Method for creating and polymerizing short fragment translocation line carrying wheat gibberellic disease-resistant gene Fhb7 and application of short fragment translocation line

Abstract

The invention belongs to the field of wheat genetic breeding, and discloses a creation and polymerization method of a short fragment translocation line carrying a wheat gibberellic disease-resistant gene Fhb7 and application of the short fragment translocation line carrying the wheat gibberellic disease-resistant gene Fhb7. According to the invention, by combining artificial climate chamber generation addition, molecular marker-assisted selection, in-situ hybridization identification and gibberellic disease resistance identification technologies, polymerization of the gibberellic disease resistance gene Fhb7 on wheat 7B and 7D chromosomes is realized; the Fhb7 is subjected to subgenome polymerization on different chromosomes, so that the resistance of the wheat to the gibberellic disease can be remarkably improved, and the Fhb7 has an important application prospect in wheat gibberellic disease resistance breeding.

Description

Technical Field

[0001] The invention belongs to the field of wheat genetic breeding, and specifically relates to the creation, polymerization method and application of a short segment translocation line carrying a wheat scab resistance gene Fhb7. Background Art

[0002] As one of the most important food crops in the world, wheat is loved by people all over the world because of its easy processing, good quality, wide variety and rich flavor. Its planting area has always ranked first among cereal crops. However, wheat is threatened by various diseases throughout its growth process, which seriously affects wheat yield and quality. Among them, wheat fusarium head blight (FHB), a fungal disease caused by Fusarium graminearum, seriously affects wheat yield and quality.

[0003] Studies have shown that discovering fusarium scab resistance genes and breeding disease-resistant wheat varieties are one of the most economical and effective ways to resist pathogens. Fhb7 from Elytium longipedum is a major fusarium scab resistance gene that has broad-spectrum resistance to Fusarium pathogens. Years of multi-point experiments have shown that the introduction of Fhb7 can not only significantly improve wheat's resistance to fusarium scab, but also has no significant adverse effects on agronomic traits such as wheat yield. In addition, there is a significant dosage effect in the expression of Fhb7. Summary of the invention

[0004] In view of the above problems existing in the existing progress, the present invention uses ph1b to induce the recombination of partial homologous chromosomes, and creates wheat-Elygropyrum longissima short segment translocation lines carrying Fhb7 on wheat chromosomes 7B and 7D, respectively, to achieve the polymerization and utilization of Fhb7 in the seventh homologous group of wheat, so that its resistance to fusarium head blight is further improved on the original basis. After the polymerization is successful, Fhb7 is transferred to the main cultivated wheat varieties in my country, in order to obtain new wheat lines with good agronomic traits and medium resistance to fusarium head blight or above.

[0005] The present invention utilizes the Chinese Spring ph1b gene to induce the recombination of partially homologous chromosomes, and creates two groups of wheat-Elygropyrum longissima short segment translocation lines Shannong K23-6 (T7BS.7BL-7el2L) and Shannong K12-30 (T7DS.7DL-7EL) carrying Fhb7 on wheat chromosomes 7B and 7D, respectively. Subsequently, Shannong K23-6 and Shannong K12-30 are used as parents to construct a hybrid combination, and a wheat-Elygropyrum longissima short segment translocation line Shannong K10-6 carrying Fhb7 on chromosomes 7B and 7D at the same time is created by molecular marker-assisted selection, and this result is confirmed by in situ hybridization (FISH / GISH). In order to evaluate the resistance of Fhb7 to Fusarium head blight in different subgenome aggregate lines of wheat, Shannong K10-6 was used as the donor parent of Fhb7, and Fhb7 was introduced into the susceptible extended wheat variety Mengmai 023 through molecular marker-assisted backcrossing. A BC2F3 population was created, which included 30 7B Fhb7+ Department, 30 7D Fhb7+ Department, 30 7B Fhb7+ +7D Fhb7+ The resistance of BC2F3 lines to fusarium head blight was evaluated in a plant growth chamber. The results showed that 21 days after inoculation, the number of diseased spikelets (NDS) of the highly resistant control variety Sumai No. 3 was 1.3, while the NDS of the lines not carrying the Fhb7 gene was 7.9; Fhb7+ System and 7D Fhb7+ The NDS of the lines are 3.1 and 2.9 respectively, while the 7B carrying two Fhb7 Fhb7+ +7D Fhb7+ The NDS of the aggregated line was only 1.2, which was not significantly different from the high-resistance control Sumai 3. Therefore, subgenomic aggregation of Fhb7 on different chromosomes can significantly improve wheat resistance to Fusarium head blight, and has important application prospects in wheat Fusarium head blight resistance breeding.

