阅读说明：本技术 一种辅助鉴定待测小麦抗旱性的方法及其专用的分子标记 (Method for assisting in identifying drought resistance of wheat to be detected and special molecular marker thereof ) 是由 毛虎德 康振生 简超 黄雪玲 程新秀 于 2021-09-29 设计创作，主要内容包括：本发明公开了一种辅助鉴定待测小麦抗旱性的方法及其专用的分子标记。以待测小麦的基因组DNA为模板,采用引物F和引物R组成的引物对进行PCR扩增,得到DNA片段；该DNA片段即为与小麦抗旱性相关的分子标记。分子标记具体可为DNA区段甲或DNA区段乙。引物F、引物R、DNA区段甲和DNA区段乙的核苷酸序列依次如SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14和SEQ ID NO:15所示。基因组中含有DNA区段乙且不含有DNA区段甲的小麦品种具有抗旱性。应用本发明提供的分子标记,能快速筛选出具有抗旱性的小麦品种,从而加速小麦品种的育种步伐。本发明具有重要的应用价值。(The invention discloses a method for assisting in identifying the drought resistance of wheat to be detected 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 the drought resistance of wheat. The molecular marker may be specifically a DNA segment A or a DNA segment B. The nucleotide sequences of the primer F, the primer R, DNA segment A and the DNA segment B are shown as SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14 and SEQ ID NO. 15 in sequence. Wheat varieties which contain DNA segment B and do not contain DNA segment A in the genome have drought resistance. By applying the molecular marker provided by the invention, the wheat variety with drought resistance can be quickly screened out, so that the breeding pace of the wheat variety is accelerated. The invention has important application value.)

1. The specific primer pair consists of two primers for amplifying specific DNA fragments; the specific DNA fragment has a target sequence of a primer pair consisting of a primer F and a primer R in a wheat genome;

the primer F is a1) or a 2):

a1) a single-stranded DNA molecule shown as SEQ ID NO. 12;

a2) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to the SEQ ID NO. 12 and has the same function as the SEQ ID NO. 12;

the primer R is a3) or a 4):

a3) a single-stranded DNA molecule represented by SEQ ID NO 13;

a4) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in SEQ ID NO. 13 and has the same function as the SEQ ID NO. 13.

2. The specific primer pair of claim 1, wherein: the specific primer pair consists of the primer F and the primer R.

3. A kit comprising a specific primer pair according to claim 1 or 2.

4. The specific primer pair of claim 1 or 2 or the use of the kit of claim 3, which is any one of the following b1) -b 4):

b1) The drought resistance of the wheat to be tested is identified in an auxiliary manner;

b2) auxiliary screening of wheat varieties with or suspected to have drought resistance;

b3) auxiliary screening of wheat varieties without or suspected to have no drought resistance;

b4) and (5) wheat breeding.

5. The DNA fragment obtained by using the genome DNA of wheat to be detected as template and adopting the specific primer pair described in claim 1 or 2 to make amplification.

6. The use of the DNA fragment of claim 5, which is any one of the following b1) -b 8):

b1) the drought resistance of the wheat to be tested is identified in an auxiliary manner;

b2) auxiliary screening of wheat varieties with or suspected to have drought resistance;

b3) auxiliary screening of wheat varieties without or suspected to have no drought resistance;

b4) breeding wheat;

b5) preparing a product for assisting in identifying the drought resistance of the wheat to be detected;

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 without or suspected to have no drought resistance;

b8) as a molecular marker.

7. A method for auxiliary identification of drought resistance of wheat to be tested comprises the following steps:

(1) carrying out PCR amplification by using the genome DNA of wheat to be detected as a template and adopting the specific primer pair of claim 2 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 detected does not have drought resistance or is suspected to have drought resistance.

8. A method for auxiliary identification of 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 detected contains the DNA segment B and does not contain the DNA segment A, the wheat to be detected has or is suspected to have drought resistance; otherwise, the wheat to be detected does not have drought resistance or is suspected to have drought resistance.

9. A method for auxiliary screening of wheat with different drought resistances comprises the following steps: carrying out PCR amplification by using the genome DNA of wheat to be detected as a template and adopting the specific primer pair of claim 2 to obtain a PCR amplification product; the wheat having the DNA fragment of about 209bp and not having the DNA fragment of 189bp in the PCR amplification product had a higher drought resistance than the wheat having the DNA fragment of 189bp and not having the DNA fragment of 209bp in the PCR amplification product.

10. A method for auxiliary screening of 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 with the genomic DNA of the wheat to be detected containing the DNA segment B and not containing the DNA segment A is higher than that of the wheat with the genomic DNA of the wheat to be detected containing the DNA segment A and not containing the DNA segment B.

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 and develop in complex and variable environments and are often stressed by adversity, wherein drought is a main adversity factor influencing and limiting the growth and development of the plants, even leads to plant death and seriously influences agricultural production. Therefore, cultivation of drought-resistant crop varieties is always one of the main targets of agricultural science and technology research.

