CN113046467A - SNP loci significantly associated with wheat stripe rust resistance and application thereof in genetic breeding - Google Patents
SNP loci significantly associated with wheat stripe rust resistance and application thereof in genetic breeding Download PDFInfo
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Abstract
The invention discloses a group of (38) SNP loci which are obviously associated with the disease resistance of wheat stripe rust and an application method thereof in heredity and breeding. These SNPs were identified by simplified genome sequencing (GBS) of 768 wheat varieties and elite lines and mapping reference genomic sequences to discover Single Nucleotide Polymorphism Sites (SNPs) and then by whole genome association analysis. The SNP locus has high accuracy, can be converted into KASP markers and SNP chips, and can be widely applied to positioning, fine mapping and candidate gene identification of wheat stripe rust resistance genes, marker-assisted selection and whole genome selective breeding of wheat stripe rust resistance, and breeding of new stripe rust resistance germplasm and new varieties.
Description
Technical Field
The invention relates to the technical field of plant molecular markers, in particular to a group of SNPs (single nucleotide polymorphisms) which are obviously associated with wheat stripe rust resistance gene loci and application thereof in stripe rust resistance inheritance and molecular breeding.
Background
Wheat is one of the important food crops. Wheat stripe rust is one of the major factors affecting wheat production, and has long been the first disease causing yield loss in almost all wheat growing areas. Wheat stripe rust mainly damages leaves, and can infect stems, spikes, leaf sheaths, glumes and the like when the leaves are serious. Chlorosis spots appeared on leaf blades at seedling stage infected by stripe rust, and the resulting summer sporophyte matured and broke out yellow powder. After the stripe rust germs infect wheat plants, a large amount of summer spore piles are generated on the surfaces of leaves, epidermal tissues are damaged, the leaf area index of the plants is reduced, the respiration of the leaves is increased, the transpiration is reduced, and the photosynthesis and the chlorophyll content are obviously reduced, so that the metabolism of the plants is abnormal, the growth and development of the wheat plants are hindered, and the yield of the wheat is greatly reduced. Stripe rust occurs earlier and is extremely serious, which often causes that plants can not be spiced and even completely harvested, and almost all wheat planting areas in China occur and cause huge yield loss, and sometimes the loss of grain yield reaches millions of tons.
Breeding disease-resistant varieties or using chemical agents for prevention and treatment are common measures for resisting the threat of wheat stripe rust. However, long-term use of chemical agents for prevention and treatment can cause a series of adverse problems such as drug resistance of pathogenic bacteria and environmental pollution; the wheat variety with stripe rust resistance is bred and used, so that the problem of environmental pollution can be prevented and treated, the genetic composition of wheat can be improved, the yield of the wheat is increased, and the method is the most economic, safe and effective measure for preventing and treating the stripe rust of the wheat at present.
Genetic research on wheat stripe rust disease resistance plays an important role in understanding genetic structure, gene mapping and cloning of stripe rust resistance and effectively improving stripe rust resistance by using the genes in breeding. Molecular marker technology is the most commonly used technology for breeding research such as inheritance of related traits, gene mapping, marker-assisted breeding, genome selection breeding and the like. The commonly used DNA molecules are labeled with RFLP (Restriction Fragment Length Polymorphism), SSR (Simple Sequence Repeat), SNP (Single Nucleotide Polymorphism), and the like. The traditional RFLP and SSR markers have the limitations of low flux, small quantity, complicated operation process and the like, and the improvement of the working efficiency is greatly limited, so that the requirements of functional genome research cannot be met. The SNP markers are extremely abundant in genome distribution, have two-state property, are easy to carry out high-throughput automatic detection, and are molecular marker technologies with the greatest application prospect in genetic research and breeding.
Currently, the high-throughput detection technology for SNP mainly comprises sequencing and DNA chip technology. The high density of SNP markers can be obtained by DNA resequencing of sample materials using sequencing techniques, but the cost of resequencing is high. The simplified genome sequencing-by-sequencing (GBS) is to firstly perform enzyme digestion and other methods on genome DNA to reduce the complexity of the genome, construct a low-complexity genome DNA library, and then perform deep sequencing by using a second-generation sequencing technology, thereby reducing the sequencing cost to a certain extent. The technology of constructing a DNA library by treating genomic DNA with two restriction enzymes and identifying SNPs by sequencing in wheat has been developed. However, the deep sequencing cost is still high for the huge genome (16G) relative to wheat, and the process of GBS data processing, sequence alignment, genotyping and the like has high requirements on data analysis, and the process can be completed only by personnel with professional bioinformatics background, so that the deep sequencing cost is difficult for general breeders to master and utilize.
Disclosure of Invention
The development of molecular markers is required in QTL mapping, gene cloning and molecular marker-assisted selective breeding processes of important traits of wheat. The traditional SSR marker has low flux and small quantity, and is a KASP (Kompetitive Allele Specific PCR) marker based on the transformation of SNP sites, namely a competitive Allele Specific PCR technology, which can carry out accurate double Allele judgment on SNPs and InDels on Specific sites in a wide range of genome DNA samples. The method has the characteristics of high stability, accuracy, low cost and high flux, can be widely applied to gene positioning, fine mapping and cloning of genes and high-flux screening of large-scale breeding materials, and has important utilization value in genetic research, molecular marker-assisted breeding and genome selective breeding.
