Wheat grain development gene TaEND1 and KASP molecular marker and application thereof

Abstract

The invention relates to molecular markers of wheat grain development genes TaEND1 and KASP and application of the molecular markers. According to the invention, a new gene TaEND1 related to wheat grain development is excavated, and time and cost are saved in breeding and breeding by developing molecular markers. The KASP molecular marker is developed for the wheat grain development defect gene TaEND1, so that the genotyping efficiency, accuracy and economical efficiency are improved, and the KASP molecular marker has remarkable advantages in operation simplicity and convenience and result acquisition speed. Compared with the prior art, the KASP technology is more suitable for large-scale screening and marker-assisted selective breeding, and is helpful for accelerating the research progress of wheat grain development genes and the development of new varieties. Due to the beneficial effects, the KASP technology becomes an indispensable tool in modern wheat breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of crop breeding, and relates to wheat grain development gene TaEND1, KASP molecular markers and applications thereof. Background Art

[0002] Common wheat (Triticum aestivum L.) is one of the most important food crops in the world. It is an important source of carbohydrates and proteins, and also an important source of trace nutrients. Wheat is used to make various staple foods such as bread, noodles, and biscuits, meeting the energy needs of billions of people in the world. With the increase in population and the improvement of living standards, the improvement of wheat yield and quality is becoming increasingly important for improving the national economy and life. Wheat grain development is a key process that affects the final yield and quality. Studying genes related to wheat grain development can help scientists better understand the biological mechanisms of grain size, morphology, and maturity, which provides a theoretical basis for precision breeding. By controlling genes related to grain development, the grain size, weight, and internal composition of wheat can be regulated, thereby optimizing wheat yield and quality to meet different market and processing needs. Modern molecular biology techniques can more effectively mark and select excellent genes related to grain development. This precise breeding method not only accelerates the cultivation of new varieties, but also improves breeding efficiency. By studying the regulation of genes during grain development, the content of starch, protein, and trace elements in grains can be optimized, the nutritional value of wheat can be improved, and global nutritional security can be promoted.

[0003] During the development of wheat endosperm, key genes such as TaSUS1, TaSUS2, TaAGP-S1-7A, and TaAGP-L-B1 and their encoded enzymes work together to regulate endosperm development and starch synthesis. Starch is the main storage material of the endosperm. During its synthesis, sucrose is converted into monosaccharides by sucrose synthase, and then catalyzed into starch by starch synthase (Hou et al., 2014; Jiang et al., 2011). The glucose pyrophosphorylase gene plays a core role in the process of converting glucose-1-phosphate to ADP-glucose, directly improving the efficiency of starch synthesis (Hou et al., 2017; Kang et al., 2013; Rose et al., 2016). Material transport is also important in starch synthesis, such as TaBT1 (ADP-glucose transporter) involved in sugar transport. Starch synthase (TaSS4) and starch branching enzyme (TaSBEIIa) determine starch structure and properties (Wang et al., 2019; Hawkins et al., 2021; Li et al., 2021). Overexpression of TubZIP28 and the role of TaTPP-7A, as well as the activities of starch debranching enzyme (DBE) and isoamylase (ISA), jointly regulate the rate and accumulation of starch synthesis (Burton et al., 2002; Satoh et al., 2008; Cao et al., 2022; Liu et al., 2023). In addition, genes such as TaGASR7-A1, TaSAP1-A1, and 1-FEH w3 refine the regulation of grain development and starch accumulation through hormone signaling, gene expression regulation, and participation in specific metabolic pathways (Chang et al., 2013; Dong et al., 2014; Zhang et al., 2015). Glutamine synthetase (TaGS2, TaGS1a) is involved in nitrogen assimilation, cytokinin oxidase (TaCKX6-D1, TaCKX6a02) and cell wall invertase (TaCwi-A1, TaCWI-5D) regulate hormone levels and cell wall reconstruction, indirectly affecting starch accumulation and grain physical properties (Zhang et al., 2012; Ma et al., 2012; Guo et al., 2013; Lu et al., 2015; Jiang et al., 2015; Hu et al., 2016). The TaNAC019 gene plays a key role in regulating starch synthesis and quality during endosperm development (Gao et al., 2021). TaPGS1 transcriptionally activates the downstream PLATZ transcription factor gene TaFL3, increasing the filling of endosperm dry matter during filling (Guo et al., 2022).These complex regulatory networks precisely control wheat grain development, the synthesis and transport of dry matter such as starch and sugar during the filling process, and affect grain weight and yield.

