CN115948599A - InDel molecular marker of RAD17 gene and detection primer and application thereof - Google Patents
InDel molecular marker of RAD17 gene and detection primer and application thereof Download PDFInfo
- Publication number
- CN115948599A CN115948599A CN202211728104.5A CN202211728104A CN115948599A CN 115948599 A CN115948599 A CN 115948599A CN 202211728104 A CN202211728104 A CN 202211728104A CN 115948599 A CN115948599 A CN 115948599A
- Authority
- CN
- China
- Prior art keywords
- molecular marker
- rape
- indel molecular
- rad17
- detecting
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of rape breeding, and particularly relates to an InDel molecular marker of an RAD17 gene, and a detection primer and application thereof. The InDel molecular marker is positioned on the first exon of the RAD17 gene, and comprises 5 segments of insertion sequences, wherein the 5 segments of insertion sequences are respectively positioned at 9663297bp, 9663298bp, 9663300bp, 9663303bp and 9663304bp of a C09 chromosome. The invention develops an InDel molecular marker based on natural cabbage type rape and artificially synthesized cabbage type rape RAD17 gene variation locus, the InDel molecular marker is stably and tightly linked with a genome, and the stability of the genome of the offspring of the artificially synthesized cabbage type rape can be speculated by detecting whether the molecular marker exists in the genome of the rape, so that the germplasm innovation efficiency of the rape is effectively improved, and the breeding process of the rape is accelerated.
Description
Technical Field
The invention relates to the technical field of rape breeding, in particular to an InDel molecular marker of RAD17 gene and a detection primer and application thereof.
Background
Rape is a general name of annual or perennial plants of Brassica (Cruciferae) aiming at seed extraction and oil extraction, is one of important oil crops which are distributed most widely in the world and have long planting history, and is also the fifth crop following rice, wheat, corn and soybean. Currently, rape cultivars are mainly of the following three types: brassica napus (Brassica napus), brassica rapa (Brassica rapa) and Brassica juncea (Brassica juncea). Compared with cabbage type rape and the like, the cabbage type rape has higher yield and stronger disease resistance, and is the main type of rape planted in China.
Brassica napus (AACC, 2n =4x = 38) is formed by natural doubling after interspecific crossing of two diploid ancestor brassica rapa cabbages (AA, 2n =2x = 20) and brassica oleracea (CC, 2n =2x = 18). Despite its short history of evolution, it exhibits diploid-like meiosis, during which normal ordering of chromosomes ensures fertility. This indicates that this "young" genome evolved rapidly and the genome reached stability. However, for artificially newly synthesized brassica napus (usually by crossing and artificially doubling Chinese cabbage and cabbage), the phenomenon of chromosome rearrangement and homologous chromosome recombination frequently occurs in the meiosis process, which is called as "genome shock", and is specifically expressed as abnormal pollen development and the like, so that the normal growth and development of the brassica napus are seriously influenced. By utilizing the abundant variation generated by 'genome vibration' in the evolution process of artificially synthesized cabbage type rape A and C genomes, excellent genes and molecular markers for controlling related characters can be screened out and applied to germplasm innovation of rape. The method has important significance for accelerating the stability of the genome formed by brassica allopolysomes so as to improve the breeding success rate and the rape quality by exploring the reason that the genome is artificially synthesized to obtain the stability of natural brassica napus.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides an InDel molecular marker of an RAD17 gene, a detection primer for detecting the InDel molecular marker and application of the detection primer in rape breeding.
The invention is realized by the following technical scheme:
the first aspect of the invention provides an InDel molecular marker of an RAD17 gene, wherein the InDel molecular marker is positioned on a first exon of the RAD17 gene, the InDel molecular marker comprises 5 segments of insertion sequences, the 5 segments of insertion sequences are respectively positioned at 9663297bp, 9663298bp, 9663300bp, 9663303bp and 9663304bp of a C09 chromosome of a wild cabbage W03, and nucleotide sequences are TTCGGAGAGAAG, TATAG, TCCCC, TGTATCGTTCCCTCCCTGCTCCTCGATTTTTTTTTTTTTTTTTTTAATTCGAATTCGAATTCGACCCCAC and TACATTAAAACCCAACCCCCACCCACCC.
