Development Method and Application of Single-Locus IP Markers Specific to the Radish Genome

Abstract

The present invention discloses a method for developing and applying specific single-locus IP markers in the radish genome. This method utilizes bioinformatics techniques to develop specific single-locus IP markers in radish based on the genomic information of radish and Brassica crops; and these markers are actually applied to the identification of exogenous introgressed fragments in the progeny of radish-Brassica interspecific hybridization. The present invention realizes the rapid identification of excellent gene fragments in the progeny of distant hybridization between radish as the donor and Brassica crops, effectively assists the precise introgression of excellent gene fragments, accurately tracks the size of the gene fragments involved at the same time, breaks the linkage drag of bad traits, and realizes the efficient creation and utilization of germplasm resources. At the same time, it provides important data support for the identification of chromosomal fragments of genes related to improved traits in hybrid progeny.

Description

Technical Field

[0001] The present invention relates to the technical field of radish molecular marker biotechnology, and particularly relates to a method for developing and applying specific single-locus IP markers in the radish genome. Background Art

[0002] The genera Raphanus and Brassica are two very important related genera in the Brassicaceae family. As early as 1924, Karpechenko began to conduct hybridization experiments between Raphanus and Brassica crops to create new species or achieve gene introgression. So far, multiple beneficial genes in radish have been introduced into Brassica crops through distant hybridization between radish and Brassica crops, such as the Ougra-CMS cytoplasmic male sterility gene and restoration gene, the TuMV resistance gene, the beet root knot nematode resistance gene, etc. At the same time, many excellent interspecific hybrid offspring of radish-Brassica crops have also been applied to the actual breeding work of Brassica crops. How to accurately identify and strengthen the screening of exogenous radish chromosome segments in the interspecific hybrid offspring of radish-Brassica is crucial, which is the necessary technical basis for breaking the linkage drag of bad genes in the improved crops.

[0003] Currently, molecular markers developed only using the genomic information of Brassica receptor crops can no longer meet the accurate identification of radish exogenous fragments in the interspecific hybrid offspring of radish-Brassica. Summary of the Invention

[0004] Aiming at the defect of the difficulty in identifying exogenous radish fragments in the interspecific hybrid offspring of radish-Brassica, the present invention provides a method for developing and applying specific single-locus IP markers in the radish genome. This method uses bioinformatics technology to develop specific single-locus IP markers in radish based on the genomic information of radish and Brassica crops; and actually applies these markers to the identification of exogenous radish introgressed fragments in the interspecific hybrid offspring of radish-Brassica.

[0005] To achieve the above object, the present invention relates to a method for developing specific single-locus IP markers in the radish genome, including the following steps:

[0006] 1) Acquisition of radish genomic information: Acquire the genomic information of multiple radish varieties;

[0007] 2) Primer design: Under the Linux system, based on any radish genomic annotation information and its corresponding reference genomic sequence information, use Perl language to write a script to batch extract the exon sequences on both sides of all gene introns, generate a file in the required format for Primer3.0; use the primer3_2.5.0 software to batch design primers;

[0008] 3) In silico PCR analysis: The e-pcr program (ePCR_2.3.9) was executed to perform in silico PCR analysis on the primers and the remaining radish genomic database, generating the in silico PCR analysis results of the primers. Through Venn diagram (https: / / bioinfogp.cnb.csic.es / tools / venny / index.html) mapping analysis, multiple IP marker primers amplified at the common single-locus of multiple radish genomes were obtained.

[0009] 4) Analysis of radish-specific single-locus IP markers: Using the above-mentioned multiple IP marker primers, in silico PCR analysis was respectively performed on the pan-genome of Brassica napus, Chinese cabbage, Brassica oleracea, and Brassica juncea genomes, and Venn diagrams were drawn through the software (http: / / bioinformatics.psb.ugent.be / cgi-bin / liste / Venn / calculate_venn.htpl). Reverse selection was performed on the in silico PCR results, that is, the radish single-locus IP marker primers expected to amplify in any of the reference genomes of Brassica napus, Chinese cabbage, Brassica oleracea, and Brassica juncea were discarded. Finally, only the marker sites that were expected to amplify a single locus in multiple radish genomes and were not expected to have PCR products in any of the reference genomes of Brassica napus, Chinese cabbage, Brassica oleracea, and Brassica juncea were retained, and finally multiple radish genome-specific single-locus IP marker primers were obtained.

