A gene for controlling spike length and spike grain number in wheat and its detection marker

Abstract

This invention discloses a gene controlling spike length and grain number per spike in wheat. This gene is located on chromosome 2B using a recombinant inbred line population and high-density SNP markers, within the 657.82–664.40 Mb region of chromosome 2B in the wheat Chinese spring reference genome. A candidate gene, named TaeEF1A, was identified based on the wheat reference genome sequence, RNA-seq, and gene function. Gene editing of TaeEF1A further validated its function in regulating spike length, spikelet number, and grain number per spike. Through gene sequencing and analysis of the gene sequence in a large number of germplasm resources, key variations affecting spike length and grain number per spike were identified in the 3'-UTR of TaeEF1A. Based on this, a detection marker was designed for this gene, which can be used to detect superior allelic variants of the TaeEF1A gene. This marker can be easily, rapidly, and with high throughput applied to marker-assisted breeding to increase spike length, spikelet number, and grain number per spike, thereby increasing yield.

Description

Technical Field

[0001] This invention belongs to the field of wheat breeding technology and relates to molecular markers for wheat spike length and grain number genes and their applications. Background Technology

[0002] Wheat is one of the staple food crops for approximately 40% of the global population. A primary goal of wheat breeding is to increase yield to meet the needs of a rapidly growing population. Wheat yield is determined by the number of spikes per unit area, the number of grains per spike, and the thousand-grain weight. Spike length is one of the most important factors in wheat spike development and production, influencing the number of spikelets and grains per spike. Studies on yield-related traits such as the number of spikelets per spike, the number of grains per spike, the harvest index, aboveground biomass, and grain yield have shown a positive correlation between spike length and cereal yield. Therefore, identifying and validating the genetic loci controlling spike length is crucial for improving wheat yield.

[0003] The rapid development of wheat genomics has facilitated the cloning of wheat genes. To date, at least eight genes controlling spike length have been cloned. However, these genes typically affect multiple traits with varying degrees of positive or negative impact, and their breeding value remains unclear. Further identification and cloning of more spike length-related genes / QTLs are needed to meet the demands of molecular design breeding. Summary of the Invention

[0004] This invention discloses a gene controlling spike length and grain number per spike in wheat. This gene was located on chromosome 2B using a recombinant inbred line population and high-density SNP markers, within the 657.82–664.40 Mb region of chromosome 2B in the wheat Chinese spring reference genome. A candidate gene, named TaeEF1A, was identified based on the wheat reference genome sequence, RNA-seq, and gene function. Gene editing of TaeEF1A further validated its function in regulating spike length, spikelet number, and grain number per spike. Key variations affecting spike length and grain number per spike were identified in the 3'-UTR of TaeEF1A, and a detection marker was designed based on this. This marker can be used to detect superior allelic variants of the TaeEF1A gene and can be easily, rapidly, and with high throughput applied to marker-assisted breeding to increase spike length, spikelet number, and grain number per spike, thereby increasing yield.

[0005] Phenotypic identification was performed on the recombinant inbred line (RIL) population of Shannong 4155 × Shimai 12. Furthermore, single-plant DNA was extracted from each line in the RIL population constructed using Shannong 4155 / Shimai 12 as parents. After quality testing of the single-plant DNA, wheat 55K microarray genotyping was performed to construct a genetic map. Combined with the three-year spike length phenotype, eight QTLs controlling spike length were identified. One major-effect stable QTL controlling wheat spike length was located in the interval of 657.82–664.40 Mb on chromosome 2B.

[0006] Furthermore, RNA-seq analysis revealed that 34 out of 47 genes in the localization region had expression levels below 5 or were not expressed. Of the remaining 13 genes, only two showed differential expression. Based on gene function and parental sequence analysis, TraesCS2B02G465300 was identified as a candidate gene and named TaeEF1A. The nucleotide sequence of TraesCS2B02G465300 is shown in SEQ ID NO.1.

[0007] Furthermore, a binary editing vector containing TaeEF1A and its homologous gene TraesCS2D02G443300 was constructed. By transforming wheat receptor JW1 with Agrobacterium, two homozygous double mutants were identified. Phenotypic identification showed that the mutant ears were shorter, further proving that TaeEF1A is a key gene controlling wheat ear length.