Aiming at the defects in the prior art, 768 parts of wheat materials are subjected to simplified genome sequencing by utilizing a mature simplified genome sequencing (GBS) technology in wheat, sequence comparison is carried out on the simplified genome sequencing and a wheat reference genome to obtain SNP sites with high density of a whole genome, the SNP sites which are obviously associated with stripe rust resistance are identified by whole genome association analysis, and the method can be widely applied to development of KASP markers, gene positioning, marker-assisted selection and whole genome selection and serve genetic research and molecular breeding of the stripe rust resistance of wheat.
The invention provides a set of SNP (Single nucleotide polymorphism) which is obviously associated with wheat stripe rust resistance, can be applied to marker-assisted selection and whole genome selective breeding of wheat stripe rust resistance, can be used for developing a single KASP (Kasan-derived protein) marker and an SNP chip, and is convenient to use in heredity and breeding groups. Can be widely applied to the positioning of wheat stripe rust resistance genes, fine mapping and cloning, the marker-assisted selection, the aggregation and the whole genome selection of single or multiple disease-resistant fundamental tones in breeding.
The technical scheme adopted by the invention is as follows:
the invention provides a group of (38) Single Nucleotide Polymorphism (SNP) sites which are obviously associated with the disease resistance of wheat stripe rust, comprising SNP flanking sequences, SNP site information and base mutation information, wherein the SNP sites are positioned on 13 different chromosomes of common wheat.
The group of SNP loci which are obviously associated with the disease resistance of wheat stripe rust disease provided by the invention comprises 38 SNP loci which are numbered as SNP 01-SNP 38, and the information is as follows:
the physical position in the table takes the Chinese spring genome IWGSC reference genome v1.1 (IWGSC, 2018) as a reference sequence;
the sequences listed in the table are shown in sequence tables SEQ ID NO. 1-SEQ ID NO. 76.
The SNP loci obviously associated with wheat stripe rust resistance provided by the invention can be applied to identification of wheat stripe rust resistance.
The SNP loci obviously associated with the wheat stripe rust resistance can be applied to the preparation of a wheat stripe rust resistance identification kit.
The SNP loci which are obviously associated with the wheat stripe rust resistance can be applied to the preparation of single detectable SNP markers or gene chips.
The SNP loci remarkably associated with the wheat stripe rust resistance can be used for preparing stripe rust resistant gene lociqYr2A.1Co-separated KASP tags 2A _19902461 or 2A _ 20010283.
The SNP loci obviously associated with the wheat stripe rust resistance can be applied to the identification and detection method of the wheat stripe rust resistance.
In the application of the detection method, KASP primers can be designed according to SNP sites, and are designed according to DNA short sequences which comprise 50bp of the upstream and downstream of the SNP sites. Specifically, a website PolyMarker (http:// www.polymarker.info /) is used for primer design, and default parameter setting of the website is adopted. The primer is added with a joint, the FAM sequence is GAAGGTGACCAAGTTCATGCT, and the HEX sequence is GAAGGTCGGAGTCAACGGATT.
After the primers are designed and synthesized, the effectiveness detection can be carried out by utilizing a separation population or a natural population, and whether the QTL identified by GWAS exists or not is verified, and the using methods are respectively as follows:
(1) wheat DNA is used as a PCR amplification template, and a synthesized KASP primer is designed to carry out PCR amplification, wherein the reaction system is 6 mu L. The reaction system specifically comprises: 20-50 ng/. mu.L of DNA 3. mu.L, 2 XKASP Master mix 3. mu.L, and KASP Assay mix (upstream and downstream primer mix) 0.0825. mu.L. Amplified in 384-well PCR instrument.
(2) The PCR amplification program is pre-denaturation at 94 ℃ for 15 min; denaturation at 94 ℃ for 20s, renaturation at 65-57 ℃ for 60s (0.8 ℃ per cycle), 10 cycles; denaturation at 94 ℃ for 20s, renaturation at 57 ℃ for 60s, 30 cycles; storing at 10 deg.C;
(3) after the PCR is finished, placing the sample in an Omega SNP typing instrument to detect the PCR typing result;
(4) analyzing and identifying, and analyzing the genotype according to the typing result.
Compared with the prior art, the research has the following advantages:
(1) the present invention identifies a set of (38) SNPs that are significantly associated with stripe rust resistance. Basically covers the present stripe rust resistant quantitative trait gene locus in the Chinese wheat germplasm.
(2) The SNP can be further converted into a single detectable SNP marker (such as KASP marker), and is used for positioning of the stripe rust resistance gene, fine mapping, cloning and high-throughput molecular marker assisted selection applied to breeding materials in a large scale, so that the efficiency of molecular breeding is improved.
(3) These SNPs can also be made into gene chips, and applied to the whole genome selection of disease resistance of breeding materials, so that the efficiency and accuracy of molecular breeding are further improved.
(4) Develops the gene locus of stripe rust resistanceqYr2A.1Co-segregating KASP markers 2A _19902461 and 2A _ 20010283) not only demonstrated that the SNPs we identified were very effective, but alsoqYr2A.1The marker assisted selection provides good high-throughput detection of the marker。
Drawings
FIG. 1 is a Manhattan plot and a QQ plot of a stripe rust resistance genome-wide association analysis.
Detailed Description
Example 1 identification of SNPs by simplified genomic sequencing
1.1 sequencing materials
768 wheat varieties (lines) were selected for simplified genome sequencing and SNP identification. The materials are mainly wheat varieties and excellent strains from main wheat producing areas in China, including Huang-Huai wheat areas, northern winter wheat areas, middle and lower Yangtze river wheat areas and southwest wheat areas.