Wheat (TriticumaestivumL) is the leading carbohydrate and protein source for people worldwide. Although the planting area and the yield of the wheat are increased dramatically and rapidly in the world, abiotic stress such as drought, high temperature, high salt and the like seriously affects the yield of the wheat, and an effective breeding means is urgently needed to be found to improve the yield of the wheat. Among many environmental stress factors, the threat of drought to agricultural production is one of the most serious worldwide problems. Research shows that the key of genetic improvement of drought tolerance of crops is the cloning and utilization of excellent drought tolerance genes. Therefore, the excavation of the wheat drought-resistant gene has important significance for cultivating drought-resistant wheat varieties and improving the wheat yield. The molecular marker-assisted selection can rapidly and accurately analyze the genetic composition of individuals on the molecular level, thereby realizing the direct selection of genotypes for molecular breeding; the method is not influenced by environmental conditions and has high selection accuracy, so that the development of the molecular marker related to wheat drought resistance has great significance.

Disclosure of Invention

The invention aims to assist in identifying the 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 has a target sequence of a primer pair consisting of a primer F and a primer R in a wheat genome;

the primer F can be a1) or a2) as follows:

a1) a single-stranded DNA molecule shown as SEQ ID NO. 12;

a2) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to the SEQ ID NO. 12 and has the same function as the SEQ ID NO. 12;

the primer R can be a3) or a4) as follows:

a3) a single-stranded DNA molecule represented by SEQ ID NO 13;

a4) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in 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 can be any one of the following b1) -b 4):

b1) the drought resistance of the wheat to be tested is identified in an auxiliary manner;

b2) auxiliary screening of wheat varieties with or suspected to have drought resistance;

b3) auxiliary screening of wheat varieties without or suspected to have no drought resistance;

b4) and (5) 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 genome extraction and/or conventional reagents for agarose gel electrophoresis may also be included in the kit.

The function of the kit can be any one of the following b1) -b 4):

b1) the drought resistance of the wheat to be tested is identified in an auxiliary manner;

b2) auxiliary screening of wheat varieties with or suspected to have drought resistance;

b3) auxiliary screening of wheat varieties without or suspected to have no drought resistance;

b4) and (5) 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 pair can be any one of the following b1) -b 4):

b1) the drought resistance of the wheat to be tested is identified in an auxiliary manner;

b2) auxiliary screening of wheat varieties with or suspected to have drought resistance;

b3) auxiliary screening of wheat varieties without or suspected to have no drought resistance;

b4) and (5) wheat breeding.

The method for identifying the drought resistance of the wheat to be detected by applying the specific primer pair or the kit in an auxiliary manner comprises the following steps: taking genome DNA of wheat to be detected as a template, and carrying out PCR amplification by adopting any one of the specific primer pairs 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 detected does not have drought resistance or is suspected to have drought resistance.

The method for applying the specific primer pair or the kit to assist in screening the wheat variety with or suspected to have drought resistance comprises the following steps: taking genome DNA of wheat to be detected as a template, and carrying out PCR amplification by adopting any one of the specific primer pairs 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 a candidate wheat variety with drought resistance, otherwise, the wheat to be detected is or is a candidate wheat variety without drought resistance.

The invention also protects a DNA fragment obtained by using the genome DNA of wheat to be detected as a template and adopting any one of the specific primer pairs for amplification. The DNA fragment is the molecular marker which is to be protected and is related to the wheat drought resistance. The molecular marker can be specifically 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 in SEQ ID NO. 15.

The invention also protects the application of the DNA fragment (i.e. the molecular marker), which can be any one of the following b1) -b 8):

b1) the drought resistance of the wheat to be tested is identified in an auxiliary manner;

b2) auxiliary screening of wheat varieties with or suspected to have drought resistance;

b3) Auxiliary screening of wheat varieties without or suspected to have no drought resistance;

b4) breeding wheat;

b5) preparing a product for assisting in identifying the drought resistance of the wheat to be detected;

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 without or suspected to have no drought resistance;

b8) as a molecular marker.

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 the drought resistance of wheat to be detected, which comprises the following steps:

(1) taking genome DNA of wheat to be detected as a template, and carrying out PCR amplification by adopting any one of the specific primer pairs 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 detected does not have drought resistance or is suspected to have drought resistance.

The invention also provides a method for assisting in identifying the drought resistance of wheat to be detected, which 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 detected contains the DNA segment B and does not contain the DNA segment A, the wheat to be detected has or is suspected to have drought resistance; otherwise, the wheat to be detected does not have drought resistance or is suspected to have drought resistance.

