CN113652423B - Method for auxiliary identification of drought resistance of wheat to be detected and special molecular marker thereof - Google Patents
Method for auxiliary identification of drought resistance of wheat to be detected and special molecular marker thereof Download PDFInfo
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Abstract
The invention discloses a method for assisting in identifying drought resistance of wheat to be tested and a special molecular marker thereof. Taking genome DNA of wheat to be detected as a template, and carrying out PCR amplification by adopting a primer pair consisting of a primer F and a primer R to obtain a DNA fragment; the DNA fragment is a molecular marker related to drought resistance of wheat. The molecular marker may specifically be a DNA segment a or a DNA segment b. The nucleotide sequences of the primer F, the primer R, DNA section A and the DNA section B are sequentially shown as SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14 and SEQ ID NO. 15. Wheat varieties containing the DNA segment B and not containing the DNA segment A in the genome have drought resistance. By applying the molecular marker provided by the invention, wheat varieties with drought resistance can be rapidly screened, so that the breeding pace of the wheat varieties is accelerated. The invention has important application value.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for assisting in identifying drought resistance of wheat to be detected and a special molecular marker thereof.
Background
Plants grow in complex and varied environments and are often subjected to adversity stress, wherein drought is a major adversity factor that affects and limits plant growth and development, and even causes death of plants, severely affecting agricultural production. Therefore, cultivation of drought-resistant crop varieties is always one of main targets of agricultural science and technology research.
Wheat (triticum aestinum l.) is the most prominent carbohydrate and protein source for people worldwide. Although the global wheat planting area and yield are increased suddenly, drought, high temperature, high salt and other abiotic stress seriously affect the wheat yield, and an effective breeding means is urgently needed to improve the wheat yield. Among many environmental stress factors, drought is the most serious worldwide problem for agricultural production. Research shows that the key of crop drought tolerance genetic improvement is cloning and utilization of excellent drought tolerance gene. Therefore, the excavation of the wheat drought-resistant gene has important significance for cultivating drought-resistant wheat varieties and improving wheat yield. The molecular marker assisted selection can rapidly and accurately analyze the genetic composition of an individual from the molecular level, so that the direct selection of genotypes is realized, and molecular breeding is carried out; the method is not influenced by environmental conditions and has high selection accuracy, so that the development of the molecular marker related to drought resistance of wheat has great significance.
Disclosure of Invention
The invention aims to assist in identifying drought resistance of wheat to be tested.
The invention firstly protects a specific primer pair, which can be composed of two primers for amplifying specific DNA fragments; the specific DNA fragment is provided with a target sequence of a primer pair consisting of a primer F and a primer R in a wheat genome;
the primer F may be a 1) or a 2) as follows:
a1 A single stranded DNA molecule shown in SEQ ID NO. 12;
a2 A DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides of SEQ ID NO. 12 and has the same function as SEQ ID NO. 12;
the primer R may be a 3) or a 4) as follows:
a3 A single stranded DNA molecule shown in SEQ ID NO. 13;
a4 A DNA molecule which is obtained by substituting and/or deleting and/or adding one or a plurality of nucleotides of SEQ ID NO. 13 and has the same function as the SEQ ID NO. 13.
The specific primer pair can specifically consist of the primer F and the primer R.
The function of the specific primer pair may be any one of the following b 1) -b 4):
b1 Auxiliary identification of drought resistance of wheat to be tested;
b2 Auxiliary screening of wheat varieties with or suspected to have drought resistance;
b3 Auxiliary screening of wheat varieties which are not provided with drought resistance or are suspected to be provided with drought resistance;
b4 Wheat breeding.
The specific primer pair is used for amplifying the following molecular markers related to the drought resistance of wheat.
The invention also provides a kit comprising any one of the specific primer pairs.
Conventional reagents for PCR amplification and/or conventional reagents for genomic extraction and/or conventional reagents for agarose gel electrophoresis may also be included in the kit.
The function of the kit may be any one of the following b 1) -b 4):
b1 Auxiliary identification of drought resistance of wheat to be tested;
b2 Auxiliary screening of wheat varieties with or suspected to have drought resistance;
b3 Auxiliary screening of wheat varieties which are not provided with drought resistance or are suspected to be provided with drought resistance;
b4 Wheat breeding.
The invention also protects the application of any one of the specific primer pairs or any one of the kits, and the specific primer pairs or the kits can be any one of the following b 1) to b 4):
b1 Auxiliary identification of drought resistance of wheat to be tested;
b2 Auxiliary screening of wheat varieties with or suspected to have drought resistance;
b3 Auxiliary screening of wheat varieties which are not provided with drought resistance or are suspected to be provided with drought resistance;
b4 Wheat breeding.
The method for assisting in identifying drought resistance of wheat to be tested by using the specific primer pair or the kit comprises the following steps of: taking genome DNA of wheat to be detected as a template, and carrying out PCR amplification by adopting any specific primer pair to obtain a PCR amplification product; then, the following judgment is made: if the PCR amplification product has a DNA fragment of about 209bp and does not have a DNA fragment of 189bp, the wheat to be detected has or is suspected to have drought resistance; otherwise, the wheat to be tested does not have drought resistance or is suspected to have drought resistance.
The method for auxiliary screening of wheat varieties with or suspected to have drought resistance by using the specific primer pair or the kit comprises the following steps: taking genome DNA of wheat to be detected as a template, and carrying out PCR amplification by adopting any specific primer pair to obtain a PCR amplification product; then, the following judgment is made: if the PCR amplification product has a DNA fragment of about 209bp and does not have a DNA fragment of 189bp, the wheat to be detected is or is candidate as the wheat variety with drought resistance, otherwise, the wheat to be detected is or is candidate as the wheat variety without drought resistance.
