CN110257545B - Molecular marker for identifying hybrid paper mulberry and application thereof - Google Patents
Molecular marker for identifying hybrid paper mulberry and application thereof Download PDFInfo
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- CN110257545B CN110257545B CN201910599077.8A CN201910599077A CN110257545B CN 110257545 B CN110257545 B CN 110257545B CN 201910599077 A CN201910599077 A CN 201910599077A CN 110257545 B CN110257545 B CN 110257545B
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
Abstract
The invention discloses a molecular marker for identifying a hybrid paper mulberry and application thereof. The molecular markers are 1) or 2) as follows: 1) a DNA molecule shown in a sequence 3 in a sequence table: 2) and (b) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 3 and has the same function as the sequence 3. The molecular marker derived from the broussonetia papyrifera provided by the invention can be applied to distinguishing the broussonetia papyrifera from other plants such as wild broussonetia papyrifera and broussonetia papyrifera, has important theoretical and practical significance for variety identification of the broussonetia papyrifera, can be used for cultivation and identification of plant varieties required by agriculture and animal husbandry and ecological environment management, and has high practical application value. The invention has wide application prospect in the field of agricultural and economic energy crops.
Description
Technical Field
The invention relates to a plant molecular marker, in particular to a molecular marker for identifying a hybrid paper mulberry and application thereof.
Background
The DNA molecular identification can be applied to paternity test, disease diagnosis, prenatal screening, Chinese herbal medicine identification, plant variety identification, transgene identification and the like. DNA molecular identification is a new biological identification system created by utilizing the specificity and species diversity of standard, sufficiently variable, easily amplifiable and relatively short DNA fragments per se, and can rapidly and automatically identify species or samples.
Broussonetia papyrifera (Broussonetia papyrifera) also known as Broussonetia papyrifera, hub, Broussonetia papyrifera, Gougui tree, Baozhi tree, Orobanchus, Dastarcus and papayas belong to nitrogen-fixing branches, Rosa, Rosales and Moraceae Broussonetia in the APGIII system (angiospermm Phylogeny Group). Except for northeast and northwest areas, other provinces in China are naturally distributed and are native tree species in China. In southeast Asia, Europe, America and the Pacific region, etc. The paper mulberry is not only a cloth making raw material, but also a paper making raw material, has better edible and medicinal values, is a good livestock and poultry feed raw material, and has good ecological and economic benefits because the paper mulberry and the paper mulberry industry have the functions of treating saline-alkali soil and stony desertification, accelerating afforestation, recycling and continuous utilization and the like.
The Shenhua research team of Chinese academy of sciences uses wild Broussonetia papyrifera as male parent and Broussonetia kazinoki as female parent, and adopts the method of combining modern breeding technology and conventional technology to make interspecific hybridization so as to obtain the original new germplasm (B.kazinoki X B.papyrifera) of hybrid Broussonetia papyrifera, and utilizes the space carrying of Shenzhou airship series for 8 years, and under the special environmental influences of space microgravity, high vacuum, weak magnetic field and multi-radiation ray mutagenesis, the cells are stimulated to produce stable gene variation which is difficult to implement on ground. After the seedlings are taken off from the Shenzhou airship, the seedlings are cut by a scientific research team of a plant institute of Chinese academy of sciences and then are cultured in specific culture media respectively, the seedlings of the hybrid paper mulberry grow naturally, and the seedlings which grow to a certain degree are planted in soil for cultivation. Through observing the growth process of the variant seedlings, varieties with excellent properties are screened out, and finally, a new hybrid broussonetia papyrifera variety which is more suitable for industrial popularization and application is cultivated and named as hybrid broussonetia papyrifera 101.
The hybrid broussonetia papyrifera is a new strain obtained by interspecific hybridization of a research team of plant research institute of Chinese academy of sciences with wild broussonetia papyrifera as male parents and broussonetia papyrifera as female parents. Market disorientation often occurs in the current market that uses wild broussonetia papyrifera and other plants to impersonate and replace hybrid broussonetia papyrifera, and covers growers, thus causing economic losses to the growers. Therefore, how to rapidly and accurately identify the hybrid broussonetia papyrifera is of great significance. At present, no DNA molecular marker specially aiming at the hybrid paper mulberry exists in the market.
Disclosure of Invention
The first purpose of the invention is to provide a molecular marker for identifying hybrid paper mulberry.
The molecular marker for identifying the hybrid paper mulberry provided by the invention is derived from the hybrid paper mulberry (Broussonetia kazinoki multiplied by B. papyrifera), and is 1) or 2):
1) DNA molecule shown in sequence 3 in the sequence table;
2) and (b) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 3 and has the same function as the sequence 3.
