KASP molecular markers for detecting wheat stripe rust resistance genes and their applications

Abstract

This invention belongs to the field of molecular biology technology and relates to the KASP molecular marker for detecting wheat stripe rust resistance genes and its application. The KASP molecular marker described in this invention... XK6B‑5 The KASP molecular marker was amplified using the specific primer set shown in SEQ ID NO. 2~4. XK6B‑5 wheat stripe rust resistance gene YrZT94.1 Closely linked, exhibiting co-separation characteristics. XK6B‑5 The polymorphism is G / T. The KASP molecular marker provided by this invention can accurately track wheat stripe rust resistance genes. YrZT94.1 This invention enables marker-assisted selection to improve the efficiency of wheat disease resistance breeding. It can be applied simply, rapidly, and with high throughput to screen wheat germplasm resources for stripe rust resistance genes. YrZT94.1 This allows for its application in molecular design breeding, greatly accelerating the wheat breeding process.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology and relates to the KASP molecular marker for detecting wheat stripe rust resistance genes and its application. Background Technology

[0002] There are two main methods for controlling wheat stripe rust: chemical fungicides and breeding and planting resistant varieties. Among these, discovering resistance genes and breeding resistant varieties are effective measures for controlling wheat stripe rust. Currently, 87 formally named wheat stripe rust resistance genes have been reported. Yr1-87 Wheat stripe rust resistance genes, including a large number of tentatively named genes, can be categorized into adult-stage resistance (APR) and all-growth-cycle resistance (ASR). All-growth-cycle resistance is effective only against specific physiological or virulent races, also known as race-specific resistance. It is generally a qualitative trait, controlled by a single major gene, and effective at all stages of wheat growth. However, due to frequent mutations of stripe rust fungi and the prevalence of new virulent physiological races, many wheat varieties relying on a single resistance source lose their resistance after only 3-5 years in production. APR genes, not targeting specific stripe rust physiological races, have a broad spectrum of resistance and do not lose resistance due to race variation. They are generally considered to possess durable resistance and are highly favored by breeders. However, most known APR genes are currently in the early mapping stage, with large mapping intervals, unclear genetic effects, and some even exhibiting linkage burden issues. Few have been successfully applied in production. Therefore, there is an urgent need to discover and utilize new resistance gene resources and corresponding molecular markers to further accelerate the process of molecularly assisted breeding for wheat resistance.

[0003] Single nucleotide polymorphisms (SNPs) refer to DNA sequence polymorphisms caused by variations in a single nucleotide, mainly including deletions, insertions, transitions, and transversions of single bases. SNP markers account for over 90% of all known polymorphisms, exhibiting a very high distribution density in the genome, and are prevalent in intergenic regions, coding regions, and non-coding regions, making them the most widely used type of DNA marker. In recent years, third-generation KASP (Kompetitive allele-specific PCR) molecular markers based on SNPs have emerged as a fluorescence-based homogenization technique. Based on specific matching of primer terminal bases, it can accurately perform biallelic genotyping of target SNPs and InDels at specific sites. It boasts extremely high accuracy and conversion rate, low cost, high throughput, and convenient and rapid result analysis, playing a crucial role in gene mapping, marker-assisted selection breeding, and germplasm resource identification. Based on this, developing KASP markers closely linked to stripe rust resistance genes is of great significance for rapid screening of rust resistance genes in wheat germplasm resources and molecular marker-assisted selection breeding, which will accelerate the breeding process of new wheat varieties resistant to stripe rust. Summary of the Invention

[0004] To address the aforementioned technical problems and deficiencies, this invention provides a KASP molecular marker for detecting wheat stripe rust resistance genes and its application. This KASP molecular marker is associated with stripe rust resistance genes. YrZT94.1 Closely linked, capable of accurately combating stripe rust genes. YrZT94.1 Screening is of great significance for the creation of wheat stripe rust resistant materials or for molecular marker-assisted selection breeding of wheat stripe rust resistant materials.

[0005] In a first aspect, the present invention provides a KASP molecular marker for detecting wheat stripe rust resistance genes, wherein the KASP molecular marker is... XK6B-5 ;

[0006] The XK6B-5 The nucleotide sequence is shown in SEQ ID NO.1.