[0010] 5) Verification of the specificity of radish genome-specific single-locus IP: According to the distribution of the amplification sites of multiple specific single-locus IP marker primers in the radish genome, 25 - 30 pairs of primers were selected on each chromosome based on the principle of a physical interval of approximately 2 Mb. Actual PCR amplification and agarose gel detection were carried out to judge the effect of the above-mentioned markers in practical applications. The materials used for detection were radish materials, Brassica napus varieties, Chinese cabbage varieties, Brassica oleracea, and Brassica juncea from home and abroad. According to the verification results, multiple pairs of single-locus IP marker primer pairs specifically amplified in radish materials but did not amplify in rapeseed, Chinese cabbage, Brassica oleracea, and Brassica juncea. The above-mentioned multiple pairs of single-locus IP marker primer pairs are a set of radish-specific single-locus IP marker primer pairs that can be actually applied.

[0011] Furthermore, in the above step 1), the radish genomic information comes from 4 radish varieties, namely Qing, Rsa10, Xin-li-mei, and RS01.

[0012] The download addresses of the genomic sequences of the above 4 radish varieties are as follows:

[0013] R10, http: / / 39.100.233.196:82 / download_genome / Brassica_Genome_data / Rsa10 / ;

[0014] Qing, https: / / ftp.ncbi.nlm.nih.gov / genomes / all / GCA / 902 / 824 / 885 / GCA_902824885.1_Qing / ; Xin-li-mei and RS01, https: / / ngdc.cncb.ac.cn / bioproject / browse / PRJCA003033.

[0015] Furthermore, in the said step 2), the radish variety is Rsa10; the primer design parameter settings are as follows:

[0016] PRIMER_OPT_SIZE = 22,

[0017] PRIMER_MIN_SIZE = 18,

[0018] PRIMER_MAX_SIZE = 24,

[0019] PRIMER_PRODUCT_SIZE_RANGE = 80 - 450.

[0020] Furthermore, in the said step 3), the electronic PCR analysis parameter settings are: -n2 -g 1 -m 60, that is, allowing 2 mismatches and 1 gap for each of the left and right primers;

[0021] The remaining radish varieties are Qing, Xin-li-mei and RS01; the number of IP marker primers is 58,412.

[0022] Furthermore, in the said step 4), the number of single-locus IP marker primers specific to the radish genome is 25,448

[0023] Furthermore, in the said step 5), there are 192 pairs of single-locus IP marker primer pairs, specifically as follows:

[0024] Single-locus IP marker information specific to the radish genome

[0025]

[0026]

[0027]

[0028]

[0029]

[0030]

[0031]

[0032] The present invention also provides an application of the specific single-locus IP markers of radish developed by the above method in the identification of exogenous radish fragments in the radish-Brassica hybrid offspring.

[0033] The present invention also provides a kit for identifying exogenous radish fragments in the radish-Brassica hybrid offspring, and the kit includes the primer pair group of the specific single-locus IP markers of radish obtained by the above method.

[0034] The present invention also provides an application of the above kit in the identification of exogenous radish fragments and restoration genes in the restorer materials of cytoplasmic male sterile lines of Brassica napus.

[0035] As a preferred scheme, the application method is as follows:

[0036] a. Using multiple pairs of specific single-locus IP markers of radish genome developed by the above method to perform PCR amplification on the hybrid Yunyouza 15 of Ogura-CMS system and its parental restorer line 16C and sterile line 81A line materials, and obtaining marker sites that have amplification in Yunyouza 15 and restorer line 16C (i.e., containing radish restoration gene fragments), but no amplification in sterile line 81A;

[0037] b. Using this marker amplification site as an anchor point, and then selecting the specific single-locus IP markers of radish genome predicted in the early stage on both sides for PCR verification of the population (24 radish varieties VS 24 Brassica crop varieties / lines); after population verification, perform PCR amplification on restorer line 16C, sterile line 81A and Yunyouza 15; and analyze the obtained results;

