CN111662966A - Construction method and application of wheat backbone parent functional gene fingerprint spectrum - Google Patents
Construction method and application of wheat backbone parent functional gene fingerprint spectrum Download PDFInfo
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Abstract
The invention discloses a method for constructing a functional gene fingerprint spectrum of a wheat backbone parent, 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 26 pairs of fluorescence labeled primers, each pair of primers comprises two forward primers with tag sequences, the sequences of the forward primers are shown as SEQ ID NO.1-52 in a sequence table, and the sequence of a universal reverse primer is shown as SEQ ID NO.52-78 in the sequence table; (3) collecting the fluorescence signal of the amplified product, and typing and detecting the product. The fingerprint map of the invention comprises 26 marker combinations composed of important functional genes for controlling the relevant characters of the wheat yield and quality, and the functional gene fingerprint map construction is carried out on the wheat backbone parent, so that the functional gene identification of the wheat backbone parent can be completed in a short time, and the application value is very high.
Description
Technical Field
The invention relates to the technical field of wheat breeding and application, in particular to a wheat backbone parent functional gene fingerprint spectrum and a construction method and application thereof.
Background
Wheat belongs to the triticum genus of the gramineae family, which is one of the earliest crops cultivated in the world. Through long-term domestication and artificial selection, the plant is one of the grain crops in the world. The wheat grains are rich in starch, more protein, a small amount of fat, various mineral elements and vitamin B, and provide necessary calories for one fifth of the world population. And thus its identification has become particularly important.
In the book of 'variety improvement and pedigree analysis', the concept of backbone parents is put forward for the first time by wheat breeding families: plays a key role in cross breeding, and a large number of large-area popularization varieties are cultivated by using the hybrid breeding material, or a plurality of parent breeding materials with wide application value are derived from the hybrid breeding material. The backbone parent has the characteristics of high combining ability besides excellent properties, and is easy to be hybridized with other parents to breed excellent varieties. The backbone parent is an important germplasm type for breeding new varieties and is the key for realizing variety upgrading and updating. According to statistics, more than 2000 varieties of wheat are bred in China in 1949-2000-year-old, and the backbone parents related to the blood relationship comprise: locust wheat, swallow 1817, east gate of Yangjiang, Chengdu optical head, Scutigerella immaculata, Bimantha 4, Beijing 8, Xinong 6028, Wuyi wheat, Nanda 2419, Afu, Abbe, Loff forest 10, ink bar 66 and the like, and the sowing area of the wheat variety bred by the backbone parents reaches 86 percent of the total popularization area in production. From the historical development point of view, crop breeding is the history of creation and effective utilization of backbone parents, the backbone parents aggregate a plurality of excellent fragments of excellent materials, the change of the backbone parents can basically reflect the direction of variety improvement at different stages, and the backbone parents are important substance carriers for recording the production level of crops in specific regions in specific historical periods. The backbone parent therefore possesses superior characteristics that address significant production problems under the prevailing conditions. Genetically, genetic material from backbone parents should have important genetic contribution to new species breeding. The gene is a basic functional unit influencing the external expression of crop traits, and the identification of important trait gene loci based on backbone parents has important significance for deeply analyzing the genetic basis and guiding germplasm innovation.
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. 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, sensitivity to mutation reaction and the like. The existing wheat fingerprint is constructed based on SSR markers, and does not construct a fingerprint through functional markers, and does not have a functional gene fingerprint of a wheat backbone parent. 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. Therefore, the fingerprint spectrum is constructed on the wheat backbone parent by using the functional marker, the excellent genes carried by the backbone parent are determined, and the method has important reference value for breeding and summarizing the formation of the backbone parent in the future.
Disclosure of Invention
In order to overcome the defects of the prior art, one of the purposes of the invention is to provide a method for constructing a functional gene fingerprint of a backbone wheat parent, wherein the fingerprint of the functional gene of the backbone wheat parent is constructed by using a functional marker identified in the backbone wheat parent and used for controlling important agronomic characters, so that scientific theoretical support is provided for a breeder to evaluate and utilize resources of the backbone wheat parent, and the defects of empirical breeding are overcome.
The invention also aims to provide a wheat backbone parent functional gene fingerprint.
The invention also aims to provide application of the wheat backbone parent functional gene fingerprint spectrum in wheat variety identification.