The invention also protects a DNA fragment obtained by amplifying the genome DNA of the wheat to be detected by using any one of the specific primer pairs as a template. The DNA fragment is the molecular marker related to the drought resistance of wheat to be protected. The molecular marker may specifically be a DNA segment a and/or a DNA segment b. The nucleotide sequence of the DNA segment A is shown as SEQ ID NO. 14. The nucleotide sequence of the DNA segment B is shown as SEQ ID NO. 15.
The invention also protects the application of the DNA fragment (namely the molecular marker) and can be any one of the following b 1) to b 8):
b1 Auxiliary identification of drought resistance of wheat to be tested;
b2 Auxiliary screening of wheat varieties with or suspected to have drought resistance;
b3 Auxiliary screening of wheat varieties which are not provided with drought resistance or are suspected to be provided with drought resistance;
b4 Wheat breeding;
b5 Preparing a product for assisting in identifying drought resistance of the wheat to be tested;
b6 Preparing a product for assisting in screening wheat varieties with or suspected to have drought resistance;
b7 Preparing a product for assisting in screening wheat varieties which do not have drought resistance or are suspected to have drought resistance;
b8 As molecular markers.
The DNA segment A and/or the DNA segment B also belong to the protection scope of the invention.
The invention also provides a method for assisting in identifying drought resistance of wheat to be tested, which can comprise the following steps:
(1) Taking genome DNA of wheat to be detected as a template, and carrying out PCR amplification by adopting any specific primer pair to obtain a PCR amplification product;
(2) After the step (1) is completed, the following judgment is carried out: if the PCR amplification product has a DNA fragment of about 209bp and does not have a DNA fragment of 189bp, the wheat to be detected has or is suspected to have drought resistance; otherwise, the wheat to be tested does not have drought resistance or is suspected to have drought resistance.
The invention also provides a method for assisting in identifying drought resistance of wheat to be tested, which can comprise the following steps:
(1) Detecting whether the genome DNA of the wheat to be detected contains a DNA segment A or a DNA segment B; the nucleotide sequence of the DNA segment A is shown as SEQ ID NO. 14; the nucleotide sequence of the DNA segment B is shown as SEQ ID NO. 15;
(2) After the step (1) is completed, the following judgment is carried out: if the genome DNA of the wheat to be tested contains a DNA section B and does not contain a DNA section A, the wheat to be tested has or is suspected to have drought resistance; otherwise, the wheat to be tested does not have drought resistance or is suspected to have drought resistance.
The invention also provides a method for assisting in screening wheat with different drought resistances, which can comprise the following steps: taking genome DNA of wheat to be detected as a template, and carrying out PCR amplification by adopting any specific primer pair to obtain a PCR amplification product; the drought resistance of wheat having a DNA fragment of about 209bp and no DNA fragment of 189bp in the PCR amplification product is higher than that of wheat having a DNA fragment of 189bp and no DNA fragment of 209bp in the PCR amplification product.
The invention also provides a method for assisting in screening wheat with different drought resistances, which comprises the following steps: detecting whether the genome DNA of the wheat to be detected contains a DNA segment A or a DNA segment B; the nucleotide sequence of the DNA segment A is shown as SEQ ID NO. 14; the nucleotide sequence of the DNA segment B is shown as SEQ ID NO. 15; the drought resistance of the wheat containing the DNA segment B and not containing the DNA segment A in the genome DNA of the wheat to be tested is higher than that of the wheat containing the DNA segment A and not containing the DNA segment B in the genome DNA of the wheat to be tested.
Any of the above-described above-mentioned heights may be statistically higher.
Experiments prove that wheat varieties containing the DNA segment B and not containing the DNA segment A in the genome DNA of the wheat have drought resistance. By applying the molecular marker provided by the invention, wheat varieties with drought resistance can be rapidly screened, so that the breeding pace of the wheat varieties is accelerated. The invention has important application value.
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FIG. 1 shows a real-time fluorescent quantitative determination of T 3 Expression level of TaPYL1 gene in wheat transformed with TaPYL1 gene.
FIG. 2 is T 3 Phenotype of the transgenic TaPYL1 wheat after PEG stress treatment and rehydration for 7 days.
FIG. 3 is T 3 Phenotype of wheat transformed with TaPYL1 gene after drought treatment and rehydration for 3 days.
FIG. 4 is T 3 Survival rate statistics results after drought treatment and rehydration of the transgenic TaPYL1 gene wheat for 3 days.
FIG. 5 shows the sequence differences of two haplotypes in drought-resistant and drought-sensitive wheat.
FIG. 6 shows PCR amplification results for two haplotypes.
FIG. 7 shows nucleotide polymorphism analysis of 120 wheat varieties.
FIG. 8 shows the difference in drought tolerance between haplotype A homozygous and haplotype B homozygous wheat materials.
FIG. 9 is a graph showing statistics of relative expression levels of TaPYL1 genes of the homozygous wheat material A and homozygous wheat material B under normal growth and drought stress.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Vector pCAMBIA3301 is described in the following literature: regulatory changes in TaSNAC8-6A are associated with drought tolerance in wheat seedlings.Plant Biotechnol J.202018 (4): 1078-1092.
The wheat variety chinese spring is described in the following literature: regulatory changes in TaSNAC8-6A are associated with drought tolerance in wheat seedlings.Plant Biotechnol J.202018 (4): 1078-1092. Hereinafter, the wheat variety chinese spring is abbreviated as chinese spring.
Wheat variety Fielder is described in the following documents: regulatory changes in TaSNAC8-6A are associated with drought tolerance in wheat seedlings.Plant Biotechnol J.202018 (4): 1078-1092.
The naming method of the polymorphic site in the following examples is the mark type + sequence number; wherein, the mark types are SNP and InDel, the SNP represents single nucleotide polymorphism, and the InDel represents insertion or deletion; the sequence number is marked as "+1" by A in the initiation codon (ATG) of the TaPYL1 gene in the Chinese spring reference genome DNA; such as SNP181 represents the presence of a single nucleotide polymorphism at base 181 downstream of A in the initiation codon (ATG); inDel-442 represents an insertion or deletion of 1 or more bases downstream of the 442 th base upstream of A in the initiation codon (ATG).