[0008] Furthermore, based on the InDel present in the 3'UTR of TaeEF1A in Shannong 4155 (a wheat variety with long spikes) and Shimai 12 (a wheat variety with short spikes), a molecular marker was designed to detect the superior haplotype of TaeEF1A. The nucleotide sequence of the InDel is as shown in SEQ ID NO.4.

[0009] The material is characterized by using the InDel5830 molecular marker, the nucleotide sequence of which is shown in SEQ ID NO.2 and SEQ ID NO.3. If the InDel5830 molecular marker amplification product band is 681 bp, then TaeEF1A in this material is consistent with Shannong 4155, representing Type I, characterized by increased spike length and number of grains per spike. If the InDel5830 molecular marker amplification product band is 979 bp, then TaeEF1A in this material is consistent with Shimai 12, representing Type II, characterized by decreased spike length and number of grains per spike.

[0010] This invention provides a method for detecting superior haplotypes of wheat TaeEF1A, comprising the following steps:

[0011] (1) DNA extraction: DNA was extracted from the wheat samples to be tested using the CTAB method.

[0012] (2) PCR amplification: Using the genomic DNA as a template, PCR amplification was performed using the molecular markers.

[0013] InDel5830 molecular marker reaction system:

[0014]

[0015] (3) Electrophoretic detection of PCR amplification products: If the band of the InDel5830 molecular marker amplification product is 681bp, then TaeEF1A in this material is of type I with increased ear length and increased number of grains. If the band of the InDel5830 molecular marker amplification product is 979bp, then TaeEF1A is of type II with decreased ear length and decreased number of grains.

[0016] The technical solution adopted in this invention is:

[0017] This invention provides a gene TraesCS2B02G465300 that controls the length of wheat spikelets, number of spikelets, and number of grains per spike. The gene is located on chromosome 2B and its nucleotide sequence is shown in SEQ ID NO.1. The gene is named TaeEF1A. This gene has application value in regulating wheat spike development and increasing wheat yield.

[0018] Preferably, the nucleotide sequence of the gene TaeEF1A is shown in SEQ ID NO.1. The protein encoded by this sequence has a conserved domain of eukaryotic translation elongation factor 1A and regulates the expression of wheat ear development-related genes by interacting with specific transcription factors.

[0019] Preferably, the TaeEF1A gene in wheat is modified using gene editing technology to obtain wheat varieties with increased spike length, spikelet number, and grain number per spike. The gene editing technology includes, but is not limited to, the CRISPR / Cas9 system.

[0020] This invention provides a molecular marker, InDel5830, for detecting superior allelic variations in the wheat TaeEF1A gene. The nucleotide sequence of the molecular marker InDel5830 is shown in SEQ ID NO.2 and SEQ ID NO.3. This molecular marker is located in the 3'-UTR region of the TaeEF1A gene and is closely linked to key variations affecting spike length and grain number per spike. It can be used to accurately distinguish different allelic variation types of the TaeEF1A gene, and its detection method has high accuracy and repeatability.

[0021] Preferably, the detection method for the molecular marker InDel5830 includes the following steps:

[0022] (1) DNA was extracted from the wheat samples to be tested using the CTAB method;

[0023] (2) Using the genomic DNA as a template, PCR amplification was performed using molecular marker primers as shown in SEQ ID NO.2 and SEQ ID NO.3;

[0024] (3) Electrophoresis detection of PCR amplification products: if the amplification product band is 681bp, then TaeEF1A in this material is of type I with increased spike length and increased number of grains per spike; if the amplification product band is 979bp, then TaeEF1A is of type II with decreased spike length and decreased number of grains per spike. This detection method can accurately identify the allelic variation type of TaeEF1A gene in wheat varieties and / or lines, providing an effective molecular-assisted selection method for wheat breeding.

[0025] Preferably, the primers are designed based on specific sequence variations in the 3'-UTR region of the TaeEF1A gene, enabling the primers to specifically amplify allele fragments related to ear length and ear grain number.

[0026] Beneficial effects:

[0027] Based on RIL population QTL mapping, this invention has discovered a new and stable QTL that is significantly associated with wheat spike length and cloned the gene. It has also developed the molecular marker InDel5830, which can be widely used for marker-assisted breeding of wheat spike length and grain number to accelerate the breeding process.