Research method
1.2.1 extraction of wheat genomic DNA
Seedling leaves were used to provide genomic DNA. The DNA extraction was carried out by the modified CTAB (butyl trimethyl ammonium bromide) method (Stewart and Via, 1993). The method comprises the following specific operations: taking young and tender wheat leaves in a 2mL centrifuge tube, freezing by using liquid nitrogen, and grinding into powder on a tissue grinder; (b) adding 800 μ L CTAB into 2mL tube, placing in 65 deg.C water bath for 90min, and shaking gently for 5-8 times during the water bath period to fully crack DNA; (c) adding 800 μ L chloroform isoamyl alcohol (volume ratio 24: 1) and shaking for 10 min; (d) centrifuging at 12000rpm for 10min, and placing 600 μ L of supernatant in a new 2mL tube (note corresponding number); (e) add 60. mu.L of 3M sodium acetate (pH = 5.2) and 600. mu.L of isopropanol (frozen at-20 ℃ C. in advance), mix with gentle shaking to see the generation of white DNA flocs, and put in a refrigerator at-20 ℃ for 1h to increase DNA yield. (f) Centrifuging at 12000rpm for 10min, pouring out supernatant, washing the precipitate with 70% ethanol (freezing in a refrigerator at-20 deg.C in advance) for 2-3 times, standing in a fume hood, and air drying; (g) add 200. mu.L of ddH2O dissolves the DNA.
Sample quality detection
And detecting by using agarose gel electrophoresis with the mass fraction of 1%, and checking an electrophoresis result by using a gel imaging system to ensure the integrity of the genome DNA. The ratio of A260/280 of the genomic DNA should be between 1.8 and 2.0, and the ratio of A260/230 should be between 1.8 and 2.2. The DNA was diluted to a working concentration of 20 ng/ul. Storing at-20 deg.C for use.
Library construction and GBS sequencing
Construction of GBS DNA libraries was performed with reference to Poland et al. (2012 b). Genomic DNA was digested with two restriction enzymes PstI and MspI (New England BioLabs, Inc., Ipswich, MA, United States). The barcode sequence was ligated to the digested DNA fragment using T4(New England BioLabs, Inc., Ipswich, MA, United States) ligase. All products from each plate were mixed and purified using QIAq rapid PCR purification kit (Qiagen, inc., Valencia, CA, United States). PCR amplification was performed using primers complementary to the barcode sequence. The PCR products were again purified using the QIAquick PCR purification kit and the concentration was determined using the Qubit ™ double-stranded DNA high-sensitivity fluorescent quantitation kit (Life Technologies, Inc., Grand Island, NY, United States). The 200-size 300-size DNA fragments were screened by agarose gel electrophoresis (Life Technologies, Inc., Grand Island, NY, United States), and the concentration of each DNA library was estimated using the Qubit 2.0 fluorescent agent and the Qubit double-stranded DNA high sensitivity fluorescence quantification kit. Fragment size-screened DNA libraries were loaded onto P1v3 chips using an Ion CHEF instrument (Ion PI Hi-Q CHEF Kit) and sequenced using an Ion Proton sequencer (Life Technologies, Inc., Grand Island, NY, United States, software version 5.10.1). This Ion Torrent system can produce sequences of various read lengths.
Site identification
The sequencing sequence was sequenced by adding 80 poly-A bases to its 3' end and then using TASSEL 5.0, so that it was possible to process sequences shorter than 64 bases by the train Analysis by Association, Evolution and Linkage (TASSEL) pipeline 5.0 (TASSEL 5.0) (Bradbury et al, 2007) rather than just discarding these short sequences. Sequence alignment is carried out by taking the IWGSC reference gene v1.1 (IWGSC, 2018) of the Chinese spring genome as a reference sequence and using TASSEL 5.0 (Bradbury et al, 2007) to identify SNP sites. All parameters are set to default settings of TASSEL 5.0. A total of 432,588 SNP sites were obtained covering approximately 14Gb of the whole genome, with an average distance between markers of 34.0 kb. Wherein 150784 sites on the A genome are spaced at an average distance of 32.9 kb; 182192 loci on the B genome with an average spacing of 28.9 kb; the D genome has 99612 sites and the average distance is 40.3 kb. The number of SNP markers on each chromosome is 10177 to 31149, and the variation range of the marker interval is 26.1-50.1 kb. These SNPs were mainly located in the intergenic region, 364203, accounting for 84.1, followed by the CDS region, 39901 (9.2%), the intron region 22215 (5.1%), the 5 'UTR region 3543 (0.8%), and the 3' UTR region 3300 (0.8%).
TABLE 1 distribution of SNP sites identified by GBS in wheat genome
Example 2 Whole genome identification of wheat stripe rust resistance quantitative trait Gene loci
2.1 materials
768 wheat materials for genotyping by GBS technique, as in example 1.
2.2 methods
2.2.1 identification of stripe rust resistance
To identify the resistance of these materials to stripe rust (YR), 768 wheat materials were planted in 2017-.
