CN110592259A - Molecular marker for detecting corn southern rust resistant gene RPPS313 and application - Google Patents
Molecular marker for detecting corn southern rust resistant gene RPPS313 and application Download PDFInfo
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- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention discloses a molecular marker for detecting a corn southern rust resistant gene RPPS313 and application thereof, wherein the molecular marker is named as A009919, is located at 1681461 th base of a corn 10 # chromosome, and has the molecular marker polymorphism of A/G; the molecular marker for detecting the maize southern rust resistant gene RPPS313 site has high specificity, the provided application method of the molecular marker is convenient to operate, the detection result is accurate and reliable, the molecular marker can be used for identification and assisted breeding of the maize RPPS313 gene, and the problems of low efficiency and long period of traditional breeding can be solved.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and relates to a molecular marker for detecting a corn southern rust resistant gene RPPS313 and application thereof.
Background
Corn (Zea mays L.) is an important food crop. Southern Rust of maize (Southern Corn Rust) is a worldwide endemic airborne disease that occurs in tropical, subtropical, temperate regions including Africa, southeast Asia, Australia, south America, Southern America, the Southern United states, the south of the United states, the Indian countries of the south of the Ring, etc. In China, the disease conditions of a plurality of corn producing areas from south to north are reported. The southern rust pathogen is Puccinia polysora Underw. Unlike common rust, southern rust, once pandemic, causes 40-80% yield loss and even no grain harvest when severe. The occurrence rule of southern rust of corn is not clear at present, so that the southern rust of corn is difficult to prevent, in addition, the disease mainly occurs at the later growth stage of corn, the plant is tall and big at the moment, and the bactericide is difficult to spray into the interior of the plant canopy while increasing the planting cost, so that the prevention and control effect is greatly reduced. Decades of practical experience in production show that the most effective measure for preventing and treating southern rust is also a main-cultivation resistant variety.
The resistance of a plurality of main cultivars in China to the southern rust of corn is poor, if Zhengdan 958 with the largest planting area in China at present has excellent comprehensive properties but is highly susceptible to southern rust, the southern rust of corn in southern areas of Huang-Huai-Hai in 07 and 08 years is generated greatly, and the yield loss of serious rust in Zhengdan 958 fields is over 10 percent when Zhengdan 958 fields are planted. In addition, in the current maize breeding in China, most of the utilized resistance sources are derived from American hybrid 78599, the resistance sources are single, and the single resistance sources do not have effective resistance to the prevalence of new physiological races, for example, the southern rust disease is successfully controlled by the Rpp9 gene in the United states for 30 years, but recent research shows that the disease-resistant inbred lines and the hybrid containing the Rpp9 in Columbia, Hawaii, Texas and the like in the United states have different degrees of susceptibility. Therefore, there is a real need to develop new germplasm resources for resisting southern rust, and to perform genetic analysis and fine positioning on disease-resistant genes of the germplasm resources until cloning while applying the germplasm resources to breeding, so as to provide a genetic basis for analyzing a molecular mechanism of corn-rust interaction. The introduction of more resistant genes or the aggregation of multiple resistance genes in new cultivars can increase the level of resistance and extend the duration of resistance in maize varieties. Therefore, more new germplasm resources for resisting southern rust need to be mined, and 7 major QTL sites for resistance to southern rust have been reported in maize by 2019. At present, Molecular Marker-assisted selection (MAS) is combined with a traditional hybridization method and applied to the cultivation of southern rust resistant corn varieties, the resistance of the southern rust resistant corn varieties is increased by polymerizing the most suitable genotype combination of disease resistant genes through Molecular Marker-assisted selection, and the improvement of the southern rust resistance can greatly improve the breeding efficiency and bring huge social and economic benefits.
Disclosure of Invention
The invention aims to provide a molecular marker for detecting a corn southern rust resistant gene RPPS313 and application thereof.
In order to realize the purpose of the invention, the technical scheme of the invention is as follows: the invention provides a molecular marker for detecting a corn southern rust resistant gene RPPS313, wherein the molecular marker is named as A009919, is located at 1681461 th base of a corn 10 # chromosome, and has the molecular marker polymorphism of A/G.
The primer combination for detecting the molecular marker A009919 comprises an upstream FAM primer, an upstream HEX primer and a downstream universal primer, wherein the sequence of the upstream FAM primer is shown as SEQ.ID.NO.1, the sequence of the upstream HEX primer is shown as SEQ.ID.NO.2, and the sequence of the downstream universal primer is shown as SEQ.ID.NO. 3.
The reagent or the kit containing the primer combination belongs to the protection scope of the invention.
The invention provides application of the primer combination in detecting a corn southern rust resistant gene RPPS 313.
