CN109536633B - SNP (Single nucleotide polymorphism) marker co-separated from corn gray spot resistant major QTL-qRgls2 and application - Google Patents
SNP (Single nucleotide polymorphism) marker co-separated from corn gray spot resistant major QTL-qRgls2 and application Download PDFInfo
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Abstract
The invention provides an SNP marker co-separated from a corn gray spot resistant major QTL-qRgls2 and application thereof. The nucleotide sequence of the SNP marker is shown as SEQ ID NO. 3; wherein, the 679 th base is a polymorphic site, and the base is A or G. The invention also provides KASP primers for detecting the SNP markers. The SNP marker and the KASP primer thereof provided by the invention can be used for identifying the resistant material of the grey leaf spot of corn with high efficiency and low cost, and can realize high-throughput detection of the disease-resistant gene of the grey leaf spot of corn.
Description
Technical Field
The invention belongs to the fields of molecular biology and plant molecular breeding, and particularly relates to an SNP (single nucleotide polymorphism) marker co-separated from a main effect QTL-qRgls2 for resisting gray leaf spot of corn and application thereof.
Background
Corn is the most important grain, feed, industrial raw material and energy crop in the world today. Gray spot is a fungal disease caused by cercospora zeae-maydis Tehon & Daniels, also known as cercospora zeae leaf spot. Gray spot disease was first discovered in the united states in the 20 th century, and is now one of the major diseases harming corn production, and is particularly serious in the southwest region of China. Long-term production practice proves that breeding and popularization of disease-resistant varieties are one of the most effective ways for preventing and treating the gray leaf spot of corn.
For the disease resistance breeding of corn, the identification of the disease resistance of breeding materials is crucial, because the occurrence of the gray leaf spot of corn has higher requirements on the air temperature and the air humidity, the natural inoculation identification and the artificial inoculation identification are limited by the factors such as seasons, regions and annual disease prevalence conditions, and the identification of the disease resistance is limited only in the specific seasons (such as spring and summer) meeting the growth of the corn and the regions suitable for the occurrence of the disease. The application of the molecular marker provides a quick and effective method for identifying disease resistance, the molecular marker tightly linked with the disease resistance QTL can be used for screening the breeding material containing the disease resistance QTL in the seedling stage, compared with natural inoculation identification and artificial inoculation identification, the screening of the disease resistance material by using the molecular marker is not limited by the influence of the growth period, planting region and year of corn, and the effect is better than that of the inoculation identification.
The molecular marker is used for screening the disease-resistant QTL of the gray leaf spot of the corn, the accuracy of the screening is determined by the tight linkage degree of the used molecular marker and the disease-resistant QTL, the higher the linkage degree of the molecular marker and the disease-resistant QTL is, the higher the screening accuracy of the molecular marker is, and otherwise, the lower the screening accuracy is. If the molecular marker is located inside the gene of the disease-resistant QTL, the molecular marker will co-segregate with the disease-resistant QTL.
At present, molecular markers such as SSR are utilized to screen disease-resistant QTL, and the method has the defects of low flux, high cost, environmental friendliness and the like, and is difficult to be applied in large range in commercial breeding. The SNP marker is used as a third-generation molecular marker, has the advantages of high flux, low cost of single marker data and the like, simultaneously has accurate and reliable genotyping data, good genetic stability and repeatability, is easy to realize automatic detection, reduces the labor cost, and can meet the integration of multiple genotyping data results.
The molecular marker high-throughput detection technology for resisting the gray leaf spot of the corn is utilized to carry out disease-resistant auxiliary breeding, and the molecular marker high-throughput detection technology plays an important role in commercial breeding.
Disclosure of Invention
The invention aims to provide an SNP marker co-separated from a corn gray spot resistance major QTL-qRgls2 and application thereof.
The technical route of the invention is as follows: obtaining an SNP marker primer combination inside a corn gray leaf spot related gene: 1. the major QTL site qRgls2 related to the resistance of the gray leaf spot of corn is known to be located on chromosome 5 of corn, the disease-resistant gene sequence located on the QTL site qRgls2 is derived from a disease-resistant material Y32, the sequence is shown as SEQ ID NO:1, the susceptible gene sequence located on the QTL site qRgls2 is derived from a susceptible material B73, and the sequence is shown as SEQ ID NO: 2. 2. And comparing the disease-resistant gene sequence and the disease-sensitive gene sequence by adopting sequence comparison software DNAMAN. 3. Selecting a single nucleotide polymorphism Site (SNP) between two sequences according to a comparison result, extracting sequences of at least 50bp at two sides of the SNP site, submitting the sequences to an Egji analysis (Shanghai) science and technology Limited company (LGC company), and designing a KASP primer for detecting the genotype of the selected SNP site. 4. The designed KASP primers were verified. Extracting DNA of fresh leaves of 92 parts of corn material, detecting SNP sites related to gray leaf spot resistance of 92 parts of corn material by using an SNP high-throughput detection platform produced by LGC company, and verifying the genotyping effect of the designed KASP primer combination on the corresponding SNP sites. For corn disease resistant material Y32, see Xu, l., Zhang, y., Shao, s., Chen, w., Tan, j., Zhu, m., … & Xu, m. (2014), High-resolution mapping and characterization of qRgls2, a major quantitative trap in a volume in space cost resistance space bmc plant, 14(1), 230.
