CN110923356B - Molecular marker primer of rice gall midge-resistant major gene Gm5, and marking method and application thereof - Google Patents
Molecular marker primer of rice gall midge-resistant major gene Gm5, and marking method and application thereof Download PDFInfo
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Abstract
The invention relates to the field of molecular genetics, in particular to a molecular marker primer of a rice gall midge-resistant major gene Gm5, a marking method and application thereof, wherein the method is used for hybridizing rice insect-resistant varieties ARC5984 (male parent) and 9311 (female parent) to obtain genotypes of F2 single plants and simultaneously combining the genotypes with F2 single plants 2:3 Genetic linkage analysis of the resistance grade against rice gall midge of the pedigree carried the resistance major gene Gm5, identified as carried by insect-resistant line ARC5984, and narrowed the Gm5 region to a 49kb fragment defined by flanking markers Z57 and Z64, and closely linked to molecular marker Z22595. The molecular marker Z22595 can effectively detect whether the insect-resistant variety ARC5984 and the derived variety (line) thereof contain the major resistance gene locus, greatly improve the selection efficiency of the rice plant resistant to the rice gall midge and obtain the rice variety (line) resistant to the rice gall midge containing Gm 5.
Description
[ technical field ] A method for producing a semiconductor device
The invention belongs to the field of molecular genetics, and particularly relates to a molecular marker primer of a rice gall midge-resistant major gene Gm5, a marking method and application thereof.
[ background of the invention ]
Rice is one of the most important crops in the world, and its production is threatened by various pathogens, pests and other harmful organisms. The midge (GM, Orseolia oryzae, Diptera: Cecidomyiae) eats the juice of the growing point of rice in the larval stage, so that the heart leaves stop growing after the growing point is damaged, and the leaf sheath stretches into a tube shape, namely the 'standard onion' appears, so that the rice can not be spilt. It is statistically expected that the damage of the rice gall midge alone can cause annual yield losses of over $ 5.5 billion (Herdt 1991). The chemical pesticides usually used for the control of the rice gall midge can cause environmental pollution or new drug-resistant varieties. Planting varieties with resistance capability is known as an environment-friendly and economical pest management method. However, the rice gall midge resistance gene must first be determined and utilized in rice breeding programs.
To date, 11 anti-gall midge genes have been identified in different cultivars of rice, of which Gm1, Gm2, Gm3, Gm4, Gm6, Gm8 and Gm11(t) have been mapped to rice chromosomes using molecular markers, while the remaining 4 gall midge resistance genes (Gm5, Gm7, Gm9 and Gm10) have not been assigned to specific chromosomes and need further prediction and identification. At least four and seven biotypes have been reported in China and India, respectively, and the relationship between the disease resistance gene of the rice gall midge and the biotype has been primarily established. For example, Gm1, Gm2, Gm4, Gm5 and Gm6 all have resistance to indian biotypes 1, while Gm2 and Gm4 also confer resistance to other indian biotypes, and Gm6 also confers resistance to indian biotypes 1 as well as to four chinese biotypes. However, none of the identified genes in the current reports confer resistance to all of the gall midge biotypes, nor do none of the gall midge biotypes exhibit virulence against all known resistance genes. In conclusion, there is a complex relationship between the gall midge biotypes and resistance genes and it is suggested that the identification of more gall midge resistance genes is important for a broad spectrum of gall midge resistance.
However, due to the complexity of insect resistance identification, it is often difficult to efficiently introduce and aggregate different insect-resistant genes using conventional breeding approaches.
[ summary of the invention ]
In view of the above, there is a need to provide a molecular marker primer of rice gall midge-resistant major gene Gm5, a marking method and applications thereof, which can predict gall midge resistance of rice plants and accelerate breeding of gall midge-resistant rice varieties by detecting a molecular marker closely linked to a gall midge-resistant major gene locus.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a primer pair for molecular markers of a rice gall midge-resistant major gene Gm5 is characterized in that the primers are Z22595:
the sequence of the upstream primer is as follows: 5'-GGAGAGGCTGTAGAAGGTGTT-3', respectively;
the sequence of the downstream primer is as follows: 5'-GGATGAAAATTAAGCTCCTCACC-3' are provided.
