Molecular markers and methods for identifying wheat root length traits

Abstract

This invention discloses molecular markers and methods for identifying wheat root length traits. The molecular markers disclosed in this invention are nucleotides in the wheat genome corresponding to positions 1024, 1067, 1453, 1801, 1818, and 2792 of SEQ ID No. 1 in the sequence listing, which are A, G, G, C, T, C (haap-5B-1), G, G, G, T, T, C (haap-5B-2), or A, C, A, C, C, G (haap-5B-3). Experiments have shown that wheat seedlings with Hap-5B-3 have longer root lengths than those with Hap-5B-1 and Hap-5B-2, and wheat seedlings with nucleotide C at position 1067 have longer root lengths than those with G at that position. Using the molecular markers of this invention, wheat varieties with superior root traits can be quickly and accurately identified, which is of great significance in the cultivation of stress-resistant wheat varieties or in research.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically molecular markers and methods for identifying wheat root length traits. Background Technology

[0002] Wheat is one of my country's three major food crops and plays a vital role in ensuring national food security. With the rapid development of molecular biology, quantitative genetics, and bioinformatics, molecular marker-assisted breeding has gradually become one of the most active research areas in agricultural science. Molecular breeding technology can be combined with traditional breeding techniques and widely applied in wheat breeding, providing an efficient pathway for crop genetic improvement and germplasm innovation. Summary of the Invention

[0003] The technical problem to be solved by this invention is how to detect the root length trait of wheat.

[0004] To solve the above-mentioned technical problems, the present invention first provides the application of substances that detect wheat molecular markers in the detection or auxiliary detection of wheat root length traits;

[0005] The wheat molecular markers are nucleotides in the wheat genome corresponding to positions 1024, 1067, 1453, 1801, 1818, and / or 2792 of SEQ ID No. 1 in the sequence listing. Specifically, the nucleotide corresponding to position 1024 of SEQ ID No. 1 is A or G; the nucleotide corresponding to position 1067 of SEQ ID No. 1 is G or C; the nucleotide corresponding to position 1453 of SEQ ID No. 1 is G or A; the nucleotide corresponding to position 1801 of SEQ ID No. 1 is C or T; the nucleotide corresponding to position 1818 of SEQ ID No. 1 is T or C; and the nucleotide corresponding to position 2792 of SEQ ID No. 1 is C or G.

[0006] In the above applications, the substance used to detect wheat molecular markers may be a primer pair named TaHT1-5B-Primer or a set of reagents for detecting wheat molecular markers.

[0007] The TaHT1-5B-Primer consists of two single-stranded DNA sequences shown in SEQ ID No. 4 and SEQ ID No. 5 of the sequence listing;

[0008] The kit of reagents for detecting the wheat molecular marker may include a primer pair named TaHT1-5B-CAPS, which consists of two single-stranded DNA molecules as shown in SEQ ID No. 2 and SEQ ID No. 3 in the sequence listing.

[0009] In the above applications, the kit may also include restriction endonucleases Hpy188 III and / or HhaI.

[0010] The complete set of reagents may be TaHT1-5B-CAPS, or may be composed of TaHT1-5B-CAPS and restriction endonuclease Hpy188 III and / or HhaI.

[0011] In the above applications, the root length of homozygous wheat whose genome corresponds to nucleotide C at position 1067 of SEQ ID No. 1 in the sequence listing is greater than or candidate to be greater than the root length of homozygous wheat whose genome corresponds to nucleotide G at position 1067 of SEQ ID No. 1 in the sequence listing;

[0012] The root length of homozygous wheat whose genome corresponds to nucleotide A at position 1453 of SEQ ID No. 1 in the sequence listing is greater than or candidate greater than the root length of homozygous wheat whose genome corresponds to nucleotide G at position 1453 of SEQ ID No. 1 in the sequence listing;

[0013] The root length of homozygous wheat whose genome corresponds to nucleotide C at position 1818 of SEQ ID No. 1 in the sequence listing is greater than or candidate greater than the root length of homozygous wheat whose genome corresponds to nucleotide T at position 1818 of SEQ ID No. 1 in the sequence listing;

[0014] The root length of homozygous wheat whose genome corresponds to nucleotide G at position 2792 of SEQ ID No. 1 in the sequence listing is greater than or candidate to be greater than the root length of homozygous wheat whose genome corresponds to nucleotide C at position 2792 of SEQ ID No. 1 in the sequence listing;

[0015] The root length of homozygous wheat whose nucleotides at positions 1024, 1067, 1453, 1801, 1818, and 2792 of SEQ ID No. 1 in the sequence listing are A, C, A, C, C, and G, respectively, is greater than or candidate greater than the root length of homozygous wheat whose nucleotides at positions 1024, 1067, 1453, 1801, 1818, and 2792 of SEQ ID No. 1 in the sequence listing are A, G, G, C, T, and C, respectively.

[0016] The root length of homozygous wheat whose nucleotides at positions 1024, 1067, 1453, 1801, 1818, and 2792 of SEQ ID No. 1 in the sequence listing are A, C, A, C, C, and G, respectively, is greater than or candidate greater than the root length of homozygous wheat whose nucleotides at positions 1024, 1067, 1453, 1801, 1818, and 2792 of SEQ ID No. 1 in the sequence listing are G, G, G, T, T, and C, respectively.