The positions of the primers or sequences in the following examples are each denoted "+1" as A in the initiation codon (ATG) of the TaPYL1 gene in the China spring reference genome DNA.
The genome sequence of TaPYL1 gene in Chinese spring reference genome DNA is shown as SEQ ID NO. 11, the 601 st to 1832 nd positions from the 5' end of SEQ ID NO. 11 are full-length cDNA sequence of TaPYL1 gene, the 719 th to 1360 th positions code for TaPYL1 protein with the amino acid sequence shown as SEQ ID NO. 1, and the 719 th position is base A in the initiation codon ATG.
EXAMPLE 1 cloning of the TaPYL1 Gene
1. Taking Chinese spring seeds, accelerating germination at 25 ℃ for 3 days, transferring the germinated seeds to nutrient soil, and culturing for two weeks at 25 ℃ to obtain Chinese spring seedlings.
2. Extracting total RNA of Chinese spring seedlings, and then carrying out reverse transcription to obtain cDNA of Chinese spring.
3. The cDNA of China spring is used as a template, and primer pairs consisting of 5'-ATGGAGCAGCAGCCTGTG-3' (SEQ ID NO: 3) and 5'-TTATTCTGCCGGCGGCGC-3' (SEQ ID NO: 4) are adopted for PCR amplification, so that a PCR amplification product of about 642bp is obtained.
4. The PCR amplification product was sequenced.
Sequencing results show that the nucleotide sequence of the PCR amplification product is shown as SEQ ID NO. 2.
The gene shown in SEQ ID NO. 2 was designated as the TaPYL1 gene. The TaPYL1 gene codes the TaPYL1 protein, and the amino acid sequence of the TaPYL1 protein is shown as SEQ ID NO. 1.
Example 2 acquisition and drought resistance identification of TaPYL1 Gene-transferred wheat
Solute of 10 XSLS Major and its concentration is CaCl 2 ·2H 2 O44g/L、KH 2 PO 4 1.7g/L、KNO 3 19g/L、MgSO 4 ·7H 2 O37g/L and NH 4 NO 3 16.5g/L, and the solvent is water.
100 XLS Minor has a solute and a concentration of CoC l2 ·6H 2 O2.5mg/L、CuSO 4 ·5H 2 O2.5mg/L、H 3 BO 3 620mg/L、KI83mg/L、MnSO 4 ·5H 2 O2230mg/L and ZnSO 4 ·7H 2 O1060mg/L, and water as solvent.
100 XFe-EDTA solute and FeSO concentration thereof 4 ·7H 2 O2.78g/L and Na 2 EDTA3.73g/L, and water as the solvent.
100 XVitamin solute and its concentration are Pyridoxine-HCl 0.05g/L, nicotinic acid 0.05g/L, thiamine-HCl 0.1g/L and myo-Inositol 10g/L, and the solvent is water.
Selection medium: 100mL of 10 XLS Major, 10mL of 100 XLS Minor, 10mL of 100 XFe-EDTA, 10mL of 100 XVitamin, 5mL of 2,4-D, 40g of Maitose, 0.5g of Glutamine, 0.75g of MgCl 2 ·6H 2 O, 1.95g MES and 5g Agarose are dissolved in a proper amount of water, and then the volume is fixed to 1L by water; sterilizing at 121deg.C for 15min; then 10g/L of Ascorbic acid, 50. Mu.L of AgNO at a concentration of 100mM are added 3 1mL of solution was concentrated at 150g/L of Timantin.
Differentiation medium: 100mL of 10 XSLS Major, 10mL of 100 XSLS Minor, 10mL of 100 XSELEDTA, 10mL of 100 XSELIATIN, 50mL of Zeatin solution at a concentration of 100mg/L, 100. Mu.L of CuSO at a concentration of 100mM 4 ·5H 2 Dissolving an O solution, 20g of Sucrose, 0.5g of MES and 3g of Gelrite in a proper amount of water, and then using water to fix the volume to 1L; sterilizing at 121deg.C for 15min; after that, 250. Mu.L of PPT at a concentration of 20g/L and 1mL of Carbenicillin at a concentration of 250g/L were added.
Rooting medium: 100mL of 10 XSLS Major, 10mL of 100 XSLS Minor, 10mL of 100 XSELEDTA, 10mL of 100 XSELIAmin, 2mL of IBA solution with the concentration of 100mg/L, 15g of Sucrose, 0.5g of MES and 3g of Gelrite are dissolved in a proper amount of water, the pH value is adjusted to 5.8, and then the volume is fixed to 1L by water; sterilizing at 121deg.C for 15min; after that, 250. Mu.L of PPT at a concentration of 20g/L and 1mL of Carbenicillin at a concentration of 250g/L were added.
1. Construction of recombinant vector pCAMBIA3301-GZ
The small DNA fragment between the restriction enzymes HindIII and EcoRI of the vector pCAMBIA3301 was replaced with the DNA molecule shown in positions 1 to 639 from the 5' -end of SEQ ID NO. 2 to obtain the recombinant vector pCAMBIA3301-GZ.
The recombinant vector pCAMBIA3301-GZ expresses TaPYL1 protein shown in SEQ ID NO. 1.
2. Acquisition of recombinant Agrobacterium tumefaciens
The recombinant vector pCAMBIA3301-GZ is transformed into agrobacterium tumefaciens EHA105 to obtain recombinant agrobacterium tumefaciens which is named EHA105/pCAMBIA3301-GZ.
The vector pCAMBIA3301 was transformed into Agrobacterium tumefaciens EHA105 to give a recombinant Agrobacterium designated EHA105/pCAMBIA3301.
