Screening of KASP primer sets for wheat with short stalks, dense spikelets and resistance to scab and its application

Abstract

The present invention discloses a KASP primer set for screening wheat with short stalks, dense spikelets and resistance to fusarium head blight and its application. The present invention uses Wheat55K wheat high-throughput gene chip to obtain genotype data, and detects two QTL regions Q.2D and Q.5A related to plant height, spikelet density and fusarium head blight resistance from Yangmai 11 on chromosomes 2D and 5A, whose tightly linked markers are AX110519154 (2D) and AX111618105 (5A), and develops two KASP marker primer sets accordingly. Experiments have proved that the KASP molecular markers of the present invention can be used for molecular marker-assisted selection breeding of wheat plant height, spikelet density and fusarium head blight resistance. The present invention provides a good tool for the effective use of two QTL regions Q.2D and Q.5A related to plant height, spikelet density and fusarium head blight resistance in breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of wheat molecular breeding methods and relates to a KASP primer set for screening short-stalked, dense-spikelet-resistant wheat and an application thereof. Background Art

[0002] Wheat fusarium rust is mainly caused by Fusarium graminearum and other fungi. Once it infects the wheat ear, it will produce and accumulate various toxins, such as deo xynivalenol (DON), nivalenol (NIV) and zearalenol (ZEN), which will then spread to the ear axis and affect the transport of nutrients and water. In severe cases, it will lead to withered white ears and shriveled grains, thereby reducing wheat yield and damaging quality. Toxin pollution will cause great harm to the health of humans and animals, becoming a major threat to food security. The manifestations of wheat resistance to Fusarium fusarium can be divided into five categories: the first category is resistance to invasion (type I), the second category is resistance to spreading (type II); the third category is resistance to kernel infection (type III), the fourth category is tolerance to FHB and trichothecenes (type IV), and the fifth category is resistance to trichothecene accumulation (type V). Among them, the first and second categories are the types that domestic and foreign scholars have been committed to studying for a long time. Several known varieties resistant to ergot disease, such as Sumai No. 3, Wangshuibai, Bai Sanyuehuang, and Xinzhongchang, have excellent resistance to ergot disease, but these materials have poor comprehensive agronomic traits and poor yield. The most common disadvantages are high plant height (above 120 cm) and sparse spikelet density. Therefore, after hybridizing these wheat materials with other wheat materials, the offspring generally have difficult-to-overcome disadvantages such as plants being too tall, easy to lodging, sparse spikelet density, and few spikelets per plant, resulting in low breeding selection efficiency and heavy workload, and it is impossible to select varieties that can be promoted and applied. The Lixiahe Agricultural Science Institute in Jiangsu Province selected parents with good comprehensive high yield and mild fusarium smut from the germplasm introduced from Central Asia and Europe and America for mating. The offspring were selected for resistance to fusarium smut and comprehensive agronomic traits, and a number of wheat varieties with resistance to fusarium smut and high yield were bred, such as Yangmai No. 4. In 1983, it was rainy in the late wheat growth period and fusarium smut recurred. Yangmai No. 4 still showed good high yield and low severity of fusarium smut (PSS%). Later, Yangmai No. 5 and Yangmai 158 were bred from Yangmai No. 4 and Zhengyin No. 1. Yangmai No. 5 and Yangmai 158 successively won the first prize of the National Science and Technology Invention Award. Our laboratory found through research that Yangmai No. 4, Yangmai 5 and Yangmai 158 do not carry the disease resistance alleles of the known major effect genes Fhb1-Fhb7 for resistance to fusarium smut, so there may be other fusarium smut resistance genes in them.Yangmai 11 is another improved variety bred from Yangmai 158. In the production demonstration in Yangzhou in 2001, the average yield per mu was 542 kg, an increase of 7.8% over Yangmai 158. This variety has good high yield and stable yield, and won the second prize of Jiangsu Science and Technology Progress. Its plant height is lower than that of Yangmai 158, the spikelet density is higher than that of general varieties, and the resistance to ergot is stronger than that of Yangmai 158. Therefore, it is of great significance to dig out the genetic loci of Yangmai 11's resistance to ergot and related agronomic traits and clarify its genetic basis for resistance to ergot and agronomic traits for wheat to improve resistance to ergot. So far, there has been no precedent for reporting the ergot resistance loci of Yangmai 11 and its correlation with agronomic traits.

[0003] Plant height (PH) and spikelet compactness (SC) are very important wheat yield-related traits. The dwarfing genes reported in wheat are mainly Rht-D1, Rht-B1 and Rht8, which are distributed in 4DS, 4BS and 2DS. Their relationship with fusarium head blight resistance has not been determined, mainly because the effects of loci on traits are different under different genetic backgrounds and inoculation types. Wheat spikelet density (total number of spikelets per spikelet / spike length) is a very complex agronomic trait, which is a comprehensive reflection of the total number of spikelets per spikelet and spike length. The reported related genetic loci are mainly concentrated in the C gene on the short arm of chromosome 2D and the Q gene on the long arm of chromosome 5A. Previous researchers have not studied the relationship between these loci and fusarium head blight resistance. So far, there are few reports on single-cause pleiotropic loci or QTL clusters for wheat fusarium head blight resistance and related agronomic traits.

[0004] KASP (Kompetitive Allele Specific PCR) is a competitive allele-specific PCR that can be applied to a wide range of genomic DNA samples (even some complex genomic DNA samples) to accurately determine the biallelic nature of SNPs and InDels at specific sites. KASP has high throughput, is fast, and is stable, making it an ideal molecular marker for breeding. In recent years, with the continuous improvement of wheat whole genome sequencing, genetic research on various wheat traits has reached the SNPs level. Therefore, the development and use of KASP markers has become one of the main approaches for wheat molecular marker-assisted breeding. This technology has been widely used in wheat breeding applications such as plant height, grain weight, and vernalization genes / locus.