The invention also provides application of the primer pair in breeding rice variety resisting against rice gall midge.
The invention also provides a method for carrying out molecular marking on the rice gall midge-resistant major gene Gm5 by applying the primer pair, which comprises the following steps: and carrying out PCR amplification on the DNA of the target material by using the primer pair Z22595, carrying out gel electrophoresis detection on an amplification product, and if a 229bp fragment can be amplified, indicating that the material contains the anti-gall midge major gene Gm 5.
The invention also provides a method for breeding a rice gall midge-resistant rice variety by applying the primer pair, which comprises the following steps: extracting total DNA of rice leaf, carrying out PCR amplification on the total DNA of the leaf by using the primer pair Z22595, carrying out gel electrophoresis detection on an amplification product, and carrying out surface treatment on the rice variety with the anti-gall midge major gene Gm5 if 229bp fragments can be amplified.
Further, the PCR amplified products were separated by 7% denaturing PAGE gel, and the amplified DNA bands were recorded by silver staining.
The invention also provides a method for screening the labeled primer, which comprises the following steps:
(1) ARC5984 is a material with high resistance to gall midge. Based on the positioning result of the early resistance major gene, the invention develops the SSR molecular marker which is closely linked with the resistance gene. In one aspect, a number of molecular markers are selected according to a relatively uniform genetic distance based on existing SSR and InDel molecular markers. On the other hand, based on the later positioning segment of the resistance gene, referring to the corresponding genome sequences of Nipponbare and 9311 of rice variety (line), STS markers are designed at the segments with difference for final fine positioning;
(2) the rice gall midge indica rice line 9311 is used as a female parent, the rice gall midge resistant variety ARC5984 is used as a male parent, a hybrid is prepared, and a 9311/ARC 5984F 2 separation population is constructed for molecular marker analysis; each F2 individual plant is selfed to obtain corresponding F 2:3 Family, used for insect resistance identification in seedling stage;
(3) extraction of parent ARC5984 and 9311 and F by CTAB method (Murray et al 1980) 2 Leaf genomic DNA of individual plants of the population. Polymorphism screening is carried out on the amphipathy through the alternative marker obtained in the step (1), PCR reaction is carried out on a BIOER amplification instrument, the amplification product is subjected to electrophoresis analysis on 7% polypropylene gel, and an SSR marker with polymorphism between parents is recorded and selected for subsequent analysis;
(4) the insect resistance identification is carried out by adopting the seedling stage insect inoculation, the rice gall midge source used in the experiment is from the Nanning paddy field, and the shallot seedlings are collected and then put on the insect-susceptible variety TN1 plant for propagation. Randomly inoculating the adult midge at the ratio of 1 head/row/16 plants in the period that the rice grows to two leaves (about 7 days), and when the insect-sensing rate of the control insect-sensing variety TN1 in the 1 row reaches more than 60 percent, the experimental data is valid, and the insect-sensing rates of all numbers are counted;
(5) according to F 2：3 And (3) respectively selecting leaf genome DNAs of 10 extreme insect-resistant single plants and 10 extreme insect-sensitive single plants to mix and construct an anti-sensitive DNA pool and a sensitive DNA pool according to the average insect-sensitive rate of the family. Meanwhile, primers with polymorphism between parents are utilized to respectively screen the resistant and sensitive DNA pools and obtain molecular markers with polymorphism between the resistant and sensitive pools, and the polymorphism markers indicate that the molecular markers are possibly linked with resistance traits. Then, according to the chromosome where the linkage marker is located, primers with polymorphism between parents on the chromosome are selected to amplify each individual plant of the F2 segregation population, and genotype data of the population is obtained. According to the linkage exchange rule, the software JoinMap 3.0 is used for constructing a partial genetic map of the rice from the group genotype data and obtaining the genetic distance of each molecular marker. Finally, binding F 2 The molecular marker genotype data of each individual plant of the population and the insect-susceptible rate of the corresponding cecidomyiia resistance identification are utilized to carry out QTL locus scanning on the target chromosome by utilizing a MapQTL 5 software composite interval mapping method；
(6) The resistance genes were finely mapped using the 9311/ARC5984 backcross generation. In the first round of fine mapping, 890 BC1F were identified 2 Screening 21 recombinants; next, 9320 BC1F are paired 2:3 The individuals were further resistance identified and rescreened with 2 markers (12M22.5 and RM28540) located in the region of interest and having polymorphisms between 9311 and ARC 5984. Genotypic and phenotypic analysis of recombinants narrowed the resistance-associated region to a fragment between markers 12M22.6 and Z75, which corresponds to a physical distance of 49kb in the Nipponbare genome.