3. Obtaining of TaPYL1 Gene-transferred wheat
1. The EHA105/pCAMBIA3301-GZ is introduced into a wheat variety Fielder by adopting an agrobacterium-mediated gene transformation method to obtain T 0 The TaPYL1 gene wheat is replaced.
The method comprises the following specific steps:
(1) EHA105/pCAMBIA3301-GZ was inoculated into YEB liquid medium containing 25mg/L spectinomycin, and shake-cultured at 28℃to OD 600nm The value is 0.5, and the recombinant agrobacterium is obtainedAnd (3) liquid.
(2) The young embryo of wheat variety Fielder was placed in a centrifuge tube (specification: 2 mL) filled with preservation solution (10 mL of 10 XSEMAjor, 1mL of 100 XSLS Minor, 1mL of 100 XSFe-EDTA, 1mL of 100 XSITAMIN, 10mL of Glucose and 0.5g of MES were dissolved in a proper amount of water, and then the volume was fixed to 1L with water), heat-treated at 46℃for 3min, and centrifuged at 2000rpm for 10min to obtain a treated young embryo.
(3) After the step (2) is completed, adding recombinant agrobacterium tumefaciens bacteria liquid into the treated young embryo, performing dark culture at 22 ℃ for 3 days, transferring to a selection medium, and performing dark culture at 28 ℃ for 7-10 days; screening with glufosinate with different concentrations, transferring to differentiation medium, transferring to rooting medium after differentiation, transferring into nutrient soil after a certain size to obtain T 0 The TaPYL1 gene wheat is replaced.
2. Taking T 0 Screening positive seedlings to obtain T by replacing TaPYL1 gene-transferred wheat 0 And (3) replacing TaPYL1 gene wheat.
3. Taking T 0 The wheat with the substitution TaPYL1 gene is subjected to selfing, positive seedlings are screened, and T is obtained 1 And (3) replacing TaPYL1 gene wheat.
4. Taking T 1 The wheat with the substitution TaPYL1 gene is subjected to selfing, positive seedlings are screened, and T is obtained 2 And (3) replacing TaPYL1 gene wheat.
5. Taking T 2 The wheat with the substitution TaPYL1 gene is subjected to selfing, positive seedlings are screened, and T is obtained 3 And (3) replacing TaPYL1 gene wheat.
The method for screening positive seedlings comprises the following steps: extracting genome DNA of wheat to be detected, taking the genome DNA as a template, adopting primer pairs consisting of 5'-TCGATGCTCACCCTGTTGTTTG-3' (SEQ ID NO: 9) and 5'-TGTATAATTGCGGGACTCTAATC-3' (SEQ ID NO: 10) to carry out PCR amplification to obtain a PCR amplification product, and carrying out Sanger sequencing; then, the following judgment is made: if a PCR amplified product contains a DNA fragment of about 871bp and the sequencing result comprises a nucleotide sequence shown as SEQ ID NO. 2, wheat seedlings corresponding to the PCR amplified product are positive seedlings.
According to the method, EHA105/pCAMBIA3301-GZ is replaced by EHA105/pCAMBIA3301, and other steps are the same, so as to obtain T 3 Substitute-transformed empty carrier wheatIs called as trans-empty vector wheat for short.
4. Real-time fluorescent quantitative detection T 3 Expression level of TaPYL1 Gene in TaPYL1 Gene-transferred wheat
1. Respectively T each 3 Seedlings which are grown to 10 days by transferring the TaPYL1 gene wheat are put into liquid nitrogen for preservation, and corresponding samples to be tested are obtained. And (5) placing seedlings grown to 10 days from the empty carrier-transformed wheat into liquid nitrogen for preservation, and obtaining corresponding samples to be tested. Taking a wheat variety Fielder, and alternately culturing at 16 ℃ for 10 days in a light-dark mode to obtain wheat seedlings to be detected; and (5) placing the wheat seedlings to be tested into liquid nitrogen for preservation, and obtaining corresponding samples to be tested.
2. Total RNA of a sample to be tested is extracted by using Trizo1 (biotupped), then first-strand cDNA is reversely transcribed, the cDNA is diluted by 100 times by using sterile water as a template, and the relative expression quantity of the TaPYL1 gene (TaActin 1 gene is an internal reference gene) is detected by real-time quantitative PCR.
Primers for detecting the TaPYL1 gene were 5'-CCGTCACCACCGTCTCCGAACT-3' (SEQ ID NO: 5) and 5'-CCTCGGCCACGGACTTGAGCT-3' (SEQ ID NO: 6).
Primers for detecting the TaActin1 gene were 5'-AAATCTGGCATCACACTTTCTAC-3' (SEQ ID NO: 7) and 5'-GTCTCAAACATAATCTGGGTCATC-3' (SEQ ID NO: 8).
The partial results are shown in FIG. 1 (WT is wheat variety Fielder, OE1-OE12 are all T 3 Wheat transformed with the TaPYL1 gene). The result shows that the relative expression quantity of the TaPYL1 gene in the wheat variety field and the transgenic carrier wheat has no obvious difference; each T is compared with the wheat variety Fielder 3 The relative expression level of TaPYL1 gene in the wheat transformed with the TaPYL1 gene is obviously increased, wherein 3T 3 The relative expression level of the TaPYL1 gene in the wheat strain transformed with the TaPYL1 gene is higher, and the relative expression level is sequentially named as OE4, OE5 and OE6, and subsequent experiments are carried out.
5. T (T) 3 Drought resistance identification of wheat transformed with TaPYL1 gene
Drought resistance identification
T with OE4 as wheat seed to be tested 3 Seed of substitution, T of OE5 3 Seed of substitution, T of OE6 3 Seeds of the wheat variety Fielder or empty vectorWheat seeds.
