Molecular marker of wheat heat-resistant gene TaFAD8 and application of molecular marker

Abstract

The invention discloses a molecular marker of a wheat heat-resistant gene TaFAD8 and application of the molecular marker. The technical problem to be solved is how to identify the heat resistance of the wheat, and a molecular marker for identifying or assisting in identifying the heat resistance of the wheat is provided. The invention specifically discloses any one of the following applications of an InDel molecular marker or a substance for detecting the InDel molecular marker: A1) an application in identification or auxiliary identification of wheat heat resistance; a2) application in preparation of products for identification or auxiliary identification of wheat heat resistance; a3) application in preparation of wheat breeding products; the InDel molecular marker is a DNA molecule of which the nucleotide sequence is 84th-3617th in a sequence 2 or a sequence 3, and the heat resistance of insertion type wheat is higher than that of deletion type wheat or is higher than that of deletion type wheat in a candidate manner. The method can be used for wheat breeding practice.

Description

Technical Field

[0001] The invention specifically relates to a molecular marker of a wheat heat-resistant gene TaFAD8 and application thereof, and belongs to the field of biotechnology. Background Art

[0002] Wheat is very sensitive to environmental temperature. High temperature stress may cause damage to its growth and development, thus affecting yield and quality. Especially in the Huanghuai winter wheat area, wheat often encounters high temperatures above 32°C in the middle and late stages of grain filling, and may even reach extreme temperatures of up to 40°C, accompanied by dry and hot wind disasters. This situation can easily cause the phenomenon of wheat "withering and ripening", which seriously restricts the stability of wheat grain production. Cultivating and promoting heat-resistant wheat varieties is one of the effective ways to solve this problem. This requires researchers to identify heat-resistant genes as soon as possible, develop molecular markers closely related to heat-resistant genes, and realize the effective use of heat-resistant genes. The heat tolerance of wheat is jointly regulated by multiple genes and is a complex quantitative trait. At the same time, it is difficult to identify heat-resistant phenotypes, and related mechanism research needs to be in-depth. In previous studies, researchers decomposed heat tolerance into quantifiable traits such as thermal sensitivity index and canopy temperature, or used chlorophyll fluorescence parameters, fatty acid content and other important factors affecting heat tolerance as standards, and found a large number of QTLs related to heat tolerance on 21 chromosomes of wheat. Although most of the current research is still at the initial QTL positioning stage, the obtained QTL interval is large and it is difficult to accurately predict the heat-resistant phenotype, but molecular marker-assisted selection is still considered to be an important strategy to improve the efficiency of wheat heat-resistant genetic improvement. Compared with traditional breeding methods, molecular marker-assisted selection can directly screen the target gene accurately, without being affected by gene expression and environmental factors, and can greatly improve the accuracy of selection, thereby accelerating the breeding process. Therefore, the development of molecular markers that are highly linked to wheat heat-resistant genes will help reduce the linkage drag effect during the selection process and improve the application effect of heat-resistant genes in actual breeding. Summary of the invention

[0003] The technical problem solved by the invention is to provide a method and application for identifying or assisting in identifying the heat resistance of wheat through molecular markers.

[0004] In order to solve the above problems, the present invention provides any of the following applications of InDel molecule markers or substances for detecting the InDel molecule markers:

[0005] A1) Application in identification or auxiliary identification of heat tolerance of wheat;

[0006] A2) Application in the preparation and identification or auxiliary identification of wheat heat-resistant products;

[0007] A3) Application in the preparation of wheat breeding products;

[0008] The InDel molecular marker is a molecular marker that can identify whether the DNA molecule with the nucleotide sequence of the 84th to 3617th position in sequence 2 and / or the DNA molecule of sequence 3 exists.

[0009] In the present application, the heat resistance index may be a chlorophyll fluorescence parameter. The chlorophyll fluorescence parameter may be F v / F m .

[0010] The substance may be a product. The detection substance may include reagents, kits and instruments for detecting the above-mentioned InDel molecular markers. Specifically, primers and / or other reagents and instruments required for in vitro amplification of nucleic acids for detecting the above-mentioned InDel molecular markers.

[0011] In order to solve the above problems, the present invention also provides a product.

[0012] The product is a substance containing the above-mentioned substance for detecting the InDel molecular marker, and is any one of the following G1)-G4):

[0013] G1) detecting the product labeled by the InDel molecule;

[0014] G2) Application in identification or auxiliary identification of wheat heat tolerance;

[0015] G3) Application in the preparation and identification or auxiliary identification of wheat heat-resistant products;

[0016] G4) in the preparation of wheat breeding products.

[0017] In order to solve the above problems, the present invention also provides a method for identifying or assisting in identifying the heat resistance of wheat.

[0018] The method comprises detecting the genotype of the above-mentioned InDel molecular marker in the wheat genome, and identifying or assisting in identifying the heat tolerance of wheat according to the genotype of the above-mentioned InDel molecular marker in the wheat genome:

[0019] The heat resistance of the wheat with the insertion type genotype is higher or has the potential to be higher than the heat resistance of the wheat with the deletion type genotype. The insertion type is a homozygous type in which sequence 3 is inserted between positions 83 and 84 of sequence 1, and the deletion type is a homozygous type in which sequence 3 is not inserted between positions 83 and 84 of sequence 1.

