Isatis indigotica genome-specific Single-locus IP labeling primer set and its application

Abstract

The invention discloses a genome-specific Single-locus IP marker primer set of Isatis indigofera and its application, and relates to the technical field of molecular biology. The primer set provided by the invention includes a total of 193 pairs of primers, and the nucleotide sequences thereof are shown in SEQ ID NO.1 to SEQ ID NO.386, respectively. The primer set is developed based on the Single-locus IP marker specific to the Isatis indigofera genome, and can be used for the identification of Isatis indigofera exogenous introduced fragments in Isatis indigofera-Brassica napus hybrid offspring, and can realize the rapid identification of excellent gene fragments of distant hybrid offspring with Isatis indigofera as a donor and Brassica genus, effectively assist the accurate introduction of excellent gene fragments, and accurately track the size of the gene fragments involved, break the undesirable linkage encumbrance, realize the efficient creation and utilization of germplasm resources, and provide data support for the identification of chromosome fragments of genes related to improved traits of hybrid offspring.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, and in particular to a specific Single-locus IP labeling primer set for identifying Isatis indigotica genome and application thereof. Background Art

[0002] Brassica Brassica ) plants and their closely related genera, is an effective way to innovate their germplasm and apply them in breeding. Brassica napus ) and turnip ( Brassica rapa ) have been used for interspecific hybridization, and many scholars at home and abroad have carried out interspecific and inter-generic hybridization. As a traditional dye and medicinal plant in my country, the roots (i.e., Radix Isatidis) and leaves (Leaf of Isatis indigotica) of the Cruciferae family have strong antiviral activity. Through distant hybridization technology, it has been widely used in the research of improving and breeding Brassica crops. Chinese scholars Zhao Heju et al. (1994) reported the hybridization of Brassica napus and Isatis indigotica ( Isatis tinctoria ) and obtained disease-resistant materials of Brassica napus. Du et al. (2009) obtained Brassica napus and Isatis indigofera ( Isatis indigotica Fort., Chinese woad, 2n=14, Ⅱ). Kang et al. (2014) created a complete set of Brassica napus-I. indigofera monosomic addition lines. All 7 addition lines except MAAL Me showed stamen carpelization. The sterile plants were continuously backcrossed with the parental Brassica napus to select a new Brassica napus cytoplasmic male sterile line inap CMS (Kang et al 2017). Mitochondrial genome sequencing found that most genes came from I. indigofera. With the continuous creation of excellent intergeneric hybrid progenies of I. indigofera-Brassica crops, it is very important to accurately identify and strengthen the screening of exogenous I. indigofera chromosome fragments in intergeneric hybrid progenies of I. indigofera-Brassica rapa, which is the necessary technical basis for breaking the linkage drag of undesirable genes in improved crops.

[0003] Distant hybrids are prone to abnormal phenomena such as linkage drag or meiotic behavior disorder due to the influence of exogenous genes, and early materials are prone to severe separation. There are also many technical means for their identification, including molecular markers, trait observation, chromosome observation (karyotype / banding analysis, in situ hybridization), specific proteins, etc. However, identification methods such as trait observation, chromosome observation, and specific proteins are susceptible to environmental factors, have low resolution, are difficult to operate, and have a narrow scope of application. It is difficult to achieve accurate identification using the above technical means alone. Molecular markers are not affected by environmental factors and have the advantages of high detection efficiency, simple operation, stability, and high throughput. They have become a necessary technical means for the identification of distant hybrid offspring and their breeding applications. At present, there are reports on the research of distant hybridization between Isatis indigotica and Brassica crops, but the research on the use of Isatis indigotica genome-specific molecular markers for the identification of its offspring is not systematic and in-depth. Most of its specific markers are a small number of differential SSR markers obtained by screening between two specific species of Isatis indigotica and Isatis indigotica, which cannot meet the universal requirements for the identification of Isatis indigotica exogenous fragments in the interspecific hybridization progenies of Isatis indigotica and Brassica, and restrict the breeding application efficiency of its related excellent distant hybridization progenies. Therefore, it is urgent to develop a method for the identification of Isatis indigotica chromosome fragments in distant hybridization progenies with high universality and strong systematicity. Summary of the invention

[0004] The invention aims to provide a set of universal Single-locus IP marker primers for identifying the genome of Isatis indigotica, which can be used for identifying the exogenous introduced fragments of Isatis indigotica in the interspecific hybridization offspring of Isatis indigotica and Brassica napus.

