CN116949199A - Ac transposase molecular marker, primer, kit and application for identifying fruit anthracnose - Google Patents
Ac transposase molecular marker, primer, kit and application for identifying fruit anthracnose Download PDFInfo
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- CN116949199A CN116949199A CN202211552870.0A CN202211552870A CN116949199A CN 116949199 A CN116949199 A CN 116949199A CN 202211552870 A CN202211552870 A CN 202211552870A CN 116949199 A CN116949199 A CN 116949199A
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- 235000013399 edible fruits Nutrition 0.000 title claims abstract description 51
- 102000008579 Transposases Human genes 0.000 title claims abstract description 20
- 108010020764 Transposases Proteins 0.000 title claims abstract description 20
- 239000003147 molecular marker Substances 0.000 title claims abstract description 10
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 22
- 241000233866 Fungi Species 0.000 claims abstract description 12
- 241000193738 Bacillus anthracis Species 0.000 claims description 19
- 238000001514 detection method Methods 0.000 claims description 12
- 230000003321 amplification Effects 0.000 claims description 10
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 10
- 238000006243 chemical reaction Methods 0.000 claims description 9
- 241001639768 Colletotrichum siamense Species 0.000 claims description 6
- 241001478662 Colletotrichum camelliae Species 0.000 claims description 5
- 238000000034 method Methods 0.000 claims description 5
- 241000223600 Alternaria Species 0.000 claims description 4
- 238000012408 PCR amplification Methods 0.000 claims description 4
- 241001523629 Pestalotiopsis Species 0.000 claims description 4
- 238000003752 polymerase chain reaction Methods 0.000 claims description 3
- 239000012807 PCR reagent Substances 0.000 claims description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 2
- 238000000137 annealing Methods 0.000 claims description 2
- 238000004925 denaturation Methods 0.000 claims description 2
- 230000036425 denaturation Effects 0.000 claims description 2
- 239000000499 gel Substances 0.000 claims description 2
- 238000003384 imaging method Methods 0.000 claims description 2
- 239000000463 material Substances 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 238000012257 pre-denaturation Methods 0.000 claims description 2
- 241000526900 Camellia oleifera Species 0.000 abstract description 14
- 108091092195 Intron Proteins 0.000 abstract description 3
- 238000011895 specific detection Methods 0.000 abstract 2
- 210000000349 chromosome Anatomy 0.000 abstract 1
- 239000010495 camellia oil Substances 0.000 description 13
- 108020004414 DNA Proteins 0.000 description 12
- 239000003921 oil Substances 0.000 description 11
- 241001529387 Colletotrichum gloeosporioides Species 0.000 description 9
- 239000000523 sample Substances 0.000 description 9
- 230000035945 sensitivity Effects 0.000 description 9
- 240000001548 Camellia japonica Species 0.000 description 8
- 241001639769 Colletotrichum fructicola Species 0.000 description 7
- 241000196324 Embryophyta Species 0.000 description 7
- 241001122767 Theaceae Species 0.000 description 7
- 235000018597 common camellia Nutrition 0.000 description 7
- 201000010099 disease Diseases 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
- 241000222199 Colletotrichum Species 0.000 description 5
- 241000894007 species Species 0.000 description 4
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 239000012154 double-distilled water Substances 0.000 description 3
- 239000008157 edible vegetable oil Substances 0.000 description 3
- 101150092328 22 gene Proteins 0.000 description 2
- XUMBMVFBXHLACL-UHFFFAOYSA-N Melanin Chemical compound O=C1C(=O)C(C2=CNC3=C(C(C(=O)C4=C32)=O)C)=C2C4=CNC2=C1C XUMBMVFBXHLACL-UHFFFAOYSA-N 0.000 description 2
- 238000013461 design Methods 0.000 description 2
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 206010003210 Arteriosclerosis Diseases 0.000 description 1
- 244000060011 Cocos nucifera Species 0.000 description 1
- 235000013162 Cocos nucifera Nutrition 0.000 description 1
- 244000147058 Derris elliptica Species 0.000 description 1
- 240000003133 Elaeis guineensis Species 0.000 description 1
- 235000001950 Elaeis guineensis Nutrition 0.000 description 1
- 241000207836 Olea <angiosperm> Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 208000011775 arteriosclerosis disease Diseases 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000002490 cerebral effect Effects 0.000 description 1
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- 230000000052 comparative effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses an Ac transposase molecular marker, a primer, a kit and application for identifying fruit anthracnose. The genome analysis is compared by utilizing the whole genome sequence information of a plurality of anthracnose fungi closely related to the anthracnose of the camellia oleifera to obtain specific chromosome sequences and candidate genes of the anthracnose of the camellia oleifera, and the candidate genes which can be used for specifically identifying the anthracnose of the camellia oleifera are selected as molecular markers according to the possible gene functions of the candidate genes and the characteristics of whether the candidate genes contain introns or not, and the specific detection is carried out on the anthracnose of the camellia oleifera by utilizing the markers. The invention can be used for diagnosing and predicting the anthracnose of the camellia oleifera and can be used for the specific detection of the fruit anthracnose.