[0005] Therefore, it is urgent to strengthen the genetic and breeding research of wheat resistance to Fusarium ergot and cultivate wheat varieties with synergistic improvements in resistance and yield. Summary of the invention

[0006] In order to overcome the defects in the prior art, a linked molecular marker is provided for molecular marker-assisted selection of wheat breeding for genotypes with short stalks, dense spikelets and resistance to fusarium head blight. The present invention uses Wheat 55KSNP wheat high-throughput gene chip to obtain genotype data, and detects two QTL regions Q.2D and Q.5A from the wheat variety Yangmai 11 that are significantly associated with fusarium head blight resistance, plant height and spikelet density. Further, by screening the effectiveness and reliability of nearby markers, it is determined that the closely linked markers of the two QTL regions are AX110519154 (2D) and AX111618105 (5A), and based on this, two KASP marker primer sets are developed to efficiently screen genotypes with short stalks, dense spikelets and resistance to fusarium head blight.

[0007] In a first aspect, the present invention provides a substance for detecting resistance to ergot disease, plant height and spikelet density, the substance being a primer combination of a set of primers X1 for detecting whether the 36th deoxyribonucleotide of a molecular marker shown in SEQ ID No.4 on chromosome 2D in the wheat genome is GG, AA, or G and A, and a set of primers X2 for detecting whether the 36th deoxyribonucleotide of a molecular marker shown in SEQ ID No.8 on chromosome 5A in the wheat genome is TT, CC, or T and C, or a detection reagent or kit containing the primer combination.

[0008] Further, the set of primers X1 contains one upstream primer and two downstream primers;

[0009] The upstream primer is designed according to the 36th deoxyribonucleotide of the sequence shown in SEQ ID No. 4 on chromosome 2D in the wheat genome and its upstream sequence;

[0010] The downstream primers are designed according to the downstream sequence of the 36th deoxyribonucleotide of the sequence shown in SEQ ID No. 4 on chromosome 2D in the wheat genome, and the 3' terminal deoxyribonucleotide of one downstream primer is T, and the 3' terminal deoxyribonucleotide of the other downstream primer is C.

[0011] Furthermore, the set of primers X1 consists of the single-stranded DNA molecule shown in SEQ ID No.1, the single-stranded DNA molecule shown in positions 22-41 of SEQ ID No.2 or its derivatives, and the single-stranded DNA molecule shown in positions 22-41 of SEQ ID No.3 or its derivatives; wherein the derivative of the single-stranded DNA molecule shown in positions 22-41 of SEQ ID No.2 is the single-stranded DNA molecule shown in positions 22-41 of SEQ ID No.2 with the 5' end connected to a specific fluorescent label sequence A; and the derivative of the single-stranded DNA molecule shown in positions 22-41 of SEQ ID No.3 is the single-stranded DNA molecule shown in positions 22-41 of SEQ ID No.3 with the 5' end connected to a specific fluorescent label sequence B.

[0012] Further, the set of primers X2 contains two upstream primers and one downstream primer;

[0013] The upstream primer is designed according to the 36th deoxyribonucleotide of the sequence shown in SEQ ID No. 8 on chromosome 5A in the wheat genome and its upstream sequence, and the 3' terminal deoxyribonucleotide of one of the upstream primers is T, and the 3' terminal deoxyribonucleotide of the other upstream primer is C;

[0014] The downstream primer is designed according to the downstream sequence of the 36th deoxyribonucleotide of the sequence shown in SEQ ID No. 8 on chromosome 5A in the wheat genome.

[0015] Furthermore, the set of primers X2 consists of the single-stranded DNA molecule shown at positions 22-41 of SEQ ID No.5 or its derivative, the single-stranded DNA molecule shown at positions 22-41 of SEQ ID No.6 or its derivative, and the single-stranded DNA molecule shown at positions 22-41 of SEQ ID No.7; wherein the derivative of the single-stranded DNA molecule shown at positions 22-41 of SEQ ID No.5 is the single-stranded DNA molecule shown at positions 22-41 of SEQ ID No.5 with the 5' end connected to a specific fluorescent label sequence A; and the derivative of the single-stranded DNA molecule shown at positions 22-41 of SEQ ID No.6 is the single-stranded DNA molecule shown at positions 22-41 of SEQ ID No.6 with the 5' end connected to a specific fluorescent label sequence B.

[0016] Furthermore, the specific fluorescent label sequence A is a fluorescent label sequence FAM, and the specific fluorescent label sequence B is a fluorescent label sequence HEX.

[0017] Furthermore, the primer set X1 consists of the upstream primer sequence SEQ ID No.1 and the downstream primer sequences SEQ ID No.2 and SEQ ID No.3; the primer set X2 consists of the upstream primer sequences SEQ ID No.5 and SEQ ID No.6 and the downstream primer sequence SEQ ID No.7.

[0018] In a second aspect, the present invention further provides the use of the substance described in the first aspect of the present invention in any of the following:

[0019] (A) Identifying or assisting in identifying the resistance of wheat to scab disease with short stalks and dense spikelets;

[0020] (B) Compare the plant height, spikelet density and resistance to fusarium wilt of the tested wheat;

[0021] (C) breeding or selecting wheat plants, strains, lines or varieties with lower plant height, higher spikelet density and relatively stronger resistance to ergot;

[0022] (D) breeding or selecting wheat plants, strains, lines or varieties with higher plant height, lower spikelet density and relatively weaker resistance to ergot;

[0023] (E) preparing a product for identifying or assisting in identifying or comparing plant height, spikelet density and resistance to fusarium head blight;

[0024] (F) preparing a product for breeding or screening wheat plants, strains, lines or varieties with lower plant height, higher spikelet density and relatively stronger resistance to ergot;

[0025] (G) preparing a product for breeding or screening wheat plants, strains, lines or varieties with higher plant height, lower spikelet density and relatively weaker resistance to ergot disease.