[0028] By applying the molecular marker InDel5830, developed in this invention and associated with wheat spike length and grain number, the genotype of the tested wheat variety (line) TaeEF1A can be determined, namely Type I (no 298bp insertion in the 3'UTR) and Type II (298bp insertion in the 3'UTR). Haplotype analysis of 428 wheat varieties / lines showed that, based on the genotypes of the tested wheat, wheat plants with the superior Type I genotype had longer spikes and higher grain numbers at maturity than those with the Type II genotype. This finding helps in screening wheat varieties with longer spikes and higher grain numbers, providing a theoretical basis for breeding large-spike, high-yielding wheat varieties and offering a tool for molecular-assisted selection.

[0029] Conventional PCR based on InDel sequences can achieve high-precision genotyping, while also possessing the advantages of high yield, low cost, high accuracy, and strong genetic stability. Therefore, conventional PCR based on specific InDel sequences can be used to breed wheat varieties with large ears and high yields, which is of great significance for high-yield wheat breeding. Attached Figure Description

[0030] Figure 1 The diagram shows the spike length phenotypes of the Shannong 4155, Shimai 12, and RIL populations. SL20 refers to 2020-2021; SL21 refers to 2021-2022; and SL22 refers to 2022-2023.

[0031] Figure 2 These are the QTL mapping results for spike length in the RIL population. SL20 refers to 2020-2021; SL21 refers to 2021-2022; and SL22 refers to 2022-2023.

[0032] Figure 3 This is the identification result of the qSL2B candidate gene. Figure A shows the qSL2B location intervals: SL20 refers to 2020-2021; SL21 refers to 2021-2022; and SL22 refers to 2022-2023. Figure B shows the nucleotide sequence analysis of TraesCS2B02G465300 in Shannong 4155 and Shimai 12.

[0033] Figure 4 These are the phenotypic identification results of homozygous gene-edited mutants. Figure A shows the mutation status of the TaeEF1A gene-edited mutant; Figure B shows the spike phenotype of the gene-edited mutant and the wild type, where WT refers to the wheat variety JW1; Figure C shows the statistical analysis of spike length, spikelet number, and grain number per spike of the gene-edited mutant and the wild type.

[0034] Figure 5 This figure shows the distribution of TaeEF1A haplotypes in wheat varieties / lines identified by the molecular marker InDel5830. Figure A shows the partial genotyping results of the molecular marker InDel5830 in 428 wheat varieties / lines; Figure B compares the spike length, spikelet number, and grain number per spike of TaeEF1A Type I and Type II haplotypes in 428 wheat varieties / lines. Detailed Implementation

[0035] The specific embodiments of the present invention will be described below with reference to the accompanying drawings and examples. However, the following examples are only used to illustrate the present invention in detail and do not limit the scope of the present invention in any way. Unless otherwise specified, the instruments and equipment involved in the following examples are conventional instruments and equipment; unless otherwise specified, the raw materials involved are commercially available conventional raw materials; unless otherwise specified, the detection methods involved are conventional methods.

[0036] Example 1. Positioning of QTL for controlling wheat spike length

[0037] 1. Experimental materials

[0038] The experimental materials were wheat varieties Shannong 4155 (a long-spike wheat variety) and Shimai 12 (a short-spike wheat variety) and their hybrids to construct a high-generation recombinant inbred line (RIL) population.

[0039] 2. Test methods

[0040] 2.1 Measurement of wheat ear length

[0041] The spike length trait of wheat was measured during the grain-filling stage of wheat. The length from the base to the top of the wheat spike (excluding the awn length) is the spike length. The spike length was measured in the RIL population of Shannong 4155 / Shimai 12.

[0042] Planting methods for the RIL population: Phenotypic identification was conducted in three experimental environments over three years. The RIL population was planted in Tai'an, Shandong Province in 2020, 2021, and 2022. Each line was planted in one row, with each row 1.5m apart and a row spacing of 25cm. 25 seeds were sown per row. At maturity, 10 main panicles were randomly selected from the middle of each line to investigate the panicle length, and the average panicle length was calculated.

[0043] 2.2 Fabrication and Processing of 55K SNP Chips

[0044] DNA was extracted from each line of the RIL population using the CTAB method and submitted to Beijing Compson Biotechnology Co., Ltd. Genotyping was performed using a high-density 55K SNP chip to obtain genotype data.

