Molecular marker primer group related to stripe rust resistance character of wheat and application of molecular marker primer group

Abstract

The invention relates to the technical field of molecular markers, and particularly discloses a molecular marker primer group related to the stripe rust resistance character of wheat and application of the molecular marker primer group. The molecular marker Qyr.saas-4a is located on a long arm of a wheat 4A chromosome, and is closely linked with stripe rust resistant major QTL Qyr.saas-4A. The invention provides a specific KASP primer group for detecting the molecular marker, and the nucleotide sequence of the specific KASP primer group is shown as SEQ ID NO.1-3. The molecular marker is a co-dominant marker, can efficiently and accurately carry out genetic typing on the stripe rust resistance character of a wheat material through a fluorescent quantitative PCR technology, and is not limited by the environment and the growth stage. The invention also relates to an application of the primer group in molecular marker-assisted breeding for breeding a new variety of stripe rust resistant wheat, the breeding selection efficiency and success rate can be obviously improved, and the limitation that the traditional breeding depends on phenotypic selection is overcome.

Description

Technical Field

[0001] This invention relates to the field of molecular marker technology, specifically to a molecular marker primer set related to wheat stripe rust resistance and its application. Background Technology

[0002] Common wheat ( Triticum aestivum Wheat (L.) is one of the world's most important crops, with its sown area accounting for 20%-30% of the country's arable land (Zhang Ruixia, 2014). It is the staple food for 35% of the population. It has been reported that wheat production should be increased by 70% to meet future food demands.

[0003] Wheat production is affected by a variety of diseases and pests, among which stripe rust is one of the most serious. In my country, stripe rust has caused more than 10 major epidemics in the past few decades, with each epidemic affecting an area of ​​over 4 million hectares. 2 The above information indicates that from 2006 to 2016, stripe rust in my country's main wheat-producing areas remained at a moderate or lower level. However, major epidemics occurred in 2017, 2020, and 2021, affecting areas of 5.4352 million hectares. 2 4,395,200 hectares 2 and 4.5 million hectares 2 (Huang et al. 2020). These data indicate that the incidence of stripe rust has been on the rise in recent years, especially in the southwestern wheat-growing areas where stripe rust is frequently prevalent. Therefore, we need to strengthen our ability to control wheat stripe rust.

[0004] Wheat resistance to stripe rust is classified into all-stage resistance (ASR) and adult-plant resistance (APR) based on its expression at different physiological stages (Caldwell. 1968; Flor. 1956). ASR genes represent race-specific resistance, functioning throughout the entire wheat growth cycle, but generally only providing immunity against a few specific physiological races of stripe rust. These genes typically possess a typical NBS-LRR domain (Ellis et al.) and are inherited as a qualitative trait. The widespread use of a single ASR gene can dramatically increase wheat resistance to stripe rust within a certain period, leading to rapid changes in the physiological races of stripe rust and ultimately causing the gene to lose its resistance. For example, the loss of resistance to genes such as Yr1, Yr9, and Yr26 resulted in a major outbreak of stripe rust in wheat production in my country (McIntosh et al. 2018). APR genes are non-race-specific resistances, usually inherited as quantitative traits, exhibiting partial resistance. In wheat seedlings, they often show susceptibility, but later, these genes are expressed in large quantities, conferring partial resistance. Because they lack specificity, they are resistant to many physiological races and are unaffected by race succession, resulting in more persistent resistance (Zeng Shimai, 1979). However, in wheat breeding, when APR genes exist alone, they confer relatively little or insufficient resistance. But when multiple APR genes are combined, the additive effect significantly enhances resistance (Babayan and Sarkisyan, 1971).

[0005] Wheat stripe rust resistance at the adult stage is a complex quantitative trait controlled by multiple quantitative trait loci (QTLs). It is characterized by low heritability, high susceptibility to environmental influences, and high selection difficulty. Therefore, traditional breeding methods suffer from long development times, high costs, and limited results. Molecular marker-assisted breeding, however, does not rely on phenotypic selection, meaning it is unaffected by environmental factors, gene interactions, or gene-environment interactions. Instead, it directly selects for genotypes, thus significantly improving breeding efficiency.

[0006] Single nucleotide polymorphism (SNP) refers to DNA sequence polymorphism caused by changes such as transitions, transversions, insertions, and deletions at a specific nucleotide position within the genome. The technique involves using known sequence information to locate SNP sites, then designing specific primers based on the identified variant sites to perform PCR amplification on genomic DNA or cDNA, obtaining specific polymorphic products based on the SNP sites. Finally, electrophoresis is used to analyze the polymorphism of the products. The advantages of SNP markers are their large number, wide distribution, uneven distribution within individual genes and the entire genome, and the ease of estimating SNP allele frequencies.

