InDel molecular marker related to wheat TaBADH-A1 gene and application thereof

Abstract

The application discloses a wheat TaBADH-A1 gene related InDel molecular marker and application thereof. The application firstly provides application of an InDel molecular marker or a substance for detecting the InDel molecular marker, wherein the InDel molecular marker is a double-stranded DNA molecule with a nucleotide sequence of 365-618 of SEQ ID No. 1; the InDel molecular marker or the substance for detecting the InDel molecular marker can be applied in identification or auxiliary identification of wheat water stress resistance, and can also be applied in wheat breeding. The molecular marker can be used as a basis for preliminary identification of drought resistance and salt tolerance of wheat, has high efficiency and applicability such as simple operation, low cost and short time consumption, and provides an efficient selection method for new varieties of wheat with water stress resistance.

Description

Technical Field

[0001] This invention relates to the field of molecular genetic breeding, specifically to the InDel molecular marker related to the wheat TaBADH-A1 gene and its application. Background Technology

[0002] Wheat is one of the most important food crops, providing 60% of the world's population with their daily needs for calories and protein. Due to global climate change, drought has become a serious environmental problem; a 40% reduction in water availability can lead to a decrease in wheat yield to 21% of its original level. Simultaneously, salinization is another serious factor reducing crop productivity, with salt affecting 40% of irrigated land, and this phenomenon is gradually intensifying; it is projected that 50% of the world's arable land will be salinized by 2050. Among all abiotic stresses, drought and salt stress are the main challenges to crop growth and development, causing enormous damage to global food production. Enhancing the drought and salt tolerance of wheat itself is one of the most economical and effective ways to solve these problems.

[0003] Water stresses such as drought and salt stress trigger osmotic regulation in plants, which is a major component of plant drought and salt tolerance mechanisms. When plants experience a certain level of drought or salt stress, osmotic regulators such as betaine, proline, soluble sugars, and inorganic ions increase. These regulators help plants maintain or absorb more water by lowering cell osmotic potential, increasing cell osmotic pressure, buffering cell redox potential, maintaining protein structure, and protecting photosynthetic organs from stress damage. Betaine, as an important osmotic regulator, also plays a role in protecting plants from abiotic stresses by lowering cell osmotic potential and increasing cell osmotic pressure. Betaine is a small, non-toxic organic metabolite that increases when plants experience a certain level of drought or salt stress, thereby lowering cell osmotic potential and increasing cell osmotic pressure, further helping plants maintain or absorb more water. Betaine aldehyde dehydrogenase (BADH) is the final step in the synthesis of betaine, and it is an important enzyme in the process of betaine synthesis. Studying the function of the superior allelic variant of the wheat betaine aldehyde dehydrogenase gene TaBADH-A1, and developing functional molecular markers, can provide excellent genetic resources and rapid selection methods for breeding wheat with improved water stress resistance.

[0004] Conventional drought and salt tolerance assessments require a large amount of wheat seeds and time. However, in the early stages of breeding, there are numerous types of individual plants or lines in lower generations, but the number of seeds for each type is extremely small, making conventional stress resistance assessments unsuitable. Furthermore, the genotypes and phenotypes of higher generations of lines are largely stable, limiting the scope or potential for stress resistance selection. Discovering and utilizing superior allelic variations in wheat stress resistance-related genes is one effective way to improve wheat stress resistance. Rapidly identifying superior allelic variations is an effective method and forms the basis for precise selection in wheat breeding. Summary of the Invention

[0005] The problem to be solved by this invention is how to identify or assist in the identification of wheat water stress resistance.

[0006] To address the above technical problems, this invention provides the application of InDel molecular markers or substances for detecting InDel molecular markers, and verifies the practicality of the markers, providing reliable molecular markers for identifying or assisting in the breeding of wheat varieties.

[0007] This invention first provides the application of InDel molecular markers or substances for detecting InDel molecular markers, wherein the InDel molecular marker is a double-stranded DNA molecule with a single-stranded nucleotide sequence of positions 365 to 618 of SEQ ID No. 1; the application is any of the following:

[0008] 1) The application of InDel molecular markers or substances that detect InDel molecular markers in the identification or auxiliary identification of wheat water stress resistance.

[0009] 2) Application of InDel molecular markers or substances that detect InDel molecular markers in wheat breeding.

[0010] The nucleotide sequence of the other strand of the InDel molecular marker is reverse complementary to positions 365 to 618 of SEQ ID No. 1.

[0011] The above SEQ ID No.1 consists of 828 nucleotides.