[0045] 2.3 Linkage Graph Construction

[0046] IciMapping V4.2 (http: / / www.isbreeding.net) software was used to remove SNPs that were not polymorphic between the two parents and redundant markers to obtain Bin markers. A genetic linkage map of the RIL population was constructed using the Bin markers.

[0047] 2.4 QTL Positioning

[0048] QTL mapping was performed using the obtained RIL population genotype and phenotypic data with IciMapping 4.2 software, and the LOD was > 2.5.

[0049] 3. Test Results

[0050] 3.1 Phenotypic Statistical Analysis of the RIL Population

[0051] The spike length of Shannong 4155 was significantly longer than that of Shimai 12. For three consecutive years, the spike length of Shannong 4155 was 11.0–11.4 cm, while that of Shimai 12 was 8.7–9.0 cm. Figure 1 A). The spikelet length varied considerably within the RIL population, ranging from 8.8–12.1 cm in 2020, 7.9–12.7 cm in 2021, and 8.1–13.1 cm in 2022. Figure 1 B). The correlation coefficients of ear length across different environments ranged from 0.55 to 0.69, with a heritability of 76%, indicating that ear length is highly heritable. Continuous distribution and cross-boundary segregation of ear length were observed in the RIL population, suggesting that ear length is a quantitative trait.

[0052] 3.2 QTL localization of spike length based on wheat 55K-SNP chip

[0053] A total of 14,691 polymorphic SNPs were identified between the two parents. After removing SNPs with significant segregation distortion, 12,951 SNPs were retained. A total of 12,263 SNPs showed reliable physical locations in the Chinese Spring reference genome V1.1 and were used to construct linkage maps.

[0054] The eight QTLs controlling spike length are located on chromosomes 2B, 2D, 3A(2), 4A, 4B, 5B, and 7A, respectively. Figure 2 The LOD values ​​ranged from 2.60 to 13.14, and the phenotypic variation (PVE) ranged from 3.5% to 20.0%. Notably, except for qSL4A, Shannong 4155 contributed positive alleles for seven QTLs (increasing spike length). qSL2B, qSL2D, qSL3A.2, and qSL5B were detected in at least two of the three years, explaining 3.61-19.96% of the phenotypic variation, and were considered environmentally stable QTLs. Among them, qSL2B was detected in all three years and explained 9.92-12.71% of the phenotypic variation, and was considered a major-effect stable QTL for spike length. The other four QTLs were detected in only one environment.

[0055] Furthermore, according to IWGSC RefSeq v1.1, qSL2B is located in the 6.58Mb interval ( Figure 3 A) is flanked by markers AX.110391939 and AX.110990121 on chromosome 2B, which contains 47 annotated genes.

[0056] Example 2. Identification of candidate genes controlling spikelet length

[0057] 1. Experimental materials: Shannong 4155 (long spike), Shimai 12 (short spike)

[0058] 2. Test methods

[0059] 2.1 RNA-seq analysis

[0060] Young spikelets of Shannong 4155 and Shimai 12 at the pistil and stamen differentiation stage were selected from the field for RNA-seq experiments. Collected samples were immediately placed in liquid nitrogen for preservation and then stored at -80℃ for later use. RNA extraction, cDNA library preparation, and sequencing were all performed by the company.

[0061] 2.2 Filtering of transcriptome data and alignment with reference sequences

[0062] The raw data obtained after sequencing were filtered to remove adapter sequences and low-quality sequencing data, resulting in high-quality clean data. FPKM values ​​of genes were extracted from the assembled transcript annotation files, and differential gene expression analysis was performed using the DESeq software package. Genes with at least one parent having an FPKM ≥ 5.0 and a fold change ≥ 2.0 were considered differentially expressed genes (DEGs) between parents.

[0063] 2.3 Gene Sequencing Analysis

[0064] The DNA sequence of TraesCS2B02G465300 from the published Chinese spring reference genome was obtained from the WheatOmics 1.0 website (wheatomics.sdau.edu.cn). Primers were designed using Primer Premier 5, and primer synthesis and first-generation sequencing were performed at Sangon Biotech (Shanghai) Co., Ltd. Sequence alignment was performed using SnapGene software.