Inoculating wheat stripe rust fungus mixed physiological race after wheat is grown in spring every year "CYR29”、“CYR32”、“Su11"and"Su14". The rust germs are inoculated by a needle tube injection method, and a small amount of spore suspension (the spores of the mixed germs are diluted to be light yellow by sterile water) is injected into the stems by a syringe (2.5 ml). During inoculation, the wheat stem is bent down by the left hand, the syringe is pushed by the right hand, the fungus liquid is injected into the wheat stem, and the water overflowing from the top end of the wheat stem is used as an effective needle. Inoculation is generally carried out after 3 pm, the temperature is gradually reduced, the evaporation capacity of the bacterial liquid is small, and spore germination and invasion are facilitated.
In 2017 and 2018, the stripe rust resistance of the materials is identified under the natural disease condition of the field in Luoyang and Guiyang. Under all experimental conditions, Huizhou red and mingxian 169 were used as susceptible control materials. Each material was planted in 1 row, 3m long, 25 cm row spacing.
In the middle and late 5 months of each year, disease resistance was identified when the affected material was sufficiently diseased. Grade 0-4, 0 = no obvious symptoms or necrotic spots; 1 = little and very small clumps of spores; 2 = small or medium, relatively dispersed sporangium; 3 = moderate to very large in sporangium, large in number; 4 = widespread megasporophyte (Sheng, 1988).
2.2.2 Whole genome Association analysis
And (3) further screening the SNP sites with the Minimum Allele Frequency (MAF) of more than 0.01 and the deletion rate of less than 80% of all the identified 432588 SNP sites to obtain 327609 SNPs. Whole genome association analysis was performed on stripe rust disease resistance. The GAPIT v.3 package was used, which uses EMMA, a Compressed Mixed Linear Model (CMLM) and a population parameters previous determined (P3D) to improve the efficiency of GWAS operation. Kinship was analyzed using EMMA algorithm, using the first 3 principal components to control population structure. Significance threshold was set at 1.0 × 10-5。
2.3 results
2.3.1 genome-wide Association analysis of stripe rust disease resistance
By GWAS of stripe rust resistance reactions measured under multiple environments, 87 associated SNP Sites (MTAs) are identified in total, and are combined into 34 stripe rust resistance QTLs according to LD. 24 of these QTLs were located within the 1.0Mb interval, each containing less than 10 annotated genes. The method shows that the QTL can be positioned in a narrower physical interval by using the high-density SNP marker identified by GBS to carry out GWAS, and great convenience is brought to the fine positioning, gene cloning and molecular marker-assisted breeding of the subsequent QTL.
2.3.2 identified SNPs associated with stripe rust resistance
For the SNPs identified by GWAS, one SNP most significantly associated in each environment is selected for each site, and 38 SNP sites significantly associated with stripe rust resistance are obtained in total, and the information of the sites is shown in Table 2. In the table, "QTL" column, the QTL name linked to SNP is indicated; in the table, a list of 'chromosomes and physical positions of SNPs' indicates the chromosomes and the physical positions of the SNPs, and the physical positions refer to IWGSC reference gene v1.1 (IWGSC, 2018) of the Chinese spring genome; in the table, "sequence and SNP variation site" is listed, and "base" in "[ ]" indicates a variation site, and some sites have only one base, indicating that the base is deleted after variation.
TABLE 2 SNP site information significantly associated with wheat stripe rust
Example 3 wheat stripe rust resistance GeneqYr2A.1Verification and mapping of
3.1 materials
The research materials comprise a parent GH9 resisting stripe rust, a parent nicotiana tabacum 19 highly susceptible to stripe rust and F constructed by hybridization and hybridization of the two2Segregating the population for a total of 273F2Individual, susceptible control, Huixian red.
3.2 methods
3.2.1 phenotypic identification of disease resistance
The same as in example 1.
3.2.2 DNA extraction
The same as in example 1.
3.2.3 KASP primer design, dilution:
according to the GWAS analysis result, the QTL for resisting stripe rust disease is compared with the QTL for resisting stripe rust diseaseqYr2A.1Two significantly related GBS-SNPs (2A _19902461 and 2A _ 20010283) two sets (three each) of KASP primers k2A19902 and k2A20010 were designed based on SNP site information. The two groups of primers, three primers and ultra pure water are diluted to 100 mu M and then the volume ratio of the forward primer-FAM-R: forward primer-HEX-S: reverse primer: ultrapure water = 6: 6: 15: 23, and storing the mixture to-20 ℃ for later use.
3.2.4 KASP PCR amplification System and procedure are as follows:
the KASP reaction system used a 6 μ L system:
the system was prepared on ice according to the following table, 3. mu.L (about 20 ng/. mu.L) of template DNA, 2 × Master mix 3. mu.L (LGC Group UK), and 0.0825. mu.L of KASP assay primer (synthesized by Shanghai Biotech).
TABLE 3 KASP reaction System
The PCR procedure was as follows:
pre-denaturation at 1.94 ℃ for 5 min;
denaturation at 2.94 ℃ for 20 s;
3.65 ℃ for 30s (0.8 ℃ per cycle), and steps 2-3 are cycled 10 times.
Denaturation at 4.94 ℃ for 20 s;
annealing at 5.57 deg.C for 30s, and circulating step 4-5 for 35 times.
Storing at 6.10 deg.C. And (5) signal detection.
3.3 results
3.3.1 isolation of stripe rust resistance in segregating populations
The results of the GWAS analysis show that,qYr2A.1is a QTL for controlling the wheat stripe rust resistance, and is positioned in the interval of 0.5-21.8 Mb on 2 AS. GH9 is polymorphic with Nicotiana 19 at these two sites. GH9 has high stripe rust resistance and Nicotiana 9 high stripe rust resistance. To further validate this QTL and develop the KASP marker linked to it, F was constructed by GH9 hybridization with Nicotiana tabacum 192Isolating the population.