The second objective of the invention is to provide a primer pair for amplifying the full length of the DNA molecule or any fragment thereof.
Further, the primer pair consists of a forward primer and a reverse primer;
the forward primer is a1) or a2) as follows:
a1) a single-stranded DNA molecule shown as a sequence 1 in a sequence table;
a2) a single-stranded DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 1 and has the same function as the sequence 1;
the reverse primer is a3) or a4) as follows:
a3) a single-stranded DNA molecule shown in a sequence 2 in a sequence table;
a4) and (b) a single-stranded DNA molecule obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 2 and having the same function as the sequence 2.
The third purpose of the invention is to provide a new application of the molecular marker or the primer pair.
The invention provides the application of the molecular marker or the primer pair in any one of the following c1) -c 8):
c1) preparing a product for identifying or assisting in identifying the hybrid broussonetia papyrifera;
c2) identifying or assisting in identifying the hybrid broussonetia papyrifera;
c3) preparing a product for identifying or assisting in identifying whether a plant sample to be detected is a hybrid broussonetia papyrifera or not;
c4) identifying or assisting in identifying whether the plant sample to be detected is a hybrid broussonetia papyrifera or not;
c5) preparing a product for distinguishing or assisting in distinguishing the hybrid paper mulberry from other plants;
c6) distinguishing or assisting in distinguishing the hybrid paper mulberry from other plants;
c7) preparing products for distinguishing or assisting in distinguishing the hybrid paper mulberry from other paper mulberry;
c8) distinguishing or assisting in distinguishing hybrid broussonetia papyrifera from other broussonetia papyrifera.
The fourth purpose of the invention is to provide a kit containing the primer pair;
the function of the kit is any one of d1) -d4) as follows:
d1) identifying or assisting in identifying the hybrid broussonetia papyrifera;
d2) identifying or assisting in identifying whether the plant sample to be detected is a hybrid broussonetia papyrifera or not;
d3) distinguishing or assisting in distinguishing the hybrid paper mulberry from other plants;
d4) distinguishing or assisting in distinguishing hybrid broussonetia papyrifera from other broussonetia papyrifera.
The kit of the present invention also includes other reagents for PCR amplification.
The other reagents for PCR amplification may include Buffer, dNTP, Taq enzyme and ddH2O。
The fifth purpose of the invention is to provide a method for identifying or assisting in identifying whether a plant sample to be detected is a hybrid broussonetia papyrifera or not.
The method for identifying or assisting in identifying whether the plant sample to be detected is the hybrid broussonetia papyrifera or not provided by the invention comprises the following steps: detecting whether the plant sample to be detected contains the molecular marker:
if the plant sample to be detected contains the molecular marker, the plant sample to be detected is or is selected as a hybrid broussonetia papyrifera;
and if the plant sample to be detected does not contain the molecular marker, the plant sample to be detected is not the hybrid paper mulberry or the candidate is not the hybrid paper mulberry.
In the method for identifying or assisting in identifying whether the plant sample to be detected is the hybrid broussonetia papyrifera or not,
the method for detecting whether the plant sample to be detected contains the molecular marker comprises the following steps: and (2) performing PCR amplification by using the genome DNA of the plant sample to be detected as a template and adopting the primer pair to obtain a PCR product, and judging whether the plant sample to be detected contains the molecular marker according to the nucleotide sequence of the PCR product:
if the nucleotide sequence of the PCR product is sequence 3, the plant sample to be detected is or is selected as a hybrid paper mulberry;
and if the nucleotide sequence of the PCR product is not the sequence 3, the plant sample to be detected is not the hybrid paper mulberry or the candidate is not the hybrid paper mulberry.
The sixth purpose of the invention is to provide a method for distinguishing or assisting in distinguishing hybrid broussonetia papyrifera from other broussonetia papyrifera.
The method for distinguishing or assisting in distinguishing the hybrid paper mulberry from other paper mulberry provided by the invention comprises the following steps: detecting whether the plant sample to be detected contains the molecular marker:
if the plant sample to be detected contains the molecular marker, the plant sample to be detected is or is selected as a hybrid broussonetia papyrifera;
and if the plant sample to be detected does not contain the molecular marker, the plant sample to be detected is or is selected as other broussonetia papyrifera.
In the above method for distinguishing or assisting in distinguishing the hybrid paper mulberry from other paper mulberry,
the method for detecting whether the plant sample to be detected contains the molecular marker comprises the following steps: and (2) performing PCR amplification by using the genome DNA of the plant sample to be detected as a template and adopting the primer pair to obtain a PCR product, and judging whether the plant sample to be detected contains the molecular marker according to the nucleotide sequence of the PCR product:
if the nucleotide sequence of the PCR product is sequence 3, the plant sample to be detected is or is selected as a hybrid paper mulberry;
and if the nucleotide sequence of the PCR product is not the sequence 3, the plant sample to be detected is or is selected as other paper mulberry.