The second aspect of the invention provides a detection primer for detecting an InDel molecular marker of 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:5, respectively.
A third aspect of the invention provides a kit for detecting the InDel molecular marker of the RAD17 gene, the kit comprising the detection primer for detecting the InDel molecular marker of the RAD17 gene as described above.
Further, the kit also comprises Green Taq Mix.
The fourth aspect of the present invention provides the use of the kit for detecting the InDel molecular marker of RAD17 gene as described above or the detection primer for detecting the InDel molecular marker of RAD17 gene as described above in rape breeding.
Further, the application comprises the application in the amplification and/or detection of the genotype of the rape RAD17 gene or the prediction and/or identification of the genome stability of the rape.
Further, the application comprises the steps of:
s1, extracting genome DNA of rape to be detected;
s2, using a nucleic acid sequence as set forth in SEQ ID NO:4-5, carrying out PCR amplification on a detection primer for detecting the InDel molecular marker of the RAD17 gene;
and S3, judging whether the rape genome is stable or not 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 was extracted by the CTAB method.
Further, in step S2, the reaction system for PCR amplification includes: 9.5 μ 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.
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; extension at 72 ℃ for 7min.
The invention has the advantages and positive effects that:
the invention develops an InDel molecular marker based on natural cabbage type rape and artificially synthesized cabbage type rape RAD17 gene variation sites, and specifically comprises 5 segments of insertion sequences which are stably and tightly linked with a genome, and by detecting whether the InDel molecular marker of the invention exists in offspring of the artificially synthesized cabbage type rape and the natural cabbage type rape or not, the stability of the genome of the offspring of the artificially synthesized cabbage type rape can be presumed, and if the 5 segments of insertion sequences exist in the genome of the offspring of the RAD17 gene, the genome of the single plants is stable; if the RAD17 gene is detected to be haplotype 1 in the genome of the later generation, namely the 5 segments of insertion sequences are deleted, the genome of the single plants is not stable, and therefore, whether the artificially synthesized brassica napus genome is stable can be quickly judged by conventional means such as PCR amplification and the like, so that the germplasm innovation efficiency of the rape is effectively improved, and the breeding process of the rape is accelerated.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the description below are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a diagram showing the information of InDel molecular marker loci in RAD17 gene according to the embodiment of the present invention;
FIG. 2 is a diagram showing agarose gel electrophoresis results of PCR amplification products of the present invention, wherein "M" in FIGS. a-c represents DNA ladder marker, "W03" is wild cabbage W03, "HC-1" and "HC-Q" are artificially synthesized Brassica napus, "ZS11," "Westar," and "Tapidor" are natural Brassica napus, "4" - "6" in FIG. a and "7" - "42" in FIG. b are natural Brassica napus, and "1" - "36" in FIG. c is a hybrid F of natural Brassica napus and artificially synthesized Brassica napus 6 And (4) a group.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, and the equipment and reagents used in the examples and test examples are commercially available without specific reference. The specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.
Various modifications to the precise description of the invention will be readily apparent to those skilled in the art from the information contained in this application without departing from the spirit and scope of the appended claims. It is to be understood that the scope of the invention is not limited to the procedures, properties, or components defined, as these embodiments, as well as others described, are intended to be merely illustrative of particular aspects of the invention. Indeed, various modifications of the embodiments of the invention which are obvious to those skilled in the art or related fields are intended to be included within the scope of the following claims.
For a better understanding of the invention, and not as a limitation on the scope thereof, all numbers expressing quantities, percentages, and other numerical values used in this application are to be understood as being modified in all instances by the term "about". Accordingly, unless expressly 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. At the very least, 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 to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below.
RAD17 is a very important early regulatory protein in response to DNA damage monitoring and replication stress signaling, and plays an important role in telomere structure stabilization and meiotic cell cycle monitoring. Research shows that the RAD17 gene plays an important role in maintaining the chromosome stability in human cells and ensuring normal repair of yeast cell meiotic double-strand breaks and other processes, and the functional defects and abnormal expression of the RAD17 gene are possibly closely related to the occurrence and development of cell canceration and tumors. In addition, the RAD17 gene also functions similarly in plants. For example, the AtRAD17 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 associated with its involvement in the detection of DNA damage and the detection of replication to maintain cellular genome stability, and its deletion or low expression may result in increased cell mutation rates. These findings provide new ideas for understanding genetic variation and genome stability, or for accelerating genome stability of artificially synthesized polyploid brassica crops and promoting formation of new germplasm resources.