The identification of the stripe rust resistance of the population shows that F2The resistance of the stripe rust is obviously separated, the disease resistance is 0-2 grade, the infection statistics is 3-4 grade, the disease resistance and infection resistance single plants respectively comprise 210 and 63 plants, and the chi square test accords with the resistance: feeling =3:1 ratio.
3.3.2 relationship between stripe rust resistance and marker genotype in segregating populations
After converting the two most significant GBS-SNPs (2A _19902461 and S2A _ 20010283) associated with the QTL into KASP markers, the population was genotyped, and the results showed that both of these KASP markers were able to classify F2And (5) carrying out group typing. ANOVA analysis is carried out on the genotype and the disease resistance performance, and the result P value<0.001, indicating that the two markers are very significant in disease resistanceCorrelation, two SNP markers genotype F identical to GH92The single plant shows disease resistance (IT is less than or equal to 1), the single plant which is the same as the tobacco grower 19 shows infection (IT is close to 3), and the disease resistance of the heterozygous individual is between the two, which indicates that a QTL for controlling the wheat stripe rust resistance exists in the interval.
The foregoing is only a preferred embodiment of this patent, and it should be noted that, for those skilled in the art, various modifications and substitutions can be made without departing from the technical principle of this patent, and these modifications and substitutions should also be regarded as the protection scope of this patent.
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<212> DNA
<213> wheat (Triticum aestivum)
<400> 8
ttggagcttc tttgcagctt cctctctatg ttgccggata aatctgcaga caaaaaggat 60
ttgttgtttt gtttgccaat aatcaaacaa ctatgatata c 101
<210> 9
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 9
ttgcctggag cgattggaaa tttagcaggc ctgcagatca tgttagatgt gagcaacaat 60
aacctcagtg gtgtgttgcc acaacaactt gggaagttgg a 101
<210> 10
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 10
ttgcctggag cgattggaaa tttagcaggc ctgcagatca tgttagatgt aagcaacaat 60
aacctcagtg gtgtgttgcc acaacaactt gggaagttgg a 101
<210> 11
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 11
ggaaatttag caggcctgca gatcatgtta gatgtgagca acaataacct cagtggtgtg 60
ttgccacaac aacttgggaa gttggagatg ctagaatttc t 101
<210> 12
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 12
ggaaatttag caggcctgca gatcatgtta gatgtgagca acaataacct tagtggtgtg 60
ttgccacaac aacttgggaa gttggagatg ctagaatttc t 101
<210> 13
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 13
ttggtcaatg gcaacaccat aatgggagtt ctgcaggtca agtcaggagg tcgactgatt 60
ggagaatggg tatgccctgc ccgcttgtaa gtgatcacat c 101
<210> 14
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 14
ttggtcaatg gcaacaccat aatgggagtt ctgcaggtca agtcaggagg ccgactgatt 60
ggagaatggg tatgccctgc ccgcttgtaa gtgatcacat c 101
<210> 15
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 15
tctaccgtcg ccatgtgctt cccatgggca agcctggaaa gggtcgagag tgcaaatcct 60
agcgtggacg gaggcacatc aagacatctg cagtggattg c 101
<210> 16
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 16
tctaccgtcg ccatgtgctt cccatgggca agcctggaaa gggtcgagag ggcaaatcct 60
agcgtggacg gaggcacatc aagacatctg cagtggattg c 101
<210> 17
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 17
tccgcttcga tctgtctttc ttttgccaac caggtctgct atttgctcat cgatgaatcg 60
gaagcgccca tcggccttgg ctgcctgcag cagcaatcgc c 101
<210> 18
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 18
tccgcttcga tctgtctttc ttttgccaac caggtctgct atttgctcat tgatgaatcg 60
gaagcgccca tcggccttgg ctgcctgcag cagcaatcgc c 101
<210> 19
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 19
acctgatagg tacggaccac tagcttaact ctctcgtgct agctgcagct agccatggcc 60
catgcaagag gcatcctcct ccccacgtac tgcctgatca g 101
<210> 20
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 20
acctgatagg tacggaccac tagcttaact ctctcgtgct agctgcagct ggccatggcc 60
catgcaagag gcatcctcct ccccacgtac tgcctgatca g 101
<210> 21
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 21
gccaccacag aacgtcgcaa catcgccgca gtcgtcgagc tcgaagcacc atgggccgct 60
gtcgcagcat atgtggcagg tcgccccacc agcctgcagc a 101
<210> 22
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 22
gccaccacag aacgtcgcaa catcgccgca gtcgtcgagc tcgaagcacc gtgggccgct 60
gtcgcagcat atgtggcagg tcgccccacc agcctgcagc a 101
<210> 23
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 23
catatcatgg tgctctgtct gtttacaccc aatggcttga tgatgatgct cgtgctgcag 60
ctgttctcac tgctagtgtt ctgcctcagt ttgcttctga g 101
<210> 24
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 24
catatcatgg tgctctgtct gtttacaccc aatggcttga tgatgatgct tgtgctgcag 60
ctgttctcac tgctagtgtt ctgcctcagt ttgcttctga g 101
<210> 25
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 25
ggcgatgcgc gtggtggtgg tgcctccctt ggcggctttc ggtgtgggaa ggaaggggag 60