It is a final object of the invention to provide a method for distinguishing or assisting in distinguishing hybrid broussonetia papyrifera from other plants.
The method for distinguishing or assisting in distinguishing the hybrid paper mulberry from other plants comprises the following steps:
1) and (2) performing PCR amplification by using the genome DNA of the plant sample to be detected as a template and adopting the primer pair to obtain a PCR product, and judging whether the plant sample to be detected is a paper mulberry or other plants according to the size of the PCR product:
if the size of the PCR product is 3649bp, the plant sample to be detected is or is selected as a paper mulberry tree;
if the size of the PCR product is 4125bp, the plant sample to be detected is or is selected as other plants;
2) and distinguishing the hybrid paper mulberry from other paper mulberry according to the method for distinguishing or assisting in distinguishing the hybrid paper mulberry from other paper mulberry.
In any of the above methods, the PCR reaction system is specifically as follows (total volume is 25 μ L): 1 μ L of DNA template, 1 μ L of each of forward and reverse primers, 10 XBuffer 2.5 μ L, 2 μ L of dNTP mix (10mmol/L each), 0.25 μ L of Taq enzyme (20U/μ L), ddH2O12.25. mu.L. The final concentration of the forward primer and the final concentration of the reverse primer in the reaction system are both 5 mu M.
The PCR reaction conditions are specifically as follows: pre-denaturation at 94 ℃ for 5 min; 36 cycles of 94 ℃ for 30s, 55 ℃ for 30s and 72 ℃ for 60 s; extension at 72 ℃ for 10 min.
In the above molecular marker or application or kit or method, the plant sample to be tested may be a leaf, root or stem of a plant to be tested.
In the above molecular marker or application or kit or method, the hybrid broussonetia papyrifera is hybrid broussonetia papyrifera No. 101;
the other plants are selected from at least one of the following: arabidopsis thaliana _ thraciana, Beta _ vulgaris _ subsp, Brassica campestris _ napus, Brassica oleracea _ oleracea, Broussonetia papyrifera, Broussonetia kazinoki, Broussonetia papyrifera, Cannabis _ sativa, Capsicum Capsicum capsulatum _ annuum, Carica papaya _ papaya, Chlamydomonas reinhardtii _ reinhardtii, Citrullus Citrullus _ lantus, Cucumis melo _ specimen, Cucumis sativus _ sativus, Dendrobii officinarum _ indica, Ectocarpus sativus _ silvestris, Brassica oleracea _ serovar, Fragaria _ veuca, Glycine _ Glycine, Glycine _ max, Glycine japonica _ soybean, Glycine japonica _ saponin, Gossima japonica _ tomato, Gomphis japonica, Glycine japonica _ Thamnolia, Glycine japonica, Glycine indica, Glycine japonica, Glycine japonica, Glycine, potato Solanum _ tuberosum, Sorghum _ bicolor, wheat Triticum _ aestivum, mung bean Vigna _ radiata, corn Zea _ mays, jujube tree Ziziphus _ jujuba;
the other broussonetia papyrifera is selected from at least one of the following: broussonetia _ kazinoki, Broussonetia _ papyrifera, wild Broussonetia.
The invention provides a molecular marker for identifying a hybrid paper mulberry based on a DNA bar code technology, designs a primer pair for amplifying the molecular marker, and successfully establishes an identification method of the hybrid paper mulberry. The molecular marker provided by the invention can distinguish the hybrid paper mulberry from other plants such as wild paper mulberry, broussonetia papyrifera and the like, can be used for identifying hybrid paper mulberry seed sources popularized all over the country at present, can protect intellectual property rights of plant houses in Chinese academy of sciences, can guarantee benefits of vast growers, has important theoretical and practical significance for identifying varieties of the hybrid paper mulberry, and has higher practical application value for cultivating and identifying plant varieties required by agriculture and animal husbandry and ecological environment management. The invention has wide application prospect in the field of agricultural and economic energy crops.
Drawings
FIG. 1 is a schematic diagram of electrophoresis in sample DNA extraction.
FIG. 2 is a schematic diagram of electrophoresis of PCR results.
FIG. 3 is a diagram showing the alignment results.
FIG. 4 is a schematic diagram of a structure tree of the evolutionary tree.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified.
The nucleotide sequences used in the following examples were all synthesized by Megi biosciences, Shanghai. The reagents used were those of NEB company unless otherwise specified, and the water used was ultrapure water.
