Wheat stem rot resistant KASP molecular marker based on TaUGT1433 gene and application

Abstract

The invention discloses a wheat stalk rot resistance KASP molecular marker based on a TaUGT1433 gene and application, and belongs to the field of molecular genetic breeding. The nucleotide sequence of the gene TaUGT1433 is shown as SEQ ID NO.1, the nucleotide sequence of the molecular marker is shown as SEQ ID NO.12, C / T mutation exists at the 101 basic group, and the genotypes of the site comprise CC, TT and CT genotypes. The invention illustrates the effect of the gene TaUGT1433 in the basal stem rot resistance. A high-throughput KASP molecular marker is developed based on the gene, allele types carried by different wheat materials can be detected, and an important molecular tool is provided for screening of wheat anti-FCR germplasm resources.

Description

Technical Field

[0001] The invention relates to the field of molecular genetic breeding, and in particular to a wheat stem rot resistance KASP molecular marker based on TaUGT1433 gene and application thereof. Background Art

[0002] Wheat stem rot (FCR) is a worldwide soil-borne disease caused by the genus Fusarium. The leaf sheaths and stems at the base of the diseased plants turn brown, and the stem nodes necrotize, causing plant death and ear whitening, which seriously affects the formation and filling of wheat grains and reduces yield. In recent years, with the large-scale implementation of farming measures such as returning straw to the fields and continuous cropping of wheat and corn, the disease has continued to spread and has become one of the important new diseases that seriously threaten wheat production.

[0003] During the process of Fusarium infecting the host, a variety of fungal toxins are released, including deoxynivalenol (DON). Studies have shown that DON helps the pathogen spread in the host. Wheat resistance to Fusarium diseases such as fusarium head blight and stem base rot can be improved by degrading or reducing the toxicity of DON. Studies have shown that plant UDP-glucosyltransferase (UGT) can convert DON into DON-3-glucoside (D3G) through glycosylation, thereby reducing the harm of DON toxin. At present, it has been determined that multiple UGT genes can enhance plant resistance to fusarium head blight by glycosylation of DON toxins, such as TaUGT1, TaUGT2, TaUGT3, TaUGT4, TaUGT5, TaUGT6 and TaUGT7, among which TaUGT3, TaUGT5, TaUGT6 and TaUGT7 have been shown to enhance host resistance to fusarium head blight in overexpressed Arabidopsis and wheat. However, the current research on UGT genes that improve wheat stem base rot resistance is still insufficient. Therefore, determining the key genes that can effectively enhance wheat resistance to stem rot is of great significance for the genetic improvement of wheat resistance to stem rot. On this basis, based on the sequence differences of the resistant and susceptible alleles of the key genes for wheat resistance to stem rot, the development of high-throughput KASP molecular markers that can distinguish different alleles is of great significance for the rapid screening of wheat materials with stronger resistance to stem rot and accelerating the genetic improvement of wheat resistance to stem rot. Summary of the invention

[0004] The purpose of the present invention is to provide a wheat stem base rot resistance KASP molecular marker based on TaUGT1433 gene and its application to solve the problems existing in the above-mentioned prior art. The KASP molecular marker provided by the present invention can quickly distinguish wheat materials with disease-resistant alleles or susceptible alleles, thereby effectively identifying wheat materials with stronger resistance to stem base rot, and providing an important molecular tool for the screening of wheat FCR-resistant germplasm resources.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The invention provides a gene TaUGT1433 related to wheat stem base rot resistance, and the nucleotide sequence of the gene TaUGT1433 is shown as SEQ ID NO.1.

[0007] The present invention also provides application of the gene TaUGT1433 in distinguishing wheat stem base rot resistance.

[0008] The present invention also provides a molecular marker for distinguishing wheat stem base rot resistance based on the gene TaUGT1433, the nucleotide sequence of the molecular marker is shown in SEQ ID NO.12, there is a C / T mutation at the 101st base, and the genotype of this site includes CC, TT and CT genotypes.