[0026] In a third aspect, the present invention further provides any of the following methods:

[0027] Method A: A method for comparing the resistance of wheat to scab disease of dwarf stalks and dense spikelets to be tested, comprising the following steps (A1) or (A2):

[0028] (A1) detecting whether the genotype of the 36th position of the molecular marker shown by SEQ ID No. 4 on chromosome 2D in the wheat genome is AA, GG or AG, and simultaneously detecting whether the genotype of the 36th position of the molecular marker shown by SEQ ID No. 8 on chromosome 5A in the wheat genome is CC, TT or CT;

[0029] (A2) determining the fusca resistant trait of the wheat to be tested as follows: when the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No. 4 on chromosome 2D in the genome is GG and the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No. 8 on chromosome 5A in the genome is TT, the wheat to be tested has the fusca resistant trait of fusca resistant trait of fusca;

[0030] Method B: A method for breeding or screening wheat plants or strains or lines or varieties with lower plant height, higher spikelet density and relatively stronger resistance to fusarium head blight, comprising the following steps:

[0031] (B1) detecting whether the genotype of the 36th position of the molecular marker shown by SEQ ID No. 4 on chromosome 2D in the wheat genome is AA, GG or AG, and simultaneously detecting whether the genotype of the 36th position of the molecular marker shown by SEQ ID No. 8 on chromosome 5A in the wheat genome is CC, TT or CT;

[0032] (B2) selecting a wheat plant to be tested in which the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No.4 on chromosome 2D in the genome is G and the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No.8 on chromosome 5A in the genome is T as a parent for breeding, and selecting a wheat plant in which the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No.4 on chromosome 2D in the genome is G and the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No.8 on chromosome 5A in the genome is T in each breeding generation, and finally obtaining a wheat plant or strain or variety with a lower plant height, a higher spikelet density and a relatively stronger resistance to ergot disease;

[0033] Method C: A method for breeding or screening wheat plants or strains or lines or varieties with higher plant height, lower spikelet density, and relatively weaker resistance to scab, comprising the following steps:

[0034] (C1) detecting whether the genotype of the 36th position of the molecular marker shown by SEQ ID No.4 on chromosome 2D in the wheat genome is AA, GG or AG, and simultaneously detecting whether the genotype of the 36th position of the molecular marker shown by SEQ ID No.8 on chromosome 5A in the wheat genome is CC, TT or CT;

[0035] (C2) selecting a wheat plant to be tested in which the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No.4 on chromosome 2D in the genome is A and the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No.8 on chromosome 5A in the genome is C as a parent for breeding, and selecting wheat plants in which the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No.4 on chromosome 2D in the genome is A and the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No.8 on chromosome 5A in the genome is C in each breeding generation, and finally obtaining wheat plants or strains or lines or varieties with higher plant height, lower spikelet density and relatively weaker resistance to ergot disease.

[0036] Furthermore, the specific operations of (A1)(B1)(C1) are:

[0037] The detection reagent or kit provided by the first aspect of the present invention is used to perform PCR amplification on the wheat genomic DNA to be tested, the amplified product is subjected to fluorescent signal scanning, the scanning data is analyzed, and then the type of the 36th deoxyribonucleotide shown in SEQ ID No.4 on chromosome 2D in the wheat gene to be tested and the type of the 36th deoxyribonucleotide shown in SEQ ID No.8 on chromosome 5A in the wheat gene to be tested are determined as follows:

[0038] If the fluorescent signal data of the amplified product of the wheat to be tested is displayed as blue, the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No.4 on chromosome 2D in the genome of the wheat to be tested is a homozygous G, or the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No.8 on chromosome 5A in the genome of the wheat to be tested is a homozygous T;

[0039] If the fluorescent signal data of the amplified product of the wheat to be tested is displayed as red, the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No.4 on chromosome 2D in the genome of the wheat to be tested is a homozygous A, or the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No.8 on chromosome 5A in the genome of the wheat to be tested is a homozygous C.

[0040] Compared with the prior art, the present invention uses Wheat55K wheat high-throughput gene chip to obtain genotype data, and detects two QTL regions Q.2D and Q.5A related to plant height, spikelet density and fusarium head blight resistance from Yangmai 11 on chromosomes 2D and 5A, and its closely linked markers are AX110519154 (2D) and AX111618105 (5A). Based on this research, the present invention converts linked SNPs into KASP markers that can be used for high-throughput, low-cost and low-error rate detection or auxiliary detection of plant height, spikelet density and fusarium head blight resistance, and further develops KASP marker special primer sets KASP_2D and KASP_5A. Experiments have proved that the KASP molecular markers of the present invention can be used for molecular marker-assisted selection breeding of wheat plant height, spikelet density and fusarium head blight resistance. The present invention provides a good tool for the effective use of two QTL regions Q.2D and Q.5A related to plant height, spikelet density and ergot resistance in breeding. These two KASP markers can quickly screen wheat plant height, spikelet density and ergot resistance, provide convenience for screening wheat materials carrying excellent allelic variations, and improve the efficiency of wheat ergot resistance breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the specific implementation manner of the present invention or the technical solution in the prior art, the drawings required for describing the specific implementation manner are briefly introduced below.

[0042] Figure 1 It is a genetic linkage map and positioning diagram, among which, Figure 1 a is a partial genetic linkage map of chromosome 2D and a schematic diagram of Q.2D positioning; Figure 1 b is a partial genetic linkage map of chromosome 5A and a schematic diagram of the location of Q.5A.