[0005] In order to achieve the above object, the present invention provides a specific Single-locus IP marker primer set that can be used to identify the genome of Isatis indigotica. The primer set comprises 193 pairs of primers, and the nucleotide sequences thereof are shown in SEQ ID NO.1 to SEQ ID NO.386, respectively.

[0006] The primer set provided by the invention can be used for identifying exogenous fragments of Isatis indigotica.

[0007] The primer set provided by the present invention can be used to assist in the precise introduction of excellent gene fragments of Isatis indigotica, break the unfavorable linkage drag of distant hybridization between Isatis indigotica and Brassica crops, realize the efficient creation and utilization of germplasm resources, and be used for the breeding of Isatis indigotica.

[0008] The present invention also provides a method for identifying exogenous fragments of Isatis indigotica, comprising the following steps:

[0009] 1) Using the above primer set to perform PCR amplification on the whole genome of the sample to be tested, a marker locus was obtained that was amplified in Isatis indigotica and Isatis oil, but not in other Brassica crops;

[0010] 2) Using the obtained marker site as an anchor point, the previously predicted single-locus IP markers unique to the whole genome of Isatis indigotica were selected on both sides for group PCR verification; after group verification, PCR amplification was performed again in the sample to be tested, and the results were analyzed;

[0011] 3) Use the above analysis results to draw a specific marker map of chromosome fragments introduced exogenously from Isatis indigotica in candidum oil, so as to achieve accurate identification of exogenously introduced fragments of Isatis indigotica in candidum oil.

[0012] Furthermore, the above-mentioned PCR, its specific conditions include:

[0013] PCR reaction system: template DNA 2.5 μL, forward and reverse primers 0.5 μL each, T3 Super PCR Mix 16.5 μL;

[0014] PCR amplification program: pre-denaturation at 98°C for 2 min; denaturation at 98°C for 10 s, annealing at 55°C for 12 s, extension at 72°C for 12 s, for a total of 35 cycles; final terminal extension at 72°C for 2 min and storage at 4°C.

[0015] The present invention also provides a kit for identifying the genome of Isatis indigotica, which comprises the above-mentioned 193 pairs of primers and can be used to assist in the precise introduction of excellent gene fragments of Isatis indigotica, break the unfavorable linkage drag of distant hybridization between Isatis indigotica and Brassica crops, realize the efficient creation and utilization of germplasm resources, and be used for the breeding of Isatis indigotica.

[0016] The Isatis indigotica whole genome-specific Single-locus IP labeling primer developed by the present invention has the following advantages:

[0017] The invention develops for the first time a Single-locus IP marker primer specific to the whole genome of Isatis indigotica, which can realize the rapid identification of excellent gene fragments of distant hybridization offspring with Isatis indigotica as a donor and Brassica crops, and is not subject to specific restrictions on crop materials within the Brassica genus.

[0018] The marker primers provided by the present invention can effectively assist in the precise introduction of excellent gene fragments and target trait genes, and at the same time accurately track the size of chromosome fragments containing excellent genes of Isatis indigotica, help break undesirable linkage drag, realize efficient creation and utilization of germplasm resources, and at the same time provide important data support for the identification of chromosome fragments of genes related to improved traits of hybrid offspring. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Wayne plots of e-PCR in four cultivars of Isatis indigotica using the primer set developed for the cultivar Isain_V1.0.

[0020] Figure 2Wayne plots of e-PCR using consensus IP-tagged primers developed for four woad cultivars in the Brassica napus pan-genome, Brassica rapa, Brassica oleracea, and Brassica juncea genomes.

[0021] Figure 3 The identification results of four Single-locus IP marker primers developed based on the Isatis indigotica genome.

[0022] Figure 4 These are the identification results of 4 pairs of labeled primers provided in the present invention in the material to be tested.

[0023] Figure 5 These are the identification results of 4 pairs of labeled primers provided in the present invention in the material to be tested.

[0024] Figure 6 It is a marker map constructed based on Isatis indigotica chromosome 1 using the marker primers provided in the present invention.