The experiment was repeated three times to average the values, and the procedure for each repetition was as follows:
1. taking wheat seeds to be detected, germinating for 3 days at room temperature, transferring to a water culture solution, and culturing alternately at 16 ℃ and in light and dark (16 h illumination/8 h darkness) for 15 days to obtain wheat seedlings to be detected.
Solute of water culture solution and concentration thereof is 0.75mM K 2 SO 4 、0.1mM KCl、0.25mM KH 2 PO 4 、0.65mM MgSO 4 、0.1mM EDTA-Fe、2mM Ca(NO 3 ) 2 、1.0μM MnSO 4 、1.0μM ZnSO 4 、0.1μM CuSO 4 And 0.005. Mu.M (NH) 4 ) 6 Mo 7 O 2 The solvent is water.
2. 20 wheat seedlings to be tested with basically consistent growth state are transferred to a water culture solution (control) or a water culture solution containing 30% PEG, and are alternately cultured at 16 ℃ and in light and dark (16 h light/8 h dark) for 10 days, so that phenotypes are observed.
The results show that the leaves of the transgenic empty vector wheat and wheat variety Fielder are obviously dried up, T 3 Leaves of wheat transformed with the TaPYL1 gene (OE 4, OE5, and OE 6) wilt seriously.
3. After the step 2 is completed, transferring 20 wheat seedlings to be tested to a water culture solution, culturing at 16 ℃ in a light-dark alternating mode (16 h illumination/8 h darkness) for 7 days, and observing the phenotype.
The results are shown in FIG. 2 (VC is the empty vector wheat, WT is the wheat variety Fielder). The results show that, compared with the wheat variety Fielder, T 3 The tolerance of the transgenic TaPYL1 gene wheat (OE 4, OE5 and OE 6) to PEG is obviously improved; the tolerance of the trans-empty vector wheat and the wheat variety Fielder to PEG is not significantly different.
(II) drought resistance identification II
T with OE4 as wheat seed to be tested 3 Seed of substitution, T of OE5 3 Seed of substitution, T of OE6 3 Seeds of the wheat variety Fielder or empty vector wheat seeds.
The experiment was repeated three times to average the values, and the procedure for each repetition was as follows:
1. 48 wheat seeds to be tested are sown in a box (35.0 cm multiplied by 20.0cm multiplied by 15.0 cm) filled with 0.6kg of nutrient soil, and are cultured for 21 days under normal conditions, so as to obtain wheat seedlings to be tested.
The phenotype of some wheat seedlings tested is shown in FIG. 3 (before drought) (WT is wheat variety Fielder).
2. After completion of step 1, drought treatment (stopping watering) was performed for 30 days, and phenotypes were observed.
The results show that the leaves of the transgenic empty vector wheat and wheat variety Fielder are obviously dried up, T 3 Leaves of wheat transformed with the TaPYL1 gene (OE 4, OE5, and OE 6) wilt seriously.
3. After completion of step 2, rehydration, phenotype was observed after 3 days and survival was counted.
Plants that appear to be capable of normal growth and seed recovery are defined as surviving plants. Survival is the percentage of the number of surviving plants to the total number of plants.
The phenotype of a part of wheat seedlings tested after rehydration is shown in FIG. 3 (after drought) (WT is wheat variety Fielder).
The survival statistics are shown in FIG. 4 (WT is wheat variety Fielder). The results show that, compared with the wheat variety Fielder, T 3 The survival rate of the wheat (OE 4, OE5 and OE 6) with the transformed TaPYL1 gene is obviously improved to 80% -85%; the survival rates of the transformed empty vector wheat and the wheat variety Fielder are not significantly different.
Thus, the TaPYL1 gene is a wheat drought-resistant gene.
Example 3, wheat drought resistance-related haplotype, molecular markers and application thereof
1. Development of molecular markers
1. Through a great deal of experiments, the inventor of the present invention found that 2 drought-resistance-related haplotypes exist at the polymorphic site upstream of the initiation codon ATG of the TaPYL1 gene in the genomic DNA of wheat, and the haplotypes are named haplotype A and haplotype B respectively, as shown in FIG. 5.
2. Preparing a specific primer pair for identifying the molecular marker related to the drought resistance of the wheat. The specific primer pair consists of a primer F: CGAAGAATTGGTGAATCATGTACTAC-3' (SEQ ID NO: 12) and primer R:5'-TAAAAAATAGAAGAGCATCTCCTAAAAG-3' (SEQ ID NO: 13).
2. Polymorphism detection
1. The drought-resistant wheat variety Pubing202 is used as a male parent, the drought-sensitive wheat variety GLUYAS EARLY is used as a female parent for hybridization, and then selfing is carried out for 2 generations, so that the segregating population Pubing202 XGLUYAS EARLY is obtained. The drought-resistant wheat variety Pubing202 is used as a male parent, the drought-sensitive wheat variety Wanmai33 is used as a female parent for hybridization, and then selfing is carried out for 2 generations, so that the segregating population Pubing202 multiplied by Wanmai33 is obtained.
2. Genotyping
(1) Extracting genomic DNA of wheat (Pubing 202, GLUYAS EARLY, wanmai33, group Pubing202 XGLUYAS EARLY or group Pubing202 XWanmai 33) to obtain genomic DNA of wheat.
(2) And (3) respectively carrying out PCR amplification by using the genomic DNA extracted in the step (1) as a template and using a primer pair consisting of the primer F and the primer R in the step one to obtain a PCR amplification product.
PCR reaction conditions: 94 ℃ for 5min;94℃30s,58℃30s,72℃ 30s,40 cycles; 72 ℃ for 5min; permanent at 16 ℃.
(3) And (3) taking part of the PCR amplification product obtained in the step (2), performing 2% agarose gel electrophoresis, then staining nucleic acid, and photographing under a gel imaging system.