[0043] Figure 2 The results of genotype amplification detection of natural wheat populations using KASP_2D marker and KASP_5A marker in Example 2. Figure 2 a is the result of KASP_2D marker amplification detection in wheat natural population genotype; Figure 2 b shows the genotype amplification detection result of KASP_5A marker in natural wheat population. DETAILED DESCRIPTION

[0044] The embodiments of the technical solution of the present invention will be described in detail below in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention and are therefore only used as examples and cannot be used to limit the scope of protection of the present invention. It should be noted that, unless otherwise specified, the technical terms or scientific terms used in this application should be the common meanings understood by technicians in the field to which the present invention belongs. As a professional agricultural research institution, the applicant has long-term preservation of relevant germplasm materials, and the relevant wheat varieties are all publicly available in the market or existing germplasm banks.

[0045] Example 1 Screening and preliminary verification of stable SNP loci significantly associated with wheat plant height, spikelet density and scab resistance

[0046] In this example, 293 recombinant inbred lines (F 6 ) was used as the material. The recombinant inbred line and its parents were planted at the Wanfu Experimental Base in Lixiahe District, Jiangsu Province for three consecutive growing seasons in 2018, 2019 and 2020. A randomized block design was adopted, with 3 rows of 30 seeds per row, 2.2m long, 0.23m between rows, two replicates, and conventional field management. Further inoculation identification of wheat fusarium resistance was carried out in the field in 2019, 2020 and 2021, and a suspension of fusarium spores (1×10 5 —5×10 5 Spore mL –1 ), single flower drip inoculation method was used. At the beginning of wheat flowering, 10 μL of spore solution was drawn with a syringe and injected into any floret of the sixth spikelet from the top spikelet to the bottom of the wheat ear. 15 spikelets were inoculated in each line. After inoculation, artificial misting was used for moisture retention (mist spraying for 5 minutes every 1.5 hours). 25 days after inoculation, the number of diseased spikelets in the inoculated ear was investigated and the diseased spikelet rate was calculated. Diseased spikelet rate (PSS) = number of diseased spikelets / total number of spikelets × 100%. The disease resistance investigation and identification standards were in accordance with the "Agricultural Industry Standard of the People's Republic of China NY / T 2954-2016: Technical Regulations for Identification of Resistance to Fusarium Head Blight in Wheat Regional Trial Varieties". At the same time, during the wheat filling and maturity period in 2019, 2020 and 2021, the plant height (PH), main stem spike length (SL) and total number of spikelets per spike (SNS) of 10 individual plants in each line were randomly surveyed, and the average value of the 10 individual plants was taken to calculate the average spikelet density of each line (SC = SNS / SL).

[0047] Genomic DNA was extracted by CTAB method, genotypes were obtained using Wheat 55K SNP chip, and genetic maps were constructed. Genotype data were pre-processed and grouped using IciMapping v4.1 software, genetic maps were constructed and corrected using JoinMap v4.0, and genetic maps were drawn using MapChart2.3 (http: / / www.wur.nl / en / show / Mapchart.htm). Inclusive composite interval mapping (ICIM) was used to detect QTLs significantly associated with plant height, spikelet density, and resistance to fusarium head blight, with the LOD threshold set to 3.0. In order to compare with previous results, the linked markers or gene sequences were aligned with the wheat reference genome sequence version 2.1 database (http: / / 202.194.139.32 / blast / blast.html).

[0048] The experiment obtained two relatively stable QTL regions, Q.2D and Q.5A, which were significantly correlated with wheat plant height, spikelet density and ergot resistance. The LOD value range of Q.2D was 5.92–14.77, and the phenotypic contribution rate was 9.28%–26.24%. The LOD value range of Q.5A was 7.44–13.16, and the phenotypic contribution rate was 12.63%–20.36%. The genotypes for reducing plant height, increasing spikelet density and enhancing ergot resistance were all from Yangmai 11. The results are shown in the table below.

[0049] Table 1 Closely linked markers and physical locations of Q.2D and Q.5A

[0050]

[0051] like Figure 1As shown in Table 1, the present application detected QTL regions Q.2D and Q.5A originating from Yangmai 11, which were significantly correlated with wheat plant height, spikelet density and ergot resistance, on 2D and 5A, respectively, through complete interval mapping. After comparison with the wheat reference genome 2.1 database, it was found that they were located on the long arms of wheat chromosomes 2D and 5A, respectively. After comparison, these two QTL regions were not in similar physical intervals with the C gene and Q gene of wheat, and previous researchers had not reported QTL clusters significantly correlated with wheat plant height, spikelet density and ergot resistance in these two intervals. Further preliminary screening of SNPs markers based on marker homology was performed, and SNP markers near the LOD peak and with the best phenotypic linkage were selected for KASP marker conversion. It was determined that AX110519154 (2D) and AX111618105 (5A) had the best genome specificity and the most significant correlation with Fusarium head blight resistance, and their flanking sequences were SEQ ID NO.4 and SEQ ID NO.8, respectively. KASP primers were designed using Primer3.0 (https: / / bioinfo.ut.ee / primer3-0.4.0 / ), and the primers were synthesized by Beijing Jiacheng Biotechnology Co., Ltd. Finally, the AX110519154 (2D) and AX111618105 (5A) markers were successfully converted into effective KASP markers KASP_2D and KASP_5A, respectively, which had the best specificity on the wheat genome and no homologous amplification sequence. The corresponding variable sites were G / A (complementary bases of C / T) and T / C, respectively. The specific sequences are shown in Table 2:

[0052] Table 2 Primer sequence information and flanking sequence information of KASP_2D and KASP_5A

[0053]