The invention also provides a method for auxiliary screening of wheat with different drought resistances, which comprises the following steps: taking genome DNA of wheat to be detected as a template, and carrying out PCR amplification by adopting any one of the specific primer pairs to obtain a PCR amplification product; the wheat having the DNA fragment of about 209bp and not having the DNA fragment of 189bp in the PCR amplification product had a higher drought resistance than the wheat having the DNA fragment of 189bp and not having the DNA fragment of 209bp in the PCR amplification product.

The invention also provides a method for auxiliary screening of 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 with the genomic DNA of the wheat to be detected containing the DNA segment B and not containing the DNA segment A is higher than that of the wheat with the genomic DNA of the wheat to be detected containing the DNA segment A and not containing the DNA segment B.

Any of the above-described elevations can be statistically elevated.

Experiments prove that the wheat variety which contains the DNA segment B and does not contain the DNA segment A in the wheat genome DNA has drought resistance. By applying the molecular marker provided by the invention, the wheat variety with drought resistance can be quickly screened out, so that the breeding pace of the wheat variety is accelerated. The invention has important application value.

Drawings

FIG. 1 shows the real-time fluorescent quantitative detection T₃The expression level of the TaPYL1 gene in the wheat transformed with the TaPYL1 gene is changed.

FIG. 2 is T₃The wheat with transferred TaPYL1 gene has the phenotype after being stressed by PEG and being rehydrated for 7 days.

FIG. 3 is T₃The wheat transformed with the TaPYL1 gene has a phenotype after being subjected to drought treatment and rehydration for 3 days.

FIG. 4 is T₃And (3) counting the survival rate of the wheat after being subjected to drought treatment and rehydration for 3 days after the transfer of the TaPYL1 gene.

FIG. 5 is a sequence difference between two haplotypes in drought and drought sensitive wheat.

FIG. 6 shows the results of PCR amplification of two haplotypes.

FIG. 7 shows nucleotide polymorphism analysis of 120 wheat varieties.

FIG. 8 is a graph of the difference in drought tolerance between haplotype A homozygous and haplotype B homozygous wheat material.

FIG. 9 is a statistical result of the relative expression levels of TaPYL1 gene under normal growth and drought stress of wheat material homozygous haplotype A and haplotype B.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The vector pCAMBIA3301 is described in the following documents: regulatory changes in TaSNAC8-6A are associated with a drop height strategy in the world seed lines plant Biotechnol J.202018(4): 1078-.

The wheat variety, chinese spring, is described in the following documents: regulation changes in TaSNAC8-6A are associated with a gravity complete in the wheat seeds plant Biotechnol J.202018(4): 1078-.

Wheat variety Fielder is described in the following literature: regulatory changes in TaSNAC8-6A are associated with a drop height strategy in the world seed lines plant Biotechnol J.202018(4): 1078-.

The naming method of the polymorphic sites in the following examples is the marker type + sequence number; wherein, the marker types comprise SNP and InDel, the SNP represents single nucleotide polymorphism, and the InDel represents insertion or deletion; the sequence number is marked as "+ 1" in A in the initiation codon (ATG) of the TaPYL1 gene in the Chinese spring reference genome DNA; e.g., SNP181 represents the presence of a single nucleotide polymorphism at base 181 downstream of A in the initiation codon (ATG); InDel-442 represents the presence of an insertion or deletion of 1 or more bases downstream of the 442 th base upstream of A in the initiation codon (ATG).

In the following examples, the position of the primer or sequence was designated as "+ 1" with the reference of "A" in the initiation codon (ATG) of the TaPYL1 gene in the genomic DNA of Chinese spring.

The genome sequence of the TaPYL1 gene in the Chinese spring reference genome DNA is shown as SEQ ID NO. 11, the 601-1832 th position of the SEQ ID NO. 11 from the 5' end is the full-length cDNA sequence of the TaPYL1 gene, the 719-1360 th position encodes the TaPYL1 protein of which the amino acid sequence is shown as SEQ ID NO. 1, and the 719 th position is the base A in the ATG of the initiation codon.

Example 1 cloning of the TaPYL1 Gene

1. Taking seeds of Chinese spring, accelerating germination for 3 days at 25 ℃, transferring the germinated seeds to nutrient soil, and culturing for two weeks at 25 ℃ to obtain Chinese spring seedlings.

2. Extracting the total RNA of the Chinese spring seedlings, and then carrying out reverse transcription to obtain the cDNA of the Chinese spring.

3. The cDNA of Chinese spring is used as a template, and a primer pair consisting of 5'-ATGGAGCAGCAGCCTGTG-3' (SEQ ID NO:3) and 5'-TTATTCTGCCGGCGGCGC-3' (SEQ ID NO:4) is adopted for PCR amplification to obtain a PCR amplification product of about 642 bp.

4. Sequencing the PCR amplification product.

The sequencing result shows 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 named the TaPYL1 gene. The gene TaPYL1 encodes TaPYL1 protein, and the amino acid sequence of the protein TaPYL1 is shown in SEQ ID NO 1.

