CN108950057B - Development and application of Ular pattern wheat powdery mildew resistance gene Pm60 specific molecular marker - Google Patents
Development and application of Ular pattern wheat powdery mildew resistance gene Pm60 specific molecular marker Download PDFInfo
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Abstract
The invention discloses development and application of a specific molecular marker of a Ular pattern wheat powdery mildew resistance gene Pm 60. The invention provides a set of primers for identifying wheat powdery mildew resistance, which comprises a primer pair A consisting of a primer 1 and a primer 2; the primer 1 is a single-stranded DNA molecule shown in a sequence 2 of a sequence table; the primer 2 is a single-stranded DNA molecule shown in a sequence 3 of a sequence table. The primer set also comprises a primer pair B consisting of a primer 3 and the primer 2; the primer 3 is a single-stranded DNA molecule shown in a sequence 1 of a sequence table. The molecular marker found by the invention has the following advantages: the specificity is good, and three genotypes with disease-resistant functions of Pm60a, Pm60b and Pm60 in the Wulare diagram wheat can be accurately identified by a PCR method; the amplified fragment is shorter, and is suitable for the application of molecular marker assisted breeding.
Description
Technical Field
The invention belongs to the field of genetic engineering and molecular biology, and particularly relates to development and application of a Wularch wheat powdery mildew resistance gene Pm60 specific molecular marker.
Background
Wheat powdery mildew is one of the main diseases of wheat, and seriously threatens the production safety of wheat. Many of the existing powdery mildew resistant genes come from related species of wheat, and in the seventies of the last century, disease resistant breeding begins to greatly utilize the Pm8 gene carried by a wheat-rye T1BL/1RS translocation line, so that the disease resistant gene in a large-scale popularization variety of wheat is single, and the toxicity frequency of the wheat powdery mildew resistant gene to the Pm8 gene is rapidly increased. One reason for the major epidemic of powdery mildew in China in 1990 and 1991 is the loss of disease resistance of the Pm8 gene (Zhouyang et al, 2004; zhuang et al, 1993; Lihongjie et al, 2011). Therefore, the polymerization of a plurality of disease-resistant genes into one material through molecular markers is an important way for realizing the durable resistance of wheat.
Wheat A genome donor wheat (Triticum urartu) is an important resource for wheat disease-resistant breeding, but only 1 cloned wheat powdery mildew-resistant gene Pm60 is from wheat in the Ular map so far, and Pm60 is reported to have three forms of alleles Pm60a (4125bp), Pm60(4365bp) and Pm60b (4605bp) (Zou et al, 2017). However, the specific marker of the Pm60 gene is not reported, and the work of identifying the Pm60 gene in the Wulare diagram wheat, assisting breeding by molecular markers and the like is limited. In conclusion, the development of a specific molecular marker of the Pm60 gene is necessary for wheat breeding for disease resistance.
Disclosure of Invention
An object of the present invention is to provide a set of primers for identifying wheat powdery mildew resistance.
The primer set provided by the invention comprises a primer pair A consisting of a primer 1 and a primer 2;
the primer 1 is (a1) or (a 2):
(a1) a single-stranded DNA molecule shown in a sequence 2 of a sequence table;
(a2) DNA molecules which are obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 2 and have the same functions as the sequence 1;
the primer 2 is (a3) or (a 4):
(a3) a single-stranded DNA molecule shown in sequence 3 of the sequence table;
(a4) and (b) the DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 3 and has the same function as the sequence 2.
The primer set also comprises a primer pair B consisting of a primer 3 and the primer 2;
the primer 3 is (a5) or (a 6):
(a5) a single-stranded DNA molecule shown in sequence 1 of the sequence table;
(a6) and (b) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 1 and has the same function as the sequence 3.
In the above-mentioned primer set, the primer set,
the molar ratio of the primer 1 to the primer 2 is 1: 1;
the molar ratio of the primer to the primer 4 is 1: 1.
another object of the present invention is to provide PCR reagents for identifying a set of primers for wheat powdery mildew resistance.
The PCR reagent provided by the invention comprises a PCR reagent A containing the primer pair A.
The PCR reagent also comprises a PCR reagent B containing the primer pair B.