[0054] Preparation of KASP_2D primers and PCR reaction

[0055] Take 30 μL (100 μM) of the upstream primer (nucleotide sequence as shown in SEQ ID NO.3), take 12 μL (100 μM) of the downstream primer (nucleotide sequence as shown in SEQ ID NO.1 and SEQ ID NO.2), add to 100 μL with sterile ultrapure water, mix thoroughly, and use as the primer working solution of KASP labeling for standby use. PCR amplification reaction system: 2 μL (about 30 ng / μL) of wheat DNA template to be tested, 0.08 μL of primer working solution, 2.5 μL of KASP Master Mix (LGC Company, KBS-1016-002), add to 5 μL with sterile ultrapure water; PCR reaction program: first step, 95℃ pre-denaturation for 12 min; second step, 95℃ denaturation for 20 s, 65–57℃ (1℃ decrease for each cycle) for 60 s, a total of 8 cycles; third step, 95℃ denaturation for 20 s, 57℃ renaturation for 1 min, 33 cycles; 4℃ storage. The experiment also set up a blank control (NTC) in which no template DNA was added to the reaction system. One or more blank controls were set up for each plate. Wheat seedlings were taken and the genomic DNA of the wheat to be tested was extracted using the CTAB method. The genomic DNA of the wheat to be tested was used as a template and PCR amplification was performed using the above KASP primer set and PCR reagent to obtain the PCR amplification product. The PCR reaction was performed on S1000 TM The PCR was performed on a Ther mal Cycler PCR instrument (Bio-Rad Laboratories Inc.), and the PCR amplification products were scanned and read with a multifunctional microplate reader (PHERAstar Plus, BMGLABTECH, Germany). The excitation wavelength of FAM was 485 nm, and the emission wavelength was 520 nm; the excitation wavelength of VIC was 535 nm, and the emission wavelength was 556 nm. The excitation wavelength of the system reference fluorescence ROX was 575 nm, and the emission wavelength was 610 nm. Kluster Caller software (KBioscience) was used for genotyping, and the KASP_2D genotype was determined based on the analysis results.

[0056] Preparation of KASP_5A primers and PCR reaction

[0057] Take 12 μL (100 μM) of each upstream primer (nucleotide sequence as shown in SEQ ID NO.1 and SEQ ID NO.2), take 30 μL (100 μM) of downstream primer (nucleotide sequence as shown in SEQ ID NO.3), add to 100 μL with sterile ultrapure water, mix thoroughly, and use as KASP-labeled primer working solution for standby use. PCR amplification reaction system: 2 μL (about 30 ng / μL) of wheat DNA template to be tested, 0.08 μL of primer working solution, 2.5 μL of KASP Master Mix (LGC Company, KBS-1016-002), add to 5 μL with sterile ultrapure water; PCR reaction procedure: first step, 92℃ pre-denaturation for 12 min; second step, 94℃ denaturation for 20 s, 64-56℃ (1℃ decrease for each cycle) for 60 s, a total of 9 cycles; third step, 94℃ denaturation for 20 s, 56℃ renaturation for 1 min, 31 cycles; 4℃ storage. The experiment also set up a blank control (NTC) in which no template DNA was added to the reaction system. One or more blank controls were set up for each plate. Wheat seedlings were taken and the genomic DNA of the wheat to be tested was extracted using the CTAB method. The genomic DNA of the wheat to be tested was used as a template and PCR amplification was performed using the above KASP primer set and PCR reagent to obtain the PCR amplification product. The PCR reaction was performed on S1000 TM The PCR was performed on a Ther mal Cycler PCR instrument (Bio-Rad Laboratories Inc.), and the PCR amplification products were scanned and read with a multifunctional microplate reader (PHERAstar Plus, BMGLABTECH, Germany). The excitation wavelength of FAM was 485 nm, and the emission wavelength was 520 nm; the excitation wavelength of VIC was 535 nm, and the emission wavelength was 556 nm. The excitation wavelength of the system reference fluorescence ROX was 575 nm, and the emission wavelength was 610 nm. Kluster Caller software (KBioscience) was used for genotyping, and the KASP_5A genotype was determined based on the analysis results.

[0058] 293 recombinant inbred lines of "Yangmai 11×Zhengmai 9023" and two parents were subjected to KASP_2D and KASP_5A amplification according to the above method. The fluorescence signal data of the KASP_2D amplification product was analyzed by Kluster Caller software and clustered at the position close to the X-axis in the fluorescence signal coordinate system of the typing result (blue), which is the same as Yangmai 11, which proves that the genotype of these wheats at the 36th base (SNP site) of the molecular marker KASP_2D flanking nucleotide sequence (such as SEQ ID NO.4) is G; while the fluorescence signal data of the amplification product was analyzed by Kluster Caller software and clustered at the position close to the Y-axis in the coordinate system (red), which is different from the typing of Yangmai 11, which proves that the genotype of these wheats at the SNP site is A. The fluorescence signal data of KASP_5A amplification products were analyzed by Kluster Caller software and clustered near the X-axis position (blue) in the typing result fluorescence signal coordinate system, which was the same as Yangmai 11, which proved that the genotype of these wheats at the 36th base (SNP site) of the molecular marker KASP_5A flanking nucleotide sequence (such as SEQ ID NO.4) was T; while the fluorescence signal data of the amplification products were analyzed by Kluster Caller software and clustered near the Y-axis position (red) in the coordinate system, which was different from the typing of Yangmai 11, which proved that the genotype of these wheats at the SNP site was C. The results of the genotype detection and phenotypic average test of some families are shown in Table 3. The material typing was good and the KASP primer set was successfully designed.