Example 2 obtaining of TaPYL1 transgenic wheat and identification of drought resistance

10 XLS Major solute and the concentration thereof is CaCl₂·2H₂O44g/L、KH₂PO₄1.7g/L、KNO₃19g/L、MgSO₄·7H₂O37g/L and NH₄NO₃16.5g/L, and the solvent is water.

100 × LS Minor solute and CoC concentration_l2·6H₂O2.5mg/L、CuSO₄·5H₂O2.5mg/L、H₃BO₃620mg/L、KI83mg/L、MnSO₄·5H₂O2230mg/L and ZnSO₄·7H₂O1060mg/L, and the solvent is water.

100 xFe-EDTA solute and FeSO concentration₄·7H₂O2.78g/L and Na₂EDTA3.73g/L, and the solvent is water.

100 xVitamin solute and its concentration is 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.

Selecting a culture 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 ₂·6H₂Dissolving O, 1.95g of MES and 5g of Agarose in a proper amount of water, and then adding water to a constant volume of 1L; sterilizing at 121 deg.C for 15 min; then 10g/L Ascorbic acid, 50. mu.L AgNO with a concentration of 100mM was added₃Solution, 1mL of Timentin with a concentration of 150 g/L.

Differentiation medium: 100mL of 10 XLS Major, 10mL of 100 XLS Minor, 10mL of 100 XFe-EDTA, 10mL of 100 Xvitamine, 50mL of Zeatin solution at a concentration of 100mg/L, and 100. mu.L of CuSO at a concentration of 100mM₄·5H₂Dissolving the O solution, 20g of Sucrose, 0.5g of MES and 3g of Gelrite in a proper amount of water, and then adding water to a constant volume of 1L; sterilizing at 121 deg.C for 15 min; then 250. mu.L of PPT at a concentration of 20g/L and 1mL of Carbenicilin at a concentration of 250g/L were added.

Rooting culture medium: dissolving 100mL of 10 XLS Major, 10mL of 100 XLS Minor, 10mL of 100 XFe-EDTA, 10mL of 100 XVitamin, 2mL of IBA solution with the concentration of 100mg/L, 15g of Sucrose, 0.5g of MES and 3g of Gelrite in proper water, adjusting the pH value to 5.8, and then adding water to the volume of 1L; sterilizing at 121 deg.C for 15 min; then 250. mu.L of PPT at a concentration of 20g/L and 1mL of Carbenicilin at a concentration of 250g/L were added.

Construction of recombinant vector pCAMBIA3301-GZ

The DNA fragment between the restriction enzymes HindIII and EcoRI of the vector pCAMBIA3301 was replaced with the DNA molecule shown in the 1 st to 639 th positions from the 5' end of SEQ ID NO:2 to obtain the recombinant vector pCAMBIA 3301-GZ.

The recombinant vector pCAMBIA3301-GZ expresses TaPYL1 protein shown in SEQ ID NO. 1.

II, obtaining recombinant agrobacterium tumefaciens

The recombinant vector pCAMBIA3301-GZ is transformed into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium, which is named as EHA105/pCAMBIA 3301-GZ.

The vector pCAMBIA3301 was transformed into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium, which was designated as EHA105/pCAMBIA 3301.

Thirdly, obtaining of TaPYL1 transgenic wheat

1. Introducing EHA105/pCAMBIA3301-GZ into wheat variety Fielder by Agrobacterium-mediated gene transformation method to obtain T₀Wheat with transfer of TaPYL1 gene is simulated.

The method comprises the following specific steps:

(1) inoculating EHA105/pCAMBIA3301-GZ into YEB liquid medium containing 25mg/L spectinomycin, and shake-culturing at 28 deg.C to OD_600nmThe value is 0.5, and the recombinant agrobacterium liquid is obtained.

(2) Placing the young embryo of wheat variety Fielder in a centrifuge tube (specification is 2mL) filled with a preservation solution (10 mL of 10 XLSMajor, 1mL of 100 XLS Minor, 1mL of 100 XFe-EDTA, 1mL of 100 XVitamin, 10mL of Glucose and 0.5g of MES are dissolved in a proper amount of water and then the volume is determined to be 1L by water), carrying out heat treatment at 46 ℃ for 3min, and centrifuging at 4 ℃ and 2000rpm for 10min to obtain the treated young embryo.

(3) After the step (2) is finished, adding recombinant agrobacterium liquid into the treated immature embryos, culturing for 3 days at 22 ℃ in the dark, then transferring to a selective medium, and culturing for 7-10 days at 28 ℃ in the dark; screening by using glufosinate-ammonium with different concentrations, transferring to a differentiation culture medium, transferring to a rooting culture medium after differentiation, transferring to nutrient soil after certain size to obtain T ₀Wheat with transfer of TaPYL1 gene is simulated.

