Method for detecting excellent allelic variation of TaTPP-1A gene related to plant height and thousand seed weight of wheat

Abstract

The invention discloses a method for detecting TaTPP-1A gene excellent allelic variation related to wheat plant height and thousand seed weight. The invention provides application of a substance for detecting single nucleotide polymorphism of an SNP981 site in a TaTPP1-1A gene on a wheat genome or a substance for identifying haplotype of wheat to be detected based on the TaTPP1-1A gene in identification or auxiliary identification of wheat plant height and / or yield. The molecular marker has important application value in the wheat molecular marker assisted breeding process.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly relates to a method for detecting excellent allelic variations of the TaTPP-1A gene related to wheat plant height and thousand-grain weight. Background Art

[0002] Wheat is one of the main food crops in China, and the level of its yield is directly related to China's food security. The plant height of wheat is closely related to the harvest index, planting density, lodging resistance and yield. Reducing the plant height can not only increase the planting density, but also increase the yield by improving the harvest index. The three components of wheat yield include the number of spikes per unit area, the number of grains per spike and the thousand-grain weight, and there is a negative correlation among them. Therefore, higher yields can be obtained by reducing the plant height to increase the number of spikes per unit area while maintaining a relatively high thousand-grain weight. Thus, it can be seen that synergistically improving the plant height and thousand-grain weight of wheat is of great significance for breeding high-yield wheat varieties. Trehalose-6-phosphate phosphatase (TPP) is used to catalyze the conversion of trehalose-6-phosphate to trehalose. Trehalose is a kind of substance that regulates the osmotic ability produced by plants under stress. The wheat trehalose-6-phosphate phosphatase 1 gene (TaTPP1) is significantly up-regulated under drought stress and is differentially expressed in both wheat leaves and roots. So far, there has been no research report on the development of molecular markers for the TaTPP1 gene and its relationship with agronomic traits.

[0003] SNP is a single nucleotide variation site located on a chromosome, while a haplotype refers to a combination of multiple nucleotide variation sites located on a chromosome that can be inherited together. At present, a variety of methods for detecting these nucleotide variation sites have been established, such as direct sequencing, CAPS markers, dCAPS markers, etc. The CAPS (Cleaved Amplified Polymorphic Sequences) molecular marker is a marker method based on PCR (Polymerase Chain Reaction) and restriction endonuclease digestion technology, which is used to detect polymorphisms in DNA sequences. The dCAPS (derived Cleaved Amplified Polymorphic Sequences) marker is derived from CAPS. When designing primers, 1-3 bases near the variation site are changed to generate a new restriction enzyme site at the variation site, and then the bases are identified by the size of the products after digestion. By using these markers, the haplotype of a variety can be directly judged by observing the electrophoresis band pattern, which has the advantages of simple operation and stable and reliable results. Summary of the Invention

[0004] The object of the present invention is to provide a method for detecting excellent allelic variations of the TaTPP-1A gene related to wheat plant height and thousand-grain weight.

[0005] In a first aspect, the present invention claims the use of substance A or substance B in any of the following:

[0006] (A1) Identifying or assisting in the identification of wheat plant height and / or yield;

[0007] (A2) Preparing a product for identifying or assisting in the identification of wheat plant height and / or yield;

[0008] (A3) Comparing the plant height and / or yield of a wheat sample to be tested;

[0009] (A4) Preparing a product for comparing the plant height and / or yield of a wheat sample to be tested;

[0010] (A5) Assisting in the breeding of wheat individual plants or lines or strains or varieties with relatively lower plant height and / or relatively higher yield;

[0011] (A6) Preparing a product for assisting in the breeding of wheat individual plants or lines or strains or varieties with relatively lower plant height and / or relatively higher yield;

[0012] (A7) Screening and eliminating wheat individual plants with relatively higher plant height and / or relatively lower yield;

[0013] (A8) Preparing a product for screening and eliminating wheat individual plants with relatively higher plant height and / or relatively lower yield;

[0014] (A9) Wheat breeding.

[0015] Wherein, the substance A is a substance for detecting the single nucleotide polymorphism of the SNP981 site in the TaTPP1-1A gene on the wheat genome.

[0016] The substance B is a substance for identifying whether the haplotype of the wheat sample to be tested based on the TaTPP1-1A gene is TaTPP1-1A-Hap1 or TaTPP1-1A-Hap2. The haplotype based on the TaTPP1-1A gene involves the polymorphisms of the SNP350 site, the SNP981 site and the SNP1382 site in the TaTPP1-1A gene on the wheat genome (note: the three SNP sites are linked).

[0017] The SNP350 site is located at the 359th position of SEQ ID No.6 in the TaTPP1-1A gene on the wheat genome, and the nucleotide at this SNP site is A or T;

[0018] The SNP981 site is located at the 990th position of SEQ ID No.6 in the TaTPP1-1A gene on the wheat genome (or the 32nd position of SEQ ID No.5), and the nucleotide at this SNP site is A or G;

[0019] The SNP1382 locus is located at the 1391st position of SEQ ID No.6 in the TaTPP1-1A gene on the wheat genome, and the nucleotide at this SNP locus is C or T.

[0020] The TaTPP1-1A-Hap1 refers to that the nucleotide at the SNP350 locus in the TaTPP1-1A gene on the wheat genome is A (homozygous), the nucleotide at the SNP981 locus is A (homozygous), and the nucleotide at the SNP1382 locus is C (homozygous).

[0021] The TaTPP1-1A-Hap2 refers to that the nucleotide at the SNP350 locus in the TaTPP1-1A gene on the wheat genome is T (homozygous), the nucleotide at the SNP981 locus is G (homozygous), and the nucleotide at the SNP1382 locus is T (homozygous).

[0022] Furthermore, the substance A and the substance B can be a primer pair or a reagent or kit containing the primer pair.

[0023] Even further, the primer pair can be primer pair 1 and / or primer pair 2;

[0024] The primer pair 1 consists of an upstream primer 1 and a downstream primer 1; the upstream primer 1 is designed according to the upstream sequence of the SNP350 locus in the TaTPP1-1A gene on the wheat genome; the downstream primer 1 is designed according to the downstream sequence of the SNP1382 locus in the TaTPP1-1A gene on the wheat genome.