Among the PCR reagents described above, in the case of PCR reagents,
the concentrations of the primer 1 and the primer 2 in the PCR reagent are both 200 nM;
or, the concentration of the primer 3 and the primer 4 in the PCR reagent is 200 nM.
A kit comprising the primer set or the PCR reagent set is also within the scope of the present invention.
The application of the above primer set or the above PCR reagent or the above kit in 1) or 2) is also within the scope of the present invention:
1) identifying wheat powdery mildew resistance;
2) preparing a product for wheat powdery mildew resistance.
The 3 rd object of the invention provides a preparation method of the kit.
The preparation method provided by the invention comprises the step of packaging each primer separately.
The 4 th purpose of the invention is to provide a method for identifying or assisting in identifying the wheat powdery mildew resistance to be detected.
The method provided by the invention is 1) or 2):
1) the method comprises the following steps:
the primer pair is used for amplifying wheat to be detected, a PCR amplification product is detected,
if an amplification product is obtained, the wheat to be detected is or is selected as anti-powdery mildew wheat;
if the amplification product is not obtained, the wheat to be detected is not or is not candidate to be anti-powdery mildew wheat;
2) the method comprises the following steps:
respectively amplifying the wheat to be detected by using the primer pair A and the primer pair B, detecting a PCR amplification product of the primer pair A and a PCR amplification product of the primer pair B,
if the primer pair A is amplified to obtain an amplification product, or the primer pair B is amplified to obtain an amplification product, the wheat to be detected is or is selected as powdery mildew resistant wheat; and if the primer pair A is not amplified to obtain an amplification product and the primer pair B is not amplified to obtain an amplification product, the wheat to be detected is not or is not candidate to be powdery mildew resistant wheat.
The size of an amplification product obtained by the primer pair A is 516bp (Pm60a),756bp (Pm60) or 996(Pm60 b);
the amplification product obtained by the amplification of the primer pair B is 591bp (Pm60a),931bp (Pm60) or 1171(Pm60 b).
The wheat to be detected is Wulare diagram wheat or cultivated one-grain wheat.
Experiments prove that the molecular marker found by the invention has the following advantages: the specificity is good, and three genotypes with disease-resistant functions of Pm60a, Pm60b and Pm60 in the Wulare diagram wheat can be accurately identified by a PCR method; the amplified fragment is shorter, and is suitable for the application of molecular marker assisted breeding.
Drawings
FIG. 1 is a schematic representation of Uvaria resistance to wheat powdery mildew.
FIG. 2 shows the positions of M-Pm60 markers on Pm60a, Pm60 and Pm60 b.
FIG. 3 shows the alignment of Pm60a, PI 662269 (disease-resistant material) and PI 662227 (susceptible material) amplification sequences.
FIG. 4 is PCR electrophoresis gel diagram amplified in partial Ural diagram wheat by using Pm60-F (2900T) and Pm60-R (3465T), Pm60-F (3000T) and Pm60-R (3465T) two pairs of Pm60 specific markers.
FIG. 5 is PCR electrophoresis gel diagram amplified in partial Ural diagram wheat by using Pm60-F (2900T) and Pm60-R (3465T), Pm60-F (3000T) and Pm60-R (3465T) two pairs of Pm60 specific markers.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The Ural chart and the PI number of the cultivated one-grain wheat are inquired through a website: https:// www.genesys-pgr.org/zh/sel/.
8 physiological species of powdery mildew: e01, E05, E07, E09, E15, E18, E20, E13-10 are described in the following documents: zhouying forest, Selenyu, Chenggao, Shengbaoju, Zhanying, 40 wheat fine variety resources resistant to powdery mildew gene derivation [ J ] plant pathology report, 2002(04): 301. supplement 305+318. Hippon peak, Vanjiao sha, Zhouying forest, Wang Lou, Baiwei, Liguang wheat variety (line) resistant to powdery mildew gene derivation and molecular marker identification [ J ] plant pathology report, 2017,47(03): 370. supplement 379.
Xue was described in the following documents: plum root bridge, house lizine, Jie, Gaoliliang, Li Xue, Jie super, Yang Cai Ming, Sunjin, Liu Shi Yong.