[0020] In order to solve the above problems, the present invention also provides a method for wheat breeding.

[0021] The method comprises selecting wheat with the insertion type genotype as described above as a parent for breeding.

[0022] In the above, the above method is applied in wheat breeding.

[0023] In the above-mentioned applications, products and methods, the purpose of breeding includes cultivating or selecting wheat with high or low heat resistance.

[0024] In the above applications, products and methods, the substance for detecting the InDel molecular marker is the following D1), D2), D3) or D4):

[0025] D1) in vitro nucleic acid amplification primers containing specific amplification InDel molecular markers;

[0026] D2) an in vitro nucleic acid amplification reagent containing the in vitro nucleic acid amplification primer described in D1);

[0027] D3) a kit containing the in vitro nucleic acid amplification primers described in D1) or the in vitro nucleic acid amplification reagents described in D2);

[0028] D4) A detection instrument containing the in vitro nucleic acid amplification primers described in D1), the in vitro nucleic acid amplification reagents described in D2) or the kit described in D3).

[0029] The in vitro nucleic acid amplification technology can be polymerase chain reaction (PCR), strand displacement amplification (SDA), ligase chain reaction (LCR) and nucleic acid sequence-dependent amplification (NASBA), rolling circle nucleic acid amplification (RCA), loop-mediated isothermal amplification (LAMP), helicase-dependent isothermal amplification technology (HDA) or Qβ replication technology.

[0030] The present application uses polymerase chain reaction (PCR) as an amplification method to perform polymorphism detection.

[0032] The specific amplification in D1) can detect the nucleotide sequence of the InDel site by the presence or absence of an amplification product or by the presence or absence of an amplification product combined with an auxiliary reagent such as a probe.

[0033] In the above-mentioned applications, methods and products, the in vitro nucleic acid amplification primer may be labeled or not labeled with a marker. The marker refers to any atom or molecule that can be used to provide a detectable effect and can be attached to a nucleic acid. Markers include but are not limited to dyes; radioactive labels, such as 32P; binding moieties, such as biotin; haptens, such as digoxin (DIG); luminescent, phosphorescent or fluorescent moieties; and fluorescent dyes alone or in combination with moieties that can inhibit or shift the emission spectrum by fluorescence resonance energy transfer (FRET). The marker can provide a signal that can be detected by fluorescence, radioactivity, colorimetry, weight determination, X-ray diffraction or absorption, magnetism, enzyme activity, etc. The marker can be a charged moiety (positive or negative charge) or, alternatively, can be charge neutral. The marker can include a nucleic acid or protein sequence or a combination thereof, as long as the sequence containing the marker is detectable. In some embodiments, the nucleic acid is directly detected without a marker.

[0034] In the above-mentioned application, product or method, the in vitro nucleic acid amplification primers include primer pair TAM-TaFAD8-7:

[0035] The primer pair TAM-TaFAD8-7 consists of TAM-TaFAD8-7F and TAM-TaFAD8-7R;

[0036] TAM-TaFAD8-7F is a single-stranded DNA whose nucleotide sequence is SEQ ID No. 6 in the sequence list;

[0037] TAM-TaFAD8-7R is a single-stranded DNA whose nucleotide sequence is SEQ ID No. 7 in the sequence list.

[0038] In order to solve the above problems, the present invention also provides a method for wheat breeding.

[0039] The method comprises replacing a sequence 1 region in the target wheat genome with a sequence 2 to obtain wheat having a higher heat resistance than the target wheat.

[0040] In order to solve the above problems, the present invention also provides the following application.

[0041] Application of the nucleotides 84 to 3617 of sequence 2 or sequence 1 in the wheat genome corresponding to different heat resistance in any of the following:

[0042] A1) Application in identification or auxiliary identification of heat tolerance of wheat;

[0043] A2) Application in the preparation and identification or auxiliary identification of wheat heat-resistant products;

[0044] A3) Application in preparing wheat breeding products.

[0045] In order to solve the above problems, the present invention also provides a DNA molecule.

[0046] The above DNA molecule is a DNA molecule having a nucleotide sequence of sequence 3.

[0047] In the above, the wheat may be at least one of the following:

[0048] The wheat may be a hybrid offspring of wheat A and wheat B, wherein wheat A is an insertion-type wheat and wheat B is a deletion-type wheat. The hybrid offspring may be F2 generation and subsequent generations, such as F2 generation, BC1F2, RIL population, etc.

[0049] The insertion type is a homozygous type in which sequence 3 is inserted between positions 83 and 84 of sequence 1, and the deletion type is a homozygous type in which sequence 3 is not inserted between positions 83 and 84 of sequence 1.

[0050] The purpose of the breeding includes cultivating wheat with strong heat resistance.

[0051] The wheat A may be TAM107. The wheat B may be Luzi238.

[0052] In the above applications, methods and products, the substance may be a reagent and / or kit and / or instrument required for determining the InDel molecular marker or genotype by at least one of the following methods: in vitro nucleic acid amplification, DNA sequencing, restriction fragment length polymorphism, single-stranded conformation polymorphism, denaturing high performance liquid chromatography and InDel chip. Among them, InDel chips include chips based on nucleic acid hybridization reaction, chips based on single base extension reaction, chips based on allele-specific primer extension reaction, chips based on "one-step" reaction, chips based on primer ligation reaction, chips based on restriction endonuclease reaction, chips based on protein DNA binding reaction, and chips based on fluorescent molecule DNA binding reaction.