2. Get T₀Substituting wheat with transfer-simulated TaPYL1 gene, screening positive seedlings to obtain T₀Wheat with transfer of TaPYL1 gene.

3. Get T₀Transferring TaPYL1 gene wheat, selfing, screening positive seedling to obtain T₁Wheat with transfer of TaPYL1 gene.

4. Get T₁Transferring TaPYL1 gene wheat, selfing, screening positive seedling to obtain T₂Wheat with transfer of TaPYL1 gene.

5. Get T₂Transferring TaPYL1 gene wheat, selfing, screening positive seedling to obtain T₃Wheat with transfer of TaPYL1 gene.

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, performing PCR amplification by using a primer pair consisting of 5'-TCGATGCTCACCCTGTTGTTTG-3' (SEQ ID NO:9) and 5'-TGTATAATTGCGGGACTCTAATC-3' (SEQ ID NO:10) to obtain a PCR amplification product, and performing Sanger sequencing; then, the following judgment is made: if a certain PCR amplification product contains a DNA fragment of about 871bp and the sequencing result comprises a nucleotide sequence shown in SEQ ID NO. 2, the wheat seedling corresponding to the PCR amplification product is a positive seedling.

Replacing EHA105/pCAMBIA3301-GZ with EHA105/pCAMBIA3301 according to the above method, and performing the same procedures to obtain T₃Plants for transferring empty carrier wheat are called empty carrier wheat for short.

Four, real-time fluorescence quantitative detection T₃Expression level of TaPYL1 gene in wheat with transferred TaPYL1 gene

1. Respectively combine each T₃And (3) placing seedlings of the wheat which have transferred the TaPYL1 gene and grow for 10 days into liquid nitrogen for preservation to obtain a corresponding sample to be detected. And (4) storing the seedlings of the wheat growing to 10 days after transferring the empty carrier in liquid nitrogen to obtain corresponding samples to be detected. Taking a wheat variety Fielder, and alternately culturing for 10 days at 16 ℃ in light and dark to obtain a wheat seedling to be detected; and (4) storing the wheat seedlings to be detected in liquid nitrogen to obtain corresponding samples to be detected.

2. Extracting total RNA of a sample to be detected by Trizo1 (Biotoped), then carrying out reverse transcription to obtain first strand cDNA, diluting the cDNA by 100 times by using sterile water as a template, and carrying out real-time quantitative PCR (polymerase chain reaction) to detect the relative expression quantity of the TaPYL1 gene (the TaActin1 gene is an internal reference gene).

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).

Part of the test results are shown in FIG. 1(WT is wheat variety Fielder, OE1-OE12 are all T₃Wheat transformed with the TaPYL1 gene). The result shows that the relative expression quantity of the TaPYL1 gene in the wheat variety Fielder and the empty vector transferred wheat has no obvious difference; compared with wheat variety Fielder, each T ₃Transgenic TaPYL1 geneThe relative expression quantity of the TaPYL1 gene in wheat is obviously increased, wherein 3T genes are₃The relative expression quantity of the TaPYL1 gene in the wheat strain transformed with the TaPYL1 gene is higher, and the wheat strain is named as OE4, OE5 and OE6 in sequence, and subsequent experiments are carried out.

Five, T₃Drought resistance identification of wheat with transfer TaPYL1 gene

(one) drought resistance identification

T with OE4 as wheat seed to be detected₃Seed generation, T of OE5₃Seed generation, T of OE6₃Generation seeds, wheat variety Fielder seeds or empty vector transferred wheat seeds.

The experiment was repeated three times to obtain an average, and the procedure for each repetition was as follows:

1. and (3) taking the wheat seeds to be detected, germinating at room temperature for 3 days, transferring to a water culture solution, and alternately culturing in light and dark at the temperature of 16 ℃ for 15 days (16h light/8 h dark) to obtain the wheat seedlings to be detected.

The solute of the water culture solution and the concentration thereof are 0.75mM K₂SO₄、0.1mM KCl、0.25mM KH₂PO₄、0.65mM MgSO₄、0.1mM EDTA-Fe、2mM Ca(NO₃)₂、1.0μM MnSO₄、1.0μM ZnSO₄、0.1μM CuSO₄And 0.005. mu.M (NH)₄)₆Mo₇O₂The solvent is water.

2. Transferring 20 wheat seedlings to be tested with basically consistent growth states to a water culture solution (control) or a water culture solution containing 30% PEG, alternately culturing in light and dark at 16 ℃ for 10 days (16h of light/8 h of dark), and observing the phenotype.

The results show that the leaves of the empty vector-transferred wheat and the wheat variety Fielder are obviously withered, T₃Wheat (OE4, OE5 and OE6) transformed with the TaPYL1 gene had severe wilting of leaves.