The invention has the following beneficial effects:
1. according to the invention, the anti-gall midge major gene Gm5 in the rice variety ARC5984 is finely positioned by using molecular markers for the first time.
2. The main effective gene locus position detected by the molecular marker developed by the invention is definite and convenient to identify. By detecting the molecular marker closely linked with the gene locus, the resistance of the rice plant to the gall midge can be predicted, and the method is used for detecting the genotype of the rice variety or strain to judge whether the variety or strain has the gall midge resistance, so as to quickly screen insect-resistant varieties (strains), and the detection is convenient and quick and is not influenced by the environment.
3. The auxiliary breeding selection target is clear, and the cost is saved. In the traditional breeding method, parents with insect-resistant genes are collected and are subjected to a series of hybridization with cultivated varieties, and the rice varieties are subjected to the identification and selection of the rice gall midge-resistant characters, so that the operation is very complicated and is influenced by the environment. In addition, before insect-resistant identification, the insect sources are firstly obtained and bred to the rice gall midge population, and meanwhile, the inoculation of the insect sources and the seedling age of rice seedlings are relatively synchronous, which brings trouble to breeding work, and if the relationship among the insect sources, the seedlings and the environment cannot be effectively treated, the phenotypic identification result of the rice gall midge is low in reliability. Therefore, the traditional insect-resistant breeding is time-consuming, difficult and high in cost. According to the invention, by detecting the major gene locus of the anti-gall midge, a single plant with high resistance to gall midge can be identified in the seedling stage, and other plants are eliminated, so that the production cost is saved, the screening efficiency of the anti-gall midge rice is greatly improved, the breeding period of the anti-gall midge rice variety is greatly shortened, and the breeding efficiency is improved.
[ description of the drawings ]
FIG. 1 is a histogram of welsh onion ratios (PSS) in the F2 population;
FIG. 2 is a preliminary mapping of rice line ARC5984 against the rice gall midge major gene Gm 5;
FIG. 3 is a diagram of the molecular marker genotype and phenotype of the recombinant individuals used for fine mapping of Gm 5;
FIGS. 4-5 are agarose gel electrophoresis detection band patterns of the amplified products of the molecular marker primer (Z22595) in different rice materials; wherein, in FIG. 4, 1-17 represent the detection banding patterns of individual strains of the 9311 and ARC5984 hybrids; in FIG. 5, 1 to 10 show the detection banding patterns of individual strains after the 9311 and ARC5984 hybrids; 11-15 indicate the detection band types 570011, 9311, ARC5833, ARC5984, MH63, respectively. Wherein, Marker is BM2000 DNA Marker, and the size of the strip from top to bottom is as follows in proper order: 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp … …
[ detailed description ] embodiments
In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, embodiments accompanying figures are described in detail below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, but rather should be construed as broadly as the present invention is capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Example 1:
acquisition of molecular markers
First, F2 population construction and phenotypic identification
(1) Indica variety ARC5984 is highly resistant to the gall midge biotypes 1, 2 and 5 (Kumar et al 1998), while ARC5984 is highly resistant to the chinese biotypes 2 and 4 (Wu et al 2014). The insect-resistant variety is from the plant protection institute of Guangxi farm academy. F obtained after 9311/ARC5984 hybridization experiments 1 Individual reconstructionSelfing to obtain F 2 Progeny, each F 2 Selfing the single plant to obtain corresponding F 2：3 And (4) family selection of insect resistance. Insect resistance identification experiments showed that ARC5984 is highly resistant to the rice gall midge population from oryza sativa;