[0006] The present invention discloses a method for creating and polymerizing a wheat-loblolly grass resistant to scab disease short segment translocation line, which is characterized by the following steps:

[0007] (1) The wheat-deploid Thinoploid Elymus ovatus chromosome substitution line 7el2 (7B) carrying Fhb7 was used as the male parent and the Chinese Spring ph1b mutant (CS ph1b) was used as the female parent to obtain F1;

[0008] (2) backcrossing the F1 generation obtained in step (1) with CS ph1b, extracting genomic DNA from the BC1F1 generation plants, and detecting them using the specific molecular markers Xpsr128, Xpsr574 and XAWJL3 of the ph1b gene and the functional marker K-Fhb7 of Fhb7; screening out individual plants that are homozygous for the ph1b gene and heterozygous for the Fhb7 gene, and obtaining a number of BC1F2 generation plants after self-pollination of these individual plants, and extracting genomic DNA from all of them;

[0009] (3) Using the functional markers of Fhb7, K-Fhb7, and PSY-E2, K-PSY, the functional markers of PSY-E2, genotyping the BC1F2 generation plants was performed to screen for a wheat-decaploid Thinoplophora elongatum short segment translocation line carrying the Fusarium fusca resistance gene Fhb7 but without the yellow pigment gene PSY-E2. After self-pollination, the line was named Shannong K23-6.

[0010] (4) The PCR amplification reaction products were subjected to polyacrylamide gel electrophoresis and agarose gel electrophoresis to analyze the genotypes of the BC1F1 generation plants, BC1F2 generation plants, and Shannong K23-6;

[0011] (5) In situ hybridization identification of Shannong K23-6;

[0012] (6) The wheat-diploid Thinopsis elongata chromosome substitution line 7E (7D) carrying Fhb7 was used as the male parent and the Chinese Spring ph1b mutant (CS ph1b) was used as the female parent to obtain F1;

[0013] (7) F1 was backcrossed with CS ph1b, and genomic DNA was extracted from the BC1F1 plants. The ph1b gene-specific molecular markers Xpsr128, Xpsr574, and XAWJL3 and the functional marker K-Fhb7 of Fhb7 were used for detection. Individual plants that were homozygous for the ph1b gene and heterozygous for the Fhb7 gene were screened, and several BC1F2 plants were obtained after self-pollination of these individual plants, and their genomic DNA was extracted from all of them.

[0014] (8) The functional marker K-Fhb7 of Fhb7 and the linkage marker KE7005 upstream of Fhb7 were used to perform genotyping on the BC1F2 generation plants, and a wheat-diploid Elymus elongatus short segment translocation line carrying K-Fhb7 but not KE7005 was selected. After self-pollination, the line was named Shannong K12-30.

[0015] (9) The PCR amplification reaction products were subjected to polyacrylamide gel electrophoresis and agarose gel electrophoresis to analyze the genotypes of BC1F1 generation plants, BC1F2 generation plants, and Shannong K12-30;

[0016] (10) In situ hybridization was performed on Shannong K12-30;

[0017] (11) Shannong K23-6 was used as the male parent and Shannong K12-30 as the female parent, and the F1 was obtained by hybridization;

[0018] (12) The F1 obtained in step (11) was self-pollinated to obtain F2, and genomic DNA was extracted from the F2 plants. The molecular marker KE7205 specific to chromosome 7E of the diploid Elymus elongatus and the molecular marker XsdauK79 specific to chromosome 7e12 of the decaploid Elymus elongatus were used for detection. A wheat-Elymus elongatus short segment translocation line carrying Fhb7 on both chromosomes 7B and 7D was selected, and self-pollination was continued. After homozygosity was stabilized, it was named Shannong K10-6;

[0019] (13) The PCR amplification products were subjected to agarose gel electrophoresis to analyze the genotypes of the F2 plants and Shannong K10-6;

[0020] (13) In situ hybridization was performed on Shannong K10-6.