One of the purposes of the invention is realized by adopting the following technical scheme:
a construction method of a wheat backbone parent 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 26 pairs of fluorescence labeled primers, each pair of primers comprises two forward primers with tag sequences, the sequences of the forward primers are shown as SEQ ID NO.1-52 in a sequence table, and the sequence of a universal reverse primer is shown as SEQ ID NO.52-78 in the sequence table;
(3) collecting the fluorescence signal of the amplified product, and typing and detecting the product.
Further, the functional genes of the wheat backbone parents comprise genes related to yield traits and genes related to quality traits: the yield trait related genes comprise 7 genes for controlling thousand seed weight, namely Sus1-7A, Sus1-7B, Sus2-2A, Sus2-2B, Cwi-4A, TGW6 and GS-D1; 3 genes for controlling the particle type, namely GASR-A1, GW2-6A and GW 2-6B; 2 genes for controlling the grain number of the panicle are CKX-D1 and MOC-7A respectively; the genes related to the quality comprise 3 genes affecting gluten strength, namely Glu-B1, Glu-A1 and Glu-D1; 3 genes influencing the hardness of grains, namely Pina-D1, Pinb-D1 and Pinb 2-B2; amylopectin affecting gene Wx-B1; influence polyphenol oxidase genes Ppo-A1 and Ppo-D1; gene Pod-A1 affecting peroxidase; the 4 genes influencing the yellow pigment content are Zds-A1, Lcy-B1, Pds-B1 and Psy-B1 respectively.
The wheat yield is three factors of the number of ears per unit area, the number of grains per ear and thousand-grain weight, the three factors are mutually dependent and mutually influenced, and the improvement and aggregation of yield-related characters and corresponding genes to achieve high yield are always the targets of continuous pursuit of breeding workers. The phenotypic traits related to the invention comprise thousand kernel weight, kernel type and spike number, wherein 7 genes for controlling thousand kernel weight are Sus1-7A, Sus1-7B, Sus2-2A, Sus2-2B, Cwi-4A, TGW6 and GS-D1 respectively; 3 genes for controlling the particle type, namely GASR-A1, GW2-6A and GW 2-6B; 2 genes for controlling the grain number of the panicle are CKX-D1 and MOC-7A respectively.
With the development of economy in China, the living demands of people are continuously improved, and the status of wheat quality improvement in breeding targets is increasingly important. Wheat quality includes nutritional quality and processing quality. The processing quality is mainly related to the factors of glutenin subunits with high and low molecular weights, polyphenol oxidase, flavochrome content, grain hardness and the like. The phenotypic traits related to the invention comprise gluten strength, grain hardness, yellow pigment content, polyphenol oxidase and peroxidase, wherein 3 genes influencing the gluten strength are Glu-B1, Glu-A1 and Glu-D1; 3 genes influencing the hardness of grains, namely Pina-D1, Pinb-D1 and Pinb 2-B2; amylopectin affecting gene Wx-B1; influence polyphenol oxidase genes Ppo-A1 and Ppo-D1; gene Pod-A1 affecting peroxidase; the 4 genes influencing the yellow pigment content are Zds-A1, Lcy-B1, Pds-B1 and Psy-B1 respectively.
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 volume of isopropanol pre-cooled at-20 ℃ was added in step D above.
Further, the PCR amplification reaction system comprises 2.2 muL of DNA sample to be detected with the concentration of 40 ng/muL, 2.5 muL of KASP LowMixture, 0.04 muL of Mg2+, 0.056 muL of primer solution and 0.204 muL of dd H2O 0.204; 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 site in different colors, displaying materials of the same allelic variation type by using the same color point through a two-dimensional clustering chart, and representing the two allelic variation of each gene by using black and white colors to construct the wheat fingerprint.
The second purpose of the invention is realized by adopting the following technical scheme:
a wheat backbone parent functional gene fingerprint spectrum is constructed by the method.
The third purpose of the invention is realized by adopting the following technical scheme:
the application of the wheat backbone parent functional gene fingerprint spectrum in the identification of wheat varieties.
Compared with the prior art, the invention has the beneficial effects that: the construction method of the wheat backbone parent functional gene fingerprint spectrum provided by the invention comprises the steps of identifying the wheat variety by utilizing a PCR amplification technology and a KASP genotyping technology based on 26 pairs of functional markers developed based on genes related to yield and quality traits, and completing identification of the wheat backbone parent functional gene variety in a short time, wherein the spectrum basically comprises important yield traits and genes related to quality traits which are concerned by breeders at present, scans and identifies the backbone parent which plays an important role in breeding, provides theoretical guarantee for development of breeding work in future, and has the advantages of time saving, high flux, rapidness, accuracy, convenience in operation, difficulty in environmental influence on identification results and the like; compared with the traditional fingerprint constructed based on SSR markers, the method is characterized in that the functional gene fingerprint is constructed on the basis of the markers developed by 26 important functional genes for controlling the yield and quality traits of wheat, whether the important genes are carried in the material can be determined by using the markers, and the method can be quickly applied to breeding practice and has high application value. The method can effectively discriminate the yield gene and the quality gene carried by the wheat backbone parent, reveal the essence of the backbone parent from the DNA level, provide technical support for reasonable utilization of excellent germplasm in the process of breeding wheat varieties, and have good application prospect.