[0053] Beneficial Effects

[0054] First, the present invention provides a molecular marker capable of identifying the heat resistance of different varieties of wheat, specifically an InDel molecular marker, wherein the InDel molecular marker is a DNA molecule having a nucleotide sequence of the 84th to 3617th position in sequence 2 or sequence 3.

[0055] Furthermore, the present invention provides a specific method for using the InDel molecule: by detecting the genotype of the wheat InDel molecule marker, the heat tolerance of wheat is identified or assisted in identification according to the wheat genotype:

[0056] The heat resistance of the wheat with the insertion type genotype is higher or has the potential to be higher than the heat resistance of the wheat with the deletion type genotype. The insertion type is a homozygous type in which sequence 3 is inserted between positions 83 and 84 of sequence 1, and the deletion type is a homozygous type in which sequence 3 is not inserted between positions 83 and 84 of sequence 1.

[0057] Finally, the present invention provides a method for breeding using the above-mentioned InDel molecules. During the hybrid breeding process, the progeny wheat with high heat resistance characteristics is screened by using InDel molecular markers. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 Schematic diagram of BSA-seq results of RIL population derived from wheat varieties Luzi 238 and TAM107.

[0059] Figure 2 is the physical position of TAM-TaFAD8-7 on wheat chromosome 2D; the physical position refers to the Chinese Spring genome IWGSCRefSeqv1.0 version.

[0060] Figure 3 This is the electrophoresis result of PCR amplification products of Luzi 238 and TAM107 labeled with TAM-TaFAD8-7.

[0061] Figure 4 The results of the heat tolerance test of wheat are shown in Figure 2. The black group is the control group, and the gray group is the treatment group. The significant difference was tested using Student's t test, with P < 0.05 indicating a significant difference, P < 0.01 indicating a very significant difference, and P < 0.001 indicating an extremely significant difference.

[0062] Figure 5 This is the result of CDS sequence alignment of TaFAD8-2D gene of TAM107 and Luzi 238. The black background indicates that the sequences of the two are identical.

[0063] Figure 6 This is the result of the amino acid sequence alignment of the TaFAD8-2D gene of TAM107 and Luzi 238. The black line part is the histidine-rich motif.

[0064] Figure 7 This is the result of the comparison of the promoter sequences of TAM107 and Luzi 238. DETAILED DESCRIPTION

[0065] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0066] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0067] SPSS 11.5 statistical software was used to process the data in the following examples. The experimental results were expressed as mean ± standard deviation and were tested using Student's t test. P < 0.05 indicated a significant difference, P < 0.01 indicated a very significant difference, and P < 0.001 indicated an extremely significant difference.

[0068] The term "gene" refers to a segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer regions) that are involved in the transcription / translation of the gene product and the regulation of said transcription / translation, as well as intervening sequences (introns) between individual coding regions (exons).

[0069] The term "allele" refers to one of multiple alternative forms of a gene or non-coding region of DNA that occupies the same position on a chromosome. The term allele can be used to describe DNA from any organism, including but not limited to bacteria, viruses, fungi, protozoa, mold, yeast, plants, humans, non-humans, animals, and archaea.

[0070] The term "genotype" refers to the general term for all the genetic combinations of a biological individual. Biological individuals include, but are not limited to, diploids, tetraploids, or other possible polyploids. For example, in diploids, the genotype can be homozygous or heterozygous. A homozygous genotype means that the two alleles are exactly the same, such as AA or aa. A heterozygous genotype means that the two alleles are different, such as Aa. Other polyploid genotypes may appear and be described based on the actual situation of the alleles.

[0071] The term "InDel molecular marker" refers to an InDel marker (full name Insertion and Deletion marker), which is an insertion or deletion event that occurs in the genome, based on the insertion or deletion of nucleotides that occurs at a specific position in the genome. Similar to alleles, there can be multiple InDel molecular marker genotypes. For example, in diploids, the InDel molecular marker genotype can be homozygous or heterozygous. A homozygous genotype refers to two alleles that are exactly the same, such as AA or aa. A heterozygous genotype refers to two different alleles, such as Aa. Other polyploid genotypes may appear and be described based on the actual conditions of the alleles.

[0072] The term "template" refers to any nucleic acid molecule that can be used for amplification according to the present invention. Non-natural double-stranded RNA or DNA can be made into double-stranded DNA and used as double-stranded DNA. Any double-stranded DNA or product containing a variety of different double-stranded DNA molecules can be used as template DNA to amplify one or more loci contained in the template DNA.

[0073] The term "primer" refers to an oligonucleotide that can be used in an amplification method such as the polymerase chain reaction (PCR) for amplifying a nucleotide sequence based on a polynucleotide sequence that corresponds to a specific genomic sequence. At least one PCR primer used to amplify a polynucleotide sequence is sequence-specific for the sequence.

[0074] The term "amplification reaction" refers to a process for copying a nucleic acid one or more times. In embodiments, the amplification method includes, but is not limited to, polymerase chain reaction, self-sustained sequence reaction, ligase chain reaction, rapid amplification of cDNA ends, polymerase chain reaction and ligase chain reaction, Q-β phage amplification, strand displacement amplification, or overlap extension splicing polymerase chain reaction. In some embodiments, a single molecule nucleic acid is amplified, for example, by digital PCR.