[0038] Results A total of 4 pairs of newly verified radish genome-specific single-locus IP markers were amplified in Yunyouza 15 and its parent restorer line 16C, but not in its parent sterile line 81A, namely Rsa10033353_intron_4, Rsa10033329_intron_17, Rsa10025941_intron_5 and Rsa10025804_intron_1. These 5 pairs of radish-specific single-locus IP markers confirmed the real existence of the exogenous radish chromosome fragment in the restorer line 16C, and the amplified fragment should be the radish chromosome sequence carrying the exogenous restorer gene RFO in the restorer line 16C introduced into the Brassica napus genome;

[0039] c. The above results were used to draw a specific marker map of the exogenous radish chromosome fragment carrying the restoration gene of the restorer line 16C; the exogenous chromosome fragment in the restorer line 16C of the Brassica napus Ogura-CMS cytoplasmic sterility line came from the segment corresponding to the 9.2068-11.0319 interval of the radish R9 genome; the terminal boundaries on both sides of the exogenous introduced fragment were predicted to be in the interval of about 847Kb between 8.3598-9.2068Mb and about 769Kb between 11.0319-11.8012Mb, respectively; thus, the accurate identification of the exogenous radish fragment and restoration gene of the restorer material 16C of the Brassica napus Ogura-CMS cytoplasmic sterility line was successfully achieved using the single-locus IP unique to the radish genome of the R9 linkage group.

[0040] Beneficial effects of the present invention:

[0041] The advantage of the present invention is that it has developed a single-locus IP marker unique to the whole genome of radish for the first time, which can realize the rapid identification of excellent gene fragments of distant hybrid offspring with radish as the donor and Brassica crops, effectively assist the precise introduction of excellent gene fragments, and accurately track the size of the gene fragments involved, breaking the undesirable linkage drag, and realizing the efficient creation and utilization of germplasm resources. At the same time, it provides important data support for the identification of chromosome fragments of genes related to improved traits of hybrid offspring. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The Venn diagram of the amplification results of radish, rapeseed pan-genome, Brassica juncea, Brassica rapa, and Brassica oleracea;

[0043] Figure 2 The validation map of the single-locus IP markers unique to radish on chromosomes 1-5 in radish, Chinese cabbage, cabbage, and mustard rapeseed;

[0044] In the figure, A is the first chromosome marker Rsa10038528_intron_1 map,

[0045] Figure B is the Rsa10007852_intron_4 map;

[0046] Figure C is the Rsa10021873_intron_4 map of chromosome 2,

[0047] Figure D is the Rsa10007500_intron_9 map;

[0048] Figure E is the Rsa10026180_intron_4 map of chromosome 3,

[0049] Figure F is the Rsa10021373_intron_4 map;

[0050] Figure G is the Rsa10018033_intron_1 map of chromosome 4,

[0051] Figure H is the Rsa10021559_intron_5 map;

[0052] Figure I is the Rsa10030129_intron_2 map of chromosome 5,

[0053] Figure J is the Rsa10013043_intron_3 map;

[0054] Number 1 represents radish, number 2 represents Brassica napus hybrid, number 3 represents Yunnan rapeseed local variety, number 4 represents Brassica juncea, number 5 represents Brassica oleracea, number 6 represents Brassica rapa, and M represents Marker;

[0055] Figure 3 It is the verification map of radish-specific single-locus IP markers on chromosomes 6-9 in radish, Brassica rapa, Brassica oleracea, and Brassica juncea;

[0056] In the figure, Figure K is the Rsa10039997_intron_5 map of chromosome 6,

[0057] Figure L is the Rsa10012457_intron_11 map;

[0058] Figure M is the Rsa10023454_intron_1 map of chromosome 7,

[0059] Figure N is the Rsa10034079_intron_2 map;

[0060] Figure O is the Rsa10011009_intron_9 map of chromosome 8,

[0061] Figure P is the Rsa10004007_intron_2 map;

[0062] Q is the map of Rsa10017151_intron_6 on chromosome 9,

[0063] R is the map of Rsa10033477_intron_4;

[0064] Number 1 represents radish, number 2 represents Brassica napus hybrid, number 3 represents local variety of Yunnan rape, number 4 represents Brassica juncea, number 5 represents Brassica oleracea, number 6 represents Chinese cabbage, and M represents Marker;

[0065] Figure 4 It is the screening map of specific IP markers on the exogenous chromosome fragment of Ogura-CMS restorer line 16C,

[0066] In the figure, M is marker; 16C is Ogura-CMS restorer line; 81A is Ogura-CMS sterile line; YYZS, Yunyouza 15;

[0067] Figure 5 It is to draw the specific marker map of the exogenous radish chromosome fragment carrying the restorer gene of 16C. Specific implementation mode

[0068] The following combines specific embodiments to further describe the present invention in detail for those skilled in the art to understand.