The paper mulberry in the following examples is paper Broussonetia kekuei 101 (' Kekuei 101 ') described in Shenshiwa, Pengyu, Setarian, Xiongwei. Innovation industry for assisting poverty-Effect and revelation of the hybrid paper mulberry poverty-England project in Shandong, J.Proc. of Chinese academy of sciences, 2018,33(9):979 + 986 ', and the public can obtain the biological material from the applicant, and the biological material is only used for repeating the related experiments of the invention and cannot be used for other purposes.
Example 1 design of molecular markers for identifying hybrid Broussonetia papyrifera and amplification primer pairs specific for the same
Molecular marker of first, hybrid broussonetia papyrifera
The present invention provides a DNA barcode (DNA barcode) derived from a hybrid paper mulberry (Broussonetia kazinoki. times.B. papyrifera), the nucleotide sequence of which is shown as sequence 3 in the sequence table, and which is named Bpmcbode. The DNA molecule shown in the sequence 3 is a molecular marker for identifying the hybrid paper mulberry.
Second, specific primer pair
Designing a primer pair for specifically amplifying the full length of the hybrid broussonetia papyrifera according to the Bpmcbode sequence which is obtained in the first step and is unique to the hybrid broussonetia papyrifera, wherein the primer sequence is as follows:
f: 5'-AACCTGGGGCGTATTGCTCAA-3' (SEQ ID NO: 1);
r: 5'-TTACCATATATATATAACACAAAATTTC-3' (SEQ ID NO: 2).
Example 2 specific detection of primer pairs for identifying hybrid Broussonetia papyrifera
1. Extraction of DNA from plant material
Total DNA of each plant material in Table 1 was extracted separately, and the extracted total DNA was detected by 1% agarose gel electrophoresis. Wherein, the agarose gel electrophoresis detection result of the total DNA extracted by taking the leaves, roots or stalks of the hybrid paper mulberry as the material is shown in figure 1. The result shows that the extracted DNA has obvious electrophoresis band which is larger than 20kb, and the obtained DNA has higher purity and is more complete.
2. PCR amplification
And (3) respectively taking the total DNA samples extracted in the step (1) as templates, and carrying out PCR amplification by using the primer pairs designed in the step (II) of the embodiment 1 to respectively obtain PCR products.
The PCR reaction system was as follows (total volume 25. mu.L): 1 μ L of DNA template, 1 μ L of each of F, R primers, 2.5 μ L of 10 XBuffer, 2 μ L of dNTP mix (10mmol/L each), 0.25 μ L of Taq enzyme (concentration 20U/. mu.L), ddH2O12.25. mu.L. F. The final concentration of the R primers in the reaction system was 5. mu.M.
The PCR reaction conditions were as follows: pre-denaturation at 94 ℃ for 5 min; then 36 cycles of 94 ℃ for 30s, 55 ℃ for 30s and 72 ℃ for 60 s; finally, extension is carried out for 10min at 72 ℃.
3. PCR product detection and sequencing
And (3) carrying out electrophoresis detection on the PCR product, then recovering by using a PCR product purification kit (Beijing all-type gold biotechnology limited), and directly sending to Shanghai Meiji bio-corporation for sequence sequencing after recovery.
The results are shown in Table 1.
TABLE 1 Chinese and Latin names of the respective plant materials and their amplification product sizes
The results show that: the hybrid paper mulberry, the broussonetia papyrifera and the wild paper mulberry are all amplified to obtain a band with the size of 3649bp (the electrophoresis detection result of the PCR product of the hybrid paper mulberry is shown in figure 2), and the other plant materials are all amplified to obtain a band with the size of 4125 bp. Therefore, the specific primer pair of the invention can effectively distinguish the paper mulberry from other species: if the PCR product size of the plant sample to be detected is 3649bp, the plant sample to be detected is a paper mulberry tree; and if the PCR product of the plant sample to be detected is 4125bp, determining that the plant sample to be detected is other plants.
In addition, the nucleotide sequence of the amplification product in the hybridization broussonetia papyrifera of the specific primer pair of the invention is shown as sequence 3, the nucleotide sequence of the amplification product in the broussonetia papyrifera is shown as sequence 4, and the nucleotide sequence of the amplification product in the wild broussonetia papyrifera is shown as sequence 5. The invention can also distinguish hybrid broussonetia papyrifera, broussonetia papyrifera and wild broussonetia papyrifera according to Bpmcbode sequences: if the nucleotide sequence of the PCR product of the plant sample to be detected is sequence 3, the plant sample to be detected is a hybrid paper mulberry; and if the nucleotide sequence of the PCR product of the plant sample to be detected is not the sequence 3, determining that the plant sample to be detected is other paper mulberry trees.
4. Alignment of sequences
The sequence results obtained were aligned with response sequences from other species using version mega 7.0. The alignment results are shown in FIG. 3.