[0075] The terms "include", "including", "have", "contain" and the like are all open terms, meaning including but not limited to.

[0076] The terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0077] When the term "identity" is used to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in MolecμLar Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48: 443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2: 482; the local homology algorithm of Pearson et al. (1988) P. The similarity search method of Proc. Natl. Acad. Sci. 85: 2444; Smith-Waterman algorithm (Meth. Mol. Biol. 70: 173-187 (1997); and BLASTP, BLASTN, and BLASTX algorithms (see Altsch μL et al. (1990) J. Mol. Biol. 215: 403-410). Computer programs that utilize these algorithms are also available and include, but are not limited to, ALIGN or Megalign (DNASTAR) software, or WU-BLAS T-2 (Altsch μL et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altsch μL et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0078] Example 1 Acquisition and development of molecular markers co-segregating with the wheat heat tolerance gene TaFAD8

[0079] 1. Comparison of CDS sequences of TaFAD8-2D gene in TAM107 and Luzi 238

[0080] TAM107 and Luzi 238 are commonly used wheat varieties, but there are differences in heat resistance between the two. Therefore, the TaFAD8-D gene related to heat resistance in the genomes of TAM107 and Luzi 238 was cloned, and the differences between the two in different varieties were compared. The CDS sequence of the TaFAD8-D gene of Luzi 238 is sequence 8. The CDS sequence of the TaFAD8-D gene of TAM107 is sequence 9.

[0081] The CDS sequence length of the TaFAD8-D gene in TAM107 and Luzi 238 is 1320bp. The CDS sequences of the TaFAD8-D genes in TAM107 and Luzi 238 were aligned with the translated amino acid sequences, and it was found that there were only 4 SNP differences in the CDS sequences, and the sequence similarity was high. TaFAD8-D encodes a protein with 439 amino acids. The amino acid sequence of the TaFAD8-D protein in Luzi 238 is sequence 10 in the sequence list, and the amino acid sequence of the TaFAD8-D protein in TAM107 is sequence 11 in the sequence list. There are two amino acid variations in the translated amino acid sequence. The functional domain in the TaFAD8-D protein is the three histidine-rich motifs of fatty acid dehydrogenase. This structure is complete in the TaFAD8-D protein sequences in TAM107 and Luzi 238, indicating that the protein functions normally. The sequence alignment results are as follows: Figure 5 and Figure 6 As shown, Figure 5 The CDS sequence alignment results of TaFAD8-D gene of TAM107 and Luzi 238. The black background indicates that the sequences of the two are identical. Figure 6 This is the result of amino acid sequence alignment of TaFAD8-D gene in TAM107 and Luzi 238. The black line part is the histidine-rich motif, indicating that the histidine-rich motif structure of TaFAD8-D protein in TAM107 and Luzi 238 is complete and the protein function is normal.

[0082] Sequence 8 is as follows:

[0083]

[0084] Sequence 9 is as follows:

[0085]

[0086] The specific sequence 10 is as follows:

[0087] MARLLLPQCCCGLTPLPLPRRAVALPPPALFPSSSGAAASRRALSLRVAVAAPARLATAEDDGSGSRAAGAQGGDEGPADGFDPGAPPPFGLADIRAAIPKHCWVKDPWRSMGYVVRDVVVVLALAAAAARLDSWLAWPVYWAAQGTMFWALFVLGHDCGHGSFSNNAKLNSVVGHILHSSILVPYNGWRISHRTHHQNHGHVENDESWHPLPEKLYRSLDSSTRKLRFALPFPMLAYPFYLWSRSPGKSGSHFHPSSDLFQPNEKKDIVTSTTCWLAMAGLLAGLTVVMGPLQILKLYAVPYWIFVMWLDFVTYLHHHGHNDKLPWYRGKAWSYLRGGLTTLDRDYGWLNKIHHDIGTHVIHHLFPQIPHYHLVEATEAAKPVLGKYYREPDKSGPFPFHLFGALARSMKSDHYVSDTGDIIYYQTDPKLAAGAHTSD*

[0088] The specific sequence 11 is as follows:

[0089] MARLLLPQCCCGLTPLPLPRRAVALPPPALFPSSSGAAASRRALSLRVAVAAPARLATAEDDGSGSRAAGAQGGDEGPADGFDPGAPPPFGLADIRAAIPKHCWVKDPWR SMGYVVRDVVVVLALAAAAARLDSWLAWPVYWAAQGTMFWALFVLGHDCGHGSFSNNAKLNSVVGHILHSSILVPYNGWRISHRTHHQNHGHVENDESWHPLPEKLYRSL DSSTRKLRFALFPMLAYPFYLWSRSPGKSGSHFHPSSDLFQPNEKKDIVTSTTCWLAMAGLLAGLTVVMGPLQILKLYAVPYWIFVMWLDFVTYLHHHGHNNKLPWYRG KAWSYLRGGLTTLDRDYGWLNNIHHDIGTHVIHHLFPQIPHYHLVEATEAAKPVLGKYYREPDKSGPFPFHLFGALARSMKSDHYVSDTGDIIYYQTDPKLAAGAHTSD*

[0090] II. Acquisition and sequence alignment of the promoter of TaFAD8-D gene in TAM107 and Luzi 238

[0091] Using the resequencing information, the promoter sequences upstream of the start codon of the TaFAD8-D gene in TAM107 and Luzi 238 were obtained respectively.