Description
Technical Field
The invention belongs to the technical field of plant anthracnose detection, and particularly relates to an Ac transposase molecular marker, a primer, a kit and application for identifying fruit anthracnose.
Background
The oil tea is evergreen shrubs or trees in camellia, is a woody edible oil tree species with long cultivation history native to China, and is called four woody oil plants in the world together with olives, oil palm and coconuts. The camellia oil pressed by camellia seeds is rich in polyphenols and various unsaturated fatty acids, has health care effects of protecting heart and cerebral vessels, preventing arteriosclerosis and the like as edible oil, and in addition, recent researches show that the camellia oil has effects of preventing melanin formation, resisting oxidization and the like, and can be widely applied to health, food and cosmetic manufacturing industries as functional oil (Chaikul, sripiut, chanpirom, sathiachawan, & Ditthawushikul, 2017; xiao et al, 2017). The oil tea has strong adaptability and wide growth area, and is distributed in the north part of the Yangtze river besides the main production area in the south of the Yangtze river and the Qinling mountain. The camellia oleifera is resistant to barren and acid soil, is simple to plant and low in management and protection cost, so that the camellia oleifera is developed greatly by utilizing the sloping field and forest land with high planting difficulty, the camellia oleifera can beautify the environment, keep water and soil, recuperate water sources and regulate climate, and has important significance for increasing the output of oil crops in China and protecting the safety of edible oil ("the camellia oleifera is planted when barren mountain is changed into a vault," 2020 ", and the method is selected from left and quiet and Zhu Mei, 2020). However, the production and quality of camellia oil are severely restricted by diseases such as root rot, southern blight, anthracnose, soft rot, bud disease, leaf blight and coal pollution of the camellia oil for a long time, and the problem of the camellia disease is increasingly prominent along with the annual expansion of the planting area of the camellia oil.
The anthracnose of the tea-oil tree is a main leaf and fruit disease seriously threatening the production of the tea-oil tree, and can cause symptoms such as falling fruits, falling buds, dead branch tips and the like. The production of the oil tea in each area is reduced by 10% -30% in the year, and the production of the oil tea can be reduced by more than 50% in the disaster area, so that serious economic loss is caused (Liu Xiaoyu et al, 2018). The method is characterized in that the fruits and leaves of the camellia anthracnose strains from different producing areas are separated and identified to obtain four species of anthrax genus such as Colletotrichum camelliae, colletotrichum gloeosporioides, colletotrichum siamense and Colletotrichum fructicola and three plant disease related fungi such as Neopetalotopsis, pestalotiopsis and Alternaria. Further, it was determined by a pathogenicity test that camellia oleifera fruit anthracnose (Colletotrichumfructicola) is the main pathogen of camellia oleifera anthracnose (Wang et al 2020). The related anthrax related relationship of the four separated oil tea anthracnose is very close, and belongs to the anthrax genus colletotrichum gloeosporioides complex group (Colletotrichum gloeosporioides species complex). At present, a simple and effective method for identifying, detecting and quantifying the fruit anthracnose is not available, and the identification of the fruit anthracnose of the camellia oleifera can only be carried out by a conventional method for separating, purifying and combining with sequencing and homologous sequence comparison.
Disclosure of Invention
In the invention, 965 specific coding sequences of the fruit anthracnose are screened from 16731 coding sequences in the genome of the fruit anthracnose by comparing the whole genome sequences of four anthracnose strains, namely colletotrichum calline, colletotrichum gloeosporioides, colletotrichum simonsite and Colletotrichum fructicola, separated from the anthracnose spots of the oil tea camellia (figure 1). 115 gene coding sequences with important functions were selected based on the sequence annotation and functional prediction provided by NCBI, with 22 gene sequences without introns serving as alternative molecular markers. Finally, one of the Ac transposase molecular markers is selected as an Ac transposase molecular marker for identifying the fruit anthracis.
