CN115948599B - InDel molecular marker of RAD17 gene, detection primer and application thereof - Google Patents
InDel molecular marker of RAD17 gene, detection primer and application thereof Download PDFInfo
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- CN115948599B CN115948599B CN202211728104.5A CN202211728104A CN115948599B CN 115948599 B CN115948599 B CN 115948599B CN 202211728104 A CN202211728104 A CN 202211728104A CN 115948599 B CN115948599 B CN 115948599B
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Abstract
The application belongs to the technical field of rape breeding, and particularly relates to an InDel molecular marker of a RAD17 gene, a detection primer and application thereof. The InDel molecular marker is positioned on a first exon of the RAD17 gene, the InDel molecular marker comprises 5 sections of insertion sequences, and the 5 sections of insertion sequences are respectively positioned at 9663297bp, 9663298bp, 9663300bp, 9663303bp and 9663304bp of a C09 chromosome. The application develops an InDel molecular marker based on natural brassica napus and artificially synthesized brassica napus RAD17 gene mutation sites, which is stably and tightly linked with a genome, and the stability of the offspring genome of the artificially synthesized brassica napus can be deduced by detecting whether the molecular marker exists in the genome of the brassica napus, so that the germplasm innovation efficiency of the brassica napus is effectively improved and the breeding process of the brassica napus is accelerated.
Description
Technical Field
The application relates to the technical field of rape breeding, in particular to an InDel molecular marker of a RAD17 gene, and a detection primer and application thereof.
Background
Rape is a collective term for annual or perennial plants of the Brassica (Brassica) family for the purpose of extracting seed oil, is one of the most widely distributed important oil crops in the world with a long history of planting, and is also the fifth largest crop following rice, wheat, corn and soybean. At present, the rape cultivars mainly comprise the following three types: brassica napus (Brassica napus), brassica napus (Brassica rapa) and Brassica juncea (Brassica juncea). The cabbage type rape has higher yield and stronger disease resistance compared with cabbage type rape and the like, and is the main cultivated rape type in China.
Brassica napus (AACC, 2n=4x=38) is formed by natural doubling after interspecific crossing of two diploid ancestor species of chinese cabbage (AA, 2n=2x=20) and cabbage (CC, 2n=2x=18). Although its history of evolution is short, it exhibits diploid meiosis, during which normally ordered chromosomes are guaranteed for fertility. This suggests that this "young" genome evolves rapidly and that the genome has reached stability. However, for the artificially newly synthesized brassica napus (usually by crossing cabbage and artificially doubling), the phenomenon of chromosomal rearrangement and homologous chromosomal recombination frequently occurs in the meiosis process, which is called "genome shake", and is specifically manifested as abnormal pollen development and the like, thereby seriously affecting the normal growth and development thereof. The abundant variation generated by 'genome vibration' in the artificial synthesized brassica napus A, C genome evolution process can be utilized to screen out excellent genes and molecular markers for controlling related characters, and the method is applied to germplasm innovation of the brassica napus. The method has important significance for accelerating the genome stabilization after the formation of the brassica allopolyploid so as to improve the breeding success rate and the rape quality by exploring the reason that the genome is obtained from artificial synthesis to natural brassica napus.
Disclosure of Invention
Aiming at the problems existing in the prior art, the application provides an InDel molecular marker of a RAD17 gene, a detection primer for detecting the InDel molecular marker and application of the detection primer in rape breeding.
The application is realized by the following technical scheme:
the first aspect of the application provides an InDel molecular marker of a RAD17 gene, wherein the InDel molecular marker is positioned on a first exon of the RAD17 gene, the InDel molecular marker comprises 5 sections of insertion sequences, the 5 sections of insertion sequences are respectively positioned at 9663297bp, 9663298bp, 9663300bp, 9663303bp and 9663304bp of a C09 chromosome of wild cabbage W03, and the nucleotide sequences are TTCGGAGAAG, TATAG, TCCCC, TGTATCGTATCGTTCCCCCTTGCTCGATTTCTTTTTTTTTCTTTAATTGAATTTCGCA and TACACTACTAAAACCCCACCC respectively.