Based on the situation, the embodiment of the invention provides an InDel molecular marker of an RAD17 gene, wherein the InDel molecular marker is positioned on the first exon of the RAD17 gene, the InDel molecular marker comprises 5 segments of insertion sequences, the 5 segments of insertion sequences are respectively positioned at 9663297bp, 9663298bp, 9663300bp, 9663303bp and 9663304bp of a C09 chromosome of wild cabbage W03, and nucleotide sequences are TTCGGAGAAG, TATAG, TCCCC, TGTATCGTTCCCTCCCTTGCTCGATTTTTTTTTTTCTTTAATTGAATTTCA and TACCACTAAACCCACCCACCCACCC. Specifically, referring to FIG. 1, the sequence TTCGGAGAGAAG (10 bp, see SEQ ID NO: 1) was inserted at 9663297bp, TATAG (5 bp) at 9663298bp, TCCCC (5 bp) at 9663300bp, TGTATCGTATCGTTCCCTGTCCTCGATTTTTTTTTTTTTTTTTTTTTTAATTGAATTTCGCA (58 bp, see SEQ ID NO: 2) at 9663303bp, and TACATTACTAAAACCCCCACCC (21 bp, see SEQ ID NO: 3) at 9663304 bp.
The invention screens 5 InDel molecular markers on the first exon of the C09 chromosome RAD17 gene by comparing and analyzing the genome sequences of artificially synthesized cabbage type rape, wild cabbage and natural cabbage type rape, and the polymorphism is a deletion/insertion sequence and has two genotypes of deletion and insertion. Taking genome of wild cabbage W03 as reference genome, the artificially synthesized cabbage type rape has disorder array difference on the first exon of RAD17 gene of C09 chromosome (for convenience of description, referred to as haplotype 1 hereinafter, i.e., RAD17 gene is haplotype 1 in the artificially synthesized cabbage type rape), and 5 insertion sequences exist at corresponding positions in natural cabbage type rape (for convenience of description, referred to as haplotype 2 hereinafter, i.e., RAD17 gene is haplotype 2 in the natural cabbage type rape). The genome of natural brassica napus is usually stable, so the 5 segments of insertion sequences, namely the InDel molecular marker provided by the invention, are stably and closely linked with the genome of brassica napus, so that the stability of the genome of the offspring of artificially synthesized brassica napus can be presumed by detecting the molecular marker of the invention, and if the RAD17 gene is haplotype 2 in the genome of the offspring, the 5 segments of insertion sequences exist, which indicates that the genome of the individual plants is stable; if the RAD17 gene is detected to be haplotype 1 in the genome of the progeny, namely the 5 insertion sequences are deleted, the genome of the single strains is not stable yet.
The InDel molecular marker developed based on the RAD17 gene can be used for identifying the genetic recombination and the genome stability of the single plant genome in the cabbage type rape filial generation, quickly judging whether the artificially synthesized cabbage type rape genome is stable or not, can be simply, conveniently, quickly and high-flux applied to breeding practice, has the advantages of simple typing and the like, and can effectively improve the rape germplasm innovation efficiency and accelerate the rape breeding process.
The invention further 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 as SEQ ID NO:4, the nucleotide sequence of the downstream primer is shown as SEQ ID NO:5, respectively.
The invention utilizes Indel molecular marker sites in RAD17 gene and specific sequences of upstream and downstream to develop a pair of primers, the upstream primer CTCCATACCCCCAATCCAG (shown in SEQ ID NO: 4) and the downstream primer TACAACCGTCCATGCAA (shown in SEQ ID NO: 5), the primers are used for amplification, amplification products (different banding patterns) with different sizes can be obtained aiming at different haplotypes, specifically, banding 1 (corresponding to haplotype 1) is detected in the artificially synthesized cabbage type rape population, the amplification product is 209bp, the genome is unstable, banding 2 (corresponding to haplotype 2) is detected in the natural cabbage type rape population, the amplification product is 305bp, the genome is stable, thus whether the genome reaches a stable state can be judged according to the length of the amplification product, the breeding efficiency of the rape with stable genome is greatly improved, and the breeding process is accelerated.