In order to achieve the aim, the invention provides a SNP marker which is co-separated from a corn gray spot disease resistant main effect QTL-qRgls2, wherein the nucleotide sequence of the SNP marker is shown as SEQ ID NO. 3; wherein, the 679 th base is a polymorphic site, and the base is A or G.
In a second aspect, the present invention provides KASP primers for detecting said SNP markers, said primers being as shown in table 1:
TABLE 1
In a third aspect, the invention provides a detection reagent or kit comprising said KASP primer.
In a fourth aspect, the invention provides any one of the following uses of the SNP markers, the KASP primers, or a detection reagent or kit comprising the KASP primers:
(1) the application in the genetic typing of the major QTL-qRgls2 gene for resisting the gray leaf spot of the corn;
(2) application in identifying or screening anti-gray spot corn materials;
(3) the application in the maize molecular marker assisted breeding.
In a fifth aspect, the present invention provides a method for identifying a maize germplasm resource containing a major QTL-qRgls2, comprising the steps of:
1) extracting a corn genome DNA to be detected as a template;
2) adding a specific KASP Primer mix and a general KASP Master mix into the template in the step 1) to perform PCR amplification;
3) and analyzing the PCR amplification product by using a fluorescence detector.
Wherein, the KASP Primer mix contains three specific primers: a forward primer 1, a forward primer 2 and a reverse primer (table 1), and different tag sequences are added to the 5' ends of the forward primer 1 and the forward primer 2, respectively. Preferably, the tag sequence added to the 5 'end of forward primer 1 is 5'-GAAGGTGACCAAGTTCATGCT-3'and the tag sequence added to the 5' end of forward primer 2 is 5'-GAAGGTCGGAGTCAACGGATT-3'.
The KASP Master mix comprises the following components: universal FRET cassette fluorescent primer, ROX internal reference dye, KlearTaq DNA polymerase, dNTP and MgCl2。
Preferably, the PCR reaction system used in step (2) is as follows: DNA template 0.8. mu.l, KASP Master mix and KASP Primer mix 0.8. mu.l.
Wherein the mixed solution is prepared by mixing 100 mu M KASP Master mix and 2 XKASP Primer mix according to the volume ratio of 35: 1.
The concentrations of the forward Primer 1, forward Primer 2 and reverse Primer in the KASP Primer mix were 12. mu.M, 12. mu.M and 30. mu.M, respectively.
The PCR reaction conditions were as follows: pre-denaturation at 94 ℃ for 15 min; performing a first-step amplification reaction, namely performing denaturation at 94 ℃ for 20 seconds, annealing at 61-55 ℃ and extending for 60 seconds, and performing 10 Touch Down cycles, wherein the annealing and extending temperature of each cycle is reduced by 0.6 ℃; the second amplification reaction, denaturation at 94 ℃ for 20 seconds, annealing at 55 ℃ and extension for 60 seconds, 26 cycles.
In a sixth aspect, the invention provides a high-throughput detection method of a corn gray spot disease-resistant gene and application thereof in breeding. The method is to realize high-throughput detection of the corn gray leaf spot disease-resistant gene by using the KASP primer shown in the table 1.
The object of the invention can be further achieved by the following technical measures.
By utilizing the developed gray spot disease resistant KASP primer combination, combining a high-flux DNA extraction method and an LGC high-flux SNP molecular marker genotyping platform, the high-flux molecular breeding of the corn gray spot disease resistance is realized. The main process is as follows: 1. and (4) sample preparation. Sampling the single corn kernel, shearing part of endosperm of the corn kernel, and continuously planting the kernel without damaging the embryo. And 2, quickly extracting the DNA. And (3) carrying out rapid DNA extraction on the single corn kernel by adopting a Hotshot method. The method for extracting DNA by Hotshot is low in reagent material cost, time consumption of the whole process is very short, and a large amount of manpower, material resources and time can be saved during large-scale extraction of DNA. 3. And (3) detecting high-throughput molecular markers. And (3) carrying out high-flux molecular marker detection on the DNA sample by utilizing a high-flux real-time fluorescence detection system and a water-bath PCR instrument produced by LGC. 4. Good sample tracking. According to the result of the molecular marker detection of the DNA, corresponding corn kernels are tracked, only excellent corn kernels are sown during breeding, and the sowing area can be saved by 50% -75%.
Further, the application in the backcross breeding of the corn gray leaf spot is as follows: 1. the corn material Y32 containing disease-resistant genes is used as a donor parent, the material which is susceptible to gray spot and has excellent other agronomic characters is used as a recurrent parent, and the disease resistance of the recurrent parent is improved by introducing the gray spot-resistant genes into the recurrent parent through backcross. 2. And (3) carrying out disease-resistant gene detection on the backcross group single plants by utilizing the developed KASP primer combination and an SNP high-throughput detection platform produced by LGC, screening out the single plants containing the disease-resistant genes, and carrying out backcross on the single plants and recurrent parents.
By the technical scheme, the invention at least has the following advantages and beneficial effects:
the SNP marker and the KASP primer thereof provided by the invention can be used for identifying the corn gray leaf spot resistant material with high efficiency and low cost. The method only needs to synthesize corresponding primers and add a fluorescent probe sequence at the 5' end, and compared with the probe method fluorescent quantitative PCR, the method greatly reduces the detection cost; and the fluorescence scanning is carried out at the reaction end point, so that PCR amplification of a large number of samples can be carried out firstly, and after the reaction is finished, the fluorescence scanning detection is carried out in a centralized manner, so that the high-flux detection is realized, and the detection efficiency is 12-24 times that of the fluorescence quantitative PCR; gel electrophoresis is not needed after the reaction is finished, a fluorescence signal is scanned, and meanwhile, the reaction system can be automatically constructed, so that time and labor are saved, and the error probability can be effectively reduced.