5. Construction of an evolutionary tree
And (4) constructing an evolutionary tree by using the strategy of NJ to the comparison result of the step 4. The constructed evolutionary tree is shown in fig. 4. The evolutionary tree result shows that the Bpmcbode sequence can distinguish hybrid broussonetia papyrifera from plants such as wild broussonetia papyrifera, broussonetia papyrifera and the like, and is successfully applied to identification of the hybrid broussonetia papyrifera.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
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aagatgccta atatttacaa cgctgacttg tgaagtacag caattattag gaaataatcg 120
agttagagct gtagctatga gtgctacaga tggtctaatg agaggaatgg aagtgattga 180
cacaggagct cctttaagtg ttccggtcgg cggagcgacg ttaggacgaa tttttaacgt 240
gcttggagag cccgttgata atttaggtcc tgtagatact cgcacaacat ctcctattca 300
tagatctgcg cctgccttta tacaattaga tacaaaatta tcgatttttg aaacaggaat 360
taaagtagta gatcttttag ccccttatcg ccgtggggga aaaatagggc tattcggggg 420
ggctggagta ggtaaaactg tactcattat ggaattgatc aacaacattg ccaaagctca 480
tggaggtgta tccgtatttg gtggagtagg tgagcgcacc cgtgaaggaa atgatcttta 540
catggaaatg aaagaatccg gcgtaattaa tgaacaaaat attgcagaat caaaagtggc 600
tctagtctac ggtcagatga atgaaccgcc gggggctcgt atgagagttg gtttaactgc 660
gctaactatg gcggagtatt tccgagatgt taatgaacaa gatgtacttc tatttatcga 720
caatatcttt cgttttgtcc aagcgggatc cgaagtatcc gccttattgg gtagaatgcc 780
ttctgctgtg ggttatcaac ccactcttag taccgaaatg ggttctttac aagaaagaat 840
tacttctacc aaagaggggt ccataacttc tattcaagca gtctatgtac ctgcggatga 900
tttgaccgat ccagctccgg ccacgacatt tgcacattta gatgctacta ccgtactatc 960
aagaggattg gccgccaaag gcatctatcc agcagtagat cctttagatt caacatcaac 1020
tatgctccaa cctcggattg ttggtgaaga acattatgaa actgcgcaaa gagttaaaca 1080
aactttacaa cgttacaaag aacttcagga cattatagct atccttgggt tggacgaatt 1140
atccgaagaa gatcgcttaa ccgtagcaag agcacgaaaa attgaacgtt tcctgtcaca 1200
accctttttt gtagcagaag tatttaccgg ttcaccaggg aaatatgttg gtctagcaga 1260
aacaattaga gggtttaaat tgattctttc tggcgaatta gatggtcttc ctgaacaggc 1320
tttttatttg gtaggtaaca ttgatgaagc tactgcgaag gcatgacttt ttattatcgt 1380
ccgaccgtta cggaggcttt tgcttcggtt caatatataa tgactgaagc taactttggt 1440
tggttaatcc gatcagttca tcgatggtcg gcaagtatga tggtactaat gatgatcctc 1500
cacgtatttc gtgtgtatct cactggtggc tttaaaaaac cgcgcgaatt gacttgggtt 1560
acaggtgtag ttttggctgt attgaccgca tcttttggtg taactggtta ttccttacct 1620
tgggaccaaa ttggttattg ggcagtaaaa attgtaacag gtgtaccgga agcgattcct 1680
ctaataggat cgcccttggt agagttatta cgtggaagtg ctagtgtggg acaatcaact 1740
ttgactcgtt tttatagttt acacactttt gtattacctc ttcttactgc cgtatttatg 1800
ttaatgcatt tcctaatgat acgtaagcaa ggtatttctg gccctttata atgactatag 1860
cccttggtaa aaaagatgaa aatgatttga tattatggat gactggttac ggagggaccg 1920
ttttgttttt gtaggttggt ccggtctatt gctctttcct tgtgcctatt tcgctttagg 1980
aggttggttc acaggtacaa cctttgtaac ttcatggtat actcatggat tagcaagttc 2040
ctatttggaa ggatgcaact tcttaactgc cgcagtttct actcctgcta atagtttagc 2100
acactctttg ttgttactat ggggtcctga agcacaaggc gattttactc gttggtgtca 2160
attaggtggc ctgtggactt ttgttgctct ccacggcgct ttcggactaa taggtttcat 2220