[0021] The molecular markers in the above step (2) are as follows:

[0022] Xpsr128, forward primer P1, sequence is SEQ ID NO.1; reverse primer P2, sequence is SEQ ID NO.2;

[0023] Xpsr574, forward primer P3, sequence is SEQ ID NO.3; reverse primer P4, sequence is SEQ ID NO.4;

[0024] XAWJL3, forward primer P5, sequence is SEQ ID NO.5; reverse primer P6, sequence is SEQ ID NO.6;

[0025] K-Fhb7, forward primer P7, sequence is SEQ ID NO.7; reverse primer P8, sequence is SEQ ID NO.8;

[0026] The molecular markers in the above step (3) are as follows:

[0027] K-PSY, forward primer P9, sequence is SEQ ID NO.9; reverse primer P10, sequence is SEQ ID NO.10.

[0028] The molecular markers in the above step (8) are as follows:

[0029] KE7005, forward primer P11, sequence is SEQ ID NO.11; reverse primer P12, sequence is SEQ ID NO.12.

[0030] The molecular markers in the above step (12) are as follows:

[0031] KE7205, forward primer P13, sequence is SEQ ID NO.13; reverse primer P14, sequence is SEQ ID NO.14;

[0032] XsdauK79, forward primer P15, sequence is SEQ ID NO.15; reverse primer P16, sequence is SEQ ID NO.16.

[0033] The wheat-Elygropyrum elongatum short segment translocation line carrying the scab resistance gene Fhb7 on wheat chromosomes 7B and 7D created by the method provided by the invention can be used in wheat scab resistance breeding.

[0034] The invention provides a method for creating a subgenomic aggregate line carrying a wheat fusarium resistant gene Fhb7 on both wheat chromosomes 7B and 7D. The method is characterized in that: a wheat-Elymus elongatus short segment translocation line Shannong K10-6, which carries Fhb7 on both chromosomes 7B and 7D, is used as a donor parent of Fhb7, a major wheat variety in my country is used as a recurrent parent, backcrossing is performed twice in a row and then self-pollinated three times, and auxiliary selection and identification are performed using a molecular marker KE7205 specific to the chromosome 7E of the diploid Elymus elongatus and a molecular marker XsdauK79 specific to the chromosome 7e12 of the decaploid Elymus elongatus, homozygous individual plants carrying Fhb7 on both chromosomes 7B and 7D are retained, and a subgenomic aggregate line carrying Fhb7 on both chromosomes 7B and 7D is obtained.

[0035] The subgenome aggregation system provided by the present invention carrying Fhb7 on chromosomes 7B and 7D at the same time can be used in:

[0036] (1) Application in breeding new wheat varieties resistant to ergot;

[0037] (2) Application for improving wheat resistance to Fusarium pathogens;

[0038] (3) Its application as a wheat ergot resistance breeding material in relevant scientific research and industrial practice.

[0039] The method provided by the invention creates wheat-loblolly grass resistant fusarium head blight short segment translocation lines Shannong K23-6, Shannong K12-30 and Shannong K10-6 carrying Fhb7 on different chromosomes, and discloses the application of the lines in wheat fusarium head blight resistance breeding.

[0040] The beneficial effects of the present invention mainly lie in: utilizing the chromosome engineering method, combining molecular marker-assisted selection and in situ hybridization identification technology, respectively creating wheat-Elygropyrum fusca resistant short segment translocation lines Shannong K23-6 and Shannong K12-30 carrying Fhb7 on chromosomes 7B and 7D, and constructing hybrid combinations using the two as parents, combining molecular marker-assisted selection and in situ hybridization identification technology, creating wheat-Elygropyrum fusca resistant short segment translocation line Shannong K10-6 carrying Fhb7 on chromosomes 7B and 7D at the same time. By hybridizing, backcrossing and selfing Shannong K10-6 with my country's main wheat variety Mengmai 023, and combining molecular marker-assisted selection, artificial climate chamber generation and ergot resistance identification techniques, a group of improved materials with resistance to ergot were created, carrying Fhb7 on both wheat chromosomes 7B and 7D. The results showed that compared with the short fragment translocation line carrying a single Fhb7 gene, subgenome aggregation of Fhb7 on different chromosomes can significantly improve wheat resistance to ergot, which has important application prospects in wheat breeding for resistance to ergot. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The figure shows the electrophoresis detection results of molecular markers K-PSY and K-Fhb7 in the BC1F2 generation population samples of the wheat-decaploid Thinopyrum elongatum short segment translocation line. Lanes 1-10 represent different plants of the BC1F2 generation, among which lane 10 is the genotype that breaks the linkage between Fhb7 and PSY-E2.

