Genes and molecular markers closely related to orange-red flower traits of rapeseed and their applications

Abstract

The present invention discloses a gene closely related to the orange-red flower trait of rapeseed, a molecular marker and an application thereof, wherein the BnaA07.PAP2 gene in orange-red flower rapeseed is shown in SEQ ID NO: 1, and a molecular marker closely related to the orange-red flower trait of rapeseed is developed based on the gene, and the sequence of the molecular marker is shown in SEQ ID NO: 2-3. The molecular marker can be directly applied to the breeding process of rapeseed flower color, and accelerates the breeding and creation of new rapeseed flower color varieties with different genetic backgrounds.

Description

Technical Field

[0001] The present invention belongs to the technical field of rapeseed molecular markers, and specifically relates to a gene and a molecular marker closely related to the orange-red flower trait of rapeseed, and applications thereof in the breeding of orange-red flower rapeseed, rapeseed flower color identification, and the like. Background Art

[0002] Flower color is one of the most important traits of plants. It has the functions of attracting pollinators, protecting flower organs, and maintaining the energy balance of flower tissues. At present, breeders have made breakthroughs in orange, pink, purple and other colorful rapeseed, and the yield and quality are comparable to ordinary rapeseed. Colorful rapeseed has greatly improved the ornamental value, economic value and social value of rapeseed.

[0003] The colorful rapeseed is a germplasm resource obtained by distant hybridization, so a group of available molecular markers are urgently needed for early auxiliary selection to improve selection efficiency, reduce the scale of field planting and later identification work, and shorten the breeding time. However, no functional markers have been used in the breeding of orange-red rapeseed so far. Summary of the invention

[0004] In order to solve the problems in the prior art, the object of the present invention is to provide a gene and a molecular marker closely related to the orange-red flower trait of rapeseed, so as to shorten the breeding time of orange-red flower rapeseed.

[0005] In order to achieve the above object, the technical solution of the present invention is as follows:

[0006] The present invention firstly utilizes white flower Brassica napus and orange flower Brassica napus to cross and develop an F2 population, performs coarse positioning of candidate genes for flower color traits, and obtains the BnaA07.PAP2 gene capable of regulating rapeseed flower color, the nucleotide sequence of which is shown in SEQ ID NO: 1 in red flower rapeseed and in SEQ ID NO: 4 in white flower rapeseed.

[0007] Based on an indel-specific marker in the BnaA07.PAP2 gene that co-segregates with rapeseed flower color, a molecular marker closely related to the orange-red flower trait of rapeseed was further obtained, which includes a forward primer with a sequence as shown in SEQ ID NO:2 and a reverse primer with a sequence as shown in SEQ ID NO:3.

[0008] The above molecular markers can be used to identify rapeseed flower color, and the identification method is: using the total DNA of the single rapeseed plant to be identified as a template, using the molecular markers shown in SEQ ID NOs: 2-3 to perform PCR amplification, and detecting the amplified products by 2% agarose gel electrophoresis. If only a 393bp band is obtained, the single rapeseed plant to be identified is a homozygous orange-red flower individual.

[0009] Furthermore, in the above technical solution, the PCR amplification system is 12 μL in total, including 2.0 μL DNA, 5.0 μL 2×Mix, 0.5 μL each of the forward primer and the reverse primer, and the balance ddH2O.

[0010] Furthermore, in the above technical solution, the PCR amplification procedure is: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 sec, annealing at 57°C for 30 sec, extension at 68°C for 2 min, for a total of 35 cycles; extension at 68°C for 10 min.

[0011] The above molecular markers can also be used to select and create new varieties of rapeseed with different flower colors, which is conducive to early auxiliary selection and shortening the breeding time.