Lankao 86 is described in the following references: (iii) Gerbridge wheat line "Lankao 86 (79)" [ J ] Henan agriculture, 1996(06):8.
Ergmai is described in the following documents: huangmiao, Sun Zhen Yu, Cao Shi Qin, Jia Qin Zhen, Liu Tai Guo, Chen Wan, 223 parts of wheat farmhouse variety field evaluation of stripe rust resistance and molecular detection of disease-resistant gene [ J ]. plant protection science, 2018,45(01):90-100.
Jing 411 is described in the following documents: old newborns, who tiger, Wangdsen, banker, Zhanghong, Zhang Yan, Zhang Yong, spring in summer, use Jing 411 as the backbone parent to cultivate high-yield new wheat varieties [ J ]. crop journal, 2009(04):1-5.
Stone 4185 is described in the following documents: "Zhu you Wide" stone 4185 wheat [ N ]. farmer daily report, 2004/10/09(006).
Konong 199 is described in the following documents: lijunming, Zhang Qiqi, Zhang love people, Wang Shi Guo, Anhui, Jijun, Wang Jing, high yield Guangjing wheat new variety-Kenong 199[ J ] wheat crop notice, 2007(02): 368.
Example 1 identification of Ular pattern wheat resistance Profile
Identification of powdery mildew resistance
1. Utilizing common wheat Xue Zao to propagate Erysiphe cichoracearum;
2. planting wheat seedlings to be identified in an aseptic greenhouse, and respectively inoculating 8 wheat powdery mildew physiological races E01, E05, E07, E09, E15, E18, E20 and E13-10 when leaves grow to one leaf and one heart; and (3) adopting a inoculation tower to fully inoculate powdery mildew fresh spores to the wheat leaves. Culturing at 22 deg.C, 16h light, 8 h dark and 70% humidity.
Reference to identification methods (Liu et al 1999), fig. 1 is a schematic representation of the identification of powdery mildew resistance of wheat, Ural chart, wherein note: grade 0 is immunity; 0; allergic necrosis reaction is present; level 1 is high resistance; grade 2 is medium resistance; grade 3 is in the middle; grade 4 is high feeling.
The resistance of 200 Wularchu wheat germplasm materials to 8 wheat powdery mildew physiological races E01, E05, E07, E09, E15, E18, E20 and E13-10 is identified, and partial results are shown in Table 1. The phenotype data was used in subsequent association analysis with the Pm60 marker.
Second, preliminary screening of Pm60 gene in Ural chart wheat
Comparison:
a linked molecular marker M-Pm60 transformed with published Pm60(M-Pm 60-P1:
CATTAACTTTGAGTTGTTGGA, respectively; M-Pm 60-P2: CGGTGATCATACCAGAATTC), and carrying out primary screening of Pm60 gene in 200 parts of Wulare diagram wheat.
The length of the labeled amplification PCR product is as follows: 1210bp is obtained as allele Pm60a, 1550bp is obtained as allele Pm60, and 1790bp is obtained as allele Pm60b (the marking position and the gene structure are shown in the detailed diagram in FIG. 2).
PCR amplification conditions: genstar Mix, 5min at 94 ℃ for pre-denaturation; 35 cycles of 94 ℃ for 35s, 57 ℃ for 35s,72 ℃ for 1min45 s; 10min at 72 ℃.
According to the size information of the PCR amplification product fragments, 9 Ural chart materials suspected to contain Pm60a, 5 Pm60b and 38 Pm60 are screened.
The results show that: the Wulare diagram wheat material capable of amplifying the alleles of Pm60 and Pm60b shows 0-grade immunity or 1-grade resistance to 6 Erysiphe necator species, while the four materials PI538727, PI 662229, PI 662274 and PI 662227 in the wheat capable of amplifying the alleles of Pm60a show susceptible traits, which are different from the results of the identification of powdery mildew resistance (Table 1).
Therefore, the Pm60a Ural chart wheat material screened and identified by the M-Pm60 marker has false positive, and the development of the Pm60 specific marker is necessary.