[0059] Table 3 Genotypes and average phenotypic values ​​of Q.2D and Q.5A in some RIL families

[0060]

[0061]

[0062]

[0063]

[0064] Table 4 T test results of RIL families carrying different genotypes

[0065]

[0066]

[0067] a Different lowercase letters after the numbers indicate extremely significant differences (P<0.01)

[0068] b The ** after the t value indicates a very significant difference (P<0.01)

[0069] As shown in Table 4, using the two-sample T test of Excel 2019, it can be seen that the Q.2D genotype of Yangmai 11 is GG, and the Q.5A genotype is TT, while the Q.2D genotype of Zhengmai 9023 is AA, and the Q.5A genotype is CC. The T test results of the RIL family show that the family with the Q.2D genotype of GG has significantly lower plant height (reduced by 6.73%), significantly higher spikelet density (increased by 7.69%), and significantly lower severity of fusarium smut (reduced by 27.77%) than the family with AA (P<0.01); the family with the Q.5A genotype of TT has significantly lower plant height (reduced by 9.99%), significantly higher spikelet density (increased by 9.79%), and significantly lower severity of fusarium smut (reduced by 18.06%) than the family with CC (P<0.01) (the statistical method is a conventional method in this field, and the details can also be referred to the content disclosed in the document "Gai Junyi, "Experimental Statistics Methods", China Agriculture Press, September 2000").

[0070] Table 5 Analysis of the aggregation effect of Q.2D and Q.5A

[0071]

[0072] a Different lowercase letters after the numbers indicate that there are significant differences between genotype combinations at least at the P < 0.05 level.

[0073] b The ** after the t value indicates that there is a very significant difference (P<0.01) relative to the genotype combination "AA+CC", and the * indicates that there is a significant difference (P<0.05) relative to the genotype combination "AA+CC"

[0074] As shown in Table 5, when the genotype of Q.2D and Q.5A was "GG+CC", the plant height of the family decreased by 4.68% (P<0.05), the spikelet density increased by 5.79% (P<0.05), and the severity of fusarium wilt decreased by 29.08% (P<0.01) compared with "AA+CC". When the genotype of Q.2D and Q.5A was "AA+TT", the plant height of the family decreased by 7.98% (P<0.05) compared with "AA+CC". When the genotype of Q.2D and Q.5A was "GG+TT", relative to "AA+CC", the plant height of the family decreased by 13.93% (P<0.01), the spikelet density increased by 15.79% (P<0.01), and the severity of ergot disease decreased by 36.81% (P<0.01). It can be seen that when the Q.2D genotype is GG and the Q.5A genotype is TT, the family has the lowest plant height, the highest spikelet density, and the lowest severity of ergot disease (P<0.01). Therefore, aggregating the Q.2D genotype GG and the Q.5A genotype TT can significantly reduce plant height, increase spikelet density, and enhance resistance to ergot disease (the statistical method is a conventional method in this field, and the details can also be found in the document "Gai Junyi, "Experimental Statistical Methods", China Agriculture Press, September 2000").

[0075] Tables 3 to 5 show that the two sets of KASP primer sets and genotype detection systems were used separately or simultaneously in the selection of materials with different plant heights, spikelet densities and resistance to ergot. The effects of Q.2D and Q.5A were significant, indicating that the KASP marker was successfully developed and can be further used for breeding material detection.

[0076] Example 2 Application of KASP primer set in breeding

[0077] Field trial: 120 wheat varieties (lines) from all over the country were used as materials and planted in the Wanfu Experimental Base in Lixiahe District, Jiangsu Province for two consecutive growing seasons in 2019 and 2020. A randomized block design was adopted, with 5 rows of 30 seeds per row, 2.2m long, 0.23m spacing, two replicates, and conventional field management. Further inoculation and identification of wheat fusarium resistance were carried out in the field in 2020 and 2021, and a suspension of fusarium spores (1×10 5 – 5×10 5 Spore mL –1), single flower drip inoculation method was used. At the beginning of wheat flowering, 10 μL of spore solution was drawn with a syringe and injected into any small flower of the sixth spikelet from the top spikelet to the bottom of the wheat ear. 15 spikelets were inoculated for each variety (line). After inoculation, artificial misting was used for moisturizing (mist spraying for 5 minutes every 1.5 hours). 25 days after inoculation, the number of diseased spikelets of the inoculated ear was investigated and the diseased spikelet rate was calculated. Diseased spikelet rate (PSS) = number of diseased spikelets / total number of spikelets × 100%. The disease resistance investigation and identification standards are in accordance with the "Agricultural Industry Standard of the People's Republic of China NY / T2954-2016: Technical Regulations for Identification of Resistance to Fusarium Head Blight of Wheat Regional Trial Varieties". At the same time, during the wheat filling and maturity period in 2020 and 2021, the plant height (PH), main stem spike length (SL) and total number of spikelets per spike (SNS) of 10 individual plants of each variety (line) were randomly surveyed, and the average value of the 10 individual plants was taken to calculate the average spikelet density of each line (SC = SNS / SL)

[0078] The KASP primer set obtained in Example 1 was used to genotype 120 nationally promoted wheat varieties (lines) planted in the wheat identification nursery of the Wanfu Experimental Base. The average values ​​of the fusarium identification results of the field trials in 2019, 2020 and two years and the genotype detection results are shown in Table 6 and Attached Figure 2 As shown. The fluorescence signal data of the amplified products were analyzed by Kluster Caller software and clustered near the Y axis in the coordinate system (red), which was different from the typing of Yangmai 11, proving that the genotype of these wheats at the SNP site was A. The fluorescence signal data of the KASP_5A amplified products were analyzed by Kluster Caller software and clustered near the X axis in the typing result fluorescence signal coordinate system (blue), which was the same as Yangmai 11, proving that the genotype of these wheats at the 36th base (SNP site) of the molecular marker KASP_5A flanking nucleotide sequence (such as SEQ ID NO.4) was T; and the fluorescence signal data of the amplified products were analyzed by Kluster Caller software and clustered near the Y axis in the coordinate system (red), which was different from the typing of Yangmai 11, proving that the genotype of these wheats at the SNP site was C.

[0079] Table 6 Genotypes and average phenotypic values ​​of Q.2D and Q.5A of 120 wheat promotional varieties (lines)

[0080]

[0081]

[0082]

[0083]

[0084]

[0085] Table 7 T test results of tested varieties (lines) carrying different genotypes

[0086]

[0087] a The ** after the number indicates that there is a very significant difference compared with "AA+CC" (P<0.01), and the * indicates that there is a significant difference compared with "AA+CC" (P<0.05).