[0065] 3. Test Results:

[0066] 3.1 RNA-seq analysis

[0067] A total of 47 genes were annotated within qSL2B (657.82–664.40 Mb). RNA-seq analysis identified two DEGs (Table 1) with a fragment number per kilobase (FPKM) > 5 in at least one parent and a Fold Change > 2 between Shannong 4155 and Shimai 12. Among them, TraesCS2B02G465600 encodes the vacuolar cation / proton exchanger (CAX). CAX proteins play a central role in controlling cellular pH, ion homeostasis, nutrient storage, metal accumulation, and stress response; therefore, we excluded it from the candidate gene pool. We considered TraesCS2B02G465300, which encodes eukaryotic translation elongation factor 1A, as the candidate gene and named it TaeEF1A.

[0068] Table 1. DEGs identified by RNA-seq

[0069]

[0070] 3.2 TraesCS2B02G465300 Sequence Analysis

[0071] DNA was extracted from Shannong 4155 and Shimai 12, and the gene sequence of TraesCS2B02G465300 was amplified and sequenced. The results showed that Shannong 4155 was identical to the Chinese spring reference genome sequence, while Shimai 12 had 10 SNPs in the promoter and a 298bp insertion in the 3'-UTR. Figure 3 B). The nucleotide sequence of TraesCS2B02G465300 in Shannong 4155 is shown in SEQ ID NO.1, and the nucleotide sequence of TraesCS2B02G465300 in Shimai 12 is shown in SEQ ID NO.5.

[0072] Example 3: Verification of TaeEF1A gene function

[0073] 1. Experimental materials: JW1 (wild type), two gene-edited double mutants KO3 and KO24.

[0074] 2. Test methods:

[0075] 2.1 Construction of Editing Carrier

[0076] A dual-target knockout vector was constructed using CRISPR / Cas9 technology for functional validation. The vector backbone was constructed using pMETaU6.1 and pBUE411. The knockout vector carries two sgRNA target sites, and gene editing gRNA sequences were designed using the CRISPRdirect website (http: / / crispr.dbcls.jp / ), with sequences of 5'-ATGTTAAGCCCCCTGTGAAT-3' and 5'-AGATGCCAGTGAAGACTTCT-3'. The PCR primers used for knockout vector construction and mutation detection are shown in Table 2; the PCR reaction system and conditions are shown in Table 3; and the enzyme digestion and ligation reaction system and conditions are shown in Table 4.

[0077] Table 2 Primers used in this study

[0078]

[0079]

[0080] Using the intermediate vector pMETaU6.1 as a template, PCR amplification was performed with TaeEF1A-F and TaeEF1A-R, and approximately 800 bp of the target band was recovered. The DNA polymerase used in the PCR system described below was Novizan 2×Phanta Flash Master Mix.

[0081] Table 3. PCR reaction system composition and conditions (knockout vector construction)

[0082]

[0083] Table 4. Enzyme digestion and ligation reaction system and conditions (construction of knockout vector)

[0084]

[0085] The recovered PCR product was ligated with pBUE411, and the ligation product was transformed into *E. coli*. The recombinant plasmid was then transformed into DH5α competent cells, and cultured on LB (Kan) plates until colonies emerged. Single colonies were selected for colony PCR identification and sequencing using pBUE411-F and pBUE411-R primers. The TaeEF1A target sequence was successfully detected in the vector, indicating that the sgRNA expression cassette was successfully constructed and successfully assembled into the pBUE411 binary expression vector, demonstrating the successful construction of the TaeEF1A CRISPR / Cas9 gene editing vector.

[0086] 2.2 Transformation of wheat receptor JW1 using edited vector

[0087] The constructed recombinant binary expression vector pBUE411-TaeEF1A was transformed into Agrobacterium EHA105, and the recipient material, wheat variety JW1, was transformed using Agrobacterium. Wheat variety JW1 is a new germplasm with good tissue culture ability bred by the Crop Research Institute of Shandong Academy of Agricultural Sciences, and is available to the public from the Crop Research Institute of Shandong Academy of Agricultural Sciences.

[0088] 2.3 Detection of gene-edited mutants

[0089] Based on the Chinese spring reference gene, amplification and sequencing primers (TaeEF1A-2B1F, TaeEF1A-2B1R, TaeEF1A-2B2F, TaeEF1A-2B2R, TaeEF1A-2D1F, TaeEF1A-2D1R, TaeEF1A-2D2F, TaeEF1A-2D2R in Table 2) were designed on TaeEF1A and its homologous genes. The preparation system and PCR reaction system are shown in Table 5. The reaction products were sent to Sangon Biotech for sequencing.