[0025] In an embodiment of the present invention, the upstream primer 1 is the DNA molecule shown in SEQ ID No.1; the downstream primer 1 is the DNA molecule shown in SEQ ID No.2.

[0026] The primer pair 2 consists of an upstream primer 2 and a downstream primer 2; the upstream primer 2 is designed according to the upstream sequence of the SNP981 locus in the TaTPP1-1A gene on the wheat genome; the downstream primer 2 is designed according to the downstream sequence of the SNP981 locus in the TaTPP1-1A gene on the wheat genome;

[0027] In an embodiment of the present invention, the upstream primer 2 is the DNA molecule shown in SEQ ID No.3; the downstream primer 2 is the DNA molecule shown in SEQ ID No.4.

[0028] Even further, the reagent or the kit may further contain the restriction endonuclease Nde I.

[0029] When the nucleotide at the SNP981 site in the TaTPP1-1A gene on the wheat genome is G, a recognition sequence for the restriction enzyme Nde I is formed.

[0030] In a second aspect, the present invention claims protection for primer pairs for identifying or assisting in the identification of wheat plant height and / or yield.

[0031] The primer pairs for identifying or assisting in the identification of wheat plant height and / or yield claimed by the present invention can be primer pair 1 and / or primer pair 2.

[0032] The primer pair 1 consists of an upstream primer 1 and a downstream primer 1; the upstream primer 1 is the DNA molecule shown in SEQ ID No.1; the downstream primer 1 is the DNA molecule shown in SEQ ID No.2.

[0033] The primer pair 2 consists of an upstream primer 2 and a downstream primer 2; the upstream primer 2 is the DNA molecule shown in SEQ ID No.3; the downstream primer 2 is the DNA molecule shown in SEQ ID No.4.

[0034] In a third aspect, the present invention claims protection for a reagent or kit containing the primer pairs described in the second aspect above.

[0035] The reagent or kit claimed by the present invention may further contain the restriction enzyme Nde I.

[0036] In a fourth aspect, the present invention claims protection for a DNA molecule.

[0037] The nucleotide sequence of the DNA molecule claimed by the present invention contains (or is) SEQ ID No.5 or SEQ ID No.6.

[0038] In a fifth aspect, the present invention claims protection for the application of the DNA molecule described in the fourth aspect above in any of the following:

[0039] (A1) Identifying or assisting in the identification of wheat plant height and / or yield;

[0040] (A2) Preparing a product for identifying or assisting in the identification of wheat plant height and / or yield;

[0041] (A3) Comparing the plant height and / or yield of a wheat sample to be tested;

[0042] (A4) Preparing a product for comparing the plant height and / or yield of a wheat sample to be tested;

[0043] (A5) Assisting in the breeding of wheat individual plants or lines or strains or varieties with relatively lower plant height and / or relatively higher yield;

[0044] (A6) A product for assisting in the breeding selection of individual wheat plants or lines or strains or varieties with relatively low plant height and / or relatively high yield;

[0045] (A7) Screening and eliminating individual wheat plants with relatively high plant height and / or relatively low yield;

[0046] (A8) A product for screening and eliminating individual wheat plants with relatively high plant height and / or relatively low yield;

[0047] (A9) Wheat breeding.

[0048] In (A9), the purpose of the breeding is to obtain wheat varieties with relatively low plant height (lodging resistance) and / or relatively high yield. The same applies hereinafter.

[0049] In the sixth aspect, the present invention claims any one of the following methods:

[0050] Method I: A method for comparing the plant height and / or yield of a wheat plant to be tested, including the following steps (B1) or (B2):

[0051] (B1) Detecting the nucleotide at the SNP981 site in the TaTPP1-1A gene on the genome of the wheat plant to be tested, determining the genotype of the wheat plant to be tested, and determining the plant height and / or yield of the wheat plant to be tested according to the genotype of the wheat plant to be tested as follows:

[0052] The plant height of the wheat plant to be tested with the G:G genotype is lower than or candidate lower than the plant height of the wheat plant to be tested with the A:A genotype;

[0053] The yield of the wheat plant to be tested with the G:G genotype is higher than or candidate higher than the yield of the wheat plant to be tested with the A:A genotype;

[0054] The SNP981 site is located at the 32nd position of SEQ ID No.5 in the TaTPP1-1A gene on the wheat genome (or the 990th position of SEQ ID No.6), and the nucleotide at this SNP site is A or G;

[0055] The G:G genotype is a homozygous type with the nucleotide G at the SNP981 site on the wheat genome;

[0056] The A:A genotype is a homozygous type with the nucleotide A at the SNP981 site on the wheat genome.

[0057] (B2) Detect the nucleotides at the SNP350 locus, SNP981 locus, and SNP1382 locus in the TaTPP1-1A gene on the genome of the wheat to be tested, determine the haplotype of the wheat to be tested based on the TaTPP1-1A gene, and determine the drought resistance and / or plant height and / or yield of the wheat to be tested according to the haplotype of the wheat to be tested as follows:

[0058] The plant height of the wheat to be tested with the haplotype TaTPP1-1A-Hap2 is lower than or candidate lower than that of the wheat to be tested with the haplotype TaTPP1-1A-Hap1;

[0059] The yield of the wheat to be tested with the haplotype TaTPP1-1A-Hap2 is higher than or candidate higher than that of the wheat to be tested with the haplotype TaTPP1-1A-Hap1;

[0060] The haplotype based on the TaTPP1-1A gene involves the polymorphisms of the SNP350 locus, SNP981 locus, and SNP1382 locus in the TaTPP1-1A gene on the wheat genome (note: the three SNP loci are linked);

[0061] The SNP350 locus is located at the 359th position of SEQ ID No.6 in the TaTPP1-1A gene on the wheat genome, and the nucleotide at this SNP locus is A or T;

[0062] The SNP981 locus is located at the 990th position of SEQ ID No.6 in the TaTPP1-1A gene on the wheat genome (or the 32nd position of SEQ ID No.5), and the nucleotide at this SNP locus is A or G;

[0063] The SNP1382 locus is located at the 1391st position of SEQ ID No.6 in the TaTPP1-1A gene on the wheat genome, and the nucleotide at this SNP locus is C or T;

[0064] The TaTPP1-1A-Hap1 refers to that the nucleotide at the SNP350 locus in the TaTPP1-1A gene on the wheat genome is A (homozygous), the nucleotide at the SNP981 locus is A (homozygous), and the nucleotide at the SNP1382 locus is C (homozygous);

[0065] The TaTPP1-1A-Hap2 refers to that the nucleotide at the SNP350 locus in the TaTPP1-1A gene on the wheat genome is T (homozygous), the nucleotide at the SNP981 locus is G (homozygous), and the nucleotide at the SNP1382 locus is T (homozygous).