[0004] End1 is a recessive mutant. From the grain phenotype, the mutant grains have less endosperm filling than the wild-type grains. The grain mutants at maturity are severely wrinkled. Biochemical analysis found that the starch content of the end1 mutant is significantly reduced. By constructing the F2 population, the End1 gene was located at a physical distance of about 3Mb between markers K114 and S69 by positional cloning. Based on resequencing and BSR data, the TaEND1 candidate gene was obtained. The function of the TaEND1 gene in controlling the normal development of wheat grains was verified by gene editing. The TaEND1 gene is a new key gene that controls grain development and subsequent experiments have proved that it is a key gene for grain starch synthesis. Since the endosperm development defects of the recessive homozygous mutants of end1 can be screened in advance in hybrid breeding work by developing molecular markers, saving hybrid breeding time.

[0005] KASP (Kompetitive Allele-Specific PCR) is a genotyping technology based on SNP (single nucleotide polymorphism), which is efficient, accurate and economical. KASP technology can quickly and accurately screen out superior and inferior haplotypes of wheat grain development gene end1. It can not only make accurate and stable selection in the early stage, but also overcome the problem of difficulty in identifying recessive genes, thereby accelerating the breeding process and improving breeding efficiency. Summary of the invention

[0006] In view of this, the object of the present invention is to provide wheat grain development gene TaEND1, KASP molecular markers and applications thereof.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] 1. Wheat grain development gene TaEND1, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0009] 2. The KASP molecular marker used to detect the wheat grain development gene TaEND1 has a mutation site at the 575th base of the wheat grain development gene TaEND1, and the genotype of this site is G / G, G / A, and A / A.

[0010] 3. A specific primer set for detecting the KASP molecular marker of the wheat grain development gene TaEND1, including the upstream primer KASP-END1 for identifying the G / G genotype (full grain type) G -F, upstream primer KASP-END1 for identifying A / A genotype (grain shrinkage type) A-F and the universal downstream primer KASP-END1-common-R, the sequences of each primer are as follows:

[0011] KASP-END1 G -F:5'-GAAGGTGACCAAGTTCATGCTgtgcctcctaccggtaaagG-3', as shown in SEQ IDNO.1;

[0012] KASP-END1 A -F:5'-GAAGGTCGGAGTCAACGGATTgtgcctcctaccggtaaagA-3', as shown in SEQ IDNO.2;

[0013] KASP-END1-common-R: 5′-acagccccacttcccaca-3′, as shown in SEQ ID NO.3.

[0014] 4. A detection kit for wheat grain development gene TaEND1, comprising the aforementioned specific primer set.

[0015] As one of the preferred technical solutions, the kit also contains 2×KASPmix (Beijing Jiacheng Biotechnology Co., Ltd.) and double distilled water.

[0016] 5. Application of the aforementioned KASP molecular marker or kit in detecting wheat grain development.

[0017] 6. Application of the aforementioned KASP molecular marker or kit in wheat breeding.

[0018] The beneficial effects of the present invention are:

[0019] Traditional SNP detection technologies, such as RFLP (restriction fragment length polymorphism) and SSCP (single-strand conformation polymorphism), are usually complicated to operate and difficult to achieve high-throughput detection. Although AFLP (amplified fragment length polymorphism) and RAPD (random amplified polymorphic DNA) can be used for gene detection, they have low specificity and are prone to non-specific amplification. Traditional genotyping technologies such as Sanger sequencing are accurate but costly and not suitable for large-scale screening. RFLP technology usually requires complex enzyme digestion and electrophoresis processes, which are cumbersome and time-consuming to operate. In contrast, KASP technology greatly reduces experimental costs while ensuring accuracy. The operating procedures of KASP technology are significantly simplified, which improves the work efficiency of the laboratory. Compared with traditional gene detection technologies, KASP technology has obvious advantages in the detection sensitivity of low-frequency alleles. Although other marker technologies can also be applied to MAS, the high throughput and high accuracy of KASP make it more advantageous in large-scale breeding. Compared with SSR (simple sequence repeats) and RFLP, KASP technology is more suitable for the needs of modern breeding.