(2) inoculating the parent strain F with the seedling stage 2:3 And (5) carrying out insect resistance identification on the families. To ensure parental generation and F 2:3 Each family in the population grows consistently, and all the test materials are soaked for germination before sowing. Each 16 seeds/row of each line (variety) was sown in a tray 58X 38X 9cm long and contained in an iron tray 5cm thick of paddy field soil. Each tray was seeded with 2 replicates per material, with the parent and TN1 randomly seeded (sensory control). When the seedlings grow to the two-leaf stage, the adult rice gall midge is randomly inoculated into a seedling tray according to the proportion of 1 head/row/16 plants, and finally, a nylon gauze is covered. Standard onion rates of seedlings were evaluated 20-25 days after infestation by the rice gall midge (Heinrichs et al 1985) according to standard evaluation systems. Specifically, the proportion of white (flat and light) onions (relative to normal seedlings, oval and green) was directly observed and quantified. Seedlings with standard onions were considered sensitive to the gall midge. The standard onion ratio (PSS) of each line was evaluated and the frequency distribution plots are shown in FIG. 1, with higher PSS values indicating more seedling susceptibility and PSS<5%, 6-15%, 15-50% and>50% of the lines were considered to exhibit high resistance, moderate susceptibility and strong susceptibility, respectively. The PSS for each row was averaged with all test rows. This experiment was repeated three times.
II, F 2 Molecular marker analysis of populations
(1) Extraction of parent and F by CTAB method (Murray et al 1980) 2 Leaf genome DNA of each individual plant of the population;
(2) selecting a certain number of molecular markers according to the relatively uniform genetic distance according to the existing SSR and InDel markers. In addition, based on the last positioning segment of the gene, the genome sequences of the segment corresponding to the rice line (variety) 9311 and Nipponbare are compared, and InDel and STS markers are designed in the segment with difference between the two segments for the final fine positioning.
The reaction system comprises the following components:
the length of the amplification product of the SSR and STS primers used in the experiment is generally between 100-300bp, and the adopted PCR reaction program is as follows:
the conditions used for different primers may vary, mainly depending on the annealing temperature, and sometimes it is necessary to adjust the annealing temperature for each pair of primers, and the specific temperature can be set by referring to the values given by the primer design software.
The amplified products were separated on a 7% denaturing PAGE gel and the amplified DNA bands were recorded by silver staining (Zhu et al 2004). Primers with polymorphism between parents in F 2 Analyzing in the population to obtain the genotype data of the population.
(3) According to the insect-susceptible rate of the F2 individual plant, leaf genome DNAs of 10 extreme insect-resistant individual plants and 10 extreme insect-susceptible individual plants are respectively selected and mixed to construct an anti-susceptible pool and a susceptible pool. Meanwhile, primers with polymorphism among parents are utilized to respectively screen a resistant DNA pool and obtain molecular markers with polymorphism, and the polymorphic markers indicate that the polymorphic markers are possibly linked with resistance. Then, based on the chromosome where the linkage marker is located, primers with polymorphisms between parents on the chromosome are selected to screen each individual of the F2 segregating population, and the PCR procedure is the same as above, to obtain population genotype data. According to the linkage exchange rule, the software JoinMap 3.0 is used for constructing a partial genetic map of the rice from the group genotype data and obtaining the genetic distance of each molecular marker. Finally, combining the molecular marker genotype data of each individual plant of the F2 population and the corresponding insect-resistant level identified by the resistance of the gall midge, and carrying out QTL locus scanning on the target chromosome by utilizing a MapQTL 5 software composite interval mapping method. As shown in fig. 2, Gm5 was initially located in the region between 12M22.6 and RM28540 by QTL locus scanning of the genotype and phenotype of 126 individual molecular markers of the F2 population, with molecular markers on the x-axis and LOD scores on the y-axis.
