Mustard type rape Bj subgenome specific single site IP marker primer group and application thereof

Abstract

The invention discloses a mustard type rape Bj subgenome specific single site IP labeled primer group and application thereof, and relates to the technical field of molecular biology. The primer group provided by the invention comprises 95 pairs of primers, and the nucleotide sequences of the primers are respectively shown as SEQ ID NO.1-190. The primer group is developed on the basis of a specific single site of the Bj subgenome of the brassica napus, and can be used for identifying an exogenous import fragment of the Bj subgenome of the brassica napus, Chinese cabbage and a filial generation of the brassica napus and the brassica napus; further, rapid identification of excellent exogenous gene segments of distant filial generations of brassica and brassica by taking brassica juncea as a donor can be realized, the size of the imported exogenous gene segments can be accurately tracked, and cross-species exchange of excellent genes derived from Bj subgenome and efficient creation and utilization of novel germplasm resources can be promoted; the method has an application prospect in genetic improvement breeding of brassica napus and various brassica crops.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology technology, and in particular to a method for identifying Brassica juncea B j A specific single site amplification marker primer set universal for subgenomic chromosome fragments and its application. Background Art

[0002] The genus Brassica in the family Cruciferae contains three basic diploid species: Brassica rapa ( B. rapa L., A r A r )、Kale( B.oleracea L., C o C o )、Black mustard( B.nigra L.,BB ), three tetraploid composite species Brassica napus ( B. napus L.,A n A n C n C n ), Brassica juncea ( B.juncea L., A j A j B j B j ) and Ethiopian mustard ( B. carinata L., B c B c C c C c ). For many years, the research on Brassica has mainly focused on the A and C genomes, while the research on the B genome, which is an important component of the Brassica genus, has lagged behind. There are many excellent traits in the B genome species of Brassica, which is one of the important gene treasures to be discovered in the Brassica genus. Among them, Brassica juncea is one of the representative species of the B genome of Brassica. Because of its good affinity for distant hybridization with Brassica napus, it is often used to expand and improve the Brassica napus genome library. At present, the black shank resistance gene / chromosome fragment of Brassica juncea (Rashid MH, Hausner G, Fernando WGD. Molecular and phenotypic identification of B-genome introgression linked to Leptosphaeria maculansresistant gene Rlm6in Brassica napus × B. juncea interspecific hybrids.Euphytica, 2018, 214(11).), yellow seed gene / chromosome fragment (Liu Zhongsong, Guan Chunyun, Chen Sheyuan, Liu Shuyan, Yang Liu. Research on the introduction of excellent traits of Brassica juncea into Brassica napus. Agricultural Science&Technology, 2010, 11(06):49-52.), pod cracking resistance gene / chromosome fragment and multilocular pod gene / chromosome fragment (Chen Cuiping, Xiao Lu, Du Dezhi. Research progress on multilocular traits of rapeseed. Chinese Journal of Oil Crops, 2018, 40(03):446-451.) have been successfully introduced into the Brassica napus genome.

[0003] With the continuous creation of hybrid offspring of Brassica rapa and Mustard, it is crucial to accurately identify exogenous chromosome fragments in the early stage and strengthen the screening of exogenous chromosome fragments in the distant hybridization offspring in the later stage. At present, the technical means for the identification of exogenous chromosome fragments have gradually developed from early cytological identification, biochemical marker identification, phenotypic identification, etc. to modern in situ hybridization identification and molecular marker identification. Among them, molecular markers are not affected by environmental factors and have the advantages of high detection efficiency, simple operation, strong stability and high throughput. They have gradually become a necessary technical means for the identification of distant hybridization offspring and their breeding applications. Although there are reports on the use of molecular markers of the Brassica B genome to identify the distant hybridization offspring of Brassica rapa (Tu Yuqin, Tang Jie, Zhang Yang, Xin Jiajia, Tu Weifeng, Ji Hongli, Dai Xinglin. Creation of new germplasm with limited inflorescence by hybridization of Brassica rapa and Brassica rapa. Journal of Plant Genetic Resources, 2020, 21(01):74-82.), there are no reports specifically on the identification of Brassica rapa B. j There are no reports on molecular markers for subgenome development. In addition, the reported molecular markers for the Brassica B genome have not yet been systematically studied for their universality, and cannot meet the current requirements for efficient identification of exogenous B genome fragments in the interspecific hybrids of Brassica. Therefore, it is necessary to develop a complete set of B. juncea-type rapeseed with high universality and quantitative advantages. j Genome-specific markers will effectively promote the breeding and improvement of Brassica crops and the process of distant hybridization breeding. Summary of the Invention