[0009] The present invention also provides a KASP primer set for detecting the molecular marker, comprising an upstream primer F1 with a nucleotide sequence as shown in SEQ ID NO.9, an upstream primer F2 with a nucleotide sequence as shown in SEQ ID NO.10, and a downstream primer R with a nucleotide sequence as shown in SEQ ID NO.11.

[0010] The present invention also provides a detection kit for the molecular marker, comprising the KASP primer set.

[0011] The present invention also provides an application of the KASP primer set or the detection kit in distinguishing wheat base stem rot resistance.

[0012] The present invention also provides a method for distinguishing wheat stem base rot resistance, comprising the following steps:

[0013] The genomic DNA of the wheat sample to be tested is used as a template, and the template is amplified by PCR using the KASP primer set or the detection kit. After the PCR amplification is completed, the fluorescent signal is read, the fluorescent signal is analyzed and converted, the genotype is identified, and the resistance of the wheat stem base rot is determined according to the genotype;

[0014] If the identified genotype is TT, the wheat sample to be tested is judged to be a susceptible material to stem base rot; if the identified genotype is CC, the wheat sample to be tested is judged to be a resistant material to stem base rot.

[0015] Furthermore, the PCR amplification system includes 2 μL 10 ng / μL DNA template, 5 μL 2×Master mix and 0.14 μL 10 μM mixed primer, and the volume ratio of upstream primer F1, upstream primer F2 and downstream primer R in the mixed primer is 12:12:30.

[0016] Furthermore, the PCR amplification

[0017] The program was a pre-denaturation stage at 95°C for 10 min; a denaturation stage at 95°C for 20 s, followed by a renaturation extension stage at 65°C for 40 s for 10 cycles, with a decrease of 1°C in each cycle; and a final denaturation stage at 95°C for 20 s, then 55°C for 40 s, for 35 cycles.

[0018] The present invention also provides an application of the KASP primer set or the detection kit in rapidly screening wheat materials with stronger resistance to stem base rot.

[0019] The present invention discloses the following technical effects:

[0020] The present invention identified a UGT gene related to wheat stem base rot resistance and named it TaUGT1433. The function of the gene in stem base rot resistance was verified by gene sequence difference analysis of wheat stem base rot resistant and susceptible materials, virus-induced gene silencing, gene overexpression analysis and other methods. On this basis, a high-throughput KASP molecular marker was developed based on the sequence difference of the resistant and susceptible alleles of TaUGT1433, which can detect the allele types carried by different wheat materials.

[0021] The KASP molecular marker provided by the present invention can quickly screen out wheat materials with disease-resistant alleles or susceptible alleles, thereby effectively identifying wheat materials with stronger resistance to stem base rot, and providing an important molecular tool for screening wheat FCR-resistant germplasm resources.

[0022] The KASP marker developed by the present invention can quickly identify the TaUGT1433 allele type carried by wheat materials, predict their disease resistance performance, improve the efficiency of gene aggregation and breeding of disease-resistant materials, save experimental costs and time, and accelerate the process of wheat disease-resistant breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 Figure 1 is a comparison of the nucleic acid sequences of TaUGT1433 gene in Chinese Spring, New Wheat 26 and 04 Zhong 36, where A is a schematic diagram of the differential nucleic acid sequences among Chinese Spring, New Wheat 26 and 04 Zhong 36; B is a schematic diagram of the two genotype structures of TaUGT1433;

[0025] Figure 2 This is a comparison of TaUGT1433b and reported UGT amino acid sequences, where the black frame is the PSPG domain; black, red, and cyan backgrounds represent sequence homology of 100%, ≥75%, and ≥50%, respectively; ellipsis indicates placeholders that cannot be matched during the alignment process;