[0017] The root length of homozygous wheat with nucleotides A, G, G, C, T, and C at positions 1024, 1067, 1453, 1801, 1818, and 2792 of SEQ ID No. 1 in the sequence listing was not different from that of homozygous wheat with nucleotides G, G, G, T, T, and C at positions 1024, 1067, 1453, 1801, 1818, and 2792 of SEQ ID No. 1 in the sequence listing.

[0018] The present invention also provides a method for detecting or assisting in the detection of wheat root length, the method comprising detecting the wheat molecular markers and determining the wheat root length according to the following method:

[0019] The root length of homozygous wheat whose genome corresponds to nucleotide C at position 1067 of SEQ ID No. 1 in the sequence listing is greater than or candidate to be greater than the root length of homozygous wheat whose genome corresponds to nucleotide G at position 1067 of SEQ ID No. 1 in the sequence listing;

[0020] The root length of homozygous wheat whose genome corresponds to nucleotide A at position 1453 of SEQ ID No. 1 in the sequence listing is greater than or candidate greater than the root length of homozygous wheat whose genome corresponds to nucleotide G at position 1453 of SEQ ID No. 1 in the sequence listing;

[0021] The root length of homozygous wheat whose genome corresponds to nucleotide C at position 1818 of SEQ ID No. 1 in the sequence listing is greater than or candidate greater than the root length of homozygous wheat whose genome corresponds to nucleotide T at position 1818 of SEQ ID No. 1 in the sequence listing;

[0022] The root length of homozygous wheat whose genome corresponds to nucleotide G at position 2792 of SEQ ID No. 1 in the sequence listing is greater than or candidate to be greater than the root length of homozygous wheat whose genome corresponds to nucleotide C at position 2792 of SEQ ID No. 1 in the sequence listing;

[0023] The root length of homozygous wheat whose nucleotides at positions 1024, 1067, 1453, 1801, 1818, and 2792 of SEQ ID No. 1 in the sequence listing are A, C, A, C, C, and G, respectively, is greater than or candidate greater than the root length of homozygous wheat whose nucleotides at positions 1024, 1067, 1453, 1801, 1818, and 2792 of SEQ ID No. 1 in the sequence listing are A, G, G, C, T, and C, respectively.

[0024] The root length of homozygous wheat whose nucleotides at positions 1024, 1067, 1453, 1801, 1818, and 2792 of SEQ ID No. 1 in the sequence listing are A, C, A, C, C, and G, respectively, is greater than or candidate greater than the root length of homozygous wheat whose nucleotides at positions 1024, 1067, 1453, 1801, 1818, and 2792 of SEQ ID No. 1 in the sequence listing are G, G, G, T, T, and C, respectively.

[0025] The root length of homozygous wheat with nucleotides A, G, G, C, T, and C at positions 1024, 1067, 1453, 1801, 1818, and 2792 of SEQ ID No. 1 in the sequence listing was not different from that of homozygous wheat with nucleotides G, G, G, T, T, and C at positions 1024, 1067, 1453, 1801, 1818, and 2792 of SEQ ID No. 1 in the sequence listing.

[0026] In the above method, the detection of the wheat molecular marker can be performed using the substance for detecting wheat molecular markers.

[0027] In the above method, detecting the wheat molecular marker using the TaHT1-5B-Primer may include: performing PCR amplification on wheat genomic DNA using the TaHT1-5B-Primer to obtain an amplification product; sequencing the amplification product to determine the nucleotides in the wheat genome corresponding to positions 1024, 1067, 1453, 1801, 1818 and / or 2792 of SEQ ID No. 1 in the sequence listing.

[0028] In the above method, detecting the wheat molecular marker using the complete set of reagents may include: performing PCR amplification of wheat genomic DNA using the TaHT1-5B-CAPS to obtain amplification products; and processing the amplification products using Hpy188. Enzyme III digestion yielded digestion product B. The amplified product was then digested with HhaI to obtain digestion product C. The sizes of digestion products B and C were detected. Based on the linkages between A at position 1024, G at position 1067, G at position 1453, C at position 1801, T at position 1818, and C at position 2792; the linkages between G at position 1024, G at position 1067, G at position 1453, T at position 1801, T at position 1818, and C at position 2792; and the linkages between A at position 1024, C at position 1067, A at position 1453, C at position 1801, C at position 1818, and G at position 2792, the sequence corresponding to SEQ ID in the wheat genome was determined according to the following method. Nucleotides at positions 1024, 1067, 1453, 1801, 1818, and / or 2792 of No. 1:

[0029] If the enzyme digestion product B contains DNA fragments of 263 bp and 129 bp in size and does not contain a DNA fragment of 392 bp in size, and the enzyme digestion product C contains DNA fragments of 312 bp and 80 bp in size and does not contain a DNA fragment of 392 bp in size, then the wheat is homozygous wheat, and the nucleotides corresponding to position 1024, 1067, and 1453 of SEQ ID No. 1 in the sequence listing are A, G, C, T, and C respectively.