[0007] Furthermore, in the KASP molecular marker for detecting wheat stripe rust resistance genes provided by this invention, the... XK6B-5 The amplification was achieved using three primers, including two forward primers and one reverse primer. Different fluorescent tags were added to the 5′ ends of the two forward primers.

[0008] The two forward primers are forward primer 1 and forward primer 2. The nucleotide sequence of forward primer 1 is shown in SEQ ID NO. 2, and the nucleotide sequence of forward primer 2 is shown in SEQ ID NO. 3.

[0009] The nucleotide sequence of the reverse primer is shown in SEQ ID NO.4.

[0010] Furthermore, in the KASP molecular marker for detecting wheat stripe rust resistance genes provided by the present invention, the fluorescent tag is a FAM fluorescent group and a HEX fluorescent group;

[0011] The universal primer sequence for the FAM fluorescent group is 5′-GAAGGTGACCAAGTTCATGCT-3′;

[0012] The universal primer sequence for the HEX fluorescent group is 5′-GAAGGTCGGAGTCAACGGATT-3′.

[0013] Furthermore, in the KASP molecular marker for detecting wheat stripe rust resistance genes provided by this invention, the KASP molecular marker is associated with the wheat stripe rust resistance gene. YrZT94.1 Linked and co-located on wheat chromosome 6BS;

[0014] The polymorphism of the KASP molecular marker is G / T.

[0015] Secondly, this invention provides a method for detecting whether wheat contains a stripe rust resistance gene. YrZT94.1 The kit contains two forward primers and one reverse primer. The two forward primers are forward primer 1 and forward primer 2. The nucleotide sequence of forward primer 1 is shown in SEQ ID NO.2, the nucleotide sequence of forward primer 2 is shown in SEQ ID NO.3, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.4.

[0016] Thirdly, this invention provides a KASP molecular marker for detecting wheat stripe rust resistance genes or for detecting whether wheat contains stripe rust resistance genes. YrZT94.1 The application of the kit includes: screening wheat varieties with stripe rust resistance and / or screening varieties containing wheat stripe rust resistance genes. YrZT94.1 Wheat lines and / or molecular markers assist in the breeding of stripe rust-resistant wheat.

[0017] Fourthly, the present invention provides a gene containing wheat resistance to stripe rust. YrZT94.1 The method for screening wheat lines includes: using the genomic DNA of the wheat material to be tested as a template, performing real-time PCR amplification on the template using primers with the KASP molecular marker for detecting wheat stripe rust gene, and determining the genotype based on the amplification product;

[0018] The primers include two forward primers and one reverse primer. The two forward primers are forward primer 1 and forward primer 2. The nucleotide sequence of forward primer 1 is shown in SEQ ID NO.2, the nucleotide sequence of forward primer 2 is shown in SEQ ID NO.3, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.4.

[0019] Furthermore, the present invention provides a wheat stripe rust resistance gene. YrZT94.1 In the screening method for wheat lines, the homozygous allele genotype with HEX fluorescence is "Y:Y", the homozygous allele genotype with FAM fluorescence is "X:X", the heterozygous genotype is "X:Y" or "Y:X", and the deletion value genotype is "?".

[0020] Furthermore, the present invention provides a wheat stripe rust resistance gene. YrZT94.1 In the screening method for wheat lines, if the genotype of the KASP molecular marker is "Y:Y", then the wheat material to be tested contains the stripe rust resistance gene. YrZT94.1 ;

[0021] If the genotype of the KASP molecular marker is "X:X", then the wheat material being tested does not contain the stripe rust resistance gene. YrZT94.1 ;

[0022] If the genotype of the KASP molecular marker is "X:Y" or "Y:X", then the wheat material being tested contains a heterozygous stripe rust resistance gene. YrZT94.1 .

[0023] Furthermore, the present invention provides a wheat stripe rust resistance gene. YrZT94.1 In the method for screening wheat lines, the PCR amplification system is as follows: 0.2 μg of the wheat material to be tested DNA, 2 μL of 2×KASPV4.0 Master mix, 0.0448 μL of mixed primers, and 1.9552 μL of ddH2O;

[0024] The mixed primers consist of: 12 μL of forward primer 1, 12 μL of forward primer 2, 30 μL of reverse primer, and 46 μL of ddH2O.