gttacgtcaa tttgaacttg ctcgatctgt tcaattgcgc ccttataatg caatcgcatt 2280
ctccggtcca attgctgttt ttgtttctgt attcctgatt tatccgttag gtcagtctgg 2340
ttggttcttt gcgcctagtt ttggtgtagc agctatattt cgattcatcc tctttttcca 2400
agggtttcat aattggacat tgaacccatt tcacatgatg ggcgttgccg gggtattggg 2460
tgctgctctg ctatgtgcta ttcatggtgc taccgtagaa aatactttat ttgaagatgg 2520
tgatggtgca aatacattcc gtgcttttaa cccaactcaa gctgaagaaa cttattcaat 2580
ggttaccgct aaccgctttt ggtcccaaat ctttggggtt gctttttcca ataaacgttg 2640
gttacatttc tttatgttat ttgtaccagt aactggttta tggatgagtg ctcttggagt 2700
agtcggtctg gctttgaatc tacgtgccta tgacttcgtt tctcaggaaa tccgtgcagc 2760
ggaagatcct gaatttgaga ctttctacac caaaaatatt ctcttaaacg agggtattcg 2820
tgcttggatg gcggctcaag atcagcctca tgaaaacctt atattccctg aggaggttct 2880
accccgtgga aacgctcttt aaatgggaca aaaaataaat ccacttggtt tcagacttgg 2940
tgcaacccaa ggtcatcatt ctctttggtt tgccaaccaa aaaattattc tccaagaaga 3000
taaaaaatac gatagtatca agaaatcgac ttgacaaaaa gaactttgca aatccttatg 3060
gacaccctaa tattcttgca gaatttatag ccggacaatt aaaaaataat tccgcaaaaa 3120
tgaaaaaagc gacaaaagga attcaaatac aaattgcagg gcgtatcgac ggaaaagaaa 3180
ttgcacgtgt cgaatggatc agagaaggta gggtgcctct acaaaccatt agggctaaaa 3240
ttgattactg ttcctataca gttggaacta tctatggggc tttaggtatc aaaatttgga 3300
tattatgagt aaagacgcta ttgctaacat actaacttct atacgaaatg ctgacatgaa 3360
taaaaaaaga acagttcgaa taccatatac taacatcact gaaaacattc ttaaaatcct 3420
tttacgagaa ggttttattg aaaaatgagg aaacatcagg aaaacaacaa tctttttttg 3480
gtttcagatc agtcgacccg gtctacgaat ctattctaat tatcaaagaa tcccaagaat 3540
tttaggcggg atagggattg taattctttc tacttctcgg ggtataatga cagacagaga 3600
agctcgacta gaaggaatcg gtggagaaat tttgtgttat atatggtaa 3649
<210>5
<211>3649
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>5
aacctggggc gtattgctca aatcattggt ccggtactgg atgtagcttt tccgccgggc 60
aagatgccta atatttacaa cgctgacttg tgaagtacag caattattag gaaataatcg 120
agttagagct gtagctatga gtgctacaga tggtctaatg agaggaatgg aagtgattga 180
cacaggagct cctttaagtg ttccggttgg cggagcgacg ttaggacgaa tttttaacgt 240
gcttggagag cccgttgata atttaggtcc tgtagatact cgcacaacat ctcctattca 300
tagatctgcg cctgccttta tccaattaga tacaaaatta tcgatttttg aaacaggaat 360
taaagtagta gatcttttag ctccttatcg ccgtggagga aaaataggac tattcggggg 420
ggctggagta ggtaaaactg tactcattat ggaattgatc aacaatattg ccaaagctca 480
tggaggtgta tccgtatttg gtggagtagg tgagcgcacc cgtgaaggaa atgatcttta 540
catggaaatg aaagaatccg gcgtaattaa tgaacaaaat attgcagaat caaaagtggc 600
tctagtctac ggtcagatga atgaaccgcc gggggctcgt atgagagttg gtttaactgc 660
gctaactatg gcggagtatt tccgagatgt taatgaacaa gatgtacttc tatttatcga 720
caatatcttt cgttttgtcc aagcgggatc cgaagtatcc gccttattgg gtagaatgcc 780
ttctgctgtg ggttatcaac ccactcttag taccgaaatg ggttctttac aagaaagaat 840
tacttctacc aaagaggggt ccataacttc tattcaagca gtttatgtac ctgcggatga 900
tttaaccgat ccagctccgg ccacgacatt tgcacattta gatgctacta ccgtactatc 960
aagaggattg gccgccaaag gcatctatcc agcagtagat cctttagatt caacgtcaac 1020
tatgctccaa cctcggattg ttggtgaaga acattatgaa actgcgcaaa gagttaaaca 1080
aactttacaa cgttacaaag aacttcagga cattatagct atccttgggt tggacgaatt 1140
