CN116162726A - Auxiliary selection molecular marker for southern rust-resistant corn and application thereof - Google Patents
Auxiliary selection molecular marker for southern rust-resistant corn and application thereof Download PDFInfo
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Abstract
The invention discloses an auxiliary selection molecular marker of southern rust resistant corn and application thereof, wherein the molecular marker is designed in a hot spot area of chromosome 10 by referring to a corn B73 genome sequence, and the auxiliary selection molecular marker of southern rust resistant corn can be effectively detected by hybridizing and backcrossing disease resistant corn inbred line K22 with a plurality of disease-sensitive inbred lines (Lu self 1509, lu self 1512, lx4201 and Lu self 1526) from different germplasm groups, and combining genotype identification through field rust resistance phenotype investigation. The molecular marker detected by the primer is closely linked with the southern rust resistance gene of the corn, can be used for identifying whether the southern rust resistance gene is contained in plants, has reliable and simple detection result, improves the germplasm screening efficiency of the southern rust of the corn, reduces the workload of field breeding, shortens the breeding period and saves the cost.
Description
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to an auxiliary selection molecular marker for southern rust-resistant corn and application thereof.
Background
Corn is the first large grain crop in China, has rich genetic resources, is an important feed and industrial raw material, has high corn yield and wide application, and has higher economic value in the aspects of serving as grains, feed, industrial and medical raw materials and the like.
The southern rust is a disease caused by multiple rust piles (P.polysoraunderw.), and mainly occurs on corn leaves, leaf sheaths, stems and bracts can be infected, after the disease, the leaves are covered by orange summer spore piles and summer spores, so that the leaves dry and die, the yield of light corn is reduced by 10-20%, the weight of the light corn is more than 30%, and serious plots are even dead, so that the conditions of digging and breeding corn varieties with high southern rust resistance are urgently needed, the occurrence of diseases is reduced, and the yield reduction of corn is reduced.
In recent years, the development of genomics and bioinformatics provides great convenience for the cultivation of new varieties of crops, wherein a molecular marker assisted breeding method is to develop a molecular marker closely linked with a functional gene or a target trait, and the existence of the target gene is determined by detecting the molecular marker so as to further predict the disease resistance trait of crop plants.
At present, in order to discover and select corn varieties with high southern rust resistance, a plurality of domestic subject groups develop disease resistance gene identification work of the southern rust resistance of the corn, and although some molecular markers with the southern rust resistance are developed, most markers have the limitations of far distance from resistance genes/loci, low effect value and the like, and do not have broad spectrum, so that development of a molecular marker with broad spectrum and high resistance effect value is urgently needed.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides an auxiliary selection molecular marker for southern rust resistant corn and application thereof, wherein after a B73 genome sequence of the corn is referred, a molecular marker is designed in a hot spot region of a 10 th chromosome, hybridization and backcrossing are carried out on a disease resistant corn inbred line K22 and a plurality of disease sensitive inbred lines (Lu self 1509, lu self 1512, lx4201 and Lu self 1526) from different germplasm groups, and the auxiliary selection molecular marker for southern rust resistant corn can be effectively detected by a field rust resistance phenotype investigation and combined genotype identification through finding out a primer RpF 1-RppR 1.
In order to achieve the above effects, the present invention adopts the following technical scheme:
in one aspect, the invention provides an auxiliary selection molecular marker for detecting southern rust resistant corn, wherein the nucleotide sequence of the molecular marker is shown as SEQ ID NO.3 and SEQ ID NO. 4;
in another aspect, the invention provides a primer for detecting auxiliary selection molecular markers of southern rust resistant corn, wherein the primer is RpF 1 and RppR1, and the nucleotide sequences of RppF1 and RppR1 are respectively shown in SEQ ID NO.1 and SEQ ID NO. 2.
In another aspect, the invention provides a reagent for detecting a co-selective molecular marker of southern rust resistant corn, the reagent comprising RppF1 and RppR1 primers.
The invention also provides a kit for detecting the auxiliary selection molecular marker of the southern rust resistant corn, which comprises the primer, a PCR amplification reagent and a PCR product purification reagent.
In another aspect, the invention provides application of the primer in preparing a gene chip for detecting auxiliary selection molecular markers of southern rust resistant corn.
The invention also provides application of the primer in detection of southern rust resistant corn.
Further, the application of the primer in detecting southern rust resistant corn comprises the following steps:
1) Extracting genome DNA in a sample to be detected;
2) Performing PCR amplification by using the genome extracted in the step 1) as a template and adopting the primer;
3) Agarose gel electrophoresis, and amplification results were checked.
Further, the amplification results are 180bp single band, 180/195bp double band, 195bp single band.
Furthermore, the 180bp single band is a southern rust resistance corn resistance gene homozygous plant amplification band; the 180/195bp double band is an amplification band of a southern rust resistant corn resistance gene heterozygous plant; the 195bp single band is a southern rust disease-susceptible corn plant amplification band.