[0012] In the above applications, the substance used to detect the InDel molecular marker may contain PCR primers for amplifying wheat genomic DNA fragments containing the wheat InDel molecular marker.

[0013] Further, the PCR primers may be primer pairs, each consisting of a forward primer and a reverse primer. The forward primer is a single-stranded DNA that specifically binds upstream of the InDel molecular marker in wheat genomic DNA, and the reverse primer is a single-stranded DNA that specifically binds downstream of the InDel molecular marker in wheat genomic DNA. Further, the forward primer sequence may specifically be a single-stranded DNA as shown in SEQ ID No. 3, and the reverse primer may specifically be a single-stranded DNA as shown in SEQ ID No. 4.

[0014] In the above applications, the wheat can be a wheat inbred line.

[0015] The present invention also provides a method for identifying or assisting in the identification of wheat water stress resistance, comprising the following steps: detecting whether the genomic DNA of the wheat to be identified contains the InDel molecular marker, and the water stress resistance of the wheat to be identified that does not contain the InDel molecular marker is higher or candidate higher than that of the wheat to be identified that contains the InDel molecular marker in its genomic DNA.

[0016] This invention provides a method for identifying or assisting in the identification of wheat water stress resistance, the method specifically including the following steps:

[0017] (1) Using the genomic DNA of the wheat material to be identified as a template, PCR amplification was performed using the primer pair to obtain the PCR product;

[0018] (2) Identify wheat water stress resistance based on whether the PCR product contains the InDel molecular marker:

[0019] The water stress resistance of the wheat to be identified whose PCR product does not contain the InDel molecular marker is higher than that of the wheat to be identified whose PCR product contains the InDel molecular marker.

[0020] Furthermore, the PCR amplification program is as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 62℃ annealing for 15 s, 72℃ extension for 15 s, 34 cycles; 72℃ extension for 5 min.

[0021] In the above applications or methods, the water stress resistance is drought stress resistance and / or salt stress resistance.

[0022] The InDel molecular markers described above also fall within the scope of protection of this invention.

[0023] This invention also provides a method for wheat breeding.

[0024] The wheat breeding method provided by the present invention includes selecting wheat whose genomic DNA does not contain the InDel molecular marker as a parent for breeding.

[0025] In this article, the breeding objectives include developing wheat that is resistant to water stress.

[0026] In this article, the wheat referred to can be a wheat inbred line or a hybrid offspring of two wheat inbred lines, such as a hybrid offspring of wheat Chinese Spring and wheat Yanfu 188. The wheat can also be a pure line.

[0027] In the above applications or methods, the water stress resistance is drought stress resistance and / or salt stress resistance.

[0028] The water stress resistance may be drought resistance during wheat germination and / or drought resistance during wheat seedling stage and / or salt resistance during wheat seedling stage.

[0029] During its evolution, domestication, and breeding, wheat underwent two hybridizations to eventually form its current form. Since the A genome is the fundamental genome in wheat evolution, it plays a central role in the evolution of polyploid wheat, resulting in its rich genetic diversity. In this study, we first predicted that the expression level of the TaBADH-A1 gene in the wheat A genome was higher than that of the TaBADH-B1 gene in the B genome and the TaBADH-D1 gene in the D genome under both untreated and drought-treated conditions. Subsequently, the polymorphism of the TabADH-A1 gene sequence was analyzed in two wheat varieties with different stress resistance. Based on sequence differences, molecular markers were designed to detect different allelic variations of the TabADH-A1 gene. Using natural populations and genetically segregating populations, the relationship between gene expression in response to stress, betaine accumulation, and drought and salt tolerance phenotypes and allelic variations was analyzed to evaluate the drought resistance and salt tolerance of different allelic variations of the TabADH-A1 gene and to explore its molecular and physiological mechanisms. Finally, molecular markers related to drought and salt tolerance of the TabADH-A1 gene were developed to resist water stress. These molecular markers can serve as a basis for preliminary identification of drought and salt tolerance in wheat, and are highly efficient and applicable due to their simple operation, low cost, and short time consumption, providing an efficient breeding method for new drought-resistant and salt-tolerant wheat varieties. Attached Figure Description

[0030] Figure 1 Predicted expression of three homologous genes of TabaDH.

[0031] Figure 2 This is a schematic diagram of the TabADH-A1 gene structure.

[0032] Figure 3 Chromosomal localization of molecular marker primers.

[0033] Figure 4 Agarose gel electrophoresis for detecting allelic variations using molecular markers.

[0034] Figure 5 Expression analysis of different allelic variants of the TabADH-A1 gene under drought (20% v / v PEG6000) and salt (200 mM NaCl) stress.