The application further comprises the following steps:
(1) extracting the corn genome DNA to be detected;
(2) using corn genome DNA to be detected as a template, and carrying out KASP reaction detection by using the primer combination;
(3) if only the base A is detected at the molecular marker locus, judging that the corn sample to be detected is a corn southern rust resistant homozygous genotype; if only the base G is detected, judging the corn sample to be detected to be a disease-sensitive homozygous genotype; if bases A and G are detected simultaneously, judging that the corn sample to be detected is a heterozygous genotype;
the PCR reaction conditions in the KASP detection in the step (2) are as follows: 15min at 94 ℃; then carrying out a first-step amplification reaction at 94 ℃ for 20sec, 61-55 ℃ for 1min, and reducing the annealing and extension temperature by 0.6 ℃ in each cycle for 10 cycles; then carrying out a second amplification reaction at 94 ℃ for 20sec and 55 ℃ for 1min for 26 cycles; after the reaction is finished, a multifunctional fluorescence reader ARAYA is used for reading fluorescence data of the KASP reaction product, and the reading result can be converted into a graph.
The invention provides application of the molecular marker in auxiliary identification of a corn southern rust resistance gene RPPS 313.
The invention provides application of the molecular marker or the primer combination or the kit containing the primer combination in improvement of maize germplasm resources.
The invention provides application of the molecular marker or the primer combination or the kit containing the primer combination in cultivation of corn with the capability of resisting southern rust of corn.
The invention has the advantages of
The invention provides a molecular marker for detecting a corn Southern rust resistant gene RPPS313, which has high locus specificity for detecting the RPPS313 gene, designs a KASP detection primer combination aiming at the single base difference in the molecular marker, can quickly identify the RPPS313 gene in a corn variety by utilizing a KASP detection technology, and can overcome the defects of long detection period, need of abundant experience and complicated operation or low sensitivity, or low specificity or poor repeatability of detection methods such as PCR, PAPD, PCR-RELP, Southern hybridization and the like of the existing morphological identification method. The application method of the molecular marker provided by the invention is convenient to operate, has accurate and reliable detection results, can be used for identification and assisted breeding of the corn RPPS313 gene, and can solve the problems of low efficiency and long period of traditional breeding.
Drawings
FIG. 1 is a typing chart of KASP detection results of molecular marker A009919 in parent and filial generation individual strains.
Detailed Description
The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.
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.
Example 1 development of molecular markers for southern rust resistance Gene RPPS313 in maize
1. A maize-susceptible southern rust inbred line PHW52 is used as a female parent, maize southern rust resistant S313 is used as a male parent, F1 generation hybrid is prepared, F1 generation individual plants are inbred to obtain seeds for constructing a PHW52 multiplied by S313F 2 segregation population, and each F2 individual plant is used for molecular marker analysis and field disease resistance identification. The maize material S313 is a good inbred line bred by inbreeding introduced germplasm from Thailand by maize research institute of Guangxi Zhuang autonomous region academy of agricultural sciences, belongs to tropical blooding, has high southern rust resistance, and is suitable for being used as a parent material for breeding new varieties, and the PHW52 is a temperate material introduced from America in China.
2. And (3) carrying out spraying inoculation on corn southern rust pathogenic bacteria on all plants in 3 periods of F2 single-plant corn at the emasculation period, 7d after the emasculation period and 14d after the emasculation period, wherein the corn southern rust pathogenic bacteria are from the Minggang base of Guangxi agricultural science institute and are collected on leaves of living plants of a high-susceptibility corn southern rust variety planted in due seasons. And (5) carrying out southern rust investigation on corn at the late stage of maturity, and recording the disease level. Grading the disease condition standard: resistance ratings of grade 1, 3, 5, 7, 9 were performed on each individual and the resistance rating of each family was calculated by weighted average for the parent material and each family in the population. Grading the disease condition standard:
level 1: the whole leaf has no scab or only has anaphylactic reaction without sporangium;
and 3, level: the whole leaf has a small amount of sporophyte, and the total leaf area is less than or equal to 25%;
and 5, stage: the whole leaf has moderate sporophyte which accounts for 26-50% of the total leaf area;
and 7, stage: the whole leaf has a large number of spore piles, and the total leaf area is 51-75%;
and 9, stage: the whole leaf has a large amount of spore piles, the total leaf area is less than 76-100%, and the leaf withers.