[0025] Figure 7 It is a marker map constructed based on chromosome 5 of Isatis indigotica using the marker primers provided in the present invention.

[0026] Figure 8 It is a marker map constructed based on chromosome 6 of Isatis indigotica using the marker primers provided in the present invention.

[0027] Fig. 9 The present invention provides a marker map constructed based on chromosome 7 of Isatis indigotica using the marker primers provided in the present invention. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] Note: The methods not specifically described in the present invention are all conventional technical methods in the art, and the reagents and consumables not specifically described are all conventional reagents and consumables in the art.

[0030] The present invention aims at the defect that it is difficult to identify the exogenous woad chromosome fragments in the interspecific hybridization progeny of woad and Brassica napus, and provides a single-locus IP marker, marker primer and application specific to the woad genome. The single-locus IP marker and marker primer specific to woad are developed based on the genome information of woad and Brassica crops by using bioinformatics technology; and these markers are actually applied to the identification of the exogenous chromosome introduced fragments of woad in the interspecific hybridization progeny of woad and Brassica napus, and the application effect is remarkable. The specific scheme is as follows: Experimental Example 1 Development of Single-locus IP marker primers specific to the woad genome

[0031] 1. A method for developing a single-locus IP-labeled primer specific to the Isatis indigotica genome, comprising the following steps:

[0032] 1.1 Acquisition of Isatis indigotica genome information

[0033] First, we obtained the information of four Isatis indigotica genomes, namely, Isain_V1.0 (https: / / www.ncbi.nlm.nih.gov / data-hub / genome / GCA_900406415.1 / ), ASM1057779V1 (https: / / www.ncbi.nlm.nih.gov / data-hub / genome / GCA_010577795.1 / ), ASM1459570v1 (https: / / www.ncbi.nlm.nih.gov / data-hub / genome / GCA_014595705.1 / ), and ASM90040638v1 (https: / / www.ncbi.nlm.nih.gov / data-hub / genome / GCA_900406385.1 / ), and developed the Single-locus IP marker based on these four genomes.

[0034] 1.2 Development of primers for Isatis indigotica genomic IP labeling

[0035] 1.2.1 Primer design

[0036] In the Linux system, a script was written in Perl language to construct a database of the FASTA format sequence of the woad variety (Isain_V1.0), extract the exon sequences on both sides of the introns of all its genes, and generate the required format file of Primer3.0. The Isain_V1.0 genome was used here because the genome sequence was spliced ​​to the chromosome level, and the splicing of the other three genome sequences did not reach the chromosome level. Primers were designed in batches using primer3_2.5.0 software. Input the Isain_V1.0 woad template sequence FASTA file (genomic sequence file), set the intron length upper limit to 500, the primer length between 18 and 24 bps, with 20 bps being the best, and the primer Tm value to 60℃~65℃.

[0037] 1.2.2 e-PCR analysis

[0038] The e-PCR program (e-PCR_2.3.9) was used to perform primer electronic PCR analysis on the Isatis indigotica Isain_V1.0 genome and three other Isatis indigotica genome databases. The allowed number of mismatched bases (-n) was set to 2, the allowed number of gaps (-g) was set to 1, and the expected value of product length was 80-1500. The amplification results were analyzed using a Venn diagram (https: / / bioinfogp.cnb.csic.es / tools / venny / index.html). The results are shown in the figure. Figure 1 As shown, 30763 Single-locusIP markers common to the four Isatis indigotica genomes were obtained.

[0039] 1.2.3 Acquisition of the Single-locus IP marker specific to Isatis indigotica