[0007] KASP is a novel genotyping technology developed by LGC (Laboratory of the Government Chemist) (http: / / www.lgcgenomics.com). It is characterized by low cost and high throughput. It performs precise bicelestem typing of SNPs and InDel sites through specific matching of primer terminal bases and has been widely used in molecular marker-assisted selection of crops such as rice, wheat, and soybean.

[0008] Currently, there are few tightly linked molecular markers related to wheat stripe rust resistance at the adult stage that can be used for practical molecular breeding. Therefore, it is of great significance to study and obtain QTLs or genes related to stripe rust resistance at the adult stage, and to use molecular biology techniques to increase stripe rust resistance at the adult stage, thereby increasing yield and ultimately achieving the goal of breeding high-yielding new wheat varieties.

[0009] Previous studies by some researchers have mapped QTLs for resistance to stripe rust at the adult stage, finding that related QTLs are widespread in wheat and distributed across various chromosomes. However, currently, there are few tightly linked molecular markers related to stripe rust resistance at the adult stage that can be used for practical molecular breeding. Therefore, obtaining QTLs or genes related to stripe rust resistance, utilizing molecular biology techniques to select suitable stripe rust-resistant plants, thereby improving crop photosynthesis and ultimately achieving the goal of breeding new wheat varieties with increased yield is of great significance in wheat breeding.

[0010] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of this disclosure, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0011] The purpose of this invention is to provide a molecular marker primer that can be used to analyze and identify wheat stripe rust resistance traits, thereby improving the problems of traditional wheat stripe rust resistance breeding relying on phenotypic selection and the scarcity of available molecular markers.

[0012] To achieve the above objectives, the present invention provides a set of molecular marker primers for detecting wheat stripe rust resistance. The primer set is a KASP competitive allele-specific PCR primer set, and its nucleotide sequence is shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3.

[0013] The molecular marker primers provided by this invention can be used to prepare a test kit for detecting wheat resistance to stripe rust.

[0014] The present invention also provides a kit for detecting wheat resistance to stripe rust, the kit comprising the molecular marker primer set as described in claim 1.

[0015] Preferably, the kit provided by the present invention further includes one or more of the following: enzymes, dNTPs, and buffers required for PCR reaction.

[0016] The molecular marker primer set or kit provided by this invention can be applied to molecular-assisted breeding of wheat against stripe rust, including the identification of wheat stripe rust traits or the screening of wheat lines resistant to stripe rust.

[0017] The present invention also provides a method for identifying the stripe rust trait of wheat, which involves using the genomic DNA of the wheat plant to be tested as a template, performing PCR amplification using the above-mentioned molecular marker primer set, and determining the stripe rust resistance trait of the wheat to be tested by detecting the fluorescence signal of the PCR amplification product.

[0018] Preferably, the PCR amplification in the above identification method comprises: 5 µL Master Mix, 1.4 µL mixed primers, and 5 ng template DNA, totaling 10 µL; the mixed primers are prepared by mixing 120 µL, 120 µL, and 300 µL of three primers at a concentration of 10 ng / µL, respectively, and adding 460 µL of ddH2O; the amplification program is as follows: 94℃ pre-denaturation for 15 min; 94℃ denaturation for 20 s, 61℃ annealing / extension for 60 s, for a total of 10 cycles; 94℃ denaturation for 20 s, 55℃ annealing / extension for 60 s, for a total of 26 cycles.

[0019] This invention discloses a molecular marker, Qyr.saas-4a, located on wheat chromosome 4A and linked to wheat stripe rust resistance. This marker is a flanking marker of the stripe rust resistance QTL Qyr.saas-4A on the long arm of wheat chromosome 4A, exhibiting high linkage. This marker can be used to detect the stripe rust resistance QTL on wheat chromosome 4A, rapidly screening plants with this locus, thus facilitating molecular-assisted breeding of disease-resistant wheat. The molecular marker Qyr.saas-4a provided by this invention is tightly linked to the stripe rust resistance QTL Qyr.saas-4A on wheat chromosome 4A, and can be used to locate the stripe rust resistance trait in wheat. This allows for the elimination of more susceptible plants during the breeding process, improving breeding efficiency and providing a foundation for research on wheat stripe rust resistance genes.

[0020] The beneficial effects of this invention are as follows: 1. This invention discloses for the first time a stripe rust-resistant QTL, Qyr.saas-4A, from wheat 'Sichuan Yonggang 2', located on the long arm of wheat chromosome 4A, which significantly increases stripe rust resistance. This QTL has high utilization value in wheat disease resistance (regulating stripe rust resistance) breeding.