cggcttggac aggggcggcg gagccgacgg cgtgccggct g 101
<210> 26
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 26
ggcgatgcgc gtggtggtgg tgcctccctt ggcggctttc ggtgtgggaa cgaaggggag 60
cggcttggac aggggcggcg gagccgacgg cgtgccggct g 101
<210> 27
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 27
ggcgagatgt gcagcaatca tgttatccaa agggctcgaa aagtgacccg gtggtgttga 60
cacgtattgc agcgggttat ccgttcgtcg aggtggttcc a 101
<210> 28
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 28
ggcgagatgt gcagcaatca tgttatccaa agggctcgaa aagtgacccg atggtgttga 60
cacgtattgc agcgggttat ccgttcgtcg aggtggttcc a 101
<210> 29
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 29
aacttggaag aagtgccagt ccgcgccgac gaggagaaga gcctcacctg tgacaccacc 60
gatgcggagc tgcagcgcat cgccactagg acagaggagg c 101
<210> 30
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 30
aacttggaag aagtgccagt ccgcgccgac gaggagaaga gcctcacctg cgacaccacc 60
gatgcggagc tgcagcgcat cgccactagg acagaggagg c 101
<210> 31
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 31
gggcatggag gggcgtcggc ggccagggct agcagtggag cagcagcacg cagcagcggt 60
gagggcgaag tgcgacggca gcagcatgcg gcaacggcaa t 101
<210> 32
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 32
gggcatggag gggcgtcggc ggccagggct agcagtggag cagcagcacg tagcagcggt 60
gagggcgaag tgcgacggca gcagcatgcg gcaacggcaa t 101
<210> 33
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 33
cagctgcagc agcagtcttc gggattcgtg gacgcgaccc cgctcgggcc ttccttccac 60
tcgtactact catcatatag gtcgtacact ccgttttgga a 101
<210> 34
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 34
cagctgcagc agcagtcttc gggattcgtg gacgcgaccc cgctcgggcc gtccttccac 60
tcgtactact catcatatag gtcgtacact ccgttttgga a 101
<210> 35
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 35
gatgtggcct ctaatcatgg agtggtggtg gcgtccaagg atggaaccga cgcgcagaat 60
tattctctct gtttggttga taatctcctg cagaagccga c 101
<210> 36
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 36
gatgtggcct ctaatcatgg agtggtggtg gcgtccaagg atggaaccga ggcgcagaat 60
tattctctct gtttggttga taatctcctg cagaagccga c 101
<210> 37
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 37
agcatatatg gtggctaact tgcgcaaatg agagcgagaa gagaagcaaa tgtttggcat 60
gatcctgctt attcttggag aagaccaaga tatcatcgag a 101
<210> 38
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 38
agcatatatg gtggctaact tgcgcaaatg agagcgagaa gagaagcaaa cgtttggcat 60
gatcctgctt attcttggag aagaccaaga tatcatcgag a 101
<210> 39
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 39
aagtggctgc agtcacagca gcagctgaac cagtcgacga agagcctgag gaagccagga 60
ggcgtttgag cctcacaatg tcctggtcag tgagtgacgg a 101
<210> 40
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 40
aagtggctgc agtcacagca gcagctgaac cagtcgacga agagcctgag caagccagga 60
ggcgtttgag cctcacaatg tcctggtcag tgagtgacgg a 101
<210> 41
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 41
ctagcatata tggtggctaa acatacgcaa atgagaatga gaagagaagg caaagcacga 60
tcgataaact atgatcaaga agtgatccta gaacaaccta c 101
<210> 42
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 42
ctagcatata tggtggctaa acatacgcaa atgagaatga gaagagaagg aaaagcacga 60
tcgataaact atgatcaaga agtgatccta gaacaaccta c 101
<210> 43
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 43
gacaaggagc ctggtgcaag catggaggac gactccatcg acacggcggc gtgggacaag 60
ctgggcatca ccgacattgg cggggcacgg gcttgcgagc t 101
<210> 44
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 44
gacaaggagc ctggtgcaag catggaggac gactccatcg acacggcggc atgggacaag 60
ctgggcatca ccgacattgg cggggcacgg gcttgcgagc t 101
<210> 45
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 45
ccgtccttga gagccttggg gaactagttc gccatgatct tctcgtcact actggccgcc 60
tcgatgctca gttcgtagag ctgcaggaac tcggtagggt c 101
<210> 46
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 46
ccgtccttga gagccttggg gaactagttc gccatgatct tctcgtcact gctggccgcc 60
tcgatgctca gttcgtagag ctgcaggaac tcggtagggt c 101
<210> 47
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 47
cagatcttga cgatctccat gaaggccccc ttcggccatg tggcgttagg aagcttgctg 60
atcatagctc cgctgcagtc cacgtgctgc ccgtcgacca c 101
<210> 48
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 48
cagatcttga cgatctccat gaaggccccc ttcggccatg tggcgttagg cagcttgctg 60
atcatagctc cgctgcagtc cacgtgctgc ccgtcgacca c 101
<210> 49
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 49
ggatgcagca gctcctgcac ccgcgccgcc gacgagggtc tgcacggcat ctgccacggt 60
ctccgcgccc gtcttctgca gatctccgcc cgcgacggcg g 101
<210> 50
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 50
ggatgcagca gctcctgcac ccgcgccgcc gacgagggtc tgcacggcat atgccacggt 60
ctccgcgccc gtcttctgca gatctccgcc cgcgacggcg g 101
<210> 51
<211> 102
<212> DNA
<213> wheat (Triticum aestivum)
<400> 51
ttttttatca tctgcccaga ctgttgctca tacagtgcaa gaatggcctg caatttgtga 60
gccccttgtg tagttgcttt catcacgata agtgaatcgt cc 102
<210> 52
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 52
ttttttatca tctgcccaga ctgttgctca tacagtgcaa gaatggcctg aatttgtgag 60