3. After the step 2 is completed, 20 wheat seedlings to be tested are transferred to a water culture solution, are cultured alternately in light and dark (16h of light/8 h of dark) for 7 days at the temperature of 16 ℃, and the phenotype is observed.

The results are shown in FIG. 2(VC for empty vector transferred wheat and WT for wheat variety Fielder). The results show that T is comparable to the wheat variety Fielder₃PEG-conjugation of wheat (OE4, OE5 and OE6) with transfer of TaPYL1 geneThe tolerance capability is obviously improved; the tolerance capacity of the empty vector transferred wheat and the wheat variety Fielder to PEG has no obvious difference.

(II) identification of drought resistance

T with OE4 as wheat seed to be detected₃Seed generation, T of OE5₃Seed generation, T of OE6₃Generation seeds, wheat variety Fielder seeds or empty vector transferred wheat seeds.

The experiment was repeated three times to obtain an average, and the procedure for each repetition was as follows:

1. sowing 48 wheat seeds to be detected in a box (35.0cm multiplied by 20.0cm multiplied by 15.0cm) filled with 0.6kg of nutrient soil, and culturing for 21 days under normal conditions to obtain the wheat seedlings to be detected.

The phenotype of some wheat seedlings to be tested is shown in FIG. 3 (before drought) (WT is wheat variety Fielder).

2. After completion of step 1, drought treatment (stopping watering) was carried out for 30 days and the phenotype was observed.

The results show that the leaves of the empty vector-transferred wheat and the wheat variety Fielder are obviously withered, T ₃Wheat (OE4, OE5 and OE6) transformed with the TaPYL1 gene had severe wilting of leaves.

3. After completion of step 2, rehydration was performed, and after 3 days the phenotype was observed and the survival was counted.

Plants that appeared to be capable of normal growth and harvest were defined as surviving plants. Survival rate is the percentage of the number of surviving plants to the total number of plants.

The phenotype of the wheat seedlings to be 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 T is comparable to the wheat variety Fielder₃The survival rate of wheat (OE4, OE5 and OE6) with the gene transferred with the TaPYL1 is remarkably improved to 80-85 percent; the survival rates of the empty vector transferred wheat and the wheat variety Fielder have no obvious difference.

Therefore, the TaPYL1 gene is a wheat drought-resistant gene.

Example 3 haplotype related to wheat drought resistance and molecular marker and application thereof

Development of molecular markers

1. Through a large number of experiments, the inventor of the invention finds that 2 haplotypes related to drought resistance exist at the polymorphic site upstream of the initiation codon ATG of the TaPYL1 gene in wheat genome DNA, and the haplotypes are respectively named as haplotype A and haplotype B, and are 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).

Second, polymorphism detection

1. A drought-resistant wheat variety Pubin 202 is taken as a male parent, a drought-sensitive wheat variety GLUYAS EARLY is taken as a female parent for hybridization, and selfing is carried out for 2 generations to obtain a separation population Pubin 202 multiplied GLUYAS EARLY. The drought-resistant wheat variety Pubin 202 is taken as a male parent, the drought-sensitive wheat variety Wanmai33 is taken as a female parent for hybridization, and 2 generations of selfing are carried out to obtain a segregation population Pubin 202 multiplied by Wanmai 33.

2. Genotyping

(1) The genomic DNA of wheat (Pubin 202, GLUYAS EARLY, Wanmai33, population Pubin 202 XGLUYAS EARLY or population Pubin 202 XWanmai 33) was extracted to obtain the genomic DNA of wheat.

(2) And (2) respectively taking the genomic DNA extracted in the step (1) as a template, and carrying out PCR amplification by using a primer pair consisting of the primer F and the primer R in the step one to obtain PCR amplification products.

And (3) PCR reaction conditions: 5min at 94 ℃; 30s at 94 ℃, 30s at 58 ℃, 30s at 72 ℃ and 40 cycles; 5min at 72 ℃; permanent at 16 ℃.

(3) And (3) taking part of the PCR amplification product obtained in the step (2), carrying out 2% agarose gel electrophoresis, then dyeing nucleic acid, and taking pictures under a gel imaging system.

The electrophoresis results of a portion of wheat are shown in FIG. 6. The results show that the banding patterns of the wheat PCR amplification products are three types: band type A (one band shown at 189bp), band type B (one band shown at 209bp) and band type C (two bands shown at 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 the nucleotide sequences of the wheat PCR amplification products are three types: sequence A (shown in SEQ ID NO:14, about 189bp), sequence B (shown in SEQ ID NO:15, about 209bp) and sequence C (shown in SEQ ID NO:14 and SEQ ID NO:15, respectively).