[0021] 2. This invention discloses a molecular marker, Qyr.saas-4a, for the accurate detection of the stripe rust resistance QTL Qyr.saas-4A in wheat 'Sichuan Yonggang 2' based on a real-time quantitative PCR platform. The marker is co-dominant, and the detection is accurate, efficient, convenient, and stable.

[0022] 3. The molecular marker Qyr.saas-4a disclosed in this invention is highly correlated with the stripe rust resistant QTL Qyr.saas-4A, exhibiting a tight linkage marker characteristic. It has high accuracy in molecular marker-assisted selection and can significantly improve the selection and identification efficiency of wheat varieties resistant to stripe rust that are adapted to different environments, with a high success rate. Attached Figure Description

[0023] Figure 1 This is a schematic diagram showing the location of the wheat stripe rust resistance QTL Qyr.saas-4A on chromosome 4A in Example 1 of the present invention.

[0024] Figure 2 The KASP typing results of the recombinant inbred lines 'Sichuan Yonggang 2' × 'China Spring' in Example 1 of this invention were detected by molecular marker Qyr.saas-4a; among them, HEX (blue, 'China Spring') fluorescence indicates a stripe rust-susceptible line, FAM (red, 'Sichuan Yonggang 2') fluorescence indicates a stripe rust-resistant line; green fluorescence indicates a heterozygous line with a stripe rust-resistant phenotype; and black fluorescence indicates a blank control.

[0025] Figure 3The KASP typing results of the progeny plants of wheat 'Sichuan Yonggang 2' × wheat variety '51106' in Example 2 of this invention were detected by the molecular marker Qyr.saas-4a; among them, FAM (blue, 'Sichuan Yonggang 2') fluorescence indicates a line resistant to stripe rust, HEX (red, '51106') fluorescence indicates a line susceptible to stripe rust; green fluorescence indicates a heterozygous line with a phenotype of resistance to stripe rust; black fluorescence is a blank control. Detailed Implementation

[0026] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Note: Unless otherwise specified, the experimental methods in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0028] Example 1: Obtaining and Validating the Molecular Marker Qyr.saas-4a 1. Construction of genetic mapping populations Using the wheat line 'Sichuan Yonggang 2' as the female parent and the wheat variety 'China Spring' as the male parent, hybridization was carried out to obtain the F1 hybrid. The F1 generation was then used to obtain a double haploid population (DH population) containing 145 lines by anther culture and chromosome elimination methods, which constituted the genetic mapping population.

[0029] 2. Identification of population-based disease resistance phenotypes The aforementioned DH population and the susceptible control 'Huixian Red' were planted in the field, with one row sown for each material and one row of 'Huixian Red' planted every 19 rows. After the susceptible varieties fully developed the disease, a field survey of stripe rust severity was conducted on each line of the DH population. Phenotypic data were collected from multiple environments (six ecopoints across three years: 2021-2022) and the best linear unbiased predictor (BLUP) values ​​from the collected environmental data were used as the resistance phenotypic values ​​for subsequent analysis.

[0030] 3. Initial QTL positioning and interval determination Genomic DNA was extracted from the parents and all lines of the DH population using the CTAB method. A wheat 40K SNP chip (an economical, medium-density SNP chip developed by Shijiazhuang Borui Biotechnology Co., Ltd., containing approximately 40,000 wheat SNP markers evenly distributed across 21 chromosomes, with an average of 1900 markers per chromosome, an average genetic distance of approximately 0.1 cM, and an average physical distance of less than 300 kb) was used for genotypic analysis of all materials, suitable for general germplasm resource diversity analysis, genetic mapping and new gene discovery, comparative genomics analysis, and variety registration and identification (fingerprint analysis). A genetic map was constructed using JoinMap 4.0. Combined with stripe rust resistance phenotypic data of the population, the Inclusive Composite Interval mapping method in QTL IciMapping 4.0 was used. QTL scanning was performed using Mapping-ADD (ICIM-ADD) with a LOD threshold set to ≥2.5. The results located a stably expressed major-effect QTL for wheat stripe rust resistance in a 3.4 cM region on the long arm of chromosome 4A, named Qyr.saas-4A. The positive-effect site of this QTL is derived from the paternal parent 'Sichuan Yonggang 2'. This QTL is located on the long arm of wheat chromosome 4A, with a physical location of 725.7 Mbp-737.1 Mbp in its RefSeqv1.0 genome version. See the schematic diagram of this QTL location. Figure 1 As shown.