[0066] Method II: A method for assisting in breeding individual wheat plants, or lines, or strains, or varieties with relatively low plant height and / or relatively high yield, comprising the following steps (C1) or (C2):

[0067] (C1) Detect the nucleotide at the SNP981 locus in the TaTPP1-1A gene on the genome of the wheat to be tested, determine the genotype of the wheat to be tested, select the wheat to be tested with the G:G genotype as a parent for breeding, and select wheat with the G:G genotype in each generation of breeding, and finally obtain individual wheat plants, or lines, or strains, or varieties with relatively low plant height and / or relatively high yield;

[0068] The SNP981 locus is located at the 32nd position of SEQ ID No. 5 (or the 990th position of SEQ ID No. 6) in the TaTPP1-1A gene on the wheat genome, and the nucleotide at this SNP locus is A or G;

[0069] The G:G genotype is a homozygous type with the nucleotide at the SNP981 locus on the wheat genome being G.

[0070] (C2) Detect the nucleotides at the SNP350 locus, SNP981 locus, and SNP1382 locus in the TaTPP1-1A gene on the genome of the wheat to be tested, determine the haplotype of the wheat to be tested based on the TaTPP1-1A gene, select the wheat to be tested with the haplotype TaTPP1-1A-Hap2 as a parent for breeding, and select wheat with the haplotype TaTPP1-1A-Hap2 in each generation of breeding, and finally obtain individual wheat plants, or lines, or strains, or varieties with relatively low plant height and / or relatively high yield;

[0071] The haplotype based on the TaTPP1-1A gene involves the polymorphisms of the SNP350 locus, the SNP981 locus, and the SNP1382 locus in the TaTPP1-1A gene on the wheat genome (Note: The three SNP loci are linked);

[0072] The SNP350 locus is located at the 359th position of SEQ ID No. 6 in the TaTPP1-1A gene on the wheat genome, and the nucleotide at this SNP locus is A or T;

[0073] The SNP981 locus is located at the 990th position of SEQ ID No. 6 (or the 32nd position of SEQ ID No. 5) in the TaTPP1-1A gene on the wheat genome, and the nucleotide at this SNP locus is A or G;

[0074] The SNP1382 locus is located at the 1391st position of SEQ ID No.6 in the TaTPP1-1A gene on the wheat genome, and the nucleotide at this SNP locus is C or T;

[0075] TaTPP1-1A-Hap2 refers to that the nucleotide at the SNP350 locus in the TaTPP1-1A gene on the wheat genome is T (homozygous), the nucleotide at the SNP981 locus is G (homozygous), and the nucleotide at the SNP1382 locus is T (homozygous).

[0076] Method III: A method for screening and eliminating wheat individual plants with relatively high plant height and / or relatively low yield, which may include the following steps (D1) or (D2):

[0077] (D1) Detect the nucleotide at the SNP981 locus in the TaTPP1-1A gene on the genome of the wheat to be tested, determine the genotype of the wheat to be tested, and screen out and eliminate the wheat to be tested with the A:A genotype;

[0078] The SNP981 locus is located at the 32nd position of SEQ ID No.5 (or the 990th position of SEQ ID No.6) in the TaTPP1-1A gene on the wheat genome, and the nucleotide at this SNP locus is A or G;

[0079] The A:A genotype is a homozygous type with the nucleotide A at the SNP981 locus on the wheat genome;

[0080] (D2) Detect the nucleotides at the SNP350 locus, SNP981 locus and SNP1382 locus in the TaTPP1-1A gene on the genome of the wheat to be tested, determine the haplotype of the wheat to be tested based on the TaTPP1-1A gene, and screen out and eliminate the wheat to be tested with the haplotype TaTPP1-1A-Hap1;

[0081] The haplotype based on the TaTPP1-1A gene involves the polymorphisms of the SNP350 locus, SNP981 locus and SNP1382 locus in the TaTPP1-1A gene on the wheat genome (note: the three SNP loci are linked);

[0082] The SNP350 locus is located at the 359th position of SEQ ID No.6 in the TaTPP1-1A gene on the wheat genome, and the nucleotide at this SNP locus is A or T;

[0083] The SNP981 locus is located at the 990th position of SEQ ID No.6 (or the 32nd position of SEQ ID No.5) in the TaTPP1-1A gene on the wheat genome, and the nucleotide at this SNP locus is A or G;

[0084] The SNP1382 locus is located at the 1391st position of SEQ ID No.6 in the TaTPP1-1A gene on the wheat genome, and the nucleotide at this SNP locus is C or T;

[0085] TaTPP1-1A-Hap1 means that the nucleotide at the SNP350 locus in the TaTPP1-1A gene on the wheat genome is A (homozygous), the nucleotide at the SNP981 locus is A (homozygous), and the nucleotide at the SNP1382 locus is C (homozygous).

[0086] Furthermore, detecting the nucleotide at the SNP981 locus in the TaTPP1-1A gene on the genome of the wheat to be tested can be carried out according to the method including the following step (E1);

[0087] (E1) First, use primer pair 1 described in the second aspect above to perform PCR amplification on the genomic DNA of the wheat to be tested to obtain amplification product 1; then use amplification product 1 as a template and use primer pair 2 described in the second aspect above to perform amplification to obtain amplification product 2; then use the restriction endonuclease NdeI to digest amplification product 2, and determine the nucleotide at the SNP981 locus in the TaTPP1-1A gene on the genome of the wheat to be tested according to the digestion result as follows: If the digestion product only contains a 222bp target fragment, then the nucleotide at the SNP981 locus in the TaTPP1-1A gene on the genome of the wheat to be tested is A (homozygous); if the digestion product does not contain a 222bp target fragment but contains 190bp and 32bp target fragments, then the nucleotide at the SNP981 locus in the TaTPP1-1A gene on the genome of the wheat to be tested is G (homozygous).

