Molecular marker related to thousand seed weight of wheat, detection primer and application of molecular marker

Abstract

The invention discloses a molecular marker related to thousand seed weight of wheat, a detection primer and application of the molecular marker and the detection primer, and belongs to the technical field of molecular marker-assisted breeding. The nucleotide sequence of the molecular marker TaDTX50-STS is as shown in SEQ ID NO. 1; basic groups from the 502 site to the 661 site of the sequence as shown in SEQ ID NO. 1 have insertion / deletion mutation. The InDel molecular marker related to the thousand seed weight character in a wheat genome is identified and named as TaDTX50-STS, and by detecting the polymorphism or genotype of the InDel molecular marker, the InDel molecular marker can be used for identifying or assisting in identifying the thousand seed weight character of wheat, assisting in breeding dominant varieties with high thousand seed weight of wheat and improving the wheat breeding efficiency.

Description

Technical Field

[0001] This invention relates to the field of molecular marker-assisted breeding technology, and in particular to a molecular marker, detection primer and its application related to the thousand-grain weight of wheat. Background Technology

[0002] Wheat yield is determined by three factors: spike number, number of grains per spike, and grain weight. Among these, grain weight has relatively high heritability and is the most stable yield component indicator for new variety breeding. In addition, grain weight is directly related to grain size and also has an important impact on grain appearance quality. Discovering grain weight genes is crucial for wheat variety improvement.

[0003] Currently, molecular marker-related research has been conducted on wheat thousand-grain weight, preliminarily locating some quantitative trait loci associated with thousand-grain weight and developing some associated molecular markers. However, existing technologies still have many shortcomings: First, the wheat genome is large and complex, and most of the located thousand-grain weight-related QTLs are located on large chromosomal segments with low positioning accuracy, resulting in weak association between linked molecular markers and target traits. This easily leads to false positives or false negatives in breeding applications, significantly reducing selection efficiency. Second, some developed molecular markers, such as the KASP molecular marker, have complex detection procedures and high costs, making it difficult to meet the needs of large-scale breeding material screening. Therefore, there is an urgent need to develop a molecular marker with high positioning accuracy, simple detection, low cost, wide applicability, and the ability to simultaneously improve wheat thousand-grain weight and quality, in order to overcome the limitations of existing technologies, further improve the efficiency and accuracy of wheat thousand-grain weight breeding, and provide strong technical support for breeding high-quality and high-yield new wheat varieties. Summary of the Invention

[0004] The purpose of this invention is to provide a molecular marker, detection primers, and their applications related to wheat thousand-grain weight, in order to solve the problems existing in the prior art. This invention identifies an InDel molecular marker in the wheat genome associated with the thousand-grain weight trait, named TaDTX50-STS. By detecting the polymorphism or genotype of this InDel molecular marker, it can be used to identify or assist in the identification of the wheat thousand-grain weight trait, assist in the breeding of superior wheat varieties with high thousand-grain weight, and improve wheat breeding efficiency.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a molecular marker TaDTX50-STS associated with the thousand-grain weight of wheat. The nucleotide sequence of the molecular marker TaDTX50-STS is shown in SEQ ID NO.1. There is an insertion / deletion mutation at bases 502-661 of the sequence shown in SEQ ID NO.1.

[0007] The present invention also provides a detection primer for the above-mentioned molecular marker TaDTX50-STS, which consists of an upstream primer as shown in SEQ ID NO.3 and a downstream primer as shown in SEQ ID NO.4.

[0008] The present invention also provides the application of the above-mentioned molecular marker TaDTX50-STS or the above-mentioned detection primers in the preparation of products for detecting wheat thousand-grain weight or wheat yield traits.

[0009] Optionally, the product may include a chip, a reagent, or a kit.

[0010] The present invention also provides a kit for detecting the thousand-grain weight trait or wheat yield trait of wheat, the kit containing the above-mentioned detection primers.

[0011] The present invention also provides the application of the above-mentioned molecular marker TaDTX50-STS, the above-mentioned detection primers, or the above-mentioned kit in the detection of the thousand-grain weight trait of wheat.

[0012] The present invention also provides a method for detecting the thousand-grain weight trait of wheat, comprising the following steps:

[0013] Genomic DNA was extracted from the wheat to be tested. Using the genomic DNA as a template, PCR amplification was performed using the above-mentioned detection primers. The thousand-grain weight trait of the wheat to be tested was determined based on the amplification product.

[0014] When the length of the amplification product is 1228 bp, the wheat to be tested is a wheat with a high thousand-grain weight trait; when the length of the amplification product is 1068 bp, the wheat to be tested is a wheat with a low thousand-grain weight trait.

