CN113528700B - KASP molecular marker kit for detecting wheat ear germination resistance, detection method and application - Google Patents
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Abstract
The invention provides a KASP molecular marker kit for detecting wheat ear germination resistance, a detection method and application, and relates to the technical field of wheat resistance breeding. The invention proves that the wsnp _ Ex _ c6590_11419735 marker is related to wheat ear germination resistance, the marker can be developed into a rapid detection molecular marker, such as KASP marker, allele of 2 ear germination resistance differences can be effectively distinguished by utilizing the marker, wherein the ear germination rate of A allele species is 2.43-52.38% lower than that of G allele, and the A allele is favorable allele of ear germination resistance, so the marker can be used for wheat ear germination resistance molecular marker assisted breeding to promote the ear germination resistance variety breeding work.
Description
Technical Field
The invention belongs to the technical field of wheat resistance breeding, and particularly relates to a KASP molecular marker kit for detecting wheat ear germination resistance, a detection method and application.
Background
Wheat head germination is one of global climate disasters, not only reduces the wheat yield and ensures that wheat grains are easy to mildew, but also seriously degrades the wheat quality and seed value and brings serious losses to farmers and agricultural production and processing. The wheat head germination causes a series of biochemical reactions to occur inside the grains, particularly the enzyme activities of carbohydrate degrading enzymes, proteolytic enzymes (such as alpha-amylase) and the like are increased, and the storage substances in the embryo and the endosperm are degraded, so that the protein content of the wheat grains is reduced, the gluten strength is reduced, the yield and the volume weight of the wheat grains are reduced, and the processing quality of the flour is deteriorated. Therefore, how to control the ear sprouting in the wheat mature period and further obtain the ear sprouting resistant variety with excellent properties becomes an important subject for improving the wheat variety in the world, but at present, the research on the wheat ear sprouting resistance related gene is less, the molecular marker of the ear sprouting resistance related gene is utilized, the breeding work of the wheat ear sprouting resistant variety can be accelerated, the ear sprouting disaster can be effectively responded, and the important significance is realized on ensuring the safety and stable yield of wheat grains.
Disclosure of Invention
In view of the above, the present invention aims to provide a KASP molecular marker kit for detecting wheat ear germination resistance, which can be directly used for genotype identification and breeding of wheat ear germination resistance, a detection method and applications thereof.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides application of a primer group for identifying a wsnp _ Ex _ c6590_11419735 marker in preparation of a molecular marker kit for detecting wheat head germination resistance.
Preferably, the molecular marker kit comprises a KASP molecular marker kit.
The invention also provides a KASP molecular marker kit for detecting wheat ear germination resistance, which comprises a specific primer group for identifying the wsnp _ Ex _ c6590_11419735 marker.
Preferably, the specific primer group comprises an upstream primer 1, an upstream primer 2 and a downstream primer, wherein the nucleotide sequence of the upstream primer 1 is shown as SEQ ID No.1, the nucleotide sequence of the upstream primer 2 is shown as SEQ ID No.2, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 3.
The invention also provides a method for detecting wheat ear germination resistance, which comprises the steps of preparing a reaction system by utilizing the wheat genome DNA and the KASP molecular marker kit, and carrying out PCR reaction;
and typing the wheat genome DNA according to the amplification result, wherein the A allele is the favorable allele of the pre-harvest sprouting resistance, and the G allele is the unfavorable allele of the pre-harvest sprouting resistance.
Preferably, the reaction system is 10 μ L and comprises: FLU-ARMS 2 x mix 5 u L, upstream primer 1 and upstream primer 2 each 0.1 u L, downstream primer 0.3 u L, wheat genome DNA 1 u L and the balance of sterile water.
Preferably, the procedure of the PCR reaction comprises a pre-denaturation and a touchdown procedure;
the temperature of the pre-denaturation is 95 ℃, and the time is 10 min;
the landing procedure was first performed for 10 cycles, each cycle comprising: denaturation at 95 ℃ for 15s, annealing at 61-55 ℃ for 1min, and decreasing the annealing temperature by 0.6 ℃ per cycle; then, denaturation is carried out for 15s at 95 ℃, extension is carried out for 1min at 55 ℃, 27-35 cycles are carried out, and operation is carried out for 1min at 30 ℃.
The invention also provides application of the KASP molecular marker kit in wheat ear germination resistance molecular marker assisted breeding.
The invention also provides a breeding method of the wheat ear germination resistance variety, which comprises the following steps: the KASP molecular marker kit is utilized to carry out genotyping on wheat parents or breeding offspring and screen varieties or strains of A alleles.