[0042] Figure 2 Figure shows the results of in situ hybridization identification of the wheat-Elygropyrum elongatum short fragment translocation line.

[0043] Figure 3 The figure shows the electrophoresis detection results of molecular markers KE7005 and K-Fhb7 in the BC1F2 generation population samples of the wheat-diploid Elymus elongatus short segment translocation line. Lanes 1-12 represent different plants of the BC1F2 generation, among which lane 4 is a genotype carrying the K-Fhb7 locus but not the KE7005 locus.

[0044] Figure 4 The figure shows the electrophoresis detection results of molecular markers KE7205 and XsdauK79 in Shannong K23-6 / Shannong K12-30 F2 population samples. Lanes 1-11 represent different individual plants of the F2 generation, among which lanes 8 and 11 are the genotypes of KE7205 and XsdauK79 loci carried at the same time.

[0045] Figure 5 Figure 3 shows the identification of resistance to Fusarium fusarium in the wheat-Elygropyrum elongatum short segment translocation line. DETAILED DESCRIPTION

[0046] Example 1 Molecular creation of wheat-decaploid Thinoplophora elongatus short segment translocation line

[0047] (1) The wheat-deploid Thinoploid Elymus ovata chromosome substitution line 7el2 (7B) carrying Fhb7 was used as the male parent and the Chinese Spring ph1b mutant (CS ph1b) was used as the female parent to obtain F1.

[0048] (2) F1 was backcrossed with CS ph1b, and genomic DNA was extracted from 725 BC1F1 plants. The specific molecular markers of ph1b gene, Xpsr128 (forward primer P1: (SEQ ID NO.1) and reverse primer P2: (SEQ ID NO.2)), Xpsr574 (forward primer P3: (SEQ ID NO.3) and reverse primer P4: (SEQ ID NO.4)) and XAWJL3 (forward primer P5: (SEQID NO.5) and reverse primer P6: (SEQ ID NO.6)) and the functional marker K-Fhb7 (forward primer P7: (SEQID NO.7) and reverse primer P8: (SEQ ID NO.8)) were used for detection. 185 plants that were homozygous for the ph1b gene and heterozygous for the Fhb7 gene were screened. After self-pollination, 16,000 BC1F2 plants were obtained and genomic DNA was extracted from all of them.

[0049] (3) Using the functional marker K-Fhb7 of Fhb7 and the functional marker K-PSY of PSY-E2 (forward primer P9: (SEQ ID NO.9) and reverse primer P10: (SEQ ID NO.10)), 16,000 BC1F2 plants were genotyped to obtain a line in which the molecular marker K-Fhb7 carried the decaploid Elymus ovatus genotype and the molecular marker K-PSY carried the wheat genotype. Finally, a potential wheat-decaploid Elymus ovatus short segment translocation line carrying the wheat fusarium ergot resistance gene Fhb7 was obtained ( Figure 1 ) and was named Shannong K23-6 after self-pollination. In situ hybridization results confirmed that the end of chromosome 7B of Shannong K23-6 did carry chromosome 7el2 fragment ( Figure 2 ).

[0050] (4) In the PCR amplification reaction system, the PCR reagent composition of the molecular markers Xpsr128, Xpsr574, XAWJL3 and K-PSY was: 1 μl DNA template, 7.5 μl 2×Taq Master Mix, 1.0 μl of each of the front and rear primers, and 4.5 μl H2O.

[0051] The PCR amplification program was 95℃ pre-denaturation for 5min; then 94℃ denaturation for 50s, 55℃ renaturation for 30s, 72℃ extension for 1min, 35 cycles; finally 72℃ extension for 10min; stored at 4℃. After the PCR amplification reaction was completed, the amplified product was loaded on 8% polyacrylamide gel for electrophoresis and detected by silver staining. After the electrophoresis, the AlphaInnotech V.1.2 gel imaging system was used to take pictures and record the genotype of each sample.

[0052] (5) In the PCR amplification reaction system, the PCR reagent composition of the molecular marker K-Fhb7 is: 1 μl DNA template, 7.5 μl 2× Green Taq Mix, 1.0 μl of each of the front and rear primers, and 4.5 μl H2O.