[0090] Table 5 PCR amplification reaction system

[0091]

[0092] 3. Test Results:

[0093] Two homozygous double mutants (AAbbdd) were identified in the positive gene-edited material. Figure 4 A, 4B), and homozygous double mutants have shorter ears, fewer spikelets, and fewer grains per ear. Figure 4 B, 4C) indicate that TaeEF1A affects the development of the wheat spike and controls the spike length, number of spikelets and number of grains per spike.

[0094] Example 4. Detection and application of wheat TaeEF1A marker

[0095] 1. Test materials

[0096] A natural population consisting of 428 wheat varieties and lines

[0097] 2. Test methods

[0098] 2.1 Measurement of wheat spike length, number of spikelets, and number of grains per spike

[0099] The natural population planting method was used to conduct phenotypic identification in two environments over two years. The plants were planted in Tai'an, Shandong Province in 2022 and 2023, with each material planted in one row, 1.5m long and 25cm apart, with 25 seeds sown per row. At maturity, 10 individual plants were randomly selected from the middle of each material to investigate the spike length, number of spikelets, and number of grains per spike, and the average values ​​of spike length, number of spikelets, and number of grains per spike were calculated.

[0100] 2.2 Extraction of DNA from Natural Populations

[0101] Materials are collected from leaf parts of wheat plants during the seedling stage. Fresh, tender leaves are typically cut, and total genomic DNA of wheat is extracted using the CTAB method. The specific steps are as follows:

[0102] (1) Cut about 1cm to 1.5cm of tender wheat leaves and place them in a 2.0mL centrifuge tube containing steel balls. Then put the sample into a vacuum freeze dryer and set the temperature to -50℃. After freezing for 12 hours, place it on a drying table and dry for 12 hours. Then take out the sample and grind it on a grinding shaker until the sample is ground into powder.

[0103] (2) Add 800 μL of CTAB extraction solution to a centrifuge tube, mix the extract solution with the sample thoroughly, and incubate in a water bath at 65°C for 80 min. Gently shake the tube 3 to 4 times during the water bath to ensure complete cell lysis.

[0104] (3) After the water bath is completed, wait for the sample to cool to room temperature, add 800 μL of chloroform:isoamyl alcohol (24:1) mixture to the centrifuge tube, and gently shake for 15 min;

[0105] (4) Use a low-temperature centrifuge, set the speed to 12000 rpm and the temperature to 4℃, centrifuge for 15 min. After obvious stratification appears in the centrifuge tube, aspirate 500 μL of supernatant and transfer it to another centrifuge tube. At this time, be careful not to aspirate the lower layer of liquid in the separated layer.

[0106] (5) Add 50 μL of 3M sodium acetate (pH=5.2) and 500 μL of isopropanol to the centrifuge tube containing the supernatant. Isoamyl alcohol should be stored in a low temperature environment (-20℃) in advance. After gently shaking and mixing, if white flocculent matter is seen, it indicates that DNA is present in the extract. Immediately place the centrifuge tube in a -20℃ environment for more than 1 hour to increase DNA yield.

[0107] (6) Use a low-temperature centrifuge, set the speed to 12000 rpm and the temperature to 4℃, centrifuge for 10 min, discard the supernatant, leave the precipitate and wash it 2-3 times with 70% ethanol. The 70% ethanol should be stored in a low-temperature environment (-20℃) in advance. After washing, place it in a fume hood and let it stand and air dry until there is no obvious alcohol smell and no obvious liquid in the tube.

[0108] (7) Add 200 μL ddH2O to dissolve the precipitate and obtain the DNA solution.

[0109] 2.3 PCR amplification

[0110] The 298bp insertion in the 3'UTR of TaeEF1A in Shimai 12 was converted into the molecular marker InDel5830. DNA was extracted from a natural population consisting of 428 varieties and lines, and PCR amplification was performed using these as templates to amplify the molecular marker InDel5830 of TaeEF1A.

[0111] 2.3.1 Amplification of InDel5830

[0112] The primer pairs used to amplify InDel5830 are as follows:

[0113] InDel5830 F5'-ATACATCATGTGAAGGAAGCTG-3' (SEQ ID NO. 2);

[0114] InDel5830 R5'-CACAGGTTAATTGCCACATC-3' (SEQ ID NO. 3).