In a second aspect, the application provides a detection primer for detecting an InDel molecular marker of a RAD17 gene, wherein the detection primer comprises an upstream primer and a downstream primer, and the nucleotide sequence of the upstream primer is shown in SEQ ID NO:4, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: shown at 5.
In a third aspect the application provides a kit for detecting an InDel molecular marker of a RAD17 gene, the kit comprising a detection primer as described above for detecting an InDel molecular marker of a RAD17 gene.
Further, the kit also comprises Green Taq Mix.
The fourth aspect of the application provides a kit for detecting the InDel molecular marker of the RAD17 gene or application of the detection primer for detecting the InDel molecular marker of the RAD17 gene in rape breeding.
Further, the use includes use in amplifying and/or detecting the genotype of the rape RAD17 gene or predicting and/or identifying the genomic stability of rape.
Further, the application comprises the steps of:
s1, extracting genome DNA of rape to be detected;
s2, using the sequence shown in SEQ ID NO:4-5, performing PCR amplification by using a detection primer for detecting the InDel molecular marker of the RAD17 gene;
s3, judging whether the rape genome is stable according to the size of the PCR amplification product, wherein the rape genome is stable when the length of the PCR amplification product is 305bp, and the rape genome is unstable when the length of the PCR amplification product is 209 bp.
Further, in step S1, genomic DNA is extracted by a CTAB method.
Further, in step S2, the reaction system for PCR amplification includes: 9.5 mu.L ddH 2 O, 12.5. Mu.L Green Taq Mix, 1. Mu.L of 10. Mu.M upstream primer, 1. Mu.L of 10. Mu.M downstream primer and 1. Mu.L of genomic DNA.
Further, in step S2, the reaction procedure of PCR amplification includes: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 58℃for 30s, extension at 72℃for 15s,35 cycles; extending at 72℃for 7min.
The application has the advantages and positive effects that:
the application develops an InDel molecular marker based on natural brassica napus and artificially synthesized brassica napus RAD17 gene mutation sites, and specifically comprises 5 sections of insertion sequences which are stably and tightly linked with genome, and the stability of genome of the offspring of the artificially synthesized brassica napus can be deduced by detecting whether the InDel molecular marker is present in the offspring of the artificially synthesized brassica napus and the natural brassica napus or not, if the RAD17 gene is detected to be present in the genome of the offspring, the 5 sections of insertion sequences are proved to be stable in the genome of the individual plants; if the RAD17 gene is detected in the offspring genome to be haplotype 1, namely the 5 sections of insertion sequences are deleted, the genome of the individual plants is not stable, and therefore whether the artificially synthesized brassica napus genome is stable or not can be rapidly judged through conventional means such as PCR amplification and the like, so that the germplasm innovation efficiency of the brassica napus is effectively improved, and the breeding process of the brassica napus is accelerated.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for the description of the embodiments will be briefly described below, and it is apparent that the drawings in the following description are only some embodiments of the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a diagram of InDel molecular marker locus information in the RAD17 gene according to an embodiment of the present application;
FIG. 2 is a diagram showing the result of agarose gel electrophoresis of PCR amplification products according to an embodiment of the present application, wherein "M" in the diagrams a-c represents DNA ladder marker, "W03" is wild cabbage W03, "HC-1" and "HC-Q" are artificially synthesized cabbage type rape, "ZS11", "Westar", "Tapidor" are naturally occurring cabbage type rape, "4" - "6" in the diagram a and "7" - "42" in the diagram b are naturally occurring cabbage type rape, "1" - "36" in the diagram c are naturally occurring cabbage type rape and artificially synthesized cabbage type rapeRape hybrid F 6 A population.
Detailed Description
The present application will be described in further detail with reference to the following examples, in which the apparatus and reagents used in the respective examples and test examples are commercially available unless otherwise specified, in order to make the objects, technical schemes and advantages of the present application more apparent. The specific embodiments described herein are to be considered in an illustrative sense only and are not intended to limit the application.