[0035] Figure 6 Analysis of betaine content in wheat with different allelic variations under drought (20% v / v PEG6000) and salt (200 mM NaCl) stress.

[0036] Figure 7 Phenotypic analysis of genetically segregating populations with different allelic variations of the TabADH-A1 gene under drought and salt stress. Detailed Implementation

[0037] 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.

[0038] Unless otherwise specified, the experimental methods used in the following examples 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 and reagents used in the following examples are commercially available.

[0039] Example 1: Development of molecular markers for the TaBADH-A1 gene

[0040] 1.1 Prediction of the expression of the three TabaDH homologs

[0041] Transcriptome data (PRJNA358808) from WheatOmics 1.0 (http: / / 202.194.139.32 / ) were used to analyze the expression levels of three homologous genes of TabaDH in the drought-resistant wheat variety Zubkov and the weakly drought-resistant wheat variety Atay85 before and after drought stress, and genes with high drought response were screened for further research.

[0042] The relative expression levels of the TabADH-A1 gene in the drought-resistant wheat variety Zubkov and the weakly drought-resistant variety Atay85 under normal water supply and drought stress treatments were 7.17, 10.34, 5.76, and 3.14 TPM, respectively, all higher than those in TabADH-B1 and TabADH-D1. Figure 1 Therefore, TabaDH-A1 was chosen as the gene for further research.

[0043] 1.2 Wheat DNA Extraction and Cloning and Sequence Analysis of the TabADH-A1 Gene

[0044] Genomic DNA was extracted from Taishan No. 1, Bima No. 4, and two populations using the CTAB method (Stein et al. 2010). The two populations were constructed as follows: parents were crossed, and the offspring were self-crossed to obtain an F6 genetic segregating population of 10 lines. Based on the existing TabaDH-A1 sequence (TraesCS6A02G371100), TabaDH-A1-specific primers were designed using Oligo 7.0 software (Table 1). A pair of specific primers was designed to amplify the TabaDH-A1 gene at positions 595608559-595614056 on chromosome 6A (Table 1).

[0045] Table 1 Primer information used in this study

[0046]

[0047] The TaBADH-A1 promoter region and full-length genomic DNA sequences of Taishan No. 1 and Bima No. 4 were cloned. Transposons were predicted using the Giri (https: / / www.girinst.org / censor / index.php) database and cis-regulatory elements were predicted using the PlantCare (http: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ) database. The TaBADH-A1 promoter region and full-length genomic DNA sequences were analyzed. Sequence alignment was performed using DNAMAN 9.0 software. The TaBADH-A1 genome consists of 15 exons and 14 introns. Figure 2 The full-length sequences of the TaBADH-A1 gene in Taishan No. 1 and Bima No. 4 are 7675 and 7919 bp, respectively.

[0048] 1.3 Development of molecular markers for the TabaDH-A1 gene

[0049] Polymorphism analysis of the TabADH-A1 gene sequence in the two cloned varieties revealed 41 single nucleotide polymorphisms (SNPs) and 4 1-bp and 2 2-bp insertions / deletions (InDels) in the promoter region of the TabADH-A1 gene; the full-length genomic DNA sequence contained 28 SNPs and 1 254-bp, 2 1-bp, and 1 2-bp insertion or deletion. The promoter region of BiMa 4 has 2 more ABREs, 1 I-Box, 1 O2-site, 1 G-Box, 1 STRE, 1 TCA-element, 1 W-box, and 1 AT-TATA-box than TaiShan 1, and lacks 1 TGA-element cis-acting element. The 254 bp fragment inserted into the third intron of BiMa 4 is a DNA miniature inverted repeat transposon element (MITE), containing one Box-4, one MRX, two W-boxes, two CAAT-boxes, and eleven TATA-boxes as cis-acting elements. SNPs in the exons do not cause amino acid mutations. Because DNA MITEs can have a significant impact on gene function, the TaBADH-A1 gene is further classified into two allelic variant types, BADH-A1a (In) and BADH-A1b (Del), based on the 254-bp DNA MITE InDel in the third intron.

[0050] According to the third intron of Tabahh-A1, 254-bp InDel ( Figure 2 Specific primers designed for both ends of the sequence are shown in Table 1. Using *Cymbidium goeringii* and its four null variants N6A-T6B, N6B-T6D, and N6D-T6A, chromosomal localization of the molecular marker primers was performed. ddH2O was used as a control. The PCR products were then subjected to agarose gel electrophoresis. Results are as follows: Figure 3 As shown: M represents DNA marker2000; 1 is N6A-T6B; 2 is N6B-T6D; 3 is N6D-T6B; 4 is ddH2O; 5 is Chinese spring. Among them, the PCR products with N6A-T6B and ddH2O as templates did not show bands, while the others all showed the expected target size bands, indicating that the marker primers were located on chromosome 6A.