[0012] The beneficial effects of the present invention are as follows: the present invention has discovered a new gene controlling the orange-red flower trait of rapeseed, and has developed specific molecular markers, which can be used to determine the flower color phenotype of rapeseed plants at an extremely early stage, and accelerate the selection and creation of new rapeseed flower color varieties with different genetic backgrounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 is a schematic diagram of the indel marker locations in the BnaA07.PAP2 gene;

[0014] Figure 2 This is the agarose gel electrophoresis detection diagram of DH_GC001 and GL570 parents and DH_GC001×GL570F1 rapeseed plants in Example 2;

[0015] Figure 3 This is the agarose gel electrophoresis detection diagram of different rapeseed materials in Example 3. DETAILED DESCRIPTION

[0016] The technical solution of the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. It should be understood that the specific implementation methods described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0017] Example 1 Fine Mapping and Cloning of the Brassica napus BnaA07.PAP2 Gene

[0018] (1) In order to locate the key gene controlling the flower color trait of orange-red rapeseed, the double haploid strain (DH line) DH_GC001 of orange-red rapeseed and the DH line GL570 of white rapeseed were obtained by microspore culture technology. F1 hybrids were obtained by forward and reverse hybridization, and the F1 was self-pollinated to obtain the F2 segregation population. The F1 was backcrossed with the parents to obtain the BC1 backcross population. Combining the phenotypes of the F2 segregation population and the BC1 backcross population, it was determined that the orange-red flower trait was a quality trait controlled by a single dominant gene and had no cytoplasmic genetic effect. Subsequently, the target interval controlling the orange-red flower trait was obtained by mixed pool sequencing (BSA sequencing) analysis, which was located within an interval of about 2Mb on chromosome A07. Finally, the InDel markers developed by BSA sequencing were combined with the F2 segregation population to precisely locate the key gene controlling the orange-red flower trait, which was located in the approximately 70Kb interval between markers I-142 and I-145. Combined with the transcriptome results of DH_GC001 and GL570, it was speculated that BnaA07.PAP2 was the key gene controlling the orange-red flower trait of Brassica napus.

[0019] (2) The genomic DNA of DH_GC001 and GL570 was amplified to obtain PCR products of 3.1 kb of the DH_GC001 promoter and 2.2 kb of the full-length gene (including 0.5 kb of the promoter); and 3.1 kb of the GL570 promoter and 2 kb of the full-length gene (including 0.3 kb of the promoter) were obtained. The primers used for amplification are as follows:

[0020] PAP2.A07.PF: 5'-TTAGGCCTTCTAAGACTTCATGTGA-3' (SEQ ID NO: 5);

[0021] PAP2.A07.PR:5'-AGCCGACAAAGAAAAGATTTAGACT-3'(SEQ ID NO:6);

[0022] PAP2.A07.gF:5'-GATGAAGAGACTAAGTGCTATGAACC-3' (SEQ ID NO:7);

[0023] PAP2.A07.gR: 5'-TCAAGTTCCAGTTTCTCCATCC-3' (SEQ ID NO: 8).

[0024] Gene amplification used the KOD-plus-standard reaction system (TOYOBO). The 50 μL PCR reaction system included: 100 ng genomic DNA template, 5 μL 10× PCR buffer for KOD-plus-, 5 μL dNTPs (2 mM), 2 μL MgSO4 (25 mM), 4 μL PCR primers (2 μL each of forward and reverse primers, each with a concentration of 10 μM) and 1 μL KOD-plus- (1 U / μl), supplemented with ddH2O to 50 μl.

[0025] The reaction procedure of PCR amplification was as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 sec, annealing at 57°C for 30 sec, extension at 68°C for 2 min, for a total of 35 cycles; extension at 68°C for 10 min, and storage at 4°C.

[0026] Take 3 μl of the amplified product, add 1 μl of loading buffer (98% deionized formamide, 10 mM EDTA, 0.005% xylene cyanol FF and 0.005% bromophenol blue), after electrophoresis on 1.5% agarose gel (containing 0.1% ethidium bromide), and photograph and observe the results using a gel imaging system (Bio-Rad, Gel DocTM XR+).

[0027] (3) The PCR product of BnaA07.PAP2 gene was amplified using the Beijing Tiangen PCR product recovery kit, and the recovered product was detected using the same agarose gel electrophoresis method as above.

[0028] (4) The purified PCR products were linked to the blunt-end PCR products using the zero-background pTOPO-Blunt-Simple blunt-end cloning kit from Adelaide.