Various modifications to the precise description of the application will be readily apparent to those skilled in the art from the information contained herein without departing from the spirit or scope of the appended claims. It is to be understood that the scope of the application is not limited to the defined processes, properties or components, as these embodiments, as well as other descriptions, are merely illustrative of specific aspects of the application. Indeed, various modifications of the embodiments of the application which are obvious to those skilled in the art or related fields are intended to be within the scope of the following claims.
For a better understanding of the present application, and not to limit its scope, all numbers expressing quantities, percentages, and other values used in the present application are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. Each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
In order that the above-recited objects, features and advantages of the present application will become more readily apparent, a more particular description of the application briefly described above will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings.
RAD17 is a very important early-stage marker protein in response to DNA damage detection and replication pressure signal transduction, and plays an important role in telomere structure stabilization and meiosis cell cycle detection. Research shows that RAD17 gene plays important roles in maintaining chromosome stability in human cells and ensuring normal repair of meiosis double strand break of yeast cells, and the functional defects and abnormal expression of the RAD17 gene may be closely related to the occurrence and development of cell canceration and tumors. Furthermore, the RAD17 gene has a similar function in plants. For example, the atad 17 gene regulates DNA damage repair and homologous recombination in arabidopsis, and the OsRAD17 and ZmRAD17 genes play a role in meiotic double strand break repair in rice and maize, respectively. Normal expression of RAD17 is closely related to its involvement in DNA damage and replication monitoring to maintain cell genome stability, and its absence or low expression may lead to an increase in the mutation rate of cells. The research results provide a new idea for understanding genetic variation and genome stability, or are used for accelerating the genome stability of artificially synthesized polyploid brassica crops and promoting the formation of new germplasm resources.
Based on the above, the embodiment of the application provides an InDel molecular marker of a RAD17 gene, wherein the InDel molecular marker is positioned on a first exon of the RAD17 gene, the InDel molecular marker comprises 5 sections of insertion sequences, the 5 sections of insertion sequences are respectively positioned at 9663297bp, 9663298bp, 9663300bp, 9663303bp and 9663304bp of a C09 chromosome of wild cabbage W03, and the nucleotide sequences are TTCGGAGAAG, TATAG, TCCCC, TGTATCGTATCGTTCCCCCTTGCTCGATTTCTTTTTTTTTCTTTAATTGAATTTCGCA and TACACTACTAAAACCCCACCC respectively. Specifically, see FIG. 1, a TTCGGAGAAG (10 bp, see SEQ ID NO: 1) sequence is inserted at 9663297bp, a TATAG (5 bp) sequence is inserted at 9663298bp, a TCCC (5 bp) sequence is inserted at 9663300bp, a TGTATCGTATCGTTCCCCCTTGCTCGATTTCTTTTTTTTTCTTTAATTGAATTTCGCA (58 bp, see SEQ ID NO: 2) sequence is inserted at 9663303bp, and a TACACTACTAAAACCCCACCC (21 bp, see SEQ ID NO: 3) sequence is inserted at 9663304 bp.
According to the application, through comparing and analyzing the genome sequences of artificially synthesized brassica napus, wild brassica oleracea and natural brassica napus, 5 InDel molecular markers are screened on the first exon of the C09 chromosome RAD17 gene, and the polymorphism is a deletion/insertion sequence, and the two genotypes are deleted and inserted. The genome of wild cabbage W03 is taken as a reference genome, the unordered sequence difference of the wild cabbage type rape on the first exon of the C09 chromosome RAD17 gene is artificially synthesized (for convenience of description, the RAD17 gene is referred to as haplotype 1 in the wild cabbage type rape), and 5 sections of insertion sequences exist at corresponding positions in the natural cabbage type rape (for convenience of description, the RAD17 gene is referred to as haplotype 2 in the natural cabbage type rape). The genome of the natural brassica napus is usually stable, so that the 5-segment insertion sequence, namely the InDel molecular marker provided by the application, is stably and tightly linked with the genome of the brassica napus, and therefore, the stability of the genome of the offspring of the artificially synthesized brassica napus can be deduced by detecting the molecular marker provided by the application, and if the RAD17 gene is detected in the genome of the offspring, namely the haplotype 2 exists, the genome of the individual plants is stable; if the RAD17 gene is detected in the offspring genome as haplotype 1, i.e., the 5-piece insert sequence is deleted, it is indicated that the genomes of these individuals have not been stabilized.