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FIG. 1 shows the use of KASP primer combination HB2-1 for backcross generations BC in example 3 of the present invention2F1The result of the detection of the individual plant (2). Wherein, 1-corn material containing gray spot resistant gene, 2-corn material without gray spot resistant gene, and the rest points represent deletion.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or the conditions as recommended by the manufacturer's instructions.
Example 1 acquisition of SNP marker associated with Gray leaf Spot
Known QTL locus qRgls2 related to resistance to the gray leaf spot of corn is located on chromosome 5 of corn, the gene sequence of the gray leaf spot resistance of a disease-resistant material Y32 is shown as SEQ ID NO. 1, and the gene sequence of the gray leaf spot sensitivity of a disease-susceptible material B73 is shown as SEQ ID NO. 2; comparing the Nucleotide sequences of SEQ ID NO:1 and 2 by using sequence comparison software DNAMAN, and selecting 4 Single Nucleotide Polymorphism (SNP) sites between the two sequences: HB2-1, HB2-2, HB2-3 and HB2-4, and at least 50bp nucleotide sequences at two sides of SNP sites HB2-1, HB2-2, HB2-3 and HB2-4 are respectively shown as SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6.
Example 2KASP primer design and validation
The obtained SNP sites HB2-1, HB2-2, HB2-3, HB2-4 and sequences at both sides thereof are shown as SEQ ID NO 3 (679 th base n is SNP site, n is a (sense) or g (anti), SEQ ID NO 4 (204 th base n is SNP site, n is g (sense) or t (anti), SEQ ID NO 5 (193 th base n is SNP site, n is a (sense) or c (anti), SEQ ID NO 6 (115 th base n is SNP site, n is g (sense) or a (anti), KASP primer design is submitted to Aigiga analysis (Shanghai) science and technology Limited, the specific primer sequences are as follows:
wherein, the 5' ends of the Primer _ FAM and the Primer _ HEX are respectively added with a tag sequence, and the tag sequence of the FAM is 5'-GAAGGTGACCAAGTTCATGCT-3', HEX and the tag sequence is 5'-GAAGGTCGGAGTCAACGGATT-3'.
The KASP primer is used for detecting corresponding SNP sites, and the application effect of the KASP primer is verified. 96 parts of corn material is selected, DNA of the fresh corn leaves is extracted by adopting an improved CTAB method, and the designed KASP primer is verified. Transferring the extracted DNA from a 96-well plate to a 384-well plate by using an LGC OKTOPURE DNA automatic extraction workstation, subpackaging and mixing a PCR reaction system into an array tape by using an LGC Intelliqube SNP high-throughput detection platform, sealing a film and sealing, wherein the PCR reaction system is as follows: 0.8ul of DNA, 0.8ul of a mixture of KASP Master mix and KASP Primer mix (100 μ M KASP Master mix and 2 XKASP Primer mix at a volume ratio of 35:1, wherein the concentrations of primers Primer _ FAM, Primer _ HEX and Primer _ common in the KASP Primer mix are 12 μ M, 12 μ M and 30 μ M, respectively). Carrying out PCR reaction in an LGC Hydrocycler16 high-throughput water bath PCR instrument, wherein the PCR reaction procedure is as follows: pre-denaturation at 94 ℃ for 15 min; performing a first-step amplification reaction, namely performing denaturation at 94 ℃ for 20 seconds, annealing at 61-55 ℃ and extending for 60 seconds, and performing 10 Touch Down cycles, wherein the annealing and extending temperature of each cycle is reduced by 0.6 ℃; the second amplification reaction, denaturation at 94 ℃ for 20 seconds, annealing at 55 ℃ and extension for 60 seconds, 26 cycles. After the PCR reaction is finished, the LGC Intelliqube SNP high-throughput detection platform is used for carrying out fluorescence scanning on array tape. Proved by verification, the KASP primer HB2-1 has good genotyping effect on the corresponding SNP locus.
Example 3 auxiliary selection Using SNP markers in the improvement of gray leaf Spot backcrossing
Disease-resistant material Y32 containing gray spot disease-resistant gene is used as donor parent, material LA2061 susceptible to gray spot disease is used as recurrent parent, disease-resistant gene is introduced into recurrent parent LA2061 by backcross, and SNP high-throughput detection platform produced by KASP primer combination HB2-1 and LGC is used for backcrossing generation BC2F1The individual plants of (2) were tested. The method specifically comprises the following steps: fresh leaves of each individual plant are taken in the field, 182 samples are obtained in total, and the DNA of the leaves is rapidly extracted by adopting a hotspot method. The LGC OKTOPURE DNA automatic extraction workstation is used for transferring the extracted DNA from a 96-well plate to a 384-well plate, an LGC Intelliqube SNP high-throughput detection platform is used for subpackaging and mixing a PCR reaction system into an array tape, a film is sealed for sealing, and the PCR reaction system and the reaction program are the same as those in the example 2.