atccgaagaa gatcgcttaa ccgtagcaag agcacgaaaa attgaacgtt tcctgtcaca 1200
accctttttt gtagcagaag tatttaccgg ttcaccaggg aaatatgttg gtctagcaga 1260
aacaattaga gggtttaaat tgatcctttc tggcgaatta gatggtcttc ctgaacaggc 1320
tttttatttg gtaggtaaca ttgatgaagc tactgcgaag gcatgacttt ttattatcgt 1380
ccgaccgtta cggaggcttt tgcttcggtt caatatataa tgactgaagc taactttggt 1440
tggttaatcc gatcagttca tcgatggtcg gcaagtatga tggtactaat gatgatcctc 1500
cacgtatttc gtgtgtatct cactggtggc tttaaaaaac cgcgcgaatt gacttgggtt 1560
acaggtgtag ttttggctgt attgaccgca tcttttggtg taactggtta ttctttacct 1620
tgggaccaaa ttggttattg ggcagtaaaa attgtaacag gtgtaccgga agcgattcct 1680
ctaataggat ctcccttggt agagttatta cgtggaagtg ctagtgtggg acaatcaact 1740
ttgactcgtt tttatagttt acacactttt gtattacctc ttcttactgc cgtatttatg 1800
ttaatgcatt tcctaatgat acgtaagcaa ggtatttctg gccctttata atgactatag 1860
cccttggtaa aaaagacgaa aatgatttga tattatggat gactggttac ggcgggaccg 1920
ttttgttttt gtaggttggt ccggtctatt gctctttcct tgtgcctatt tcgctttagg 1980
aggttggttc acaggtacaa cctttgtaac ttcatggtat actcatggat tagcaagttc 2040
ctatttggaa ggatgcaact tcttaactgc tgcagtttcg actcctgcta atagtttagc 2100
acactctttg ttgttactat ggggtcctga agcacaaggc gattttactc gttggtgtca 2160
attaggcggc ctgtggactt ttgttgctct ccacggcgct ttcgcactaa taggtttcat 2220
gttacgtcaa tttgaacttg ctcgatctgt tcaattgcgc ccttataatg caatcgcatt 2280
ctccggtcca attgctgttt ttgtttctgt attcctgatt tatccgttag gtcagtctgg 2340
ttggttcttt gcgcctagtt ttggtgtagc agctatattt cgattcatcc tctttttcca 2400
agggtttcat aattggacat tgaacccatt tcacatgatg ggcgttgccg gggtattggg 2460
tgctgctctg ctatgtgcta ttcatggtgc taccgtagaa aatactttat ttgaagatgg 2520
tgatggtgca aatacattcc gtgcttttaa cccaactcaa gctgaagaaa cttattcaat 2580
ggttaccgct aaccgctttt ggtcccaaat ctttggggtt gctttttcca ataaacgttg 2640
gttacatttc tttatgttat ttgtaccagt aactggttta tggatgagtg ctcttggagt 2700
agtcggtctg gctttgaatc tacgtgccta tgacttcgtt tctcaggaaa tccgtgcagc 2760
ggaagatcct gaatttgaga ctttctacac caaaaatatt ctcttaaacg agggtattcg 2820
tgcttggatg gcggctcaag atcagcctca tgaaaacctt atattccctg aggaggttct 2880
accccgtgga aacgctcttt aaatgggaca aaaaataaat ccacttggtt tcagacttgg 2940
tgcaacccaa agtcatcatt ctctttggtt tgccaaccaa aaaattattc tacaagaaga 3000
taaaaaatac gatagtatca agaaatcgac ttgacaaaaa gaactttgca aatccttatg 3060
gacaccctaa tattcttgca gaatttatag ccggacaatt aaaaaataat tccgcaaaaa 3120
tgaaaaaagc gacaaaagga attcaaatac aaattgcagg gcgtatcgac ggaaaagaaa 3180
ttgcacgtgt cgaatggatc agagaaggta gggtgcctct acaaaccatt agggctaaaa 3240
ttgattactg ttcctataca gttcgaacta tctatggggc tttaggtatc aaaatttgga 3300
tattatgagt aaagacgcta ttgctaacat actaacttct atacgaaatg ctgacatgaa 3360
taaaaaaaga acagttcgaa taccatatac taacatcact gaaaacattc ttaaaatcct 3420
tttacgagaa ggttttattg aaaaatgagg aaacatcagg aaaacaacaa tctttttttg 3480
gtttcagatc agtcgacccg gtctacgaat ctattctaat tatcaaagaa tcccaagaat 3540
tttaggcggg atggggattg taatcctttc tacttctcgg ggtataatga cagacagaga 3600
agctcgacta gaaggaatcg gtggagaaat tttgtgctat atatggtaa 3649
Claims (10)
1. A molecular marker for identifying a hybrid paper mulberry is a DNA molecule shown as a sequence 3 in a sequence table;
the hybrid paper mulberry is hybrid paper mulberry 101.
