A type of mustard-like rapeseed B j Subgenome-specific single-site IP marker primer set and its application

Abstract

This invention discloses a mustard-type rapeseed B j This invention relates to a subgenome-specific single-site IP marker primer set and its applications, falling within the field of molecular biology. The primer set provided by this invention comprises 95 primer pairs, with nucleotide sequences shown in SEQ ID NO. 1~190. This primer set is based on Brassica oleracea var. chinensis (Brassica oleracea var. chinensis). j Developed from a single site specific to the subgenome, it can be used for the B-type rapeseed in Brassica napus, Chinese cabbage, and hybrids of Brassica napus and Brassica oleracea. j Identification of exogenous subgenomic fragments enables rapid identification of superior exogenous gene fragments in the offspring of distant hybridization between rapeseed and Brassica species, as well as accurate tracking of the size of the introduced exogenous gene fragments, promoting the development of gene sequences derived from B. j The cross-species exchange of superior genes from subgenomes and the efficient creation and utilization of new germplasm resources have promising applications in the genetic improvement breeding of rapeseed and various Brassica crops.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, specifically to a method for identifying rapeseed B (a type of rapeseed). j A set of universal, specific, single-site amplification marker primers for subgenomic chromosome segments and their applications. Background Technology

[0002] Brassica genus of the Brassicaceae family contains three diploid basic species, including Chinese cabbage (Brassica napus). B. rapa L., A r A r ), cabbage ( B.oleracea L., C o C o ), black mustard B.nigra L.,BB), three tetraploid complex species of Brassica napus ( B.napus L.,A n A n C n C n ), mustard-type rapeseed ( 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, research on the Brassica genus has mainly focused on the A and C genomes, while research on the B genome, an important component of the genus, has lagged behind. Many desirable traits exist in Brassica B genome species, making it one of the important gene pools of the genus yet to be explored. Among them, rapeseed (Brassica rapa) is one of the representative species of the Brassica B genome, and due to its good compatibility with Brassica napus (Brassica napus var. alopecuroides), it is often used to expand and improve the Brassica napus genome library. Currently, the black shank resistance gene / chromosomal fragment of rapeseed (Rashid MH, Hausner G, Fernando WGD. Molecular and phenotypic identification of B-genome introgression linked to Leptosphaeria maculans resistant gene) is being studied. Rlm6In Brassica napus × B. juncea interspecific hybrids.Euphytica, 2018, 214(11).), the yellow seed gene / chromosome fragment (Liu Zhongsong, Guan Chunyun, Chen Sheyuan, Liu Shuyan, Yang Liu. Research on the introduction of superior traits of mustard-type rapeseed into Brassica napus-type rapeseed (English). Agricultural Science & Technology, 2010, 11(06):49-52.), the anti-cracking pod gene / chromosome fragment and the multi-locular pod gene / chromosome fragment (Chen Cuiping, Xiao Lu, Du Dezhi. Research progress on multi-locular traits in 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 Brassica napus-Mustela hybrid offspring, accurate identification in the early stage and enhanced screening in the later stage of distant hybrid offspring have become crucial. Currently, the technical means for identifying exogenous chromosome fragments have gradually evolved from early cytological identification, biochemical marker identification, and phenotypic identification to modern in situ hybridization identification and molecular marker identification. Among these, molecular markers, due to their independence from environmental factors and advantages such as high detection efficiency, simple operation, strong stability, and high throughput, have gradually become an essential technical means for the identification of distant hybrid offspring and their breeding applications. Although there are research reports on the use of Brassica napus B genome molecular markers to identify distant hybrid offspring of Brassica napus (Tu Yuqin, Tang Jie, Zhang Yang, Xin Jiajia, Tu Weifeng, Ji Hongli, Dai Xinglin. Creation of new determinate inflorescence germplasm from Brassica napus-Mustela hybridization. Journal of Plant Genetic Resources, 2020, 21(01):74-82.), there are still many studies specifically targeting Brassica napus B genome markers. j No molecular markers for subgenome development have been reported. Furthermore, the reported molecular markers for the Brassica genus B genome have not yet undergone systematic and universal studies, failing to meet the current requirement for efficient identification of exogenous B genome fragments in the progeny of a wide range of interspecific hybrids within the Brassica genus. Therefore, developing a comprehensive set of molecular markers for the B genome of rapeseed with high universality and quantitative advantages is crucial. j Genome-specific markers will strongly promote the breeding improvement and distant hybridization of Brassica crops. Summary of the Invention

[0004] The purpose of this invention is to provide a method for identifying rapeseed type B. j A set of universal, single-site amplification marker primers specific to the subgenome can be used for progeny B of interspecific hybrids between mustard-type and rapeseed-type rapeseed. j Identification of subgenomic segments introduced into chromosomes.