[0030] If the enzyme digestion product B contains a DNA fragment of 392 bp and does not contain DNA fragments of 263 bp and 129 bp, and the enzyme digestion product C contains DNA fragments of 312 bp and 80 bp and does not contain a DNA fragment of 392 bp, then the wheat is homozygous wheat, and the nucleotides corresponding to position 1024, 1067, and 1453 of SEQ ID No. 1 in the sequence listing are G, T at position 1801, T at position 1818, and C at position 2792 in its genomic DNA.

[0031] If the enzyme digestion product B contains DNA fragments of 263 bp and 129 bp in size and does not contain a DNA fragment of 392 bp in size, and the enzyme digestion product C contains a DNA fragment of 392 bp in size and does not contain DNA fragments of 312 bp and 80 bp in size, then the wheat is homozygous wheat, and the nucleotides corresponding to position 1024, position 1067, position 1453, position 1801, position 1818, and position 2792 in its genomic DNA are A, C, and G, respectively.

[0032] If the enzyme digestion product B contains DNA fragments of 263bp, 129bp, and 392bp, and the enzyme digestion product C contains DNA fragments of 312bp and 80bp, but does not contain a DNA fragment of 392bp, then the wheat is a heterozygous wheat. One chromosome corresponds to nucleotide A at position 1024, G at position 1067, G at position 1453, C at position 1801, T at position 1818, and C at position 2792 in SEQ ID No. 1 of the sequence listing, and the other chromosome corresponds to nucleotide G at position 1024, G at position 1067, G at position 1453, T at position 1801, T at position 1818, and C at position 2792 in SEQ ID No. 1 of the sequence listing.

[0033] If the enzyme digestion product B contains DNA fragments of 263 bp and 129 bp in size and does not contain a DNA fragment of 392 bp in size, and the enzyme digestion product C contains DNA fragments of 312 bp, 80 bp, and 392 bp in size, then the wheat is a heterozygous wheat. One chromosome corresponds to nucleotide A at position 1024, G at position 1067, G at position 1453, C at position 1801, T at position 1818, and C at position 2792 in SEQ ID No. 1 of the sequence listing, and the other chromosome corresponds to nucleotide A at position 1024, C at position 1067, A at position 1453, C at position 1801, C at position 1818, and G at position 2792 in SEQ ID No. 1 of the sequence listing.

[0034] If the enzyme digestion product B contains DNA fragments of 263bp, 129bp, and 392bp in size, and the enzyme digestion product C contains DNA fragments of 312bp, 80bp, and 392bp in size, then the wheat is a heterozygous wheat. One chromosome corresponds to nucleotides G at position 1024, G at position 1067, G at position 1453, T at position 1801, T at position 1818, and C at position 2792 in SEQ ID No. 1 of the sequence listing, and the other chromosome corresponds to nucleotides A at position 1024, C at position 1067, A at position 1453, C at position 1801, C at position 1818, and G at position 2792 in SEQ ID No. 1 of the sequence listing.

[0035] The substance used to detect wheat molecular markers is also within the scope of protection of this invention.

[0036] The wheat molecular markers mentioned above are also within the scope of protection of this invention.

[0037] This invention also provides any of the following applications:

[0038] The application of wheat molecular markers described in X1) in wheat breeding;

[0039] X2) The application of wheat molecular markers in the detection or auxiliary detection of wheat root length traits;

[0040] X3) The application of the substances used to detect wheat molecular markers in wheat breeding;

[0041] X4) The application of the substance for detecting wheat molecular markers in the preparation of wheat breeding products;

[0042] X5) The application of the substances used to detect wheat molecular markers in the preparation of products for detecting or assisting in the detection of wheat root length traits;

[0043] The application of the method for detecting or assisting in the detection of wheat root length described in X6) in wheat breeding;

[0044] X7) The application of detecting substances in the wheat genome corresponding to nucleotides 1024, 1067, 1453, 1801, 1818 and / or 2792 of SEQ ID No. 1 in the sequence listing in the selection of wheat with excellent root length traits;

[0045] X8) The application of detecting substances in the wheat genome corresponding to nucleotides 1024, 1067, 1453, 1801, 1818 and / or 2792 of SEQ ID No. 1 in the sequence listing in the preparation and breeding of wheat products with excellent root length traits.

[0046] This invention also provides a method for wheat breeding, the method comprising I, II, III, IV or V:

[0047] I. Detect nucleotides at positions 1024, 1067, 1453, 1801, 1818, and 2792 of the wheat genome corresponding to SEQ ID No. 1 in the sequence listing, and select wheat varieties whose nucleotides at positions 1024, 1067, 1453, 1801, 1818, and 2792 of the wheat genome are A, C, A, C, C, and G, respectively, as parents for breeding;

[0048] II. Detect the nucleotide at position 1067 of SEQ ID No. 1 in the wheat genome, and select wheat with nucleotide C at position 1067 of SEQ ID No. 1 in the wheat genome as the parent for breeding;

[0049] III. Detect the nucleotide at position 1453 of the wheat genome corresponding to SEQ ID No. 1 in the sequence listing, and select wheat with nucleotide A at position 1453 of the wheat genome corresponding to SEQ ID No. 1 in the sequence listing as the parent for breeding;

[0050] IV. Detect the nucleotide at position 1818 of SEQ ID No. 1 in the wheat genome, and select wheat with nucleotide C at position 1818 of SEQ ID No. 1 in the wheat genome as the parent for breeding;

[0051] V. Detect the nucleotide at position 2792 of the wheat genome corresponding to SEQ ID No. 1 in the sequence listing, and select wheat whose nucleotide at position 2792 of the wheat genome corresponding to SEQ ID No. 1 in the sequence listing is G as the parent for breeding.