[0088] Table 7 The results of the two-sample T test using Excel 2019 showed that among the 120 wheat varieties (lines), when the Q.2D genotype was AA and the Q.5A genotype was CC, the variety (line) had the highest plant height, the lowest spikelet density, and the highest severity of fusarium scurf (P<0.01); when the Q.2D genotype was GG and the Q.5A genotype was CC, compared with "AA+CC", the plant height of the variety (line) decreased by 3.86% (P<0.01), the spikelet density increased by 4.17% (P<0.05), and the severity of fusarium scurf decreased by 33.16% (P<0.01); when the Q.2D genotype was AA and the Q.5A genotype was TT, compared with "AA+CC", the plant height of the variety (line) decreased by 2.07% (P<0.05), the spikelet density increased by 4.17% (P<0.05), and the severity of fusarium scurf decreased by 29.33% (P<0.05). % (P<0.05); when the Q.2D genotype was GG and the Q.5A genotype was TT, relative to "AA+CC", the plant height of the variety (line) decreased by 5.52% (P<0.01), the spikelet density increased by 7.29% (P<0.01), and the severity of fusarium scab decreased by 54.36% (P<0.01); when the Q.2D genotype was GG and the Q.5A genotype was TT, the variety (line) had the lowest plant height, the highest spikelet density, and the lowest severity of fusarium scab (P<0.01), indicating that the aggregation of Q.2D genotype GG and Q.5A genotype TT can significantly reduce plant height, increase spikelet density, and enhance fusarium scab resistance (the statistical method is a conventional method in the art, and the specific details can also be found in the document "Gai Junyi, "Experimental Statistical Methods", China Agricultural Press, September 2000"). The above two sets of KASP primer sets and genotype detection systems can be used separately or simultaneously in molecular marker-assisted selection breeding for wheat plant height, spikelet density and fusarium head blight.

[0089] It can be concluded from the above experimental results that: by performing PCR amplification on wheat genomic DNA through the two sets of KASP primer sets of the present invention, it can be directly determined through KASP typing whether the genotype of Yangmai 11 with short stems, dense spikelets and resistance to ergot disease is carried, the detection method is simple to operate, the detection result is very intuitive, and the detection effect is obvious and effective. The use of these two sets of KASP primer sets can greatly improve the efficiency of molecular marker-assisted selection breeding of short stem, dense spikelets and resistance to ergot disease in wheat.

[0090] Unless otherwise specifically stated, the numerical value set forth in these embodiments does not limit the scope of the present invention. In all examples shown and described here, unless otherwise specified, any specific value should be interpreted as merely exemplary, rather than as restriction, and therefore, other examples of exemplary embodiments may have different values. SEQUENCE LISTING <110> Jiangsu Lixiahe Region Agricultural Science Research Institute <120> Screening of KASP primer sets for wheat with short stalks, dense spikelets and resistance to scab and its application <130> 202206 <160> 8 <170> PatentIn version 3.3 <210> 1 <211> 20 <212> DNA <213> Artificial sequence <400> 1 cgagcctccc atatccgtct 20 <210> 2 <211> 41 <212> DNA <213> Artificial sequence <400> 2 gaaggtgacc aagttcatgc tccagctagg ctcttcacac t 41 <210> 3 <211> 41 <212> DNA <213> Artificial sequence <400> 3 gaaggtcgga gtcaacggat tccagctagg ctcttcacac c 41 <210> 4 <211> 71 <212> DNA <213> Wheat (Triticum aestivum L.) <220> <221> misc_feature <222> (36)..(36) <223> R is the base A or G <400> 4 ttaaggggta tcggtctagc cgggcattta ggaccrgtgt gaagagccta gctggatctt 60 tttttatggt c 71 <210> 5 <211> 41 <212> DNA <213> Artificial sequence <400> 5 gaaggtgacc aagttcatgc ttggacacccg aagtagttcc c 41 <210> 6 <211> 41 <212> DNA <213> Artificial sequence <400> 6 gaaggtcgga gtcaacggat ttggacaccg aagtagttcc t 41 <210> 7 <211> 20 <212> DNA <213> Artificial sequence <400> 7 tctgcgggca catcagttag 20 <210> 8 <211> 71 <212> DNA <213> Wheat (Triticum aestivum L.) <220> <221> misc_feature <222> (36)..(36) <223> R represents the base is C or T <400> 8 gcagccctaa ctgatatgga caccgaagta gttccrgtaa taaccaacta actgatgtgc 60 ccgcagagaa g 71

Claims

1. A substance for detecting resistance of wheat to scab, characterized in that: The substance is a primer combination of a set of primers X1 for detecting whether the 36th deoxyribonucleotide of the molecular marker shown in SEQ ID No.4 on chromosome 2D in the wheat genome is GG, AA, or G and A, and a set of primers X2 for detecting whether the 36th deoxyribonucleotide of the molecular marker shown in SEQ ID No.8 on chromosome 5A in the wheat genome is TT, CC, or T and C, or a detection reagent or kit containing the primer combination; The primer set X1 consists of the following primers: (1) a single-stranded DNA molecule shown in SEQ ID No. 1; (2) a single-stranded DNA molecule shown in positions 22 to 41 of SEQ ID No. 2 or a specific fluorescent label sequence A connected to the 5' end of the single-stranded DNA molecule shown in positions 22 to 41 of SEQ ID No. 2; and (3) a single-stranded DNA molecule shown in positions 22 to 41 of SEQ ID No. 3 or a specific fluorescent label sequence B connected to the 5' end of the single-stranded DNA molecule shown in positions 22 to 41 of SEQ ID No. 3; The set of primers X2 consists of the following primers: (1) a single-stranded DNA molecule shown at positions 22-41 of SEQ ID No.5 or a specific fluorescent label sequence A connected to the 5' end of the single-stranded DNA molecule shown at positions 22-41 of SEQ ID No.5; (2) a single-stranded DNA molecule shown at positions 22-41 of SEQ ID No.6 or a specific fluorescent label sequence B connected to the 5' end of the single-stranded DNA molecule shown at positions 22-41 of SEQ ID No.6; and (3) a single-stranded DNA molecule shown at positions 22-41 of SEQ ID No.