The InDel molecular marker developed based on the RAD17 gene can be used for identifying the genetic recombination and the genome stability of a single plant genome in the hybrid offspring of the brassica napus, quickly judging whether the genome of the artificially synthesized brassica napus is stable or not, can be simply, quickly and high-flux applied to breeding practice, has the advantages of simplicity in typing and the like, and can effectively improve the germplasm innovation efficiency of the brassica napus and accelerate the breeding process of the brassica napus.
In yet another embodiment, the application provides a detection primer for detecting the InDel molecular marker of the RAD17 gene, wherein the detection primer comprises an upstream primer and a downstream primer, and the nucleotide sequence of the upstream primer is shown in SEQ ID NO:4, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: shown at 5.
According to the application, a pair of primers, namely an upstream primer CTCCCCATACCTCCCAATCG (SEQ ID NO: 4) and a downstream primer TACAACCGTCCGATCTGCAA (SEQ ID NO: 5), are developed by utilizing Indel molecular marker loci and upstream and downstream specific sequences in the RAD17 gene, and amplified products (different banding patterns) with different sizes can be obtained for different haplotypes by utilizing the primers for amplification, specifically, the banding pattern 1 (corresponding to the haplotype 1) is detected in an artificially synthesized brassica napus population, the amplified product is 209bp, the genome is unstable, the banding pattern 2 (corresponding to the haplotype 2) is detected in a natural brassica napus population, the amplified product is 305bp, and the genome is stable, so that whether the genome reaches a stable state or not can be judged according to the length of the amplified product, the brassica napus breeding efficiency of the genome stability is greatly improved, and the breeding process is accelerated.
The method for analyzing the length of the amplified product includes agarose gel electrophoresis, sequencing, etc., and the present application is not particularly limited thereto, and may be performed according to a well-known procedure in the art.
Yet another embodiment of the present application provides a kit for detecting InDel molecular markers of RAD17 gene, comprising the detection primer as described above.
The advantages of the kit for detecting the InDel molecular marker of the RAD17 gene compared with the prior art are the same as those of the detection primer for detecting the InDel molecular marker of the RAD17 gene compared with the prior art, and are not described in detail herein.
Optionally, the kit further comprises Green Taq Mix, which mainly comprises Taq DNA Polymerase, dntps and an optimized buffer system, purchased from nuezan biotechnology limited, under the trade designation: p131.
The embodiment of the application also provides a kit for detecting the InDel molecular marker of the RAD17 gene or application of the detection primer for detecting the InDel molecular marker of the RAD17 gene in rape breeding.
The advantages of the application over the prior art are the same as those of the kit for detecting the InDel molecular marker of the RAD17 gene or the detection primer for detecting the InDel molecular marker of the RAD17 gene as described above over the prior art, and are not described in detail herein.
In particular, the above-mentioned applications include applications in amplifying and/or detecting the genotype of the rape RAD17 gene or predicting and/or identifying the genomic stability of rape.
Specifically, the application comprises the steps of:
s1, extracting genome DNA of rape to be detected;
s2, using the sequence shown in SEQ ID NO:4-5, performing PCR amplification by using the detection primer;
s3, judging whether the rape genome is stable according to the size of the PCR amplification product, wherein the rape genome is stable when the length of the PCR amplification product is 305bp, and the rape genome is unstable when the length of the PCR amplification product is 209 bp.