[0115] The PCR reaction system is shown in Table 6.

[0116] Table 6 PCR amplification reaction system

[0117]

[0118] 2.4 Agarose gel electrophoresis detection

[0119] 2.4.1 Preparation of agarose gel

[0120] Take 1g of agarose powder and add it to 100ml of 1×TAE electrophoresis buffer to prepare 100mL of 1% agarose gel.

[0121] 2.4.2 Electrophoresis detection

[0122] Use a pipette to inject 5 μL of sample into the gel wells and perform electrophoresis at 100V for 30 min. After electrophoresis, observe the DNA bands in a gel imaging system.

[0123] 3. Test Results:

[0124] 3.1 Variation types of wheat TaeEF1A

[0125] InDel5830 can amplify a 681bp product in Shannong 4155, and the wheat variety / strain consistent with Shannong 4155 is Type I. It can amplify a 979bp product in Shimai 12, and the wheat variety / strain consistent with Shimai 12 is Type II. Figure 5 A).

[0126] 3.2 Application of the wheat TaeEF1A detection marker in natural populations

[0127] After genotyping the natural population using InDel5830, two alleles of TaeEF1A were identified. Figure 5 A). The majority of germplasm materials are Type I (52.6%), followed by Type II (47.4%). Figure 5 A). Statistical analysis of the panicle length, number of spikelets, and number of grains per panicle for Type I and Type II materials revealed that Type I had a longer panicle length, more spikelets, and more grains per panicle. Figure 5 B) indicates that Type I is a superior haplotype. The molecular marker InDel5830 we developed can easily and quickly distinguish Type I and can be applied to molecular marker-assisted selection breeding to breed varieties with increased ear length and number of grains per ear.

Claims

1. A gene, TraesCS2B02G465300, controlling wheat spike length, spikelet number, and grain number per spike, characterized in that... The gene is located on chromosome 2B and its nucleotide sequence is shown in SEQ ID NO.

1. The gene is named TaeEF1A and has application value in regulating wheat spike development and increasing wheat yield.

2. The gene TaeEF1A according to claim 1, characterized in that, The nucleotide sequence of the gene TaeEF1A is shown in SEQ ID NO.

1. The protein encoded by this sequence has a conserved domain of eukaryotic translation elongation factor 1A and regulates the expression of wheat ear development-related genes by interacting with specific transcription factors.

3. A method for high-yield wheat breeding using the TaeEF1A gene as described in claim 1, characterized in that, The TaeEF1A gene in wheat is modified using gene editing technology to obtain wheat varieties with increased spike length, spikelet number, and grain number per spike. The gene editing technology includes, but is not limited to, the CRISPR / Cas9 system.

4. A molecular marker, InDel5830, for detecting superior allelic variations in the wheat TaeEF1A gene, characterized in that... The nucleotide sequence of the molecular marker InDel5830 is shown in SEQ ID NO.2 and SEQ ID NO.

3. This molecular marker is located in the 3'-UTR region of the TaeEF1A gene and is closely linked to key variations affecting ear length and ear grain number. It can be used to accurately distinguish different allelic variants of the TaeEF1A gene, and its detection method has high accuracy and repeatability.

5. The detection method of the molecular marker InDel5830 according to claim 4 includes the following steps: (1) DNA was extracted from the wheat samples to be tested using the CTAB method; (2) Using the genomic DNA as a template, PCR amplification was performed using molecular marker primers as shown in SEQ ID NO.2 and SEQ ID NO.3; (3) Electrophoresis detection of PCR amplification products: if the amplification product band is 681bp, then TaeEF1A in this material is of type I with increased spike length and increased number of grains per spike; if the amplification product band is 979bp, then TaeEF1A is of type II with decreased spike length and decreased number of grains per spike. This detection method can accurately identify the allelic variation type of TaeEF1A gene in wheat varieties and / or lines, providing an effective molecular-assisted selection method for wheat breeding.

6. The molecular marker InDel5830 according to claim 4, characterized in that, The primers were designed based on specific sequence variations in the 3'-UTR region of the TaeEF1A gene, enabling the primers to specifically amplify allele fragments related to spike length and number of grains per spike.

Images