It should be noted that the method for analyzing the length of the amplification product includes agarose gel electrophoresis or sequencing, and the present invention is not limited thereto, and may be performed according to procedures known in the art.
In another embodiment, the invention provides a kit for detecting the InDel molecular marker of the RAD17 gene, wherein the kit comprises the detection primer.
The advantages of the kit for detecting the InDel molecular marker of the RAD17 gene relative to the prior art are the same as the advantages of the detection primer for detecting the InDel molecular marker of the RAD17 gene relative to the prior art, and are not repeated herein.
Optionally, the kit further comprises Green Taq Mix, which mainly comprises Taq DNA Polymerase, dntps and an optimized buffer system, purchased from norkexin biotechnology limited, having the product numbers: p131.
The embodiment of the invention also provides the application 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 in rape breeding.
The advantages of the application over the prior art are the same as the advantages 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 over the prior art, and are not described in detail herein.
Specifically, the applications include the applications in the amplification and/or detection of the genotype of the rape RAD17 gene or the prediction and/or identification of the rape genome stability.
Specifically, the application comprises the following steps:
s1, extracting genome DNA of rape to be detected;
s2, using a nucleic acid sequence as set forth in SEQ ID NO:4-5, carrying out PCR amplification;
and S3, judging whether the rape genome is stable or not 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.
In the invention, the haplotype of a single plant in a population is identified by utilizing the InDel molecular marker in offspring of artificially synthesized cabbage type rape, a 305bp strip appears in an amplification result of natural cabbage type rape, the Indel molecular marker is contained, a 209bp strip appears in the amplification result of the artificially synthesized cabbage type rape, the Indel molecular marker is deleted, and the amplification results of the filial generations of the artificially synthesized cabbage type rape and the natural cabbage type rape present three types: 1) A 305bp band; 2) A 209bp band; 3) The 305bp and 209bp bands appeared simultaneously. The PCR amplification result is that the genome with the type 1 (the amplification product is 209 bp) is unstable, and the method can be further applied to the creation of new germplasm resources; the PCR result shows that the genome is stable when the banding pattern 2 (the amplification product is 305 bp) is obtained, the method can be further applied to genetic breeding, and the simultaneous existence of two banding patterns shows that the offspring can also separate the characters. 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 using the CTAB method.
Optionally, in step S2, the reaction system for PCR amplification includes: 9.5 μ 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, and 35 cycles; extension at 72 ℃ for 7min.
The invention will be further illustrated with reference to the following specific examples. The following examples are examples of experimental procedures not specified under specific conditions, generally according to the conditions recommended by the manufacturer.
1. InDel molecular marker locus identification
3 parts of artificially synthesized cabbage type rape, 295 parts of natural cabbage type rape, 8 natural cabbage type rape and artificially synthesized cabbage type rape hybrid F 6 The generation population (1389 total) was genotyped. 1687 parts of the above-mentioned sample materials were normally sown and planted in Wuchang test base of the institute of oil crops, academy of agricultural sciences, china. Leaf tissue sampling was performed during the seedling stage and samples were rapidly stored at-20 ℃. Leaf whole genome DNA of each material was extracted and sequenced by CTAB method.
Taking the genome of wild cabbage W03 as a reference genome, referring to FIG. 1, comparing the genome analysis to find that the sequence of the artificially synthesized cabbage type rape and the wild cabbage W03 has no difference in the first exon of the RAD17 gene, and designating the genotype of the RAD17 gene without variation sites as haplotype 1, selecting 4 typical natural cabbage type rape, wherein the genome sequence of the 4 typical natural cabbage type rape has 5 insertion variations at the same positions, and the genotypes are haplotype 2 and haplotype 5 insertion sequences respectively: the insertion sequence at 9663297bp is TTCGGAGAAG (10 bp, see SEQ ID NO: 1), the insertion sequence at 9663298bp is TATAG (5 bp), the insertion sequence at 9663300bp is TCCCC (5 bp), the insertion sequence at 9663303bp is TGTATCGTATCGTTCCCTGTCTCGATTTTTTTTTTTTTCTTTAATTGAATTTCGCA (58 bp, see SEQ ID NO: 2) and the insertion sequence at 9663304bp is TACATAAACCCCCACCC (21 bp, see SEQ ID NO: 3).
