult environmental influence on identification results and the like; compared with the traditional fingerprint constructed based on SSR markers, the method disclosed by the invention is used for constructing the functional gene fingerprint of the wheat variety based on the markers developed by some important functional genes for controlling the adaptability and resistance traits in the wheat, whether the material carries the important genes can be determined by using the markers, and the method can be quickly applied to breeding practice and has very high application value. The method can effectively discriminate functional genes carried by wheat resources, reveal excellent genes of the varieties from the DNA level, provide technical support for reasonable utilization of excellent germplasm in the process of breeding the wheat varieties, and have good application prospect.
The above embodiments are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited thereby, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are within the protection scope of the present invention.
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<120> wheat KASP functional gene fingerprint and construction method and application thereof
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<211>41
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>1
gaaggtgacc aagttcatgc tcccatggcc atctccagct g 41
<210>2
<211>41
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
gaaggtcgga gtcaacggat tcccatggcc atctccagct a 41
<210>3
<211>42
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
gaaggtgacc aagttcatgc tcatggccat ctcgagctgc tc 42
<210>4
<211>42
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>4
gaaggtcgga gtcaacggat tcatggccat ctcgagctgc ta 42
<210>5
<211>53
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>5
gaaggtgacc aagttcatgc tagagttttc caaaaagata gatcaatgta aat 53
<210>6
<211>52
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>6
gaaggtcgga gtcaacggat tgagttttcc aaaaagatag atcaatgtaa ac 52
<210>7
<211>43
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>7
gaaggtgacc aagttcatgc tggcagctaa tgtggggtag tca 43
<210>8
<211>43
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>8
gaaggtcgga gtcaacggat tggcagctaa tgtggggtag tct 43
<210>9
<211>44
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>9
gaaggtgacc aagttcatgc tatcattcga attgctagct ccgg 44
<210>10
<211>44
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>10
gaaggtcgga gtcaacggat tatcattcga attgctagct ccgc 44
<210>11
<211>43
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>11
gaaggtgacc aagttcatgc tcaaggaagt atgagcagcg gtt 43
<210>12
<211>43
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>12
gaaggtcgga gtcaacggat taagaggaaa catgttgggg tcc 43
<210>13
<211>44
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>13
gaaggtgacc aagttcatgc tggtggaaca gatgcaacta aagg 44
<210>14
<211>44
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>14
gaaggtcgga gtcaacggat tggtggaaca gatgcaacta aaga 44
<210>15
<211>41
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>15
gaaggtgacc aagttcatgc ttcagcagac ttcgactcgc a 41
<210>16
<211>41
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>16
gaaggtcgga gtcaacggat ttcagcagac ttcgactcgc g 41
<210>17
<211>46
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>17
gaaggtgacc aagttcatgc tcgtgcatgc agcctacgca tacgta 46
<210>18
<211>46
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>18
gaaggtcgga gtcaacggat tcgtgcatgc agcctacgca tacgtg 46
<210>19
<211>48
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>19
gaaggtgacc aagttcatgc tcgctcttat attagtttac ggagggag 48
<210>20
<211>44
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>20
gaaggtcgga gtcaacggat tctcattttg atagctctag ctaa 44
<210>21
<211>44
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>21
gaaggtgacc aagttcatgc tcctgcgcac tttcttcttc ctgt 44
<210>22
<211>43
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>22
gaaggtcgga gtcaacggat tctgcgcact ttcttcttcc tgg 43
<210>23
<211>41
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>23
gaaggtgacc aagttcatgc tctcccccct tccttctgtc c 41
<210>24
<211>41
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>24
gaaggtcgga gtcaacggat tctcccccct tccttctgtc t 41
<210>25
<211>52
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>25
gaaggtgacc aagttcatgc tctacactag tactactttg agacaatttt tt 52
<210>26
<211>50
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>26