[0026] Figure 3 This is the evolutionary tree of TaUGT1433 and other UGTs, among which TaUGT1, TaUGT2, TaUGT3, TaUGT4, TaUGT5, TaUGT6, TaUGT7, TraesCS5A02G149600 and TraesCS5B02G148300 are from wheat; DOGT1 is from Arabidopsis; TuG1812G0500001685 is from Urartu wheat; TRITD5Av1G113020 and TRITD 5Bv1G093730 is from durum wheat; TRIDC5AG025050 and TRIDC5BG026280 are from wild emmer wheat; AET5Gv20385300 is from Aegilops tauschii; BRADI_5g03300 is from Brachypodium distichum; Os04t0204100 is from rice; HvUGT13248 is from barley; and SORBI_3006G024000 is from sorghum; Zm00001eb337300 is from maize;

[0027] Figure 4 Figure 2 is a diagram of BSMV-VIGS-induced gene silencing at the base of wheat stem and its effect on stem base rot resistance, where A is the virus spot situation on wheat leaves and the stem base rot resistance phenotype; B is the gene silencing situation detected by RT-qPCR; C is the severity of stem base rot; WT: blank control in VlGS experiment; BMSV0: blank vector control in VIGS experiment; BMSV pds : PDS gene treatment in VIGS experiments; BMSV ugt :Silencing of TaUGT1433; Different letters indicate significant differences among materials (P<0.05);

[0028] Figure 5 Detection and expression analysis of wheat TaUGT1433b transgenic plants, where A is the detection of TaUGT1433b transgenic plant vectors; B is the expression analysis of TaUGT1433b in three transgenic lines; * indicates that there is a significant difference between the transgenic line and the wild-type Fielder (P<0.05);

[0029] Figure 6Overexpression of wheat TaUGT1433b improves plant resistance to stem rot, where A is the phenotype of the control Fielder and three overexpression lines against stem rot; B is the disease index of stem rot in the seedling stage of Fielder and three overexpression lines; * indicates a significant difference between the transgenic lines and the wild type (P<0.05);

[0030] Figure 7 Root growth (A) and root length (B) of wild-type and transgenic lines under 10 μM DON treatment; ** indicates that the root length under DON treatment is significantly different from that under control conditions (P<0.01);

[0031] Figure 8 Haplotype analysis and geographical distribution of TaUGT1433, where A is the genotyping of molecular marker K5D2220; B is the grouping of 321 Chinese common wheat varieties / lines typed with molecular marker K5D2220. DETAILED DESCRIPTION

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0034] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0035] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0036] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0037] In the previous study, the present invention conducted a GWAS analysis on 358 Chinese wheat varieties / lines collected independently (the population was disclosed in the literature "Jin J J. Identification of a novel genomic region associated with resistance to Fusarium crown rot in wheat" and "Gao Y. Dissecting the genetic basis of grain morphology traits in Chinese wheat by genome wide association study"), and found that a gene encoding UGT showed significant differential expression in wheat stem base rot resistant and susceptible varieties, indicating that it may play an important role in the stem base rot infection process. The gene was named TaUGT1433, and its nucleotide sequence is shown in SEQ ID NO.1.

[0038] SEQ ID NO.1 (TaUGT1433 gene sequence):

[0039]

[0040] On this basis, the present invention further illustrates its role in wheat resistance to stem rot by comparing the sequence differences of TaUGT1433 gene between resistant and susceptible materials of wheat stem rot, virus-induced gene silencing, gene overexpression and other methods to verify the function of the gene, and develops a high-throughput KASP molecular marker that can distinguish different alleles based on the sequence differences of resistant and susceptible alleles. The following examples are used for detailed demonstration.

[0041] The disease-resistant variety (04zhong36) and the susceptible variety (Xinmai 26) in the examples were provided by the Cotton Research Institute of the Chinese Academy of Agricultural Sciences and the Xinxiang Academy of Agricultural Sciences of Henan Province, respectively.