[0088] Furthermore, detecting the nucleotides at the SNP350 locus, the SNP981 locus and the SNP1382 locus in the TaTPP1-1A gene on the genome of the wheat to be tested can be carried out according to the method including the following step (E2);

[0089] (E2) First, use primer pair 1 described in the second aspect above to perform PCR amplification on the genomic DNA of the wheat to be tested, obtaining amplification product 1; sequence the amplification product 1, and determine the nucleotides at the SNP350 site, the SNP981 site, and the SNP1382 site in the TaTPP1-1A gene on the genome of the wheat to be tested according to the sequencing results.

[0090] In each of the above-related aspects, the yield is reflected as the thousand-grain weight.

[0091] In each of the above-related aspects, the nucleotide sequence of the TaTPP1-1A gene on the wheat genome is as shown in SEQ ID No. 6.

[0092] In the implementation cases of the present invention, the wheat to be tested is specifically selected from the wheat varieties shown in Table 2.

[0093] Experiments have proved that TaTPP1-1A-Hap2 (the nucleotide at the SNP981 site is G) is an excellent haplotype with short stalks and high yield, which helps to improve the plant type and yield in the wheat breeding process, laying an experimental foundation for subsequent molecular marker-assisted selection of high-yield wheat. The present invention has important application value in the process of wheat molecular marker-assisted breeding. Description of the Drawings

[0094] Figure 1 For the single nucleotide polymorphism of TaTPP1-1A and the development of functional markers. Among them, A is the structural diagram of the TaTPP1-1A gene; B is the SNP site in the TaTPP1-1A genomic sequence; C is the dCAPS marker TaTPP1-1A-NdeI-dCAPS developed according to the TaTPP1-1A SNP site; D is the electrophoresis detection result after the NdeI endonuclease digestion of the second-round PCR product (the one marked with A has A at the 981st SNP site, and the one marked with G has G at the 981st SNP site), and Marker is DNA Marker I.

[0095] Figure 2 For the comparison of phenotypic agronomic traits of two haplotypes of TaTPP1-1A. Among them, A is the comparison of plant heights of two haplotypes of TaTPP1-1A in 10 environments of natural population 2; B is the comparison of thousand-grain weights of two haplotypes of TaTPP1-1A in 10 environments of natural population 2. One-way ANOVA in SPSS 26.0 is used for variance analysis and significance test; *, **, *** respectively indicate that the phenotypes are significantly different at the 0.05, 0.01, and 0.001 probability levels.

[0096] Figure 3Geographical distribution of TaTPP1-1A haplotype in ten major wheat-growing regions of China. The small map in the lower right corner shows the South China Sea Islands.

[0097] Figure 4 Frequency distribution of TaTPP1-1A haplotype in wheat varieties released in different years. Detailed implementation manners

[0098] The present invention will be further described in detail below in conjunction with the detailed implementation manners. The provided embodiments are only for clarifying the present invention and not for limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements and do not constitute any limitation to the present invention in any way.

[0099] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0100] The nucleotide sequence of the TaTPP1-1A gene on the wheat genome involved in the following embodiments is shown in SEQ ID No. 6.

[0101] Example 1: TaTPP1-1A sequence polymorphism, molecular marker development and association analysis

[0102] I. TaTPP1-1A sequence polymorphism

[0103] Clone the gDNA of TaTPP1-1A in 32 wheat polymorphic populations (Table 1, these wheat materials are recorded in the article "Du, Y., Li, C., Mao, X., Wang, J., Li, L., Yang, J., Zhuang, M., Sun, D., & Jing, R. (2022). TaERF73 is associated with root depth, thousand-grain weight and plant height in wheat over a range of environmental conditions. Food and Energy Security, 11, e325. https: / / doi.org / 10.1002 / fes3.325", which is publicly available from the applicant and can only be used for repeating the experiments of the present invention and not for other purposes), and amplify and sequence it using the gDNA-specific primers TaTPP1-1A-gF / gR.

[0104] TaTPP1-1A-gF: 5’-GTTCTGTTTATGGATTTGAAGACGA-3’ (SEQ ID No.1);

[0105] TaTPP1-1A-gR: 5’-TTTCTCTGGAGGGAGCTGGT-3’ (SEQ ID No.2).

[0106] The amplified product was 2980 bp, as shown in SEQ ID No.6.

[0107] Table 1. Polymorphic natural population

[0108] Number Name Number Name Number Name Number Name 1 Jin 2148-7 9 Overbearing Whip 17 04-030 25 Spring 049th-5-1 2 Linkang 5108 10 Jimai 41 18 Beijing 14 26 Spring 454th-50-1 3 Chang 6878 11 Jimai 6 19 Beijing 10 27 Jing 411 4 Changle 5 12 Cangzhou Wheat 20 An 85 Zhong 124-1 28 Dan R8093 5 Baiqi Wheat 13 Yanzhen 1 21 Beijing 8686 29 Fengkang 13 6 Changwu 131 14 Purple Stalk and White Awn First 22 04-044 30 Jinghe 8922 7 Dali 1 15 Neixiang 188 23 Spring 229th-25 31 Chinese Spring 8 Red Monk 16 White Rough Wheat 24 Jingpin 10 32 PANDAS

[0109] It was found that the gDNA region sequence of TaTPP1-1A was rich in polymorphisms, with 3 linked SNP sites ( Figure 1 A and B in the figure). Among them, the SNP site at position 981 (A / G) was located in the fifth exon region, belonging to a non-synonymous mutation, resulting in the amino acid change from serine (Ser, S) to glycine (Gly, G); the SNP site at position 350 (A / T) was located in the first intron region and did not affect the protein sequence; the SNP site at position 1382 (C / T) was located in the 7th exon region and was a synonymous mutation, not causing amino acid changes. These variant sites divided TaTPP1-1A into 2 haplotypes, TaTPP1-1A-Hap1 and TaTPP1-1A-Hap2. Among them, the physical positions of the above 3 SNP sites were based on the Chinese Spring genome v1.1 as the reference genome.