[0040] ① Using the above 30763 IP markers, e-PCR analysis was performed on the Isatis indigotica genome (Isatis indigotica), Brassica napus pan-genome (Brassica napus pan-genome), Brassica olerace pan-genome (B. rapa pan-genome) and Brassica juncea genome (Brassica juncea). The software was used to draw a Venn diagram (http: / / bioinformatics.psb.ugent.be / cgi-bin / liste / Venn / calculate_venn.htpl) to reversely select the e-PCR results, that is, the single-locus IP marker sites of Isatis indigotica that were expected to be amplified in any of the reference genomes of Brassica napus pan-genome, Brassica rapa pan-genome, Brassica oleracea pan-genome and Brassica juncea were discarded. Finally, only the marker sites that were amplified in all four Isatis indigotica genomes and were not expected to have PCR products in any of the reference genomes of Brassica napus pan-genome, Brassica rapa pan-genome, Brassica oleracea pan-genome and Brassica juncea were retained. Finally, 16,796 single-locus IP markers unique to the Isatis indigotica genome were obtained. The Venn diagram analysis results after e-PCR analysis are shown in Figure 2. Figure 2 ② Using the Single-locus IP markers unique to the 16,796 Isatis indigotica genomes, a pair of primers was selected every 2 Mb according to the size of each chromosome for ePCR again. Here, the hash database constructed with the -w 3 parameter was used. ③ The primers obtained from the secondary ePCR were selected again according to the chromosomes, and 10 pairs of primers were selected for each chromosome for blast comparison.

[0041] 1.3 Verification of the Single-locus IP marker unique to Isatis indigotica

[0042] Primers were selected based on the final blast results. According to the size of each chromosome, the markers corresponding to the selected primers were evenly distributed on the chromosome as much as possible. 20 to 30 pairs of primers were selected for each chromosome, and a total of 245 single-locus IP markers were selected. The marker primers corresponding to the above 245 markers were actually verified in Isatis oleracea, Brassica napus, Brassica juncea, Cabbage and Brassica pekinensis.

[0043] 1.3.1 Test materials

[0044] Twelve samples of Isatis indigotica were collected from different provinces and cities in China (see Table 1 for details), including 1 Isatis indigotica, 1 Brassica rapa, 1 Cabbage, 3 Brassica juncea, 3 Brassica rapa conventional varieties and 3 Brassica rapa hybrid varieties. The Isatis indigotica material was Isatis indigotica No. 1 provided by Huazhong Agricultural University in Wuhan, and the other Brassica crop materials were provided by the Rapeseed Center of the Institute of Economic Crops, Yunnan Academy of Agricultural Sciences (see Table 2 for details). The breeding of Cangyou No. 1 (as described in the reference - Cui Cheng, Li Haojie, Zhang Jinfang, etc. Breeding of Cangyou No. 2 and a preliminary study on its dual-purpose value for oil and vegetables [J]. Chinese Journal of Oil Crops) was a hybrid plant obtained by somatic cell fusion of Brassica napus Huashuang No. 3 (2n= 38, AACC) and Isatis indigotica (2n= 14, II). Cytological identification showed that the hybrid plant contained all the chromosomes of the parents (2n= 52, AACCII). Subsequently, the hybrid plant was identified by backcrossing with Huashuang No. 3 three times, that is, a monomeric addition line (2n=39, AACC+1I1-7) with a single Isatis indigotica chromosome was identified and screened in the BC3F1 population. The monomeric addition lines were further screened in the BC4F1 population, and all 7 different addition lines identified were stored on MS medium. Finally, the pollen of the addition line g (a monomeric addition line (2n = 39, AACC + 1Ig) with an additional specific chromosome (g) of Isatis indigotica (2n = 14, II) as the male parent) was used to pollinate Huashuang No. 3, and plants with 39 chromosomes were selected for self-pollination. The plants were observed generation by generation, and finally in 2019, a plant line material with good specificity, consistency and stability was obtained, completing the breeding of Isatis oil No. 1.

[0045] Table 1 Statistical table of the source information of 12 Isatis indigotica materials

[0046]

[0047] Table 2 Material information of Brassica napus, Brassica juncea, Brassica oleracea and Brassica rapa

[0048]

[0049] 1.3.2 Test methods

[0050] DNA extraction: The genomic DNA of the above 24 materials were extracted using the CTAB small sample method.

[0051] IP labeling primer synthesis: Shanghai Biotech Co., Ltd.

[0052] PCR reaction and electrophoresis detection: PCR amplification was performed using T3 Super PCR Mix from Qingke Biotechnology Co., Ltd.

[0053] The PCR reaction system was 20 μL: 2.5 μL template DNA, 0.5 μL forward and reverse primers, and 16.5 μL T3 Super PCR Media.

[0054] PCR amplification program: pre-denaturation at 98°C for 2 min; denaturation at 98°C for 10 s, annealing at 55°C for 12 s, extension at 72°C for 12 s, for a total of 35 cycles; finally, terminal extension at 72°C for 2 min and storage at 4°C.