3. Polymorphic markers between S313/PHW52 were screened on a 56k maize chip, and about 20 SNP molecular markers were selected for initial gene mapping at intervals of about 10M on 10 chromosomes of maize. Secondly, 30 Xre-sequencing is carried out on S313 and PHW52 to obtain whole genome sequence information of two inbred lines, and one-to-one comparison is carried out to obtain all SNP locus information between S313 and PHW 52. And finally, selecting SNP sites which are at least 100bp apart from other SNP sites and have no insertion or deletion in 100bp in a positioning interval based on the initial positioning result of the gene, and then obtaining flanking sequences of the SNP sites according to a reference genome B73 to design primers and develop alternative SNP molecular markers for fine positioning.
4. Genomic DNA from leaves of each individual of the parent and F2 population was extracted by the CTAB method (Murray & Thompson, 1980Rapid isolation of high-molecular-weight plant DNA. nucleic Acids Res 8: 4321-4325). The alternative molecular markers are used for carrying out polymorphism screening on the amphiphilic parents, KASP reaction is carried out on a Douglas platform SOLELLEX high-throughput PCR instrument, an amplification product is scanned and analyzed on a multifunctional fluorescent reader ARAYA, and SNP markers with polymorphism among parents are recorded and selected for subsequent analysis.
The system and reaction procedure of the KASP reaction are described with reference to the method on the LGC company website (http:// www.lgcgenomics.com /) (KASP genetic chemistry User guide and manual);
reaction system:
reaction procedure:
5. according to the disease-resistant series of F2 individuals, leaf genome DNAs of 30 extreme disease-resistant individuals and 30 extreme susceptible individuals are respectively selected to be mixed to construct an anti-susceptible DNA pool. 192 molecular markers which are polymorphic among parents and are uniformly distributed on 10 chromosomes of corn are selected to carry out detection on the resistance and the susceptibility pools, and only 1 molecular marker A006273 has polymorphism among the resistance pools, which indicates that the polymorphic marker is linked with the disease resistance trait. Since the linked marker is located on the short arm of chromosome 10, 19 primers with polymorphisms between parents on the short arm of the chromosome were selected for KASP reaction marker typing of individual strains of the F2 population. The reaction system and procedure were as above, and population genotype data was obtained. The group genotype data was then mapped locally using the mapping software, JoinMap 4.0. And finally, carrying out QTL positioning on the target chromosome by utilizing a composite interval mapping method of WinQTLCart2.5 software by combining the molecular marker genotype data of each single plant of the F2 population and the disease-resistant grade obtained by corresponding field resistance identification of the corn southern rust. A highest peak position exists between the molecular marker Affx-91298359 (physical position 0.12M) and the marker Afx-91182449 (physical position 2.03M) (interval size is about 2M), the LOD value is 77.2, and the explained phenotype value is 83.1 percent, so that 1 major gene locus for controlling the southern rust of corn exists between the two positions, and the gene is named as RPPS 313.
5. On the basis of the results of QTL scanning analysis, a large S313 XPHW 52F 2 population of 1520 plants is further planted, all individual leaves are sampled and stored at low temperature before castration, the disease resistance of each individual plant is identified and recorded after 3 times of pathogen spraying inoculation, the corresponding leaves of 386 high rust-sensitive individual plants are taken out for DNA extraction, and the number of exchanged individual plants of 11 markers AX-86277269 (physical position 0.21M), AX-86255577 (physical position 0.64M), AX-86255581 (physical position 0.84M), A01009 (physical position 1.21M), A005 (physical position 1.24M), A010010 (physical position 1.36M), A009917 (physical position 915.39M), A010011 (physical position 1.45M), A009919 (physical position 1.68M), A009920 (physical position 1.71M) and A010013 (physical position 1.93M) is 24, 21, 17, 14, 12, 2, 3, 10 and 22 respectively. According to the reduction trend of the number of the exchanged individuals, the exchanged individuals of A009919 are the least. Therefore, it can be determined that A009919 is the marker most closely linked to the gene for resisting southern rust of maize S313, and is located at 1681461 th base of chromosome 10 of maize, and the sequences of the primers used for detecting molecular marker A009919 are shown in SEQ ID No.1, SEQ ID No.2 and SEQ ID No. 3.
Example 2 validation of molecular markers for southern rust resistance in maize RPPS313
1. The experimental material is 95 parts of high-susceptibility maize southern rust inbred line PHW52, high-resistance maize southern rust inbred line S313 and S313/PHW 52F 2 group material; the primer sequences of the used molecular marker A009919 are shown as SEQ.ID.NO.1, SEQ.ID.NO.2 and SEQ.ID.NO. 3.
2. Extracting genome DNA of a corn leaf to be detected by using a CTAB method, and amplifying by using a primer of a molecular marker A009919 according to the KASP method; after the reaction is finished, a multifunctional fluorescence reader ARAYA is used for reading fluorescence data of the KASP reaction product, and the reading result can be converted into a graph.