Thirdly, screening 9311/ARC 5984F by using molecular marker 3 Recombinant single-plant fine positioning gene Gm5
According to the positioning result of QTL, the molecular markers 12M22.6 and RM28540 on both sides of the primary positioning are used for screening F 3 And (5) obtaining a single plant with recombination between two markers. And simultaneously, screening more primers with polymorphism among parents in a primary positioning segment, combining the genotype and the phenotype of the recombinant single strain, and inspecting the condition of co-segregation of the marker and the resistance phenotype. As shown in fig. 3, black, white and gray columns represent ARC5984, 9311 and heterozygous marker genotypes, respectively; l1 to L14 represent recombinant individuals; n represents the total number of recombinant individuals selected. Gm5 was located in the 49kb region between Z57 and Z64, and molecular marker Z22595 was tightly linked to the resistance gene.
Example 2:
validation of molecular markers
1. Materials and methods
(1) Material
Negative varieties: the susceptible strain (species) 9311 and MH63 were conventional rice materials stored in the laboratory.
Positive variety: highly insect-resistant strains (species) ARC5984, 570011 and ARC5833, were conventional rice material kept in this laboratory.
The ARC 5984X 9311 cross combines 28 progeny.
Molecular marker primer: z22595
(2) Method for producing a composite material
The genomic DNA of the leaf of the rice sample was extracted by CTAB extraction, the sample DNA was amplified with primer Z22595, and the amplified product was separated by 3% agarose gel electrophoresis (same procedure as in example 1).
2. As a result:
the genomic DNAs of 33 different samples of rice lines 570011, 9311, ARC5984, etc. were PCR-amplified as described above. As shown in FIGS. 4 to 5, in FIG. 4, 1 to 17 indicate the detection bands of the individual strains after the 9311 and ARC5984 hybrids; in FIG. 5, 1 to 10 show the detection banding patterns of individual strains after the 9311 and ARC5984 hybrids; 11-15 indicate the detection band patterns of 570011, 9311, ARC5833, ARC5984, MH63, respectively; the results show that 229bp fragments consistent with the ARC5984 banding pattern were amplified in all positive samples, while fragments consistent with the ARC5984 banding pattern size were not amplified in all negative samples. Therefore, the molecular marking method provided by the invention can accurately screen out the sample containing the major gene of the anti-gall midge, thereby greatly improving the selection efficiency of the anti-insect rice material.
In conclusion, the molecular marker Z22595 can effectively detect whether the major resistance gene locus is contained in the insect-resistant variety ARC5984 and derivatives thereof, greatly improve the selection efficiency of the rice plant resistant to the gall midge and obtain the rice variety (line) resistant to the gall midge containing Gm 5.
The above examples are merely illustrative of several embodiments of the present invention, and the description thereof is more specific and detailed, but not to be construed as limiting the scope of the invention.
Sequence listing
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Claims (5)
1. The primer pair for the rice gall midge-resistant major gene Gm5 molecular marker is characterized in that the primer pair is Z22595:
the sequence of the upstream primer is as follows: 5'-GGAGAGGCTGTAGAAGGTGTT-3', respectively;
the sequence of the downstream primer is as follows: 5'-GGATGAAAATTAAGCTCCTCACC-3' are provided.
2. Use of a primer pair according to claim 1 for breeding a rice variety resistant to gall midge.
3. A method for carrying out molecular marking on a rice gall midge-resistant major gene Gm5 by using a primer pair as described in claim 1, which is characterized by comprising the following steps: and carrying out PCR amplification on the DNA of the target material by using the primer pair Z22595, carrying out gel electrophoresis detection on an amplification product, and if a 229bp fragment can be amplified, indicating that the material contains the anti-gall midge major gene Gm 5.
4. A method for breeding a rice variety resistant to gall midge by using the primer pair of claim 1, wherein the method comprises: extracting total DNA of rice leaf, carrying out PCR amplification on the total DNA of the leaf by using the primer pair Z22595, carrying out gel electrophoresis detection on an amplification product, and carrying out surface treatment on the rice variety with the anti-gall midge major gene Gm5 if 229bp fragments can be amplified.
5. The method of claim 3, wherein the PCR amplified product is separated by 7% denaturing PAGE gel and the amplified DNA bands are recorded by silver staining.
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