[0051] The results of detection using molecular marker primers on natural populations and genetically segregating populations are as follows: Figure 4M represents DNA marker 2000; 1-10 represent partial materials from the natural and segregating populations. The amplified fragment lengths exhibited polymorphism, with the molecular marker amplified fragments being 828 bp (574+254) and 574 bp, respectively. These fragment sizes were consistent with the expected sizes, confirming the effectiveness of the molecular marker. Sequencing revealed them to be BADH-A1a (SEQ ID No. 1 in the sequence listing) and BADH-A1b (SEQ ID No. 2 in the sequence listing), respectively.

[0052] Among them, the 254-bp InDel variant sequence is the InDel molecular marker for wheat water stress resistance. The nucleotide sequence of the InDel molecular marker for wheat water stress resistance is shown as positions 365 to 618 of SEQ ID No. 1 in the sequence listing.

[0053] 1.4 Analysis of TabADH-A1 gene expression under drought and salt stress with different allelic variants

[0054] Since MITE inserted in the third intron may affect gene expression, we selected Yanfu 188 and Bima 4, which contain the BADH-A1a allele, and Taishan 1 and Zhongguochun, which contain the BADH-A1b allele, to investigate whether there is a difference in gene expression between the two allele types TaBADH-A1. The specific steps are as follows:

[0055] 1) Taishan No. 1 and Bima No. 4, different stress-resistant varieties used for cloning, were selected. Yanfu 188, containing the BADH-A1a allelic variant, was selected, and five lines containing the BADH-A1a allelic variant were randomly selected from the genetic segregating population and named lines 1 to 5. Zhongguo Chun, containing the BADH-A1b allelic variant, was selected, and five lines containing the BADH-A1b allelic variant were randomly selected from the genetic segregating population and named lines 6 to 10. All were cultured in a Hogrange nutrient solution at 22℃ in a light incubator with a relative humidity of 60% and a photoperiod of 8 hours in darkness and 16 hours in light. Subsequently, at the four-leaf stage, the wheat was treated with a Hogrange nutrient solution containing NaCl (200 mM) or PEG6000 (20% v / v).

[0056] 2) Leaf samples were collected at 0h, 1h, 6h, 12h, and 24h of stress treatment. RNA was extracted using TRIzol (Beijing Qingke), and then cDNA was synthesized using EasyScript one-step gDNA removal and cDNA Synthesis SuperMix (TransGen Biotech, Beijing). Real-time quantitative PCR (qRT-PCR) was performed using Perfect Start™ Green qPCR Supermix (TransGen Biotech, Beijing), with TaActin as an internal control gene. All qRT-PCR analyses used three technical replicates and three biological replicates. -ΔΔCt The relative expression level of a gene can be calculated.

[0057] Four wheat varieties were subjected to drought and salt stress respectively when they reached the four-leaf stage, and the results were as follows: Figure 5 a and Figure 5 As shown in Figure b, under drought and salt stress treatment for 0 h, the expression level of the TaBAHD-A1 gene in wheat varieties carrying the BADH-A1b allelic variant was higher than that in wheat varieties carrying the BADH-A1a allelic variant. With the increase of drought and salt stress duration, the expression pattern of the TaBADH-A1 gene in the four varieties was consistent, showing a trend of first increasing and then decreasing. Moreover, before and after stress, the expression level of the TaBAHD-A1 gene in wheat varieties carrying the BADH-A1b allelic variant was consistently higher than that in wheat varieties carrying the BADH-A1a allelic variant.

[0058] Considering the differences in genetic background among different wheat varieties, the above results were further verified using a segregating population constructed with Chinese Spring and Yanfu 188 as parents. Five lines containing the BADH-A1a allelic variation were randomly selected from the segregating population with relatively small differences in genetic background. Figure 5 c and Figure 5 The strains numbered 1-5 in the middle d section and 5 strains containing BADH-A1b allelic variation ( Figure 5 c and Figure 5 The strains numbered 6-10 in the middle d series. Under drought and salt stress, the expression of the TabADH-A1 gene showed a trend of first increasing and then decreasing. Figure 5 c and Figure 5 (d) Before and after stress, the expression level of the TaBADH-A1 gene in lines carrying the BADH-A1b allelic variant was higher than that in the BADH-A1a allelic variant. Therefore, the deletion of the 254-bp DNA MITE in intron 3 can enhance the expression of the TaBADH-A1 gene.