ccccttgtgt agttgctttc atcacgataa gtgaatcgtc c 101
<210> 53
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 53
catgtccact gcaaggaggt gcgacaagat aagctggcag caatcaaggt cgccaacacc 60
cagaggcatg acttccgatc tttcatggag acttttattg c 101
<210> 54
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 54
catgtccact gcaaggaggt gcgacaagat aagctggcag caatcaaggt tgccaacacc 60
cagaggcatg acttccgatc tttcatggag acttttattg c 101
<210> 55
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 55
tctcatattc accaacattt tcacaaattc atgacaacat ttaaatccgt gaacatcttc 60
agatcggaag tcggtgctta tttcgagagc gagcgctgca g 101
<210> 56
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 56
tctcatattc accaacattt tcacaaattc atgacaacat ttaaatccgt caacatcttc 60
agatcggaag tcggtgctta tttcgagagc gagcgctgca g 101
<210> 57
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 57
aagttctttg gcaagccgga gcccgtgtat gagcgcctca tactcggcca cgttgttgga 60
ggcggcgaag tggatctgca gcgcatatct gagcttgtcg c 101
<210> 58
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 58
aagttctttg gcaagccgga gcccgtgtat gagcgcctca tactcggcca tgttgttgga 60
ggcggcgaag tggatctgca gcgcatatct gagcttgtcg c 101
<210> 59
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 59
gtaactgagg gaatactacg gaatataaca ggtaatgtgt ctgcaggttg acgcaccctt 60
cacacaaacc tctccacgcc ctacccggcc gctaagagaa t 101
<210> 60
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 60
gtaactgagg gaatactacg gaatataaca ggtaatgtgt ctgcaggttg gcgcaccctt 60
cacacaaacc tctccacgcc ctacccggcc gctaagagaa t 101
<210> 61
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 61
gctgtctgag tagaacatcc ttgggtacac ccagaggggc tgcagcttgc ataaccagtt 60
tcttttgtgc tctaacttta catctccctc gatacccaat c 101
<210> 62
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 62
gctgtctgag tagaacatcc ttgggtacac ccagaggggc tgcagcttgc gtaaccagtt 60
tcttttgtgc tctaacttta catctccctc gatacccaat c 101
<210> 63
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 63
tctctgcggt tactgttctt ctgttcctgc agctgtctcc gtccagtgct gcttgaacat 60
gatgagtagc actagcacta gcacgtctag gaattacaag a 101
<210> 64
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 64
tctctgcggt tactgttctt ctgttcctgc agctgtctcc gtccagtgct ccttgaacat 60
gatgagtagc actagcacta gcacgtctag gaattacaag a 101
<210> 65
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 65
agaaaagcta aaagagacac gaaaaagaac atatacaaaa aaataacgaa atgatccaaa 60
gtataaaagg ctatatgtgt gccttatgaa gcaaaggctg c 101
<210> 66
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 66
agaaaagcta aaagagacac gaaaaagaac atatacaaaa aaataacgaa ctgatccaaa 60
gtataaaagg ctatatgtgt gccttatgaa gcaaaggctg c 101
<210> 67
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 67
tcccacaccg ccgtcttcac ctgcaccacc ggaagagcat catcatcacc gtttccttgg 60
aagaagctgc agcctaatcg gcgccaccaa agaggttgta c 101
<210> 68
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 68
tcccacaccg ccgtcttcac ctgcaccacc ggaagagcat catcatcacc ttttccttgg 60
aagaagctgc agcctaatcg gcgccaccaa agaggttgta c 101
<210> 69
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 69
gcactcgtgc tcgacgtcac ccacccacgc ttaggtgtac ctggctctga tgtccgtgcc 60
atggatgccg cagcacgagc tgcagaagcc ctcgacgact a 101
<210> 70
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 70
gcactcgtgc tcgacgtcac ccacccacgc ttaggtgtac ctggctctga cgtccgtgcc 60
atggatgccg cagcacgagc tgcagaagcc ctcgacgact a 101
<210> 71
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 71
cactctcaca tgcatgctca tgtgtgtgta tgcatgcatg agcgtgactc ctctgcagtg 60
gatttgtacg taggtataca caggccacaa aatctacctt g 101
<210> 72
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 72
cactctcaca tgcatgctca tgtgtgtgta tgcatgcatg agcgtgactc ctctgcagtg 60
gatttgtacg taggtataca caggccacaa aatctacctt g 101
<210> 73
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 73
gatattcttc aaatggccac aaggtgtact gtgcggattg atttcacgaa actgcagggg 60
tgtatctgta aatgttcaac aggtgactgg tctagctggt g 101
<210> 74
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 74
gatattcttc aaatggccac aaggtgtact gtgcggattg atttcacgaa tctgcagggg 60
tgtatctgta aatgttcaac aggtgactgg tctagctggt g 101
<210> 75
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 75
tactatggac ttgcatgcca attcatggac tcaccgagcc atctatcggc aaaacgaatt 60
cagatgacca aagagttaac tggcaatccg tataattcag a 101
<210> 76
<211> 101
<212> DNA
<213> wheat (Triticum aestivum)
<400> 76
tactatggac ttgcatgcca attcatggac tcaccgagcc atctatcggc caaacgaatt 60
cagatgacca aagagttaac tggcaatccg tataattcag a 101
Claims (7)
1. A group of SNP loci which are obviously associated with the disease resistance of wheat stripe rust are characterized in that: the SNP loci comprise 38 SNP loci with the numbers of SNP 01-SNP 38, and the information is as follows:
the physical position in the table takes the Chinese spring genome IWGSC reference genome v1.1 (IWGSC, 2018) as a reference sequence;
the sequences listed in the table are shown in sequence tables SEQ ID NO. 1-SEQ ID NO. 76.