Drawings
FIG. 1 is functional gene fingerprint of 14 parts of wheat backbone parent variety obtained by the method of 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 the functional gene fingerprint of the first wheat backbone parent in China, the invention utilizes 14 backbone parents which make great contribution to the wheat breeding in China between 1949 and 2000-plus, and the molecular markers are respectively as follows: locust wheat, swallow tail 1817, Wuyi wheat, Bimantha 4, Dongmen, Scutigerella, Xinong 6028, Nanda 2419, Chengdu bared, Abbe, Afu, Beijing No. 8, inkpad 66 and Loff forest No. 10. These backbone parents have extraordinary influence on wheat breeding in China, for example: cultivating 88 excellent varieties of backbone parent locust wheat; in addition, the backbone parent of 56-60 years of the last century, namely Nanda 2419, Beijing No. 8, Abbe, Aff and the like, well solves the problem of rust resistance, and increases the average yield of wheat in China by 1 time. The visible backbone parent has great promotion effect on the wheat breeding process in China, and has important significance in constructing the fingerprint spectrum. The functional genes and phenotypes involved in the fingerprint of the present invention are shown in table 1.
TABLE 1
(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 2
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) Performing PCR amplification on the DNA extracted in the step (1), wherein the used primers comprise 26 pairs of fluorescence labeled primers, each pair of primers comprises two forward primers with tag sequences, the sequences of the forward primers are shown as SEQ ID NO.1-52 of the sequence table, and the tag sequences are as follows: FAM (5 'AAGGTGACCAAAGTTCATGCT 3') and HEX (5 'AAGGTCGGAGTCAACGGATT 3') are universal reverse primers, and the sequences of the universal reverse primers are shown in sequence tables SEQ ID NO.52-78 so as to ensure the total amplification length<120bp, detailed in Table 3, 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 and result interpretation of data are realized through QuantStaudio real-time PCR Software v1.3(Applied Biosystems by Life Technologies), different allelic variation at the same site presents different colors, materials with the same allelic variation type are displayed through a two-dimensional cluster map by using points with the same color, a negative control (ddH2O) is used as a judgment base point, 14 backbone parents present points with different colors on corresponding coordinates according to the allelic variation types carried by the backbone parents, and on the basis of the 26 gene loci, fingerprint maps of 14 wheat backbone parents are constructed, wherein two allelic variations of each gene are expressed by two colors of black and white, and the results are shown in Table 3 and FIG. 1.
TABLE 3
The construction method of the wheat backbone parent functional gene fingerprint spectrum provided by the invention comprises 26 genes related to yield and quality traits, utilizes the functional markers developed by the differential sites thereof, utilizes the PCR amplification technology and the KASP genotyping technology to identify wheat varieties, can finish the identification of the wheat backbone parent functional gene varieties in a short time, basically comprises the important agronomic traits concerned by current breeders, scans and identifies the backbone parents which play a major role in breeding, provides theoretical guarantee for the development of future breeding work, and has the advantages of time saving, high flux, rapidness, accuracy, convenient operation, difficult environmental influence on the identification result and the like; compared with the traditional fingerprint constructed based on SSR markers, the method is based on the markers for developing 26 important functional genes for controlling the yield and quality traits of the wheat, the functional gene fingerprint is constructed on the backbone parent variety of the wheat, whether the backbone parent carries the important genes can be determined by using the markers, and the method can be quickly applied to breeding practice and has high application value. The method can effectively discriminate the functional genes carried by the backbone parents of the wheat, reveals the reason for the formation of the backbone parents from the DNA level, provides technical support for reasonable utilization of excellent germplasm in the process of breeding wheat varieties, and has 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.