Development of specific marker of III, Wulare diagram Pm60
Sequencing was performed on PCR products amplified from M-Pm60 of 5 disease-resistant materials PI 662269, PI 662251, PI 428219, PI 428208 and 01C0104213 and 4 disease-sensitive materials PI538727, PI 662229, PI 662274 and PI 662227, and sequence analysis showed that: the sequences of the 5 disease-resistant materials are consistent, the similarity with Pm60a is 100%, and the 5 disease-resistant materials are presumed to be Pm60 a. On the other hand, the sequences of 4 susceptible materials were identical (sequence 4), the similarity with Pm60a was 98%, and some SNP differences were observed (shown in FIG. 3). Therefore, the development of specific labeling of Pm60 can be carried out by utilizing the difference.
The two pairs of molecular markers are as follows:
pm60-F (2900T) CTCACAGTTCCACACTGATAT (SEQ ID NO: 1)
Pm60-R (3465T) CTCCATCAATCTCAAGTTCTTCG (SEQ ID NO: 3);
PCR amplification procedure: genstar Mix, 5min at 94 ℃ for pre-denaturation; 35 cycles of 94 ℃ for 35s, 55 ℃ for 35s,72 ℃ for 1min10 s; 10min at 72 ℃;
pm60-F (3000T) TGTATATTAATGGGTATAATAG (SEQ ID NO: 2)
Pm60-R (3465T) CTCCATCAATCTCAAGTTCTTCG (SEQ ID NO: 3)
PCR amplification System procedure: genstar Mix, 5min at 94 ℃ for pre-denaturation; 35 cycles of 94 ℃ for 35s, 52 ℃ for 35s and 72 ℃ for 1 min; 10min at 72 ℃;
Genomic DNA of the Ural chart shown in Table 1 was extracted as a template, and PCR amplification was performed using the above-mentioned marker 1 and marker 2, respectively.
The marker M-Pm60 was used as a control.
The results are shown in FIG. 4 and Table 1, where 1-5(Pm60a, against disease) is PI 662269, PI 662251, PI 428219, PI 428208,01C0104213-3,6-9 (susceptible) PI538727, PI 662229, PI 662274, PI 66222710-14(Pm60b, against disease) is PI 428203, PI 428215, PI 428204, PI 662230, PI 42831515-22 (Pm60, against disease) is PI 428309, PI 428310, PI 538737, PI 428276, PI 428306, PI 538740, PI 428296, PI 428288; no bands are amplified in wheat PI538727, PI 662229, PI 662274 and PI 662227 of the infected Wulare pattern by utilizing two pairs of molecular markers specific to Pm60, and the materials with the amplified bands are disease-resistant materials (Table 1).
Therefore, the banding patterns of the two pairs of specific molecular markers can be associated with disease-resistant phenotypes, and have higher specificity compared with the M-Pm60 marker. In addition, the amplified fragment is smaller, and the Pm60a, Pm60 and Pm60b can be distinguished more obviously.
Meanwhile, sequencing the amplified products of the 5 Pm60b material markers selected by the screening shows that the amplified products are all 100% similar to Pm60 b.
Sequencing analysis was performed on 15 labeled amplification products screened with Pm60 material (PI 662224, PI 538737, CITR17664, PI 428309, PI 428310, PI 538742, PI 428325, PI 428325, PI 538751, PI 428254, PI 428260, PI 428335, PI 662248, IG45292, PI 428308), and the results showed 100% similarity to Pm 60. In addition, no band is amplified in the tested wheat material of the susceptible Ural chart by the specific marker Pm60, and the materials with the amplified bands have higher resistance to 8 wheat powdery mildew physiological races (Table 1).
TABLE 1 Wulare chart wheat results of identifying resistance to powdery mildew different physiological races and identifying Pm60 marker
Note:
M-Pm60 is: the currently disclosed markers M-Pm60-P1/M-Pm 60-P2;
the label 1 is: pm60-F (2900T) and Pm60-R (3465T);
the label 2 is: pm60-F (3000T) and Pm60-R (3465T);
Therefore, a method for judging or assisting in judging the wheat powdery mildew resistance to be detected can be established and is 1) or 2):
1) the method comprises the following steps:
amplifying the DNA of the wheat to be detected by Pm60-F (3000T) and Pm60-R (3465T), detecting the PCR amplification product,
if the PCR amplification product is obtained, the wheat to be detected contains the Pm60 gene or the allele thereof, and the wheat to be detected is or is candidate to be the powdery mildew resistant wheat;
if the PCR amplification product is not obtained, the wheat to be detected does not contain the Pm60 gene or the allele thereof, and the wheat to be detected is not or is not candidate to be powdery mildew resistant wheat;
the PCR amplification product is 516bp (Pm60a),756bp (Pm60) or 996(Pm60 b).