[0004] The present invention aims to provide a method for identifying Brassica juncea B j A subgenome-specific universal single-site amplification marker primer set for the hybridization of Brassica juncea and Brassica oleracea. j Identification of subgenomic introduced chromosomal segments.

[0005] In order to achieve the above object, the present invention provides a mustard type rapeseed Bj A subgenome-specific single-site IP marker primer set comprises 95 pairs of primers, and the nucleotide sequences thereof are shown in SEQ ID NO.1 to SEQ ID NO.190, respectively.

[0006] The marker primer set provided by the present invention can be used for identifying exogenous fragments of Brassica juncea, and is particularly useful for Brassica juncea B j Identification of exogenous fragments of subgenomic origin.

[0007] The marker primer set provided by the present invention can be used for the hybrid offspring of Brassica juncea B j Identification of subgenomic exogenously introduced fragments.

[0008] The present invention also provides a method for preparing a method for preparing a B. j A kit for subgenome identification, comprising the above-mentioned 95 pairs of primers.

[0009] The primer set or kit provided by the present invention can be used in any of the following, including: The offspring of distant hybridization between rapeseed and Brassica crops are derived from B j Rapid identification of high-quality gene fragments in subgenomes; Used to assist mustard rape B j Accurate introduction of high-quality gene fragments from subgenomes; Achieve efficient creation and identification of Brassica crop germplasm resources such as Brassica napus; Used for breeding of mustard rapeseed.

[0010] The present invention also provides a B gene in the offspring of distant hybridization between rapeseed and Brassica crops. j The method for identifying subgenomic exogenous fragments includes the following: 1) PCR amplification of the whole genome of the sample to be tested was performed using the aforementioned 95 primer pairs to obtain marker loci that were amplified in Brassica juncea but not in other Brassica crops; 2) Using the obtained marker site as the anchor point, select the previously predicted B on both sides of it j Subgenome-specific markers are verified by PCR; after group verification, PCR amplification is performed again in the sample to be tested, and the results are analyzed; 3) Use the obtained analysis results to draw the B. j Subgenomic exogenous introduction of chromosome fragment-specific marker maps to achieve the B. juncea type in the offspring of sugarcane-mustard hybrids j Accurate identification of subgenomic exogenously introduced fragments.

[0011] Preferably, the PCR amplification in the above identification method comprises the following amplification conditions: PCR reaction system: 2.5 μL template DNA, 0.5 μL forward and reverse primers, 0.5 μL each, T3 Super PCR Mix 16.5 μL; 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.

[0012] The present invention has the following advantages: The present invention is the first to develop Brassica juncea B j Subgenome-specific single site amplification marker primers can achieve the hybridization of Brassica juncea with Brassica rapa as the donor and the Brassica rapa offspring B j Rapid identification of high-quality subgenomic gene fragments.

[0013] The marker primer provided by the present invention can effectively assist the PCR of Brassica juncea B. j The precise introduction of subgenomic chromosome fragments and the j Accurately track the size of subgenomic chromosome fragments to achieve efficient creation and utilization of germplasm resources, while providing important data support for the identification of chromosome fragments for improved traits in hybrid offspring. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The figure shows the Venn diagram of single loci amplified from three Brassica juncea genomes in the present invention.

[0015] Figure 2 Venn diagram showing IP signatures for e-PCR products missing from four Brassica crop genomes.

[0016] Figure 3 Brassica juncea B j The electrophoresis results of the preliminary screening of single-site specific markers in the genome, where B01-2541, B05-1853, and B07-0497 are primer numbers, J01-J04 represent four Brassica juncea varieties, and B01-B04 represent two Brassica rapa varieties, one Brassica rapa variety, and one Brassica oleracea variety, respectively.