[0042] Example 1 Gene sequence analysis and function verification

[0043] 1. Gene sequence analysis

[0044] The DNA of the resistant variety (04zhong36) and the susceptible variety (Xinmai 26) was extracted by the hexadecyltrimethylammonium bromide (CTAB) method. According to the sequence information of the TaUGT1433 gene in the reference genome (Chinese Spring v1.1), specific primers (upstream primer: CACTGCCTGGACGCCTATT, SEQ ID NO.2, downstream primer: GTCGGCCACTCTAAACTTCTG, SEQ ID NO.3) were designed, and the gene sequence of TaUGT1433 was amplified using high-quality DNA of the resistant and susceptible varieties as templates. The PCR product was then sent to the company for sequencing, and the sequence differences between the resistant and susceptible materials were compared using the software DNAMAN. The results showed that compared with the susceptible material, the gene sequence of TaUGT1433 in the resistant material had one SNP difference in the intron region and the exon region, in which the stop codon (TGA) in the exon region was replaced by a tryptophan codon (TGG). Therefore, the genotype corresponding to Xinmai 26 was named TaUGT1433a, and the genotype corresponding to 04zhong36 was named TaUGT1433b ( Figure 1 ). The translation analysis of TaUGT1433b showed that the gene can completely encode a polypeptide containing 475 amino acids.

[0045] The nucleotide sequence of the TaUGT1433b gene is shown in SEQ ID NO.4, and the amino acid sequence encoded by the gene is shown in SEQ ID NO.5.

[0046] SEQ ID NO.4:

[0047]

[0048] SEQ ID NO.5:

[0049] METTVPAATATTSSSSGHGAGGGAARVLLLPYPGAQGHTNPMLQLGRRLAYHGLRPTLVATRYVLSTTPPPGAPFGVAAISDGCDAGGMASCPDMAEYVSRLAAVGSETLRELLLSEAR AGRPVRVLVYDAHLAWARRVAQASGVAAAAFFSQPCAVDVVYGELWAGRLALPATDGRALLARGALGVELGPEDMPPFAAVPESQPTFLKVSVGQFEGLEDADDVLVNSFRDLEPKEVE YMELTWRAKMVGPTLPSFYLDDDRLPSNKSYGFDFFNGDALCMDWLEKQSNSSVVLVSYGTVSDYDATQLEELGNGLCNSGKPFLWVVRSNEEHKLSKELKEKCEEIGLIVSWCPQLEV LAHRAIGCFVTHCGWNSTLEALANGVPLVGIPHWADQPTIAKYVESAWSMGVRVRKDNNGYLKLEEVERCIREVMDIEKKDEYKRNAVKWMQKAKEAMQEGGSSDKNIAEFATKYSSI.

[0050] The amino acid sequences of UGT proteins related to resistance to stem base rot and head blight that have been published were searched and downloaded, and the amino acid sequences were compared using DNAMAN. The results showed that the amino acid sequence encoded by TaUGT1433b had high similarity with the amino acid sequences encoded by HvUGT13248, Bradi5g03300, and Os04g0206600, which were 89.3%, 73.3%, and 70.4%, respectively, and was exactly the same as the amino acid sequence encoded by AET5Gv20385300, but had low similarity with the remaining UGT proteins ( Figure 2 ). Further amino acid sequence multiple alignment analysis showed that TaUGT1433 has a conserved PSPG-box functional domain at the C-terminus, and this segment has a high similarity with other UGTs.

[0051] Based on the amino acid sequence encoded by TaUGT1433b, all homologous genes in Arabidopsis and Poaceae were searched in the Ensembl Plants database by BLAST, and their amino acid sequences were downloaded. The amino acid sequences were multiple aligned using the Neighbor-Joining calculation method using MEGA7 software. The results showed that TaUGT1433b was classified into the same group as AET5Gv20385300, while other UGT proteins including homologous genes on chromosomes 5A and 5B were classified into different groups ( Figure 3 ).