After the PCR reaction is finished, the LGC Intelliqube SNP high-throughput detection platform is used for carrying out fluorescence scanning on array tape, and the result is shown in figure 1, wherein the upper purple points represent the corn material containing the gray spot disease resistant gene, the lower right red points represent the corn material without the gray spot disease resistant gene, and the gray points represent the deletion (namely, the genotype of the sample cannot be distinguished). Selecting DNA containing the disease-resistant gene, tracing to a single plant in the field, and carrying out backcross on the single plant containing the disease-resistant gene. At the same time, agarose gel electrophoresis is utilized to carry out gray spot resistant gene detection on 182 single plants, and the detection result and KASPPrimer and method for producing the sameThe detection results are consistent.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
<110> Yuanyongping agricultural high-tech. Ltd
<120> SNP marker co-separated from corn gray leaf spot resistance major QTL-qRgls2 and application
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ccagaaccaa ctcataagtc ataagtttta ccttgtgttt ttgcaggcta tgtgagggcg 3540
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ctgcagttta gctgaccatt ttgttgtcta atgcatgctt tagggaagaa gttccatgac 4140
attgttggaa gtgcttacta tgtcgcacca gaagtactaa aacgacggtc tggtcctgag 4200
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agaaacccac tttatgcagg tgttcagttt aataacaccc accatctttc agatttctaa 4620
tgttcagtgt tagacagact tcattaagat gcaccttaag atgattgtaa gtagtaaaag 4680
tgctttgcac ttttgttaac ttttgagtct gaagatgact tgtggtacct atgacctcaa 4740
gaaaccaagg cattgccatt ggaatagcta attcgaatga gcttcagata tggctatctg 4800
ttttagtttt ggacatctga ctcaacttta taggataata ctatattagc aatctttgag 4860
gtcattgtct cagccaaaat aagttgcggt ctctttttta ctgtcctaag cagcaatatg 4920
gtttccattt tcattatacc agcaacttcc acctttttct tgctatttaa atatctttat 4980
gcattttatc agcaaggaca tgatacgatc gtatatgtga tattctacat cttttcactt 5040
ctcataatta ggttctaagg aacaagcctg attttcgtaa gaggccttgg tcaagcatca 5100
gcccaggtgc taaagatttt gttaaaaggt tactagtgaa gaatccaagg gccaggctaa 5160
cagctgctca agctctctgt aagttttggt atttttcatt aatttactag cctagtcatg 5220
atgatcagat tcaccttctc tatgtgagaa cagagaacac atatacatct ggcagtatgc 5280
ctttcaatca gttatgacaa tgtaaatatg caaagaccga tgttttttct atcctgcacc 5340
attttagaac attaatgggg aaaaaccaca atatattagg aaaaatgttt aattatgtcc 5400
tggtcacttg aaatgaacat ataccactga ggttttctag ttctcatgcg ttcttataat 5460
gatctaataa gtcagtggag gtttgctgcc caccacccct acatttgtat tgtgaattac 5520
tatcatcttt actgatcctg attgttcttg atatgttaag cacatccgtg ggtaagagaa 5580
ggaggggaag catccgatat ccccgtcgac atatctgtgt tatcaaacat gcgtcagttt 5640
gtcaagtaca gccgtttcaa gcaattcgcg cttcgggtaa ttacagtgat tacaaaaaac 5700
aacactgcat cgtttatttt ttcctcacaa tatttcctcg tggcatggtc aggctctggc 5760
gagcaccctt aacgaggaag agctatcaga tctgaaggat cagtttgatg caattgatat 5820
cgataaaagt ggatcgatta gtatcgagga aatgcgtcat gtaggttctg ttagtgtttg 5880
ctgatgaaaa tgccttagat cctgaactac tctgcggtgc tgattaatct gtgcatgttt 5940
cggtaggccc ttgcaaagga tcttccctgg agattgaagg gtccccgtgt gctggagatt 6000
attcaagcag taagtttgag ccttcttctg gatccagccc tttctttgtt accccccttg 6060
tttccaagaa aatagctggc cttgttctga gggtataacc aaaactgcat cttattttgt 6120
ggtagattga cagcaacact gatgggctcg tggacttcaa ggagtttgtt gcggcaactc 6180
tccatatcca ccagatggcg gagctcgact cagaaaggtg gggcatacgc tgccaagctg 6240
ctttcagtaa gtttgatctt gacggtgatg gatatatcac gccggaggaa ctcagaatgg 6300
taattttcta ctcctgtctt gtttccatgt tgcttcacca acgaatgcac agttcacata 6360
acccttatta tcatcactgc ttcccatgaa taactagctg gctcgaccat catgagattc 6420
agtacttgcg ccctgtgcac ttggttttgg tcccgcttgt tagaatgaag taatttatca 6480
atggaagcgc tgtaatattt taatcagcgt ttagatttga taaagataaa acatgttcat 6540
tgtttgtgcc aagaaatcca cttacacaga tactgagagt tgcaccgtag ataacgctaa 6600