The beneficial effects are that:
1) The molecular marker detected by the primer is closely linked with the southern rust resistance gene of the corn, can be used for identifying whether the plant contains the southern rust resistance gene, and has reliable and simple detection result.
2) The molecular marker detected by the primer provided by the invention identifies conventional breeding materials, improves the germplasm screening efficiency of southern rust of corn, reduces the workload of field breeding, shortens the breeding period and saves the cost.
Drawings
FIG. 1 is a graph showing the amplification electrophoresis results of the RpF 1 and RppR1 primers in the disease-resistant maize inbred K22, the infectious inbred 1509, the infectious inbred 1512, the infectious inbred lx4201 and the infectious inbred 1526.
FIG. 2 is a graph showing the results of amplification electrophoresis of plants after hybridization and backcrossing of the RpF 1 and RppR1 primers with K22 and Lu self 1509, lu self 1512, lx4201 and Lu self 1526. 1509, 1512, 4201 and 1526 are respectively the names of the self-bred lines of self-bred 1509, self-bred 1512, lx4201 and self-bred 1526.
Fig. 3 is a physical diagram of southern rust disease-resistant plants and disease-sensitive plants in the field.
Detailed Description
The invention will be further illustrated with reference to specific examples, but the invention is not limited to these examples only. Further, it is understood that various changes and modifications may be made by those skilled in the art after reading the teachings of the present invention, and such equivalents are intended to fall within the scope of the claims appended hereto.
The test methods for specific experimental conditions are not noted in the examples below, and are generally performed under conventional experimental conditions or under experimental conditions recommended by the manufacturer.
The following biomaterials are involved in the examples:
disease resistant maize inbred lines K22, robusta 1509, robusta 1512, lx4201 and robusta 1526, wherein disease resistant inbred lines are provided by the university of agriculture in china Yan Jianbing professor topic group, and other inbred lines are provided and maintained by the subject unit.
Synthesis of RpF 1 and RpR 1 primers Synthesis by Beijing Optimu Biotech Co., ltd
PCR mix was purchased from Nanjinozan Biotechnology Co.2X Taq Plus Master Mix (Dye Plus), and other reagents and instrumentation are commercially available.
Examples:
(1) The disease-resistant maize inbred line K22 is hybridized and backcrossed with a plurality of disease-resistant inbred lines from different germplasm groups, namely, a robust self 1509, a robust self 1512, an lx4201 and a robust self 1526, and plant seeds of the backcrossed plants are harvested.
(2) The maize B73 genomic sequence was downloaded at the Maizesequence website and primers were designed in the 10 th chromosomal region.
(3) The alkaline cooking method extracts the following seed (endosperm) DNA: and (3) carrying out hybridization and backcrossing on K22, the robust self 1509, the robust self 1512, the lx4201, the robust self 1526 and K22 and the robust self 1509, the robust self 1512, the lx4201 and the robust self 1526 respectively to obtain plant seeds.
(4) And (3) configuring a reaction system:
and (3) performing PCR amplification by using the DNA extracted in the step (2) as a template and the nucleotide sequences shown in RpF 1 and RppR1 as primers, wherein the primer sequences of RpF 1 and RppR1 are shown in table 1.
TABLE 1 RpF 1 and RppR1 primer sequences
| Primer(s) | Primer sequence (5 '-3') | Sequence numbering |
| RppF1 | CGAAATTCACGACTAGTGGGTTCATC | SEQ ID NO.1 |
| RppR1 | GCGTAAACGGTCAGAACCACAT | SEQ ID NO.2 |
The PCR amplification system was amplified with PCR mix and the PCR reaction system was 20. Mu.l, comprising the following components:
the components are sequentially added into a centrifuge tube with the volume of 0.5ml, and the PCR amplification reaction procedure is as follows: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30s, annealing at 56℃for 30s, extension at 72℃for 30s, for a total of 35 cycles; then overextended at 72℃for 7min.
(3) Agarose gel (3%) electrophoresis
(1) Washing distilled water of an appliance for electrophoresis, and erecting a comb;
(2) preparing agarose gel with proper concentration according to the size of the DNA fragment to be separated, accurately weighing a certain amount of agarose, adding the agarose into a conical flask, and adding about 30ml of electrophoresis buffer (TAE or TBE);
(3) heating and melting in a microwave oven, cooling to about 40 ℃, fully and uniformly mixing, and pouring into an electrophoresis tank;
(4) solidifying at room temperature for about 40min, carefully pulling out the comb, placing gel into electrophoresis tank, and preparing sample application
(5) Adding electrophoresis buffer solution into electrophoresis tank, and spreading on gel surface without air bubbles in sample application hole
(6) The sample is prepared before sample application, 5ul of sample is added (in eight connecting pipes), 1ul of 6xLoding buffer and 1ul of dye are mixed, the mixed sample is slowly injected into the sample application hole by robbing, and the attention is paid to not to series holes
(7) According to the positive and negative electrodes (red, positive, black and negative), the power supply is turned on, the voltage is 40-60V, the time is 30-40min, and whether electrophoresis is stopped can be judged according to the position of bromophenol blue
(8) And after the electrophoresis is finished, the power supply is turned off, gel imaging is carried out, the size of the fragments is determined by comparing with a marker, and the amplification results are shown in fig. 1 and 2.