[0031] 4. Development and screening of tightly linked KASP molecular markers To obtain practical molecular markers for breeding, flanking SNP markers of the located Qyr.saas-4A region were physically located, and candidate genes located within this region were screened. By comparing the sequences of the parents 'Sichuan Yonggang 2' and 'Zhongguo Chun' in these candidate gene regions, polymorphic SNP sites were identified. For these polymorphic sites, competitive allele-specific KASP molecular marker primers were designed using DNAMAN software. A total of 10 pairs (30 primers, sequences detailed in Table 1, numbered from top to bottom as SEQ ID NO. 1~30) were designed. Developing efficient KASP molecular markers requires the following steps:

[0032] (I) Design primers for amplifying candidate gene sequences on the target homologous chromosome (4A) of a specific wheat genotype. Although a reference genome of hexaploid wheat 'Chinese Spring' is currently available, due to possible chromosomal structural variations during wheat evolution (Ma J, Stiller J, Wei Y, Zheng YL, Devos KM, Doležel J, Liu C (2014) Extensive Pericentric Rearrangements in the Bread Wheat (Triticum aestivum L.) Genotype “Chinese Spring” Revealed from Chromosome Shotgun Sequence Data. Genome Biol Evol 6:3039-3048), the gene sequence structure and polymorphic sites of different wheat genotypes may differ significantly. The simplest method to obtain gene sequences of a specific wheat genotype efficiently, rapidly, and at low cost is homologous sequence cloning. Because wheat has three partially homologous chromosome sets, isolating sequences specific to a particular homologous chromosome is very difficult (Bagge M, Xia X, Lübberstedt T (2007) Functionalmarkers in wheat. Curr Opin Plant Biol 10:211-216), requiring the design of primers specific to a particular chromosome set. Based on a thorough understanding of comparative genomics and bioinformatics techniques, sequence extraction, alignment, and analysis were performed on reference genomes of hexaploid wheat donor diploid parents *Triticum urartu* and *Triticum aestivum*, tetraploid wild *Erinum spp.*, and hexaploid 'Chinese Spring', to obtain polymorphic sites specific to a particular chromosome, thereby designing specific primers to amplify the target region. After primer design, comparative genomics techniques are needed to further analyze primer specificity, annealing temperature, amplification length, etc., to determine primer usability.

[0033] (II) Amplification of the target wheat genotype using specific primers. During amplification, skilled molecular biology techniques are required to optimize amplification conditions, followed by cloning and sequencing to obtain the gene sequence of the target region.

[0034] (III) Obtaining polymorphic sites in candidate gene sequences of the target homologous chromosome. After obtaining the two parental candidate gene sequences, further detailed analysis of the sequences is required to detect the presence of polymorphic sites. If no polymorphic sites are found, it is necessary to return to step I and select other possible candidate regions for isolation and cloning.

[0035] (IV) Design of upstream and downstream KASP primers for polymorphic sites. After obtaining the polymorphic sites, it is necessary to design KASP-specific primers. As mentioned earlier, wheat is an allohexaploid plant, so it is still necessary to use skilled bioinformatics techniques to analyze the ABD chromosome sequence in order to obtain KASP primers specific to the target chromosome.

[0036] (V) Optimization of KASP primer amplification conditions. After primer synthesis, the primer amplification conditions need to be further adjusted and optimized based on experience in order to achieve a significant differentiation effect between the parents.

[0037] In conclusion, although KASP marker technology has been widely used in diploid species, obtaining efficient KASP markers in hexaploid wheat is by no means easy for those skilled in the art.

[0038] After multiple cloning and sequencing, primer design and amplification, a total of 10 pairs of KASP primers were designed (see Table 1), and the marker Qyr.saas-4a was found to be tightly linked to the stripe rust-resistant QTL Qyr.saas-4A.

[0039] Table 1. Sequences of 10 KASP molecular marker primers Of the 10 pairs of KASP primers designed, one molecular marker, Qyr.saas-4a, was ultimately obtained. This marker showed high co-segregation of genotype and disease-resistant phenotype in resistant and susceptible parents and in the population. The KASP genotyping results of the primers corresponding to the molecular marker Qyr.saas-4a are shown below. Figure 2 As shown. Therefore, Qyr.saas-4a was identified as a molecular marker closely linked to QTL Qyr.saas-4A. The specific KASP molecular marker primer sequences for this marker are shown in SEQ ID NO. 1~3.