[0005] To achieve the above objectives, the present invention provides a mustard-type rapeseed Bj A subgenome-specific single-site IP marker primer set, comprising 95 primer pairs, the nucleotide sequences of which are shown in SEQ ID NO.1 to SEQ ID NO.190.

[0006] The marker primer set provided by this invention can be used for the identification of exogenous fragments in rapeseed, especially in rapeseed B. j Identification of exogenous fragments of subgenomic origin.

[0007] The marker primer set provided by this invention can be used in the hybrid progeny of mustard-type rapeseed B. j Identification of exogenous subgenomic fragments.

[0008] The present invention also provides a method for use in rapeseed B (a type of mustard). j A kit for subgenomic identification, which contains the 95 pairs of primers mentioned above.

[0009] The primer set or kit provided by this invention can be used in any of the following, comprising:

[0010] The offspring of distant hybridization between rapeseed and Brassica oleracea crops originated from B. j Rapid identification of superior gene fragments in subgenomes;

[0011] Used to assist mustard-type rapeseed B j Precise introduction of superior gene fragments from subgenomes;

[0012] To achieve efficient creation and identification of Brassica genus germplasm resources, such as Brassica napus;

[0013] Used for breeding of mustard-type rapeseed.

[0014] This invention also provides a B-type offspring from a distant hybridization of rapeseed and a Brassica genus crop. j Methods for identifying exogenous subgenomic fragments include the following:

[0015] 1) Using the above 95 pairs of primers, PCR amplification was performed on the whole genome of the sample to be tested to obtain marker sites that were amplified in rapeseed but not in other crops of the Brassica genus.

[0016] 2) Using the obtained marker sites as anchor points, select B sites predicted earlier on both sides of them. j Subgenome-specific markers were validated by PCR; after population validation, PCR amplification was performed again on the test samples, and the results were analyzed.

[0017] 3) Use the obtained analysis results to draw a diagram of rapeseed type B. j Subgenomic exogenous introduction of chromosome fragment-specific marker mapping to achieve the generation of mustard-type rapeseed B in the offspring of sweet potato-mustard hybrids.j Accurate identification of exogenous fragments introduced into the subgenome.

[0018] Preferably, the PCR amplification in the above identification method includes the following amplification conditions:

[0019] PCR reaction system: 2.5 μL template DNA, 0.5 μL each of forward and reverse primers, 16.5 μL T3 Super PCR Mix;

[0020] PCR amplification program: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 55℃ annealing for 12 s, 72℃ extension for 12 s, for a total of 35 cycles; final extension at 72℃ for 2 min, and storage at 4℃.

[0021] The present invention has the following advantages:

[0022] This invention is the first to develop mustard-type rapeseed B j Subgenome-specific single-site amplification marker primers can be used to achieve the amplification of progeny B from distant hybridization between mustard-type rapeseed and Brassica napus. j Rapid identification of superior gene fragments in subgenomes.

[0023] The marker primers provided by this invention can effectively assist rapeseed B-type rapeseed in production. j Precise importation of subgenomic chromosome fragments, while simultaneously targeting B... j Accurate tracking of subgenomic chromosome fragment sizes enables efficient creation and utilization of germplasm resources, while providing important data support for chromosome fragment identification in hybrid offspring to improve traits. Attached Figure Description

[0024] Figure 1 This is a Venn diagram of three single sites of genome amplification in rapeseed of the mustard type in this invention.

[0025] Figure 2 Venn diagram of IP markers without product in e-PCR of four Brassica genus crops.

[0026] Figure 3 Mustard-type rapeseed B j Preliminary screening electrophoresis results of single-site specific markers in the genome: B01-2541, B05-1853, and B07-0497 are primer numbers; J01-J04 are 4 mustard-type rapeseed samples; and B01-B04 are 2 Brassica napus samples, 1 Chinese cabbage sample, and 1 Chinese cabbage sample.