tcggcagtat cctaatcgag attttctttc aaggtgcagc accctgggtt gaagggatct 6660
atcgagccgc tgctggagga ggccgacatc gacaaagacg gcaagataag cctgtccgag 6720
ttccgcaagc tcctacggac agcgagcatg agcaacgtac ccagcccaag ggggccccca 6780
aaccctcagg ctctgtgaat tccggctcgg ccactaggga ggagcaagct taggaagttg 6840
ccatacaata gccatgtgtt ctttgggttc ttcagagtgc catgtgatgt ttctggtttt 6900
tagcatccag gttatgtgtg c 6921
<210> 2
<211> 7116
<212> DNA
<213> corn (Zea mays)
<400> 2
gtggcttcct ctcttcccct cctcccttgg ccccgtctcg tctcctcccc ctcaccttcc 60
tctccacctt cctccctccc acccctccgc gcctcgcccc accgcgccaa ccaaccaaca 120
cggcgtccca gcctgcctat ataccgctcc ccccgcgccc ccacacgcgc aaatccatat 180
gctcagctcc cgcctcctcc catccccgga ccccggaccc cggccatggg cgcttgcttc 240
tcctccgcct ctgccgcccc cgccggcgcc gccgtcgacg agcgccgccc gtccaaggag 300
ggcgacggca agaagaggcg ccgcgccgcc ggggcatcgc cggatgccgc ggcgcccgtg 360
cgcgtggagt tcggctacga gagggacttc gaggcgcgct acgaggtcgg ccgcctgctc 420
ggccacggcc agttcggcta caccttcgcc gccaccgacc gcggctctgg ggaccgcgtt 480
gccgtcaagc gcatcgacaa ggccaaggtg agctgccgcc tgcccccccg caccccaagc 540
cgccgcgctg tccctgtctc tgtctctcct actagtagta gtagctggtg gtgattccga 600
gcgcgtcttt ggtctggtgc atcgaaccac ttgtgcttgg tgcatttcga ggggattcgg 660
tgtaattccg tgcaaattag ggatttctct cctgttgctt tccgaggttt aggtgtttcg 720
attgggacgc gattggagcc gttcatttta ggacatttcc ggtgcctttt gggaggcgtt 780
tagctcaacg agtagctcac tcacatttct agctgtttgg ccgcttcatt tctcccaagc 840
tttcgttgtt tgccggtggt tctgagctgc gggatcttga cgttggccag agaggtggtt 900
tcgacattca ggcatctcgg atgacctctt agtttggcac tacagctcta ttatttcggg 960
aacgacgtgt tgctcagtgc gcacctcatt catggaagtg gcaaggtcgc ttgtctgcag 1020
aacggggaag gtgcttttca tctggctatt catggaaaac gacttgttca gttgccctac 1080
taataatttc aataagattg cctgcctcct tgaatggttg gggcttggaa ggttcctgtc 1140
gaagaaaaag tcaggaaaga taacaattgc gcacttgcag tggacaacgc ttccctgtct 1200
tctatgctat aggtggacag catttttcta ggtataatta atttgacctt caaacatatg 1260
tatactaacc aacgcggttt tgattccatc aaatgttttg gactctctct gctgaactgt 1320
caaagttact tcatggggca aaatgtcaaa ttttctggaa ccttccgtag tatattttgg 1380
aaatgagtgt ttattgtgtc attggaaata ccgttcatgt gtctgtgaca gaatgtgtca 1440
ctagaaagct gaattggtgt tgtccttgtc aaaaaggcac taaacacgag tctgaaaatt 1500
aggcctgttc ttggtaaggg aaggaatctg agcatcaatg ctgataggaa tagactctgt 1560
ctgtcaatat tgttaacttg tttatagggc ttcgagtttt caacttttga ggcagataag 1620
taggatacct cttttgatca tgatatataa catattctta tatacctcaa gccttgcact 1680
gttaagttaa tgtggcatcc tttctagaga tcatgacctc aagttgcata tggatgccaa 1740
taatatcgac accaagtgaa catcagtgtc tgtggaatat gccgaaagca gccaacgtgc 1800
cattactgaa ttttcatatg attattatat tctgtttaga tttatttacg tcggaacaca 1860
gtgagatggt aacgtaatga atcaaaatag gctataaaca tgcaattcaa catatcatta 1920
tcatgcccaa gtgttttgtc attctatctt tattcgtcca agaaggacaa gcctggtgca 1980
ttgttgaggg aaccagttct tctgcagtac ttctagggag gtaaaaattc aacaccgttg 2040
gatgcagatc tatcgaaccc agggactttg tgcttccagt gaaaagttat atggacccat 2100
aggccagagg atgtgagagt tttacctctc tggaagttat atgcgctagc attagtgtgg 2160
tcatcaatgg gatcaaagat gagctccacc tttggtgtag agctggagct aggggactct 2220
agcatcctgg cgctccaatc ttccatccag tgaactctgt tttttgggtc tagtaggtca 2280
agggtccagt tattttttct ttctgctgta aagtctctag ttaaggtgtg agttttgtat 2340
ggtgtttttt cgaggtttcc ccaaacctca ccttttttcc ttcttaatat aatgatatgc 2400
agctttcctg cgtattcgag aaaagaaagt tttatctctc tggaagttaa ctgcagagga 2460
acttgttaca ttgttgagag ttgtctcacc gagtcaccag gtcgctggtt caaagcagtc 2520
tctccacatt tatggggaag gcttgcctcg gtttatccct tcccaagact ctacttgtgg 2580
gagactctgg cattgggtct gtcctatgcc gttgaagcgc taggttcgtt tatcccttcc 2640
ctatacccac ttgtcagagc ctccaacact gagtctgccc taagcttcca agttccaaca 2700
ctgggtctgc cctaggccgt cgaagcgtta tatgattgcc atgtactgtt atgctttgtt 2760
gccttcacat attttccgtt cgaaatcatc tccttgttgc cttcacatat tgccttgttg 2820
ctttcacata ttttctgttc cacgtcatac ttagaagtta gaacacgtga tttatgccaa 2880
ttaagattat tattttatat aacagatgac ccgccctgtt gctgtggagg atgtgaaaag 2940