[0020] The present invention has discovered a new gene TaEND1 related to wheat grain development, and saves time and cost in breeding and selection by developing molecular markers. KASP technology can efficiently detect the polymorphism of TaEND1 gene, especially in large-scale sample screening. Because its fluorescent probe is simple and the experimental operation is economical, it can significantly reduce the detection cost, especially in breeding projects. This is particularly important for breeding projects that need to screen a large number of samples. KASP technology is easy to operate, only requires PCR amplification and fluorescence signal detection, the experimental process is simple, and the results are easy to interpret. This allows non-professional technicians to quickly master KASP technology, and has low requirements on the DNA quality and quantity of samples, which is suitable for plant tissue samples from different sources. This means that even samples from the field or in poor environmental conditions can be accurately genotyped by KASP technology, and have strong adaptability. This provides a guarantee for early screening and breeding optimization of wheat grain development defect genes. The TaEND1 molecular marker developed by KASP technology can play a key role in marker-assisted selection breeding (MAS). Breeders can use these markers to quickly screen superior and inferior haplotypes carrying the END1 gene, thereby improving grain development traits and increasing wheat quality and yield.

[0021] The development of KASP molecular markers for the wheat grain development defect gene TaEND1 not only improves the efficiency, accuracy and economy of genotyping, but also has significant advantages in ease of operation and speed of obtaining results. Compared with previous technologies, KASP technology is more suitable for large-scale screening and marker-assisted selection breeding, which helps to accelerate the research progress of wheat grain development genes and the development of new varieties. These beneficial effects make KASP technology an indispensable tool in modern wheat breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0023] Figure 1 Comparison diagram of wild-type and mutant grains, where (a) is a comparison diagram of WT and end1 grain length and width, and (b) is a comparison diagram of WT and end1 mature grain longitudinal and transverse sections;

[0024] Figure 2 The nucleic acid sequence of the TaEND1 gene and the sequence variation site in the present invention;

[0025] Figure 3 The KASP marker detection results of TaEND1 in the single plant segregation population, mutants and hybrid parents of the present invention are shown. DETAILED DESCRIPTION

[0026] The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0027] Example 1: Acquisition of KASP molecular markers of TaEND1

[0028] The applicant successfully cloned TaEND1, a key gene controlling wheat grain development, through positional cloning technology. TaEND1 encodes a branched-chain amino acid aminotransferase. The 575th base of its coding region has a SNP mutation from G to A, which causes the 192nd amino acid to mutate from glycine to aspartic acid, which in turn causes serious impact on wheat grain development. Figure 2 The nucleic acid sequence of wheat grain development gene TaEND1 is shown in Figure 2. Based on the above key variant SNP sites, two pairs of primers were designed:

[0029] The first pair of primers targets the wild-type (full-type) allele KASP-END1 at the SNP site G ;,

[0030] The second pair of primers targets the mutant (shrunken) allele KASP-END1 A .

[0031] Primer optimization: Ensure the specificity and amplification efficiency of primers, and improve the specificity and sensitivity of PCR reaction by optimizing primer sequence and annealing temperature. Use specialized primer design software (such as Primer3) for optimization.