[0025] The concentrations of forward primer 1, forward primer 2, and reverse primer were 100 μM.

[0026] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0027] This invention provides a gene for resistance to wheat stripe rust. YrZT94.1 Closely linked SNP tags, based on which to develop and YrZT94.1Tightly linked KASP markers, exhibiting co-segregation characteristics, can accurately identify the presence of wheat stripe rust resistance genes. YrZT94.1 This allows for the elimination of susceptible plants during the breeding process, improving breeding efficiency and playing a significant role in the creation of wheat stripe rust resistant materials or molecular marker-assisted selection breeding for wheat stripe rust resistance. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 Genes for wheat resistance to stripe rust YrZT94.1 Location results on wheat chromosome 6BS.

[0030] Figure 2 for XK6B-5 Mark the genotyping results of natural populations.

[0031] Figure 3 for XK6B-5 The genotype-phenotype association results for natural population typing are labeled. (A) represents the IT value, (B) represents the DS value, and A represents the presence of the stripe rust resistance gene. YrZT94.1 The wheat material to be tested, B, does not contain the stripe rust resistance gene. YrZT94.1 The wheat material to be tested. Detailed Implementation

[0032] The technical solution of the present invention will be described below with reference to embodiments. However, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental and detection methods described in each embodiment are conventional methods; the reagents and materials described are commercially available unless otherwise specified. Unless otherwise specified, all percentages in the following embodiments refer to mass percentages. Unless otherwise specified, all proportions in the following embodiments refer to mass ratios.

[0033] Example 1

[0034] This embodiment provides YrZT94.1 The positioning and development of closely linked KASP tags.

[0035] A recombinant inbred line (RIL) was constructed by continuously self-pollinating wheat germplasm ZTR94 (a wheat variety with excellent stripe rust resistance bred from a cross between the Indian wheat variety PI 183527 and the Australian wheat variety AvS) and Kenong 9204. This RIL formed the initial mapping population. Phenotypic segregating families were selected from this population as remaining heterozygous lines to construct the fine mapping population. The entire experiment was conducted in the field in Yangling District, Shaanxi Province. The specific steps were as follows: the RIL population was row-sown with 20 seeds per row; the fine mapping population was sown individually; the fine mapping population was then tagged for sampling, phenotypic identification, and genotyping to screen for recombinants, thereby narrowing the mapping interval. Phenotypic identification used two evaluation indicators: infection type (IT) and disease severity (DS). The IT (Indexed Testing) standard used a 0-9 grade identification standard (Line and Qayoum 1992), while the DS (Discrimination Data Set) standard used 0, 1%, 5%, 10%, 20%, 30%, 40%, 60%, 80%, and 100%. Genomic DNA was extracted from leaves of the parental ZTR94, Kenong 9204, F2 secondary segregating populations, and the RIL population using the CTAB (Cetyltrimethylammonium bromide) method. DNA quality control was performed using agarose gel electrophoresis, and DNA quantification was measured using a Thermo Fisher Scientific NanoDrop™ One / OneC ultra-micro UV spectrophotometer. Qualified DNA was sent to Shijiazhuang Borui Technology Co., Ltd. for 16K SNP microarray typing, and a genetic map was constructed using IciMapping V4.2. The Inclusive Composite Interval Mapping-ADD (ICIM-ADD) method in QTL IciMapping V4.2 was used, with a LOD threshold of ≥2.5. The stripe rust phenotype was used to detect QTLs and locate wheat stripe rust resistance genes. YrZT94.1 and calculate YrZT94.1 The genetic distance between the location and molecular markers was initially determined using RIL populations and secondary segregating populations (population size 2589). YrZT94.1 Located within a 0.329 cM region on the wheat 6BS chromosome, corresponding to a physical location of 104.6–118.7 Mb in the Chinese Spring reference genome 2.0, and co-segregating with the KASP marker XK6B-5 ( Figure 1 ).

[0036] In this embodiment, the molecular markers used were derived from the 16K microarray and resequencing data of the parental ZTR94 and KENON 9204. The obtained SNP sites were converted into KASP markers using the Polymarker website and synthesized at Shanghai Sangon Biotech Co., Ltd.