[0052] In this invention, the wheat can be any of the 32 wheat varieties and any wheat material in Table 2 or its offspring. The 32 wheat varieties are PANDAS, An85zhong124-1, Yanzhan 1, Bawangbian, Beijing 10, Beijing 14, Cangzhou Wheat, Changwu 131, Chang 6878, Dali 1, DanR8093, Fengkang 13, Jimai 41, Jimai 6, Jin 2148-7, Jinghe 8922, Linkang 5108, Baiqi Wheat, Changle 5, Hongheshang, Beijing 8686, 04-044, 04-030, Chun 22 9th-25, Zigan Baimangxian, Jingpin 10, Chun 04 9th-5-1, Chun 45 9th-50-1, Neixiang 188, Jing 411, China Spring, and White Rough Wheat.

[0053] This invention, through genetic variation analysis of the TaHT1-5B gene in a natural wheat population, identified six SNPs associated with wheat root length, located at positions 1024, 1067, 1453, 1801, 1818, and 2792 of SEQ ID No. 1. These six SNPs exhibit three haplotypes: haplotype Hap-5B-1 (SNPs A, G, G, C, T, C), haplotype Hap-5B-2 (SNPs G, G, G, T, T, C), and haplotype Hap-5B-3 (SNPs A, C, A, C, C, G). All three haplotypes of these six SNPs are linked. Association analysis and natural population validation confirmed that, among the homozygous types of these three haplotypes, the maximum root length at the seedling stage of Hap-5B-3 is significantly longer than that of Hap-5B-1 and Hap-5B-2. Nucleotide 1067 in the genomic DNA, corresponding to sequence 1 in the sequence listing, is also associated with wheat root length. Wheat seedlings with nucleotide C at position 1067 have significantly longer maximum roots than wheat with nucleotide G at that position. Experiments have demonstrated that by detecting the wheat haplotype of this invention and the nucleotide corresponding to position 1067 in the genomic DNA of sequence 1 in the sequence listing, wheat varieties with superior root traits can be quickly and accurately identified. This invention provides a novel method for marker-assisted selection breeding of wheat and has significant implications for the cultivation of stress-resistant wheat varieties or for related research.

[0054] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way. Attached Figure Description

[0055] Figure 1 This diagram illustrates the SNPs in the TaHT1-5B genome sequence and shows partial detection results for one marker in this invention. a is a schematic diagram of the TaHT1-5B gene structure; b is an electrophoretic detection result of partial enzyme digestion product B; c is an electrophoretic detection result of partial enzyme digestion product C.

[0056] Figure 2 The statistical results of the maximum root length of the three haplotypes of the TaHT1-5B gene in the natural population 1 of wheat natural variant population are presented. Detailed Implementation

[0057] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials, reagents, instruments, etc., used in the following embodiments are commercially available. All quantitative experiments in the following embodiments were performed in at least three replicates, and the results were averaged.

[0058] Example 1: Obtaining the polymorphic sites and haplotypes of the root-related gene TaHT1-5B

[0059] I. Obtaining TaHT1-5B gene polymorphic sites and haplotypes

[0060] 1. Acquisition of TaHT1-5B gene polymorphic sites

[0061] (1) Based on the characteristics of the wheat TaHT1-5B genomic DNA sequence, specific primers for its genome were designed. The primer sequences are as follows:

[0062] TaHT1-5B-F (forward primer): 5′-CTCCTCCGCGGTTAACTCG-3′ (SEQ ID No. 4 in the sequence listing);

[0063] TaHT1-5B-R (reverse primer): 5′-TTCCTCCCAGAATCGAAACG-3′ (SEQ ID No. 5 in the sequence listing).

[0064] The recognition sequences of TaHT1-5B-F and TaHT1-5B-R are located upstream and downstream of the TaHT1-5B gene, respectively.

[0065] (2) Using the genomic DNA of 32 wheat materials (all from the National Germplasm Resource Bank) as templates, PCR amplification was performed using the primers in step (1) to obtain PCR amplification products. Then, the obtained PCR amplification products were sequenced and sequence aligned.

[0066] The PCR amplification system used (20 μL) consisted of: 11.0 μL ddH2O, 4.0 μL 5×PCR buffer, 1 μL each of forward primer (5 μmol / L) and reverse primer (5 μmol / L), 1.6 μL dNTPs (2.5 mmol / L), 0.4 μL transfastpfu enzyme (5 U), and 1 μL template DNA (20 ng / μL). Both the 5×PCR buffer and transfastpfu enzyme (5 U) were products of Beijing TransGen Biotech Co., Ltd.

[0067] The PCR amplification conditions were: 95℃ for 5 min; 95℃ for 30 s, 60℃ for 30 s, 72℃ for 4 min, for 35 cycles; 72℃ for 10 min, and stored at 4℃.