[0027] Figure 4 Mustard-type rapeseed B jThe results of small-group validation of single-site specific markers in the genome are as follows: B04-1141 and B03-2551 are primer numbers; J01-J12 are 12 mustard-type rapeseed; B01-B02 are 2 Chinese cabbage; B03-B04 are 2 cabbage; B05-B11 are 2 conventional varieties of cabbage-type rapeseed and 5 hybrid varieties of cabbage-type rapeseed; and B12 is 1 mustard-type rapeseed (a hybrid of cabbage-type rapeseed and mustard-type rapeseed).

[0028] Figure 5 Exogenous B of mustard-type rapeseed in the distant hybrids of sweet and mustard j Genomic fragment size identification results, where the left side of the chromosome represents the marker location; the right side represents the marker name; + indicates screening of rapeseed B (Brassica napus) type. j Genome-specific markers; - indicates not detected. Detailed Implementation

[0029] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Note: Unless otherwise specified, the experimental methods in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0031] This invention targets exogenous mustard-type rapeseed B in the offspring of interspecific hybridization between rapeseed and mustard. j Due to insufficient technology for identifying subgenomic chromosome segments, a method for identifying mustard-type rapeseed B is proposed. j This study focuses on the development, primers, and applications of subgenome-specific single-site amplification markers for rapeseed (Brassica rapa) using bioinformatics techniques. Based on the genomic information of Brassica crops, single-site amplification markers and primers specific to rapeseed (Brassica rapa) were developed. These markers were then applied to the interspecific hybrid progeny of rapeseed (Brassica rapa) and Brassica napus (Brassica napus) – specifically, rapeseed (Brassica rapa) type B. j The application has shown significant effectiveness in the identification of exogenous chromosome fragments introduced into subgenomic genomes. The specific protocol is as follows:

[0032] Example 1: Development of Specific Single-Site Markers and Marker Primers

[0033] Mustard-type rapeseed B j The development of subgenomic universal specific single-site markers and marker primers involves the following steps:

[0034] 1. Acquisition of genomic information used in marker development

[0035] The pangenome of *Brassica napus* (type 1) is available for download at http: / / cbi.hzau.edu.cn / cgi-bin / rape / download_ext; reference genome data for *Brassica oleracea* and *Brassica oleracea* are from the Brassicaceae database BRAD (http: / / brassicadb.org / brad / ); genome information for three *Brassica rapa* (type 1) genomes are available for download from v1.5, Canus_v.1.4, and T84-66_v1.

[0036] v1.5:

[0037] http: / / brassicadb.org / brad / datasets / pub / Genomes / Brassica_juncea / V1.5 / ;

[0038] Canus_v.1.4:

[0039] https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCA_015484525.1 / ;

[0040] T84-66_v1:

[0041] https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCA_001687265.1 / .

[0042] 2. Primer design and e-PCR analysis

[0043] Under Linux, a Perl script was used to construct a database of FASTA format sequences from the rapeseed V1.5 genome, extracting exon sequences flanking introns of all genes and generating the required format files for Primer 3.0. Primers were designed in batches using Primer 3 2.5.0 software. The V1.5 template sequence FASTA file (genome sequence file) was input, with an upper limit of 500 intron length, primer length between 18-24 bps (20 bps being optimal), and a primer Tm value of 60℃-65℃. The e-PCR program (e-PCR_2.3.9) was executed to validate primers in the rapeseed genome (V1.5) and the remaining two rapeseed genomes (Canus_v.1.4, T84-66_v1) databases. The allowed number of mismatched bases (-n) was set to 2, the allowed number of gaps (-g) to 1, the expected product length to be 80-1500, and the number of amplification sites to 1. Union analysis using Venn diagrams (https: / / bioinfogp.cnb.csic.es / tools / venny / index.html) yielded 69,678 primers identifying specific single-site amplification markers common to the genomes of the three rapeseed species. The Venn diagrams for these single-site amplification in the three rapeseed genomes are shown below. Figure 1 As shown.