[0017] Figure 4 Brassica juncea B j Verification results of a small population of single-site-specific markers in the genome, where B04-1141 and B03-2551 are primer numbers, J01-J12 are 12 Brassica juncea varieties, B01-B02 are 2 Brassica rapa varieties, B03-B04 are 2 Cabbages, B05-B11 are 2 conventional Brassica rapa varieties and 5 Brassica rapa hybrid varieties, and B12 is 1 Brassica juncea (a hybrid of Brassica rapa and Brassica juncea).

[0018] Figure 5It is the exogenous B of Brassica juncea in the offspring of the distant hybridization between sugarcane and mustard. j The results of genome fragment size identification, where the left side of the chromosome is the marker position; the right side is the marker name; + means that Brassica juncea B was screened j Genome-specific markers; - means not detected by screening. DETAILED DESCRIPTION

[0019] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0020] Note: Unless otherwise noted, the experimental methods in the following examples are conventional methods, performed according to the techniques and conditions described in literature in the field or according to product specifications. Materials and reagents used in the following examples, unless otherwise noted, are commercially available.

[0021] The present invention is directed to the exogenous mustard-type rapeseed B in the hybrid offspring of rapeseed sweet and mustard. j The identification technology of subgenomic chromosome segments is insufficient, and a B. j Development of subgenome-specific single-site amplification markers, marker primers, and their applications. Using bioinformatics techniques, based on the genome information of Brassica crops, we developed single-site amplification markers and marker primers specific to Brassica juncea; and applied these markers to the hybrid offspring of Brassica juncea and Brassica napus, Brassica juncea B. j The application effect is significant in the identification of subgenomic exogenous chromosome introduced fragments. The specific scheme is as follows:

[0022] Example 1 Development of specific single site markers and marker primers Brassica juncea B j The development of subgenome-wide specific single-site markers and marker primers includes the following steps: 1. Acquisition of genomic information used in marker development The Brassica crop Brassica napus pan-genome (sequence download address: http: / / cbi.hzau.edu.cn / cgi-bin / rape / download_ext); the reference genome data for Brassica rapa and B. oleracea are from the Brassicaceae database BRAD (http: / / brassicadb.org / brad / ); the genome information for three Brassica juncea varieties, from v1.5, Canus_v.1.4, and T84-66_v1, is available for download at: v1.5: http: / / brassicadb.org / brad / datasets / pub / Genomes / Brassica_juncea / V1.5 / ; Canus_v.1.4: https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCA_015484525.1 / ; T84-66_v1: https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCA_001687265.1 / .

[0023] 2. Primer design and e-PCR analysis A Perl script was written on a Linux system to construct a database using the Brassica juncea V1.5 genome FASTA format sequence. Exon sequences flanking introns of all genes were extracted and generated into the required Primer3.0 format. Primers were then batch-designed using Primer3 2.5.0 software. The V1.5 template sequence FASTA file (genomic sequence file) was input, with an intron length limit of 500, primer lengths between 18 and 24 bp, with 20 bp being optimal, and a primer Tm of 60°C-65°C. Primers were validated using the e-PCR program (e-PCR_2.3.9) against the Brassica juncea genome (V1.5) and two other Brassica juncea genomes (Canus_v.1.4 and T84-66_v1). The permissible number of mismatches (-n) was set to 2, the permissible number of gaps (-g) was set to 1, the expected product length was set to 80-1500, and the number of amplification sites was set to 1. A total of 69,678 specific single-site amplification marker primers common to the three B. juncea genomes were obtained by union analysis using a Venn diagram (https: / / bioinfogp.cnb.csic.es / tools / venny / index.html). The Venn diagrams of single-site amplification markers for the three B. juncea genomes are shown in Figure 2. Figure 1 shown.