[0029] (5) E. coli transformation (CaCl2 transformation method): Place the CaCl2 competent cells stored at -80°C on ice for 5-10 minutes to allow them to thaw; add 10 μL of the ligation solution to 50 μL of the CaCl2 competent cells, gently pipette the mixture several times with a sterilized pipette tip, and place it on ice for 30 minutes to allow them to fully contact; place the system in a 42°C water bath for heat shock for 90 seconds, quickly take it out and place it on ice for 5 minutes to allow it to fully cool; add 500 μL or 1 ml of LB system without any antibiotics and shake it at 37°C for 1 hour; centrifuge the revived competent cells at 6000×g in a centrifuge for 30 seconds; pour off most of the supernatant, leaving 100 μL of the suspended bacterial solution, evenly spread it on a plate culture medium supplemented with kanamycin (concentration of 50 mg / L), blow dry, and incubate it upside down in a 37°C incubator for 12-16 hours.

[0030] (6) Single clones were selected and sent to Qingke Company for sequencing using M13F; M13R. Among them, the sequence of DH_GC001-BnaA07.PAP2 is shown in SEQ ID NO: 1, and the sequence of GL570-BnaA07.PAP2 is shown in SEQ ID NO: 4.

[0031] Through the above two sequence comparison analysis, it was found that compared with GL570, DH-GC001 has a 211 bp fragment inserted in the promoter 184 bp away from the start codon. Figure 1 shown.

[0032] Example 2 Acquisition of specific molecular markers

[0033] Based on the Indel differences in Example 1, specific primers for cloning the BnaA07.PAP2 gene were designed, and their sequences are shown below:

[0034] INDEL142-F:5'-AGTCTAAATCTTTTCTTTTGTCGGCT-3' (SEQ ID NO:2);

[0035] INDEL142-R:5'-ATTGTGGCTTGACAAATCCTTCG-3'(SEQ ID NO:3)

[0036] The molecular marker was used to detect rapeseed plants of DH_GC001, GL570 and DH_GC001×GL570F1, specifically: extracting total DNA from a single rapeseed plant as a template, using the molecular marker for PCR amplification, and detecting the amplified product by 2% agarose gel electrophoresis. The PCR amplification system is shown in the following table:

[0037]

[0038] The PCR amplification reaction program was as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 sec, annealing at 57°C for 30 sec, extension at 68°C for 2 min, for a total of 35 cycles; extension at 68°C for 10 min, and storage at 4°C.

[0039] Test results such as Figure 2 As shown in the figure, it can be seen that when only the 393bp band is obtained, the single strain of rapeseed to be identified is a homozygous red flower individual; when only the 182bp band is obtained, the single strain of rapeseed to be identified is a homozygous white flower individual; if both the 393bp and 182bp bands appear at the same time, the single strain of rapeseed to be identified is a heterozygous individual (presenting pink flowers in the present generation and separation in the next generation). That is, it is confirmed that the Indel marker is co-segregated with the rapeseed flower color, and the primers of this example can realize the identification of the rapeseed flower color.

[0040] Example 3

[0041] Using the specific primers in Example 2, this example detected 96 rapeseed materials (the material names are shown in the table below), and the amplification system and electrophoresis detection method were consistent with those in Example 2. The detection results are shown in Figure 3 .

[0042]

[0043]

[0044]

[0045] Note: The rapeseed varieties listed in the table are all biological materials available to the public.

[0046] The test results showed that only orange-flowered rapeseed had a clear band at the 393bp band, while yellow-flowered and white-flowered rapeseed had bands at the 182bp band. Subsequent testing found that some materials that had no detected bands were due to low DNA concentrations.

[0047] In summary, the molecular marker detection method provided by the present invention is simple and can be used for early and accurate judgment of the orange-red flower phenotype of rapeseed plants, which is conducive to accelerating the breeding of new rapeseed flower color varieties.