Has the advantages that: the invention provides application of a primer group for identifying a wsnp _ Ex _ c6590_11419735 marker in developing a molecular marker kit for detecting wheat ear germination resistance, wherein T test analysis is carried out on genotype data of the wsnp _ Ex _ c6590_11419735 marker and wheat ear germination character identification data, and the result shows that the average value of the ear germination rate of an A allele is obviously lower than that of a G allele except 2016, which indicates that the A allele is favorable for ear germination resistance, and the ear germination rate can be reduced by 2.43-52.38%. The invention proves that the wsnp _ Ex _ c6590_11419735 marker is related to wheat ear germination resistance, the marker can be developed into a rapid detection molecular marker, such as KASP marker, the marker can be used for effectively distinguishing alleles of 2 ear germination resistance differences, wherein the A allele is favorable allele of ear germination resistance, and therefore, the marker can be used for wheat ear germination resistance molecular marker assisted breeding to promote the breeding work of ear germination resistance varieties.
Drawings
FIG. 1 is a wheat ear germination experiment, which comprises the steps of collecting and preserving wheat ears, starting the wheat ear germination experiment and ending the wheat ear germination experiment in sequence from left to right;
FIG. 2 shows the results of the KASP molecular marker typing where Allle 1 is the A Allele and Allle 2 is the G Allele.
Detailed Description
The invention provides application of a primer group for identifying a wsnp _ Ex _ c6590_11419735 marker in preparation of a molecular marker kit for detecting wheat head germination resistance.
The invention adopts a wheat whole ear germination experiment to perform 5-year ear germination resistance phenotype identification on a natural population consisting of 240 parts of common wheat, combines 90k chip genotype data of the population, and utilizes Tassel and Gapit software to perform whole gene association analysis on ear germination resistance, and the result shows that 39 significant SNP markers related to the ear germination resistance are obtained, and the total number of the significant SNP markers is 25 QTLs, wherein QPhs.hbaas-7BS (wheat 7B chromosome 40.1-54.6Mb region) can explain 1.9-5.4% of ear germination resistance variation, the QTLs contain 4 significant SNP markers, and wsnp _ Ex _ c6590_11419735 marker is one of the significant SNP markers; meanwhile, Qgr.cas-7BS.1 also contains a wsnp _ Ex _ c6590_11419735 marker (Zuo J, Lin CT, Cao H, et al.2019.genome-wide association study and quantitative trait locus mapping of seed and normal in common where L. (Triticum aestivum L.). Planta250(1): 187) 198.), the genotype data of the wsnp _ Ex _ c6590_11419735 marker and the identification data of the germination trait are subjected to T test analysis, and the average value of the germination rate of the A allele is remarkably lower than that of the G allele except 2016 year, which indicates that the A allele is favorable for the germination resistance, the germination rate can be reduced by 2.43-46.25%, and the npe _ Ex _ c6590_11419735 marker and the related ear germination resistance marker can be developed and used for rapid identification of the germination resistance marker. The molecular marker kit of the present invention preferably comprises a KASP molecular marker kit.
The invention also provides a KASP molecular marker kit for detecting wheat ear germination resistance, which comprises a specific primer group for identifying the wsnp _ Ex _ c6590_11419735 marker.
The specific primer group preferably comprises an upstream primer 1, an upstream primer 2 and a downstream primer, wherein the nucleotide sequence of the upstream primer 1 is shown as SEQ ID NO. 1: 5'-GAAGGTGACCAAGTTCATGCTAAACGGTGTCGGGCCTTCA-3', the nucleotide sequence of the upstream primer 2 is shown in SEQ ID NO. 2: 5'-GAAGGTCGGAGTCAACGGATTAAACGGTGTCGGGCCTTCG-3', the nucleotide sequence of the downstream primer is shown in SEQ ID NO. 3: 5'-AAGAGGGGGTCCGATACAGTTATC-3' are provided.
The KASP molecular marker kit of the invention preferably also comprises other reagents for preparing a reaction system, such as FLU-ARMS 2 x mix and sterile water. The source of the FLU-ARMS 2 × mix in the present invention is not particularly limited, and is preferably obtained from Videgene (accession No. Cat. BGH 1001RV3).
The invention also provides a method for detecting wheat ear germination resistance, which comprises the steps of preparing a reaction system by utilizing the wheat genome DNA and the KASP molecular marker kit, and carrying out PCR reaction;
and typing the wheat genome DNA according to the amplification result, wherein the A allele is the favorable allele of the pre-harvest sprouting resistance, and the G allele is the unfavorable allele of the pre-harvest sprouting resistance.