2) The method comprises the following steps:
respectively amplifying DNA of wheat to be detected by Pm60-F (3000T), Pm60-R (3465T), Pm60-F (2900T) and Pm60-R (3465T), detecting PCR amplification products of Pm60-F (3000T) and Pm60-R (3465T) and PCR amplification products of Pm60-F (2900T) and Pm60-R (3465T),
if Pm60-F (3000T) and Pm60-R (3465T) are amplified to obtain a target product, or Pm60-F (2900T) and Pm60-R (3465T) are amplified to obtain a target product, the wheat to be detected contains a Pm60 gene or an allele thereof, the wheat to be detected contains a Pm60 gene or an allele thereof, and the wheat to be detected is or is candidate to be powdery mildew resistant wheat;
if the Pm60-F (3000T) and Pm60-R (3465T) amplification and Pm60-F (2900T) and Pm60-R (3465T) amplification do not obtain corresponding target products, the wheat to be detected does not contain the Pm60 gene or allele thereof, and the wheat to be detected is not or is not candidate to be powdery mildew resistant wheat;
the target product of the Pm60-F (3000T) and Pm60-R (3465T) amplification is 516bp (Pm60a),756bp (Pm60) or 996(Pm60 b).
The target products of the amplification of the Pm60-F (2900T) and the Pm60-R (3465T) are 591bp (Pm60a),931bp (Pm60) or 1171(Pm60 b).
Example 2 application of Pm60 marker in judging powdery mildew resistance of wheat to be detected
Identification of powdery mildew resistance
The one-grain wheat to be cultivated in table 2 was identified by the powdery mildew resistance identification method of the one in example 1, and the results are shown in table 2.
II, judging powdery mildew resistance of wheat to be detected by Pm60 marker
1. And (3) extracting the genome DNA of the to-be-tested cultivated one-grain wheat in the table 2 by using a CTAB method.
2. PCR amplification
The genomic DNA of the to-be-tested cultivated one-grain wheat in the table 2 is used as a template, and the markers 1(Pm60-F (3000T), Pm60-R (3465T)) and 2(Pm60-F (2900T), Pm60-R (3465T)) in the first embodiment 1 are respectively used for PCR amplification, and the specific steps are as follows 1) or 2):
1) the method comprises the following steps:
amplifying the DNA of the wheat to be detected by using Pm60-F (3000T) and Pm60-R (3465T), detecting a PCR amplification product,
if the PCR amplification product is obtained, the wheat to be detected contains the Pm60 gene or the allele thereof, and the wheat to be detected is or is candidate to be the powdery mildew resistant wheat;
if the PCR amplification product is not obtained, the wheat to be detected does not contain the Pm60 gene or the allele thereof, and the wheat to be detected is not or is not candidate to be powdery mildew resistant wheat;
the PCR amplification product is 516bp (Pm60a),756bp (Pm60) or 996(Pm60 b).
2) The method comprises the following steps:
respectively amplifying DNA of wheat to be detected by Pm60-F (3000T), Pm60-R (3465T), Pm60-F (2900T) and Pm60-R (3465T), detecting PCR amplification products of Pm60-F (3000T) and Pm60-R (3465T) and PCR amplification products of Pm60-F (2900T) and Pm60-R (3465T),
if Pm60-F (3000T) and Pm60-R (3465T) are amplified to obtain a target product, or Pm60-F (2900T) and Pm60-R (3465T) are amplified to obtain a target product, the wheat to be detected contains a Pm60 gene or an allele thereof, the wheat to be detected contains a Pm60 gene or an allele thereof, and the wheat to be detected is or is candidate to be powdery mildew resistant wheat;
if the Pm60-F (3000T) and Pm60-R (3465T) amplification and Pm60-F (2900T) and Pm60-R (3465T) amplification do not obtain corresponding target products, the wheat to be detected does not contain the Pm60 gene or allele thereof, and the wheat to be detected is not or is not candidate to be powdery mildew resistant wheat;
the target products of the Pm60-F (3000T) and Pm60-R (3465T) amplification are 516bp (Pm60a),756bp (Pm60) or 996(Pm60 b).