gaaggtcgga gtcaacggat tacactagta ctactttgag acaattttaa 50
<210>27
<211>47
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>27
gaaggtgacc aagttcatgc tggtatgcca tttaacataa tcatgaa 47
<210>28
<211>47
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>28
gaaggtcgga gtcaacggat tggtatgcca tttaacataa tcatgat 47
<210>29
<211>42
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>29
gaaggtgacc aagttcatgc tcgtgtcttg gacctgagca at 42
<210>30
<211>42
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>30
gaaggtcgga gtcaacggat tcgtgtcttg gacctgagca ac 42
<210>31
<211>42
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>31
gaaggtgacc aagttcatgc tcagatcccc ggttctctca ag 42
<210>32
<211>42
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>32
gaaggtcgga gtcaacggat tcagatcccc ggttctctca aa 42
<210>33
<211>40
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>33
gaaggtgacc aagttcatgc tttgggctca cgtcgtgcaa 40
<210>34
<211>40
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>34
gaaggtcgga gtcaacggat ttgtctgttt cgctgggatg 40
<210>35
<211>40
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>35
gaaggtgacc aagttcatgc tggagcaggt ccagatcgcg 40
<210>36
<211>41
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>36
gaaggtcgga gtcaacggat tcggagcagg tccagatcgc a 41
<210>37
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>37
tcgggtacaa ggtgcgggcg 20
<210>38
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>38
cgggtacaag gtgcgcgcc 19
<210>39
<211>29
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>39
gttagtagtg atggtccaat aatgccaaa 29
<210>40
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>40
cacaggcttt cctatcattc gt 22
<210>41
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>41
gcctgaacgc ctagcctgtg ta 22
<210>42
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>42
gcctcccact acactgggc 19
<210>43
<211>30
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>43
gtgagtgtta tatgaaacta atgatccatt 30
<210>44
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>44
aggatctcgt tggccttgac g 21
<210>45
<211>25
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>45
tgatccatgc acgcatcagc gatcg 25
<210>46
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>46
cttcttccga agtgtatcat atg 23
<210>47
<211>29
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>47
tttcaccttg tgatatggat tgccttgat 29
<210>48
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>48
aggaagacgg cccgagcttt 20
<210>49
<211>29
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>49
aacaaactcc agtgtaaaca ccacagttt 29
<210>50
<211>27
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>50
tactatatgg gagcattatt tttttcc 27
<210>51
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>51
tgacctgagt cccgtcaaga 20
<210>52
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>52
cccccaaatg atcgagaata 20
<210>53
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>53
cttccagttt ctgctgccat 20
<210>54
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>54
gaagctccgg tagatggagg cta 23
Claims (10)
1. A method for constructing a wheat KASP functional gene fingerprint is characterized by comprising the following steps:
(1) extracting DNA of a wheat variety to be tested;
(2) carrying out PCR amplification on the DNA extracted in the step (1), wherein the used primers comprise 18 pairs of KASP fluorescence labeling primers, each pair of primers comprises two forward primers with label sequences, the sequences of the forward primers are shown as SEQ ID NO.1-36 of the sequence table, and the sequence of a universal reverse primer is shown as SEQ ID NO.37-54 of the sequence table;
(3) and (3) based on the intensity of the fluorescence signal of the product in the amplification process, carrying out typing detection on the target product.
2. The method of claim 1, wherein the wheat KASP functional genes comprise wheat adaptability-associated genes: genes Rht-B1 and Rht-D1 for controlling plant height, and genes Vrn-A1, Vrn-B1 and Vrn-D1 for influencing vernalization of wheat; a photoperiod gene Ppd-D1 affecting wheat flowering; wheat resistance-related genes: 4 anti-pre-sprouting genes are PHS1, Sdr-B1, VP-1B and MFT-A1 respectively, and the anti-drought genes are 1-fehw3 and Dreb-B1; leaf rust-resistant genes Lr14a, Lr34 and Lr 68; the gene Yr15 for resisting stripe rust, the gene Fhb1 for resisting gibberellic disease and a 1BL/1RS translocation line.