The primary aim of the invention is to provide an Ac transposase molecular marker for identifying fruit anthracnose, which is one segment of Ac transposase with the gene number CGGC 5-v 012077, and the specific sequence is as follows:
>ANPB02000007.1_cds_KAF4479514.1_12077
ATGGACCATCTTCCTCTTTTTTTCACCAGGGGCCAAGCCTACGTTAAGGCCGAACTTCACAGTGCCCTCACCAAGATCCATATTG
GGTTTGATCTGTGGACTTCACCCAACAACTACGCCTACCTTGCAGTTACTGCCCACTTCGTCAACAATATGGGCCAGCACAAGTC
TCGTCTCATCGCCTTCAACCACATGAGTGGCGACCACAGTGGTCTCAACCTATCAAACAACATCTACGAGACTCTTCAGCAGTGG
GAGATCACTAGCCAAGTGGGTGTGGTGGTCTGTGACAACGCCAGCAATAACGATACCTGTGTCTTCGCCCTCTTCAAGAGACTTA
ACCCCACGATGAATCAGCTCGACTGTCAGGCAAGGAGAATGCGATGCTACGGCCACATCCTCAACCTGGTAGCACGGGCTCTTCT
CTTTGGTGCTGATCGCGAAGTCTTTGAGGCTGAGTCACTCTTCTACCAGACTGTCCACCATGAGGAAGAAGATCTGAGACTCTGG
AGGAAGACTGGGCCAGTTAGCAAGCTTCGCAACATCGTGAAGTTCATCAGGGCTTCCCCTCAGCGATCTGAGCGATTCAGGAAAG
CAGCCCAGGAAGTTGATGCTGGGTCAGACTTTGAGCTATTCGCCCAAGGGTCTAAGGAGTCACATCTCCTCCTCAACAACGAGAC
AAGATGGAATTCCACCTACCTCATGATCCATCGAGCTCTTCAGAAGAGGGCAGAGATTGAGACCTACGTCAACTGGGTTCAGGAG
CAGGATGTTGCCACCAGGAGGATACCTGACGATGACCTCCTTTCTTCAGAAGACTGGAAAGTCTTGGTTGAGATTAGATCTATCC
TTGAGCCTCTCTACCTGCAGACGAAGAGGACAGAGGGATGGGGTAAAGGGGATGGGCATGGTCGTCTCTGGGAAGTCATGACTGG
CATGGAGTACCTCCTTGAGCATCTAGAGGAGTGGAAGAGTCTATACAACTCCATACTCCATCCTATTGATTCTCAACAGGAAGAT
GAGTCGTCTACGATCGATCTGACTGCTGATGAAGAGTCCCTACCATCACAGACTCGATCAGGGCGTCCCATTCGATCTTCAGTCG
ACAGTCAGACCCAGCCTCTTCAGGAATCTATACTTCCACAACATGTAAGGGAGGACTGGAGCCAACGTACTGCTCGATTCAGGGA
TCTCTCTTCCTCTTACCAGGAGCATCTTCGTACTTCTGTTGAGTTGGCATGGCAGAAGCTCTCCTCCTATTATACGAAGTTAGAG
GAGTCCCCTTTGTTTGCTGCTTCTGTTATTCTCCACCCTTCACTTGGTATCTCATACCTTGAGGCAGTCTGGGATGAAGGGGTTC
AGCTTGAGTGGGTTCGTGATGCAAAGAAGGGGCTTAGGGATTACTTTGACCGCTGGTATAGGTCAGAGGAAGAGTCTGATGACCC
TACGGCTGTTTTTGAGATCACACTTCCATCCCATGAAGACAGCCACTTCAGGCAATGGGTACAGAGTAAACGTGGTTGTGAGACT
TCTCGACAGCAGGATGAGCTTGAGACATACCTCAGGCAACCTCCTCAACCCACAGGCGATCCTATTGAGTGGTGGAGAGACCATA
AGTCAACGTACCCTCTACTTAGTAGACTAGCACTTGATGTGATGGCAACACCAGCTATGGCTACTGACTGTGAGAGGGCATTCAG
TACTGCGAAGTTGACCTTGACGTCGCAAAGGCACTCAATAAAGCCTCAGACTATGGGTCAATTGCAGCTGACGAAGAATTGGCTAAAGGGCAGAGTTATGCCTGTGGGGAGTGAGGTTAGCTCATTGATGGGATCATGA, SEQ NO.1.