The target products of the amplification of the Pm60-F (2900T) and the Pm60-R (3465T) are 591bp (Pm60a),931bp (Pm60) or 1171(Pm60 b).
The marker M-Pm60 was used as a control.
The results are shown in FIGS. 5(1-3: PI 662251(Pm60a), PI 428309(Pm60), PI 428204(Pm60b), 4-9(TmPm60, disease resistant): TRI 1418, TRI 4275, TRI 28871, TRI 4312, TRI 4323, PI 265008, 10-17 (disease resistant): TRI 644, TRI 2381, TRI 18421, TRI 18425, TRI 18426, TRI 18429, TRI 18422, TRI 19070, 18-24 (susceptible disease): TRI 19069, TRI 19315, TRI 19319, TRI 19313, TRI 4308, TRI 4322, TRI 12749) and Table 2,
table 2 shows the resistance of the cultivated one-grain wheat to different physiological races of powdery mildew and the identification result of Pm60 marker
Note:
M-Pm60 is: the currently disclosed markers M-Pm60-P1/M-Pm 60-P2;
the label 1 is: pm60-F (2900T) and Pm60-R (3465T);
the label 2 is: pm60-F (3000T) and Pm60-R (3465T);
As can be seen, 6 pieces of material with PCR amplified bands (TRI 1418, TRI 4275, TRI 28871, TRI 4312, TRI 4323, PI 265008) were co-screened, the fragment size is indicated as Pm60, and the PCR product sequence of these 6 pieces of material was shown to be identical by sequencing (SEQ ID NO: 5). The similarity of the gene with Pm60 is 99%, and the SNP variation of A → G exists at position 3487, which leads to the change of threonine Thr at position 1163 of the Pm60 protein to alanine Ala, but the 6 materials all have higher resistance to the detected 4 physiological races of wheat powdery mildew (see Table 2). The M-Pm60 marker has an amplification band in a plurality of disease-resistant and disease-sensitive materials, the sequence analysis of PCR products of the materials with the sizes indicated as Pm60a, 3 disease-resistant (TRI 1774, TRI 1990, TRI 17946) and 4 disease-sensitive (TRI 11488, TRI 14261, TRI 17026 and TRI 4322) finds that the sequences detected between the disease-resistant and disease-sensitive materials have no difference (sequence 6), the similarity with Pm60a is 98%, the similarity with the sequence amplified by the M-Pm60 marker of the wheat disease-sensitive material of the Ular map (sequence 4) is 99.42%, and the sequence analysis and amplification results show that the interference of the similar sequences can be eliminated by using the specific Pm60 molecular marker (FIG. 5). The results show that the two specific molecular markers of Pm60 also have certain application value in screening and identifying Pm60 homologous genes of other wheat species except the Wulare diagram wheat.
Example 3 potential of specific marker Pm60 molecular marker assisted breeding in common wheat
The method of the second embodiment 2 is adopted to detect 11 parts of common wheat Xuehao, Jing 411, Shi 4185, China spring, chancellor, Filder, Kenong 199, Lankou 86, Ergmai, foreign white wheat and Zhengnong 17 infected with E13-10 strains at the seedling stage, and any strip can not be amplified, which indicates that the wheat is not powdery mildew resistant wheat containing Pm60 gene, the Pm60 gene and the developed Pm60 specific marker can be applied to disease resistance improvement and molecular marker-assisted breeding of the materials.