[0048] The above description is a preferred embodiment of the present invention, which cannot be used to limit the scope of rights of the present invention. It should be pointed out that for ordinary technicians in this technical field, any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention. Sequence Listing <110> Huazhong Agricultural University <120> Genes and molecular markers closely related to orange-red flower traits of rapeseed and their applications <160> 8 <170> SIPOSequenceListing 1.0 <210> 1 <211> 5223 <212> DNA <213> BnaA07.PAP2 sequence (DH_GC001) <400> 1 ttaggccttc taagacttca tgtgattaga ggtgttaatc tcgccattag agattctaag 60 agtagcgatc cttacgtcat tgttcgtatg ggccaacagg tatattccag actcctaact 120 cttcacgaat ttcgagttg tgatcagtt tcttaggttc cgtatatatt caggttgtc 180 ccgtttttt atatggatct ttggtcac caatctggtt gtgctgctca gattttctga 240 tgtatctgat gagagaattt cagttaaaaa gtgaatatta tcaacttc gtgttatat 300 gagataaaa aaaatgagac agaacataca tatatacgtg tgtcaatt aggttttgatt 360 caacgcgagt tttaagtttc aacacgtcaa atgtagttga gttttactaa tgagaaatttc 420 ctaggattg attttatcat tcaaatagt agttcttgtt tctgaccttg tcacaccat 480 aaaatgat caatttatt ttacatgtac aaatttacac ttgaatatat ggtgttatgt 540 ttttctcttt ttgacagaa attacgaact cgtgtggtga aaagaattt gatccagaa 600 tggaacgaag acttgacact ctctatttct gatccaattc ttcctatcaa aatcgtaaat 660 ggatcaaca ttacacaag ttgctctaag ttttgagaa cagacagaa cataacaag 720 aagtgggtat ttttctgtg atgatacaga tggtttacga tagggactgg ttctcaaggg 780 atgataagat gggagatgcg ttttttcgca ttgatccatt ccttgaagcc atcaggatcc 840 aaaaccagtt cagaggactc cccgaaggaa ctgtaataat gaagatacag gcaagcaggc 900 agaactgtct atcagaaaag agcaagattg tgtggaacaa tggaaagatt gtccagaata 960 tgttccttaa gctccagaac gttgagtgtg gagaggtcga gcttcagctt gagtggatcg 1020 atgtctcagg tcttctgagt ataaatgagc atgaggttgt tgcttactag atctattata 1080 ttattacatc aagtttgtta aaaagtatac tccctcgttt caatacatg agcatgagga 1140 tgttgcttac tagatctatt atcttattac atcaaattag ctgacatttt caattttcaa 1200 cttaaaattt attaactttt tatgttatat taccaaaata tgtattcttc attctatttt 1260 atcattggtg gaattgtgtt tagataaata attagtgatc tttttgttta gaaaatataa 1320 atattgaatg tttctttaat ctatctgcac aattctaaaa cgatacatga ttaaaaaccg 1380 gagggagtac tattatgag atccgaagga gagggaggct tctctccaat aatcagggtg 1440 tctatttgtt ccctctggtt ttgtgttctt attctaacaa gataacataa caaaaagata 1500 acatagcacg tttgttgta gggacttata gagaaagag atgagatga aaccaagtag 1560 atagcaaag atgagacggt gaactgatt tttcgctcat cttacacatc ttcactaca 1620 tatttatact aaaagcag agtaatgaac tcaatttcc ctaccctttc acacgttata 1680 gagccacata ataactacca ctctagctct agcatact agctccacac tttaggaaaa 1740 gaaacaagca actccaagga ctttaacaag catatgagat gaaaaatcac gtcatccaag 1800 aaagcagct caagggaat atccttatta tctgagaac gtaggaat cgaagaaaaa 1860 gaaaattaga tgggaatgat ggcaaggat ttcagcagaa ccatgaaag gttttgagaa 1920 agccacaag aaatctttc agccagggtt tggaagtct cgagatcgtg agataattca 1980 aaaaaaaaaaaaaaaaag aagacagttc aaatgaagaa acttgttg tggtattca 2040 catcaattgt ttaagggaa gctgaacgat gtaacggaga aagccttact tcaatttca 2100 aatgaatgag aacctcagg aagaacagtc aaagaatc tgaaactaga aagcagattc 2160 acatgctgaa tagtgaacc aatgatctgg agagattct ctctgttaga agggtaggca 2220 agtccaactt tggactcggt tacaaagggg tgcaaaacta ftaaaaat agagtttgtt 2280 cggactaaga cttaagataa gaggttagct atgatacagg aagtaagtat