3. And (4) analyzing results: the results show that FAM fluorescent blue spots representing A bases can be detected in disease-resistant samples in fields, HEX fluorescent red spots representing G bases can be detected in disease-sensitive samples (see figure 1), the original image of the detection result is a color image, and only a black and white image can be submitted in consideration of patent application, so that the applicant marks genotypes represented by small circles in the image. As can be seen from the figure, the disease resistance of the 1 st well S313 in the 1 st column is blue, the disease susceptibility of the 2 nd well PHW52 in the 1 st column is blue, the blank control of the 3 rd well in the 3 rd column is blank, 5 of the 93F 2 samples show pink representing no detection, and the remaining 88F 2 samples are red, and the detection result is consistent with the result of the inventor in the field test. Therefore, the molecular marking method provided by the invention can accurately screen the major gene with resistance to southern rust, can predict whether the corn plant has resistance to southern rust, and greatly quickens the selection progress of the southern rust resistant corn material.
Sequence listing
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Claims (8)
1. A molecular marker for detecting a corn southern rust resistant gene RPPS313 is characterized in that the molecular marker is named as A009919, is located at 1681461 th base of a corn 10 # chromosome, and has the molecular marker polymorphism of A/G.
2. The primer combination for detecting the molecular marker as claimed in claim 1, which comprises an upstream FAM primer, an upstream HEX primer and a downstream universal primer, wherein the sequence of the upstream FAM primer is shown as SEQ ID No.1, the sequence of the upstream HEX primer is shown as SEQ ID No.2, and the sequence of the downstream universal primer is shown as SEQ ID No. 3.
3. A reagent or kit comprising the primer combination of claim 2.
4. Use of the primer combination of claim 2 in detecting the anti-southern rust gene RPPS313 in corn.
5. Use according to claim 4, characterized in that it comprises the following steps:
(1) extracting the corn genome DNA to be detected;
(2) using corn genome DNA to be detected as a template, and carrying out KASP reaction detection by using the primer combination;
(3) if only the base A is detected at the molecular marker locus, judging that the corn sample to be detected is a corn southern rust resistant homozygous genotype; if only the base G is detected, judging the corn sample to be detected to be a disease-sensitive homozygous genotype; if bases A and G are detected simultaneously, judging that the corn sample to be detected is a heterozygous genotype;
the use according to claim 5, wherein the PCR conditions in the KASP assay in step (2) are: 15min at 94 ℃; then carrying out a first-step amplification reaction at 94 ℃ for 20sec, 61-55 ℃ for 1min, and reducing the annealing and extension temperature by 0.6 ℃ in each cycle for 10 cycles; then carrying out a second amplification reaction at 94 ℃ for 20sec and 55 ℃ for 1min for 26 cycles; after the reaction is finished, a multifunctional fluorescence reader ARAYA is used for reading fluorescence data of the KASP reaction product, and the reading result can be converted into a graph.
6. Use of the molecular marker of claim 1 for assisting in the identification of the southern rust resistance gene RPPS313 in maize.
7. Use of the molecular marker of claim 1 or the primer combination of claim 2 or the kit of claim 3 for the improvement of maize germplasm resources.
8. Use of the molecular marker of claim 1 or the primer combination of claim 2 or the kit of claim 3 for breeding corn with resistance to southern rust of corn.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113372424A (en) * | 2020-06-22 | 2021-09-10 | 北京市农林科学院 | Corn southern rust resistance gene and application thereof |
| CN113897352A (en) * | 2020-06-22 | 2022-01-07 | 北京市农林科学院 | Corn southern rust resistance gene close linkage marker and application thereof |
| CN114277173A (en) * | 2021-12-27 | 2022-04-05 | 中国农业科学院作物科学研究所 | Molecular marker closely linked with corn southern rust resistance major QTL and application thereof |
| CN114982630A (en) * | 2022-05-06 | 2022-09-02 | 宿州学院 | Molecular marker assisted breeding method for corn with high southern rust resistance |
| CN116970735A (en) * | 2023-09-22 | 2023-10-31 | 三亚中国检科院生物安全中心 | Rapid detection method and kit for maize rust recombinant polymerase |
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| WO2008021225A2 (en) * | 2006-08-14 | 2008-02-21 | Monsanto Technology Llc | Maize polymorphisms and methods of genotyping |
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| CN116970735A (en) * | 2023-09-22 | 2023-10-31 | 三亚中国检科院生物安全中心 | Rapid detection method and kit for maize rust recombinant polymerase |
| CN116970735B (en) * | 2023-09-22 | 2024-01-19 | 三亚中国检科院生物安全中心 | Rapid detection method and kit for maize rust recombinant polymerase |
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