The application utilizes the InDel molecular marker to identify the haplotype of a single plant in a group in the offspring of artificially synthesized brassica napus, the natural brassica napus amplification result shows a 305bp band, the InDel molecular marker is contained, the artificially synthesized brassica napus amplification result shows a 209bp band, the InDel molecular marker is deleted, and the hybridization offspring of the artificially synthesized brassica napus and the natural brassica napus shows three band types: 1) A 305bp band; 2) A 209bp band; 3) The 305bp and 209bp bands occur simultaneously. The PCR amplification result shows that the genome of the band 1 (the amplification product is 209 bp) is unstable, and the PCR amplification method can be further applied to the creation of new germplasm resources; the PCR result is band type 2 (the amplified product is 305 bp), which shows that the genome is stable, the PCR method can be further applied to genetic breeding, and the existence of two band types shows that the offspring can be separated in character. The detection method of the embodiment is simple and convenient to operate, easy to realize standardization, low in cost and wide in applicability.
Alternatively, in step S1, genomic DNA is extracted by CTAB method.
Optionally, in step S2, the reaction system for PCR amplification includes: 9.5 mu.L ddH 2 O, 12.5. Mu.L Green Taq Mix, 1. Mu.L 10. Mu.M upstream primer, 1. Mu.L 10. Mu.M downstream primer and 1. Mu.L genomic DNA.
Optionally, in step S2, the reaction procedure of PCR amplification includes: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 58℃for 30s, extension at 72℃for 15s,35 cycles; extending at 72℃for 7min.
The application will be further illustrated with reference to specific examples. The experimental methods, which do not address specific conditions in the following examples, are generally in accordance with the conditions recommended by the manufacturer.
1. InDel molecular marker locus identification
For 3 parts of artificially synthesized brassica napus, 295 parts of natural brassica napus, 8 natural brassica napus and artificially synthesized brassica napus hybrid F 6 The generation population (1389 parts total) was genotyped. The total of 1687 sample materials were planted for normal seeding at the institute of oil crop research, academy of agricultural sciences, china, and the Wuchang test base. Leaf tissue sampling was performed during the seedling stage and the samples were rapidly placed at-20℃for storage. The whole genome DNA of each material was extracted and sequenced using CTAB method.
With the genome of wild cabbage W03 as a reference genome, referring to FIG. 1, comparing genome analysis to find that the artificially synthesized cabbage rape has no difference compared with the sequence of wild cabbage W03 on the first exon of the RAD17 gene, the genotype of the RAD17 gene with no mutation site is called as haplotype 1, 4 typical natural cabbage rape are selected, the genome sequence of the 4 typical natural cabbage rape has 5 sections of insertion mutation at the same position, and the genotype is haplotype 2,5 sections of insertion sequences are respectively: the insert at 9663297bp is TTCGGAGAAG (10 bp, see SEQ ID NO: 1), the insert at 9663298bp is TATAG (5 bp), the insert at 9663300bp is TCCC (5 bp), the insert at 9663303bp is TGTATCGTATCGTTCCCCCTTGCTCGATTTCTTTTTTTTTCTTTAATTGAATTTCGCA (58 bp, see SEQ ID NO: 2) and the insert at 9663304bp is TACACTACTAAAACCCCACCC (21 bp, see SEQ ID NO: 3).
The haplotype molecular marker primer designed by using the five mutation sites can be used for RAD17 gene haplotype identification and genome stability analysis.
2. Detection primer design
According to the Primer design principle, a pair of detection primers for detecting the InDel molecular markers are designed at the upper and lower streams of the 5 InDel molecular markers by using a Primer 5, wherein the detection primers comprise an upstream Primer sequence and a downstream Primer sequence, and specifically comprise:
an upstream primer: CTCCCCATACCTCCCAATCG (see SEQ ID NO: 4);
a downstream primer: TACAACCGTCCGATCTGCAA (see SEQ ID NO: 5).