caaccaagat 2340 catctattgt gaaaccagta agaaccataa tggaggctac ttctgtggta atacatgcac 2400 atatcaagat atacttacaa atttcaaaac agaatcactc catatttgac tttgctttcc 2460 aatctttact gctttaagta acttgttaac tagtctgatc tggtttagcc tagcttcatt 2520 tcacctttgc tttttatttc actctctctt tcattcctct tctattaaca ggcttatttt 2580 actgagccgg aatttatata ctaacaaaaa ggctaacgtt aattatgaaa atctctaatc 2640 aaatgactat gatgaatttc accaatctta ttgttttaat atcaaataag ggtcaagtaa 2700 tatattttgt taactctctg ccaaaagtac atactcaaca acgcgagacg tttaaaata 2760 cgaaacaatt ttgatatcat agaacaattt gcaaaagtag agatttgtgt attcataata 2820 attgtagctg gtttttgtgt tcacaaattt gttgacagaa aaaaaaagta cactgcaaat 2880 tgtgttctca tacatccacc ctctctccta attctattat aactctggtg cgtgaagttg 2940 ttccctcttt tagtgatata accatcttcc accaatcatt ataactagta cttctctcc 3000 taattgttag gagacatgtt tctctttaaa gtcctgaaaa agatgaagag actaagtgct 3060 atgaaccttt tctctaatag tctaaatctt ttctttgtcg gctatttagt ctaaatcagt 3120 agtagcagaa cataaataac taaattagca aattaaacac gatactaaat tagcatcgta 3180 tcagatatca acgctctggt cgctgcattc gattctgttt cctttagctg gattccacga 3240 gagagaaaca aagatgccga ttggttggct aaacaatgtt tatcttttca tcaagatttg 3300 tacgctttga atgctaccta acgttatggt tgagtaatga atgttttgtg tttcaaaaaa 3360 aaaaaaaaca cgacaacaaa acattaaatg tggatgttag acatgcacgt cactcccctt 3420 tgcccgtcac ttgtataaat aaagtcctcg aaggatttgt caagccacaa tagaaaccat 3480 tttcctcaat cctttaccta tatatatata acttttataa actttggtta gatatttata 3540 ttatatcg ctggtccatg gagggtatgt ccaaagggtt gaaaaaaggt gcatggactg 3600 ctgaagaaga taatctcttg aggcaatgca ttgataagta tggagaaggg aaatggcacc 3660 aagttccttt aagagctggt atgtcttttt ttttaac ataaaagctg gtatgctact 3720 tttattaatt ttcacaca cacacaca catatatac taatagtac gtatattctt 3780 tttattttc agtacattta ttctctttct ctctgtctag tattaggaaa ttattaca 3840 ccggggtaca caatcattgt tttctttc gttttaatga aggaatcata gattcatatg 3900 ttcaatgtt ttcatgaaa aaaaaacatt tgcgttctc atgtttaatt acaaagcgag 3960 aaaatgtcaa ctctctttat tgatcgtcg ttttcttt tttttgaa aaagctctt 4020 ttgattaggg aacttttctg cacgaacccg tgtgttgtg gggaatatgt tgtttattct 4080 ggggaacttt gatccttcgt gataaaattt tactccttct gttctttaat ataaaatgtt 4140 ttgggagaaa cacatatatt aaaaaaacta ttttgtct agaaaatgtc attaaaacta 4200 taaattata gtgttcaacc aattacaaa tagactatta aaatatgatt agtttcacag 4260 ttttaata agtaaagtt acttataaaa tttgaaaca ttttatata tgaataattg 4320 gaaccaatcaa attccgcaa acatcatatt tttaggaca gagggagtag gagacccaa 4380 tgcagttttt gctcgttctt ttaatatat taatgtca ttattggttt tgtaggtcta 4440 aatcggtgca ggaagagttg tagataaga tggttgaact atttgaagcc aagtatcaag 4500 agaggaaaac tcactctga tgaagttgat cttcttattc gccttcataa gcttttagga 4560 aacaggttta cattcagac acaaattca ctttattcg tatcctcatt cggtctaatc 4620 taatcatttg atttgtttt ttttgataa aaagtactta atttattc atatgtaaat 4680 gatccattac tagtcatat ataccctaa ttttcaat gcatgcttag gtggtcttta 4740 attgctgta gattacccgg tcggaccgcc aatgacgtca aaaattactg gaacacccat 4800 ttgagtaaga accatgaacc agttgtaag acccagatga aaagagaaa cattccttgc 4860 tcttatacca caccagccca aaaaatcgac gtttcaac ctcgacctcg atccttcacc 4920 gttaacaacg gctgcagcca taataatggc atgccagaag ctgacattgt tcctctatgc 4980 cttggacaca acgaactaa taatgttttct gaaaata tcacatgtaa acgaatgat 5040 gataaatctg agcttgttag tcatttaatg gatggtcaga ataggtggtg ggaaagtttg 5100 ctagatgaga gccaagatcc agctgcgctc tttccagaaa