The reaction system of the present invention preferably includes, in an amount of 10. mu.L: FLU-ARMS 2 x mix 5 u L, upstream primer 1 and upstream primer 2 each 0.1 u L, downstream primer 0.3 u L, wheat genome DNA 1 u L and the balance of sterile water. The working concentration of the upstream primer 1 and the upstream primer 2 is preferably 0.1 mu mol/L, the working concentration of the downstream primer is preferably 0.3 mu mol/L, and the concentration of the wheat genome DNA is preferably 2.5-25 ng/mu L.
The procedure of the PCR reaction according to the invention preferably comprises a pre-denaturation and descent procedure;
the temperature of the pre-denaturation is 95 ℃, and the time is 10 min;
the landing procedure was first performed for 10 cycles, each cycle comprising: denaturation at 95 ℃ for 15s, annealing at 61-55 ℃ for 1min, and decreasing the annealing temperature by 0.6 ℃ per cycle; then, denaturation is carried out for 15s at 95 ℃, extension is carried out for 1min at 55 ℃, 27-35 cycles are carried out, and operation is carried out for 1min at 30 ℃. The method checks the parting result when the wheat head operates for 1min at 30 ℃, and judges the germination resistance of the wheat head according to the parting result.
The invention also provides application of the KASP molecular marker kit in wheat ear germination resistance molecular marker assisted breeding.
The invention also provides a breeding method of the wheat ear germination resistance variety, which comprises the following steps: the KASP molecular marker kit is utilized to carry out genotyping on wheat parents or breeding offspring and screen varieties or strains of A alleles.
In the process of the breeding method, the screening method preferably comprises the steps of preparing a reaction system by using the KASP molecular marker kit and the wheat genome DNA, carrying out PCR reaction, and typing the wheat genome DNA according to the amplification result, wherein the reaction system and the PCR reaction are preferably the same as those described above, and are not repeated herein.
The following examples are provided to describe in detail the KASP molecular marker kit for detecting wheat ear germination resistance, the detection method and the application of the kit, but they should not be construed as limiting the scope of the present invention.
Example 1
The method comprises the steps of performing 5-year ear sprouting resistance phenotype identification on a natural population consisting of 240 parts of common wheat by adopting a wheat whole ear sprouting experiment (the wheat material information is shown in table 1), combining 90k chip genotype data of the population, and performing whole gene association analysis on ear sprouting resistance by using Tassel and Gapit software, wherein the results show that 39 significant SNP markers related to the ear sprouting resistance are obtained, and the total number of 25 QTLs is shown, wherein QPhs.hbaas-7BS (wheat 7B chromosome 40.1-54.6Mb region) can explain 1.9-5.4% of ear sprouting resistance variation (table 2), the QTLs contain 4 significant SNP markers, and wsnp _ Ex _ c6590_11419735 markers are one of the markers.
The method for identifying the germination resistance phenotype of the ear of grain comprises the following steps:
collecting and storing materials: after the wheat material is physiologically mature (i.e. the ear and leaves are faded and yellow), 10 ears (containing 10cm of lower ear) of disease-free and defect-free wheat are collected, naturally dried indoors for 7d and stored in a freezer at the temperature of 20 ℃ below zero for later use, so as to avoid damaging the dormancy characteristic of wheat grains and influencing the experimental result.
Drying before germination: the wheat ears stored in a freezer at-20 ℃ are taken out 2 days ahead of time, inserted into small holes (the hole diameter is 0.6cm, the hole distance is 1.6cm) on a stainless steel tray, dried at room temperature for 2 days, and then moved into a greenhouse for ear germination experiments.
Germination experiment: the germination of the whole ear is carried out in a greenhouse by adopting automatic rainfall simulation equipment, the automatic rainfall simulation equipment sprays for 20min every 4h, the temperature is set to be 20 ℃, after continuous treatment is carried out for 5d, the germination ears are independently packaged, and are stored in a freezer at the temperature of-20 ℃ for manual counting, and the result is shown in figure 1.
And (3) germination standard: and counting the number of germinated grains and ungerminated grains by artificial threshing, and recording the length of the seeds with the bud length exceeding 1/2 as germination.
The germination rate was 10 germinated grains × 100%/(number of germinated grains of 10 ears + number of ungerminated grains of 10 ears).
Development of KASP molecular markers: according to the physical position information marked by wsnp _ Ex _ c6590_11419735, 100bp gene sequences upstream and downstream of the site are obtained from an Ensembl database for designing primers, and a Marchant-Yoshiyama (Wuhan) biological company is entrusted to synthesize specific primers: SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3.