Haplotype molecular marker primers designed by the five variation sites can be used for RAD17 gene haplotype identification and genome stability analysis.
2. Design of detection primers
According to a Primer design principle, a pair of detection primers for detecting the InDel molecular markers are designed on the upstream and downstream of the 5 InDel molecular markers by using Primer 5, and comprise an upstream Primer sequence and a downstream Primer sequence, and the method specifically comprises the following steps:
an upstream primer: CTCCCATATACCCCCAATCG (see SEQ ID NO: 4);
a downstream primer: TACAACCGTCCGTCATGCAA (see SEQ ID NO: 5).
3. Validation of the reliability of InDel molecular markers in a population
And (3) taking the sample whole genome DNA as a template, carrying out PCR amplification by using a detection primer for detecting the InDel molecular marker, and carrying out agarose gel electrophoresis on a PCR amplification product to detect and distinguish different allelic variation. 3 parts of artificially synthesized cabbage type rape, 295 parts of natural cabbage type rape, 8 natural cabbage type rape and artificially synthesized cabbage type rape hybrid F 6 The generation group (1389 parts) was identified. The PCR amplification reaction system (total volume 25. Mu.L) included: 9.5 μ L ddH 2 O, 12.5 muL Green Taq Mix, 1 muL 10 muM upstream primer, 1 muL 10 muM downstream primer and 1 muL genomic DNA; the reaction procedure comprises: pre-denaturation at 94 deg.C for 5min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 15s,35 cycles; extension at 72 ℃ for 7min.
The results of the above amplification were analyzed by agarose gel electrophoresis, and the results are shown in FIG. 2; wherein, in the graphs a, b and c, "M" represents DNA ladder marker, "W03" is wild cabbage W03, "HC-1" and "HC-Q" are artificially synthesized cabbage type rape, "ZS11," "Westar," and "Tapidor" are natural cabbage type rape, "4" - "6" in the graph a and "7" - "42" in the graph b are natural cabbage type rape,"1" - "36" in FIG. c is a hybrid F of natural Brassica napus and artificially synthesized Brassica napus 6 And (4) a group.
The agarose gel electrophoresis result shows that the amplification product of wild cabbage and artificially synthesized cabbage type rape is 209bp and shows banding pattern 1 (corresponding to haplotype 1, hap1), the amplification product of natural cabbage type rape is 305bp and shows banding pattern 2 (corresponding to haplotype 2, hap2), and the hybridization F between wild cabbage and artificially synthesized cabbage type rape 6 Three band types of band type 1, band type 2 and band types 1 and 2 appear in the generation group, the agarose gel electrophoresis result is counted, and the statistical result is shown in table 1.
TABLE 1 genotyping of oilseed rape using InDel molecular markers for the RAD17 gene
| Hap1 | Hap2 | Hap1+Hap2 | Total number of samples | |
| Artificially synthesized |
3 | 0 | 0 | n＝3 |
| Natural |
6 | 289 | 0 | n＝295 |
| [ Natural cabbage type rape 1X artificially synthesized cabbage type rape] |
1 | 7 | 1 | n＝9 |
| [ natural cabbage type rape 2X artificially synthesized cabbage type rape] |
2 | 62 | 1 | n＝65 |
| [ natural cabbage type rape 3X artificially synthesized cabbage type rape] |
2 | 218 | 0 | n＝220 |
| [ Natural cabbage type rape 4X artificially synthesized cabbage type rape] |
2 | 82 | 2 | n＝86 |
| [ Natural cabbage type rape 5X artificially synthesized cabbage type rape] |
1 | 140 | 1 | n＝142 |
| [ Natural cabbage type rape 6X artificially synthesized cabbage type rape]F 6 | 0 | 294 | 0 | n＝294 |
| [ 7X Artificial Synthesis of Natural Brassica napus] |
28 | 116 | 15 | n＝159 |
| [ Natural cabbage type rape 8X artificially synthesized cabbage type rape] |
1 | 413 | 0 | n＝414 |
As can be seen from Table 1, 3 parts of the artificially synthesized Brassica napus are haplotype 1, the amplification product does not contain the InDel molecular marker provided by the invention, 98% of the natural Brassica napus is haplotype 2, 2% of the natural Brassica napus is haplotype 1, most of the amplification products contain the InDel molecular marker provided by the invention, and the genome of the natural Brassica napus is stable, so that haplotype 2 and the genome of the Brassica napus are stably and closely linked.