Sequence listing
<110> Zunyi university of medical science
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<213> Artificial Sequence (Artificial Sequence)
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<213> Artificial Sequence (Artificial Sequence)
<400>23
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<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>24
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<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>25
gaaggtgacc aagttcatgc tcatattgca atctctatga ggctag 46
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<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>26
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<211>42
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>27
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<210>28
<211>42
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>28
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<210>29
<211>41
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>29
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<210>30
<211>41
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>30
gaaggtcgga gtcaacggat tgatttgatc catgccctct t 41
<210>31
<211>46
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>31
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<210>32
<211>46
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>32
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<210>33
<211>45
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>33
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<210>34
<211>45
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>34
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<210>35
<211>42
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>35
gaaggtcgga gtcaacggat tgcggtgtcc ttgagcttct ca 42
<210>36
<211>53
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>36
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<210>37
<211>53
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>37
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<210>38
<211>42
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>38
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<210>39
<211>42
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>39
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<210>40
<211>44
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>40
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<210>41
<211>44
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>41
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<210>42
<211>49
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>42
gaaggtgacc aagttcatgc tctgcgcttc accaacggtg ttgacgtcg 49
<210>43
<211>49
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>43
gaaggtcgga gtcaacggat tctgcgcttc accaacggtg ttgacgtca 49
<210>44
<211>39
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>44
gaaggtgacc aagttcatgc tatgcatgca tgcatgcgt 39
<210>45
<211>44
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>45
gaaggtcgga gtcaacggat tcgtcgatag tctcatgcat atgc 44
<210>46
<211>42
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>46
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<210>47
<211>42
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>47
gaaggtcgga gtcaacggat tgccaagaaa tgtcgctctc at 42
<210>48
<211>41
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>48
gaaggtgacc aagttcatgc tataactgct caccccccac c 41
<210>49
<211>41
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>49
gaaggtcgga gtcaacggat tcacggtaga ggagccggtt c 41
<210>50
<211>43
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>50
gaaggtgacc aagttcatgc tcggaaagct tagaaatgca cgg 43
<210>51
<211>43
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>51
gaaggtcgga gtcaacggat tcggaaagct tagaaatgca cgt 43
<210>52
<211>25
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>52
cgaagaagct tggcctggat agtat 25
<210>53
<211>24
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>53
ttcttctctc gttggcctta tcgc 24
<210>54
<211>30
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>54
tactaaaaag gtattaccca agtgtaactt 30
<210>55
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>55
atgaaggccc tcttcctcat agg 23
<210>56
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>56
gtcacctggc ccacaaaatg 20
<210>57
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>57
tgttttggtg gtggtgaaga tga 23
<210>58
<211>27
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>58
accctagtat tgtaccttag ttcaaac 27
<210>59
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>59
caaacccacg cagggacaag t 21
<210>60
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>60
tactggcctg gcggtacatg at 22
<210>61
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>61
gcctaatttg attcttccac ac 22
<210>62
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>62
aagccgacgc ggattttgaa 20
<210>63
<211>26
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>63
cgaaagcgtc gaaccaagga atcctc 26
<210>64
<211>27
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>64
ggcagaaatg tattagcaaa caaaacc 27
<210>65
<211>28
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>65
aaggaagtcc gggctcatgg tggggtca 28
<210>66
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>66
ctgtcgttca acatcattgt ctg 23
<210>67
<211>24
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>67
atggttatgc ttgaatggaa gagc 24
<210>68
<211>24
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>68
gctgtggatg cggctcaggg cgcg 24
<210>69
<211>27
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>69
actgctgagt acaatgccgc gatccca 27
<210>70
<211>25
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>70
catcgaattg aagaaaagtt cacgc 25
<210>71
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>71
cgagctaggg tttgttgcga gc 22
<210>72
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>72
aggcttgtca aaacgtgggg tcc 23
<210>73
<211>28
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>73
gtgaagtaga cttgacccgt aacttgat 28
<210>74
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>74
aacttttcac ggtgaacag 19
<210>75
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>75
caagaatttt gggacggagg ga 22
<210>76
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>76
atatgtaggg caggaagggc 20
<210>77
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>77
cacctctaat ccaatgcgat cc 22
Claims (10)
1. A construction method of a wheat backbone parent functional gene fingerprint spectrum is characterized by comprising the following steps:
(1) extracting DNA of the backbone parent material of the wheat to be tested;
(2) carrying out PCR amplification on the DNA extracted in the step (1), wherein the used primers comprise 26 pairs of fluorescence labeled primers, each pair of primers comprises two forward primers with tag sequences, the sequences of the forward primers are shown as SEQ ID NO.1-52 in a sequence table, and the sequence of a universal reverse primer is shown as SEQ ID NO.52-78 in the sequence table;
(3) collecting the fluorescence signal of the amplified product, and typing and detecting the product.