[0092] The promoter nucleotide sequence upstream of the start codon of TaFAD8-D gene in Luzi 238 is sequence 12;

[0093] The promoter nucleotide sequence upstream of the start codon of the TaFAD8-D gene in TAM107 is sequence 13;

[0094] The promoter sequences of TAM107 and Luzi 238 were compared. Figure 7 As shown, the underline indicates the position of the start codon ATG.

[0095] There were multiple SNP differences in the promoter sequence upstream of the start codon of the TaFAD8-D gene in TAM107 and Luzi 238. It was also found that a 3534bp DNA fragment was inserted in the promoter region of the TaFAD8-D gene in TAM107.

[0096] Sequence 12 is as follows:

[0097]

[0098] Sequence 13 is as follows:

[0099]

[0100] 3. Molecular marker development

[0101] Using the DNA resequencing results of TAM107 and Luzi 238, primers were designed near the Indel differential sites discovered with the help of BatchPrimer3 v1.0 to develop a specific marker TAM-TaFAD8-7 (physical location such as Figure 2 ). TAM-TaFAD8-7 is composed of a primer pair TAM-TaFAD8-7F and TAM-TaFAD8-7R; wherein TAM-TaFAD8-7F is a single-stranded DNA molecule as shown in SEQ ID NO.6, and TAM-TaFAD8-7R is a single-stranded DNA molecule as shown in SEQ ID NO.7.

[0102] Sequence 6 (SEQ ID NO. 6) is as follows: AGGAGGAGGGAAATAGGGCC.

[0103] Sequence 7 (SEQ ID NO. 7) is as follows: AGAAGATACCACACCAACACC.

[0104] Using DNA from TAM107 and Luzi 238 as templates, primers TAM-TaFAD8-7 were used for PCR amplification.

[0105] The PCR amplification program was as follows: pre-denaturation at 98°C for 5 min; denaturation at 98°C for 15 s, annealing at 59°C for 15 s, extension at 68°C for 2 min, 34 cycles; and extension at 68°C for 10 min.

[0106] The PCR amplification system was 10 μL, and its components were: 1 μL 100 ng / μL genomic DNA, 5 μL 2×Gflex PCRBuffer (Mg2+, dNTP plus), 1.0 μL 10 ng / μL upstream primer, 1.0 μL 10 ng / μL downstream primer, 0.5 μL TksGflex DNA Polymerase (Takara, Dalian) and 1.5 μL dd H2O.

[0107] The amplified products were separated on a 2% agarose gel, with a voltage of 150V and electrophoresis for 10 minutes. The results were recorded and sequenced using a gel imaging system. DNA fragments with a length of 300bp and / or 3834bp were amplified. The amplified band with a length of 300bp is also called A band, the amplified band with a length of 3834bp is also called B band, and the amplified bands with a length of 300bp and 3834bp are also called AB bands.

[0108] The amplified product of Luzi 238 was A-type, and the amplified product of TAM107 was B-type (the PCR results of TAM107 and Luzi 238 are shown in Figure 3 shown).

[0109] The amplified product is only 300 bp in length, which is sequence 4, as follows:

[0110] AGAAGATACCACACCAACACCACCAAGTTGTGTGAGCCACACAGACGGAAAGGAGCCCTCGTGTCCGGCTCGTGGTCGCGGGATGGCGAGGCCCACGCAGCCCGGCCCACCGCGCCGTGGCCACCCACTATTATATGCTCGGATACGGCC GCCTCACCCCACCCGCCGGCGCAGCAAGCAGAGCGGCAGCAGCAGCAATGGCGCGCCTGCTCCTCCCCCAATGCTGCTGCGGCCTCACGCCCCTGCCCCTCCCGCGCCGCGCCGTCGCGCTCCCTCCCCGGGCCCTATTTCCCTCCTCCT.

[0111] The amplified product is only 3834 bp sequence 5, which is as follows:

[0112]

[0113] If the amplified product is only a 300bp band, the wheat to be tested is a deletion type; if the amplified product is only a 3834bp band, the wheat to be tested is an insertion type; if the amplified product contains both 300bp and 3834bp bands, the wheat to be tested is a heterozygous type.

[0114] The insertion haplotype is sequence 3 (SEQ ID No. 3) in the genome, that is, there is an insertion of 3534 nucleotides of SEQ ID No. 3 between the 84th and 3617th positions of sequence 2; the deletion haplotype is that there is no such insertion at the 84th to 3617th positions of sequence 2 (SEQ ID No. 2) in the genome.

[0115] The two homologous chromosomes of the deletion type (also known as the deletion genotype or deletion type) genome have the following characteristics: there is no nucleotide between the 84th and 3617th positions of sequence 2 (SEQ ID No. 2) in the genome, and the 3534 nucleotides of SEQ ID No. 3 are inserted. Wheat with the deletion type genotype is a deletion type plant.