[0032] The primer sequences of the two KASP molecular markers developed by the applicant are as follows:

[0033] KASP-END1 G -F was used to identify the G / G genotype (full grain genotype) with the following primer sequence: 5'-GAAGGTGACCAAGTTCATGCTgtgcctcctaccggtaaagG-3',

[0034] KASP-END1 A -F The primer sequence used to identify the A / A genotype (grain shrinkage genotype) is as follows: 5'-GAAGGTCGGAGTCAACGGATTgtgcctcctaccggtaaagA-3'

[0035] The sequence of the universal primer KASP-END1-common-R is as follows: 5'-acagccccacttcccacA-3'

[0036] Example 2 Identification of polymorphism of KASP of TaEND1 in population and parents

[0037] 1. Experimental Materials

[0038] Experimental materials: The applicant used wheat material Jinmai 47 and mutant TaEND1 ( Figure 1 ).

[0039] Segregation population: Using wheat material Jinmai 47 and mutant TaEND1 as parents, a segregation population was constructed, which included multiple F2 or F3 generation offspring.

[0040] Genomic DNA extraction: Extract genomic DNA from the leaf tissue of each wheat material using the conventional CTAB method to ensure high DNA quality and appropriate concentration. The specific steps are as follows:

[0041] 1. Place fresh wheat leaves in a corresponding 2 ml centrifuge tube, add 6 mm steel beads, grind with liquid nitrogen, and add 500 μL 2×CTAB extraction solution (reducing agent has been added).

[0042] 2. Place the centrifuge tube in a 65°C water bath for 30 min-1 h, gently shaking the centrifuge tube every 10 min or so.

[0043] 3. After the water bath is completed, cool for 2 minutes, add 0.5 mL of chloroform-isoamyl alcohol (volume ratio 24:1) mixture, shake vigorously to mix, and centrifuge at 10000-12000 rpm for 5-10 minutes.

[0044] 4. Take the supernatant from the centrifuge tube in the previous step and transfer it to a new 1.5 ml centrifuge tube. Note: After centrifugation in the previous step, there should be three layers: the upper layer is the aqueous phase, the middle layer is the protein and plant fragments, and the lower layer is the organic phase. Avoid touching the protein and organic phase when aspirating. If you accidentally aspirate the middle and lower layers, or the middle layer is thick, it is recommended to aspirate less of the aqueous phase, or add 500uL of chloroform-isoamyl alcohol mixture after aspiration, re-mix and centrifuge to obtain the supernatant.

[0045] 5. Add an equal volume of isopropanol to the supernatant and mix thoroughly by inversion (or add 2 volumes of anhydrous ethanol and 1 / 10 volume of 3M sodium acetate at pH 5.2). Place at -20°C for precipitation for more than 30 minutes, centrifuge at 12,000 rpm for 10 minutes, and discard the supernatant.

[0046] 6. Wash the precipitate once with 500 μL of 75% ethanol aqueous solution, centrifuge at 12000 r / min for 3-5 min, discard the supernatant, dry the precipitate naturally, dissolve it in 50 μL of deionized water, and incubate at 37°C for 30 min.

[0047] 7. Store at -20℃.

[0048] CTAB extraction solution (2×) formula:

[0049] CTAB (cetyltrimethylammonium bromide): 2% (w / v)

[0050] NaCl (sodium chloride): 1.4M

[0051] Tris-HCl (pH 8.0): 100 mM

[0052] EDTA (ethylenediaminetetraacetic acid, pH 8.0): 20 mM

[0053] PVP (Polyvinylpyrrolidone): 2% (w / v) (sometimes used to treat samples high in polyphenols or polysaccharides)

[0054] β-Mercaptoethanol: 0.2% (v / v) (add before use to prevent DNA oxidation)

[0055] 2. PCR amplification and genotype identification

[0056] KASP reaction system: The KASP reaction system is set up as follows:

[0057] Each reaction system contains genomic DNA, KASP primer mixture, PCR buffer, MgCl2, dNTPs, and TaqDNA polymerase. The PCR reaction system is as follows:

[0058] 2×KASP reaction mixture: 4.0 μl

[0059] KASP primer mix: 0.1 μl

[0060] Template DNA (concentration about 100 ng / μl): 1.0 μl

[0061] Double distilled water: 2.9 μl

[0062] The KASP primer mix ratios are as follows:

[0063] KASP-END1 G -F (100 μM): 12 μL

[0064] KASP-END1 A -F (100 μM): 12 μL

[0065] KASP-common-R (100 μM): 30 μL

[0066] Double distilled water: 46 μl

[0067] Total system: 100 μl

[0068] PCR amplification conditions: KASP reaction uses the following PCR touch down amplification procedure:

[0069] 1. Pre-denaturation: 95°C for 10 minutes;

[0070] 2. Denaturation: 95°C for 20 seconds;

[0071] 3. Annealing and extension: 61°C-55°C (0.6°C drop per cycle) for 40 seconds;

[0072] 4. Repeat 10 times

[0073] 5. Denaturation: 95°C for 20 seconds;

[0074] 6. Annealing / extension: 55°C 40 seconds

[0075] 7. Repeat 30 times;

[0076] Fluorescence detection: After PCR amplification, the fluorescence signal of the KASP reaction was detected by QuantStudio 3 real-time fluorescence quantitative PCR to identify the distribution of different alleles.

[0077] 4. Genotyping of Segregating Populations

[0078] Genotype classification: According to the color of the fluorescence signal, the samples are divided into three categories: wild type, heterozygous and mutant, corresponding to different allele combinations.

[0079] Data statistics: The distribution ratio of each genotype in the segregating population was counted to verify the association between the TaEND1 gene and grain development. The effectiveness of the KASP molecular marker in the segregating population was verified by analyzing the effects of different genotypes on grain traits.

[0080] exist Figure 3 In the middle, blue is KASP-END G (Full) haplotype, red is KASP-END A (Shriveled) haplotype, green is KASP-END G / A (Heterozygous) haplotype.

[0081] 5. Results Analysis and Application

[0082] Association analysis between genotype and traits: Association analysis was performed based on the genotype results of each segregating population and the phenotypic data of wheat grain development. By comparing the differences in grain development traits among wild type, heterozygote and mutant ( Figure 1 ), confirming the role of TaEND1 gene in wheat grain development.

[0083] Application of KASP markers: Based on KASP molecular markers, individuals carrying excellent TaEND1 genotypes can be screened quickly and accurately, providing a molecular marker-assisted selection (MAS) tool for the rapid selection of excellent varieties in the wheat breeding process.

[0084] Implementation Effect

[0085] The KASP molecular marker technology has the advantages of high specificity and high sensitivity, and can effectively distinguish the segregated populations of different TaEND1 genotypes. By applying it to wheat breeding, it can accelerate the screening and fixation of excellent traits, greatly shorten the breeding cycle, and improve breeding efficiency.

[0086] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. Wheat grain development gene TaEND1, It is characterized in that Its nucleotide sequence is shown in SEQ ID NO.

1.

2. A KASP molecular marker for detecting the wheat grain development gene TaEND1 according to claim 1, It is characterized in that The mutation site is the 575th base of the wheat grain development gene TaEND1, and the genotype of this site is G / G, G / A, and A / A.

3. The specific primer set for the KASP molecular marker according to claim 2, It is characterized in that Contains upstream primer KASP-END1 for identification of G / G genotype G -F, upstream primer KASP-END1 for identification of A / A genotype A -F and the universal downstream primer KASP-END1-common-R, the sequences of each primer are as follows: KASP-END1 G -F: 5'-GAAGGTGACCAAGTTCATGCTgtgcctcctaccggtaaagG-3', as shown in SEQ ID NO.1; KASP-END1 A -F: 5'-GAAGGTCGGAGTCAACGGATTgtgcctcctaccggtaaagA-3', as shown in SEQ ID NO.2; KASP-END1-common-R: 5′-acagccccacttcccaca-3′, as shown in SEQ ID NO.

3.

4. The detection kit for the wheat grain development gene TaEND1 according to claim 1, It is characterized in that Comprising the specific primer set described in claim 3.

5. Use of the KASP molecular marker according to claim 2 or the kit according to claim 4 in detecting wheat grain development.

6. Use of the KASP molecular marker according to claim 2 or the kit according to claim 4 in wheat breeding.

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