[0069] Example 1

[0070] The development method of single-locus IP markers specific to the radish genome includes the following steps:

[0071] 1.1 Obtaining radish genome information

[0072] Obtain 4 radish genome information, which are from radish varieties Qing, Rsa10, Xin-li-mei and RS01 respectively. The download addresses of the 4 radish genome sequences are:

[0073] Rsa10: http: / / 39.100.233.196:82 / download_genome / Brassica_Genome_data / Rsa10 / ;

[0074] Qing: https: / / ftp.ncbi.nlm.nih.gov / genomes / all / GCA / 902 / 824 / 885 / GCA_902824885.1_Qing / ;

[0075] Xin-li-mei and RS01: https: / / ngdc.cncb.ac.cn / bioproject / browse / PRJCA003033.

[0076] 1.2 Development of IP markers for radish genome

[0077] 1.2.1 Primer design

[0078] Under the Linux system, a script was written in Perl language to construct a database from the FASTA format sequence of the Rsa10 radish variety genome, extract the exon sequences on both sides of all gene introns, and generate the file format required by Primer3.0; the primer3_2.5.0 software was used to design primers in batches. The FASTA file (genome sequence file) of the Rsa10 radish template sequence was input, the upper limit value of intron length was set to 200, the primer length was between 18 - 24 bps, the best was 20 bps, and the primer Tm value was 60°C - 65°C.

[0079] 1.2.2 e-PCR analysis

[0080] The e-PCR program (e-PCR_2.3.9) was executed to verify the primers in the Rsa10 radish genome and 4 radish genome databases. The number of allowed mismatched bases (-n) was set to 2, the number of allowed gaps (-g) was set to 1, and the expected product length was 80 - 1500. The amplified bands were analyzed through a Venn diagram (https: / / bioinfogp.cnb.csic.es / tools / venny / index.html) to obtain 58,412 IP marker primers common to the 4 radish genomes.

[0081] 1.2.3 Obtaining primers for radish-specific single-locus IP markers

[0082] Using the above 58,412 IP-tagged primers, e-PCR analysis was performed on the Brassica napus pan-genome, Chinese cabbage, Brassica oleracea, and Brassica juncea genomes, and a Venn diagram was drawn using software (http: / / bioinformatics.psb.ugent.be / cgi-bin / liste / Venn / calculate_venn.htpl). Reverse selection was performed on the in silico PCR results, that is, the radish single-locus IP-tagged sites that were expected to amplify in any of the reference genomes of Brassica napus, Chinese cabbage, Brassica oleracea, and Brassica juncea were discarded. Finally, only the marker sites that amplified single loci in all four radish genomes and did not produce PCR products in any of the reference genomes of Brassica napus, Chinese cabbage, Brassica oleracea, and Brassica juncea were retained. Finally, 25,448 single-locus IP tags unique to the radish genome were obtained ( Figure 1 ).

[0083] 1.3 Experimental verification of radish-specific single-locus IP tags in radish, rapeseed, Brassica oleracea, and Chinese cabbage

[0084] According to the physical distance of every 2 M on each chromosome as the standard, 25 - 30 pairs were evenly selected and experimentally verified in radish, Brassica napus, Chinese cabbage, Brassica oleracea, and Brassica juncea respectively.

[0085] 1.3.1 Experimental materials

[0086] In this example, a total of 24 radish materials from home and abroad (Table 1), 12 domestic popularized Brassica napus hybrid materials, 6 Yunnan local rapeseed varieties, 2 Brassica oleracea varieties, 2 Chinese cabbage varieties, and 2 Brassica juncea materials (Table 2) were involved.