SEQ ID NO:14：

cgaagaattggtgaatcatgtactactatgttttaagatggcgtttagtaaaataaaaaagtagtgaagttatttcggtggccagaaatgaaatgaatttgataaaataaattactccactaaatttCCTaaatttagtggagtaaaaatagtccattaatcttttaggagatgctcttctatttttta

SEQ ID NO:15：

Cgaagaattggtgaatcatgtactactatgttttaagatggcgtttagtaaaataaaaaagtagtgaagttatttcggtggccagaaatgaaatgaatttgataaaataaattactccactaaatttaggagatcagttatgtacaatcaaaatttagtggagtaaaaatagtccattaatcttttaggagatgctcttctatttttta

If the banding pattern of the PCR amplification product of the wheat to be detected is banding pattern A or the nucleotide sequence is sequence A, the wheat to be detected is homozygous for haplotype A; if the banding pattern of the PCR amplification product of the wheat to be detected is banding pattern B or the nucleotide sequence is sequence B, the wheat to be detected is homozygous for haplotype B; if the banding pattern of the PCR amplification product of the wheat to be detected is banding pattern C or the nucleotide sequence is sequence C, the wheat to be detected is the wheat heterozygous for the haplotype A and the haplotype B.

The results of the genotype identification are shown in tables 1 and 2.

TABLE 1 genotype of parents and survival statistics after drought treatment

Parent strain	Genotype(s)	Survival rate (%)
			Pubing202	Haplotype B homozygous	96.2
GLUYASEARLY	Homozygous for monomeric nail	9.4
			Wanmai33	Homozygous for monomeric nail	0.0

TABLE 2 genotype of drought-resistant and drought-sensitive hybrid segregating populations and survival statistics after drought treatment

Note: the lower case letters are significance of different genotypes of plants in a population in a way that P is less than 0.05 through one-way ANOVA, the difference is not significant when the same lower case letters are contained, and the difference is significant when the same lower case letters are not contained; capital letters are significance of different genotypes of plants in a population in P <0.01 through one-way ANOVA, the difference is not significant when the plants contain the same capital letters, and the difference is extremely significant when the plants do not contain the same capital letters. In Table 2, the segregation ratios of genotypes of each population are in accordance with Mendelian's Law of inheritance.

3. Phenotypic identification of drought resistance

The experiment was repeated three times and the mean was taken for statistical analysis. The method comprises the following specific steps:

randomly taking 90 grains of wheat seeds homozygous for haplotype A and 90 grains of wheat seeds homozygous for haplotype B from a segregating population (Pubin 202 multiplied by GLUYAS EARLY or Pubin 202 multiplied by Wanmai33), transplanting the wheat seeds homozygous for haplotype B into cultivation pots (with the length multiplied by the width multiplied by the depth multiplied by 0.70 multiplied by 0.50 multiplied by 0.18 m and containing cultivation medium mixed by 5 kg of vermiculite and 5 kg of turfy soil) after pregermination, planting 180 plants in each pot, respectively cultivating the plants in a greenhouse for 21 days under the conditions of 16h of light and 8h of darkness, 14 ℃/night 12 ℃ and 60% of air humidity, and carrying out drought treatment (namely, no watering) on the plants. Rehydration is carried out for one week after the relative water content of the soil (namely the volume percentage content of the water in the soil measured by a soil moisture meter SU-LA (W) produced by Beijing alliance Weiwei science and technology Limited) reaches 0%. And (3) counting the survival rate of each genotype plant in each parent and each group after 3 days of rehydration (defining the plant which can not turn green on the overground part after 3 days of rehydration as a dead plant, defining the plant which can turn green on the overground part after 3 days of rehydration as a survival plant, and the survival rate is the percentage of the survival plant to the total number of plants).

The statistical results are shown in tables 1 and 2. The results show that the drought resistance of wheat homozygous for haplotype B is greater than that of wheat homozygous for haplotype A, and the greater is statistically. Therefore, in wheat breeding, a haplotype B homozygous for high drought resistance should be selected for breeding.

Example 4 discovery of haplotypes associated with drought resistance of 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 a drop height strategy in the world seed lines plant Biotechnol J.202018(4): 1078-.

TABLE 3

1. Phenotypic identification of drought resistance

Planting a natural variation group consisting of 120 parts of wheat varieties in a cultivation pool, and subpackaging the cultivation pool with nutrient soil and vermiculite as cultivation substrates. Each pool is divided into 120 districts, and each district can plant 12 seedlings. And stopping watering when the three true-leaf seedlings are old, carrying out drought treatment, continuing until the relative water content of the soil (namely the volume percentage content of water in the soil measured by a soil moisture meter SU-LA (W) produced by Beijing Allen Chuangwei science and technology Limited) is reduced to 0, then rehydrating for 7 days, and counting the survival rate after 3 days of rehydration (defining the plant which cannot turn green on the overground part after 3 days of rehydration as a dead plant, defining the plant which can turn green on the overground part after 3 days of rehydration as a survival plant, wherein the survival rate is the percentage of the survival plant in the total number of the plants).

The drought phenotype data used in the statistical analysis are all the means of independent repeat experiments.