ctacagcaat aaaaaagggc 5160 gcaacctccg cgtttgacgt tgagcaactt tggagcctgt tggatggaga aactggaact 5220 tga 5223 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <400> 2 agtctaaatc ttttctttgt cggct 25 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <400> 3 attgtggctt gacaaatcct tcg 23 <210> 4 <211> 5010 <212> DNA <213> BnaA07.PAP2 sequence (GL570) <400> 4 ttaggccttc taagacttca tgtgattaga ggtgttaatc tcgccattag agattctaag 60 agtagcgatc cttacgtcat tgttcgtatg ggccaacagg tatattccag actcctaact 120 cttcacgaat ttcgagtttg tgattcagtt tcttaggttc cgtatatatt caaggttgtc 180 ccgttttttt atatggatct ttttggtcac caatctggtt gtgctgctca gattttctga 240 tgtatctgat gagagaattt cagttaaaaa gtgaatatta tcacttatc gtgttatat 300 gagataaaa aaaatgagac agaacataca tatatacgtg tgtcaatt aggttttgatt 360 caacgcgagt tttaagtttc aacacgtcaa atgtagttga gttttactaa tgagaaatttc 420 ctaggattg attttatcat tcaaatagt agttcttgtt tctgaccttg tcacaccat 480 aaaatgat caatttatt ttacatgtac aaatttacac ttgaatatat ggtgttatgt 540 ttttctcttt ttgacagaa attacgaact cgtgtggtga aaagaattt gatccagaa 600 tggaacgaag acttgacact ctctatttct gatccaattc ttcctatcaa aatcgtaaat 660 ggatcaaca ttacacaag ttgctctaag ttttgagaa cagacagaa cataacaag 720 aagtgggtat ttttctgtg atgatacaga tggtttacga tagggactgg ttctcaaggg 780 atgataagat gggagatgcg tttttcgca ttgatccatt ccttgaagcc atcaggatcc 840 aaaaccagtt cagaggactc cccgaaggaa ctgtataat gagatacag gcaagcaggc 900 agaactgtct atcagagag agcagattg tgtgaacaa tggaagat gtccagaata 960 tgttccttaa gctccagaac gttgagtgtg gagaggtcga gcttcagctt gagtggatcg 1020 atgtctcagg tcttctgagt ataaatgagc atgaggttgt tgcttactag atctattata 1080 ttattacatc aagtttgtta aaaagtatac tccctcgttt caatacatg agcatgagga 1140 tgttgcttac tagatctatt atcttattac atcaaattag ctgacatttt caattttcaa 1200 cttaaaattt attaactttt tatgttatat taccaaaata tgtattcttc attctatttt 1260 atcattggtg gaattgtgtt tagataaata attagtgatc tttttgttta gaaaatataa 1320 atattgaatg tttctttaat ctatctgcac aattctaaaa cgatacatga ttaaaaaccg 1380 gagggagtac tattatgag atccgaagga gagggaggct tctctccaat aatcagggtg 1440 tctatttgtt ccctctggtt ttgtgttctt attctaacaa gataacataa caaaaagata 1500 acatagcacg ttttgttgta gggacttata gagaaaagag aatgagatga aaccaagtag 1560 atagcaaaag atgagacggt gaaactgatt tttcgctcat cttacacatc ttcaactaca 1620 tatttatact aataaagcag agtaatgaac tcaatttttcc ctaccctttc acacgttata 1680 gagccacata ataactacca ctctagctct aggcataact agctccacac tttaggaaaa 1740 gaacaagca actccaagga ctttaacaag catatgagat gaaaatcac gtcatccaag 1800 aaaagcagct caaagggaat atcctttata tctgaagaac gtaaggaaat cgaagaaaaa 1860 gaaattaga tgggaatgat ggcaaaggat ttcagcagaa ccatgaaaag gttttgagaa 1920 agccaacaag aaatcttttc ccaaggcgtt tggaaagtct cgagatcgtg agataattca 1980 aaaaaaaaaa gaagaaaaag aagacagttc aaatgaagaa acttgttatg tgggtattca 2040 catcaattgt ttaaaggaa gctgaacgat gtaacggagaa aagccttact tcaatttcaa 2100 aatgaatgag aacctcaagg aagaacagtc taagaaaatc tgaaactaga aagcagattc 2160 acatgctgaa tagtgaaacc aatgatctgg agaagaattct ctctgttaga agggtaggca 2220 agtccaactt tggactcggt tacaaagggg tgcaaaacta cattaaaata gagtttgttc 2280 ggactaagac ttaagataag aggttagcta tgatacagga agtaagtatc aaccaagatc 2340 2400 tatcaagata tacttacaaa tttcaaaaca gaatcactcc atatttgact ttgctttcca 2460 atctttactg ctttaagtaa cttgttaact agtctgatct ggtttagcct agcttcattt 2520 cacctttgct ttttatttca