KASP reaction conditions
The PCR reaction system is as follows: FLU-ARMS 2 Xmix (product number Cat. BGH1001RV3, product of Videgene corporation) 5. mu.L, 0.1. mu.L each of the forward primer 1 and the primer 2, 0.3. mu.L of the reverse primer, 1. mu.L of the DNA template, and sterile water was added to a total volume of 10. mu.L.
The PCR program was pre-denatured at 95 ℃ for 10min, followed by a touchdown program for 10 cycles: denaturation at 95 ℃ for 15s, extension at 61-55 ℃ for 1min (decreasing at 0.6 ℃ per cycle), followed by 27 cycles: denaturation at 95 ℃ for 15s, extension at 55 ℃ for 1min, final operation at 30 ℃ for 1min for fluorescence detection, examination of genotyping results, appropriate increase in circulation according to the genotyping results (denaturation at 95 ℃ for 15s, extension at 55 ℃ for 1min), and final operation at 30 ℃ for 1min for examination of the genotyping results.
TABLE 1240 wheat Material ear sprouting phenotype data and wsnp _ Ex _ c6590_11419735 labelling results
Note: "-" indicates a data miss.
TABLE 2 QPhs, hbaas-7BS related information
T test analysis is carried out on the genotype data marked by wsnp _ Ex _ c6590_11419735 and the identification data of the ear sprouting traits (Table 3), and the results show that the average value of the ear sprouting rate of the A allele is obviously lower than that of the G allele except 2016 years, which indicates that the A allele is the favorable allele of the ear sprouting resistance, and the ear sprouting rate can be reduced by 2.43-46.25%.
TABLE 3240 comparison of the sprouting averages of different alleles between Material years
Note:**indicating a significant difference at the 0.01 level.
In view of the above analysis, which shows that the wsnp _ Ex _ c6590_11419735 marker is associated with resistance to ear sprouting, the marker can be developed as a rapid detection molecular marker, such as KASP marker, for identification of ear sprouting resistance genotypes and breeding efforts. The development and utilization of related molecular markers are not found at present.
Example 2
222 parts of wheat population material (Table 4) were used, comprising 120 parts of cultivar and 102 parts of local cultivar. The population is sown in a testing field of a grain crop institute of agricultural academy of sciences of Hubei province in 2017 and 2019, is managed in a conventional field, and is harvested in 2018 and 2020.
TABLE 4222 wheat material ear sprouting phenotype data and KASP primer detection results
Note: in the serial numbers, A is a cultivated variety, and B is a local variety;
"-" indicates a data miss.
The same phenotype identification of ear germination resistance as in example 1 was performed, and some results are shown in fig. 2, and the molecular marker test results are shown in table 5, wherein the average ear germination rate of the material containing the a allele is significantly lower than that of the material containing the G allele, the reduction range of the average ear germination rate is 34.86-52.38% between different years, which indicates that the a allele is a favorable allele for ear germination resistance, and the results confirm that the KASP molecular marker can be used for identifying ear germination resistance.
TABLE 5222 comparison of sprouting averages of different alleles of panicles between different years for materials
Note:*indicating a significant difference at the 0.05 level,**indicating a significant difference at the 0.01 level.
Analysis of the frequency of distribution of both alleles in cultivars and cultivars (Table 6) revealed that the ear germination resistance favorable allele A accounted for 45.05% and was slightly lower than the G allele in the population. The A allele accounts for 60.78% in the local variety and is higher than 31.67% in the cultivated variety, which indicates that the A allele accounts for high ratio in the local variety, while the A allele accounts for the increased position in the cultivated variety to improve the germination resistance of the variety.
TABLE 6 allelic distribution
In conclusion, the marker can be used to effectively distinguish the alleles of 2 differences in ear sprouting resistance, wherein the A allele is the favorable allele of ear sprouting resistance. The marker can be used for wheat ear germination resistance molecular marker assisted breeding and promoting ear germination resistance variety breeding work.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
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<110> institute of food crops of academy of agricultural sciences of Hubei province
<120> KASP molecular marker kit for detecting wheat ear germination resistance, detection method and application
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Application publication date: 20211022 Assignee: Hubei huichuzhi Biotechnology Co.,Ltd. Assignor: HUBEI ACADEMY OF AGRICULTURAL SCIENCES INSTITUTE OF FOOD CROPS Contract record no.: X2022420000059 Denomination of invention: Kasp molecular marker kit for detecting wheat ear germination resistance and its detection method and Application Granted publication date: 20220517 License type: Common License Record date: 20220714 |