It is a second object of the present invention to provide amplification primers designed according to the specific sequence of the molecular marker.
The primer, preferably the primer sequence is as follows:
AcTrans-F: GGCGTCCCATTCGATCTTCA; see SEQ NO.2;
AcTrans-R: AGCAGCAAACAAAGGGGACT; see SEQ NO.3.
A third object of the invention is to provide the application of the primer in identifying fruit anthracnose. The specific application steps are as follows:
(1) Extracting total DNA of fungi or fungi/host plant mixed materials as a template;
(2) PCR amplification was performed using primers:
(3) Detecting the PCR product by agarose gel electrophoresis, and reading the strip by a gel imaging system;
(4) According to the product obtained in the step (3), if a specific strip with the length of 212bp appears, indicating that fruit anthrax exists in the detection sample; if there is no specific amplification of 212bp in length, this indicates that no fruit anthrax is present in the sample.
Further, the PCR reaction system was 20. Mu.l in total, and the specific components were that PCR was performed in 20. Mu.l of a reaction system consisting of 1. Mu.l of 20 ng/. Mu.l of genomic DNA, 10. Mu.l of SuperTaqPCR-Mix and 10. Mu.mol of each of the forward and reverse primers.
Further, the PCR amplification procedure is that after 5 minutes of pre-denaturation at 95 ℃; denaturation at 95 ℃ for 15 seconds, annealing temperature 69 ℃, extension temperature 72 ℃,10 seconds, 35 cycles; final extension was then carried out at 72 ℃ for 5 minutes; the reaction was terminated.
The fourth object of the invention is to provide a kit for identifying fruit anthracnose, which comprises PCR reagents and the primers.
The invention has the advantages that:
the invention can distinguish four species of colletotrichum, colletotrichum gloeosporioides, colletotrichum siamense, colletotrichum fructicola and other anthracis and three plant disease related fungi of Neopetalotoiopsis, pestalotiopsis, alternaria and the like with high specificity and high sensitivity. Especially, the related anthrax related to the four oil tea anthracnose is very close in relationship, and belongs to the anthrax genus colletotrichum gloeosporioides complex (Colletotrichum gloeosporioides species complex) which is difficult to distinguish. The invention provides a new strategy for accurately identifying the tea-oil camellia fruit anthracnose.
Meanwhile, compared with the same type of detection method or probe, the invention has the advantage in detection sensitivity, and can be used for detecting and identifying the fruit anthracnose with high sensitivity.
Description of the drawings:
fig. 1: comparing the coding sequences of the genome genes of the anthracnose related anthracnose fungi;
fig. 2: the coding gene CGGC5_v012077 of the fruit anthracnose Ac transposase does not contain an intron region;
fig. 3: the specific primer of the coding gene of the fruit anthracnose Ac transposase has specific amplification in the fruit anthracnose;
DW is Double Distilled Water double distilled water, and double distilled water is taken as a PCR amplification template as a negative control, so that the specificity of the amplification result is proved.
Fig. 4: the detection sensitivity of the gene-specific primer of the fruit anthracnose Ac transposase to fruit anthracnose spores;
fig. 5: sensitivity of the gene-specific primer encoding the fruit anthracnose Ac transposase to detection of fruit anthracnose in camellia oleifera leaf samples.
mock is a blank control, demonstrating no non-specific amplification; TUB is the tea-oil tree Bata-tublin, is a plant genome internal reference, and proves that the four fruit anthracnose gradient dilution mixed tea-oil tree genome DNA samples contain equivalent tea-oil tree genome DNA.
The specific embodiment is as follows:
the following examples are intended to further illustrate the invention, but not to limit it.
Examples: in order to specifically identify the fruit anthracnose (Colletotrichum fructicola), the invention uses a comparative genomics method to find the specific sequence of the fruit anthracnose different from other fungi as a molecular marker. By comparing the whole genome sequences of four anthrax species isolated from camellia oleifera anthracnose spots Colletotrichum camelliae, colletotrichum gloeosporioides, colletotrichum siamense and Colletotrichum fructicola, 965 sequences specific to anthrax fructicola were selected from 16731 sequences in the genome of anthrax fructicola (fig. 1). Based on the sequence annotation and functional prediction provided by NCBI, 115 gene coding sequences with important functions were selected, of which 22 gene sequences without introns (Table 1) were used as alternative molecular markers.