[0024] 3. Brassica juncea B j Development of subgenomic universal specific single site marker primers, as follows: To improve the versatility of specific primers, e-PCR analysis was performed using the aforementioned 69,678 marker primers in the Brassica napus pan-genome, Brassica rapa, and Brassica oleracea genomes. Venn diagrams were generated using software (http: / / bioinformatics.psb.ugent.be / cgi-bin / liste / Venn / calculate_venn.htpl). The e-PCR results were subjected to a reverse selection process, discarding markers amplified at a single locus in the Brassica juncea genome that were expected to amplify in any of the Brassica juncea, Brassica rapa, or Brassica oleracea reference genomes. Only 36,157 markers amplified in all three Brassica juncea genomes and not expected to produce PCR products in any of the Brassica juncea, Brassica rapa, or Brassica oleracea reference genomes were retained. This resulted in a Venn diagram of e-PCR-free IP markers for four Brassica crop genomes (see Figure 1). Figure 2 shown.

[0025] ② Using 36157 selected rapeseed B j The distribution of genome-specific single-site amplification markers was analyzed by selecting a primer pair every 2 Mb on each chromosome and performing e-PCR again. The hash database constructed with the -w 3 parameter was used.

[0026] ③The single site amplification primers obtained by the secondary e-PCR were then blasted again, and 20 pairs of primers were selected for each chromosome.

[0027] 4. Brassica juncea B j Specific verification of genome-specific single site amplification markers: Primers were selected based on the final blast comparison results. 15 primer pairs were selected for each chromosome for actual PCR amplification and agarose gel analysis to determine the effectiveness of the above markers in practical applications. The details are as follows: The experimental materials were 12 samples of Brassica juncea, 2 samples of Brassica rapa conventional varieties, 6 samples of Brassica rapa hybrid varieties, 2 samples of Chinese cabbage, and 2 samples of Brassica oleracea from various prefectures in Yunnan Province (see Table 1 for details). Table 1 List of test materials

[0028] The test methods include the following: DNA extraction: The genomic DNA of the above 24 materials were extracted using the CTAB sample method.

[0029] IP-labeled primer synthesis: Shanghai Sangon Technology Co., Ltd.

[0030] PCR reaction and electrophoresis detection: PCR amplification was performed using T3 Super PCR Mix from Qingke Biotechnology Co., Ltd.

[0031] 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 Mix.

[0032] 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, and 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.

[0033] 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 was photographed and stored using a gel imaging system (UVI Platinum / Explorer).

[0034] The above selection was used to distribute the rapeseed B j A total of 120 pairs of single-site marker primers on the 8 chromosomes of the subgenome were amplified in 12 Brassica juncea, 6 Brassica oleracea hybrids, 2 Brassica oleracea conventional varieties, 2 Brassica oleracea and 2 Brassica rapa listed in Table 1. Among them, 95 pairs of marker primers showed the presence of the B. j The characteristics of subgenome-specific amplification are that the specific bands were amplified only in Brassica juncea varieties, but not in Brassica rapa, Brassica oleracea, and Brassica rapa. Some electrophoresis test results are shown in Figure 3 、 Figure 4 ,in, Figure 3 3 pairs of Brassica juncea B j Preliminary screening of subgenome-specific single-site amplification markers, Figure 4 This study validated two pairs of single-locus amplification markers in 12 Brassica juncea and 12 Brassica (A / C genotype) varieties. Detailed information for the 95 primer pairs is shown in Table 2. Each pair is numbered as SEQ ID NOs. 1-2, 3-4, and 189-190, respectively.

[0035] Table 2 95 pairs of Brassica juncea B j Subgenome-specific markers and marker primers

[0036] Example 2 Verification of the applicability of single site labeling and labeling primers For Brassica juncea B jApplication of subgenomic-specific single locus markers in identifying the progeny of Glycine max-Mustard hybrids j The applications of genomic chromosome fragments are as follows: The experimental materials and population construction are as follows: Experimental materials: Brassica juncea 109 (siliques with horizontal angles) and Brassica oleracea 337 (siliques with vertical angles).

[0037] Population construction: Hybridization was performed using Brassica juncea as the female parent (109) and Brassica oleracea as the male parent (337). Subsequently, backcrossing was performed using Brassica oleracea 337 as the recurrent parent to construct the BC1, BC2, and BC3 populations.