[0068] Sequence analysis revealed that the obtained PCR product contained TaHT1-5B genomic DNA as shown in SEQ ID No. 1, and that the TaHT1-5B genomic DNA (sequence of SEQ ID No. 1) contained 6 SNP sites. Figure 1 The positions are: position 1024 (G and A polymorphisms), position 1067 (G and C polymorphisms), position 1453 (G and A polymorphisms), position 1801 (T and C polymorphisms), position 1818 (T and C polymorphisms), and position 2792 (G and C polymorphisms) of the TaHT1-5B gene.

[0069] 2. Obtaining the TaHT1-5B genotype

[0070] Sequence analysis revealed that the six SNP sites of the TaHT1-5B gene have three haplotypes in the natural wheat variant population, which were named haplotype Hap-5B-1, haplotype Hap-5B-2, and haplotype Hap-5B-3, respectively; the nucleotides at positions 1024, 1067, 1453, 1801, 1818, and 2792 of each haplotype are shown in Table 1.

[0071] Table 1. Nucleotides of TaHT1-5B gene haplotypes at each SNP site

[0072]

[0073] In these 32 wheat materials, linkage was also found in the nucleotides of all 6 SNP sites of the three haplotypes in Table 1.

[0074] The names of these 32 wheat materials are as follows: PANDAS, An85zhong124-1, Yanzhan No.1, Bawangbian, Beijing No.10, Beijing No.14, Cangzhou Wheat, Changwu 131, Chang 6878, Dali No.1, DanR8093, Fengkang 13, Jimai 41, Jimai No.6, Jin 2148-7, Jinghe 8922, Linkang 5108, Baiqi Wheat, Changle No.5, Hongheshang, Beijing 8686, 04-044, 04-030, Chun 229th-25, Zigan Baimangxian, Jingpin No.10, Chun 04 9th-5-1, Chun 45 9th-50-1, Neixiang 188, Jing 411, China Spring, White Rough Wheat.

[0075] II. Acquisition of Molecular Markers and Their Application in Haplotype Identification

[0076] 1. Obtaining molecular markers

[0077] The SNP sites at positions 1024bp and 1067bp of the TaHT1-5B gene were designated as TaHT1-5B-1024-CAPS and TaHT1-5B-1067-CAPS markers, respectively, and primers capable of detecting each marker were designed. First, gene DNA fragments detecting the TaHT1-5B-1024-CAPS and TaHT1-5B-1067-CAPS markers were obtained by PCR amplification. The target DNA fragments were then amplified using a pair of PCR primers, the sequences of which are as follows:

[0078] TaHT1-5B-CAPS-F (forward primer): 5′-GGCTGAAACCAATACACACTTAATAC-3′ (SEQ ID No. 2);

[0079] TaHT1-5B-CAPS-R (reverse primer): 5′-AGGCTGCTACCTTCACTTCGA-3′ (SEQ ID No. 3).

[0080] 2. Identification of wheat haplotypes

[0081] (1) When the wheat to be tested is a wheat with a homozygous TaHT1-5B gene, the steps for identifying the haplotype of the wheat to be tested using the molecular markers and primers in step 1 are as follows:

[0082] Using wheat genomic DNA as a template, PCR amplification was performed using primers TaHT1-5B-CAPS-F and TaHT1-5B-CAPS-R from step 1, yielding PCR amplification product A. The nucleotide sequence of PCR amplification product A corresponds to positions 758 to 1149 of the TaHT1-5B gene.

[0083] The PCR amplification system (10 μL) consisted of: 5.0 μL Taq Mix, 3.6 μL ddH2O, 0.2 μL each of forward primer (10 μmol / L) and reverse primer (10 μmol / L), and 1.0 μL genomic DNA (200 ng / μL). Taq Mix was a product of Beijing Zhuangmeng International Biotechnology Co., Ltd.

[0084] The PCR amplification conditions were as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, for 35 cycles; 72℃ final extension for 10 min.

[0085] (2) The PCR amplification product A obtained in step (1) was digested with restriction endonucleases Hpy188 III and HhaI, respectively, to obtain digested products B and C. Electrophoresis results of some digested product B are shown below. Figure 1 In step b, if the enzyme digestion product B consists of two DNA fragments of 263 bp and 129 bp, it indicates that the 1024th nucleotide of the wheat TaHT1-5B gene being tested is A, i.e., A homozygous; if the enzyme digestion product B consists of one DNA fragment of 392 bp, it indicates that the 1024th nucleotide of the wheat TaHT1-5B gene being tested is G, i.e., G homozygous; if the enzyme digestion product B consists of three DNA fragments of 263 bp, 129 bp, and 392 bp, it indicates that the 1024th nucleotide of the wheat TaHT1-5B gene being tested is A and G, i.e., AG heterozygous. The electrophoretic detection results of some enzyme digestion products C are shown below. Figure 1 If the enzyme digestion product C consists of two DNA fragments of 312 bp and 80 bp, it indicates that the 1067th nucleotide of the wheat TaHT1-5B gene being tested is G, i.e., G homozygous; if the enzyme digestion product C consists of one DNA fragment of 392 bp, it indicates that the 1067th nucleotide of the wheat TaHT1-5B gene being tested is C, i.e., C homozygous; if the enzyme digestion product C consists of three DNA fragments of 312 bp, 80 bp, and 392 bp, it indicates that the 1067th nucleotide of the wheat TaHT1-5B gene being tested is G and C, i.e., GC heterozygous.