Sequence listing
<110> institute of genetics and developmental biology of Chinese academy of sciences
Development and application of <120> Ular pattern wheat powdery mildew resistance gene Pm60 specific molecular marker
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 21
<212> DNA
<213> Artificial sequence
<400> 1
ctcacagttc cacactgata t 21
<210> 2
<211> 22
<212> DNA
<213> Artificial sequence
<400> 2
tgtatattaa tgggtataat ag 22
<210> 3
<211> 23
<212> DNA
<213> Artificial sequence
<400> 3
ctccatcaat ctcaagttct tcg 23
<210> 4
<211> 1221
<212> DNA
<213> Artificial sequence
<400> 4
gagaggctac agatgggagc aacaagcact gctgagatgc aatccactgt aagttctcgt 60
agttgacagg agcctgcagg cgataacagg ttggtccttg ctaccaccaa ttccgagaac 120
aaatccgctg atacagaacg gctaagacaa tttcctctgt tgtagatctt caaactgttg 180
aggttgactg tgatgagagg attgaaacca tccactgtta aattctcaga gttcaccagt 240
ataagatcgg tgagacaagt gaggttcgag agctgagcca ttgactccat gcttgactct 300
ccatcaatct caagttcctc aagggaagca ggcaaaggat tgatgatggt gtgagctgct 360
tcgatgggcc gccgaaagaa gttgccacat tcttttactg taagtgattg gaggcacgcg 420
aggtcctgca gtccaatgca cgtgatgctg catcttctga catctagtct tcttagggat 480
ttcagattgc tgagcggagc cattgactgc atgcctgact ctccttcaat ctcaagtgtc 540
tcgagggaag cagggaaaag cttgatggtg tgagctactt cggtatgcca tggaaagaag 600
atgccacagt tgtatactgt aagatattgg aggcacgcga ggtcctggag tccatggcac 660
gtgaagctgc atcttctgac atctagtctt cttagggatt tcagattgtt cagctctgtc 720
attgaaatgt gtgatacatc tgtaatactc attttctcta cttttcgcac gttatgcaag 780
tccacctcac cattataccc attaatatac atgttgttcc ctgcataata agtcagctct 840
gcggaactat gtctcacatc acaaactatc attgtggagc tgtgaggcat gggagggaga 900
gacaactttg ggcaatcaaa aatttcaagt ttcgacagat ggttataaca gccagcagag 960
tactcccgca ggaagggtag cgtacggaga ttggggcatg acctgcaata aatagtttca 1020
agccttgcaa acgaatcatt aggcacccca acccactcaa taagttcagg caatgaatca 1080
aggacaatca gcttcaactg caaaaaactt ttgtttgtag cgccaccaaa gacatggcgg 1140
atctcactaa cttcataaat gttgctcaat gtgagtgatg tgagctgcgg caagtactca 1200
aaaggaggaa gaggacccaa g 1221
<210> 5
<211> 782
<212> DNA
<213> Artificial sequence
<400> 5
tcgcagagct gacttattct gcagggaaca acatgtatat taatgggtat aatagtggtg 60
aggtggactt gcataacctg cgaaaagtag agaaaatgag tattacagat gtatcacaca 120
tttcaatgac agagctgaac aatctgaaat ccctaagaag actagatgtc agaagatgca 180
gcttcacgtg ccatggactc caggacctcg tgtgcctcca atatcttaca gtatacaact 240
gtggcgtctt ctttccatgg cctaccgaag cagctcacac catcaagctt ttccctgctt 300
ccctcgagac acttgagatt gaaggagagt caggcatgca gtcaatggct ctgctcagca 360
atctgaaatc cctaaggaga ctagatgtca gaagatgcag catcacgtgc catggactgc 420
aggacctcgc atgcctccaa tcacttacag tacaagactg tggcaacttc tttccatggc 480
ctaccgaagc agctcacacc gtcaatcctt tccctcacac catcaagcct ttccctgctt 540
ccctcgaggc acttgagatt gaaggagagt taggcatgca gccagtggct ttgctcagca 600
atctgaaatc cctaagaaga ctagatgtca gaagatgcag catcacgtgc catggactgc 660
aggacctcgc gtgcctccaa tcagttacag taaaagaatg tggcaacttc tttctgcggc 720
ccatcgaagc agctcacacc atcatcaatc ctttgcctgc ttccctcgaa gaacttgagt 780
tt 782
<210> 6
<211> 1220
<212> DNA
<213> Artificial sequence
<400> 6
gaggctacag atgggagcaa caagcactgc tgagatgcaa tccactgtaa gttctcgtag 60
ttgacaggag cctgcaggcg ataacaggtt ggtccttgct accaccaatt ccgagaacaa 120
atccgctgat acagaacggc taagacaatt tcctctgttg tagatcttca aactgttgag 180
gttgactgtg atgagaggat tgaaaccatc cactgttaaa ttctcagagt tcaccagtat 240