[0040] Example 2: Verification of the application of molecular marker primers in breeding This embodiment verifies the effectiveness of the Qyr.saas-4a molecular marker and its primers in tracking and selecting disease-resistant QTLs in actual breeding populations, as detailed below: (1) Using the stripe rust resistant common wheat line 'Sichuan Yonggang 2' as the female parent and the disease-susceptible common wheat line '51106' as the male parent, hybridization was carried out. In the offspring lines, the molecular marker Qyr.saas-4a was used to track the stripe rust resistant QTL Qyr.saas-4A. For specific samples, please refer to Table 2 below.

[0041] (2) The obtained offspring were subjected to Qyr.saas-4a marker detection. The specific method was as follows: DNA was extracted from a single lineage; it was used as a template, and PCR amplification was performed using the specific primer pair corresponding to the molecular marker as primers, and the fluorescence value was read.

[0042] The PCR amplification system described above is as follows: 5 µL Master Mix, 1.4 µL mixed primers, 5 ng template DNA, and double-distilled water to a total volume of 10 µL. At least three independent blanks containing double-distilled water instead of DNA template must also be added. The mixed primers are prepared by adding three primers at concentrations of 10 ng / µL to 120 µL, 120 µL, and 300 µL respectively, followed by the addition of 460 µL of ddH2O.

[0043] The PCR amplification program was as follows: 94℃ pre-denaturation for 15 min; 94℃ denaturation for 20 s, 61℃ annealing / extension for 60 s, for a total of 10 cycles; 94℃ denaturation for 20 s, 55℃ annealing / extension for 60 s, for a total of 26 cycles; after completion, fluorescence readings were performed.

[0044] KASP classification results are shown below Figure 3 As shown, the genotype of plants that showed HEX (blue) fluorescence identical to 'Sichuan Yonggang 2' was designated as A, representing stripe rust resistant lines. The genotype of plants that showed FAM (red) fluorescence, similar to '51106', was designated as B, representing stripe rust susceptible lines. The genotypes and field phenotypic values ​​for stripe rust resistance of each line are shown in Table 2. Plants of the same type as 'Sichuan Yonggang 2' containing the stripe rust resistant QTL Qyr.saas-4A had an average stripe rust resistance severity of 38.7, significantly lower than the stripe rust resistance of plants of the '51106' type (average 90.6). A total of 28 new stripe rust resistant lines were selected. The actual results were consistent with the expected results, indicating that the stripe rust-resistant QTL Qyr.saas-4A of the present invention does indeed have a significant high stripe rust resistance effect; at the same time, the molecular marker Qyr.saas-4a of the present invention can be used to track and identify the stripe rust-resistant QTL Qyr.saas-4A.

[0045] Table 2. Genotype and phenotype correspondence of the 'Sichuan Yonggang 2' × '51106' hybrid generation QYR.SAAS-4A Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A molecular marker primer set for detecting wheat stripe rust resistance, characterized in that, The primer set is a KASP competitive allele-specific PCR primer set, and its nucleotide sequence is shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.

3.

2. The application of the molecular marker primers as described in claim 1 in the preparation of a wheat stripe rust resistance test kit.

3. A kit for detecting wheat resistance to stripe rust, characterized in that, The kit contains the molecular marker primer set as described in claim 1.

4. The reagent kit according to claim 3, characterized in that, The kit also includes one or more of the following: enzymes, dNTPs, and buffers required for PCR reactions.

5. The application of the molecular marker primer set as described in claim 1 or the kit as described in claim 3 in molecular-assisted breeding of wheat for resistance to stripe rust.

6. The application according to claim 5, characterized in that, The applications include the identification of wheat stripe rust traits or the screening of stripe rust-resistant wheat lines.

7. A method for identifying wheat stripe rust traits, characterized in that, The stripe rust resistance trait of the wheat under test was determined by PCR amplification using the molecular marker primer set described in claim 1, with the genomic DNA of the wheat plant as a template, and by detecting the fluorescence signal of the PCR amplification product.

8. The identification method according to claim 7, characterized in that, The PCR amplification system consisted of 5 µL MasterMix, 1.4 µL of mixed primers, and 5 ng template DNA, totaling 10 µL. The mixed primers were prepared by mixing 120 µL, 120 µL, and 300 µL of three primers at a concentration of 10 ng / µL, respectively, and then adding 460 µL of ddH2O.

9. The identification method according to claim 7, characterized in that, The PCR amplification program is as follows: 94℃ pre-denaturation for 15 min; 94℃ denaturation for 20 s, 61℃ annealing / extension for 60 s, for a total of 10 cycles; 94℃ denaturation for 20 s, 55℃ annealing / extension for 60 s, for a total of 26 cycles.

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