agaagtgaag attcttaaag cacttaaagg acatcagaat attgttcact tctacaatgc 3000
atttgaggat gattcatacg tgtacattgt gatggagtaa gtaggcccat acacctgttc 3060
ctgctaatag agcatatcga ttttgctatg acttttttcc ctaaagtttt aacatgaaca 3120
atatctatcc tgtttacaga atcctagaca ctaaaatgtc atttctaatt atcaattatt 3180
ctatagctaa accagatgca atcctgattt atttttctta acgtatggat atattggact 3240
tttctttcaa acctgcattt tgaatttgat tacagggaac tataacacta attcagaact 3300
ctatcatgtt taacattttt cttgcattgt tctatgtttg tcaacttgac gcacttctta 3360
gataatataa catcatcttc cacagtcacc attagttagg accttggacc ttcatggttc 3420
cgaaatttag ctaagaatgg tatacatggt catgtgattt caaatagatg ttcctatatg 3480
ccagaaccaa ctcatgagtc ataagtttta ccttgtgttt ttgcaggcta tgtgagggcg 3540
gtgaactatt agatcggatt ttggcaaagt aagtagataa gatccccatc tctttgtttc 3600
ccgtacctca ttcttcgcca ttaaatttat agatttttgt gctgtaaaat cagattgctt 3660
tatgttgttt gtctgctttg tttgatttct agttgctcgt tcaagatcct ttacttaatg 3720
gtgtgcgtgt tttgacagaa agaatagccg ctatagtgag aaagatgctg cagtggtagt 3780
ccgccaaatg ctcaaagtag ctgctgaatg ccatctgcgt gggttagttc accgagatat 3840
gaagcctgag gtagaaatca aatacttcaa tctctttgca cacagtaagc atttggtgat 3900
atttcactac ttcctcaggt catgtaagac tgtacctatt ttccttccca gaacttcctt 3960
ttcaaatcga acaaggagga ttcaccacta aaggcgacag attttggttt gtcagatttc 4020
attaagccag gtatctactt ggggccatct gaatctgtcg ggaatctgat aggggcaagt 4080
ctgcagttta gctgaccatt ttgttgtcta atgcatgctt tagggaagaa gttccatgac 4140
attgttggaa gtgcttacta tgtcgcacca gaagtactaa aacgacggtc tggtcctgag 4200
tcagatgttt ggagcatagg agtcataacc tacattttgc tctgtgggag gcgccctttt 4260
tgggataaga ccgaagacgg tatattcaag gaggtaagtg gatggatttt gcataccatg 4320
tgcttacatg taaaatatgc ttggttagag tgctgtacca gggatcagcg ttttcagcgt 4380
gctgatactg ttttgtacaa tgtgtttcta ctttctacgt catatagcag tgtttctttg 4440
ttaactattt cagtgtcaaa ctatttgtcg tgtcacaact cagcagtata attttactat 4500
tttgaacact gtaaacctgc ctggtcaggt tatccttcag taatttctct actagctacc 4560
agaaacccac tttatgcagg tgttcagttt aataacaccc accatctttc agatttctaa 4620
tgttcagtgt tagacagact tcattaagat gcaccttaag atgattgtaa gtagtaaaag 4680
tgctttgcac ttttgttaac ttttgagtct gaagatgact tgtggtacct atgacctcaa 4740
gaaaccaagg cattgccatt ggaatagcta attcgaatga gcttcagata tggctatctg 4800
ttttagtttt ggacatctga ctcaacttta taggataata ctatattagc aatctttgag 4860
gtcattgtct cagccaaaat aagttgcggt ctctttttta ctgtcctaag cagcaatatg 4920
gtttccattt tcattatacc agcaacttcc acctttttct tgctatttaa atatctttat 4980
gcattttatc agcaaggaca tgatacgatc gtatatgtga tattctacat cttttcactt 5040
ctcataatta ggttctaagg aacaagcctg attttcgtaa gaggccttgg tcaagcatca 5100
gcccaggtgc taaagatttt gttaaaaggt tactagtgaa gaatccaagg gccaggctaa 5160
cagctgctca agctctctgt aagttttggt atttttcatt aatttactag cctagtcatg 5220
atgatcagat tcaccttctc tatgtgagaa cagagaacac atatacatct ggcagtatgc 5280
ctttcaatca gttatgacaa tgtaaatatg caaagaccga tgttttttct atcctgcacc 5340
attttagaac attaatgggg aaaaaccaca atatattagg aaaaatgttt aattatgtcc 5400
tggtcacttg aaatgaacat ataccactga ggttttctag ttctcatgcg ttcttataat 5460
gatctaataa gtcagtggag gtttgctgcc caccacccct acatttgtat tgtgaattac 5520
tatcatcttt actgatcctg attgttcttg atatgttaag cacatccgtg ggtaagagaa 5580
ggaggggaag catccgatat ccccgtcgac atatctgtgt tatcaaacat gcgtcagttt 5640
gtcaagtaca gccgtttcaa gcaattcgcg cttcgggtaa ttacagtgat tacaaaaaac 5700
aacactgcat cgtttatttt ttcctcacaa tatttcctcg tggcatggtc aggctctggc 5760
gagcaccctt aacgaggaag agctatcaga tctgaaggat cagtttgatg caattgatat 5820
cgataaaagt ggatcgatta gtatcgagga aatgcgtcat gtaggttctg ttagtgtttg 5880
ctgatgaaaa tgccttagat cctgaactac tctgcggtgc tgattaatct gtgcatgttt 5940
cggtaggccc ttgcaaagga tcttccctgg agattgaagg gtccccgtgt gctggagatt 6000
attcaagcag taagtttgag ccttcttctg gatccagccc tttctttgtt accccccttg 6060
tttccaagaa aatagctggc cttgttctga gggtataacc aaaactgcat cttattttgt 6120
ggtagattga cagcaacact gatgggctcg tggacttcaa ggagtttgtt gcggcaactc 6180
tccatatcca ccagatggcg gagctcgact cagaaaggtg gggcatacgc tgccaagctg 6240