[0015] Optionally, the PCR amplification reaction system is as follows: 1.5 μL genomic DNA, 7.5 μL 2×PCR mix, 1 μL each of upstream and downstream primers, and deionized water to a final volume of 15 μL.

[0016] Optionally, the PCR amplification reaction program is as follows: 94℃ for 5 min; 36 cycles: 94℃ for 35 s, 62℃ for 30 s, 72℃ for 40 s; 72℃ for 10 min; and storage at 16℃.

[0017] The present invention also provides the application of the above-mentioned molecular marker TaDTX50-STS, the above-mentioned detection primers or the above-mentioned kits in the auxiliary breeding of wheat lines with high thousand-grain weight or high-yield traits.

[0018] The present invention discloses the following technical effects:

[0019] This invention identifies an InDel molecular marker in the wheat genome associated with the thousand-grain weight trait, named TaDTX50-STS, whose nucleotide sequence is shown in SEQ ID NO.1. An insertion / deletion mutation exists at bases 502-661 of the sequence shown in SEQ ID NO.1. By detecting the polymorphism or genotype of this InDel molecular marker, it can be used to identify the thousand-grain weight trait in wheat, assist in the breeding of superior wheat varieties with high thousand-grain weight, improve wheat breeding efficiency, and shorten the breeding process.

[0020] This invention develops genotyping primers based on the InDel molecular marker. Compared with high-throughput genotyping technologies based on sequencing and microarrays, the operation process and equipment requirements for genotyping using this marker are relatively simple. The products are amplified by a PCR instrument, and genotyping can be achieved using an agarose gel electrophoresis device and a UV lamp. The detection cost is low, and the scale of the test samples can be flexibly adjusted, providing new genetic resources and new approaches for breeding high-yield and high-quality wheat. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 Sequence alignment diagram of the molecular marker TaDTX50-STS in the genomes of wheat varieties Doumai and Shi 4185;

[0023] Figure 2 The results are the amplification products of 154 samples of some varieties from natural populations in the Huang-Huai wheat region; M is the DNA Marker; lanes 1-5, 7-10 and 12 are 10 samples with BB genotype, and lanes 6 and 11 are 2 samples with AA genotype.

[0024] Figure 3 The results are statistical analysis of the genotype and phenotypic data of 154 natural wheat varieties in the Huang-Huai wheat region. Detailed Implementation

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0027] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0028] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0029] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0030] The 154 natural populations of wheat from the Huang-Huai region in the following examples have been documented in: Li F, Wen W, Liu J, Zhang Y, Cao S, He Z, Rasheed A, Jin H, Zhang C, Yan J, Zhang P, Wan Y, Xia X (2019) Genetic architecture of grain yield in bread wheat based on genome-wide association studies. BMC Plant Biology 19(1):168. This biological material is available to the public from the applicant and is intended solely for the replication of experiments of this invention and may not be used for any other purpose.

[0031] The wheat strains in the following examples are available to the public from the applicant. These biological materials are for the purpose of repeating the experiments of this invention and may not be used for any other purpose.

[0032] The wheat variety Shi 4185 in the following examples is a biological material that the public can obtain from the applicant. This biological material is only used to repeat the experiments of the present invention and cannot be used for other purposes.

[0033] The following examples use EXCEL statistical software to process the data. The experimental results are expressed as mean ± standard error. The two-sample equal variance hypothesis test is used, and P < 0.05 (*) indicates that there is a significant difference.

[0034] Example 1: Discovery of the TaDTX50-STS Molecular Marker

[0035] 1. Identification of allelic variations in TaDTX50-5DL

[0036] Doumai is a large-grained winter wheat variety with a thousand-grain weight of approximately 52 grams, but its pedigree is unknown. Shi 4185 is a winter wheat variety bred by the Shijiazhuang Academy of Agricultural Sciences in Hebei Province, with smaller grains and a thousand-grain weight of approximately 38 grams. Significant differences exist between Doumai and Shi 4185 in thousand-grain weight, grain length, and grain width, making them suitable for constructing recombinant inbred line populations for thousand-grain weight-related trait studies. A major thousand-grain weight-related QTL was identified using the Doumai / Shi 4185 recombinant inbred line population. The allele from Doumai increased thousand-grain weight, flag leaf length, and grain length, indicating that this allele has significant breeding value. The QTL was located using map-based cloning and gene annotation was performed. Based on genome resequencing and transcriptome analysis of the parents Doumai and Shi 4185, two candidate genes associated with thousand-grain weight were predicted. The candidate gene TaDTX50-5DL was identified through transgenic overexpression and gene editing. Using genomic DNA from wheat varieties Doumai and Shi 4185 as templates, PCR amplification and first-generation sequencing identified a 160-nucleotide insertion approximately 11 kb upstream of the start codon of the TaDTX50-5DL gene (i.e., a 160 bp insertion downstream of chromosome 412,065,374 bp on chromosome 5D in the Chinese Spring Reference Genome 1.1). Since variations in the promoter region of the TaDTX50-5DL gene are crucial for regulating gene expression, a molecular marker was developed based on this allelic variant site for genotyping. This molecular marker at the allelic variant site was named the molecular marker TaDTX50-STS.