7.

2. The substance according to claim 1, characterized in that The set of primers X1 contains one upstream primer and two downstream primers; The upstream primer is designed according to the 36th deoxyribonucleotide of the sequence shown in SEQ ID No. 4 on chromosome 2D in the wheat genome and its upstream sequence; The downstream primers are designed according to the downstream sequence of the 36th deoxyribonucleotide of the sequence shown in SEQ ID No. 4 on chromosome 2D in the wheat genome, and the 3' terminal deoxyribonucleotide of one downstream primer is T, and the 3' terminal deoxyribonucleotide of the other downstream primer is C.

3. The substance according to claim 1, characterized in that The set of primers X2 contains two upstream primers and one downstream primer; The upstream primer is designed according to the 36th deoxyribonucleotide of the sequence shown in SEQ ID No. 8 on chromosome 5A in the wheat genome and its upstream sequence, and the 3' terminal deoxyribonucleotide of one of the upstream primers is T, and the 3' terminal deoxyribonucleotide of the other upstream primer is C; The downstream primer is based on the sequence shown in SEQ ID No.8 on chromosome 5A in the wheat genome. The downstream sequence of the 36th deoxyribonucleotide in the column is designed.

4. The substance according to claim 1, characterized in that The specific fluorescent label sequence A is a fluorescent label sequence FAM, and the specific fluorescent label sequence B is a fluorescent label sequence HEX.

5. The substance according to claim 4, characterized in that The primer set X1 consists of the upstream primer sequence SEQ ID No.1 and the downstream primer sequences SEQ ID No.2 and SEQ ID No.3; the primer set X2 consists of the upstream primer sequences SEQ ID No.5 and SEQ ID No.6 and the downstream primer sequence SEQ ID No.

7.

6. Use of the substance according to any one of claims 1 to 5 in any of the following: (A) Identifying or assisting in identifying the resistance of wheat to scab disease with short stalks and dense spikelets; (B) Compare the plant height, spikelet density and resistance to fusarium wilt of the tested wheat; (C) breeding or selecting wheat plants, strains, lines or varieties with lower plant height, higher spikelet density and relatively stronger resistance to ergot; (E) preparing a product for identifying or assisting in identifying or comparing plant height, spikelet density and resistance to fusarium head blight; (F) preparing a product for breeding or screening wheat plants, strains, lines or varieties with lower plant height, higher spikelet density and relatively stronger resistance to ergot disease.

7. Either of the following methods: Method A: A method for comparing the resistance of wheat to scab disease of dwarf stalks and dense spikelets to be tested, comprising the following steps (A1) or (A2): (A1) detecting whether the genotype of the 36th position of the molecular marker shown by SEQ ID No. 4 on chromosome 2D in the wheat genome is AA, GG or AG, and simultaneously detecting whether the genotype of the 36th position of the molecular marker shown by SEQ ID No. 8 on chromosome 5A in the wheat genome is CC, TT or CT; (A2) determining the fusca resistant trait of the wheat to be tested as follows: when the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No. 4 on chromosome 2D in the genome is GG and the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No. 8 on chromosome 5A in the genome is TT, the wheat to be tested has the fusca resistant trait of fusca resistant trait of fusca; Method B: A method for breeding or screening wheat plants or strains or lines or varieties with lower plant height, higher spikelet density and relatively stronger resistance to fusarium head blight, comprising the following steps: (B1) detecting whether the genotype of the 36th position of the molecular marker shown by SEQ ID No. 4 on chromosome 2D in the wheat genome is AA, GG or AG, and simultaneously detecting whether the genotype of the 36th position of the molecular marker shown by SEQ ID No. 8 on chromosome 5A in the wheat genome is CC, TT or CT; (B2) selecting a wheat plant to be tested in which the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No.4 on chromosome 2D in the genome is G and the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No.8 on chromosome 5A in the genome is T as a parent for breeding, and selecting a wheat plant in which the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No.4 on chromosome 2D in the genome is G and the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No.8 on chromosome 5A in the genome is T in each breeding generation, and finally obtaining a wheat plant or strain or variety with a lower plant height, a higher spikelet density and a relatively stronger resistance to ergot disease.

8. The method according to claim 7, characterized in that The (A1) (B1) The specific operation is: The method comprises the following steps: using the substance described in any one of claims 1 to 5 to perform PCR amplification on the wheat genomic DNA to be tested, scanning the amplified product for fluorescent signals, analyzing the scanning data, and then determining the type of the 36th deoxyribonucleotide shown in SEQ ID No. 4 on chromosome 2D in the wheat gene to be tested and the type of the 36th deoxyribonucleotide shown in SEQ ID No. 8 on chromosome 5A in the wheat gene to be tested as follows; If the fluorescent signal data of the amplified product of the wheat to be tested is displayed as blue, the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No.4 on chromosome 2D in the genome of the wheat to be tested is a homozygous G, or the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No.8 on chromosome 5A in the genome of the wheat to be tested is a homozygous T; If the fluorescent signal data of the amplified product of the wheat to be tested is displayed as red, the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No.4 on chromosome 2D in the genome of the wheat to be tested is a homozygous A, or the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No.8 on chromosome 5A in the genome of the wheat to be tested is a homozygous C.

Images