aagatcggtg agacaagtga ggttcgagag ctgagccatt gactccatgc ttgactctcc 300
atcaatctca agttcctcga gggaagcagg caaaggattg atgatggtgt gagctgcttc 360
gatgggccgc cgaaagaagt tgccacattc ttttactgta agtgattgga ggcacgcgtg 420
gtcctgcagt ccatggcacg tgatgctgca tcttctgaca tctagtcttc ttagggattt 480
cagattgctg agcggagcca ttgactgcat gcctgactct ccttcaatct caagtgtctc 540
gagggaagca gggaaaagct tgatggtgtg agctacttcg gtatgccatg gaaagaagat 600
gccacagttg tatactgtaa gatattggag gcacgcgagg tcctggagtc catggcacgt 660
gaagctgcat cttctgacat ctagtcttct tagggatttc agattgttca gctctgtcat 720
tgaaatgtgt gatacatctg taatactcat tttctctact tttcgcacgt tatgcaagtc 780
cacctcacca ttatacccat taatatacat gttgttccct gcataataag tcagctctgc 840
ggaactatgt ctcacatcac aaactaccat tgtggagctg tgaggcatgg gagggagaga 900
caactttggg caatcaaaaa tttcaagttt cgacagatgg ttataacggc cagcagagta 960
ctcccgcagg aagggtagcg tacggagatt ggggcatgac ctgcaataaa tagtttcaag 1020
ccttgcaaac gaatcattag gcaccccaac ccactcaata agttcaggca atgaatcaag 1080
gacaatcagc ttcaactgca aaaaactttt gtttgtagcg ccaccaaaga catggcggat 1140
ctcactaact tcataaatgt tgctcaatgt gagtgatgtg agctgcggca agtactcaaa 1200
aggaggaaga gtgacccaag 1220
Claims (9)
1. The complete set of primers for identifying the wheat powdery mildew resistance comprise a primer pair A consisting of a primer 1 and a primer 2;
the primer 1 is a single-stranded DNA molecule shown in a sequence 2 of a sequence table;
the primer 2 is a single-stranded DNA molecule shown in a sequence 3 of a sequence table.
2. The set of primers according to claim 1, wherein: the set of primers also comprises a primer pair B consisting of a primer 3 and the primer 2;
the primer 3 is a single-stranded DNA molecule shown in a sequence 1 of a sequence table.
3. The set of primers according to claim 1 or 2, characterized in that:
the molar ratio of the primer 1 to the primer 2 is 1: 1;
the molar ratio of the primer 3 to the primer 2 is 1: 1.
4. PCR reagents for identifying sets of primers for wheat powdery mildew resistance comprising PCR reagents comprising a primer pair according to any one of claims 1 to 3.
5. The PCR reagent according to claim 4, wherein:
the concentrations of the primer 1 and the primer 2 in the PCR reagent are both 200 nM;
or, the concentration of the primer 3 and the primer 2 in the PCR reagent is 200 nM.
6. A kit comprising a set of primers according to any one of claims 1 to 3 or PCR reagents according to any one of claims 4 to 5.
7. Use of a set of primers according to any one of claims 1 to 3 or of a PCR reagent according to any one of claims 4 to 5 or of a kit according to claim 6 in 1) or 2) as follows:
1) identifying wheat powdery mildew resistance;
2) preparing a product for wheat powdery mildew resistance.
8. A method for preparing the kit according to claim 7, comprising the step of packaging each primer individually.
9. A method for identifying or assisting in identifying wheat powdery mildew resistance to be detected comprises the following steps: amplifying wheat to be tested with the primer pair of any one of claims 1 to 3, detecting the PCR amplification product,
if an amplification product is obtained, the wheat to be detected is or is selected as anti-powdery mildew wheat;
if the amplification product is not obtained, the wheat to be detected is not or is not candidate to be anti-powdery mildew wheat.
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