[0052] 2. BSMV-VIGS functional verification of TaUGT1433b

[0053] In order to verify the effect of gene TaUGT1433b on wheat resistance to stem rot, the gene was silenced using the BSMV-VIGS system and inoculated for identification. The gene silencing fragment was predicted through the website https: / / vigs.solgenomics.net / . Primers were designed according to the target sequence, and the adapter sequence was added to its end to amplify the target fragment of the target gene.

[0054] The primers designed according to the target sequence are as follows:

[0055] Upstream primer: TTCTTCCGTTGCTAGC GTTTGGCCCAACCTTGCCA, SEQ ID NO.6;

[0056] Downstream primer: TTTTTTTTTAGCTAGC TTCCGGAATTGCACAGTCCA, SEQ ID NO.7.

[0057] The amplified target fragment is as follows (SEQ ID NO.8):

[0058] TTCTTCCGTTGCTAGC GTTGGCCCAACCTTGCCATCATTCTACCTCGACGATGATCGCCTACCATCCAATAAATCATACGGTTTCGACTTTTTCAACGGTGATGCACTGTGCATGGATTGGCTAGAGAAGCAAAGCAATTCATCCGTTGTGCTGGTGTCCTATGGGACTGTCTCTGATTATGATGCAACCCAACTAGAGGAGCTTGGCAATGGACTGTGCAATTCCGGAA GCTAGCTAAAAAAAAA .

[0059] Among them, the underlined italic parts in the upstream and downstream primers and the amplified target fragment are linker sequences.

[0060] The obtained target fragment was combined with the linearized BMSV γ The vector (the vector comes from Nanjing Agricultural University and is disclosed in the document "Wang X. Establishment of an effective virus induced gene silencing system with BSMV in Haynaldia villosa") was connected to construct a vector BMSV carrying the target gene ugt . The reaction liquid of the recombinant BSMV virus carrying the target gene was inoculated onto the second leaf of wheat in the three-leaf stage by friction inoculation. One week after inoculation, virus spots appeared on the new leaves. At this time, the disease-resistant material 04-36 was inoculated with Fusarium graminearum using the diseased grain method. About 15 days after inoculation, the wheat leaves showed obvious whitening and the base of the stem turned brown. At this time, samples were taken for quantitative identification, and the incidence of stem base rot in the materials was counted. The results showed that the expression level of TaUGT1433b was significantly reduced, indicating that the target gene had been successfully silenced ( Figure 4 B). Compared with the control, after silencing TaUGT1433b, the stem base rot resistance of 04zhong36 was significantly reduced ( Figure 4 C), indicating that TaUGT1433b plays an important role in wheat resistance to stem rot.

[0061] 3. Acquisition and expression analysis of wheat TaUGT1433b transgenic lines

[0062] The TaUGT1433b gene was transferred into the vector PWMB110 to construct an overexpression vector, and then transformed into wheat using the Agrobacterium-mediated method. After the transgenic T0 generation plants grew to a certain stage, the genomic DNA was screened using PCR detection technology to confirm the positive transgenic plants. Subsequently, the seeds of each positive transgenic plant were harvested and sown separately. The T1 and T2 generation plants were sampled individually and the homozygosity of the target gene at the DNA level was detected ( Figure 5 A). After the homozygous transgenic lines were determined, RNA was extracted and the transcription level of TaUGT1433b gene in transgenic plants was detected by RT-qPCR technology ( Figure 5 B). Based on the above results, the transgenic lines OETaUGT133b-L1, OETaUGT133b-L3 and OETaUGT133b-L4 (T3) that can be stably inherited and efficiently expressed were selected for the subsequent functional verification work.

[0063] 4. Phenotypic evaluation of resistance of TaUGT1433b transgenic wheat to stem rot

[0064] The stable expression and homozygous transgenic T3 lines obtained by the above steps and wild-type wheat Fielder (provided by Professor Jie Chaojie of China Agricultural University, obtained in September 2020) were used to identify stem base rot. The results showed that compared with the control group, the degree of stem browning of the transgenic lines OETaUGT133b-L1, OETaUGT133b-L3 and OETaUGT133b-L4 was significantly reduced, and the disease resistance index was also significantly reduced ( Figure 6 ). Therefore, TaUGT1433b plays a certain role in wheat stem rot resistance and can inhibit the damage of Fusarium graminearum to wheat stems to a certain extent.