The survival statistics are shown in table 3.

Drought resistance of wheat varieties is divided into three types according to the survival rate results in table 3: the wheat variety with the survival rate more than or equal to 40 percent is drought-resistant type, the wheat variety with the survival rate less than 10 percent is drought-sensitive type, and the wheat variety with the survival rate less than or equal to 10 percent is intermediate type.

The typing results are shown in Table 3.

2. Polymorphic site identification

(1) 120 parts of wheat varieties of about 2.0kb of genome fragments of a coding region, a5 'untranslated region and a 3' end untranslated region of the TaPYL1 gene are sequenced respectively, and sequencing primers are F: 5'-cctctctttagccatcccttggtat-3' (SEQ ID NO:16) and R: 5'-cagcacctcaggaatcacacctat-3' (SEQ ID NO: 17). The sequencing results were aligned using MEGA5.0(http:// www.megasoftware.net /). The nucleotide polymorphisms were analyzed based on the alignment results to obtain 7 polymorphic sites, 5 SNPs and 2 InDels (shown in FIG. 7), with a Minimum Allele Frequency (MAF) of 0.05 or more.

3. Association analysis of polymorphic sites with drought resistance phenotype

120 wheat varieties were divided into haplotype A homozygous, haplotype B homozygous, haplotype A heterozygous and haplotype B heterozygous according to the method of example 3. The results are shown in Table 3.

Statistical analysis was further performed on the drought tolerant phenotype of the two haplotype wheat materials and the results are shown in FIG. 8. The results show that the survival rate of the wheat homozygous for the haplotype B is obviously higher than that of the wheat homozygous for the haplotype A.

4. The expression level of the TaPYL1 gene in the haplotype B homozygous wheat material is obviously higher than that of the haplotype A homozygous wheat material

To determine the differences in the contribution of gene expression to drought resistance, the expression levels of the TaPYL1 gene were analyzed for 46 wheat varieties (see table 4) under normal growth and drought stress. The results are shown in fig. 9, the expression level of the TaPYL1 gene in 22 parts of wheat material homozygous for haplotype b is significantly higher than that in 24 parts of wheat material homozygous for haplotype a.

TABLE 4

The steps for analyzing the relative expression level of the TaPYL1 gene in 46 wheat varieties are as follows:

sterilizing wheat seeds for 10min by 1 per thousand (v/v) of Topsin-M (Rotam Crop Sciences Ltd.), rinsing for 3 times by using deionized water, placing on a culture dish paved with filter paper, accelerating germination for 3 days at 22 ℃, and transferring the germinated seeds to nutrient soil. The water cut treatment was performed at the 3 rd leaf stage, and at 90% and 58% Relative Leaf Water Content (RLWC), the leaves were taken separately, total RNA was extracted from less than three seedlings by the TRIZOL (Biotopped) method, followed by decontamination of the genome with DNAse I (Takara), concentration was measured with Nanodrop1000(Thermo Scientific product, USA), and 5. mu.g was collectively subjected to 0.8% agarose gel electrophoresis. Mu.g of total RNA was used to synthesize cDNAs using 1. mu.g of Oligo (dT)23 as a primer by recombinant M-MLV reverse transcriptase (Promega). And analyzing the relative expression level of the TaPYL1 gene in 46 wheat varieties by adopting fluorescent real-time quantitative PCR.

With specific primer F2: 5'-CCGTCACCACCGTCTCCGAACT-3' (SEQ ID NO:18) and R2: 5'-CCTCGGCCACGGACTTGAGCT-3' (SEQ ID NO:19) was used to quantify the gene TaPYL 1. Taking a gene TaActin1 as an internal reference, and taking primers as FC 2: 5'-AAATCTGGCATCACACTTTCTAC-3' (SEQ ID NO:20) and RC 2: 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) with 3 replicates per parallel run. The method reported by Livak KJ and Schmittgen TD (2001), 2^-ΔΔCTRelative expression levels were calculated.

ΔΔC_T＝(C_T.Target－C_T.TaActin1)_{Time x}－(C_T.Target－C_T.TaActin1)_{Time 0}

Time x denotes an arbitrary Time point, Time₀Represents 1-fold amount of target gene expression after TaActin1 correction.

The relative expression level of the TaPYL1 gene of the 46 wheat varieties obtained was analyzed by SPSS12.0 software for differential significance.

The results are shown in FIG. 9. The result shows that the expression level of the TaPYL1 gene in the haplotype B homozygous wheat material is obviously higher than that of the haplotype A homozygous wheat material under the drought stress condition.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the 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 reference to specific embodiments, it will be appreciated that the invention can 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 use of some of the essential features is possible within the scope of the claims attached below.

<110> northwest agriculture and forestry science and technology university

<120> method for auxiliary identification of drought resistance of wheat to be detected and special molecular marker thereof

<160>21

<170> PatentIn version 3.5

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