[0055] The PCR amplification products were separated by 1% agarose gel electrophoresis at a constant voltage of 160 V for 20 min. After electrophoresis, the gel imaging system (UVI Platinum / Explorer) was used to take pictures and save them.

[0056] 1.3.3 Test results

[0057] The 245 pairs of Single-locus IP marker primers selected and distributed on the 7 chromosomes of Isatis indigotica were used for amplification detection in the above 12 Isatis indigotica materials, 6 Brassica napus, 3 Brassica juncea, 1 Isatis oil, 1 Brassica oleracea, and 1 Brassica rapa. Among them, 193 pairs of Single-locus IP markers (see Table 3) showed the characteristics of Isatis indigotica-specific amplification, that is, specific bands were amplified only in Isatis indigotica varieties, and no amplification was found in Brassica napus, Brassica oleracea, Brassica juncea, and Brassica rapa. Some electrophoresis detection results are shown in Figure 3 , Figure 4 ,in, Figure 3 This is a preliminary screening diagram of 4 pairs of single-locus IP markers unique to the Isatis indigotica genome. Figure 4 It is the identification result of the labeled primer in all the tested materials except rapeseed oil.

[0058] Table 3 Single-locus IP markers unique to the Isatis indigotica genome

[0059]

[0060] Note: All primer sequences in Table 1 are numbered in sequence, and specifically each primer set is numbered in order of arrangement as SEQ ID NO.1~2, SEQ ID NO.3~......384, SEQ ID NO.385~386.

[0061] Experimental Example 2 Application Verification

[0062] This experimental example is to study the practical application of the single-locus IP labeled primer pair unique to Isatis indigotica in detecting exogenous fragments of Isatis indigotica oil, as follows:

[0063] The experimental materials were the same as those in Experimental Example 1, including Isatis indigo No. 1, Isatis indigo, Brassica napus, Brassica juncea, Brassica oleracea and Brassica rapa. The 193 pairs of Isatis indigo-specific Single-locus IP marker primers obtained in Experimental Example 1 were used to screen for linkage markers of Isatis indigo exogenously introduced fragments in Isatis indigo No. 1 and map linkage markers, so as to effectively identify Isatis indigo exogenous chromosome fragments in Brassica napus, as follows:

[0064] (a) Screening of Isatis indigo-specific markers in the Isatis indigo genome

[0065] The genomic DNA of all materials was extracted by CTAB small sample method, and PCR and electrophoresis detection were the same as in Experimental Example 1. Some screening electrophoresis results are shown in Figure 5 , Figure 5 The results of identification of 4 pairs of marker primers in the above 24 materials are shown. A total of 42 pairs of marker primers were amplified in Isatis indigotica and Isatis oil, and no marker sites were amplified in Brassica napus, Brassica juncea, Cabbage and Brassica rapa (see Table 4 for marker positions and sequences). Among them, 26 specific markers on chromosome 6 of Isatis indigotica were amplified in Isatis oil 1. The above 26 markers cover the entire chromosome 6 of Isatis indigotica. This result is consistent with the results of GISH analysis and identification of Isatis indigotica hybrid progeny by previous researchers (see reference [1] Yang Han, Kang Lei, Li Pengfei, et al. Creation and cytological analysis of the disomic addition line of Brassica napus-Isatis indigotica [J]. Chinese Journal of Oil Crops). In addition, 5, 7 and 4 specific markers were detected on chromosomes 1, 5 and 7 of Isatis indigotica, respectively, which were not detected in previous reports.

[0066] Table 4 Statistics of linked markers of exogenous fragments introduced into Isatis indigotica

[0067]

[0068] (b) Construction of specific marker map of chromosome fragments introduced exogenously into Isatis indigotica in candied oil