[0116] The two homologous chromosomes of the genome of the insertion type (also called insertion genotype or insertion type) have the following characteristics: there is an insertion of 3534 nucleotides of SEQ ID No. 3 between the 84th and 3617th positions of sequence 2 (SEQ ID No. 2) in the genome. Wheat with the insertion type genotype is an insertion type plant.

[0117] The amplified product is only 300 bp in length, which is Sequence 1, as follows:

[0118] AGAAGATACCACACCAACACCACCAAGTTGTGTGAGCCACACAGACGGAArGGAGCCCTCGTGTCCGGCTCGTGGTCGCGGGATGsCGAGGCCCACKyAGCCCGGCCCACCGCGCCGTGGCCACCCACTATTATATGCTCGGATACGGCC GCCTCACCCCACCCGCCGGCGCAGCAAGCAGAGCGGCAGCAGCAGCAATGGCGCGCCTGCTCCTCCCCCAATGCTGCTGCGGCCTCACGCCCCTGCCCCTCCCGCGCCGCGCCGTCGCGCTCCCTCCCCGGGCCCTATTTCCCTCCTCCT.

[0119] The amplified product is only 3834 bp sequence 2, which is as follows:

[0120]

[0121] As described above, the present invention has developed a molecular marker related to heat tolerance of wheat, which is an Indel molecular marker. The Indel molecular marker can identify two haplotypes, namely an insertion haplotype and a deletion haplotype.

[0122] The insertion sequence is sequence 3 (SEQ ID No. 3), that is, sequence 3 is inserted between the 83rd and 84th positions of sequence 1. After the insertion of sequence 3, sequence 1 on the genome is mutated into sequence 2. Among them, sequence 3 is the 84th to 3617th positions of sequence 2, that is, there is a nucleotide sequence of 3534 nucleotides of sequence 3 (SEQ ID No. 3) inserted between the 84th to 3617th positions of sequence 2; the deletion haplotype is that there is no insertion of sequence 3 at the 83rd to 84th positions of sequence 1 (SEQ ID No. 1) in the genome.

[0123] Sequence 3 is as follows:

[0124]

[0125] The two homologous chromosomes of the deletion type (also known as the deletion genotype or deletion type) genome have the following characteristics: there is no nucleotide between the 83rd and 84th positions of sequence 1 (SEQ ID No. 1) in the genome, and there is an insertion of 3534 nucleotides of sequence 3 (SEQ ID No. 3). Wheat with a deletion genotype is a deletion type plant.

[0126] The two homologous chromosomes of the genome of the insertion type (also called insertion genotype or insertion type) have the following characteristics: there is an insertion of 3534 nucleotides of sequence 3 (SEQ ID No.3) between the 83rd and 84th positions of sequence 1 (SEQ ID No.1) in the genome. Wheat with the insertion type genotype is an insertion type plant.

[0127] One homologous chromosome of the genome of the heterozygous type (also known as heterozygous genotype or heterozygous type) has the following characteristics: there is an insertion of 3534 nucleotides of sequence 3 (SEQ ID No.3) between the 83rd and 84th positions of sequence 1 (SEQ ID No.1) in the genome; the other homologous chromosome has the following characteristics: there is no insertion of 3534 nucleotides of SEQ ID No.3 between the 83rd and 84th positions of sequence 1 (SEQ ID No.1) in the genome. Wheat with a heterozygous genotype is a heterozygous individual plant.

[0128] The heat tolerance of the wheat with the insertion type genotype is higher or has the potential to be higher than the heat tolerance of the wheat with the deletion type genotype.

[0129] Example 2 Validation of Molecular Markers

[0130] 1. Source of Materials

[0131] RIL populations constructed using heat-tolerant wheat material TAM107 and heat-sensitive wheat material Luzi 238 were used for testing. Luzi 238 is recorded in the non-patent literature "Wang Xiaowei et al., Development and Application of Wheat AFLP-SCAR Markers [J]. Journal of Wheat Crops, 2008(05):738-744." TAM107 is recorded in the non-patent literature "Wang Fei et al., Differences in membrane permeability and lipid composition of heat-tolerant and heat-sensitive wheat genotypes under high temperature stress [J]. Journal of Agricultural Biotechnology, 2013. DOI:10.3969 / j.issn.1674-7968.2013.08.003". The public can obtain it from the applicant to repeat this experiment, but it cannot be used for other purposes.

[0132] 2. Identification of Chlorophyll Fluorescence Parameters Phenotype of RIL Population

[0133] RIL populations derived from Luzi 238 and TAM107 were sown in the greenhouse (Luzi 238 and TAM107 were sown normally, with Luzi 238 as the female parent and TAM107 as the male parent, and seeds were harvested by hybridization. After normal sowing and harvesting of the F1 generation, the F7 generation population was obtained by single-seed propagation for subsequent research, as shown in Table 2). Under the conditions of day / night: 12h / 12h, 22℃ / 18℃, after 7 days of normal growth, each RIL line was evenly divided into two groups, and leaves of about 3 cm were taken from each group as samples. One group was used as the control group. After 30 minutes of dark adaptation at room temperature, the chlorophyll fluorescence parameters were detected by the FluorCam closed chlorophyll fluorescence imaging system (Photon Systems Instruments, Czech); the other group was used as the treatment group, which was protected from light and treated in a 42℃ water bath for 1 hour, and the chlorophyll fluorescence parameters of the leaves were detected using the above method. The results of the heat tolerance test of the parents are shown in the figure. Figure 4 The data of individual plants are shown in Table 2. SPSS11.5 statistical software was used to process the data results. The experimental results were expressed as mean ± standard deviation. Student's t test was used. P < 0.05 indicated significant difference, P < 0.01 indicated extremely significant difference, and P < 0.001 indicated extremely significant difference. The results are shown in Table 1.