[0087] Table 1 Information table of 24 radish materials from home and abroad involved in the experiment

[0088]

[0089]

[0090] Table 2 Statistics of 24 related materials of Brassica napus, Brassica juncea, Brassica oleracea, and Chinese cabbage

[0091]

[0092]

[0093] 1.3.2 Experimental methods

[0094] DNA extraction: The genomic DNA of the above 48 materials was extracted using the CTAB small-sample method.

[0095] Synthesis of IP primers: Synthesized by Shanghai Sangon Biotech Co., Ltd.

[0096] PCR reaction and electrophoresis detection: Taq enzyme Mixture (P111) of Nanjing Novoprotein Scientific Co., Ltd. was used for PCR amplification. The PCR reaction system was as follows: 2×Taq Master Mix 10 μL, forward and reverse primers (10 μmol / L) 0.5 μL each, DNA template (50 ng / μL) 2 μL, ddH 2 O 7 μL, total system 20 μL. The PCR amplification program was as follows: 95°C (3 min); 95°C (15 s), 60°C (-0.5°C / cycle) (15 s), 72°C (20 s), 9 cycles; 95°C (15 s), 55°C (15 s), 72°C (20 s), 30 cycles; 72°C (10 min); stored at 4°C. The PCR amplification products were separated by 2% agarose gel electrophoresis at a constant voltage of 160 V for 20 min. After electrophoresis, they were photographed and saved using a gel imaging system (UVIPlatinum / Explorer).

[0097] 1.3.3 Test results

[0098] Using 260 pairs of single-locus IP primers selected and evenly distributed on 9 chromosomes of radish, the amplification and detection results in 24 radish varieties, 12 Brassica napus var. oleifera hybrids, 6 Brassica napus var. oleifera varieties in Yunnan region, 2 Brassica juncea varieties, 2 Brassica oleracea varieties and 2 Brassica rapa varieties were as follows. Most of the primers showed the characteristics of specific amplification in radish, that is, specific bands were amplified only in radish varieties, and no amplification occurred in Brassica napus var. oleifera, Brassica oleracea, Brassica juncea and Brassica rapa ( Figure 2 , Figure 3 ). Finally, 192 pairs of radish-specific single-locus IP marker primers were verified. Among them, there were 24 radish-specific single-locus IP markers on chromosome 1, 22 on chromosome 2, 20 on chromosome 3, 23 on chromosome 4, 24 on chromosome 5, 23 on chromosome 6, 19 on chromosome 7, 18 on chromosome 8, and 22 on chromosome 9 (see Table 4 for details). At the same time, the sizes of the amplified fragments of the above single-locus IP markers were also basically consistent with the expected results.

[0099] Table 3 Information on radish genome-specific single-locus IP markers verified by PCR

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107] Example 2

[0108] Application of the specific single-locus IP marker primer pair of radish in detecting the alien chromosome fragment of Ogura-CMS restorer line 16C in Brassica napus, and the specific method is as follows:

[0109] The Brassica napus Ogura-CMS restorer line material 16C is a primary progeny material of a distant hybridization between Brassica napus and radish obtained by the Rapeseed Research Center of Yunnan Academy of Agricultural Sciences through exchanges with foreign counterparts. Through continuous backcrossing and self-crossing for more than 10 years and intensive screening, a stable-fertility double-low Ogura-CMS restorer line has been obtained and has been used in the breeding of Brassica napus hybrid varieties. To verify the actual effect of the specific single-locus IP marker of radish in identifying the alien chromosome fragment and to explore the size of the alien chromosome fragment carried by the Rfo restorer gene in 16C, this example takes the Ogura-CMS sterile line 81A, the restorer line 16C and their hybrid Yunyouza 15 as the research objects, uses 192 pairs of specific single-locus IP markers of radish to screen for Rfo gene-linked markers, draws a genetic linkage map of the Rfo gene, and realizes the effective identification of the alien chromosome fragment of radish in Brassica napus.

[0110] 2.1 Experimental materials

[0111] Experimental materials: The Ogura-CMS system hybrid Yunyouza 15 and its parental restorer line 16C and sterile line 81A lines. The above materials were provided by the Rapeseed Research Center of the Institute of Cash Crops, Yunnan Academy of Agricultural Sciences. The Rfo gene sequence (GenBank accession: AJ550021) was downloaded from the NCBI website (https: / / www.ncbi.nlm.nih.gov).