[0037] KASP markup XK6B-5Resequencing data from parental ZTR94 and Kenon 9204, and YrZT94.1 Co-separation.

[0038] XK6B-5 The nucleotide sequence is shown in SEQ ID NO.1: 5′-GTTTTGCCTAGTTCTCCATGGGCGCTTCTAGTTCC[G / T]TCCACGACGGGCATCGAGCAATTGTTTTCCGAGGT-3′.

[0039] XK6B-5 The sequence of the forward primer 1 is shown in SEQ ID NO.2, 5′-GAAGGTGACCAAGTTCATGCTtcgatgcccgtcgtggaC-3′;

[0040] XK6B-5 The sequence of the forward primer 2 is shown in SEQ ID NO.3, 5′-GAAGGTCGGAGTCAACGGATTtcgatgcccgtcgtggaA-3′;

[0041] XK6B-5 The reverse primer sequence is shown in SEQ ID NO.4 as 5′-gcaatccactcgaccagtca-3′.

[0042] The universal primer for FAM fluorescence is 5′-GAAGGTGACCAAGTTCATGCT-3′. The FAM fluorescent group is observed and read at an excitation wavelength of 485 nm and an emission wavelength of 520 nm.

[0043] The universal primer for HEX fluorescence is 5′-GAAGGTCGGAGTCAACGGATT-3′. The HEX fluorescent group is observed at an excitation wavelength of 535 nm and an emission wavelength of 556 nm.

[0044] Example 2

[0045] This embodiment provides a method for genotyping.

[0046] Identifying whether wheat materials contain stripe rust resistance genes YrZT94.1 The method includes the following steps:

[0047] S1. Primer synthesis: In the primer mixture, the concentrations of forward primer 1, forward primer 2 and reverse primer are 100 μM, of which the amount of forward primer 1 and forward primer 2 added is 12 μL, the amount of reverse primer added is 30 μL, and the amount of ddH2O added is 46 μL.

[0048] S2. Genomic DNA was extracted from the wheat material to be tested using the CTAB method and used as an amplification template.

[0049] S3. Using genomic DNA from the wheat material to be tested as a template, KASP molecular markers were used for amplification. XK6B-5 Primers were used to amplify the genomic DNA of the wheat material to be tested by PCR to obtain the amplification product.

[0050] S4. The reaction system used to amplify the KASP molecular marker is as follows: 0.2 μg of wheat DNA to be tested, 2 μL of 2×KASPV4.0 Master mix, 0.0448 μL of mixed primers, and 1.9552 μL of ddH2O.

[0051] S5. The reaction procedure for amplifying the KASP molecular marker is as follows: pre-denaturation at 94 °C for 15 min; denaturation at 94 °C for 20 s, annealing at 65 °C for 60 s, 10 cycles, with the annealing temperature decreasing by 0.6 °C per cycle; denaturation at 94 °C for 20 s, annealing at 55 °C for 60 s, 32 cycles.

[0052] S6. Transfer the amplification product to a microplate reader to read the fluorescence data, and then import the fluorescence data into KlusterCaller software for analysis to obtain genotype data. The genotype of a homozygous allele with HEX fluorescence is “Y:Y”, the genotype of a homozygous allele with FAM fluorescence is “X:X”, the genotype of a heterozygous allele is “X:Y” or “Y:X”, and the genotype with a deletion value is “?”.

[0053] In KASP mark XK6B-5 In the test, if the genotype is "Y:Y", then the wheat material being tested contains the stripe rust resistance gene. YrZT94.1 (Denotes it as A). If the genotype is "X:X", then the wheat material being tested does not contain the stripe rust resistance gene. YrZT94.1 (Referred to as B). If the genotype is "X:Y" or "Y:X", then the wheat material being tested contains a heterozygous stripe rust resistance gene. YrZT94.1 .

[0054] Example 3

[0055] This embodiment provides molecular markers. XK6B-5 Selecting genes resistant to stripe rust YrZT94.1 Applications in [the field].