[0044] 3. Mustard-type rapeseed B j The development of universal, specific, single-site marker primers for subgenomes is detailed below:

[0045] ① To improve the universality of specific primers, e-PCR analysis was performed on the pan-genome of *Brassica napus*, *Brassica rapa*, and *Brassica oleracea* using the aforementioned 69,678 marker primers. Venn diagrams were constructed using software (http: / / bioinformatics.psb.ugent.be / cgi-bin / liste / Venn / calculate_venn.htpl). Reverse selection was performed on the e-PCR results, discarding single-site amplification marker sites in the *Brassica rapa* genome that were expected to amplify in any of the *Brassica rapa*, *Brassica rapa*, and *Brassica oleracea* reference genomes. Only 36,157 markers that amplified in the three *Brassica rapa* genomes and did not contain the expected PCR products in any of the *Brassica rapa*, *Brassica rapa*, and *Brassica oleracea* reference genomes were retained. This yielded Venn diagrams of IP markers without e-PCR products for the four *Brassica* genus genomes. Figure 2 As shown.

[0046] ② Using 36,157 selected mustard-type rapeseed B jThe distribution of genome-specific single-site amplification markers was analyzed. A pair of primers was selected for each 2Mb of chromosome for e-PCR. Here, a hash database constructed using the -w 3 parameter was used.

[0047] ③Then the single-site amplification primers obtained from the second e-PCR were subjected to blast comparison again, and 20 pairs of primers were selected for each chromosome.

[0048] 4. Mustard-type rapeseed B j Specificity verification of genome-specific single-site amplification markers:

[0049] Primers were selected based on the final BLAST alignment results. Fifteen primer pairs were selected for each chromosome for actual PCR amplification and agarose gel electrophoresis to determine the effectiveness of the markers in practical applications, as detailed below:

[0050] The experimental materials were obtained from various prefectures and cities in Yunnan Province: 12 mustard-type rapeseed materials, 2 conventional varieties of Brassica napus, 6 hybrid varieties of Brassica napus, 2 Chinese cabbage, and 2 cabbage varieties (see Table 1 for details).

[0051] Table 1. List of Experimental Materials

[0052]

[0053] The test methods include the following:

[0054] DNA extraction: Genomic DNA from all 24 samples was extracted using the CTAB small sample method.

[0055] IP-tagged primers were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0056] PCR reaction and electrophoresis detection: PCR amplification was performed using T3 Super PCR Mix from Qingke Biotechnology Co., Ltd.

[0057] The PCR reaction system consisted of 20 µL of: 2.5 µL template DNA, 0.5 µL each of forward and reverse primers, and 16.5 µL of T3 Super PCRMix.

[0058] PCR amplification program: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 55℃ annealing for 12 s, 72℃ extension for 12 s, for a total of 35 cycles; final extension at 72℃ for 2 min, and then stored at 4℃.

[0059] 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.

[0060] Using the above selection method, rapeseed B, which is distributed in mustard-type rapeseed, was selected. j A total of 120 single-site marker primer pairs were used on the eight chromosomes of the subgenome. These primer pairs were amplified and detected in 12 mustard-type rapeseed accessions, 6 Brassica napus hybrids, 2 conventional Brassica napus accessions, 2 Brassica napus varieties, and 2 Chinese cabbage accessions (see Table 1). 95 of these marker primer pairs showed B markers in mustard-type rapeseed. j The characteristics of subgenome-specific amplification are that specific bands are amplified only in mustard-type rapeseed varieties, and no amplification is observed in Brassica napus, Brassica oleracea, and Chinese cabbage. Some electrophoresis results are shown below. Figure 3 , Figure 4 ,in, Figure 3 For 3 pairs of mustard-type rapeseed B j Preliminary screening diagram of subgenome-specific single-site amplification markers Figure 4 This study validated two pairs of single-site amplification markers in 12 rapeseed (Brassica oleracea) and 12 Brassica (A / C genome) crops. Details of the 95 primer pairs are shown in Table 2. The 95 primer pairs were sequentially numbered as SEQ ID NO. 1-2, 3-4, and 189-190.

[0061] Table 2 95 pairs of mustard-type rapeseed B j Subgenome-specific markers and marker primers

[0062]

[0063] Example 2: Verification of the applicability of single-site markers and marker primers

[0064] For mustard-type rapeseed B j Subgenome-specific single-site markers in identifying progeny of distant hybrids of sweet and mustard j The applications of genomic chromosome fragments are as follows:

[0065] The experimental materials and population construction are as follows:

[0066] Experimental materials: Mustard-type rapeseed 109 (horn fruit angle horizontal type) and Brassica napus type rapeseed 337 (horn fruit angle drooping type).