[0112] 2.2 Experimental methods

[0113] 2.2.1 DNA extraction

[0114] Genomic DNA of the parents and their population materials was extracted using the CTAB small-sample method.

[0115] 2.2.2 PCR Reaction and Electrophoresis Detection

[0116] PCR amplification was performed using Taq enzyme Mixture (P111) from Nanjing Novoprotein Science and Technology Co., Ltd. The PCR reaction system was as follows: 10 μL of 2× Taq Master Mix, 0.5 μL each of forward and reverse primers (10 μmol / L), 2 μL of DNA template (50 ng / μL), 7 μL of ddH 2 O, with a total volume of 20 μL. The PCR amplification program was as follows: 95°C (3 min); 95°C (15 s), 60°C (-0.5°C / cycle) (15 s), 72°C (20 s), for 9 cycles; 95°C (15 s), 55°C (15 s), 72°C (20 s), for 30 cycles; 72°C (10 min); store at 4°C. The PCR amplification products were separated by 2% agarose gel electrophoresis at a constant voltage of 160 V for 20 min. After electrophoresis, they were photographed and saved using a gel imaging system (UVI Platinum / Explorer).

[0117] 2.3 Test Results

[0118] 2.3.1 Screening of Radish-Specific single-locus IP Markers Using 192 pairs of radish genome-specific single-locus IP markers, PCR amplification was performed on the Ogura-CMS hybrid Yunyouza 15 and its parental restorer line 16C and sterile line 81A. As a result, a pair of marker loci, Rsa10025823_intron_3, which amplified in Yunyouza 15 and 16C but not in 81A, was obtained. The amplified locus of this marker was the sequence of the 10912208 - 10912415 segment on chromosome R09. Using this amplified locus as an anchor point, previously predicted radish genome-specific single-locus IP markers were selected again on both sides for population (24 radish varieties VS 24 Brassica crop varieties / lines) PCR verification; after population verification, PCR amplification was performed on 16C, 81A, and Yunyouza 15. As a result, 4 pairs of radish genome-specific single-locus IP markers amplified in both 16C and Yunyouza 15 but not in 81A, namely Rsa10033353_intron_4, Rsa10033329_intron_17, Rsa10025941_intron_5, and Rsa10025804_intron_1( Figure 4, (Table 4). These five pairs of radish-specific single-locus IP markers, on the one hand, confirmed the true existence of the exogenous radish chromosome fragment in 16C, and the amplified fragments should be the radish chromosome sequences carrying the introgressed restorer gene Rfo into the Brassica napus genome in 16C; on the other hand, they can be used for molecular marker-assisted introgression of the Rfo gene in the future.

[0119] Table 4 Statistical table of radish-specific single-locus IP markers linked to Rfo

[0120]

[0121] 2.3.2 Specific marker map of the exogenous radish chromosome fragment carried by the restorer gene of Ogura-CMS restorer line 16C

[0122] To further clarify the size and boundaries of the exogenous radish chromosome fragment carried by the exogenous restorer gene in 16C, a specific marker map of the exogenous radish chromosome fragment carried by the restorer gene of 16C was drawn based on the above results ( Figure 5 ). As Figure 5 can be seen, the outermost markers on both sides of this exogenous chromosome fragment are Rsa10033353_intron_4 and Rsa10025804_intron_1; Rsa10033477_intron_4 is the non-differential marker locus closest to the Rsa10033353_intron_4 marker, and Rsa10025700_intron_3 is the non-differential marker locus closest to the Rsa10025804_intron_1 marker. So far, the results have preliminarily determined that the exogenous chromosome fragment in 16C comes from the corresponding section in the 9.2068 - 11.0319 Mb interval of the radish R9 genome, about 1.8251 Mb in size; the terminal boundaries on both sides of the exogenous introgressed fragment are predicted to be in the intervals of about 847 Kb between 8.3598 - 9.2068 Mb and about 769 Kb between 11.0319 - 11.8012 Mb, providing data support for subsequent exploration of the location of this exogenous chromosome in the Brassica napus genome and the development of co-dominant molecular markers. This example once again proves the effectiveness and feasibility of radish-specific single-locus IP markers in detecting and locating alien chromosome fragments.

[0123] Other parts not described in detail are all prior arts. Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all embodiments. People can also obtain other embodiments based on this embodiment without creative efforts, and these embodiments all belong to the protection scope of the present invention.