[0086] Therefore, the following method can be used to genotype the wheat to be tested and determine whether the haplotype of the wheat is Hap-5B-1, Hap-5B-2, or Hap-5B-3:

[0087] 1) Extract genomic DNA from the wheat to be tested;

[0088] 2) Using the genomic DNA from step 1) as a template, PCR amplification was performed using primers TaHT1-5B-CAPS-F / R to obtain PCR amplification product A.

[0089] 3) Digest the PCR amplification product A obtained in step 2) with restriction endonucleases Hpy188 III and HhaI respectively to obtain digested products B and C;

[0090] If enzyme digestion product B contains DNA fragments of 263bp and 129bp in size (which can be determined by electrophoresis and sequencing) and does not contain a DNA fragment of 392bp, and enzyme digestion product C contains DNA fragments of 312bp and 80bp in size and does not contain a DNA fragment of 392bp, then the wheat to be tested is a homozygous wheat of the Hap-5B-1 haplotype.

[0091] If enzyme digestion product B contains a DNA fragment of 392 bp and does not contain DNA fragments of 263 bp and 129 bp, and enzyme digestion product C contains DNA fragments of 312 bp and 80 bp and does not contain a DNA fragment of 392 bp, then the wheat to be tested is a homozygous wheat of the Hap-5B-2 haplotype.

[0092] If enzyme digestion product B contains DNA fragments of 263bp and 129bp in size but not a DNA fragment of 392bp in size, and enzyme digestion product C contains a DNA fragment of 392bp in size but not a DNA fragment of 312bp and 80bp in size, then the wheat to be tested is a homozygous wheat of the Hap-5B-3 haplotype.

[0093] If enzyme digestion product B contains DNA fragments of 263bp, 129bp, and 392bp in size, and enzyme digestion product C contains DNA fragments of 312bp and 80bp in size but does not contain a DNA fragment of 392bp in size, then the wheat to be tested is a heterozygous wheat of haplotypes Hap-5B-1 and Hap-5B-2.

[0094] If enzyme digestion product B contains DNA fragments of 263bp and 129bp in size but not a DNA fragment of 392bp in size, and enzyme digestion product C contains DNA fragments of 312bp, 80bp, and 392bp in size, then the wheat to be tested is a heterozygous wheat of haplotypes Hap-5B-1 and Hap-5B-3.

[0095] If enzyme digestion product B contains DNA fragments of 263bp, 129bp, and 392bp in size, and enzyme digestion product C contains DNA fragments of 312bp, 80bp, and 392bp in size, then the wheat to be tested is a heterozygous wheat of haplotypes Hap-5B-2 and Hap-5B-3.

[0096] Example 2: Association analysis between wheat haplotypes and root length traits

[0097] I. Genotyping of Natural Population 1 and its Association with Root Length Trait

[0098] Genotyping of natural population 1 (Table 2) was performed using the TaHT1-5B-1024-CAPS and TaHT1-5B-1067-CAPS markers from Example 1, and association analysis between haplotypes and root length traits was conducted. The specific steps are as follows:

[0099] 1. Haplotype detection

[0100] The wheat varieties in natural population 1, consisting of 323 hexaploid wheat accessions, were used as test wheat. The haplotypes of the wheat were identified according to step 2 of Example 1. All wheat varieties in natural population 1 were obtained from the National Germplasm Resource Bank.

[0101] The haplotype detection results are shown in Table 2. All wheat varieties were homozygous. Then, the genomic DNA of each wheat variety was amplified using the primer pairs TaHT1-5B-F and TaHT1-5B-R from Example 1. The amplification system and conditions were the same as in Example 1. The results showed that the sequences of the PCR products of each wheat variety, except for the six SNP sites (positions 1024, 1067, 1453, 1801, 1818, and 2792 of SEQ ID No. 1), were all SEQ ID No. 1. All six SNP sites satisfied the linkage relationships of the three haplotypes shown in Table 1, indicating that the TaHT1-5B-1024-CAPS and TaHT1-5B-1067-CAPS markers of this invention can be used to detect haplotypes Hap-5B-1, Hap-5B-2, and Hap-5B-3 wheat.

[0102] Table 2. Haplotype statistics of various wheat materials in natural population 1

[0103]

[0104]

[0105]

[0106]

[0107]

[0108] Here, "data miss" indicates missing data.

[0109] 2. Correlation analysis between haplotype and root length trait

[0110] Seedling root length traits were studied using seed germination bags as growth containers. Seeds from natural population 1 were placed on top of the seed germination bags to absorb water and germinate. The maximum root length was measured on day 8.

[0111] The p-value of a marker (TaHT1-5B-1067-CAPS, i.e., the nucleotide at position 1067 of the TaHT1-5B gene) formed in natural population 1 and the root-related trait (maximum root length at seedling stage) was 0.0049, which is less than 0.05. This indicates that TaHT1-5B-1067-CAPS is significantly associated with the number of wheat seedling roots. The maximum root length of seedlings of wheat with position C at position 1067 of the TaHT1-5B gene (i.e., wheat homozygous for C at this position) was significantly longer than that of wheat with position G at this position (i.e., wheat homozygous for G at this position). This suggests that this marker can be used to breed wheat varieties with longer maximum root length at the seedling stage.