Further, by crossing the natural Brassica napus with the artificially synthesized Brassica napus F 6 The genome stability of the offspring was estimated by PCR amplification in the generation population (1389 copies in total), 2.7% of which was haplotype 1, indicating that the genomes of these individuals did not reach stability; 95.9% of the individuals are haplotype 2, which indicates that the genomes of the individuals have reached stability; 1.4% of the total of both Hap1 and Hap2 (two bands in the electrophoresis) indicates thatThe individual plants of (2) may also show segregation of progeny.
In conclusion, the InDel molecular marker developed based on the RAD17 gene variation sites of the natural brassica napus and the artificially synthesized brassica napus is stably and tightly linked with the genome, and whether the genome of the progeny of the artificially synthesized brassica napus and the natural brassica napus is stable or not can be presumed by detecting the haplotype of the progeny of the artificially synthesized brassica napus and the natural brassica napus, so that the germplasm innovation efficiency of the rape is effectively improved, and the breeding process of the rape is accelerated.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. An InDel molecular marker of an RAD17 gene is positioned on the first exon of the RAD17 gene, and comprises 5 segments of insertion sequences, wherein the 5 segments of insertion sequences are respectively positioned at 9663297bp, 9663298bp, 9663300bp, 9663303bp and 9663304bp of a C09 chromosome of a wild cabbage W03, and the 5 segments of nucleotide sequences of the insertion sequences are TTCGGAAG, TATAG, TCCCC, TGTATCGTTCCCTTCCTTGCTCGATTTTTTTTTTTTTTTTTTTCTTTAATTGAATTTCGA and TACATTACTAACCAACCCACCCC.
2. The detection primer for detecting the InDel molecular marker of the RAD17 gene is characterized by comprising an upstream primer and a downstream primer, wherein 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:5, respectively.
3. A kit for detecting the InDel molecular marker of RAD17 gene, comprising the detection primer of claim 2 for detecting the InDel molecular marker of RAD17 gene.
4. The kit for detecting the InDel molecular marker of RAD17 gene according to claim 3, wherein the kit further comprises Green Taq Mix.
5. The use of the detection primer of claim 2 for detecting the InDel molecular marker of the RAD17 gene in oilseed rape breeding.
6. Use of the detection primers for detecting the InDel molecular marker of the RAD17 gene in oilseed rape breeding according to claim 5, characterized in that said use comprises the use in amplifying and/or detecting the genotype of the oilseed rape RAD17 gene or in predicting and/or identifying the genome stability of oilseed rape.
7. The application of the detection primer of the InDel molecular marker for detecting RAD17 gene in rape breeding according to claim 6, which comprises the following steps:
s1, extracting genome DNA of rape to be detected;
s2, using a nucleic acid sequence as set forth in SEQ ID NO:4-5 for detecting the InDel molecular marker of the RAD17 gene to carry out PCR amplification;
and S3, judging whether the rape genome is stable or not 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.
8. The use of the detection primer for detecting the InDel molecular marker of RAD17 gene in rape breeding according to claim 7, wherein in step S1, the genomic DNA is extracted by CTAB method.
9. The application of the detection primer for detecting the InDel molecular marker of the RAD17 gene in rape breeding according to claim 7, wherein in the step S2, the reaction system for PCR amplification comprises: 9.5 μ L ddH 2 O, 12.5. Mu.L LGreen Taq Mix, 1. Mu.L 10. Mu.M forward primer, 1. Mu.L 10. Mu.M reverse primer and 1. Mu.L genomic DNA.