Claims

1. A method for developing single-locus IP markers specific to the radish genome, characterized in that: It includes the following steps: 1) Obtaining radish genome information: Obtain the genome information of multiple radish varieties; the radish genome information comes from 4 radish varieties, namely Qing, Rsa10, Xin-li-mei, and RS01; 2) Primer design: Under the Linux system, based on the genomic annotation information of any one radish variety among multiple radish varieties and its corresponding reference genome sequence information, use Perl language to write scripts to batch extract the exon sequences on both sides of all gene introns, and generate the format file required by Primer3.0; use primer3_2.5.0 software to batch design primers; 3) In silico PCR analysis: Execute the e-pcr program to perform in silico PCR analysis on the primers and the rest of the radish genome database, and generate the in silico PCR analysis results of the primers; obtain multiple IP marker primers that amplify common single loci in multiple radish genomes through Venn diagram mapping analysis; 4) Analysis of single-locus IP markers specific to radish: Use the above multiple IP marker primers to perform e-PCR analysis on the pan-genome of Brassica napus, Chinese cabbage, Brassica oleracea, and Brassica juncea genomes respectively, and draw a Venn diagram through software; perform reverse selection on the in silico PCR results, that is, discard the radish single-locus IP marker primers that are expected to have amplification in any reference genome of the pan-genome of Brassica napus, Chinese cabbage, Brassica oleracea, and Brassica juncea genomes. Finally, only retain the marker loci that are expected to amplify a single locus in multiple radish genomes and are not expected to have PCR products in any reference genome of the pan-genome of Brassica napus, Chinese cabbage, Brassica oleracea, and Brassica juncea genomes. Finally, obtain multiple single-locus IP marker primers specific to the radish genome; 5) Verification of the specificity of single-locus IP specific to the radish genome: According to the distribution of the amplification sites of multiple specific single-locus IP marker primers in the radish genome, select 25-30 pairs of primers on each chromosome based on the principle of a physical interval of about 2 Mb; Perform actual PCR amplification and agarose gel detection to judge the effect of the above markers in actual applications; the materials used for detection are selected from any one or more of radish materials, Brassica napus varieties, Chinese cabbage varieties, Brassica oleracea, and Brassica juncea from home and abroad; according to the verification results, there are multiple pairs of single-locus IP marker primer pairs that specifically amplify in radish materials but have no amplification in Brassica napus varieties, Chinese cabbage varieties, Brassica oleracea, and Brassica juncea; the above multiple pairs of single-locus IP marker primer pairs are a set of single-locus IP marker primer pairs specific to radish that can be actually applied.

2. The method for developing single-locus IP markers specific to the radish genome according to claim 1, characterized in that: In the step 2), the radish variety is Rsa10; the primer design parameter settings are: PRIMER_OPT_SIZE = 22, PRIMER_MIN_SIZE = 18, PRIMER_MAX_SIZE = 24, PRIMER_PRODUCT_SIZE_RANGE = 80 - 450.

3. The development method of the unique single-locus IP marker of the radish genome according to claim 2, characterized in that: In step 3), the electronic PCR analysis parameters are set as: -n 2 -g 1 -m 60, that is, allowing 2 mismatches and 1 gap for each of the left and right primers; The remaining radish varieties are Qing, Xin-li-mei and RS01; the number of IP marker primers is 58412.

4. The development method of the unique single-locus IP marker of the radish genome according to claim 1, characterized in that: In step 4), the number of primers of the unique single-locus IP marker of the radish genome is 25448.

5. The development method of the unique single-locus IP marker of the radish genome according to claim 1, characterized in that: In step 5), there are 192 pairs of single-locus IP marker primer pairs.

6. Application of the unique single-locus IP marker of radish obtained by the development method according to claim 1 in the identification of exogenous radish fragments in the radish-Brassica hybrid offspring.

7. A kit for identifying exogenous radish fragments in the radish-Brassica hybrid offspring, characterized in that: The kit includes the primer pair group of the unique single-locus IP marker of radish obtained by the method according to claim 1.

8. Application of the kit according to claim 7 in the identification of exogenous radish fragments and restoration genes in the cytoplasmic male sterile line restoration material of Brassica napus.

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