[0056] Using the KASP molecular marker described in Example 1 XK6B-5 Genotyping of the RIL population using the method described in Example 2 showed that the RIL population could be divided into two types ( Figure 2 ).like Figure 2As shown, if the genotype is "Y:Y", then the wheat material being tested contains the stripe rust resistance gene. YrZT94.1 Let A be the genotype. If the genotype is "X:X", then the wheat material being tested does not contain the stripe rust resistance gene. YrZT94.1 Let it be denoted as B. For example... Figure 3 As shown, in the mark XK6B-5 Contains genes that resist stripe rust YrZT94.1 The average IT value of the plants was 4.4, which was significantly lower than that of plants without the stripe rust resistance gene. YrZT94.1 The plants had an average IT value of 6.3 and contained a gene for resistance to stripe rust. YrZT94.1 The average DS of the plants was 35.8, which was significantly lower than that of plants without the stripe rust resistance gene. YrZT94.1 The average DS of the plants was 59.2, consistent with the expected results. The molecular markers of this invention... XK6B-5 Able to quickly YrZT94.1 Selection and germplasm resource screening can be applied to YrZT94.1 Molecular-assisted selection breeding can accelerate the breeding process.

[0057] As described above, the basic principles, main features, and advantages of the present invention have been well described. The above embodiments and specifications are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the protection scope defined by the present invention.

Claims

1. A set of primers for KASP molecular markers for the detection of wheat resistance to stripe rust genes, characterized in that, The KASP molecular marker is XK6B-5 located on the 6BS chromosome of wheat, and the polymorphism is G / T; The XK6B-5 The nucleotide sequence is shown in SEQ ID NO.1; The XK6B-5 obtained by amplification with three primers, the three primers including two forward primers and one reverse primer, the 5' ends of the two forward primers being added with different fluorescent tags, respectively; The two forward primers are forward primer 1 and forward primer 2, the nucleotide sequence of forward primer 1 is shown as SEQ ID NO. 2, and the nucleotide sequence of forward primer 2 is shown as SEQ ID NO. 3; The nucleotide sequence of the reverse primer is shown as SEQ ID NO.

4.

2. The primer of claim 1, wherein The fluorescent tag is a FAM fluorescent group and a HEX fluorescent group; The universal primer sequence of the FAM fluorescent group is 5'-GAAGGTGACCAAGTTCATGCT-3'; The universal primer sequence of the HEX fluorescent group is 5'-GAAGGTCGGAGTCAACGGATT-3'.

3. A kit for detecting the presence of an anti-rust disease gene in wheat, characterized in that, YrZT94.1 The kit comprises two forward primers and one reverse primer, the two forward primers are forward primer 1 and forward primer 2, the nucleotide sequence of forward primer 1 is shown as SEQ ID NO. 2, the nucleotide sequence of forward primer 2 is shown as SEQ ID NO. 3, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO.

4. ​ 4. A method of screening for a wheat line containing a wheat resistance gene to stripe rust, characterized in that, YrZT94.1 Comprising: Using the genomic DNA of the wheat material to be tested as a template, performing fluorescent quantitative PCR amplification on the template by using the primer pair according to any one of claims 1-2, and judging the genotype according to the amplification product; ​ The system of the PCR amplification is: 0.2 μg of the DNA of the wheat material to be tested, 2 μL of 2xKASPV4.0 Master mix, 0.0448 μL of mixed primers, and 1.9552 μL of ddH2O; The mixed primers are: 12 μL of forward primer 1, 12 μL of forward primer 2, 30 μL of reverse primer, and 46 μL of ddH2O; The concentration of forward primer 1, forward primer 2 and reverse primer is 100 μM; The homozygous allele genotype with HEX fluorescence is "Y:Y", the homozygous allele genotype with FAM fluorescence is "X:X", the heterozygous genotype is "X:Y" or "Y:X", and the missing value genotype is "?"; If the genotype of the KASP molecular marker is "Y:Y", the wheat material to be tested contains the stripe rust resistance gene YrZT94.1 ; If the genotype of the KASP molecular marker is "X:X", the wheat material to be tested does not contain the stripe rust resistance gene YrZT94.1 ; If the KASP molecular marker genotype is "X:Y" or "Y:X", the wheat material to be tested contains a heterozygous resistance to stripe rust gene YrZT94.1 YrZT94.1 .

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