[0053] The PCR amplification program was 95°C pre-denaturation for 5 min; then 94°C denaturation for 30 s, 60°C renaturation for 30 s, 72°C extension for 30 s, 35 cycles; finally 72°C extension for 10 min; and storage at 4°C. After the PCR amplification reaction was completed, the amplified product was loaded into a 1% agarose gel for electrophoresis. After the electrophoresis was completed, the AlphaInnotech V.1.2 gel imaging system was used to take pictures and record the genotype of each sample.

[0054] (6) In situ hybridization identification: Select plump seeds and place them in a culture dish with double-layer wet filter paper for germination at 23°C. After turning white, synchronize them at 4°C for 24 hours, then transfer them to a constant temperature culture at 23°C for 24 hours. Cut the root tips of appropriate length to prepare the metaphase chromosomes of mitosis, and use the decaploid Elytium longipedum-specific sequence as a probe for detection. Chromosomes were observed using an Olympus BX53 fluorescence microscope, and images were captured using a SPOT CCD DP72 and processed using Photoshop.

[0055] Example 2 Molecular creation of wheat-diploid Thinopiota elongata short segment translocation line

[0056] (1) The wheat-diploid Thinopsis elongata chromosome substitution line 7E (7D) carrying Fhb7 was used as the male parent and the Chinese Spring ph1b mutant (CS ph1b) was used as the female parent to obtain F1.

[0057] (2) F1 was backcrossed with CS ph1b, and genomic DNA was extracted from 610 BC1F1 plants. The specific molecular markers of ph1b gene, Xpsr128 (forward primer P1: (SEQ ID NO.1) and reverse primer P2: (SEQ ID NO.2)), Xpsr574 (forward primer P3: (SEQ ID NO.3) and reverse primer P4: (SEQ ID NO.4)) and XAWJL3 (forward primer P5: (SEQID NO.5) and reverse primer P6: (SEQ ID NO.6)) and the functional marker K-Fhb7 (forward primer P7: (SEQID NO.7) and reverse primer P8: (SEQ ID NO.8)) were used for detection. 128 plants that were homozygous for the ph1b gene and heterozygous for the Fhb7 gene were screened. After self-pollination, 11,000 BC1F2 plants were obtained and genomic DNA was extracted from all of them.

[0058] (3) Using the functional marker K-Fhb7 of Fhb7 and the linked marker KE7005 upstream of Fhb7 (forward primer P11: (SEQ ID NO.11) and reverse primer P12: (SEQ ID NO.12)), 11,000 BC1F2 plants were genotyped to obtain lines in which the molecular marker K-Fhb7 carried the diploid Elymus elongatus genotype and the molecular marker KE7005 carried the wheat genotype ( Figure 3 ), and finally obtained a potential wheat-diploid Thinopyrum elongatum short segment translocation line carrying the wheat fusarium fusarium resistance gene Fhb7 ( Figure 3 ) and was named Shannong K12-30 after self-pollination. The results of in situ hybridization confirmed that the end of chromosome 7D of Shannong K12-30 did carry the chromosome 7E fragment ( Figure 2 ).

[0059] (4) In the PCR amplification reaction system, the PCR reagent composition of the molecular markers Xpsr128, Xpsr574 and XAWJL3 was: 1 μl DNA template, 7.5 μl 2×Taq Master Mix, 1.0 μl of each of the front and rear primers, and 4.5 μl H2O.

[0060] The PCR amplification program was 95℃ pre-denaturation for 5min; then 94℃ denaturation for 50s, 55℃ renaturation for 30s, 72℃ extension for 1min, 35 cycles; finally 72℃ extension for 10min; stored at 4℃. After the PCR amplification reaction was completed, the amplified product was loaded on 8% polyacrylamide gel for electrophoresis and detected by silver staining. After the electrophoresis, the AlphaInnotech V.1.2 gel imaging system was used to take pictures and record the genotype of each sample.

[0061] (5) In the PCR amplification reaction system, the PCR reagent composition of molecular markers K-Fhb7 and KE7005 is: 1 μl DNA template, 7.5 μl 2× Green Taq Mix, 1.0 μl of each of the front and rear primers, and 4.5 μl H2O.

[0062] The PCR amplification program was 95°C pre-denaturation for 5 min; then 94°C denaturation for 30 s, 60°C renaturation for 30 s, 72°C extension for 30 s, 35 cycles; finally 72°C extension for 10 min; and storage at 4°C. After the PCR amplification reaction was completed, the amplified product was loaded into a 1% agarose gel for electrophoresis. After the electrophoresis was completed, the AlphaInnotech V.1.2 gel imaging system was used to take pictures and record the genotype of each sample.