10. The use of the detection primer for detecting the InDel molecular marker of RAD17 gene in rape breeding according to claim 7, wherein the reaction procedure of PCR amplification in step S2 comprises: pre-denaturation at 94 deg.C for 5min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 15s,35 cycles; extension at 72 ℃ for 7min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211728104.5A CN115948599B (en) | 2022-12-29 | 2022-12-29 | InDel molecular marker of RAD17 gene, detection primer and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211728104.5A CN115948599B (en) | 2022-12-29 | 2022-12-29 | InDel molecular marker of RAD17 gene, detection primer and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115948599A true CN115948599A (en) | 2023-04-11 |
| CN115948599B CN115948599B (en) | 2023-09-08 |
Family
ID=87282338
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211728104.5A Active CN115948599B (en) | 2022-12-29 | 2022-12-29 | InDel molecular marker of RAD17 gene, detection primer and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115948599B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
| 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 |
| KR102358788B1 (en) * | 2020-09-16 | 2022-02-07 | 충남대학교 산학협력단 | Molecular marker and for discriminating low temperature-tolerant cabbage cultivar and uses 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 |
-
2022
- 2022-12-29 CN CN202211728104.5A patent/CN115948599B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Non-Patent Citations (8)
Also Published As
| Publication number | Publication date |
|---|---|
| CN115948599B (en) | 2023-09-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113151561B (en) | Molecular marker BnC04Y2498 for identifying dwarf cabbage type rape and application thereof | |
| AU2017321184B2 (en) | Melon plants with improved traits | |
| CN112795691B (en) | Molecular marker linked with corn stem thickness and application thereof | |
| CN111321241B (en) | Molecular marker of wheat thousand-grain weight and grain length gene TaGS3-4A and application thereof | |
| KR20180077873A (en) | SNP markers for selection of marker-assisted backcross in watermelon | |
| US20170022574A1 (en) | Molecular markers associated with haploid induction in zea mays | |
| CN111719012A (en) | dCAPS molecular marker primer pair for identifying dehydration rate genotype of corn kernel and application | |
| JP2007054020A (en) | METHOD FOR IDENTIFYING RICE BLAST RESISTANCE WITH Pb1 GENE-LINKED MOLECULAR MARKER AS INDICATOR | |
| CN112218526A (en) | Methods for haploidy embryo genotyping | |
| CN113736910A (en) | Linkage molecular marker of peanut single plant pod number main effect QTL site qPN7 and application thereof | |
| US20230250446A1 (en) | Tomato plants with resistance to mi-1 resistance-breaking root-knot nematodes | |
| AU2014268142B2 (en) | Disease resistance loci in onion | |
| CN114480718B (en) | Primer group and detection kit for rice high temperature resistant genotyping based on KASP technology and application of primer group and detection kit | |
| WO2010146357A1 (en) | Oil palm and processes for producing it | |
| CN115948599B (en) | InDel molecular marker of RAD17 gene, detection primer and application thereof | |
| US10172305B2 (en) | Diagnostic molecular markers for seed lot purity traits in soybeans | |
| US11001851B2 (en) | Tomato plants with improved traits | |
| Henareh et al. | Genetic Structure and Mixed Linear Model-Based Association Analysis for Morphological Traits in a Collection of Tomato Landraces from Iran and Turkey | |
| CN112980993B (en) | SNP molecular marker linked with major QTL site qPSIIB10 for resisting aspergillus flavus infection of peanuts and application thereof | |
| CN116397034B (en) | Molecular marker related to chicken antler crown character and application thereof | |
| US20190256933A1 (en) | Guayule Molecular Markers And Primers And Use Of Molecular Markers, Primers And Rate Of Apomixis For Guayule Identification, Characterization And Breeding | |
| CN115820895B (en) | Molecular marker closely linked with chlorophyll content of corn and application thereof | |
| CN115820897B (en) | Molecular marker closely linked with corn female spike and sword leaf length and application thereof | |
| CN109750117B (en) | Functional molecular marker of tomato anthocyanin synthesis related gene Aft and application thereof | |
| Lübberstedt et al. | Linkage |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |