1. A rapid typing identification method for an S gene of Chinese wolfberry based on targeted sequencing is characterized in that a probe is designed according to the S genes of a plurality of reference Chinese wolfberry varieties, the designed probe composition is utilized to perform hybridization capture and sequencing analysis on the gene of a Chinese wolfberry sample to be detected, the sequencing result is compared with the S gene of the reference Chinese wolfberry variety, whether the S gene of the Chinese wolfberry sample to be detected is matched with the S gene of the reference Chinese wolfberry variety or not is judged, and the reference Chinese wolfberry variety matched with the S gene of the Chinese wolfberry sample to be detected is determined.

2. The rapid typing identification of the S gene of lycium barbarum based on targeted sequencing according to claim 1, wherein the S gene of the reference lycium barbarum variety is the S gene of a known lycium barbarum variety; the S genes of a plurality of reference medlar varieties are shown as SEQIDNO 1-15.

3. The rapid typing identification of the S gene of Lycium chinense Miller based on the target sequencing of claim 2, wherein the probe composition designed based on the S gene of the reference variety of Lycium chineer comprises 50 probe sequences, wherein the 50 probe sequences are represented by SEQ ID NO 16-65.

4. The rapid typing and identification of the S gene of lycium barbarum based on targeted sequencing according to claim 1, wherein the designed probe composition is used for hybridization capture and sequencing analysis of the gene of lycium barbarum sample to be tested, comprising the following steps:

step 1, preparation of a sample library:

obtaining a sequencing library after wolfberry genome DNA is subjected to breaking, end repairing, joint connection and joint product purification, and then performing library amplification and library purification;

step 2, probe hybridization: hybridizing and capturing the purified library DNA and the probe combination of claim 1, and eluting to remove unbound DNA to obtain hybridized DNA;

step 3, enrichment by PCR

Carrying out PCR amplification on the hybridized DNA to obtain a PCR amplification product;

step 4, PCR purification

Purifying PCR amplification products;

step 5 sequencing

And (3) carrying out gene sequencing on the purified PCR amplification product by adopting a second-generation sequencing technology to obtain a sequencing result of the S gene of the wolfberry sample to be detected.

5. The rapid typing identification of Lycium barbarum based on target sequencing according to claim 4,

step 1, preparing a sample library, comprising the following steps:

step 1.1, accurately quantifying the genomic DNA of the medlar;

step 1.2, cutting the wolfberry genome DNA by enzyme digestion to obtain a DNA fragment;

step 1.3, performing end repair on the interrupted genome DNA;

step 1.4, connecting the DNA fragment with the repaired tail end to a connector;

step 1.5, purifying a product connected with a joint;

step 1.6 library amplification of adaptor-ligated DNA

And step 1.7, purifying the amplified library to obtain a sample library.

Background

Lycium barbarum L belongs to Lycium of Solanaceae, and is a special economic crop with high economic benefit and wide application. The lycium plants have wide self-incompatibility, and the compatibility characteristics of the lycium plants have great influence on the high yield and stable yield of the lycium. The self-incompatibility character of the lycium plants is mainly controlled by an S-RNase (S) gene, most clones have two genotypes, and the self-incompatibility is expressed by homotypic combination. Accurate understanding of the germplasm S gene is helpful for guiding breeding practice and pollination tree configuration, and the problem that normal fructification cannot be realized due to self incompatibility is avoided. The traditional identification of S-RNase (S) genotype is mainly realized by several methods of hybridization pollination test, DNA sequencing and solid-state chip detection, wherein,

the traditional test method of the field hybridization pollination test is as follows: the method applies the field 'hybrid pollination test' among different varieties and judges the S gene carried by each variety and the S gene type of the variety by the fruit setting rate after hybridization. Namely, the fruit setting rate after hybridization is used for judging the S gene carried by each variety and the S genotype of the variety, and the method is the only method for early research of hybridization affinity among the varieties. This method has the advantage of intuitiveness and operability, but when the S genotype of the parent or parents is unknown or there is no suitable test variety, the S genotype of the variety cannot be determined.

The conventional cloning sequencing test method is as follows: designing primers according to the sequence characteristics of the conserved region, amplifying DNA sequences containing all hypervariable regions, performing homology search and sequence comparison on the sequences by using bioinformatics software and various online resources on sequencing results, and determining the S gene type of the sequences to be searched. One experimenter can complete 8 germplasm tests in about 1.5 weeks, and 1000 germplasm tests take about 125 × 1.5 to 180 weeks, and about 3.5 years.

The test method of the traditional solid-state chip detection technology is as follows: designing a corresponding detection probe contained in a target amplification fragment according to a target gene, orderly solidifying a large number of nucleic acid fragments on the surface of a chip substrate (such as a glass slide, a silicon chip and other carriers) by adopting methods of light guide in-situ synthesis or micro-spotting and the like to form dense two-dimensional molecular arrangement, then hybridizing with target molecules in a marked biological sample to be detected, and carrying out rapid, parallel and efficient detection and analysis on the intensity of a hybridization signal through a specific instrument (such as laser confocal scanning), thereby obtaining the genetic information of the sample and determining the gene type of the sample. The initial open cost of the solid-state chip is more than 50 ten thousand yuan, after a new gene is found, new detection object information is difficult to add, professional detection equipment is needed in the later period, and the detection cost of a single sample is up to more than 100 yuan.

In summary, a method for rapidly identifying the S genotype of the Chinese wolfberry needs to be developed, so that the identification time is shortened, and the identification cost is reduced.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a rapid typing and identifying method of a Chinese wolfberry S gene based on targeted sequencing.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

a rapid typing identification of a Chinese wolfberry S gene based on targeted sequencing is characterized in that a probe is designed according to the S genes of a plurality of reference Chinese wolfberry varieties, the designed probe composition is utilized to perform hybridization capture and sequencing analysis on the gene of a Chinese wolfberry sample to be detected, the sequencing result is compared with the S gene of the reference Chinese wolfberry variety, whether the S gene of the Chinese wolfberry sample to be detected is matched with the S gene of the reference Chinese wolfberry variety or not is judged, and the reference Chinese wolfberry variety matched with the S gene of the Chinese wolfberry sample to be detected is determined.

Further, the S gene of the reference Chinese wolfberry variety adopts the S gene of a known Chinese wolfberry variety; the S genes of a plurality of reference medlar varieties are shown as SEQIDNO 1-15.

Further, the probe composition designed according to the S gene of the reference medlar variety comprises 50 probe sequences, wherein the 50 probe sequences are shown as SEQIDNO 16-65

Further, the designed probe composition is used for carrying out hybridization capture and sequencing analysis on the gene of the wolfberry sample to be detected, and the method comprises the following steps:

step 1, preparation of a sample library:

obtaining a sequencing library after wolfberry genome DNA is subjected to breaking, end repairing, joint connection and joint product purification, and then performing library amplification and library purification;

step 2, probe hybridization: hybridizing and capturing the purified library DNA and the probe combination of claim 1, and eluting to remove unbound DNA to obtain hybridized DNA;

step 3, enrichment by PCR

Carrying out PCR amplification on the hybridized DNA to obtain a PCR amplification product;

step 4, PCR purification

Purifying PCR amplification products;

step 5 sequencing

Carrying out gene sequencing on the purified PCR amplification product by adopting a second-generation sequencing technology to obtain a sequencing result of the S gene of the wolfberry sample to be detected;

further, step 1, preparation of a sample library, comprising the following steps:

step 1.1, accurately quantifying the genomic DNA of the medlar;

step 1.2, cutting the wolfberry genome DNA by enzyme digestion to obtain a DNA fragment;

step 1.3, performing end repair on the interrupted genome DNA;

step 1.4, connecting the DNA fragment with the repaired tail end to a connector;

step 1.5, purifying a product connected with a joint;

step 1.6 library amplification of adaptor-ligated DNA

And step 1.7, purifying the amplified library to obtain a sample library.

Compared with the prior art, the invention has the following beneficial effects:

1. according to the DNA complementary principle, one or more probes covering target SNP are designed at each site to be detected, the probes modified by Biotin (Biotin) can be hybridized with a target region in a denatured resequencing library to form a double chain, a magnetic bead coated by streptavidin is used for adsorbing molecules carrying the Biotin, and the genotype of the target SNP is finally obtained through elution, amplification and sequencing.

2. Specific amplification primers are designed aiming at a plurality of SNP sites to be detected, and rapid typing identification of the Chinese wolfberry S-RNase gene is realized by utilizing a targeted sequencing typing (GBTS) technology.

3. By adopting the technology of the invention, the detection of up to 5 ten thousand sites can be simultaneously finished in a single tube, thereby greatly increasing the detection flux and reducing the detection cost.

Detailed Description

The invention is further illustrated by the following examples.

Example 1:

a method for identifying an S gene of Chinese wolfberry based on targeted sequencing comprises the following steps: in this embodiment, 8 parts of the sample of lycium barbarum is selected for the test, and the 8 parts of the sample of lycium barbarum are respectively: ly99-01, Ly100-01, Ly101-01, Ly102-01, Ly103-01, Ly105, Ly106 and Ly 107;

step 1, collecting the S gene of a known Chinese wolfberry variety as the S gene of a reference Chinese wolfberry variety, and designing a probe according to the S gene sequence of the reference Chinese wolfberry variety

Step 1.1, collecting the S genes of known Chinese wolfberry varieties as the S genes of reference Chinese wolfberry varieties, wherein the number of the reference Chinese wolfberry varieties is 15, and taking the DNA sequences of the S genes of the 15 reference Chinese wolfberry varieties as transcript sequences; the 15 reference medlar varieties and the S gene sequences thereof are as follows: the sequence of the medlar ID:1 is shown as SEQIDNO: 1; the sequence of the medlar ID:11 is shown as SEQIDNO: 2; 12-1 of medlar ID, the sequence of which is shown as SEQIDNO 3; 12-2 of medlar ID, the sequence of which is shown as SEQIDNO 4; the sequence of the medlar ID:2 is shown as SEQIDNO: 5; the sequence of the medlar ID:3 is shown as SEQIDNO: 6; the sequence of the medlar ID:4 is shown as SEQIDNO: 7; the sequence of the medlar ID:5 is shown as SEQIDNO: 8; the sequence of the medlar ID:6 is shown as SEQIDNO: 9; the sequence of the medlar ID:7 is shown as SEQIDNO: 10; the sequence of the Chinese wolfberry ID: HM195023 is shown as SEQIDNO: 11; HM195027, and its sequence is shown in SEQ ID NO: 12; the wolfberry fruit ID: HM195029, the sequence of which is shown as SEQIDNO: 13; the sequence of the medlar ID:8 is shown as SEQIDNO: 14; the sequence of the medlar ID:9 is shown as SEQIDNO: 15;

step 1.2, comparing DNA sequences of 15 reference medlar variety S genes, and searching a homologous region and a specific region, wherein the specific region can be used for distinguishing different S genes;

2.3, designing probes for 15 reference medlar variety S genes in a full-coverage mode, wherein the length of each probe is 120bp, and only one probe with the same sequence among different S genes is reserved; in the embodiment, 104 probes are designed, and the sequences of the 104 probes are shown as SEQIDNO 14-64;

step 2, preparing a sample library,

obtaining a sequencing library after wolfberry sample genome DNA is subjected to breaking, end repairing, joint connection and joint product purification, and then performing library amplification and library purification; the specific operation is as follows:

step 2.1, accurately quantifying the genomic DNA of the medlar;

by using2.0dsDNA to carry on the accurate quantification to the genomic DNA of fructus Lycii;

2.2, cutting the wolfberry genome DNA by enzyme digestion to obtain a DNA fragment;

and 2.3, performing end repair on the interrupted genome DNA:

end repair system: 1ng-200ng of DNA fragment; end repair buffer 7 ul; end repair enzyme 1.2ul; the total volume was 20 ul. 20min at 25 ℃; 20min at 72 ℃;

step 2.4, connecting the DNA fragment with the repaired tail end to a connector;

a joint connection system: adding 1ul of Ultra DNA ligase and 1ul of Adaptor into the terminal repair system, and keeping the temperature at 25 ℃ for 20min

Step 2.5, purifying the product after the joint is connected

(1) Adding magnetic beads with the volume 0.5 times of that of the joint connection system, blowing and mixing the mixture up and down by using a pipettor, standing the mixture for 2min, adsorbing the mixture by using a magnetic frame until the solution is clarified, and taking the supernatant and transferring the supernatant into a new tube;

(2) adding magnetic beads with the volume 0.7 times that of the joint connection system, blowing and mixing the mixture up and down by using a pipettor, standing the mixture for 2min, adsorbing the mixture by using a magnetic frame until the solution is clarified, and removing the supernatant;

(3) adding a magnetic bead suspension with the volume 1 time that of the joint connection system, resuspending magnetic beads, standing for 2min, adsorbing by a magnetic frame until the solution is clarified, and removing the supernatant;

(4) add 100 u l 80% ethanol, use the magnetic shelf to adsorb the magnetic bead on different two sides repeatedly, make the magnetic bead get the abundant washing. Adsorbing with magnetic frame for 2min, removing supernatant, and standing at room temperature until ethanol is completely volatilized.

(6) Mu.l of Elution Buffer was added, the magnetic beads were sufficiently suspended, and the mixture was allowed to stand at room temperature for 2min to elute the DNA. Adsorbing the magnetic beads by using a magnet, and adsorbing the obtained supernatant DNA solution into a new tube to obtain a sequencing library; wherein, the Elution Buffer is 10mM Tris-HCl, and the pH value is 8.0-8.5;

step 2.6 library amplification

After purification and air drying, 10 mu L, barcode of PCR mix (Shijiazhuang Boruidi biotechnology, Inc., GenoBaits for short) was added into the PCR centrifuge tube, and the total amount of ultrapure water was diluted to 20 mu L, and the PCR tube was placed into a PCR apparatus: 2min at 98 ℃; 30s at 98 ℃, 30s at 50 ℃ and 40s at 72 ℃ for 8-12 cycles; 4min at 72 ℃.

The barcode of 8 parts of medlar samples is respectively as follows: TTGGTATTGT, GAACAAGCGG, AACAACAGGA, GGTCGGCTTG, TGGTGTGAGA, GGTGTGGTAT, TGTGTCTCGG, ACACCACCTA, respectively;

step 2.7 library purification

And taking out the PCR centrifuge tube from the magnetic frame, adding 35 mu L of Tris-HCl, shaking and mixing uniformly, standing for 5min, slightly centrifuging, placing in the magnetic frame until the solution is clear, and removing the supernatant to a new tube. Performing qualitative or quantitative detection by agarose gel electrophoresis and ultraviolet spectrophotometer, and storing at-20 deg.C after qualified.

Step 3, probe hybridization: hybridizing and capturing the purified library DNA and the probe combination of claim 1, and eluting to remove unbound DNA to obtain hybridized DNA;

step 3.1, preparation of a probe:

adding 0.6-1.0 mu g of purified library DNA, 5 mu L of confining liquid, 2 mu L of confining liquid II of sequencer and 300ng of probe combination described in the embodiment into a 1.5ml PCR centrifuge tube, and placing the mixture at the temperature of less than or equal to 60 ℃ for vacuum concentration until the mixture is completely dried to obtain a probe premix;

step 3.2 DNA library hybrid Capture

Adding the probe premix into a hybridization buffer solution, uniformly mixing by pipetting or vortexing, centrifuging at 12,000rpm for 1min, standing at room temperature for 5min, uniformly mixing by pipetting or vortexing, slightly centrifuging, transferring all liquid into a 0.2ml centrifuge tube, and placing the centrifuge tube into a PCR instrument for thermal cycle incubation: hybridization at 95 ℃ for 10min (hot-cover temperature 105 ℃) and 65 ℃ (hot-cover temperature 75 ℃, overnight);

step 3.3 binding of the hybridized fragments to magnetic hybridized beads

Standing the hybrid magnetic bead solution at room temperature for 10min before use, performing vortex oscillation for 15s, mixing uniformly, adding 50 mu L of each reaction into a 0.2ml centrifuge tube, placing the centrifuge tube in a magnetic frame until the solution is clear, and removing supernatant; taking out the magnetic frame, adding 150 mu L of hybrid magnetic bead cleaning buffer solution I, carrying out vortex oscillation for 10s, placing the magnetic frame until the solution is clarified, removing supernatant, and repeating for 2 times; adding 16 mu L of the hybridization solution, sucking and beating for 10 times, uniformly mixing, and putting into a PCR instrument for carrying out: the temperature of the hot cover is 75 ℃; 45min at 65 ℃ with shaking for 5s every 12 min.

Step 3.4 elution to remove unbound DNA

Elution at 65 ℃: adding 100 mu L of hybrid magnetic bead cleaning buffer solution I preheated at 65 ℃ into each tube, performing vortex oscillation for 5s, uniformly mixing, and centrifuging for 5 s; placing in a magnetic frame until the solution is clear, and removing the supernatant; adding 150 μ L of 65 deg.C pre-heated hybridization magnetic bead washing buffer solution I, slowly sucking up and down for 10 times (to avoid air bubble generation), standing for 2min, placing in a magnetic frame until the solution is clear, removing supernatant, and repeating for 1 time.

Elution at room temperature: continuously adding 150 mu L of room temperature hybrid magnetic bead cleaning buffer solution I, and shaking for 2 min; placing in a magnetic frame until the solution is clear, and removing the supernatant; adding 150 mu L of room temperature hybridization magnetic bead cleaning buffer solution II, shaking for 1min, placing in a magnetic frame until the solution is clear, and removing supernatant; adding 150 mu L of room temperature hybridization magnetic bead washing buffer solution III, shaking for 30s, placing in a magnetic frame until the solution is clear, and removing the supernatant.

Resuspending magnetic beads: after removing the sample from the magnetic holder, 20. mu.L of ultrapure water was added thereto and the mixture was pipetted 10 times.

Step 4, enrichment by PCR

Carrying out PCR amplification on the hybridized DNA to obtain a PCR amplification product;

to the PCR tube, 10. mu.L of hybridized DNA, 15. mu.L of PCR Mix (GenoBaits Co.), 1.2. mu.L of sequencer-compatible Primer Mix (GenoBaits Co.) and 3.8. mu.L of ultrapure water were added, mixed, and placed in a PCR apparatus for: the hot lid temperature was 105 ℃; 45s at 98 ℃; 15s at 98 ℃, 30s at 50 ℃ and 40s at 72 ℃ for 10-18 cycles; 1min at 72 ℃; hold at 4 ℃.

Step 5, PCR purification

Purifying PCR amplification products;

adding 45 mu L of purified liquid into each PCR amplification product, shaking and uniformly mixing, standing for 5min, slightly centrifuging, placing in a magnetic frame until the solution is clear, and removing the supernatant; adding 100 μ L of 80% ethanol, incubating at room temperature for 30s, and removing the supernatant; uncovering the cover for 10min until the ethanol is volatilized to be dry. And taking out the PCR centrifuge tube from the magnetic frame, adding 35 mu L of Tris-HCl, shaking and mixing uniformly, standing for 5min, slightly centrifuging, placing in the magnetic frame until the solution is clear, and removing the supernatant to a new tube. Performing qualitative or quantitative detection by agarose gel electrophoresis and ultraviolet spectrophotometer, and storing at-20 deg.C after qualified.

Step 6 sequencing

Carrying out gene sequencing on the purified PCR amplification product by adopting a second-generation sequencing technology to obtain a sequencing result of the S gene of the wolfberry sample to be detected;

step 7, sequence alignment of S Gene

And comparing the sequencing result of the S gene of the to-be-detected Chinese wolfberry sample obtained by the second-generation sequencing with the S genes of 15 reference Chinese wolfberry varieties, judging whether the S gene of the to-be-detected Chinese wolfberry sample is matched with the S gene of the reference Chinese wolfberry variety or not, and finding out the reference Chinese wolfberry variety matched with the S gene of the to-be-detected Chinese wolfberry sample.

In the process of comparing S gene sequences, different S genes are distinguished through the numbers of reads and the value of Coverage, and the following principles are specifically followed:

1. removing reads aligned to multiple positions and having the same alignment value (such reads occur because there is the same reads between gene sequences and thus these reads cannot be used to distinguish gene sequences)

2. Preserving reads that are uniquely aligned and that differ in alignment between genes, which aids in distinguishing gene sequences;

3. according to the analysis principle, the depth is more than or equal to 10X, the result is covered, the gene is indicated to be covered, and the Coverage result of 8 medlar samples measured in the example is as follows:

1) and the sample to be detected Ly 99-01: and Chinese wolfberry variety ID:1, the matching result of the S gene is: coverage (111) is 0.6928571, Reads _ number (111) is 163; and Chinese wolfberry variety ID: the matching result of the S gene of 6 is: coverage (111) is 1, Reads _ number (111) is 552; and (4) conclusion: the two chromosomes of the sample Ly99-0 to be detected have the Chinese wolfberry variety ID:1 and variety ID of medlar: 6 in the S gene;

2) and the sample to be detected Ly 100-01: and Chinese wolfberry variety ID:2, the matching results for the S gene are: coverage (111) is 0.95, Reads _ number (111) is 421; and Chinese wolfberry variety ID: the matching result for the S gene of HM195023 is: coverage (111) is 1, Reads _ number (111) is 812; and (4) conclusion: the two chromosomes of the sample Ly100-01 to be detected have the Chinese wolfberry variety ID:2 and variety ID of Lycium barbarum: the S gene of HM 195023;

3) and the sample to be detected Ly 101-01: and Chinese wolfberry variety ID:7, the matching result of the S gene is: coverage (111) is 0.6952381, Reads _ number (111) is 227; and Chinese wolfberry variety ID: the matching result for the S gene of HM195023 is: coverage (111) is 1, Reads _ number (111) is 737; and (4) conclusion: the two chromosomes of the sample Ly101-01 to be detected have the Chinese wolfberry variety ID:7 and variety ID of medlar: the S gene of HM 195023;

4) and the sample to be detected Ly 102-01: and Chinese wolfberry variety ID:2, the matching results for the S gene are: coverage (111) is 0.95, Reads _ number (111) is 516; and Chinese wolfberry variety ID:3, the matching result of the S gene is: coverage (111) is 1, Reads _ number (111) is 469; and (4) conclusion: the two chromosomes of the sample Ly102-01 to be detected have the Chinese wolfberry variety ID:2 and variety ID of Lycium barbarum: 3, the S gene;

5) and the sample to be detected Ly 103-01: and Chinese wolfberry variety ID: the matching result for the S gene of 18 is: coverage (111) is 1, Reads _ number (111) is 1074; and Chinese wolfberry variety ID:2, the matching results for the S gene are: coverage (111) is 0.95, Reads _ number (111) is 427; and (4) conclusion: the two chromosomes of the sample Ly103-01 to be detected have the Chinese wolfberry variety ID:2 and variety ID of Lycium barbarum: 3, the S gene;

6) and the sample to be tested Ly 105: and Chinese wolfberry variety ID:1, the matching result of the S gene is: coverage (111) is 0.7047619, Reads _ number (111) is 348; and Chinese wolfberry variety ID: the matching result of the S gene of 9 is: coverage (111) is 1, Reads _ number (111) is 816; and (4) conclusion: the two chromosomes of the sample Ly105 to be detected have the Chinese wolfberry variety ID:1 and variety ID of medlar: 9, the S gene;

7) and the sample to be detected Ly 106: and Chinese wolfberry variety ID:1, the matching result of the S gene is: coverage (111) is 0.7047619, Reads _ number (111) is 297; and Chinese wolfberry variety ID: the matching result for the S gene of HM195027 is: coverage (111) is 1, Reads _ number (111) is 1023; and (4) conclusion: the two chromosomes of the sample Ly106 to be detected have the variety ID of the medlar: 1 and variety ID of medlar: the S gene of HM 195027;

8) and the sample to be detected Ly 107: and Chinese wolfberry variety ID:1, the matching result of the S gene is: coverage (111) is 0.702381, Reads _ number (111) is 435; and Chinese wolfberry variety ID: the matching result for the S gene of 8 is: coverage (111) is 1, Reads _ number (111) is 332; and (4) conclusion: the two chromosomes of the sample Ly106 to be detected have the variety ID of the medlar: 1 and variety ID of medlar: 8 in the genome of a human.