(4) Analysis of results
(1) As shown in FIG. 1, the amplification bands of the RpF 1 and RppR1 primers are 180bp in the disease-resistant maize inbred line K22, and 195bp in the disease-resistant inbred lines of Lu-self 1509, lu-self 1512, lx4201 and Lu-self 1526.
(2) As shown in FIG. 2, the amplified bands of the RpF 1 and RppR1 primers in the plant seeds obtained after the backcrossing are respectively 180bp single band, 180/195bp double band and 195bp single band. The 180bp sequence and 195bp sequence are shown in Table 2:
table 2 180bp sequence and 195bp sequence
And (3) carrying out field character investigation on the plants obtained after backcrossing, wherein the disease-resistant plants and disease-sensitive plants of the southern rust in the field are shown in figure 3, and the statistical results of the resistance and genotype of the southern rust in the population are shown in table 3.
TABLE 3 resistance to southern rust and genotype statistics in isolated populations
Note that: the resistance locus is not marked, and the resistance locus is marked as heterozygous (Heterozygate, hetero-) or homozygous (homozygate, homo-); the anti-rust property is marked as "+", and the anti-rust property is marked as "-".
By combining with genotype identification, if the amplified product is 180bp single band, the corn to be detected has southern rust resistance gene and is a disease-resistant plant; the amplified product is 195bp single band, so that the corn to be detected does not have a southern rust resistance gene and is a disease-sensitive plant; the amplified product is 180/195bp double band, so that the corn material to be detected has southern rust resistance gene, belongs to heterozygote and is disease-resistant plant. Therefore, the amplification results of the RpF 1 and RpR 1 are consistent with the susceptibility of southern rust, and the plant genotype can be accurately reflected, so that disease-resistant plants can be accurately screened.
The above examples are intended to illustrate the explanation and specific embodiments and implementations of the present invention and are not intended to limit the present invention, and those skilled in the art, after having read the present specification, may make modifications as necessary without creative contribution to the invention, but are protected by patent laws within the scope of the claims of the present invention.
Claims (9)
1. An auxiliary selection molecular marker for detecting southern rust resistant corn is characterized in that the nucleotide sequence of the analysis marker is shown as SEQ ID NO.3 and SEQ ID NO. 4.
2. A primer for detecting the auxiliary selection molecular marker according to claim 1, wherein the primers are RppF1 and RppR1; the nucleotide sequences of RpF 1 and RppR1 are respectively shown in SEQ ID NO.1 and SEQ ID NO. 2.
3. A reagent for detecting a co-selective molecular marker of southern rust resistant corn, comprising the primer of claim 2.
4. A kit for detecting a co-selective molecular marker of southern rust resistant corn, comprising the primer of claim 2, a PCR amplification reagent, and a PCR product purification reagent.
5. Use of the primer according to claim 2 for the preparation of a gene chip for detecting a co-selective molecular marker of southern rust resistant corn.
6. Use of the primer of claim 2 for detecting southern rust resistant corn.
7. The use according to claim 6, characterized by the steps of:
1) Extracting genome DNA in a sample to be detected;
2) Performing PCR amplification by using the primer of claim 2 with the genome extracted in the step 1) as a template;
3) Agarose gel electrophoresis, and amplification results were checked.
8. The use according to claim 7, wherein the amplification result is 180bp single band, 180/195bp double band, 195bp single band.
9. The use according to claim 8, wherein the 180bp single band is a southern rust resistant maize resistance gene homozygous plant amplification band; the 180/195bp double band is an amplification band of a southern rust resistant corn resistance gene heterozygous plant; the 195bp single band is a southern rust disease-susceptible corn plant amplification band.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116941525A (en) * | 2023-08-22 | 2023-10-27 | 河南农业大学 | Improved method for improving southern rust resistance of corn hybrid by increasing resistance due to multiple factors |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116941525A (en) * | 2023-08-22 | 2023-10-27 | 河南农业大学 | Improved method for improving southern rust resistance of corn hybrid by increasing resistance due to multiple factors |
| CN116941525B (en) * | 2023-08-22 | 2024-05-28 | 河南农业大学 | Improved method for improving southern rust resistance of corn hybrid by increasing resistance due to multiple factors |
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