[0069] In order to further clarify the size and boundaries of the exogenous chromosome fragments of Isatis indigotica in oil, the specific markers upstream and downstream of the above differential markers were further screened, and the above results were combined to draw a specific marker map of the exogenous chromosome fragments of Isatis indigotica. The results are shown in Figure 6 , 7 , 8, 9. Among them, Figure 6 is a marker map constructed based on chromosome 1. Figure 7This is a marker map constructed based on chromosome 5. Figure 8 This is a marker map constructed based on chromosome 6. Fig. 9 is a marker map constructed based on chromosome 7. The Isatis indigotica marked in the above map is 12 Isatis indigotica materials, the Brassica crops are 11 Brassica materials, and the Isatis oil is Isatis oil No. 1; "+" means that the marker is amplified in this material; "-" means that the marker is not amplified in this material. Figure 6 , 7 , 8, 9 show that the markers on both sides of the exogenous fragment on chromosome 1 are I01_1211 and I01_1255, I01_1172 is the closest non-differential marker site to I01_1211, and I01_1267 is the closest non-differential marker site to I01_1255. This result shows that the inserted fragment of Isatis indigotica is a segment corresponding to the 13.18-14.34Mb interval on the chromosome between markers I01_1211 and I01_1255, with a size of about 1.16Mb; the markers on both sides of the exogenous fragment on chromosome 5 are I05_1048 and I05_1143, I05_0997 is the closest non-differential marker site to I05_1048, and I05_1171 is the closest non-differential marker site to I05_1143. This result shows that the inserted fragment of Isatis indigotica is The markers I05_1048 and I05_1143 were located in the corresponding segment of the 10.83-13.44Mb interval on the chromosome, with a size of about 2.61Mb; all 26 pairs of markers on chromosome 6 showed differences, and this result also verified the previous GISH analysis and identification results in the hybrid offspring of Isatis indigotica, that is, the complete Isatis indigotica chromosome 6 was introduced into Isatis oil; the markers at the ends of the exogenous fragment on chromosome 7 were I07_0060 and I07_0193, I07_0016 was the non-differential marker site closest to I07_0060, and I07_0244 was the non-differential marker site closest to I07_0193. This result showed that the Isatis indigotica insertion fragment was located between markers I07_0060 and I07_0193 in the corresponding segment of the 0.44-1.54Mb interval on the chromosome, with a size of about 1.11Mb. Thus, the results not only verified the results of previous GISH analysis and identification that the complete chromosome 6 of Isatis indigotica was introduced into Isatis indigotica oil, but also identified three new exogenous chromosome fragments from Isatis indigotica oil, which were located on Isatis indigotica chromosomes 1, 5 and 7, with sizes of approximately 1.16Mb, 2.61Mb and 1.11Mb respectively. This experimental example once again proved the effectiveness and feasibility of Isatis indigotica-specific Single-locus IP marker in identifying Isatis indigotica exogenous chromosome fragments.

[0070] In summary, the 193 markers and primers screened by the present invention are Single-locus IP markers and marker primers unique to the genome of Isatis indigotica, which can be used for specific identification of the genome of Isatis indigotica, can be used for identification and tracing of germplasm resources of Isatis indigotica, and can also be used for the improvement of Isatis indigotica high-quality genes or target trait genes, which has guiding significance for the breeding of Isatis indigotica. At the same time, the primer set can be used for detecting the target to be tested by PCR, for rapid identification of excellent gene fragments of the offspring of Isatis indigotica and Brassica crops in distant hybridization, and the primer set can be used for developing a kit for the identification of the Isatis indigotica genome. At the same time, the marker primers provided by the present invention can be used for the accurate introduction of effectively assisting excellent gene fragments of Isatis indigotica, breaking the poor linkage encumbrance of distant hybridization of Isatis indigotica and Brassica crops, realizing the efficient creation and utilization of germplasm resources, and can also be used for aspects such as breeding of Isatis indigotica.

[0071] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. A single-locus IP marker primer set specific to the Isatis indigotica genome, characterized in that: The primer set comprises 193 pairs of primers, and the nucleotide sequences thereof are shown in SEQ ID NO.1 to SEQ ID NO.386 respectively.

2. Use of the primer set as claimed in claim 1 in identifying exogenously introduced fragments of Isatis indigotica in interspecific hybridization progenies of Isatis indigotica and Brassica napus.

3. Use of the primer set according to claim 1 in any of the following, comprising: Used to assist the precise introduction of chromosome fragments of Isatis indigotica; Realize the efficient creation and identification of cabbage oil germplasm resources.

4. A kit for identifying the genome of Isatis indigotica, characterized in that: The kit comprises the primer set as claimed in claim 1.

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