[0063] (6) In situ hybridization identification: Select plump seeds and place them in a culture dish with double-layer wet filter paper for germination at 23°C. After turning white, synchronize them at 4°C for 24 hours, then transfer them to a constant temperature culture at 23°C for 24 hours. Cut the root tips of appropriate length to prepare the metaphase chromosomes of mitosis, and use the diploid Elytium longipedum-specific sequence as a probe for detection. Chromosomes were observed using an Olympus BX53 fluorescence microscope, and images were captured using a SPOT CCD DP72 and processed using Photoshop.

[0064] Example 3 Polymerization and utilization of Fhb7 in wheat-Elygropyrum elongatum short segment translocation line

[0065] (1) The wheat-decaploid Elymus elongatus short segment translocation line Shannong K23-6 carrying Fhb7 on chromosome 7B was used as the male parent, and the wheat-diploid Elymus elongatus short segment translocation line Shannong K12-30 carrying Fhb7 on chromosome 7D was used as the female parent to obtain F1.

[0066] (2) F2 was obtained after self-pollination of F1, and genomic DNA was extracted from F2 plants. The molecular marker KE7205 (forward primer P13: (SEQ ID NO.13) and reverse primer P14: (SEQ ID NO.14)) specific to chromosome 7E of diploid Elymus elongatus and the molecular marker XsdauK79 (forward primer P15: (SEQ ID NO.15) and reverse primer P16: (SEQ ID NO.16)) specific to chromosome 7e12 of decaploid Elymus elongatus were used for detection. A wheat-Elymus elongatus short segment translocation line carrying Fhb7 on both chromosomes 7B and 7D was created ( Figure 4 ), continued self-pollination, and was named Shannong K10-6 after homozygosity was stabilized. The results of in situ hybridization confirmed that the ends of chromosomes 7B and 7D of Shannong K10-6 did carry exogenous chromosome fragments ( Figure 2 ).

[0067] (3) In the PCR amplification reaction system, the PCR reagent composition is: 1 μl DNA template, 7.5 μl 2× Green TaqMix, 1.0 μl each of the front and back primers, and 4.5 μl H2O.

[0068] The PCR amplification program was 95°C pre-denaturation for 5 min; then 94°C denaturation for 30 s, 60°C renaturation for 30 s, 72°C extension for 30 s, 35 cycles; finally 72°C extension for 10 min; and storage at 4°C. After the PCR amplification reaction was completed, the amplified product was loaded into a 1% agarose gel for electrophoresis. After the electrophoresis was completed, the AlphaInnotech V.1.2 gel imaging system was used to take pictures and record the genotype of each sample.

[0069] (4) In situ hybridization identification: Select plump seeds and place them in a culture dish with double-layer wet filter paper for germination at 23°C. After turning white, synchronize them at 4°C for 24 hours, then transfer them to a constant temperature culture at 23°C for 24 hours. Cut the root tips of appropriate length to prepare the metaphase chromosomes of mitosis, and use the diploid and decaploid Elytium longipedum-specific sequences as probes for detection. Chromosomes were observed using an Olympus BX53 fluorescence microscope, and images were captured using a SPOT CCD DP72 and processed using Photoshop.

[0070] Example 4 Transfer and Utilization of Fhb7 Subgenome Polymerization System

[0071] (1) The wheat-Elygropyrum short segment translocation line Shannong K10-6, which carries Fhb7 on both wheat chromosomes 7B and 7D, was used as the donor parent of Fhb7. Mengmai 023, a major wheat variety in my country, was used as the recurrent parent. After two backcrosses, the line was selfed three times. The molecular marker KE7205 (forward primer P13: (SEQ ID NO.13) and reverse primer P14: (SEQ ID NO.14)) specific to the 7E chromosome of diploid Elygropyrum and the molecular marker XsdauK79 (forward primer P15: (SEQ ID NO.15) and reverse primer P16: (SEQ ID NO.16)) specific to the 7e12 chromosome of decaploid Elygropyrum were used for auxiliary selection and identification. Thirty 7B chromosomes were included in the BC2F3 generation population of each genetic background. Fhb7+ Department, 30 7D Fhb7+ Department, 30 7B Fhb7+ +7D Fhb7+ lines and 30 lines that do not carry the Fhb7 gene.