[0110] II. Development of molecular markers for TaTPP1-1A

[0111] According to the SNP site differences in the TaTPP1-1A genomic sequences of 32 polymorphic materials (Table 1), a dCAPS marker named TaTPP1-1A-NdeI-dCAPS was designed using the online website dCAPS Finder 2.0 based on the SNP variation at position 981 (A / G). By introducing a mismatched base T in the primer, the variation at position 981 (A / G) could be cut by the restriction enzyme NdeI (CA▼TATG) ( Figure 1 C in the figure), and the second-round PCR amplification was carried out using the molecular marker primers TaTPP1-1A-NdeI-dCAPS-F / R (the primers used in the first-round PCR amplification were TaTPP1-1A-gF / gR), and the length of the target fragment was 222 bp; then the PCR product was digested with the restriction enzyme NdeI, and different haplotypes of TaTPP1-1A were judged according to the fragment sizes of the digested products.Figure 1 In D). If the SNP locus at position 981 is A, it cannot be cut by NdeI endonuclease, and the digestion product has only one band of 222 bp. This haplotype is named TaTPP1-1A-Hap1 (since the three SNP loci are linked, the SNP350 locus is A and the SNP1382 locus is C here); if the SNP locus at position 981 is G, it can be cut by NdeI endonuclease, and the digestion products are 190 bp and 32 bp. This haplotype is named TaTPP1-1A-Hap2 (since the three SNP loci are linked, the SNP350 locus is T and the SNP1382 locus is T here).

[0112] TaTPP1-1A-NdeI-dCAPS-F: 5’-ATTTTAATTGTTCACTTGTTTTCACTCATAT-3’ (SEQ ID No.3);

[0113] TaTPP1-1A-NdeI-dCAPS-R: 5’-ATCCTTGCACCTTCGATGTCC-3’ (SEQ ID No.4).

[0114] The theoretical amplification product is a 222-bp target band, and the nucleotide sequence is shown in SEQ ID No.5.

[0115] III. Association analysis between TaTPP1-1A-NdeI-dCAPS and agronomic traits

[0116] Using the molecular marker TaTPP1-1A-NdeI-dCAPS to scan 389 wheat materials in natural population 2 (Table 2. Since the wheat materials are all cultivated species, they are usually considered highly homozygous plant materials, and the haplotypes are all homozygous. The same below), it is found that the haplotypes TaTPP1-1A-Hap1 and TaTPP1-1A-Hap2 in natural population 2 account for 49.61% and 50.39% respectively.

[0117] The genotypes of TaTPP1-1A for 389 wheat materials in natural population 2 are summarized in Table 2 (these wheat materials are described in the article "Shi H, Chen M, Gao L, Wang Y, Bai Y, Yan H, Xu C, Zhou Y, Xu Z, Chen J, Tang W, Wang S, Shi Y, Wu Y, Sun D, Jia J, Ma Y. Genome-wide association study of agronomic traits related to nitrogen use efficiency in wheat. Theor Appl Genet. 2022 Dec;135(12):4289-4302. doi: 10.1007 / s00122-022-04218-5. Epub 2022 Sep 22. PMID: 36136127.", which is available to the public from the applicant and can only be used for repeating the experiments of the present invention and not for other purposes). Figure 1 In D, the electrophoresis detection results of the PCR products of some materials digested by NdeI endonuclease are shown. Among them, the one marked with A is homozygous for A at the SNP locus at position 981 (corresponding to the haplotype TaTPP1-1A-Hap1; the digestion product has only one band of 222 bp), and the one marked with G is homozygous for G at the SNP locus at position 981 (corresponding to the haplotype TaTPP1-1A-Hap2; the digestion products are 190 bp and 32 bp).

[0118] Table 2. Genotypes of TaTPP1-1A in natural population 2

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125] The 981st SNP allele variation (A / G) of TaTPP1-1A was associated with the agronomic traits in 10 environments of natural population 2 (Table 2) using the general linear model (GLM) in Tassel 5.0 software, and a total of 2 agronomic traits, plant height and 1000-grain weight, were associated (Table 3). The results showed that the two haplotypes of TaTPP1-1A (TaTPP1-1A-Hap1 and TaTPP1-1A-Hap2) were significantly or extremely significantly correlated with plant height in 10 environments (E1, E2, E3, E4, E5, E6, E7, E8, E9, E10); the two haplotypes of TaTPP1-1A (TaTPP1-1A-Hap1 and TaTPP1-1A-Hap2) were significantly or extremely significantly correlated with 1000-grain weight in 7 environments (E1, E2, E5, E6, E7, E8, E10).

[0126] Table 3. Association analysis of two haplotypes of TaTPP1-1A with phenotypic agronomic traits of natural population 2

[0127] Environment Plant Height (P value) 1000-Grain Weight (P value) E1 0.00194** 0.000070622*** E2 0.000047971*** 0.00010413*** E3 0.000056839*** n.s. E4 0.000050942*** n.s. E5 0.03457* 0.014* E6 0.00677** 0.01842* E7 0.00564** 0.00118** E8 0.0164* 0.00065837*** E9 0.04578* n.s. E10 0.03263* 0.00503**

[0128] Note: E1: 2018-SX-LN; E2: 2018-SX-N; E3: 2019-SX-LN; E4: 2019-SX-N; E5: 2020-SX-LN; E6: 2020-SX-N; E7: 2019-HB-LN; E8: 2019-HB-N; E9: 2020-HB-LN; E10: 2020-HB-N. The naming method is planting year-planting location-nitrogen treatment method. Among them, SX is the Shenfeng experimental field of Shanxi Agricultural University, HB is the experimental field in Zhaoxian, Hebei, N is the normal nitrogen treatment (i.e., nitrogen application, the nitrogen application rate is 18 kg / 667 m 2 (calculated as pure nitrogen), and 30%, 40% and 30% of the total nitrogen fertilizer amount are applied during irrigation before winter, jointing stage and heading stage respectively), LN is the low nitrogen treatment (i.e., no nitrogen is applied during the whole growth process of wheat). *, **, *** indicate that the phenotypes are significantly different at the 0.05, 0.01, 0.001 probability levels respectively.