ctctctcttt cattcctctt ctattaacag gcttatttta 2580 ctgagccgga atttatatac taacaaaaag gctaacgtta attatgaaaa tctctaatca 2640 aatgactatg atgaatttca ccaatcttat tgttttaata tcaaataagg gtcaagtaat 2700 atattttgtt aactctctgc caaaagtaca tactcaacaa cgcgagacgt tttaaaatac 2760 gaaacaattt tgatatcata gaacaatttg caaaagtaga gatttgtgta ttcataataa 2820 ttgtagctgg tttttgtgtt cacaaatttg ttgacagaaa aaaaaagtac actgcaaatt 2880 gtgttctcat acatccaccc tctctcctaa ttctattata actctggtgc gtgaagttgt 2940 tccctctttt agtgatataa ccatcttcca ccaatcatta atactagtac tttctctcct 3000 aattgttagg agacatgttt ctctttaaag tcctgaaaaa gatgaagaga ctaagtgcta 3060 tgaacctttt ctctaatagt ctaaatcttt tctttgtcgg ctatttagtc taaatcagta 3120 gtagcagaac attaatact aaattagcaa attackacacg acaaaac atttaatgtg 3180 gatgttagac atgcacgtca ctccccttg cccgtcactt gtataaataa agtcctcgaa 3240 ggatttgtca agccacaata gaaaccattt tcctcaatcc tttacctata tatatatac 3300 ttttataaac tttggttaga tatttatatt atatatcgct ggtccatgga gggtatgtcc 3360 aaagggttga aaaaggtgc atggactgct gagagata atctcttgag gcaatgcatt 3420 gataagtatg gagaagggaa atggcaccaa gttcctttaa gagctggtat gtcttttttt 3480 tttataacat agagctggt atgctacttt tattaattt cacacaca cacacacaca 3540 tatataacta ataagtacgt atattctttt tattttcag tacatttatt ctctttctct 3600 ctgtctagta ttaggaatt aattaacacc ggggtacaca atcattgtt ttctttcgt 3660 tttaatgaag gatcataga ttcatatgtt ctaatgtttt tcatgaaaa aaaacatttg 3720 cgttcttcat gttttac aaagcgagaa atgtcaact ctctttattg attcgtcgtt 3780 tttcttttt tttgaaaaa gagctcttt gattagtgaa cttttctgca cgaacccgtg 3840 ggtttgtggg gatatgttg tttattctg ggaacttga tccttcgtga taaaatttta 3900 ctccttctgt tctttaatat aagatgtttt ggtaaaaca catatattaa aaaaactatt 3960 ttttgtctag aaatgtcat taaaactata aattaagt gttcaaccaa ttacaaata 4020 gactattaaa atatgattag ttcacagtt tttaaag taaaagttac tttaaaattt 4080 gaaaacattt tatatattgg aaaattggaa catcaaatt cagtaaaaca tcatattttt 4140 aggaacagag ggagtaggag acgcaatgc agttttgct cgttcttttta atatattaa 4200 atgtcaatta ttggttttgt aggtctaaat cggtgcagga agagttgtag actaagatgg 4260 ttgaactatt tgaagccaag tatcagaga ggaaaactca actctgatga agttgatctt 4320 cttattcgcc ttcataagct tttaggaac aggtttacat tcagacaca attcaactt 4380 tatttcgtat cctcattcgg tctaatctaa tcatttgatt tgtttttt ttgataaaaa 4440 gtacttaaat ttatttcata tgtaatgat ccattactaa gtcatatata tccctaattt 4500 ttcaaatgca tgcttaggtg gtctttaatt gctggtagat tacccggtcg gaccgccaat 4560 gacgtcaaaa attactggaa cacccatttg agtaagaaac atgaaccagg ttgtaagacc 4620 cagatgaaaa agagaaacat tccttgctct tataccacac cagcccaaaa aatcgacgtt 4680 ttcaaacctc gacctcgatc cttcaccgtt aacaacggct gcagccataa taatggcatg 4740 ccagaagctg acattgttcc tctatgcctt ggacacaacg atactaataa tgtttctgaa 4800 aatataatca catgtaacaa agatgatgat aaatctgagc ttgttagtca tttaatggat 4860 ggtcagaata ggtggtggga aagtttgcta gatgagagcc aagatccagc tgcgctcttt 4920 ccagaaacta cagcaataaa aaagggcgca acctccgcgt ttgacgttga gcaactttgg 4980 agcctgttgg atggagaaac tggaacttga 5010 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <400> 5 ttaggccttc taagacttca tgtga 25 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <400> 6 agccgacaaa gaaaagattt agact 25 <210> 7 <211> 26 <212> DNA <213> Artificial Sequence <400> 7 gatgaagaga ctaagtgcta tgaacc 26 <210> 8 <211> twenty two <212> DNA <213> Artificial Sequence <400> 8 tcaagttcca gtttctccat cc 22