[0112] The statistical results of root length traits of three haplotype homozygous wheat species formed in natural population 1 are as follows: The average maximum root lengths at the seedling stage for haplotypes Hap-5B-1, Hap-5B-2, and Hap-5B-3 were 13.36 cm, 13.23 cm, and 14.31 cm, respectively. The maximum root length at the seedling stage for haplotype Hap-5B-3 was significantly longer than that for haplotypes Hap-5B-1 and Hap-5B-2. Figure 2 This indicates that the haplotype of the present invention is related to the maximum root length of wheat seedlings, and can be used to breed wheat varieties with longer maximum root lengths during the seedling stage.

[0113] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Application of substances that detect molecular markers in wheat in the detection or auxiliary detection of wheat root length traits; The wheat molecular markers are nucleotides in the wheat genome corresponding to positions 1024, 1067, 1453, 1801, 1818, and 2792 of SEQ ID No. 1 in the sequence listing. Specifically, the nucleotide corresponding to position 1024 of SEQ ID No. 1 is A or G; the nucleotide corresponding to position 1067 of SEQ ID No. 1 is G or C; the nucleotide corresponding to position 1453 of SEQ ID No. 1 is G or A; the nucleotide corresponding to position 1801 of SEQ ID No. 1 is C or T; the nucleotide corresponding to position 1818 of SEQ ID No. 1 is T or C; and the nucleotide corresponding to position 2792 of SEQ ID No. 1 is C or G.

2. The application according to claim 1, characterized in that: The substance used to detect wheat molecular markers is named TaHT1. - Primer pairs for 5B-Primer or a complete set of reagents for detecting the wheat molecular marker; The TaHT1 - 5B-Primer consists of two single-stranded DNA molecules, as shown in SEQ ID No. 4 and SEQ ID No. 5 in the sequence listing; The kit for detecting the wheat molecular marker includes a primer pair named TaHT1-5B-CAPS, which consists of two single-stranded DNA molecules as shown in SEQ ID No. 2 and SEQ ID No. 3 in the sequence listing.

3. The application according to claim 2, characterized in that: The kit also includes restriction endonucleases. Hpy 188III and / or Hha I.

4. The application according to any one of claims 1-3, characterized in that: The root length of wheat whose genome corresponds to nucleotide C at position 1067 of SEQ ID No. 1 in the sequence listing is greater than or can be greater than the root length of wheat whose genome corresponds to nucleotide G at position 1067 of SEQ ID No. 1 in the sequence listing; The root length of wheat whose genome corresponds to nucleotide A at position 1453 of SEQ ID No. 1 in the sequence listing is greater than or can be greater than the root length of wheat whose genome corresponds to nucleotide G at position 1453 of SEQ ID No. 1 in the sequence listing; The root length of wheat whose genome corresponds to nucleotide C at position 1818 of SEQ ID No. 1 in the sequence listing is greater than or can be greater than the root length of wheat whose genome corresponds to nucleotide T at position 1818 of SEQ ID No. 1 in the sequence listing; The root length of wheat whose genome corresponds to nucleotide G at position 2792 of SEQ ID No. 1 in the sequence listing is greater than or can be greater than the root length of wheat whose genome corresponds to nucleotide C at position 2792 of SEQ ID No. 1 in the sequence listing; The root length of wheat whose nucleotides at positions 1024, 1067, 1453, 1801, 1818, and 2792 of SEQ ID No. 1 in the sequence listing are A, C, A, C, C, and G, respectively, is greater than or candidate greater than the root length of wheat whose nucleotides at positions 1024, 1067, 1453, 1801, 1818, and 2792 of SEQ ID No. 1 in the sequence listing are A, G, G, C, T, and C, respectively. The root length of wheat whose nucleotides at positions 1024, 1067, 1453, 1801, 1818, and 2792 of SEQ ID No. 1 in the sequence listing are A, C, A, C, C, and G, respectively, is greater than or candidate greater than the root length of wheat whose nucleotides at positions 1024, 1067, 1453, 1801, 1818, and 2792 of SEQ ID No. 1 in the sequence listing are G, G, G, T, T, and C, respectively.