[0065] 5. Overexpression of TaUGT1433b reduces the inhibition of DON toxin on root growth of wheat seedlings

[0066] Root growth experiments were performed on transgenic lines OETaUGT133b-L1, OETaUGT133b-L3 and OETaUGT133b-L4 and wild-type Fielder to evaluate the effect of overexpressing TaUGT1433b on DON toxin inhibition of root growth in wheat seedlings. Experimental methods refer to "Bethke G, Huang YD, Hensel G, Heinen S, Liu CC, Wyant SR, LiX, Quin MB, Mccormick S, Morrell PL, Dong YH, Kumlehn J, Salvi S, Berthiller F, Muehlbauer G J. (2023). UDP-glucosyltransferase HvUGT13248 confers type II resistance to Fusarium graminearum in barley. Plant Physiology 193: 2691-2710". The experimental results showed that after treatment with 10 μM DON, the root growth of both wild-type and transgenic strains was significantly inhibited ( Figure 7 However, compared with the wild type, the root growth inhibition rate of the transgenic lines was significantly reduced ( Figure 7 B), indicating that overexpression of TaUGT1433b can alleviate the inhibitory effect of DON toxin on root growth.

[0067] Example 2 KASP molecular marker for wheat resistance to stem rot based on TaUGT1433 gene

[0068] According to the results of gene sequence analysis in Example 1, based on the SNP differences in the exon region of the DNA of 04zhong36 and Xinmai26, a KASP molecular marker K5D2220 was designed, and the primer sequence for amplifying the molecular marker is as follows:

[0069] Upstream primer F1:

[0070] 5'- GAAGGTGACCAAGTTCATGCT GCAATGGTGGGTTGGTCAGCT-3' (SEQ ID NO.9);

[0071] Upstream primer F2:

[0072] 5'- GAAGGTCGGAGTCAACGGATT CAATGGTGGGTTGGTCAGCC-3' (SEQ ID NO. 10);

[0073] Downstream primer R:

[0074] 5'-GTACCTCTAGTGGGTATTCCGCATT-3' (SEQ ID NO. 11).

[0075] The underlined italic part in the upstream primer is the linker sequence, and the bold base T (for SEQ ID NO. 9) and C (for SEQ ID NO. 10) at the 3' end are typing site sequences.

[0076] The sequence of molecular marker K5D2220 is as follows:

[0077] GACCTCCTCTAGCTTTAGATATCCATTATTGTCCTTCCGTACACGCACACCCATGCTC

[0078] CATGCACTCTCCACATACTTTGCAATGGTGGGTTGGTCAGCRCAATGCGGAATACCCACT

[0079] AGAGGTACACCATTAGCAAGTGCCTCTAATGTCGAGTTCCATCCACAATGGGTAACAAA

[0080] GCAACCTGCATGTATGTCAGGAA (SEQ ID NO. 12).

[0081] Among them, the bold "R" is a SNP site, that is, the 101st base of the sequence shown in SEQ ID NO.12 is a SNP site, and its base is T or C (the base of the complementary chain is A or G).

[0082] The base of the susceptible material Xinmai 26 at the above SNP site is A, and the base of the resistant material 04 Zhong36 at the above SNP site is G ( Figure 8 A), using molecular marker K5D2220 genotyping to detect the distribution of resistance to wheat stem rot in a natural population consisting of 321 Chinese wheat varieties (lines). The steps are as follows:

[0083] (1) Extract DNA from 321 natural wheat populations;

[0084] (2) Using the sample DNA as a template, PCR amplification was performed using the primers designed above (upstream primer F1, upstream primer F2, downstream primer R).