The embodiments described above are only preferred embodiments of the invention and are not exhaustive of the possible implementations of the invention. Any obvious modifications to the above would be obvious to those of ordinary skill in the art, but would not bring the invention so modified beyond the spirit and scope of the present invention.

Sequence listing

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gaaagccgtt tcaaaatgta tacaggatgc cagaaaaata agtgagctgg acgaacgctg 180

gcctcaactg aaatacgact acgagtttgg tatagacgaa caatatctct ggaaaaagga 240

attcctaaaa catggaagct gtggtataaa gcggtaccca caacctgcat attttgattt 300

agccatgaat ttaaaagaca agtttgatct cttgagtact ctcagaaatc atgggattac 360

tcctggttca acttatcagc ttgatgatat cgaaaaggcc ataaagacag tttctataga 420

<210> 8

<211> 423

<212> DNA

<213> matrimony vine (unknown)

<400> 8

gtcgtcgtca acccaccact gcgttatata aagttcagag taagttacca aactaacttt 60

ctcagatgct cgtaattcat tcattcaaat ttcaccttag tgttcgttca tatttttatt 120

gaatgaagcc gtttcaaaat gtatacagga tcccagagaa gtaactgagc tggacaatcg 180

ctggcctcaa ctgaaatact ctaaattcga tggtacagat agacaacctc tctggagacg 240

tgaattccta aaacatggaa gctgtggtat aaataggtac aaacaacctg catattttga 300

tttagctatg aatttaaagg acaagtttga tctcttgagt actctcagaa atcatggaat 360

tactcctggt tcaacttatc agcttgatga tatcgaaaaa gccgtaatga cagtttctat 420

aaa 423

<210> 9

<211> 420

<212> DNA

<213> matrimony vine (unknown)

<400> 9

gtgggctacc caaaagtgcg ttatgatatc atcaaggtaa attaccaaac taattttctc 60

aaatgttcgt aattcattga ttcaaatttc accttagcgt ttgttcacgc atttattgaa 120

gaaagccgtt tcaaaatgta tacaggatgc cagaaaaata agtgagctgg acgaacgctg 180

gcctcaactg aaatacgaca accagtttgg tatagacgaa caatatctct ggaaaaagga 240

attcctaaaa catggaagct gtggtataaa gctgtaccca caacctgcat attttgattt 300

agccatgaat ttaaaagaca agtttgatct cttgagtact ctcagaaatc atgggattac 360

tcctggttca acttatcagc ttgatgatat cgaaaaggcc ataaagacag tttctataga 420

<210> 10

<211> 420

<212> DNA

<213> matrimony vine (unknown)

<400> 10

gtgggctgcc caccagtgtg ttataatgac atcacggtaa attaccaaac taattttctc 60

aaatgttcgt aattcattta ttcaaatttc accttagcgt ttgttcatac atttattgaa 120

tgaagccgtt tcaaaatgta tacaggatgc cagaaaaata agtgagctga acaaacgctg 180

gcctcaactg aaatacaagg acgacgttgg tatagacaaa caatatctct ggaaaaagga 240

attcctaaaa catggaagct gtggtataaa gcggtaccaa caacctgcat attttgattt 300

agccatgaat ttaaaagaca agtttgatct cttgagtact ctcagaaatc atgggattac 360

tcctggttca acttatcagc ttgatgatat cgaaaaggcc ataaagacag tttctataaa 420

<210> 11

<211> 378

<212> DNA

<213> matrimony vine (unknown)

<400> 11

aacaagaaca cactactgaa taactgcgcc cctggtgcaa catatcataa gatagacgat 60

ccaggtatgt tcaaacagat ggacgatcgg tggacagaac taacctcaga tgtaaaagat 120

agtaaaaaat atcaacgatt ctgggaacat gaattcttaa agcatggaac gtgttgtgag 180

ggtcatgata ctgaagaagc atattttaaa ttagccatgc gcttaaaaga cagatttgat 240

cttttgacaa ttctcagagc tagtggaatt attcctggaa attattattc cattgacagc 300

attcagaaag ccatcgaggg agttactcga gcggttccaa atctatattg taatcctgat 360

ccaaataacc caagaatg 378

<210> 12

<211> 363

<212> DNA

<213> matrimony vine (unknown)

<400> 12

aactacagca gaacgctaaa tttttgcgac cgcagtaaga tttataataa attcacggat 60

gacaaagaga agagtgatct gtacgaacgc tggcctgact tgaccatcac tgaatttgat 120

tgtttagaca agcaagcttt ctggagccgt gaatacataa agcatggcac gtgttgttca 180

gacaagtttg accgtgtgca atattttact ttagccatgg ccttgaaaga caagttcgat 240

cttttgaaat ctctaagaaa tcatggaatt attcgtggat attcttatac cgtccaaaag 300

atcaatagca ccataaaggc tattactcga gggtatccaa atctcacgtg cgctggatta 360

agg 363

<210> 13

<211> 381

<212> DNA

<213> matrimony vine (unknown)