The result of electrophoresis of a part of wheat is shown in FIG. 6. The results show that the band patterns of the wheat PCR amplification products are three: band A (showing one band, 189 bp), band B (showing one band, 209 bp) and band C (showing two bands, 189bp and 209bp, respectively).
(4) And (3) taking part of the PCR amplification product obtained in the step (2) and sequencing.
Sequencing results show that three nucleotide sequences of the wheat PCR amplification products are available: sequence A (SEQ ID NO:14, about 189 bp), sequence B (SEQ ID NO:15, about 209 bp) and sequence C (both sequences shown as SEQ ID NO:14 and SEQ ID NO:15, respectively).
SEQ ID NO:14：
cgaagaattggtgaatcatgtactactatgttttaagatggcgtttagtaaaataaaaaagtagtgaagttatttcggtggccagaaatgaaatgaatttgataaaataaattactccactaaatttCCTaaatttagtggagtaaaaatagtccattaatcttttaggagatgctcttctatttttta
SEQ ID NO:15：
Cgaagaattggtgaatcatgtactactatgttttaagatggcgtttagtaaaataaaaaagtagtgaagttatttcggtggccagaaatgaaatgaatttgataaaataaattactccactaaatttaggagatcagttatgtacaatcaaaatttagtggagtaaaaatagtccattaatcttttaggagatgctcttctatttttta
If the band type of the PCR amplified product of the wheat to be detected is band type A or the nucleotide sequence is sequence A, the wheat to be detected is homozygous wheat of haplotype A; if the band type of the PCR amplified product of the wheat to be detected is band type B or the nucleotide sequence is sequence B, the wheat to be detected is homozygous of haplotype B; if the band type of the PCR amplified product of the wheat to be detected is band type C or the nucleotide sequence is sequence C, the wheat to be detected is the wheat heterozygous with monomer type A and monomer type B.
The genotyping results are shown in tables 1 and 2.
TABLE 1 genotype of parents and survival statistics after drought treatment
| Parent strain | Genotype of the type | Survival (%) |
| Pubing202 | Monomer type B homozygote | 96.2 |
| GLUYASEARLY | Monomer type nail homozygosis | 9.4 |
| Wanmai33 | Monomer type nail homozygosis | 0.0 |
TABLE 2 genotype of drought-resistant and drought-sensitive hybrid segregating populations and survival statistics after drought treatment
Note that: the lower case letters are significance of plants with different genotypes in the population at P <0.05 through one-way ANOVA, and the plants with different genotypes in the population contain the same lower case letters and have no significant differences in the lower case letters; capitalization is the significance of plants of different genotypes in a population at P <0.01 through one-way ANOVA, and the difference is not significant when the plants contain the same capitalization, and the difference is extremely significant when the plants do not contain the same capitalization. In Table 2, the genotyping ratios for each population were in accordance with Mendelian's genetic law.
3. Drought resistance phenotype identification
The experiment was repeated three times and the mean value was taken for statistical analysis. The method comprises the following specific steps:
from the segregating group (Pubing 202 XGLUYAS EARLY or Pubing202 XWanmai 33), 90 grains of the homozygous wheat seeds of the monomer type A and 90 grains of the homozygous wheat seeds of the monomer type B are randomly taken, transplanted into a cultivation basin (the length x width x depth is 0.70 x 0.50 x 0.18 m, the cultivation matrix containing 5 kg of vermiculite and 5 kg of turfy soil is mixed), 180 plants are planted in each basin, and the plants are respectively cultivated in a greenhouse for 21 days under the conditions of 16h illumination and 8h darkness in a photoperiod, the temperature is 14 ℃ in the daytime at 12 ℃ at night and the air humidity is 60 percent, and are subjected to drought treatment (namely, are not watered). And rehydrating after the relative water content of the soil (namely the volume percent of the water in the soil measured by a soil moisture meter SU-LA (W) manufactured by Beijing alliance Innovative technology Co., ltd.) reaches 0 percent for one week. The survival rate of each genotype plant in each parent and population was counted 3 days after rehydration (the plants whose aerial parts failed to turn green after 3 days of rehydration were defined as dead plants; the plants whose aerial parts could turn green after 3 days of rehydration were defined as surviving plants; the survival rate was the percentage of surviving plants to total plant number).
The statistical results are shown in tables 1 and 2. The result shows that the drought resistance of the homozygous wheat of the monomer type B is greater than that of the homozygous wheat of the monomer type A, and the greater is statistically. Therefore, in wheat breeding, a haplotype B homozygous wheat with higher drought resistance should be selected for breeding.
Example 4 discovery of haplotypes related to drought resistance in wheat
The names of 120 wheat varieties in this example are detailed in column 2 of Table 3, and 120 wheat varieties are described in the following documents: regulatory changes in TaSNAC8-6A are associated with drought tolerance in wheat seedlings.Plant Biotechnol J.202018 (4): 1078-1092.
TABLE 3 Table 3
1. Drought resistance phenotype identification
Planting natural variation group composed of 120 parts of wheat variety in a cultivation pool, using nutrient soil and vermiculite as cultivation substrates, and sub-packaging in the two pools. Each pool is divided into 120 cells, and 12 seedlings can be planted in each cell. And stopping watering when three pieces of plants are She Miaoling, and performing drought treatment until the relative water content of soil (namely, the volume percent of water in the soil measured by a soil moisture meter SU-LA (W) manufactured by Beijing Union Innovative and Wei industry technologies Co., ltd.) is reduced to 0, and rehydrating the soil for 7 days, wherein the survival rate is counted after rehydration for 3 days (the plants with the overground parts failing to turn green after rehydration for 3 days are defined as dead plants, the plants with the overground parts capable of turning green after rehydration for 3 days are defined as survival plants, and the survival rate is the percentage of the survival plants to the total planting number).
The drought phenotype data used in the statistical analysis are all the average values of independent repeated tests.
The survival statistics are shown in Table 3.