[0129] Analysis of variance was performed on the plant heights of the two haplotypes, and it was found that the plant height of haplotype TaTPP1-1A-Hap1 was higher than that of haplotype TaTPP1-1A-Hap2 in 10 environments, and it reached a significant or extremely significant level in environments E1, E2, E3, E4, E5, E6, E7, E8, E9 and E10 ( Figure 2In A), variance analysis was performed on the thousand-grain weight of the two haplotypes, and it was found that the thousand-grain weight of haplotype TaTPP1-1A-Hap1 was lower than that of haplotype TaTPP1-1A-Hap2 in 10 environments, and significant or highly significant levels were reached in environments E1, E2, E4, E5, E6, E7, E8, and E10 ( Figure 2 In B).

[0130] IV. Geographic Distribution of TaTPP1-1A Haplotypes

[0131] Wheat cultivation in China spreads throughout the country. According to the natural conditions such as climate, terrain, and soil type in different regions of China, the wheat planting areas in China are divided into ten major wheat regions (I-X), namely I Northern Winter Wheat Region, II Huanghuai Winter Wheat Region, III Middle and Lower Yangtze River Winter Wheat Region, IV Southwest Winter Wheat Region, V South China Winter Wheat Region, VI Northeast Spring Wheat Region, VII Northern Spring Wheat Region, VIII Northwest Spring Wheat Region, IX Qinghai-Tibet Winter and Spring Wheat Region, and X Xinjiang Winter and Spring Wheat Region.

[0132] To study the distribution of TaTPP1-1A haplotypes in wheat regions of China, the distribution frequencies of the two haplotypes TaTPP1-1A-Hap1 and TaTPP1-1A-Hap2 of TaTPP1-1A in the wheat natural population 2 (Table 2) were statistically analyzed ( Figure 3 ). The results showed that in wheat regions IV, V, and IX, the frequency of TaTPP1-1A-Hap1 was lower than that of TaTPP1-1A-Hap2. On the contrary, in the remaining wheat regions, the frequency of TaTPP1-1A-Hap1 was higher than that of TaTPP1-1A-Hap2, and TaTPP1-1A-Hap2 was mainly concentrated in regions with low latitudes or high altitudes and had a relatively high distribution.

[0133] V. Frequency Distribution of TaTPP1-1A Haplotypes in Varieties of Different Eras

[0134] To verify whether excellent allelic variations are positively selected by breeders and the natural environment, the present invention performed a statistical analysis on the haplotypes of varieties from different years in natural population 3 (Table 4, these materials are recorded in the article "Du, Y., Li, C., Mao, X., Wang, J., Li, L., Yang, J., Zhuang, M., Sun, D., & Jing, R. (2022). TaERF73 is associated with root depth, thousand-grain weight and plant height in wheat over a range of environmental conditions. Food and Energy Security, 11, e325. https: / / doi.org / 10.1002 / fes3.325", which is publicly available from the applicant and can only be used for repeating the experiments of the present invention and not for other purposes). The natural population 3 was scanned using the molecular marker TaTPP1-1A-NdeI-dCAPS to verify whether the haplotype TaTPP1-1A-Hap2 was positively selected during the wheat variety improvement process.

[0135] Table 4. Genotypes of TaTPP1-1A in natural population 3

[0136]

[0137]

[0138]

[0139]

[0140]

[0141] The results are as Figure 4 shown, indicating that: the haplotype TaTPP1-1A-Hap2 was 50% during the 1940s. After that, over time, the frequency of the haplotype TaTPP1-1A-Hap2 showed a gradually decreasing trend and tended to fluctuate stably. Moreover, the frequency distribution of the haplotype TaTPP1-1A-Hap2 over the years was significantly lower than that of the haplotype TaTPP1-1A-Hap1, indicating that there is still a large room for selection of the excellent haplotype TaTPP1-1A-Hap2 during the wheat breeding process.

[0142] Comprehensive correlation analysis, variance analysis, and geographical and chronological distribution frequencies indicate that TaTPP1-1A-Hap2 is an excellent haplotype with short plant height and high yield, which contributes to the improvement of plant type and yield during wheat breeding, laying an experimental foundation for subsequent molecular marker-assisted selection of high-yield wheat, and can attract attention in future wheat breeding.

[0143] The above has detailed the present invention. For those skilled in the art, without departing from the purpose and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses, or improvements to the present invention, including changes made using conventional techniques known in the art that depart from the scope disclosed in this application.

Claims

1. The use of substance A or substance B in any of the following: (A1) Identifying or assisting in the identification of wheat plant height and / or yield; (A2) preparing a product for identifying or assisting in identifying wheat plant height and / or yield; (A3) comparing plant height and / or yield of the tested wheat; (A4) preparing a product for comparing plant height and / or yield of wheat to be tested; (A5) assisting in the selection of wheat plants or lines or strains or varieties with relatively lower plant height and / or relatively higher yield; (A6) preparing a product for assisting in breeding wheat plants or lines or strains or varieties with relatively low plant height and / or relatively high yield; (A7) screening and eliminating wheat plants with relatively high plant height and / or relatively low yield; (A8) preparing a product for screening and eliminating wheat plants with relatively high plant height and / or relatively low yield; (A9) Wheat breeding; The substance A is a substance used to detect the single nucleotide polymorphism of the SNP981 site in the TaTPP1-1A gene on the wheat genome; The substance B is a substance used to identify whether the haplotype of the wheat to be tested based on the TaTPP1-1A gene is TaTPP1-1A-Hap1 or TaTPP1-1A-Hap2; the haplotype based on the TaTPP1-1A gene involves the polymorphism of the SNP350 site, the SNP981 site and the SNP1382 ​​site in the TaTPP1-1A gene on the wheat genome; The SNP350 site is located at position 359 of SEQ ID No.6 in the TaTPP1-1A gene on the wheat genome, and the nucleotide at the SNP site is A or T; The SNP981 site is located at position 990 of SEQ ID No. 6 in the TaTPP1-1A gene on the wheat genome, and the nucleotide at the SNP site is A or G; The SNP1382 ​​site is located at position 1391 of SEQ ID No.6 in the TaTPP1-1A gene on the wheat genome, and the nucleotide at the SNP site is C or T; The TaTPP1-1A-Hap1 means that the nucleotide at the SNP350 site in the TaTPP1-1A gene on the wheat genome is A, the nucleotide at the SNP981 site is A, and the nucleotide at the SNP1382 ​​site is C; The TaTPP1-1A-Hap2 means that the nucleotide at the SNP350 site in the TaTPP1-1A gene on the wheat genome is T, the nucleotide at the SNP981 site is G, and the nucleotide at the SNP1382 ​​site is T.