<400> 13

aacaactccg ttatgctgaa taactgcgtg ggcaaccaaa aagtgggtta tgatatcatc 60

atggatgtca gaaaactaag tgagctggac aaacgctggc ctcaactgaa atacgactac 120

caaactggta tagacgaaca atatctctgg aaaaaggaat tcctaaaaca tggaagctgt 180

ggtataaagc tgtacccaca acctgcatat tttgatttag ccatgaattt aaaagacaag 240

tttgatctct tgagtactct cagaaatcat gggattactc ctggttcaac ttatctgctt 300

catgatatcg aaaaggccat aaagacagtt tctataaagg ttcctagcct caagtgcatt 360

gaaaaatatc ctggagatgt g 381

<210> 14

<211> 420

<212> DNA

<213> matrimony vine (unknown)

<400> 14

ccggtcaacc gaaaattccg ttataatacg atcaaggtaa attaccaaac taattttctc 60

aaatgttctt aattcattca ttcaaatttc atcttagcgt ttgttcatat atttattgaa 120

tgaagccgtt tcaaaatgta tacaggatca gaggaaagtg aatgagctgg acaaacgttg 180

gcctcaactg aaatacgacg aaaactttgg tagaaataag caatatctct gggaaaatga 240

attcctaaaa catggaagct gtagtataca gcgttacaaa caacctgcat attttgattt 300

agcaatgaat ttaaaagaca agtttgatct cttgagtact ctcagaaatc atggaattac 360

tcctggttca acttatgacc ttgatgatat cgaaaaggcc ataaagacag tttctataaa 420

<210> 15

<211> 420

<212> DNA

<213> matrimony vine (unknown)