Table 1 compares the specific intronless genes of 22 fruit anthrax obtained by genomic analysis
The NCBI on-line primer design tool blast primer is utilized to design primers for the 22 intron-free specific gene coding sequences of the fruit anthracnose, colletotrichum fructicum, colletotrichum camelliae, colletotrichum gloeosporioides and Colletotrichum siamense genomic DNA are used as templates, the amplification specificity of the designed primers is detected by utilizing common PCR, wherein the specific primers designed for the Ac transposase of the fruit anthracnose only have specific amplification products in the fruit anthracnose, and the specific primers are used for subsequent experiments.
Specific primers were designed for the screened candidate molecular markers of fruit anthracnose and tested for amplification specificity in 4 species of anthracnose genus such as Colletotrichum fructicola, colletotrichum camelliae, colletotrichum gloosporioides and Colletotrichum siamense, and three species of anthracnose-related fungi such as Neopetalotopsis, pestalotiopsis and Alternaria (FIG. 3). The primers designed for Ac transposase encoding genes (number CGGC 5-v 012077, figure 2) are found to have specific amplified bands only in fruit anthracnose and no bands in other anthracnose related fungi by using the genome DNA of the fungi as a template through a common PCR amplification specificity detection experiment.
The genome DNA of the conidium of the fruit anthracnose is taken as a template by gradient dilution, and the fruit of the invention is utilizedThe anthrax Ac transposase encoding gene specific primer is subjected to ordinary PCR, and the detection sensitivity of the primer is tested. The results are shown in FIG. 4, and the number of the conidium of the fruit anthrax in each PCR reaction system is respectively 1 to 10 5 . The result shows that the detection sensitivity of the gene-specific primer of the fruit anthracnose Ac transposase coding gene in the common PCR can reach 10 3 Individual spores per microliter of sample or 5pg of genomic DNA sample.
The genomic DNA of the conidium of the fruit anthracnose subjected to gradient dilution is respectively mixed with the genomic DNA of the tea-oil camellia leaves, the mixed DNA is used as a template, the ordinary PCR is carried out by using the primer specific to the coding gene of the Ac transposase of the fruit anthracnose, and the detection sensitivity of the primer in the mixed sample of the fruit anthracnose and the tea-oil camellia is tested. The results are shown in FIG. 5, and the number of the conidium of the fruit anthrax in each PCR reaction system is 10 respectively 2 To 10 5 . The result shows that the detection sensitivity of the primer pair specific to the gene encoding the fruit anthracnose Ac transposase in the fruit anthracnose and oil tea mixed sample can reach 10 4 Individual spores per microliter of sample or 50pg of genomic DNA sample.
Claims (9)
1. An Ac transposase molecular marker for identifying fruit anthracnose is characterized in that one segment of Ac transposase with a gene number CGGC5_v012077 has the following specific sequence:
ATGGACCATCTTCCTCTTTTTTTCACCAGGGGCCAAGCCTACGTTAAGGCCGAACTTCACAGTGCCCTCACCAAGATCCATATTGGGTTTGATCTGTGGACTTCACCCAACAACTACGCCTACCTTGCAGTTACTGCCCACTTCGTCAACAATATGGGCCAGCACAAGTCTCGTCTCATCGCCTTCAACCACATGAGTGGCGACCACAGTGGTCTCAACCTATCAAACAACATCTACGAGACTCTTCAGCAGTGGGAGATCACTAGCCAAGTGGGTGTGGTGGTCTGTGACAACGCCAGCAATAACGATACCTGTGTCTTCGCCCTCTTCAAGAGACTTAACCCCACGATGAATCAGCTCGACTGTCAGGCAAGGAGAATGCGATGCTACGGCCACATCCTCAACCTGGTAGCACGGGCTCTTCTCTTTGGTGCTGATCGCGAAGTCTTTGAGGCTGAGTCACTCTTCTACCAGACTGTCCACCATGAGGAAGAAGATCTGAGACTCTGGAGGAAGACTGGGCCAGTTAGCAAGCTTCGCAACATCGTGAAGTTCATCAGGGCTTCCCCTCAGCGATCTGAGCGATTCAGGAAAGCAGCCCAGGAAGTTGATGCTGGGTCAGACTTTGAGCTATTCGCCCAAGGGTCTAAGGAGTCACATCTCCTCCTCAACAACGAGACAAGATGGAATTCCACCTACCTCATGATCCATCGAGCTCTTCAGAAGAGGGCAGAGATTGAGACCTACGTCAACTGGGTTCAGGAGCAGGATGTTGCCACCAGGAGGATACCTGACGATGACCTCCTTTCTTCAGAAGACTGGAAAGTCTTGGTTGAGATTAGATCTATCCTTGAGCCTCTCTACCTGCAGACGAAGAGGACAGAGGGATGGGGTAAAGGGGATGGGCATGGTCGTCTCTGGGAAGTCATGACTGGCATGGAGTACCTCCTTGAGCATCTAGAGGAGTGGAAGAGTCTATACAACTCCATACTCCATCCTATTGATTCTCAACAGGAAGATGAGTCGTCTACGATCGATCTGACTGCTGATGAAGAGTCCCTACCATCACAGACTCGATCAGGGCGTCCCATTCGATCTTCAGTCGACAGTCAGACCCAGCCTCTTCAGGAATCTATACTTCCACAACATGTAAGGGAGGACTGGAGCCAACGTACTGCTCGATTCAGGGATCTCTCTTCCTCTTACCAGGAGCATCTTCGTACTTCTGTTGAGTTGGCATGGCAGAAGCTCTCCTCCTATTATACGAAGTTAGAGGAGTCCCCTTTGTTTGCTGCTTCTGTTATTCTCCACCCTTCACTTGGTATCTCATACCTTGAGGCAGTCTGGGATGAAGGGGTTCAGCTTGAGTGGGTTCGTGATGCAAAGAAGGGGCTTAGGGATTACTTTGACCGCTGGTATAGGTCAGAGGAAGAGTCTGATGACCCTACGGCTGTTTTTGAGATCACACTTCCATCCCATGAAGACAGCCACTTCAGGCAATGGGTACAGAGTAAACGTGGTTGTGAGACTTCTCGACAGCAGGATGAGCTTGAGACATACCTCAGGCAACCTCCTCAACCCACAGGCGATCCTATTGAGTGGTGGAGAGACCATAAGTCAACGTACCCTCTACTTAGTAGACTAGCACTTGATGTGATGGCAACACCAGCTATGGCTACTGACTGTGAGAGGGCATTCAGTACTGCGAAGTTGACCTTGACGTCGCAAAGGCACTCAATAAAGCCTCAGACTATGGGTCAATTGCAGCTGACGAAGAATTGGCTAAAGGGCAGAGTTATGCCTGTGGGGAGTGAGGTTAGCTCATTGATGGGATCATGA。
2. an amplification primer designed according to the specific sequence of the molecular marker of claim 1.
3. The primer of claim 2, wherein the primer sequence is designed as follows:
AcTrans-F：GGCGTCCCATTCGATCTTCA；
AcTrans-R：AGCAGCAAACAAAGGGGACT。
4. use of the primer of claim 2 or 3 for identifying fruit-borne anthrax.
5. The use according to claim 4, wherein,
(1) Extracting total DNA of fungi or fungi/host plant mixed materials as a template;
(2) PCR amplification was performed using primers:
(3) Detecting the PCR product by agarose gel electrophoresis, and reading the strip by a gel imaging system;
(4) According to the product obtained in the step (3), if a specific strip with the length of 212bp appears, indicating that fruit anthrax exists in the detection sample; if there is no specific amplification of 212bp in length, this indicates that no fruit anthrax is present in the sample.
6. The method according to claim 5, wherein the PCR reaction system is 20. Mu.l in total, and the PCR reaction is performed in 20. Mu.l of a reaction system consisting of 1. Mu.l of 20 ng/. Mu.l of genomic DNA, 10. Mu.l of SuperTaqPCR-Mix and 10. Mu.mol of forward and reverse primers, respectively, 1. Mu.l.
7. The use according to claim 5, wherein,
the PCR amplification procedure is that the PCR amplification procedure is carried out after 5 minutes of pre-denaturation at 95 ℃; denaturation at 95 ℃ for 15 seconds, annealing temperature 69 ℃, extension temperature 72 ℃,10 seconds, 35 cycles; final extension was then carried out at 72 ℃ for 5 minutes; the reaction was terminated.
8. The use according to claim 5 for distinguishing between
Colletotrichum camelliae Colletotrichumgloosporioides, colletotrichum siamense species of anthrax, neopetalotopsis, pestalotiopsis and Alternaria.
9. A kit for identifying anthrax fruit, comprising PCR reagents and the primer of claim 2 or 3.
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