3. Verification of reliability of InDel molecular markers in populations
And (3) taking the whole genome DNA of the sample as a template, carrying out PCR amplification by using a detection primer for detecting InDel molecular markers, and carrying out agarose gel electrophoresis on PCR amplification products to detect and distinguish different allelic variations. For 3 parts of artificially synthesized brassica napus, 295 parts of natural brassica napus, 8 natural brassica napus and artificially synthesized brassica napus hybrid F 6 The generation population (1389 parts) was identified. The PCR amplification reaction system (total volume 25. Mu.L) included: 9.5 mu.L ddH 2 O, 12.5. Mu.L Green Taq Mix, 1. Mu.L 10. Mu.M upstream primer, 1. Mu.L 10. Mu.M downstream primer and 1. Mu.L genomic DNA; the reaction procedure included: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 58℃for 30s, extension at 72℃for 15s,35 cycles; extending at 72℃for 7min.
Agarose gel electrophoresis analysis is carried out on the amplification result, and the result is shown in figure 2; wherein "M" in the diagrams a, b and c represents DNA ladder marker, "W03" is wild cabbage W03, "HC-1" and "HC-Q" are artificially synthesized cabbage type rape, "ZS11", "Westar", "Tapidor" are natural cabbage type rape, "4" - "6" in the diagram a and "7" - "42" in the diagram b are natural cabbage type rape, "1" - "36" in the diagram c are natural cabbage type rape and artificially synthesized cabbage type rape hybrid F 6 A population.
The agarose gel electrophoresis result shows that the amplified products of wild cabbage and artificially synthesized cabbage type rape are 209bp in size, and present band type 1 (corresponding to haplotype 1, hap 1), the amplified products of natural cabbage type rape are 305bp in size, present band type 2 (corresponding to haplotype 2, hap 2), and the cabbage type rape hybridizes with artificially synthesized cabbage type rape F 6 The generation of the population has three types of bands 1, 2 and 1 and 2, and the agarose gel electrophoresis results are counted and shown in table 1.
TABLE 1 genotyping of canola plants using InDel molecular markers of RAD17 Gene
| Hap1 | Hap2 | Hap1+Hap2 | Total number of samples | |
| Artificially synthesized cabbage type rape | 3 | 0 | 0 | n＝3 |
| Natural cabbage type rape | 6 | 289 | 0 | n＝295 |
| [ Natural brassica napus 1X artificially synthesized brassica napus ]]F 6 | 1 | 7 | 1 | n＝9 |
| [ Natural cabbage type rape 2X artificially synthesized cabbage type rape ]]F 6 | 2 | 62 | 1 | n＝65 |
| [ Natural cabbage type rape 3X artificially synthesized cabbage type rape ]]F 6 | 2 | 218 | 0 | n＝220 |
| [ Natural cabbage type rape 4X artificially synthesized cabbage type rape ]]F 6 | 2 | 82 | 2 | n＝86 |
| [ Natural cabbage type rape 5X artificially synthesized cabbage type rape ]]F 6 | 1 | 140 | 1 | n＝142 |
| [ Natural cabbage type rape 6X artificially synthesized cabbage type rape ]]F 6 | 0 | 294 | 0 | n＝294 |
| [ Natural cabbage type rape 7X artificially synthesized cabbage type rape ]]F 6 | 28 | 116 | 15 | n＝159 |
| [ Natural cabbage type rape 8X artificially synthesized cabbage type rape ]]F 6 | 1 | 413 | 0 | n＝414 |
As shown in Table 1, 3 parts of artificially synthesized brassica napus are haplotype 1, the amplified products do not contain the InDel molecular markers provided by the application, 98% of the natural brassica napus are haplotype 2, 2% of the natural brassica napus are haplotype 1, most of the amplified products contain the InDel molecular markers provided by the application, and the genome of the natural brassica napus is stable, so that the haplotype 2 is stably and tightly linked with the genome of the brassica napus.
Further, by crossing F with natural brassica napus and artificial brassica napus 6 PCR amplification was performed in the generation population (1389 parts total) to infer genome stability of the offspring, 2.7% of which were haplotype 1, indicating that the genomes of these individuals were not stable; 95.9% are haplotype 2, indicating that the genomes of these individuals have reached stability; 1.4% contains both Hap1 and Hap2 (there are two bands on the result of electrophoresis), it is shown that such a single plant also has the phenomenon of offspring separation.