3. The method for constructing a wheat KASP functional gene fingerprint according to claim 1, wherein the process of extracting DNA in the step (1) is as follows:
a: preparing CTAB buffer solution, and preheating in a 65 ℃ water bath;
b: taking a proper amount of wheat tissue, cooling by liquid nitrogen, crushing, adding the CTAB buffer solution preheated in the step A, uniformly mixing, and heating in a water bath;
c: cooling the mixture obtained in the step B to room temperature, adding a mixed solvent of chloroform and isoamylol, uniformly mixing, centrifuging, taking supernate, adding RNaseA, uniformly mixing, and standing at room temperature for a period of time;
d: and D, adding precooled isopropanol into the mixture obtained in the step C, uniformly mixing, standing for precipitation, centrifuging, taking the precipitate, adding an ethanol solution for washing, and drying to obtain the DNA sample to be detected.
4. The method for constructing a fingerprint of a wheat KASP functional gene according to claim 3, wherein the CTAB buffer solution in the step A is prepared by the following steps: to each liter of CTAB buffer was added 20g CTAB, 200mL of 1.0M Tris-HCl buffer at pH 8.0, 81g NaCl, 40mL of 0.5M EDTA, 10g of 1% PVP.
5. The method for constructing a wheat KASP functional gene fingerprint as claimed in claim 3, wherein the volume ratio of chloroform to isoamyl alcohol in the step C is 24: 1; 0.7 times the volume of the mixture of isopropanol pre-cooled at-20 c was added in step D above.
6. The method for constructing a fingerprint of a wheat KASP functional gene according to claim 1, wherein the PCR amplification reaction system comprises: 2.2 muL of DNA sample to be tested with concentration of 40 ng/muL, 2.5 muL of KASP Low mix, Mg2+0.04. mu.L, primer solution 0.056. mu.L, ddH2O0.204 μ L; the KASP amplification program was: pre-denaturation at 94 ℃ for 15 min; then, denaturation is carried out for 20s at the temperature of 95 ℃; gradient annealing at 65 ℃ and extending for 25s for 10 cycles, each cycle decreasing by 1 ℃; finally, denaturation is carried out for 10s at 95 ℃; annealing at 57 deg.C and extending for 1min for 30 cycles; storing at 4 ℃.
7. The method for constructing a fingerprint of a wheat KASP functional gene according to claim 6, wherein the composition of the primer solution in the PCR amplification in the step (2) is as follows: in a volume of 100. mu.L, each of 12. mu.L of two forward primers at a concentration of 100. mu.M, 30. mu.L of a universal reverse primer, ddH2O 46μL。
8. The method for constructing a wheat KASP functional gene fingerprint as claimed in claim 7, wherein in the step (3), after the PCR reaction is completed, the fluorescence signals of the amplified products are collected, the products are subjected to typing detection, different allelic variations at the same locus show different colors, the materials of the same allelic variation type are displayed by two-dimensional clustering charts with the same color points, the genetic diversity of 18 gene loci is counted, and two allelic variations of each gene are represented by black and white colors, so as to construct a wheat fingerprint.
9. A wheat KASP functional gene fingerprint constructed by the method of any one of claims 1 to 8.
10. The use of a wheat KASP functional gene fingerprint according to claim 9 in the identification of wheat varieties.
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| CN113637789A (en) * | 2021-08-13 | 2021-11-12 | 四川农业大学 | Wheat stripe rust resistant gene YRTD121 linked KASP molecular marker, primer, kit and application |
| WO2023232152A1 (en) * | 2022-08-24 | 2023-12-07 | 江苏省农业科学院 | Multiplex kasp marker primer set for wheat plant height major genes and use thereof |
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| CA3049172A1 (en) * | 2016-12-21 | 2018-06-28 | Institute Of Crop Sciences, The Chinese Academy Of Agricultural Sciences | Plant grain trait-related protein, gene, promoter and snps and haplotypes |
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| CA3049172A1 (en) * | 2016-12-21 | 2018-06-28 | Institute Of Crop Sciences, The Chinese Academy Of Agricultural Sciences | Plant grain trait-related protein, gene, promoter and snps and haplotypes |
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| CN113637789A (en) * | 2021-08-13 | 2021-11-12 | 四川农业大学 | Wheat stripe rust resistant gene YRTD121 linked KASP molecular marker, primer, kit and application |
| WO2023232152A1 (en) * | 2022-08-24 | 2023-12-07 | 江苏省农业科学院 | Multiplex kasp marker primer set for wheat plant height major genes and use thereof |
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Application publication date: 20200918 |