[0059] 1.5 Analysis of betaine content in wheat varieties (lines) with different allelic variations under drought and salt stress.

[0060] In addition to the materials selected for gene expression analysis, Ning 9940 carrying the BADH-A1a allelic variant and Longfumai 18 carrying the BADH-A1b allelic variant were also selected. Ten lines from the genetic segregating populations constructed using Taishan 1, Longfumai 18, Zhongguochun, Bima 4, Yanfu 188, Ning 9940, and Zhongguochun and Yanfu 188 as parents were cultured in the same manner as described above. Subsequently, at the four-leaf stage, all wheat varieties were treated with Hoagland's nutrient solution containing NaCl (200 mM) or PEG6000 (20% v / v). Leaf samples were collected under stress treatments of 0 h, 1 h, 6 h, 12 h, 24 h, and 48 h, and betaine content was determined according to the Solarbio Betaine Content Detection Kit instructions (catalog number: BC3130).

[0061] The results are as follows Figure 6 As shown, under drought stress, the betaine content in wheat varieties (lines) containing the allelic variant BADH-A1b decreased slightly after 1 hour of drought stress, and then gradually increased; the betaine content in wheat varieties (lines) containing the allelic variant BADH-A1a also decreased slightly after 1 hour of drought stress, then increased, reached a peak at 12 hours, and then began to decrease. Figure 6 (a) Under salt stress, the betaine content in wheat varieties (lines) containing allelic variants BADH-A1a and BADH-A1b decreased slightly at 1 h, but peaked at 6 h, and then began to decline. However, at 48 h, the betaine content in wheat varieties (lines) containing the allelic variant BADH-A1b increased again. Figure 6 (b)

[0062] Regardless of drought or salt stress, wheat varieties (lines) containing the BADH-A1b allelic variant consistently had higher betaine content than those containing the BADH-A1a allelic variant, and the difference in betaine content between the two wheat varieties (lines) reached its maximum at 48 hours of stress. Figure 6 (a and b).

[0063] Furthermore, using genetically segregating populations with relatively small differences in genetic background and different allelic variations, the betaine content of lines containing different allelic variations was determined after 48 hours of drought and salt stress. Under drought stress, five randomly selected lines containing the BADH-A1b allelic variation ( Figure 6 The betaine contents of the five strains (c 6-10) were 24, 23, 23, 25, and 24 μmol / g (dry weight, DW), respectively. Five strains containing the allelic variant BADH-A1a (…) Figure 6The betaine contents of the five strains (c1-5) were 17, 16, 16, 16, and 16 μmol / g (DW), respectively. Under salt stress, the betaine contents of the five strains containing the allelic variant BADH-A1b were... Figure 6 The betaine contents of the five strains (6-10) were 23, 22, 24, 25, and 23 μmol / g (DW), respectively. Five strains containing the allelic variant BADH-A1b (…) Figure 6 The betaine contents of the d lines 1-5 were 17, 16, 16, 16, and 15 μmol / g (DW), respectively. Therefore, under drought and salt stress, the betaine content of wheat varieties (lines) containing the allelic variant BADH-A1b was consistently significantly higher than that of wheat varieties (lines) containing the allelic variant BADH-A1a.

[0064] Example 2: Application of InDel molecular markers for wheat water stress resistance

[0065] 2.1 Genotyping of InDel254

[0066] This embodiment utilizes the wheat water stress resistance InDel molecular marker (InDel254) developed in Example 1 to identify drought resistance during wheat germination. This wheat water stress resistance InDel molecular marker is located at positions 365 to 618 of the double-stranded DNA molecule shown in SEQ ID No. 1. The specific method is as follows:

[0067] Genomic DNA was extracted from 121 wheat varieties (lines) listed in Table 2. Using the genomic DNA as a template, PCR was performed on different wheat varieties (lines) using primers 6AM-F (5'-CGCAAGTCAATGGGCTGTGTAT-3') and 6AM-R (5'-CAAGACAGGAGGGGAATGGGAAAT-3'). The PCR system consisted of 10 μL of 2×Rapid Taq Master Mix, 7 μL of ddH2O, primers 6AM-F (10 μM), 6AM-R (10 μM), and DNA (300 ng / μL). -1 The template was 1 μL. The first cycle of the PCR amplification program was 95℃ for 3 min, 95℃ for 15 s, 62℃ for 15 s, and 72℃ for 15 s. Then, 34 cycles were performed at 95℃ for 15 s, 62℃ for 15 s, and 72℃ for 15 s. Finally, the temperature was 72℃ for 5 min.