In conclusion, the InDel molecular marker developed based on the natural brassica napus and artificially synthesized brassica napus RAD17 gene mutation sites is stably and tightly linked with the genome, and whether the genome of the offspring of the artificially synthesized brassica napus and the natural brassica napus reaches stability can be presumed by detecting the haplotype of the offspring of the artificially synthesized brassica napus and the natural brassica napus, so that the germplasm innovation efficiency of the brassica napus is effectively improved, and the breeding process of the brassica napus is accelerated.
The foregoing description of the preferred embodiments of the application is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the application.
Claims (8)
1. A detection primer for detecting an InDel molecular marker of a RAD17 gene, wherein the detection primer comprises an upstream primer and a downstream primer, and the nucleotide sequence of the upstream primer is shown as SEQ ID NO:4, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: shown at 5.
2. A kit for detecting an InDel molecular marker of a RAD17 gene, comprising the detection primer for detecting an InDel molecular marker of a RAD17 gene according to claim 1.
3. The kit for detecting InDel molecular markers of RAD17 gene according to claim 2, further comprising Green Taq Mix.
4. Use of a detection primer for detecting InDel molecular markers of RAD17 gene according to claim 1 in brassica napus breeding, wherein said use comprises the use in detecting the genotype of brassica napus RAD17 gene or in predicting or identifying the genomic stability of brassica napus.
5. The use of a detection primer for detecting InDel molecular markers of RAD17 gene according to claim 4 in breeding of brassica napus, comprising the steps of:
s1, extracting genome DNA of brassica napus to be detected;
s2, using the sequence shown in SEQ ID NO:4-5, performing PCR amplification by using a detection primer for detecting the InDel molecular marker of the RAD17 gene;
s3, judging whether the genome of the brassica napus is stable according to the size of the PCR amplification product, wherein the genome of the brassica napus is stable when the length of the PCR amplification product is 305bp, and the genome of the brassica napus is unstable when the length of the PCR amplification product is 209 bp.
6. The use of the detection primer for detecting the InDel molecular marker of RAD17 gene according to claim 5 in cabbage type rape breeding, wherein in step S1, genomic DNA is extracted by a CTAB method.
7. The use of the detection primer for detecting InDel molecular markers of RAD17 gene according to claim 5 in brassica napus breeding, wherein in step S2, the reaction system of PCR amplification comprises: 9.5 mu.L ddH 2 O, 12.5. Mu.L Green Taq Mix, 1. Mu.L of 10. Mu.M upstream primer, 1. Mu.L of 10. Mu.M downstream primer and 1. Mu.L of genomic DNA.
8. The use of a detection primer for detecting InDel molecular markers of RAD17 gene according to claim 5 in brassica napus breeding, wherein in step S2, the reaction procedure of PCR amplification comprises: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 58℃for 30s, extension at 72℃for 15s,35 cycles; extending at 72℃for 7min.
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| CN108950052A (en) * | 2018-08-17 | 2018-12-07 | 中国农业科学院油料作物研究所 | With the InDel label of cabbage type rape floral leaf gene linkage and application |
| KR102358788B1 (en) * | 2020-09-16 | 2022-02-07 | 충남대학교 산학협력단 | Molecular marker and for discriminating low temperature-tolerant cabbage cultivar and uses thereof |
| CN112111594A (en) * | 2020-10-22 | 2020-12-22 | 青海省农林科学院 | InDel molecular marker for identifying multi-chamber character of brassica napus as well as primer group and application thereof |
| KR20220056806A (en) * | 2020-10-28 | 2022-05-06 | 서울대학교산학협력단 | DNA marker for discriminating genotype of Brassica rapa, Brassica oleracea and their interspecific hybrid and uses thereof |
| CN112593005A (en) * | 2020-12-29 | 2021-04-02 | 南京农业大学 | Molecular marker of leaf rolling site on brassica napus and application thereof |
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| GenBank: HG994373.1.GenBank.2021,ORIGIN. * |
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