[0038] The specific verification method is as follows: DNA extraction: The genomic DNA of the above-mentioned single plants of the Glycine max hybrid and the single plants of the backcross BC1, BC2 and BC3 populations were extracted using the CTAB small sample method.

[0039] IP marker primer synthesis: The markers used in this experiment were 95 pairs of Brassica juncea B selected in Example 1. j Subgenome-specific markers and corresponding marker primers were synthesized by Shanghai Sangon Technology Co., Ltd.

[0040] PCR reaction and electrophoresis detection: PCR amplification was performed using T3 Super PCR Mix from Qingke Biotechnology Co., Ltd.

[0041] 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.

[0042] 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, and 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.

[0043] 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 was photographed and stored using a gel imaging system (UVI Platinum / Explorer).

[0044] First, 95 pairs of Brassica juncea B jGenome-specific marker primers were used to detect the individual plants of BC1, BC2 and BC3 populations. The results showed that a pair of marker loci B06-0962 were amplified in the oblique-type plants in the three backcross generations, but not in the vertical-type plants. Then, B06-0962 was used as an anchor marker to further select other predicted B markers within 5M of the B06-0962 marker on chromosome B6. j PCR verification of subgenome-specific single-site amplification markers ultimately led to accurate identification of Brassica juncea B. j The specific size of the exogenous genomic fragment is the segment corresponding to the 5.90-10.56Mb interval of chromosome B06, which is 4.66Mb. The end markers on both sides of the exogenous fragment on chromosome B06 are B06-0706 and B06-1157, B06-0682 is the closest non-differential site to B06-0706, and B06-1179 is the closest non-differential site to B06-1157. There are 5 differential markers in total. j The results of the construction of the genome exogenous fragment size and specific marker map are as follows Figure 5 This example once again proves that Brassica juncea B j Application of subgenome-specific single-site IP markers and their marker primers in identifying B. juncea in sugarcane-mustard distant hybridization j Validity and feasibility of subgenomic exogenous chromosome fragments.

[0045] In summary, the 95 markers and marker primers screened by the present invention are B. j Subgenome-specific single-site IP markers and marker primers can be used for the B generation of distant hybrids between Brassica juncea and Brassica crops. j The specific identification of excellent sub-genome gene fragments can realize the efficient creation and identification of Brassica crop germplasm resources such as Brassica napus. At the same time, this set of primers can be used to detect the target by PCR. This primer set can be used to develop a primer for Brassica juncea B. j The kit for subgenomic identification. Furthermore, the marker primers provided by the present invention can be used to effectively assist in the precise introduction of superior gene fragments from Brassica juncea, enabling efficient creation and utilization of germplasm resources. It can also be used in aspects such as Brassica juncea breeding, and has significant application value.

[0046] Although the present invention has been described in detail through the above preferred embodiments, it should be understood that the above description is not intended to limit the present invention. After reading the above description, various modifications and substitutions of the present invention will become apparent to those skilled in the art. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A mustard-type rapeseed B j The subgenome-specific single site IP labeling primer set is characterized by: The labeled primer set comprises 95 pairs of primers, and the nucleotide sequences thereof are shown as SEQ ID NO.1 to SEQ ID NO.190, respectively.

2. Use of the labeled primer set according to claim 1 in identifying exogenous fragments in Brassica juncea.

3. The use according to claim 2, characterized in that The exogenous fragment is Brassica juncea B j Subgenomic origin.

4. The marker primer set according to claim 1 is used to detect the expression of B. j Application in identification of exogenously introduced subgenomic fragments.

5. A method for the preparation of rapeseed B j A kit for subgenome identification, characterized in that The kit comprises the primer set according to claim 1.

6. Use of the labeled primer set according to claim 1 or the kit according to claim 5 in any one of the following, comprising: The offspring of distant hybridization between rapeseed and Brassica crops are derived from B j Rapid identification of high-quality gene fragments in subgenomes; Used to assist mustard rape B j Precise introduction of high-quality subgenomic gene fragments; To achieve efficient creation and identification of Brassica crop germplasm resources.

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