ctttcagtaa gtttgatctt gacggtgatg gatatatcac gccggaggaa ctcagaatgg 6300
taattttcta ctcctgtctt gtttccatgt tgcttcacca acgaatgcac agttcacata 6360
acccttatta tcatcactgc ttcccatgaa taactagctg gctcgaccat catgagattc 6420
agtacttgcg ccctgtgcac ttggttttgg tcccgcttgt tagaatgaag taatttatca 6480
atggaagcgc tgtaatattt taatcagcgt ttagatttga taaagataaa acatgttcat 6540
tgtttgtgcc aagaaatcca cttacacaga tactgagagt tgcaccgtag ataacgctaa 6600
tcggcagtat cctaatcgag attttctttc aaggtgcagc acactgggtt gaagggatct 6660
atcgagccgc tgctggagga ggccgacatc gacaaagacg gcaagataag cctgtccgag 6720
ttccgcaagc tcctacggac agcgagcatg agcaacgtac ccagcccaag ggggccccca 6780
aaccctcagg ctctgtgaat tccggctcgg ccactaggga ggagcaagct taggaagttg 6840
ccatacaata gccatgtgtt ctttgggttc ttcagagtgc catgtgatgt ttctggtttt 6900
tagcatccag gttatgtgtg cagtgcagcc ccgagtgagt ttcgaagtaa atattcagtg 6960
ctttcttttt ctttccggaa gagtgagagg tggaggtcaa aatggtaggc aagactcgcc 7020
ttcttctttc ctttacactg tacagtgata ctgaaatatg tacgattttt attataactg 7080
ttcgtcgcaa taaagttatt tggagaagtg aggatt 7116
<210> 3
<211> 1069
<212> DNA
<213> corn (Zea mays)
<400> 3
gtggcttcct ctcttcccct cctcccttgg ccccgtctcg tctcctcccc ctcaccttcc 60
tctccacctt cctccctccc acccctccgc gcctcgcccc accgcgccaa ccaaccaaca 120
cggcgtccca gcctgcctat ataccgctcc ccccgcgccc ccacacgcgc aaatccatat 180
gctcagctcc cgcctcctcc catccccgga ccccggaccc cggccatggg cgcttgcttc 240
tcctccgcct ctgccgcccc cgccggcgcc gccgtcgacg agcgccgccc gtccaaggag 300
ggcgacggca agaagaggcg ccgcgccgcc ggggcatcgc cggatgccgc ggcgcccgtg 360
cgcgtggagt tcggctacga gagggacttc gaggcgcgct acgaggtcgg ccgcctgctc 420
ggccacggcc agttcggcta caccttcgcc gccaccgacc gcggctctgg ggaccgcgtt 480
gccgtcaagc gcatcgacaa ggccaaggtg agctgccgcc tgcccccccg caccccaagc 540
cgccgcgctg tccctgtctc tgtctctcct actagtagta gtagctggtg gtgattccga 600
gcgcgtcttt ggtctggtgc atcgaaccac ttgtgcttgg tgcatttcga ggggattcgg 660
tgtaattccg tgcaaattng ggatttctct cctgttgctt tccgaggttt aggtgtttcg 720
attgggacgc gattggagcc gttcatttta ggacatttcc ggtgcctttt gggaggcgtt 780
tagctcaacg agtagctcac tcacatttct agctgtttgg ccgcttcatt tctcccaagc 840
tttcgttgtt tgccggtggt tctgagctgc gggatcttga cgttggccag agaggtggtt 900
tcgacattca ggcatctcgg atgacctctt agtttggcac tacagctcta ttatttcggg 960
aacgacgtgt tgctcagtgc gcacctcatt catggaagtg gcaaggtcgc ttgtctgcag 1020
aacggggaag gtgcttttca tctggctatt catggaaaac gacttgttc 1069
<210> 4
<211> 400
<212> DNA
<213> corn (Zea mays)
<400> 4
gttttgtatg gtgttttttc gaggtttccc caaacctcac cttttttcct tcttaatata 60
atgatatgca gctttcctgc gtattcgaga aaagaaagtt ttatctctct ggaagttaac 120
tgcagaggaa cttgttacat tgttgagagt tgtctcaccg agtcaccagg tcgctggttc 180
aaagcagtct ctccacattt atgnggaagg cttgcctcgg tttatccctt cccaagactc 240
tacttgtggg agactctggc attgggtctg tcctatgccg ttgaagcgct aggttcgttt 300
atcccttccc tatacccact tgtcagagcc tccaacactg agtctgccct aagcttccaa 360
gttccaacac tgggtctgcc ctaggccgtc gaagcgttat 400
<210> 5
<211> 401
<212> DNA
<213> corn (Zea mays)
<400> 5
tcccgcttgt tagaatgaag taatttatca atggaagcgc tgtaatattt taatcagcgt 60
ttagatttga taaagataaa acatgttcat tgtttgtgcc aagaaatcca cttacacaga 120
tactgagagt tgcaccgtag ataacgctaa tcggcagtat cctaatcgag attttctttc 180
aaggtgcagc acnctgggtt gaagggatct atcgagccgc tgctggagga ggccgacatc 240
gacaaagacg gcaagataag cctgtccgag ttccgcaagc tcctacggac agcgagcatg 300
agcaacgtac ccagcccaag ggggccccca aaccctcagg ctctgtgaat tccggctcgg 360
ccactaggga ggagcaagct taggaagttg ccatacaata g 401
<210> 6
<211> 259
<212> DNA
<213> corn (Zea mays)
<400> 6
acagtcacca ttagttagga ccttggacct tcatggttcc gaaatttagc taagaatggt 60
atacatggtc atgtgatttc aaatagatgt tcctatatgc cagaaccaac tcatnagtca 120
taagttttac cttgtgtttt tgcaggctat gtgagggcgg tgaactatta gatcggattt 180
tggcaaagta agtagataag atccccatct ctttgtttcc cgtacctcat tcttcgccat 240
taaatttata gatttttgt 259
<210> 7
<211> 47
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
gaaggtgacc aagttcatgc tgattcggtg taattccgtg caaatta 47