[0072] (2) In the PCR amplification reaction system, the PCR reagent composition is: 1 μl DNA template, 7.5 μl 2× Green TaqMix, 1.0 μl each of the front and back primers, and 4.5 μl H2O.

[0073] The PCR amplification program was 95°C pre-denaturation for 5 min; then 94°C denaturation for 30 s, 60°C renaturation for 30 s, 72°C extension for 30 s, 35 cycles; finally 72°C extension for 10 min; and storage at 4°C. After the PCR amplification reaction was completed, the amplified product was loaded into a 1% agarose gel for electrophoresis. After the electrophoresis was completed, the AlphaInnotech V.1.2 gel imaging system was used to take pictures and record the genotype of each sample.

[0074] (3) In 2024, the resistance of the BC2F3 generation population under the background of Mengmai 023 was identified in the artificial climate chamber of Shandong Agricultural University. At the early stage of wheat flowering, 10 μL of spore suspension was taken with a pipette and injected between the palea and lemma of the unilateral floret at 1 / 3 from top to bottom of the ear; after inoculation, a No. 4 ziplock bag was put on and kept moist for 48-72 hours. When brown spots appeared on the inoculated florets, the ziplock bag was removed; 21 days after inoculation, the number of diseased spikelets (NDS) and disease severity (DS) were counted, DS = number of diseased spikelets / total number of spikelets × 100%. The obtained data were statistically analyzed using the statistical software SPSS18.0, and the differences between treatments were expressed using the Duncan test method.

[0075] (3) The results showed that the number of diseased spikelets (NDS) of the highly resistant control variety Sumai 3 was 1.3, while the NDS of the control variety without Fhb7 was 8.9; Fhb7+ System and 7D Fhb7+ The NDS of the lines are 2.2 and 2.3 respectively, while the 7B carrying two Fhb7 Fhb7+ +7D Fhb7+ The NDS of the polymerized line was only 1.2, which was not significantly different from the high-resistance control Sumai 3 (Table 1, Figure 5 ). Therefore, subgenomic aggregation of Fhb7 on different chromosomes can significantly improve wheat resistance to Fusarium head blight and has important application prospects in wheat Fusarium head blight resistance breeding.

[0076] Table 1 Identification of Fusarium head blight resistance in Fhb7 subgenome aggregate lines under Mengmai 023 background

[0077]

[0078]

[0079] Note: Different lowercase letters after the phenotypic values ​​in a column indicate significant differences at P < 0.05.

Claims

1. A method for creating and polymerizing a short fragment translocation line carrying the wheat scab resistance gene Fhb7, characterized in that Here are the steps: (1) The wheat-decaploid Thinoploid elytris chromosome substitution line 7el2 (7B) carrying Fhb7 was used as the male parent and the Chinese Spring ph1b mutant was used as the female parent to obtain F1; (2) backcrossing the F1 generation obtained in step (1) with the Chinese spring ph1b mutant, extracting genomic DNA from the BC1F1 plants, and detecting them using the specific molecular markers Xpsr128, Xpsr574 and XAWJL3 of the ph1b gene and the functional marker K-Fhb7 of Fhb7; screening out individual plants that are homozygous for the ph1b gene and heterozygous for the Fhb7 gene, and obtaining a number of BC1F2 plants after self-pollination of these individual plants, and extracting genomic DNA from all of them; (3) Using the functional markers of Fhb7, K-Fhb7, and PSY-E2, K-PSY, the functional markers of PSY-E2, genotyping the BC1F2 generation plants was performed to screen for a wheat-decaploid Thinoplophora elongatum short segment translocation line carrying the Fusarium fusca resistance gene Fhb7 but without the yellow pigment gene PSY-E2. After self-pollination, the line was named Shannong K23-6. (4) The PCR amplification reaction products were subjected to polyacrylamide gel electrophoresis and agarose gel electrophoresis to analyze the genotypes of the BC1F1 generation plants, BC1F2 generation plants, and Shannong K23-6; (5) In situ hybridization identification of Shannong K23-6; (6) Using the wheat-diploid Thinopsis elongata chromosome substitution line 7E (7D) carrying Fhb7 as the male parent and the Chinese Spring ph1b mutant as the female parent, the F1 was obtained by hybridization; (7) F1 was backcrossed with the Chinese Spring ph1b mutant, and genomic DNA was extracted from the BC1F1 plants. The ph1b gene-specific molecular markers Xpsr128, Xpsr574, and XAWJL3 and the functional marker K-Fhb7 of Fhb7 were used for detection. Individual plants that were homozygous for the ph1b gene and heterozygous for the Fhb7 gene were screened, and several BC1F2 plants were obtained after self-pollination of these individual plants, and their genomic DNA was extracted from all of them. (8) The functional marker K-Fhb7 of Fhb7 and the linkage marker KE7005 upstream of Fhb7 were used to perform genotyping on the BC1F2 generation plants, and a wheat-diploid Elymus elongatus short segment translocation line carrying K-Fhb7 but not KE7005 was selected. After self-pollination, the line was named Shannong K12-30. (9) The PCR amplification reaction products were subjected to polyacrylamide gel electrophoresis and agarose gel electrophoresis to analyze the genotypes of BC1F1 generation plants, BC1F2 generation plants, and Shannong K12-30; (10) In situ hybridization was performed on Shannong K12-30; (11) Shannong K23-6 was used as the male parent and Shannong K12-30 as the female parent, and the F1 was obtained by hybridization; (12) The F1 obtained in step (11) was self-pollinated to obtain F2, and genomic DNA was extracted from the F2 plants. The molecular marker KE7205 specific to chromosome 7E of the diploid Elymus elongatus and the molecular marker XsdauK79 specific to chromosome 7e12 of the decaploid Elymus elongatus were used for detection. A wheat-Elymus elongatus short segment translocation line carrying Fhb7 on both chromosomes 7B and 7D was selected, and self-pollination was continued. After homozygosity was stabilized, it was named Shannong K10-6; (13) The PCR amplification products were subjected to agarose gel electrophoresis to analyze the genotypes of the F2 plants and Shannong K10-6; (14) In situ hybridization was performed on Shannong K10-6.