2. Use of the set of SNP sites of claim 1 significantly associated with wheat stripe rust resistance in the identification of wheat stripe rust resistance.
3. Use of the set of SNP sites significantly associated with wheat stripe rust resistance according to claim 1 in preparation of a wheat stripe rust resistance identification kit.
4. Use of a set of SNP sites significantly associated with wheat stripe rust resistance according to claim 1 in the preparation of a single detectable SNP marker or gene chip.
5. The method for preparing the SNP loci which are significantly associated with the wheat stripe rust resistance according to the set of the SNP loci of claim 1qYr2A.1Co-separated KASP tags 2A _19902461 or 2A _ 20010283.
6. The application of the set of SNP loci significantly associated with wheat stripe rust resistance according to claim 1 in a wheat stripe rust resistance identification and detection method.
7. Use in a detection method according to claim 6, characterized in that the detection method comprises the steps of:
designing KASP primers according to the SNP sites, and designing KASP primers according to DNA short sequences containing 50bp of upstream and downstream of the SNP sites; specifically, a website http:// www.polymarker.info/is used for primer design, and default parameter setting of the website is adopted; adding a joint in front of the primer, wherein the FAM sequence is GAAGGTGACCAAGTTCATGCT, and the HEX sequence is GAAGGTCGGAGTCAACGGATT;
after the primers are designed and synthesized, the effectiveness detection can be carried out by utilizing a separation population or a natural population, whether the QTL identified by GWAS exists or not can be verified, and the method can also be used for gene mapping, marker-assisted selection and the like, and the using methods are respectively as follows:
(1) using wheat DNA as a PCR amplification template, designing a synthesized KASP primer, and carrying out PCR amplification with a reaction system of 6 mu L; the reaction system specifically comprises: 20-50 ng/. mu.L of DNA 3. mu.L, 2 XKASP Master mix 3. mu.L, and KASP Assay mix upstream and downstream primer mixture 0.0825. mu.L; amplifying in a 384-well PCR instrument;
(2) the PCR amplification program is pre-denaturation at 94 ℃ for 15 min; denaturation at 94 deg.C for 20s, and renaturation at 65-57 deg.C for 60 s; the reduction per cycle is 0.8 ℃; 10 cycles; denaturation at 94 ℃ for 20s, renaturation at 57 ℃ for 60s, 30 cycles; storing at 10 deg.C;
(3) after the PCR is finished, placing the sample in an Omega SNP typing instrument to detect the PCR typing result;
(4) analyzing and identifying, and analyzing the genotype according to the typing result.
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|---|---|---|---|---|
| CN108531638A (en) * | 2018-04-26 | 2018-09-14 | 中国科学院遗传与发育生物学研究所 | Molecular labeling with wheat stripe rust resistance new gene YrQz close linkages and its application |
| CN109706263A (en) * | 2019-02-22 | 2019-05-03 | 四川农业大学 | Chain SNP marker and application with wheat stripe rust resisting ospc gene QYr.sicau-1B-1 |
| CN111635957A (en) * | 2020-07-20 | 2020-09-08 | 湖北省农业科学院粮食作物研究所 | Molecular marker for detecting wheat stripe rust resistance QTL and application of molecular marker in disease-resistant breeding |
| CN112481275A (en) * | 2020-12-11 | 2021-03-12 | 四川农业大学 | Wheat stripe rust resistant gene yrZ15-1370 and molecular marker and application thereof |
-
2020
- 2020-12-23 CN CN202011543041.7A patent/CN112481410A/en not_active Withdrawn
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2021
- 2021-04-14 CN CN202110397710.2A patent/CN113046467B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108531638A (en) * | 2018-04-26 | 2018-09-14 | 中国科学院遗传与发育生物学研究所 | Molecular labeling with wheat stripe rust resistance new gene YrQz close linkages and its application |
| CN109706263A (en) * | 2019-02-22 | 2019-05-03 | 四川农业大学 | Chain SNP marker and application with wheat stripe rust resisting ospc gene QYr.sicau-1B-1 |
| CN111635957A (en) * | 2020-07-20 | 2020-09-08 | 湖北省农业科学院粮食作物研究所 | Molecular marker for detecting wheat stripe rust resistance QTL and application of molecular marker in disease-resistant breeding |
| CN112481275A (en) * | 2020-12-11 | 2021-03-12 | 四川农业大学 | Wheat stripe rust resistant gene yrZ15-1370 and molecular marker and application thereof |
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| CN113046467B (en) | 2023-07-18 |
| CN112481410A (en) | 2021-03-12 |
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