[0067] Population construction: Brassica rapa type was used as the female parent (109) and Brassica napus type was used as the male parent (337) for hybridization. Subsequently, Brassica napus type 337 was used as the recurrent parent for backcrossing to construct BC1, BC2 and BC3 populations.

[0068] The specific verification method is as follows:

[0069] DNA extraction: Genomic DNA from single plants of the above-mentioned sweet mustard-hybrid hybrid progeny and single plants of the backcross BC1, BC2 and BC3 populations was extracted using the CTAB small sample method.

[0070] IP marker primer synthesis: The markers used in this experiment were the 95 pairs of rapeseed B markers screened in Example 1. j Subgenome-specific markers were used, and the corresponding marker primers were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0071] PCR reaction and electrophoresis detection: PCR amplification was performed using T3 Super PCR Mix from Qingke Biotechnology Co., Ltd.

[0072] The PCR reaction system consisted of 20 µL of: 2.5 µL template DNA, 0.5 µL each of forward and reverse primers, and 16.5 µL T3 Super PCRMix.

[0073] PCR amplification program: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 55℃ annealing for 12 s, 72℃ extension for 12 s, for a total of 35 cycles; final extension at 72℃ for 2 min, and then stored at 4℃.

[0074] 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.

[0075] Firstly, 95 pairs of mustard-type rapeseed B were used. j Genome-specific marker primers were used to detect individual plants in the BC1, BC2, and BC3 populations. The results showed a marker locus B06-0962 that amplified in oblique-growing plants but not in drooping plants across the three backcross generations. Subsequently, using B06-0962 as an anchor marker, other predicted B6 chromosomes within a 5M radius of the B06-0962 marker were further selected. j Subgenome-specific single-site amplification markers were validated by PCR to accurately identify mustard-type rapeseed B. j The specific size of the exogenous genomic fragment corresponds to the 5.90-10.56 Mb region on chromosome B06, with a size of 4.66 Mb. The exogenous fragment on chromosome B06 is marked with B06-0706 and B06-1157 at its flanking ends. B06-0682 is the closest indifferential locus to B06-0706, and B06-1179 is the closest indifferential locus to B06-1157, for a total of 5 differential markers. Specifically, in the offspring of a distant hybrid of rapeseed and mustard, mustard-type rapeseed B...j The results of genome exogenous fragment size and specific marker map construction are as follows: Figure 5 As shown. This embodiment further demonstrates that mustard-type rapeseed B j Subgenome-specific single-site IP markers and their marker primers in identifying rapeseed B in sweet potato-mustard distant hybrids j The effectiveness and feasibility of subgenomic exogenous chromosome fragments.

[0076] In summary, the 95 markers and their primers screened by this invention are from rapeseed B. j Subgenome-specific single-site IP markers and marker primers can be used for the progeny B of distant hybrids between rapeseed and Brassica oleracea crops. j The specific identification of superior gene fragments in subgenomic regions enables the efficient creation and identification of Brassica genus germplasm resources, such as Brassica napus. Simultaneously, this primer set can be used for PCR detection of target genes, and this primer set can be used to develop a primer sequence for Brassica napus B... j A kit for subgenomic identification. Furthermore, the marker primers provided by this invention can be used to effectively assist in the precise introduction of superior gene fragments from rapeseed, enabling the efficient creation and utilization of germplasm resources. They can also be used in breeding of rapeseed, demonstrating significant application value.

[0077] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A type of mustard-like rapeseed B j Subgenome-specific single-site IP marker primer set, characterized in that, The labeled primer set consists of 95 pairs of primers, and their nucleotide sequences are shown in SEQ ID NO.1 to SEQ ID NO.190, respectively.

2. The marker primer set as described in claim 1 in rapeseed B j Applications in the identification of exogenous fragments from subgenomic origins.

3. The marker primer set as described in claim 1 in the hybrid progeny of mustard-type rapeseed B j Applications in the identification of exogenous subgenomic fragments.

4. A method for use in mustard-type rapeseed B j A kit for subgenomic identification, characterized in that, The kit contains the primer set as described in claim 1.

5. The use of the labeled primer set as described in claim 1 or the kit as described in claim 4 in any of the following, comprising: The offspring of distant hybridization between rapeseed and Brassica oleracea species are derived from rapeseed B. j Rapid identification of gene fragments in subgenomes; Used to assist mustard-type rapeseed B j Precise introduction of subgenomic gene fragments.

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