2. The use according to claim 1, characterized in that: The substance A and the substance B are a primer pair or a reagent or a kit containing the primer pair; The primer pair is primer pair 1 and / or primer pair 2; The primer pair 1 consists of an upstream primer 1 and a downstream primer 1; the upstream primer 1 is designed according to the upstream sequence of the SNP350 site in the TaTPP1-1A gene on the wheat genome; the downstream primer 1 is designed according to the downstream sequence of the SNP1382 ​​site in the TaTPP1-1A gene on the wheat genome; Furthermore, the upstream primer 1 is the DNA molecule shown in SEQ ID No. 1; the downstream primer 1 is the DNA molecule shown in SEQ ID No. 2; The primer pair 2 consists of an upstream primer 2 and a downstream primer 2; the upstream primer 2 is designed according to the upstream sequence of the SNP981 site in the TaTPP1-1A gene on the wheat genome; the downstream primer 2 is designed according to the downstream sequence of the SNP981 site in the TaTPP1-1A gene on the wheat genome; Furthermore, the upstream primer 2 is a DNA molecule shown by SEQ ID No.3; and the downstream primer 2 is a DNA molecule shown by SEQ ID No.

4.

3. The use according to claim 2, characterized in that: The reagent or the kit also contains restriction endonuclease Nde I.

4. A primer pair for identifying or assisting in identifying wheat plant height and / or yield, characterized in that: The primer pair is primer pair 1 and / or primer pair 2; The primer pair 1 consists of an upstream primer 1 and a downstream primer 1; the upstream primer 1 is a DNA molecule shown in SEQ ID No. 1; the downstream primer 1 is a DNA molecule shown in SEQ ID No. 2; The primer pair 2 consists of an upstream primer 2 and a downstream primer 2; the upstream primer 2 is a DNA molecule shown in SEQ ID No.3; the downstream primer 2 is a DNA molecule shown in SEQ ID No.

4.

5. A reagent or kit containing the primer pair according to claim 4, characterized in that: The reagent or the kit also contains restriction endonuclease Nde I.

6. A DNA molecule, the nucleotide sequence of which comprises SEQ ID No. 5 or SEQ ID No.

6.

7. Use of the DNA molecule according to claim 6 in any of the following: (A1) Identifying or assisting in the identification of wheat plant height and / or yield; (A2) preparing a product for identifying or assisting in identifying wheat plant height and / or yield; (A3) comparing plant height and / or yield of the tested wheat; (A4) preparing a product for comparing plant height and / or yield of wheat to be tested; (A5) assisting in the selection of wheat plants or lines or strains or varieties with relatively lower plant height and / or relatively higher yield; (A6) preparing a product for assisting in breeding wheat plants or lines or strains or varieties with relatively low plant height and / or relatively high yield; (A7) screening and eliminating wheat plants with relatively high plant height and / or relatively low yield; (A8) preparing a product for screening and eliminating wheat plants with relatively high plant height and / or relatively low yield; (A9) Wheat breeding.