[0037] The sequence of this molecular marker is shown in SEQ ID NO.1, with an insertion of 160 bases after the 501st base (in bold); the sequence with 160 bases missing is shown in SEQ ID NO.2.

[0038] SEQ ID NO.1:

[0039]

[0040] SEQ ID NO.2:

[0041]

[0042] Example 2: Development of a specific primer set based on the molecular marker TaDTX50-STS

[0043] Given that variations in the promoter region of the TaDTX50-5DL gene are crucial for regulating its expression, a primer set for identifying the molecular marker TaDTX50-STS was developed based on differences in sequence length.

[0044] Using genomic DNA from wheat varieties Doumai and Shi 4185 as templates, PCR amplification was performed using primer sets. The specific steps are as follows:

[0045] 1) Obtain the 1228bp sequence of the TaDTX50-5DL gene approximately 11kb upstream of the start codon from the genome resequencing data of soybean, wheat and Shi 4185, and perform sequence alignment using DNAMAN software.

[0046] 2) Based on the alignment results, determine the specific sites of the target DNA segment and design specific primers accordingly:

[0047] STS-F: 5'-CCTCTCGGCCTACATACATAGTATCC-3' (SEQ ID NO. 3);

[0048] STS-R: 5'-TATAGACACGTTCGTAATCCTGCC-3' (SEQ ID NO. 4).

[0049] 3) Establish the PCR detection system (15 μL): 1.5 μL of genomic DNA (concentration approximately 100 ng / μL) as template, 7.5 μL of 2×PCR mix (PowerPol 2×PCR Mix with Dye V2, ABclonal), 1 μL each of forward and reverse primers STS-F / STS-R (primer concentration 10 μM), and 4 μL of deionized water. PCR was performed in a 96-well plate.

[0050] 4) Establish PCR amplification program: 94℃ for 5 min; 36 cycles: 94℃ for 35 s, 62℃ for 30 s, 72℃ for 40 s; 72℃ for 10 min; store at 16℃.

[0051] 5) The size of the PCR amplification products was detected by 1.2% agarose gel electrophoresis.

[0052] PCR amplification using STS-F / STS-R yielded a 1228 bp fragment for the genomic DNA of the wheat variety Doumai, and a 1068 bp fragment for the genomic DNA of the wheat variety Shi 4185. Sequencing alignment revealed a 160 nucleotide insertion in the PCR product of the wheat variety Doumai (see...). Figure 1 The amplified fragment was consistent with the expected size, confirming the effectiveness of this molecular marker in distinguishing between high and low thousand-grain weights in wheat.

[0053] Specifically, the genotyping criteria for the wheat molecular marker TaDTX50-STS are as follows: if the PCR amplification product band size is 1068 bp, the tested wheat belongs to genotype BB, which is a germplasm resource with low thousand-grain weight; if the PCR amplification product band size is 1228 bp, the tested wheat belongs to genotype AA, which is a germplasm resource with high thousand-grain weight. The thousand-grain weight trait of genotype AA wheat is superior to that of genotype BB wheat.

[0054] This invention defines the genotype containing the molecular marker TaDTX50-STS as the insertion type (abbreviated as AA genotype). Wheat varieties with genotype AA have a high thousand-grain weight, and the nucleotide sequence of the amplification product is shown in SEQ ID NO.1. The genotype not containing the molecular marker TaDTX50-STS is defined as the deletion type (abbreviated as BB genotype). Wheat varieties with genotype BB have a low thousand-grain weight, and the nucleotide sequence of the amplification product is shown in SEQ ID NO.2.

[0055] Example 3: Application of the molecular marker TaDTX50-STS and its specific primer set in the identification of the thousand-grain weight trait in wheat.

[0056] 1. Field phenotypic identification and data analysis of 154 natural wheat populations in the Huang-Huai region

[0057] 154 natural wheat varieties from the Huang-Huai wheat region were planted in Anyang, Henan Province and Suixi, Anhui Province, using a completely randomized block design with three replicates, three-row plots, row length 1.5 meters, row width 0.2 meters, and 50 grains / row. Field management practices followed local wheat field management standards. Thousand-grain weight was measured and recorded using a Wanshen SC-G seed analyzer.

[0058] 2. Analysis of genotypes and genetic effects of wheat varieties in the Huang-Huai wheat region using the molecular marker TaDTX50-STS.