[0134] F v / F m The detection was performed in a FluorCam chlorophyll fluorescence imager. The light pulse (PPFD 0.09 μmol m -2 ) provides the minimum chlorophyll fluorescence (F0) measurement, and the subsequent saturation light pulse (PPFD 5500 μmol m -2 ) provides the maximum chlorophyll fluorescence (F m ) is the measured value of the variable fluorescence (F v ) is calculated as F m –F0, chlorophyll fluorescence parameter calculated as F v / F m All fluorescence parameters measured in this way were averaged over the entire leaf sample.

[0135] Decrease (%) = (control group F v / F m - Treatment Group F v / F m )*100%.

[0136] 3. Verification of TAM-TaFAD8-7 markers

[0137] Extract genomic DNA from the leaves of the wheat material in step 2.

[0138] The RIL population derived from TAM107 and Luzi 238 sown in the greenhouse was tested for chlorophyll fluorescence parameters and then genomic DNA was extracted. A leaf of about 1 cm in length was taken from each RIL line as a sample to extract genomic DNA:

[0139] 1) Take an appropriate amount of wheat leaves, place them in a 2.0 mL centrifuge tube with a 6 mm steel ball, cover it with a lid, cool it in liquid nitrogen, and then put it into a grinder for grinding;

[0140] 2) Add 800uL of CTAB extract to the sample, mix thoroughly, place it in a 65°C water bath, and mix by inversion every 10 minutes for three times;

[0141] 3) After taking out the sample, add 600uL of 24:1 (chloroform: isoamyl alcohol ratio) extraction solution, invert and mix for 5 minutes, and let stand at room temperature for 5 minutes;

[0142] 4) After standing and stratifying, centrifuge at 12000 rpm for 10 min;

[0143] 5) After centrifugation, pipette 500uL of supernatant into a 1.5mL centrifuge tube, add an equal volume of pre-cooled isopropanol, invert to mix, and place in a refrigerator at -20℃ for 30min;

[0144] 6) Cool the centrifuge to 4°C in advance and centrifuge at 12,000 rpm for 10 min. Discard the supernatant and add 1 mL of 75% ethanol. Flick to suspend and centrifuge at 12,000 rpm for 2 min. Repeat once.

[0145] 7) Discard the supernatant, leave to dry at room temperature, and then add deionized water to dissolve;

[0146] 8) Adjust the concentration so that the DNA concentration of each RIL line is 200 ng / μL and store at -20°C.

[0147] In the treatment group, 23 RIL lines with high chlorophyll fluorescence parameters and 23 RIL lines with low chlorophyll fluorescence parameters were selected, and the DNA of the above groups was mixed into two extreme pools. DNA samples of the extreme pools were resequenced by Shenzhen BGI Genomics Co., Ltd. Through bioinformatics analysis, the genetic interval associated with wheat heat tolerance was determined. The BSA-seq results showed that among the 21 wheat chromosomes, a strong difference signal peak appeared at 34Mb-44Mb on the short arm of chromosome 2D, indicating that this interval is a candidate interval that may affect wheat heat tolerance (results are shown in Figure 2). Figure 1 According to the annotation information of the Chinese Spring reference genome, this candidate interval contains the gene TaFAD8 that affects wheat heat tolerance (the results are shown in Figure 2 shown).

[0148] 4. Genotype identification of RIL population

[0149] The DNA template obtained above was amplified using primer pair TAM-TaFAD8-7 (primer pair TAM-TaFAD8-7 consists of TAM-TaFAD8-7F and TAM-TaFAD8-7R; wherein, TAM-TaFAD8-7F is the single-stranded DNA molecule shown in SEQ ID NO.6, and TAM-TaFAD8-7R is the single-stranded DNA molecule shown in SEQ ID NO.7).

[0150] The PCR amplification program was as follows: pre-denaturation at 98°C for 5 min; denaturation at 98°C for 15 s, annealing at 59°C for 15 s, extension at 68°C for 2 min, 34 cycles; and extension at 68°C for 10 min.

[0151] The PCR amplification system was 10 μL, and its components were: 1 μL 100 ng / μL genomic DNA, 5 μL 2×Gflex PCRBuffer (Mg2+, dNTP plus), 1.0 μL 10 ng / μL upstream primer, 1.0 μL 10 ng / μL downstream primer, 0.5 μL TksGflex DNA Polymerase (Takara, Dalian) and 1.5 μL dd H2O.

[0152] The amplified products were separated on a 2% agarose gel at 150 V for 10 min. The results were recorded using a gel imaging system. DNA fragments with a length of 300 bp and / or 3834 bp were amplified.

[0153] If the amplified product is only a 300bp band, the wheat to be tested is a deletion type; if the amplified product is only a 3834bp band, the wheat to be tested is an insertion type; if the amplified product contains both 300bp and 3834bp bands, the wheat to be tested is a heterozygous type.