[0068] The PCR amplification products were subjected to 2% agarose gel electrophoresis. The results showed that, in Table 2, the PCR products of 12 wheat varieties (lines), namely Taishan No. 1, Longfumai 18, Luomai 23, Longmai 26, Chang 6359, Longmai 32, Lumai 14, Pingliang 40, Beimai No. 7, Canghe 030, Laizhou 953, and Gaoyuan 602, were all DNA bands of about 500 bp (DNA fragments, specific PCR products); the PCR products of the remaining 109 wheat varieties (lines) in Table 2 were all DNA bands of about 750 bp (DNA fragments, specific PCR products).

[0069] Specific PCR products from each wheat variety (line) were recovered. The results showed that the DNA fragments of approximately 500 bp for the 12 wheat varieties (lines) listed in Table 2—Taishan 1, Longfumai 18, Luomai 23, Longmai 26, Chang 6359, Longmai 32, Lumai 14, Pingliang 40, Beimai 7, Canghe 030, Laizhou 953, and Gaoyuan 602—were all 574 bp in size. The nucleotide sequence of this DNA fragment is SEQ ID No. 2 in the sequence listing. The genotype of wheat varieties (lines) whose PCR products contain the DNA fragment with the nucleotide sequence of SEQ ID No. 2 in the sequence listing was defined as the deletion type (allelic variation BADH-A1b). The remaining 109 wheat varieties (lines) in Table 2 all had DNA fragments of approximately 750 bp in size, all 828 bp in size. The nucleotide sequence of this DNA fragment is SEQ ID No. 1 in the sequence listing.

[0070] The DNA fragment from positions 365 to 618 of SEQ ID No. 1; the PCR product containing the nucleotide sequence listed in the sequence listing.

[0071] The genotype of wheat varieties (lines) for the DNA fragment at positions 365 to 618 of SEQ ID No. 1 is defined as the insertion type (allelic variation BADH-A1a).

[0072] 2.2 Phenotypic typing of drought resistance at germination stage in wheat varieties (lines) with different allelic variations

[0073] The drought resistance of 121 wheat varieties (lines) during germination was assessed. 100 seeds were placed in a 12×12×5cm sieve. 3 In the germination box, a single layer of filter paper was used as the germination bed. The treatment group received 12 mL of 20% (v / v) PEG6000 solution (solvent: distilled water), while the control group received 12 mL of distilled water. 100 seeds constituted one replicate, with each group (treatment and control) replicated four times. The standard for wheat seed germination was defined as when the length of the wheat radicle was equal to the length of the wheat seed, and the plumule length reached half the seed length.

[0074] The seeds were cultured in the dark at 20℃ for 168 hours, and the number of germinating seeds was observed. The relative germination rate was calculated by dividing the number of germinating seeds in the treatment group by the number of germinating seeds in the control group. The relative germination rate was used to reflect the drought resistance of wheat during the germination period.

[0075] The relative germination rates of treatment and control groups of 121 wheat varieties (lines) were studied. The results showed that the average relative germination rate of wheat varieties (lines) containing the BADH-A1a allelic variant was 51.2%, while the average relative germination rate of wheat varieties (lines) containing the BADH-A1b allelic variant was 67.3% (Table 2). The average relative germination rate of wheat varieties (lines) containing the BADH-A1b allelic variant was significantly higher than that of wheat varieties (lines) containing the BADH-A1a allelic variant, indicating that BADH-A1b can improve drought resistance during wheat germination. This demonstrates that the InDel molecular marker InDel254 can be used to identify drought resistance during wheat germination; wheat containing InDel254 exhibits lower drought resistance during germination than wheat without InDel254.

[0076] Table 2.1 Genotypes, relative germination rates, drought resistance levels, and origins of 121 wheat varieties.

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084] Note: In the table, drought resistance levels are: HR represents extremely strong; R represents strong; MR represents moderate; S represents weak; HS represents extremely weak.

[0085] Table 3 shows the relative germination rate and the number of varieties (lines) with different allelic variations in wheat varieties (lines).

[0086]

[0087] Statistical analysis was performed using the LSD method, with different letters indicating significance (P<0.01).

[0088] 2.3 Phenotypic typing of water stress resistance at the four-leaf stage in wheat varieties (lines) with different allelic variations

[0089] Construction of a genetic segregating population using wheat Chinese Spring and wheat Yanfu 188 as parents: The parents were hybridized, and the hybrid offspring were then self-crossed to obtain a genetic segregating population consisting of 10 lines, numbered from line 1 to line 10.