8. Either of the following methods: Method I: A method for comparing plant height and / or yield of wheat to be tested, comprising the following steps (B1) or (B2): (B1) detecting the nucleotide at the SNP981 site in the TaTPP1-1A gene on the genome of the wheat to be tested, determining the genotype of the wheat to be tested, and determining the plant height and / or yield of the wheat to be tested according to the genotype of the wheat to be tested as follows: The plant height of the wheat to be tested of the G:G genotype is lower than or is lower than the plant height of the wheat to be tested of the A:A genotype; The yield of the wheat to be tested of the G:G genotype is higher or is candidate higher than the yield of the wheat to be tested of the A:A genotype; The SNP981 site is located at position 32 of SEQ ID No. 5 in the TaTPP1-1A gene on the wheat genome, and the nucleotide at the SNP site is A or G; The G:G genotype is a homozygous type in which the nucleotide at the SNP981 site on the wheat genome is G; The A:A genotype is a homozygous type in which the nucleotide at the SNP981 site on the wheat genome is A; (B2) detecting nucleotides at SNP350, SNP981 and SNP1382 ​​in the TaTPP1-1A gene on the genome of the wheat to be tested, determining the haplotype of the wheat to be tested based on the TaTPP1-1A gene, and determining the plant height and / or yield of the wheat to be tested according to the haplotype of the wheat to be tested as follows: The plant height of the wheat to be tested with the haplotype TaTPP1-1A-Hap2 is lower than or is likely to be lower than the plant height of the wheat to be tested with the haplotype TaTPP1-1A-Hap1; The yield of the wheat to be tested with the haplotype TaTPP1-1A-Hap2 is higher or has a candidate higher yield than the yield of the wheat to be tested with the haplotype TaTPP1-1A-Hap1; The haplotype based on the TaTPP1-1A gene involves the polymorphisms of the SNP350 site, the SNP981 site and the SNP1382 ​​site in the TaTPP1-1A gene on the wheat genome; The SNP350 site is located at position 359 of SEQ ID No.6 in the TaTPP1-1A gene on the wheat genome, and the nucleotide at the SNP site is A or T; The SNP981 site is located at position 990 of SEQ ID No. 6 in the TaTPP1-1A gene on the wheat genome, and the nucleotide at the SNP site is A or G; The SNP1382 ​​site is located at position 1391 of SEQ ID No.6 in the TaTPP1-1A gene on the wheat genome, and the nucleotide at the SNP site is C or T; The TaTPP1-1A-Hap1 means that the nucleotide at the SNP350 site in the TaTPP1-1A gene on the wheat genome is A, the nucleotide at the SNP981 site is A, and the nucleotide at the SNP1382 ​​site is C; The TaTPP1-1A-Hap2 refers to the nucleotide at the SNP350 site in the TaTPP1-1A gene on the wheat genome is T, the nucleotide at the SNP981 site is G, and the nucleotide at the SNP1382 ​​site is T; Method II: A method for assisting in the selection of wheat plants or lines or strains or varieties with relatively low plant height and / or relatively high yield, comprising the following steps (C1) or (C2): (C1) detecting the nucleotide at the SNP981 site in the TaTPP1-1A gene on the genome of the wheat to be tested, determining the genotype of the wheat to be tested, selecting the wheat to be tested with a G:G genotype as a parent for breeding, and selecting wheat with a G:G genotype in each breeding generation, and finally obtaining wheat plants or lines or strains or varieties with relatively low plant height and / or relatively high yield; The SNP981 site is located at position 32 of SEQ ID No. 5 in the TaTPP1-1A gene on the wheat genome, and the nucleotide at the SNP site is A or G; The G:G genotype is a homozygous type in which the nucleotide at the SNP981 site on the wheat genome is G; (C2) detecting nucleotides at SNP350, SNP981 and SNP1382 ​​in the TaTPP1-1A gene on the genome of the wheat to be tested, determining the haplotype of the wheat to be tested based on the TaTPP1-1A gene, selecting the wheat to be tested with a haplotype of TaTPP1-1A-Hap2 as a parent for breeding, and selecting wheat with a haplotype of TaTPP1-1A-Hap2 in each breeding generation, and finally obtaining wheat plants or strains or lines or varieties with relatively low plant height and / or relatively high yield; The haplotype based on the TaTPP1-1A gene involves the polymorphisms of the SNP350 site, the SNP981 site and the SNP1382 ​​site in the TaTPP1-1A gene on the wheat genome; The SNP350 site is located at position 359 of SEQ ID No.6 in the TaTPP1-1A gene on the wheat genome, and the nucleotide at the SNP site is A or T; The SNP981 site is located at position 990 of SEQ ID No. 6 in the TaTPP1-1A gene on the wheat genome, and the nucleotide at the SNP site is A or G; The SNP1382 ​​site is located at position 1391 of SEQ ID No.6 in the TaTPP1-1A gene on the wheat genome, and the nucleotide at the SNP site is C or T; The TaTPP1-1A-Hap2 refers to the nucleotide at the SNP350 site in the TaTPP1-1A gene on the wheat genome is T, the nucleotide at the SNP981 site is G, and the nucleotide at the SNP1382 ​​site is T; Method III: A method for screening and eliminating wheat plants with relatively high plant height and / or relatively low yield, comprising the following steps (D1) or (D2): (D1) detecting the nucleotide at the SNP981 site in the TaTPP1-1A gene on the genome of the wheat to be tested, determining the genotype of the wheat to be tested, and screening out the wheat to be tested with the A:A genotype; The SNP981 site is located at position 32 of SEQ ID No. 5 in the TaTPP1-1A gene on the wheat genome, and the nucleotide at the SNP site is A or G; The A:A genotype is a homozygous type in which the nucleotide at the SNP981 site on the wheat genome is A; (D2) detecting nucleotides at SNP350, SNP981 and SNP1382 ​​in the TaTPP1-1A gene on the genome of the wheat to be tested, determining the haplotype of the wheat to be tested based on the TaTPP1-1A gene, and screening out the wheat to be tested with a haplotype of TaTPP1-1A-Hap1; The haplotype based on the TaTPP1-1A gene involves the polymorphisms of the SNP350 site, the SNP981 site and the SNP1382 ​​site in the TaTPP1-1A gene on the wheat genome; The SNP350 site is located at position 359 of SEQ ID No.6 in the TaTPP1-1A gene on the wheat genome, and the nucleotide at the SNP site is A or T; The SNP981 site is located at position 990 of SEQ ID No. 6 in the TaTPP1-1A gene on the wheat genome, and the nucleotide at the SNP site is A or G; The SNP1382 ​​site is located at position 1391 of SEQ ID No.6 in the TaTPP1-1A gene on the wheat genome, and the nucleotide at the SNP site is C or T; The TaTPP1-1A-Hap1 means that the nucleotide at the SNP350 site in the TaTPP1-1A gene on the wheat genome is A, the nucleotide at the SNP981 site is A, and the nucleotide at the SNP1382 ​​site is C.

9. The method according to claim 8, characterized in that: Detecting the nucleotide at the SNP981 site in the TaTPP1-1A gene on the wheat genome to be tested is performed according to a method comprising the following steps (E1); (E1) firstly using the primer pair 1 described in claim 4 to perform a first round of PCR amplification on the genomic DNA of the wheat to be tested to obtain an amplified product 1; then using the amplified product 1 as a template, using the primer pair 2 described in claim 4 to perform amplification to obtain an amplified product 2; then using the restriction endonuclease NdeI to digest the amplified product 2, and according to the digestion result, determining the nucleotide at the SNP981 site in the TaTPP1-1A gene on the wheat genome to be tested as follows: if the digestion product contains only a 222 bp target fragment, the nucleotide at the SNP981 site in the TaTPP1-1A gene on the wheat genome to be tested is A; If the enzyme digestion product does not contain the 222 bp target fragment but contains the 190 bp and 32 bp target fragments, the nucleotide at the SNP981 site in the TaTPP1-1A gene on the wheat genome to be tested is G; or Detecting the nucleotides at the SNP350 site, the SNP981 site and the SNP1382 ​​site in the TaTPP1-1A gene on the wheat genome to be tested is performed according to a method comprising the following step (E2); (E2) performing a first round of PCR amplification on the genomic DNA of the wheat to be tested using the primer pair 1 described in claim 4 to obtain an amplified product 1; sequencing the amplified product 1, and determining the nucleotides at the SNP350 site, the SNP981 site, and the SNP1382 ​​site in the TaTPP1-1A gene on the genome of the wheat to be tested based on the sequencing results.

10. The use or primer pair or reagent or kit or method according to any one of claims 1 to 5 and 7 to 9, characterized in that: The yield is expressed as thousand-grain weight.

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