Drought resistance of wheat varieties is classified into three types according to the survival rate results in table 3: the wheat variety with the survival rate of more than or equal to 40 percent is drought-resistant, the wheat variety with the survival rate of less than 10 percent is drought-sensitive, and the wheat variety with the survival rate of less than or equal to 10 percent is intermediate.
The typing results are shown in Table 3.
2. Polymorphic site identification
(1) The coding region, 5 'untranslated region and 3' untranslated region of the TaPYL1 gene in 120 wheat varieties were sequenced to about 2.0kb genomic fragment, and the sequencing primers were F:5'-cctctctttagccatcccttggtat-3' (SEQ ID NO: 16) and R:5'-cagcacctcaggaatcacacctat-3' (SEQ ID NO: 17). Sequencing results were aligned using MEGA5.0 (http:// www.megasoftware.net /). The nucleotide polymorphism was analyzed according to the alignment result, and 7 polymorphic sites were obtained, of which there were 5 SNPs and 2 InDels (shown in FIG. 7), and the Minimum Allele Frequency (MAF) was 0.05 or more.
3. Correlation analysis of polymorphic loci and drought resistance phenotypes
120 wheat varieties were classified as haplotype A homozygous, haplotype B homozygous, haplotype A and haplotype B heterozygous according to the method of example 3. The results are shown in Table 3.
Further statistical analysis was performed on the drought tolerance phenotype of the two haplotype wheat materials, and the results are shown in fig. 8. The results show that the survival rate of the homozygous wheat of the haplotype B is significantly higher than that of the homozygous wheat of the haplotype A.
4. The expression level of TaPYL1 gene in the haplotype type B homozygous wheat material is obviously higher than that of the haplotype type A homozygous wheat material
To determine the differences in contribution of gene expression to drought resistance, 46 wheat varieties (see table 4) were analyzed for expression levels of the TaPYL1 gene under normal growth and drought stress. As a result, as shown in FIG. 9, the expression level of TaPYL1 gene in 22 parts of the haplotype B homozygous wheat material was significantly higher than that in 24 parts of the haplotype A homozygous wheat material.
TABLE 4 Table 4
The procedure for analyzing the relative expression level of the TaPYL1 gene in 46 wheat varieties was as follows:
wheat seeds were sterilized with 1%o (v/v) Topsin-M (Rotam Crop Sciences Ltd.) for 10min, rinsed 3 times with deionized water, and finally placed on a petri dish with filter paper laid thereon for germination acceleration at 22℃for 3 days, and the germinated seeds were transferred to nutrient soil. Cutting off water treatment is carried out when the seedling age is 3, when the Relative Leaf Water Content (RLWC) is 90% and 58%, respectively taking leaves, extracting total RNA from not less than three seedlings by TRIZOL (Biotopped) method, then eliminating genome pollution by DNAse I (Takara), then measuring concentration by Nanodrop1000 (Thermo Scientific product, USA), uniformly taking 5 mug and carrying out 0.8% agarose gel electrophoresis. 1. Mu.g of total RNA was used to synthesize cDNAs using recombinant M-MLV reverse transcriptase (Promega) and 1. Mu.g of Oligo (dT) 23 as a primer. The relative expression level of the TaPYL1 gene in 46 wheat varieties was analyzed by fluorescence real-time quantitative PCR.
Using specific primer F2:5'-CCGTCACCACCGTCTCCGAACT-3' (SEQ ID NO: 18) and R2:5'-CCTCGGCCACGGACTTGAGCT-3' (SEQ ID NO: 19) quantitative analysis of the gene TaPYL1 was performed. Taking a gene TaActin1 as an internal reference, and taking a primer FC2:5'-AAATCTGGCATCACACTTTCTAC-3' (SEQ ID NO: 20) and RC2:5'-GTCTCAAACATAATCTGGGTCATC-3' (SEQ ID NO: 21).
real-Time fluorescent quantitative PCR was performed on a real-Time fluorescent quantitative PCR apparatus Applied Biosystems Step One Real-Time PCR System (ABI, USA), and 3 replicates were set for one parallel test. Methods reported using Livak KJ and Schmittgen TD (2001), i.e. 2 -ΔΔCT The relative expression level was calculated.
ΔΔC T ＝(C T.Target －C T.TaActin1 ) Time x －(C T.Target －C T.TaActin1 ) Time 0
Time x represents any point in Time, time 0 Represents 1-fold amount of target gene expression corrected by TaActin 1.
The relative expression levels of the TaPYL1 gene of the 46 wheat varieties obtained were subjected to differential significance analysis by SPSS12.0 software.
The results are shown in FIG. 9. The result shows that under drought stress condition, the expression level of TaPYL1 gene in the haplotype B homozygous wheat material is obviously higher than that of the haplotype A homozygous wheat material.
The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
<110> university of agriculture and forestry science and technology in northwest
<120> method for auxiliary identification of drought resistance of wheat to be tested and special molecular marker thereof
<160>21
<170> PatentIn version 3.5
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Ser Leu Val Ala Gln Arg Ile Glu Ala Pro Pro Ala Ala Val Trp Ala
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Ile Val Arg Arg Phe Asp Cys Pro Gln Val Tyr Lys His Phe Ile Arg
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Ser Cys Ala Leu Arg Pro Asp Pro Glu Ala Gly Asp Glu Leu Arg Pro
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Gly Arg Leu Arg Glu Val Ser Val Ile Ser Gly Leu Pro Ala Ser Thr
100 105 110
Ser Thr Glu Arg Leu Asp Leu Leu Asp Asp Ala Arg Arg Ala Phe Gly
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Phe Thr Ile Thr Gly Gly Glu His Arg Leu Arg Asn Tyr Arg Ser Val
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Thr Thr Val Ser Glu Leu Ser Pro Ala Ala Pro Ala Glu Ile Cys Thr
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Val Val Leu Glu Ser Tyr Val Val Asp Val Pro Asp Gly Asn Ser Glu
165 170 175
Glu Asp Thr Arg Leu Phe Ala Asp Thr Val Val Arg Leu Asn Leu Gln
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Lys Leu Lys Ser Val Ala Glu Ala Asn Ala Ala Ala Ala Ala Thr Thr
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Ala Pro Pro Ala Glu
210
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Claims (7)
1. The application of the specific primer pair is any one of the following b 1) to b 4):
b1 Auxiliary identification of drought resistance of wheat to be tested;
b2 Auxiliary screening of wheat varieties with or suspected to have drought resistance;
b3 Auxiliary screening of wheat varieties which are not provided with drought resistance or are suspected to be provided with drought resistance;
b4 Wheat breeding;
the specific primer pair consists of a primer F and a primer R;
the primer F is a single-stranded DNA molecule shown in SEQ ID NO. 12;
the primer R is a single-stranded DNA molecule shown as SEQ ID NO. 13.