[0154] The amplified band of the deletion type (also known as deletion genotype or deletion type) wheat is band A, the amplified band of the insertion type (also known as insertion genotype or insertion type) wheat is band B, and the amplified band of the heterozygous type (also known as heterozygous genotype or heterozygous type) wheat is band AB.

[0155] The heat tolerance of the wheat with the insertion type genotype is higher or has the potential to be higher than the heat tolerance of the wheat with the deletion type genotype.

[0156] The amplified product of Luzi 238 was of type A, and that of TAM107 was of type B. Some RIL populations derived from the two were of type A, and some were of type B.

[0157] The chlorophyll fluorescence parameters and genotypes of the treatment group and the control group were statistically analyzed, and the results are shown in Tables 1 and 2. The results showed that there was no significant difference in the chlorophyll fluorescence parameters of the two band-type RIL populations in the control group; there were significant differences in the chlorophyll fluorescence parameters of the RIL populations in the treatment group, and the average chlorophyll fluorescence parameters of the B-band type RIL population were significantly higher than the average chlorophyll fluorescence parameters of the A-band type RIL population.

[0158] Table 1 shows the statistical results of chlorophyll fluorescence parameters of RIL lines in the control group and treatment group

[0159]

[0160] The above data showed that the heat resistance of the RIL line with the TAM107 genotype was higher or candidate higher than that of the RIL line with the Luzi 238 genotype, which was consistent with the genotype identification results.

[0161] Table 2 Genotypes and related phenotypes of 136 samples of Luzi 238, TAM107 and their derived RIL populations

[0162]

[0163]

[0164]

[0165]

[0166] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Any of the following applications of InDel molecule labeling or detecting a substance labeled with the InDel molecule: A1) Application in identification or auxiliary identification of heat tolerance of wheat; A2) Application in the preparation and identification or auxiliary identification of wheat heat-resistant products; A3) Application in the preparation of wheat breeding products; The InDel molecular marker is a molecular marker that can identify whether the DNA molecule with the nucleotide sequence of the 84th to 3617th position in sequence 2 and / or the DNA molecule of sequence 3 exists.

2. A product, characterized in that The product is a substance containing the substance for detecting the InDel molecular marker according to claim 1, and is any one of the following G1)-G4): G1) detecting the product marked by the InDel molecule; G2) Application in identification or auxiliary identification of wheat heat tolerance; G3) Application in the preparation and identification or auxiliary identification of wheat heat-resistant products; G4) in the preparation of wheat breeding products.

3. A method for identifying or assisting in identifying heat resistance of wheat, characterized in that: The method comprises detecting the genotype of the InDel molecular marker of claim 1 in the wheat genome, and identifying or assisting in identifying the heat tolerance of wheat according to the genotype of the InDel molecular marker of claim 1 in the wheat genome: The heat resistance of the wheat with the insertion type genotype is higher or has the potential to be higher than the heat resistance of the wheat with the deletion type genotype. The insertion type is a homozygous type in which sequence 3 is inserted between positions 83 and 84 of sequence 1, and the deletion type is a homozygous type in which sequence 3 is not inserted between positions 83 and 84 of sequence 1.

4. A method for wheat breeding, comprising selecting wheat having the insertion type genotype as claimed in claim 3 as a parent for breeding.

5. The use according to claim 1, the product according to claim 2, the method according to claim 3 or 4, characterized in that: The purpose of the breeding includes cultivating or selecting wheat with high or low heat tolerance.

6. The use according to claim 1, the product according to claim 2, the method according to claim 4 or the use, product or method according to claim 5, characterized in that: The substance for detecting the InDel molecule marker is the following D1), D2), D3) or D4): D1) in vitro nucleic acid amplification primers containing specific amplification InDel molecular markers; D2) an in vitro nucleic acid amplification reagent containing the in vitro nucleic acid amplification primer described in D1); D3) a kit containing the in vitro nucleic acid amplification primers described in D1) or the in vitro nucleic acid amplification reagents described in D2); D4) A detection instrument containing the in vitro nucleic acid amplification primers described in D1), the in vitro nucleic acid amplification reagents described in D2) or the kit described in D3).

7. The use, product or method according to claim 6, characterized in that: The in vitro nucleic acid amplification primers include primer pair TAM-TaFAD8-7: The primer pair TAM-TaFAD8-7 includes TAM-TaFAD8-7F and TAM-TaFAD8-7R; TAM-TaFAD8-7F is a single-stranded DNA whose nucleotide sequence is SEQ ID No. 6 in the sequence list; TAM-TaFAD8-7R is a single-stranded DNA whose nucleotide sequence is SEQ ID No. 7 in the sequence list.

8. A method for wheat breeding, characterized in that: The sequence 1 region in the target wheat genome is replaced with sequence 2 to obtain wheat with stronger heat resistance than the target wheat.

9. Application of the nucleotides 84 to 3617 of sequence 2 or sequence 1 in the wheat genome corresponding to different heat resistance in any of the following: A1) Application in identification or auxiliary identification of heat tolerance of wheat; A2) Application in the preparation and identification or auxiliary identification of wheat heat-resistant products; A3) Application in preparing wheat breeding products.

10. A DNA molecule, characterized in that The DNA molecule is a DNA molecule whose nucleotide sequence is sequence 3.

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