2. The primer pair for amplifying the molecular marker of claim 1, which consists of a single-stranded DNA molecule shown in a sequence 1 in a sequence table and a single-stranded DNA molecule shown in a sequence 2 in the sequence table.
3. Use of the molecular marker of claim 1 or the primer pair of claim 2 in any one of c1) or c2) as follows:
c1) preparing a product for identifying a hybrid broussonetia papyrifera;
c2) identifying the hybrid broussonetia papyrifera;
the hybrid paper mulberry is hybrid paper mulberry 101.
4. A kit containing the primer pair according to claim 2;
the function of the kit is to identify the hybrid paper mulberry;
the hybrid paper mulberry is hybrid paper mulberry 101.
5. A method for identifying whether a plant sample to be detected is a hybrid broussonetia papyrifera or not comprises the following steps: detecting whether a plant sample to be tested contains the molecular marker of claim 1:
if the plant sample to be tested contains the molecular marker in the claim 1, the plant sample to be tested is a hybrid paper mulberry;
if the plant sample to be tested does not contain the molecular marker of claim 1, the plant sample to be tested is not a hybrid broussonetia papyrifera;
the hybrid paper mulberry is hybrid paper mulberry 101.
6. A method for distinguishing a hybrid broussonetia papyrifera from other broussonetia papyrifera, comprising the steps of: detecting whether a plant sample to be tested contains the molecular marker of claim 1:
if the plant sample to be tested contains the molecular marker in the claim 1, the plant sample to be tested is a hybrid paper mulberry;
if the plant sample to be tested does not contain the molecular marker of claim 1, the plant sample to be tested is other broussonetia papyrifera;
the hybrid paper mulberry is hybrid paper mulberry 101.
7. The method of claim 6, wherein: the method for detecting whether a plant sample to be detected contains the molecular marker in claim 1 comprises the following steps: using the genome DNA of a plant sample to be detected as a template, carrying out PCR amplification by using the primer pair of claim 2 to obtain a PCR product, and judging whether the plant sample to be detected contains the molecular marker of claim 1 according to the nucleotide sequence of the PCR product.
8. The method according to claim 6 or 7, characterized in that: the other broussonetia papyrifera is selected from at least one of the following: broussonetia _ kazinoki, Broussonetia _ papyrifera, wild Broussonetia.
9. A method of distinguishing a hybrid broussonetia papyrifera from other plants, comprising the steps of:
1) using the genome DNA of a plant sample to be detected as a template, carrying out PCR amplification by using the primer pair of claim 2 to obtain a PCR product, and judging whether the plant sample to be detected is a paper mulberry or other plants according to the size of the PCR product:
if the size of the PCR product is 3649bp, the plant sample to be detected is a paper mulberry tree;
if the size of the PCR product is 4125bp, the plant sample to be detected is other plants;
2) distinguishing a hybrid paper mulberry from other paper mulberry according to the method of claim 6;
the hybrid paper mulberry is hybrid paper mulberry 101.
10. The method of claim 9, wherein: the other plants are selected from at least one of the following: arabidopsis thaliana _ thaliana, Beta _ vulgaris _ subsp, Brassica napus _ napus, Brassica oleracea _ oleracea, Cannabis _ sativa, Capsicum Capsicum _ annuum, Carica papaya _ papaya, Chlamydomonas reinhardtii, Citrullus Citrullus _ lantus, Cucumis melo _ melo, Cucumis Cucumis _ sativus, Dendrobium officinale, Dendrobii officinalis _ officinalis, Ectocarpus sativus, Fragaria Phaneralis _ purpurea, Glycine max, Glycine soyae _ soyas, Gossypium ramosum _ Reissondi, Helianthus annuus _ annuius, rubber tree _ sea, Saccharomyces cerevisiae, Solanum nigrum _ polysaccharide _ seed, Solanum nigrum _ Solanum _ serous, Solanum nigrum _ Solanum _ serovar, Solanum nigrum _ serovar _ serotina, Solanum nigrum _ serotina, Solanum _ indica, Solanum _ serotina nigrum _ serotina, Solanum _ indica, Solanum _ serotina _ indica, Solanum _ serotina, Solanum _ indica, Solanum _ serovar _ indica, and Solanum _ indica, Solanum _ serovar _ indica, Solanum _ serovar _ indica, Solanum _ serovar _ root, Solanum _ serovar _ indica, Solanum _ serovar _ indica, Solanum _ serovar _ root, Solanum _ serovar _, Wheat Triticum _ aestivum, mung bean Vigna _ radiata, corn Zea _ mays and jujube tree Ziziphus _ jujuba;
the other broussonetia papyrifera is selected from at least one of the following: broussonetia _ kazinoki, Broussonetia _ papyrifera, wild Broussonetia.
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