[0090] The genotypes of InDel254 in lines 1 to 10 were determined according to the method in step 2.1.

[0091] At the four-leaf stage, lines containing different allelic variations from a genetic segregating population constructed using Chinese Spring and Tobacco 188 as parents were subjected to water stress treatments such as drought and high salinity. Lines containing the BADH-A1a allelic variation and lines containing the BADH-A1b allelic variation from the genetic segregating population were planted in pots containing 165g of nutrient soil (substrate:vermiculite = 3:1), with the pots measuring 10×10cm at the top, bottom, and height. 2 7.5×7.5cm 2 Four wheat seedlings were planted in each pot, 7.5 cm in diameter. They were grown in a light-controlled incubator. Water stress was applied when the seedlings reached the four-leaf stage. Under drought stress, the wheat was not watered for 15 days, then re-watered for 5 days. Under salt stress, the wheat was irrigated with a 700 mM NaCl solution (distilled water) for 25 days (irrigated every 5 days); a control group was also included, irrigated with distilled water for 25 days. The survival rate of the wheat was recorded. Each experiment was repeated three times, with 36 wheat plants used per line per experiment.

[0092] The results are as follows Figure 7 As shown, under drought stress, the growth of some wheat varieties (lines) at the four-leaf stage is as follows: Lines 6-10, containing the allelic variant BADH-A1b, grew better than lines 1-5, which contained the allelic variant BADH-A1a, and their leaves were green. Figure 7 (a) After 5 days of rehydration, the survival rates of lines 6-10 containing the BADH-A1b allelic variant were 49.3%, 61.7%, 52.3%, 59.0%, and 51.0%, respectively, while the survival rates of lines 1-5 containing the BADH-A1a allelic variant were 38.7%, 37.7%, 36.7%, 35.7%, and 32.0%, respectively. Figure 7 (a) The results showed that lines containing BADH-A1b had higher drought resistance than lines containing BADH-A1a. This indicates that the InDel molecular marker InDel254 can be used to identify drought resistance in wheat seedlings, and wheat seedlings containing InDel254 had lower drought resistance than those without InDel254.

[0093] Under salt stress, the growth of some wheat varieties (lines) at the four-leaf stage was as follows: the leaves of lines 1-5 containing the BADH-A1a allelic variant were more withered than those of lines 6-10 containing the BADH-A1b allelic variant. Figure 7 (b) After 25 days of salt stress, the survival rates of lines 1-5 containing the BADH-A1a allele were 56.0%, 71.3%, 59.0%, 66.7%, and 72.0%, respectively, while the survival rates of lines 6-10 containing the BADH-A1b allele were 91.7%, 85.0%, 89.7%, 86.7%, and 88.0%, respectively. Figure 7 (b) The results showed that the lines containing BADH-A1b had higher salt tolerance than the lines containing BADH-A1a. This indicates that the InDel molecular marker InDel254 can be used to identify salt tolerance in wheat seedlings, and wheat seedlings containing InDel254 had lower salt tolerance than those without InDel254.

[0094] The present invention has been described in detail above. For those skilled in the art, 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. Although specific embodiments have been given, 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. Some of the essential features can be applied within the scope of the following appended claims. SEQUENCE LISTING <110> Chifeng College <120> InDel molecular markers associated with the wheat TaBADH-A1 gene and their applications <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 828 <212> DNA <213> Wheat (Triticum aestivum) <400> 1 cgcaagtcaa tgggctgtgt attaaatttt ttttgaaaca ccatgggctg tgcattaatt 60 gtgcctgctc agttggtcaa tcagtcaact tcttactagt attttacttc ctattatggg 120 tgccaaatga atgccagatt tgtcagtgat ccagtagga atcacataaa caagctctaa atgatactcg agaatattg tcagtttttg atgcatttga tttccttttc cttcagatga ttgagcggaa atctgatctg gctaggctcg aggcacttga ttgcgggaag cctcttgatg aagcggcatg ggacatggta tgcgatccac cttttcactg tctgcatgca ttgtattttt 360 gtactccctc cggtcctttt tactctgcac attaggtttg tctaaagtca acctcatcca 420 actttgacca agtttataca aaaaattatt aacattcaca taacaacaca tttattaata gatccatctc ggatatatt ttcatattta ttagatgtta tagatgtta tattttctaa father gtcaaactta gcaaactttg acttcagtca aagctaatgt gcggagtaaa aaggaccgga gggagtacac aggatgtact gatgtgcact atgttctttt tatcaggacg 660 atgtcgccgg ctgctttgag ttcttcgcag gtcatgctga agccttggac aaaaggcaaa 720 atgctgcagt tgctctcccg gagatttta aatgccatct tag cctattggtg 780 tagttgctct aatcacacct tggtatttcc cattcccctc ctgtcttg 828 <210> 2 <211> 574 <212> DNA <213> Wheat (Triticum aestivum ) <400> 2 cgcaagtcaa tgggctgtgt attaattttt ttttgaaaca ccatgggctg tgcattaatt 60 gtgcctgctc agttggtcaa tcagtcaact tcttactagt attttacttc ctattatggg 120 tgccaaatga atgccagatt tgtcagtgat ccagtaggaa atcacataaa caagctctaa 180 aggatactcg agaatatatg tcagtttttg atgcatttga tttccttttc cgtcagatga 240 ttgagcggaa atctgatctg gctaggctcg aggcacttga ttgcgggaag cctcttgatg 300 aagcggcatg ggacatggta tgcgatccac cttttcactg tctgcatgca ttgtattttt 360 gtacacagga tgtactgatg tgcactatgt tctttttatc aggacgatgt cgccggctgc 420 tttgagttct tcgcaggtca tgctgaagcc ttggacaaaa ggcaaaatgc tgcagttgct 480 ctcccggaga attttaaatg ccatcttaag aaggaaccta ttggtgtagt tgctctaatc 540 acaccttggt atttcccatt cccctcctgt cttg 574 <210> 3 <211> twenty two <212> DNA <213> Artificial Sequence <400> 3 cgcaagtcaa tgggctgtgt at 22 <210> 4 <211> twenty four <212> DNA <213> Artificial Sequence <400> 4 caagacagga ggggaatggg aaat 24