2. Use of a kit comprising a specific primer pair as defined in claim 1, as any one of the following b 1) -b 4):
b1 Auxiliary identification of drought resistance of wheat to be tested;
b2 Auxiliary screening of wheat varieties with or suspected to have drought resistance;
b3 Auxiliary screening of wheat varieties which are not provided with drought resistance or are suspected to be provided with drought resistance;
b4 Wheat breeding.
3. The use of a DNA fragment amplified using the specific primer pair of claim 1 using genomic DNA of wheat to be tested as a template is any one of the following b 1) to b 8):
b1 Auxiliary identification of drought resistance of wheat to be tested;
b2 Auxiliary screening of wheat varieties with or suspected to have drought resistance;
b3 Auxiliary screening of wheat varieties which are not provided with drought resistance or are suspected to be provided with drought resistance;
b4 Wheat breeding;
b5 Preparing a product for assisting in identifying drought resistance of the wheat to be tested;
b6 Preparing a product for assisting in screening wheat varieties with or suspected to have drought resistance;
b7 Preparing a product for assisting in screening wheat varieties which do not have drought resistance or are suspected to have drought resistance;
b8 As a molecular marker for assisting in identifying drought resistance of wheat.
4. A method for assisting in identifying drought resistance of wheat to be tested comprises the following steps:
(1) Taking genome DNA of wheat to be detected as a template, and carrying out PCR amplification by adopting the specific primer pair in claim 1 to obtain a PCR amplification product;
(2) After the step (1) is completed, the following judgment is carried out: if the PCR amplification product has a 209bp DNA fragment and does not have a 189bp DNA fragment, the wheat to be detected has or is suspected to have drought resistance; otherwise, the wheat to be tested does not have drought resistance or is suspected to have drought resistance.
5. A method for assisting in identifying drought resistance of wheat to be tested comprises the following steps:
(1) Detecting whether the genome DNA of the wheat to be detected contains a DNA segment A or a DNA segment B; the nucleotide sequence of the DNA segment A is shown as SEQ ID NO. 14; the nucleotide sequence of the DNA segment B is shown as SEQ ID NO. 15;
(2) After the step (1) is completed, the following judgment is carried out: if the genome DNA of the wheat to be tested contains a DNA section B and does not contain a DNA section A, the wheat to be tested has or is suspected to have drought resistance; otherwise, the wheat to be tested does not have drought resistance or is suspected to have drought resistance.
6. A method for assisting in screening wheat with different drought resistances comprises the following steps: taking genome DNA of wheat to be detected as a template, and carrying out PCR amplification by adopting the specific primer pair in claim 1 to obtain a PCR amplification product; the drought resistance of wheat having a DNA fragment of 209bp and not having a DNA fragment of 189bp in the PCR amplification product is higher than that of wheat having a DNA fragment of 189bp and not having a DNA fragment of 209bp in the PCR amplification product.
7. A method for assisting in screening wheat with different drought resistances comprises the following steps: detecting whether the genome DNA of the wheat to be detected contains a DNA segment A or a DNA segment B; the nucleotide sequence of the DNA segment A is shown as SEQ ID NO. 14; the nucleotide sequence of the DNA segment B is shown as SEQ ID NO. 15;
the drought resistance of the wheat containing the DNA segment B and not containing the DNA segment A in the genome DNA of the wheat to be tested is higher than that of the wheat containing the DNA segment A and not containing the DNA segment B in the genome DNA of the wheat to be tested.
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| CN114774579B (en) * | 2022-06-02 | 2023-03-28 | 西北农林科技大学 | Haplotype related to drought resistance of wheat TaDTG6 gene and molecular marker and application thereof |
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| CN112176083A (en) * | 2019-07-05 | 2021-01-05 | 中国科学院遗传与发育生物学研究所 | Functional molecular marker of wheat powdery mildew resistance related gene Pm41 and application thereof |
| CN112501335A (en) * | 2020-12-03 | 2021-03-16 | 宁夏农林科学院农作物研究所（宁夏回族自治区农作物育种中心） | Method for identifying drought resistance of wheat and special complete set of reagent thereof |
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| CN112176083A (en) * | 2019-07-05 | 2021-01-05 | 中国科学院遗传与发育生物学研究所 | Functional molecular marker of wheat powdery mildew resistance related gene Pm41 and application thereof |
| CN112501335A (en) * | 2020-12-03 | 2021-03-16 | 宁夏农林科学院农作物研究所（宁夏回族自治区农作物育种中心） | Method for identifying drought resistance of wheat and special complete set of reagent thereof |
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| 小麦TaPYL基因家族的分析及功能鉴定;史梦梦;《中国优秀硕士学位论文全文数据库 农业科技辑》;20210115(第1期);第18页第2.2.5节，第21页第一段，第28页第3.2.3节，第34页第4.1.6节，第35页第4.2.1节 * |
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