2. The method for constructing the functional gene fingerprint of the wheat backbone parent according to claim 1, wherein the functional genes of wheat comprise genes related to yield traits and genes related to quality traits: the yield trait related genes comprise 7 genes for controlling thousand seed weight, namely Sus1-7A, Sus1-7B, Sus2-2A, Sus2-2B, Cwi-4A, TGW6 and GS-D1; 3 genes for controlling the particle type, namely GASR-A1, GW2-6A and GW 2-6B; 2 genes for controlling the grain number of the panicle are CKX-D1 and MOC-7A respectively; the genes related to the quality comprise 3 genes affecting gluten strength, namely Glu-B1, Glu-A1 and Glu-D1; 3 genes influencing the hardness of grains, namely Pina-D1, Pinb-D1 and Pinb 2-B2; amylopectin affecting gene Wx-B1; influence polyphenol oxidase genes Ppo-A1 and Ppo-D1; gene Pod-A1 affecting peroxidase; the 4 genes influencing the yellow pigment content are Zds-A1, Lcy-B1, Pds-B1 and Psy-B1 respectively.
3. The method for constructing a functional gene fingerprint of a backbone parent of wheat according to claim 1, wherein the DNA extraction process 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 leaf tissues, 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 the fingerprint of the functional gene of the backbone parent of wheat as claimed in claim 3, wherein the CTAB buffer solution is prepared 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.
5. The method for constructing the functional gene fingerprint of the backbone parent of wheat as claimed in claim 3, wherein the volume ratio of chloroform to isoamyl alcohol in the step C is 24: 1; 0.7 volume of isopropanol pre-cooled at-20 ℃ was added in step D above.
6. The method for constructing the fingerprint of the functional genes of the backbone parents of wheat as claimed in claim 1, wherein the PCR amplification reaction system comprises 2.2 μ L of DNA sample to be tested with a concentration of 40ng/μ L, 2.5 μ L of KASP Low mix, and Mg2+0.04. mu.L, primer solution 0.056. mu.L, dd H2O0.204 μ L; the amplification procedure 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 extension for 25s, 10 cycles eachCircularly reducing the temperature 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 functional gene of a backbone parent of wheat according to claim 6, wherein the primer solution in the PCR amplification in the step (2) comprises: 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。
8. The method for constructing a fingerprint of a functional gene of a backbone wheat as claimed in claim 7, wherein in step (3) after the PCR reaction is completed, fluorescence signals of the amplified product are collected, typing detection is performed on the product, different allelic variations at the same site are in different colors, materials of the same allelic variation type are displayed by two-dimensional clustering charts at the same color point, and two allelic variations of each gene are represented by black and white colors, thereby constructing a wheat fingerprint.
9. A wheat backbone parent functional gene fingerprint map, which is constructed by the method of any one of claims 1 to 8.
10. The use of the fingerprint of functional gene of wheat backbone parent of claim 9 in wheat variety identification.
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| CN108913698A (en) * | 2018-07-25 | 2018-11-30 | 安徽农业大学 | One kind CAPS label relevant to wheat ear germinating resistance/perception and its application |
| CN113249513A (en) * | 2021-06-08 | 2021-08-13 | 中国农业大学 | Molecular marker for identifying or assisting in identifying wheat SDS-sedimentation value and application thereof |
| CN113817859A (en) * | 2021-08-24 | 2021-12-21 | 江汉大学 | MNP (MNP) marker locus, primer composition and kit for wheat variety identification and application of MNP marker locus, primer composition and kit |
| CN116855504A (en) * | 2023-05-11 | 2023-10-10 | 四川农业大学 | Wheat red glume red stalk gene RgM G52 and KASP molecular marker and application thereof |
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| CN108913698A (en) * | 2018-07-25 | 2018-11-30 | 安徽农业大学 | One kind CAPS label relevant to wheat ear germinating resistance/perception and its application |
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| CN113817859A (en) * | 2021-08-24 | 2021-12-21 | 江汉大学 | MNP (MNP) marker locus, primer composition and kit for wheat variety identification and application of MNP marker locus, primer composition and kit |
| CN113817859B (en) * | 2021-08-24 | 2023-10-13 | 江汉大学 | MNP (MNP) marking site for wheat variety identification, primer composition, kit and application of MNP marking site |
| CN116855504A (en) * | 2023-05-11 | 2023-10-10 | 四川农业大学 | Wheat red glume red stalk gene RgM G52 and KASP molecular marker and application thereof |
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