Claims

1. Application of the BnaA07.PAP2 gene in orange-red flower Brassica napus to regulate flower color of Brassica napus, characterized in that: The nucleotide sequence of the BnaA07.PAP2 gene is shown in SEQ ID NO:

1.

2. A molecular marker closely related to the orange-red flower trait of Brassica napus, characterized in that: The molecular markers are Indel markers shown in SEQ ID NO: 1 and SEQ ID NO:

4.

3. Use of the molecular marker as claimed in claim 2 in identifying flower color of Brassica napus.

4. The use according to claim 3, characterized in that: The total DNA of the Brassica napus single strain to be identified was used as a template, and PCR amplification was performed using a forward primer with a sequence as shown in SEQ ID NO: 2 and a reverse primer with a sequence as shown in SEQ ID NO: 3, and the amplified product was detected.

5. The use according to claim 4, characterized in that: The PCR amplification system has a total volume of 12 μL, including 2.0 μL of DNA, 5.0 μL of 2×Mix, 0.5 μL of each of the forward primer and the reverse primer, and the balance of ddH2O.

6. The use according to claim 4, characterized in that: The PCR amplification procedure is: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 sec, annealing at 57°C for 30 sec, extension at 68°C for 2 min, for a total of 35 cycles; extension at 68°C for 10 min.

7. The use according to claim 4, characterized in that: If only a 393 bp band is obtained, the Brassica oleracea strain to be identified is a homozygous orange-red flower individual.

8. The use according to claim 4, characterized in that: The detection was performed by 2% agarose gel electrophoresis.

9. Use of the molecular marker as claimed in claim 2 in breeding new varieties of Brassica napus with different flower colors.

10. A detection kit, characterized in that: It comprises a forward primer having a sequence as shown in SEQ ID NO:2 and a reverse primer having a sequence as shown in SEQ ID NO:3.

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