[0059] Genomic DNA was extracted from 154 wheat varieties from the Huang-Huai wheat region as templates. PCR amplification was performed using the identification primer set STS-F / STS-R for the molecular marker TaDTX50-STS, following the PCR system and procedure described in Example 2. The genotypes of the molecular marker TaDTX50-STS were then confirmed by 1.2% agarose gel electrophoresis. Agarose gel electrophoresis results for some varieties are shown below. Figure 2 As shown.

[0060] Based on genotyping results and phenotypic data, the t-test using Excel statistical software was used to determine the genetic effect of the molecular marker TaDTX50-STS on thousand-grain weight. The genetic analysis results for thousand-grain weight are shown in Table 1 and... Figure 3 As shown in Table 1 and Figure 3 This is a statistical comparison of the thousand kernel weight between the two genotypes AA and BB. * indicates a significant difference (P < 0.05).

[0061] Table 1. Relationship between TaDTX50-STS genotype and wheat thousand-grain weight phenotype.

[0062]

[0063] Note: The statistical analysis uses the two-sample equal variance hypothesis test method.

[0064] Table 1 shows that the thousand-grain weight (mean 48.10 ± 1.17 g) of the AA genotype wheat is superior to that of the BB genotype wheat (mean 44.49 ± 0.45 g). Among these, the AA homozygous TaDTX50-STS genotype wheat has a higher thousand-grain weight than the BB homozygous wheat. Since the number of AA genotype wheat lines in natural populations is relatively small, using the molecular markers of this invention to breed wheat lines with high thousand-grain weight is of great significance for increasing wheat yield, expanding the dominant population, and accelerating the breeding process.

[0065] In summary, in the AA homozygous wheat genome, there is a 160-nucleotide insertion approximately 11kb upstream of the start codon in the TaDTX50-5DL gene on both chromosomes (wheat genotype is the allelic AA genotype); this type of germplasm resource belongs to the high-quality germplasm resource with high thousand-grain weight. In the BB homozygous wheat genome, there is no 160-nucleotide insertion approximately 11kb upstream of the start codon in the TaDTX50-5DL gene on both chromosomes (wheat genotype is the allelic BB genotype); this type of germplasm resource belongs to the low-quality germplasm resource with low thousand-grain weight. When breeding high-quality germplasm resources with high thousand-grain weight, wheat with the AA genotype (TaDTX50-STS molecular marker), i.e., wheat with the AA homozygous genotype, should be preferentially selected as parents for breeding.

[0066] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A molecular marker TaDTX50-STS associated with wheat thousand-grain weight, characterized in that, The nucleotide sequence of the molecular marker TaDTX50-STS is shown in SEQ ID NO.1; the sequence shown in SEQ ID NO.1 contains insertion / deletion mutations at bases 502-661.

2. A detection primer for the molecular marker TaDTX50-STS as described in claim 1, characterized in that, It consists of an upstream primer as shown in SEQ ID NO.3 and a downstream primer as shown in SEQ ID NO.

4.

3. The application of the molecular marker TaDTX50-STS as described in claim 1 or the detection primer as described in claim 2 in the preparation of products for detecting wheat thousand-grain weight or wheat yield traits.

4. The application according to claim 3, characterized in that, The products include chips, reagents, or reagent kits.

5. A kit for detecting the thousand-grain weight trait or wheat yield trait in wheat, characterized in that, The kit contains the detection primers as described in claim 2.

6. The application of the molecular marker TaDTX50-STS as described in claim 1, the detection primer as described in claim 2, or the kit as described in claim 5 in the detection of the thousand-grain weight trait of wheat.

7. A method for detecting the thousand-grain weight trait of wheat, characterized in that, Includes the following steps: Genomic DNA was extracted from the wheat to be tested. Using the genomic DNA as a template, PCR amplification was performed using the detection primers described in claim 2. The thousand-grain weight trait of the wheat to be tested was determined based on the amplification product. When the length of the amplification product is 1228 bp, the wheat to be tested is a wheat with a high thousand-grain weight trait; when the length of the amplification product is 1068 bp, the wheat to be tested is a wheat with a low thousand-grain weight trait.

8. The method according to claim 7, characterized in that, The PCR amplification reaction system consisted of: 1.5 μL genomic DNA, 7.5 μL 2×PCR mix, 1 μL each of forward and reverse primers, and deionized water to a final volume of 15 μL.

9. The method according to claim 7, characterized in that, The PCR amplification reaction program was as follows: 94℃ for 5 min; 36 cycles: 94℃ for 35 s, 62℃ for 30 s, 72℃ for 40 s; 72℃ for 10 min; and storage at 16℃.

10. The application of the molecular marker TaDTX50-STS of claim 1, the detection primer of claim 2, or the kit of claim 5 in the assisted breeding of wheat lines with high thousand-grain weight or high-yield traits.

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