Claims

1. An application for detecting InDel-labeled substances, characterized in that, The InDel molecular marker is a double-stranded DNA molecule with a single-stranded nucleotide sequence of positions 365 to 618 of SEQ ID No. 1; the application is any of the following: 1) Application in identifying or assisting in the identification of wheat water stress resistance. 2) Application in breeding wheat for resistance to water stress; The substance contains PCR primers for amplifying wheat genomic DNA fragments including the InDel molecular marker; The PCR primers are primer pairs, each consisting of a forward primer and a reverse primer. The forward primer is a single-stranded DNA that specifically binds upstream of the InDel molecular marker in wheat genomic DNA, and the reverse primer is a single-stranded DNA that specifically binds downstream of the InDel molecular marker in wheat genomic DNA. The sequence of the forward primer is shown in SEQ ID No. 3, and the sequence of the reverse primer is shown in SEQ ID No.

4. The genotype of wheat varieties whose PCR products contain a DNA fragment whose nucleotide sequence is SEQ ID No. 2 in the sequence listing is defined as the deletion type allelic variant BADH-A1b; The genotype of wheat varieties whose PCR products contain DNA fragments whose nucleotide sequences are from positions 365 to 618 of SEQ ID No. 1 in the sequence listing is defined as the insertion type allelic variant BADH-A1a; The betaine content of wheat varieties containing the allelic variant BADH-A1b was consistently significantly higher than that of wheat varieties containing the allelic variant BADH-A1a. The average relative germination rate of wheat varieties containing the allelic variant BADH-A1b was significantly higher than that of wheat varieties containing the allelic variant BADH-A1a.

2. The application according to claim 1, characterized in that, The wheat in question is a wheat inbred line.

3. A method for identifying or assisting in the identification of wheat water stress resistance, characterized in that, Includes the following steps: The method involves detecting whether the genomic DNA of the wheat to be identified contains the InDel molecular marker as described in claim 1. The water stress resistance of the wheat to be identified that does not contain the InDel molecular marker in its genomic DNA is higher than that of the wheat to be identified that contains the InDel molecular marker in its genomic DNA. The water stress resistance is drought stress resistance and / or salt stress resistance.

4. A method for identifying or assisting in the identification of wheat water stress resistance, characterized in that, Includes the following steps: (1) Using the wheat genomic DNA to be identified as a template, PCR amplification was performed using the primer pair described in claim 1 to obtain the PCR product; (2) The resistance of wheat to water stress is identified based on whether the PCR product contains the InDel molecular marker as described in claim 1; The water stress resistance of the wheat to be identified whose PCR product does not contain the InDel molecular marker as described in claim 1 is higher than that of the wheat to be identified whose PCR product contains the InDel molecular marker as described in claim 1. The water stress resistance is drought stress resistance and / or salt stress resistance.

5. A method for breeding wheat with resistance to water stress, the method comprising selecting wheat whose genomic DNA does not contain the InDel molecular marker described in claim 1 as a parent for breeding.

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