2. The method for creating and polymerizing a short segment translocation line carrying the wheat scab resistance gene Fhb7 according to claim 1, characterized in that: The molecular markers in step (2) are as follows: Xpsr128, forward primer P1, sequence is SEQ ID NO.1; reverse primer P2, sequence is SEQ ID NO.2; Xpsr574, forward primer P3, sequence is SEQ ID NO.3; reverse primer P4, sequence is SEQ ID NO.4; XAWJL3, forward primer P5, sequence is SEQ ID NO.5; reverse primer P6, sequence is SEQ ID NO.6; K-Fhb7, forward primer P7, sequence is SEQ ID NO.7; reverse primer P8, sequence is SEQ ID NO.8; The molecular markers in step (3) are as follows: K-PSY, forward primer P9, sequence is SEQ ID NO.9; reverse primer P10, sequence is SEQ ID NO.

10. The molecular markers in step (8) are as follows: KE7005, forward primer P11, sequence is SEQ ID NO.11; reverse primer P12, sequence is SEQ ID NO.

12. The molecular markers in step (12) are as follows: KE7205, forward primer P13, sequence is SEQ ID NO.13; reverse primer P14, sequence is SEQ ID NO.14; XsdauK79, forward primer P15, sequence is SEQ ID NO.15; reverse primer P16, sequence is SEQ ID NO.

16.

3. A method for creating and polymerizing a short fragment translocation line carrying the wheat scab resistance gene Fhb7, characterized in that: The wheat-Elygropyrum short segment translocation line Shannong K10-6, which carries Fhb7 on chromosomes 7B and 7D simultaneously, created according to claim 1 or 2, is used as the donor parent of Fhb7, and the main cultivated wheat varieties in my country are used as the recurrent parents. After two consecutive backcrossings, the line is self-pollinated three times. Auxiliary selection and identification are performed using the molecular marker KE7205 specific to the chromosome 7E of the diploid Elygropyrum and the molecular marker XsdauK79 specific to the chromosome 7e12 of the decaploid Elygropyrum, and homozygous plants carrying Fhb7 on chromosomes 7B and 7D are retained, thereby obtaining a subgenome aggregation line carrying Fhb7 on chromosomes 7B and 7D simultaneously.

4. Use of the subgenomic aggregation system carrying Fhb7 on chromosomes 7B and 7D as claimed in claim 3 in the following aspects: (1) Application in breeding new wheat varieties resistant to ergot; (2) Application for improving wheat resistance to Fusarium pathogens; (3) Its application as a wheat ergot resistance breeding material in relevant scientific research and industrial practice.

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