<210> 8
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
gaaggtcgga gtcaacggat tcggtgtaat tccgtgcaaa ttg 43
<210> 9
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
cctcggaaag caacaggaga gaaat 25
Claims (9)
1. The SNP marker co-separated with the main effect QTL-qRgls2 for resisting the gray leaf spot of the corn is characterized in that the nucleotide sequence of the SNP marker is shown as SEQ ID NO. 3; wherein, the 679 th base is a polymorphic site, and the base is A or G.
2. KASP primer for detecting SNP marker according to claim 1, wherein the primer is as follows:
3. a detection reagent or kit comprising the primer of claim 2.
4. Use of the SNP marker of claim 1, the KASP primer of claim 2 or the detection reagent or kit of claim 3 for genotyping the major QTL-qRgls2 for gray leaf spot resistance in maize.
5. Use of the SNP marker of claim 1, the KASP primer of claim 2, or the detection reagent or kit of claim 3 to identify or screen anti-gray leaf spot corn material.
6. Use of the SNP marker of claim 1, the KASP primer of claim 2, or the detection reagent or kit of claim 3 in maize molecular marker assisted breeding.
7. A method for identifying a maize germplasm resource containing a major QTL-qRgls2, comprising the steps of:
1) extracting a corn genome DNA to be detected as a template;
2) adding a specific KASP Primer mix and a general KASP Master mix into the template in the step 1) to perform PCR amplification;
3) analyzing the PCR amplification product by using a fluorescence detector;
wherein, the KASP Primer mix contains three specific primers: a forward primer 1, a forward primer 2 and a reverse primer, which are defined as described in claim 2; and, the 5' ends of the forward primer 1 and the forward primer 2 are respectively added with different tag sequences;
the KASP Master mix comprises the following components: universal FRET cassette fluorescent primer, ROX internal reference dye, KlearTaq DNA polymerase, dNTP and MgCl2。
8. The method of claim 7, wherein the PCR reaction system used in step (2) is as follows: DNA template 0.8. mu.l, KASP Master mix and KASP Primer mix 0.8. mu.l;
wherein the mixed solution is formed by mixing 100 mu M of KASP Master mix and 2 XKASP Primer mix according to the volume ratio of 35: 1;
the concentrations of the forward Primer 1, forward Primer 2 and reverse Primer in the KASP Primer mix were 12. mu.M, 12. mu.M and 30. mu.M, respectively.
9. The method according to claim 7 or 8, wherein the PCR reaction conditions used in step (2) are as follows: pre-denaturation at 94 ℃ for 15 min; performing a first-step amplification reaction, namely performing denaturation at 94 ℃ for 20 seconds, annealing at 61-55 ℃ and extending for 60 seconds, and performing 10 Touch Down cycles, wherein the annealing and extending temperature of each cycle is reduced by 0.6 ℃; the second amplification reaction, denaturation at 94 ℃ for 20 seconds, annealing at 55 ℃ and extension for 60 seconds, 26 cycles.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811593457.2A CN109536633B (en) | 2018-12-25 | 2018-12-25 | SNP (Single nucleotide polymorphism) marker co-separated from corn gray spot resistant major QTL-qRgls2 and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811593457.2A CN109536633B (en) | 2018-12-25 | 2018-12-25 | SNP (Single nucleotide polymorphism) marker co-separated from corn gray spot resistant major QTL-qRgls2 and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109536633A CN109536633A (en) | 2019-03-29 |
| CN109536633B true CN109536633B (en) | 2021-09-03 |
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| High-resolution mapping and characterization of qRgls2, a major quantitative trait locus involved in maize resistance to gray leaf spot;Ling Xu 等;《BMC Plant Biology》;20141231;第14卷;第230篇第1-10页 * |
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