5. A method for detecting or assisting in the detection of wheat root length, comprising detecting the wheat molecular markers described in claim 1, and determining wheat root length according to the following method: The root length of wheat whose genome corresponds to nucleotide C at position 1067 of SEQ ID No. 1 in the sequence listing is greater than or can be greater than the root length of wheat whose genome corresponds to nucleotide G at position 1067 of SEQ ID No. 1 in the sequence listing; The root length of wheat whose genome corresponds to nucleotide A at position 1453 of SEQ ID No. 1 in the sequence listing is greater than or can be greater than the root length of wheat whose genome corresponds to nucleotide G at position 1453 of SEQ ID No. 1 in the sequence listing; The root length of wheat whose genome corresponds to nucleotide C at position 1818 of SEQ ID No. 1 in the sequence listing is greater than or can be greater than the root length of wheat whose genome corresponds to nucleotide T at position 1818 of SEQ ID No. 1 in the sequence listing; The root length of wheat whose genome corresponds to nucleotide G at position 2792 of SEQ ID No. 1 in the sequence listing is greater than or can be greater than the root length of wheat whose genome corresponds to nucleotide C at position 2792 of SEQ ID No. 1 in the sequence listing; The root length of wheat whose nucleotides at positions 1024, 1067, 1453, 1801, 1818, and 2792 of SEQ ID No. 1 in the sequence listing are A, C, A, C, C, and G, respectively, is greater than or candidate greater than the root length of wheat whose nucleotides at positions 1024, 1067, 1453, 1801, 1818, and 2792 of SEQ ID No. 1 in the sequence listing are A, G, G, C, T, and C, respectively. The root length of wheat whose nucleotides at positions 1024, 1067, 1453, 1801, 1818, and 2792 of SEQ ID No. 1 in the sequence listing are A, C, A, C, C, and G, respectively, is greater than or candidate greater than the root length of wheat whose nucleotides at positions 1024, 1067, 1453, 1801, 1818, and 2792 of SEQ ID No. 1 in the sequence listing are G, G, G, T, T, and C, respectively.

6. The method according to claim 5, characterized in that: The detection of the wheat molecular markers described in claim 1 is performed using any of the substances described in claims 1-3 for detecting wheat molecular markers.

7. Any of the following applications: X1) The application of wheat molecular markers as described in claim 1 in wheat root length trait breeding; X2) The application of the wheat molecular markers described in claim 1 in the detection or auxiliary detection of wheat root length traits; X3) The application of any of the substances for detecting wheat molecular markers as described in claims 1-3 in wheat root length trait breeding; X4) The application of the substance for detecting wheat molecular markers as described in any of claims 1-3 in the preparation of wheat root length trait breeding products; X5) The use of any of the substances for detecting wheat molecular markers as described in claims 1-3 in the preparation of products for detecting or assisting in the detection of wheat root length traits; X6) Application of the method according to claim 5 or 6 in wheat root length trait breeding; X7) The application of detecting substances in the wheat genome corresponding to nucleotide 1067 of SEQ ID No. 1 in the sequence listing in the selection of wheat with excellent root length traits; X8) The application of detecting substances in the wheat genome corresponding to nucleotide 1453 of SEQ ID No. 1 in the sequence listing in the selection of wheat with excellent root length traits; X9) The application of detecting substances in the wheat genome corresponding to nucleotide 1818 of SEQ ID No. 1 in the sequence listing in the selection of wheat with excellent root length traits; The application of X10) detection of substances in the wheat genome corresponding to nucleotide 2792 of SEQ ID No. 1 in the sequence listing in the selection of wheat with excellent root length traits; X11) The application of detecting substances in the wheat genome corresponding to nucleotides 1024, 1067, 1453, 1801, 1818 and 2792 of SEQ ID No. 1 in the sequence listing in the selection of wheat with excellent root length traits; X12) The application of detecting substances in the wheat genome corresponding to nucleotide 1067 of SEQ ID No. 1 in the sequence listing in the preparation and breeding of wheat products with excellent root length traits; X13) The application of detecting substances in the wheat genome corresponding to nucleotide 1453 of SEQ ID No. 1 in the sequence listing in the preparation and breeding of wheat products with excellent root length traits; X14) The application of detecting substances in the wheat genome corresponding to nucleotide 1818 of SEQ ID No. 1 in the sequence listing in the preparation and breeding of wheat products with excellent root length traits; X15) The application of detecting substances in the wheat genome corresponding to nucleotide 2792 of SEQ ID No. 1 in the sequence listing in the preparation and breeding of wheat products with excellent root length traits; The application of X16 in detecting substances in the wheat genome corresponding to nucleotides 1024, 1067, 1453, 1801, 1818 and 2792 of SEQ ID No. 1 in the sequence listing in the preparation and selection of wheat products with excellent root length traits.

8. Breeding methods for wheat root length trait, including I, II, III, IV, or V: I. Detect nucleotides at positions 1024, 1067, 1453, 1801, 1818, and 2792 of the wheat genome corresponding to SEQ ID No. 1 in the sequence listing, and select wheat varieties whose nucleotides at positions 1024, 1067, 1453, 1801, 1818, and 2792 of the wheat genome are A, C, A, C, C, and G, respectively, as parents for breeding; II. Detect the nucleotide at position 1067 of SEQ ID No. 1 in the wheat genome, and select wheat with nucleotide C at position 1067 of SEQ ID No. 1 in the wheat genome as the parent for breeding; III. Detect the nucleotide at position 1453 of the wheat genome corresponding to SEQ ID No. 1 in the sequence listing, and select wheat with nucleotide A at position 1453 of the wheat genome corresponding to SEQ ID No. 1 in the sequence listing as the parent for breeding; IV. Detect the nucleotide at position 1818 of SEQ ID No. 1 in the wheat genome, and select wheat with nucleotide C at position 1818 of SEQ ID No. 1 in the wheat genome as the parent for breeding; V. Detect the nucleotide at position 2792 of the wheat genome corresponding to SEQ ID No. 1 in the sequence listing, and select wheat whose nucleotide at position 2792 of the wheat genome corresponding to SEQ ID No. 1 in the sequence listing is G as the parent for breeding.

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