[0085] Amplification system: 2 μL 10 ng / μL DNA sample, 5 μL 2×Master mix and 0.14 μL mixed primer, the final concentration of the mixed primer is 10 μM, and the volume ratio of upstream primer F1, upstream primer F2 and downstream primer R is 12:12:30.

[0086] Amplification program: pre-denaturation stage at 95°C for 10 min; denaturation stage at 95°C for 20 s, then annealing extension stage at 65°C for 40 s for 10 cycles, with a decrease of 1°C in each cycle; final denaturation stage at 95°C for 20 s, 55°C for 40 s, for 35 cycles.

[0087] (3) After the reaction is completed, the fluorescence data of the PCR reaction product is directly read using a real-time fluorescence quantitative PCR instrument, and the results of the fluorescence scanning are automatically converted into a graph.

[0088] The results showed that K5D2220 could divide the population into two groups, and the mean disease index between the two groups was significantly different ( Figure 8 The base type of the susceptible group is A, and the base type of the resistant group is G. The geographical distribution of the two base types of SNP-2220 was analyzed using the Wheat Genome Variation Joint Database (http: / / wheat.cau.edu.cn / WheatUnion / ). The results showed that the proportion of the disease-resistant allele type was higher in Tibet and other places in my country, while the proportion was relatively small in the Huanghuai wheat region.

[0089] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A gene TaUGT1433 associated with wheat stem rot resistance, characterized in that: The nucleotide sequence of the gene TaUGT1433 is shown in SEQ ID NO.

1.

2. Use of the gene TaUGT1433 described in claim 1 in distinguishing wheat stem base rot resistance.

3. A molecular marker for distinguishing wheat stem rot resistance based on the gene TaUGT1433 according to claim 1, characterized in that: The nucleotide sequence of the molecular marker is shown in SEQ ID NO.

12. There is a C / T mutation at the 101st base, and the genotypes of this site include CC, TT and CT genotypes.

4. A KASP primer set for detecting the molecular marker according to claim 3, characterized in that: It comprises an upstream primer F1 with a nucleotide sequence as shown in SEQ ID NO.9, an upstream primer F2 with a nucleotide sequence as shown in SEQ ID NO.10 and a downstream primer R with a nucleotide sequence as shown in SEQ ID NO.

11.

5. A molecular marker detection kit according to claim 3, characterized in that: Comprising the KASP primer set according to claim 4.

6. Use of the KASP primer set according to claim 4 or the detection kit according to claim 5 in distinguishing wheat base stem rot resistance.

7. A method for distinguishing wheat stem rot resistance, characterized in that: The steps include: Using the genomic DNA of the wheat sample to be tested as a template, using the KASP primer set described in claim 4 or the detection kit described in claim 5 to perform PCR amplification on the template, reading the fluorescent signal after the PCR amplification is completed, analyzing and converting the fluorescent signal, identifying the genotype, and judging the resistance of wheat to stem base rot according to the genotype; If the identified genotype is TT, the wheat sample to be tested is judged to be a susceptible material to stem base rot; if the identified genotype is CC, the wheat sample to be tested is judged to be a resistant material to stem base rot.

8. The method according to claim 7, characterized in that The PCR amplification system includes 2 μL 10 ng / μL DNA template, 5 μL 2×Master mix and 0.14 μL 10 μM mixed primers, wherein the volume ratio of upstream primer F1, upstream primer F2 and downstream primer R in the mixed primers is 12:12:

30.

9. The method according to claim 7, characterized in that: The PCR amplification program is as follows: a pre-denaturation stage at 95°C for 10 min; a denaturation stage at 95°C for 20 s, followed by a renaturation extension stage at 65°C for 40 s for 10 cycles, with a decrease of 1°C in each cycle; and a final denaturation stage at 95°C for 20 s, then 55°C for 40 s, for 35 cycles.

10. Use of the KASP primer set according to claim 4 or the detection kit according to claim 5 in rapid screening of wheat materials with stronger resistance to stem base rot.

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