<400> 15

gtgggctacc caaaagtgcg ttatgatatc atcatggtaa attaccaaac taattttctc 60

aaatgttcgt aattcattga ttcaaatttc accttagcgt ttgttcatgc atttattgaa 120

gaaagccgtt tcaaaatgta tacaggatcc cagaaaaata agtgagctgg acgaacgctg 180

gcctcaactg aaatacgaca agcagtttgg tatagacgaa caatatctct ggaaaaagga 240

attcctaaaa catggaagct gtggtataaa gcggtaccca caacctgcat attttgattt 300

agccgtgaat ttaaaagaca agtttgatct cttgagtact ctcagaaatc atgggattac 360

tcctggttca acttatcagc ttgatgatat cgaaaaggcc ataaagacag tttctataga 420

<210> 16

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 16

ccggtcaagc aaaaattggg ttataaaacg atcacggtaa attaccaaac taattttctc 60

aaatgttctt aattcattca ttcaaatttc accttagcgt ttgctcatat atttattgaa 120

<210> 17

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 17

acagcttcga taaccaatat ctctggaaaa aagaattcct aaaacatgga agctgtagta 60

tagagcgtta caaacaacct gcatattttg atttagcaat gaattcaaaa gacaagtttg 120

<210> 18

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 18

aaaacatgga agctgtagta tagagcgtta caaacaacct gcatattttg atttagcaat 60

gaattcaaaa gacaagtttg atctcttgag tactctcaga aatcatggaa ttactcctgg 120

<210> 19

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 19

gcatattttg atttagcaat gaattcaaaa gacaagtttg atctcttgag tactctcaga 60

aatcatggaa ttactcctgg ttcaacttat gaccttggtg atatcgaaaa ggccataaag 120

<210> 20

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 20

attttgattt agcaatgaat tcaaaagaca agtttgatct cttgagtact ctcagaaatc 60

atggaattac tcctggttca acttatgacc ttggtgatat cgaaaaggcc ataaagacag 120

<210> 21

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 21

atcacggtaa attaccaaac taattttctc aaatgttctt aattcattca ttcaaatttc 60

accttagcgt ttgctcatat atttattgaa tgaagccgtt tcaaaatgta tacaggatca 120

<210> 22

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 22

ttcaaatttc accttagcgt ttgctcatat atttattgaa tgaagccgtt tcaaaatgta 60

tacaggatca gaggaaagtg agtgagctgg acaaacgttg gcctcaactg aaacacgaga 120

<210> 23

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 23

gtgggctacc cagaagtgag ttatgatatc atcagggtaa attaccaaac taattttctc 60

aaatgttcgt aattcattga ttcaaatttc accttagcgt ttgttcatgc atttattgaa 120

<210> 24

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 24

atttattgaa tgaagccgtt tcaaaatgta tacaggatgc cagaaaaata agtgagctgg 60

acaaacgctg gcctcaactg aaatacgact accagtttgg tatagacgaa caatatctct 120

<210> 25

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 25

agtgagctgg acaaacgctg gcctcaactg aaatacgact accagtttgg tatagacgaa 60

caatatctct ggaaaaagga attcctaaaa catggaagct gtggtataaa gcggtaccca 120

<210> 26

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 26

accagtttgg tatagacgaa caatatctct ggaaaaagga attcctaaaa catggaagct 60

gtggtataaa gcggtaccca caacctgcat attttgattt agccatgaat ttaaaagaca 120

<210> 27

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 27

attcctaaaa catggaagct gtggtataaa gcggtaccca caacctgcat attttgattt 60

agccatgaat ttaaaagaca agtttgatct cttgagtact ctcagaaatc atgggattac 120

<210> 28

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 28

caacctgcat attttgattt agccatgaat ttaaaagaca agtttgatct cttgagtact 60

ctcagaaatc atgggattac tcctggttca acttatcagc ttgatgatat cgaaaaggcc 120

<210> 29

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 29

atcagggtaa attaccaaac taattttctc aaatgttcgt aattcattga ttcaaatttc 60

accttagcgt ttgttcatgc atttattgaa tgaagccgtt tcaaaatgta tacaggatgc 120

<210> 30

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 30

ttcaaatttc accttagcgt ttgttcatgc atttattgaa tgaagccgtt tcaaaatgta 60

tacaggatgc cagaaaaata agtgagctgg acaaacgctg gcctcaactg aaatacgact 120

<210> 31

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 31

ccggtcgacc gacaattccg ttatgatacg atcacggtaa attaccaaac taattttctc 60

aaatgttctt aattcattca ttcaaatttc accttagcgt ttgttcatat atttattgaa 120

<210> 32

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 32

acaatatctc tggaaaaatg aattcctaaa acatggaagc tgtggtatag agcggtacaa 60

acaacctgca tattttgatt tagccatgaa tttaaaagac aagtttgatc tcttgagtac 120

<210> 33

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 33

agcggtacaa acaacctgca tattttgatt tagccatgaa tttaaaagac aagtttgatc 60

tcttgagtac tcttagaaat aatgggatta ctcctggttc aacttatcag cttgatgata 120

<210> 34

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 34

caacctgcat attttgattt agccatgaat ttaaaagaca agtttgatct cttgagtact 60

cttagaaata atgggattac tcctggttca acttatcagc ttgatgatat cgaaaaggcc 120

<210> 35

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 35

gtcgtcgtca acccaccact gcgttatata aagttcagag taagttacca aactaacttt 60

ctcagatgct cgtaattcat tcattcaaat ttcaccttag tgttcgttca tatttttatt 120

<210> 36

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 36

tatttttatt gaatgaagcc gtttcaaaat gtatacagga tcccagagaa gtaactgagc 60

tggacaatcg ctggcctcaa ctgaaatact ctaaattcga tggtacagat agacaacctc 120

<210> 37

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 37

gtaactgagc tggacaatcg ctggcctcaa ctgaaatact ctaaattcga tggtacagat 60

agacaacctc tctggagacg tgaattccta aaacatggaa gctgtggtat aaataggtac 120

<210> 38

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 38

ctaaattcga tggtacagat agacaacctc tctggagacg tgaattccta aaacatggaa 60

gctgtggtat aaataggtac aaacaacctg catattttga tttagctatg aatttaaagg 120

<210> 39

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 39

tgaattccta aaacatggaa gctgtggtat aaataggtac aaacaacctg catattttga 60

tttagctatg aatttaaagg acaagtttga tctcttgagt actctcagaa atcatggaat 120

<210> 40

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 40

aaacaacctg catattttga tttagctatg aatttaaagg acaagtttga tctcttgagt 60

actctcagaa atcatggaat tactcctggt tcaacttatc agcttgatga tatcgaaaaa 120

<210> 41

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 41

caacctgcat attttgattt agctatgaat ttaaaggaca agtttgatct cttgagtact 60

ctcagaaatc atggaattac tcctggttca acttatcagc ttgatgatat cgaaaaagcc 120

<210> 42

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 42

taagttacca aactaacttt ctcagatgct cgtaattcat tcattcaaat ttcaccttag 60

tgttcgttca tatttttatt gaatgaagcc gtttcaaaat gtatacagga tcccagagaa 120

<210> 43

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 43

tcattcaaat ttcaccttag tgttcgttca tatttttatt gaatgaagcc gtttcaaaat 60

gtatacagga tcccagagaa gtaactgagc tggacaatcg ctggcctcaa ctgaaatact 120

<210> 44

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 44

aactttggta gaaataaaca atatctctgg aaaaatgaat tcctaaaaca tggaagctgt 60

agtataaagc ggtaccaaca gcctgcatat tttgatttag ccatgaattt aaaagacaag 120

<210> 45

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 45

gaaataaaca atatctctgg aaaaatgaat tcctaaaaca tggaagctgt agtataaagc 60

ggtaccaaca gcctgcatat tttgatttag ccatgaattt aaaagacaag tttgatctct 120

<210> 46

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 46

tcctaaaaca tggaagctgt agtataaagc ggtaccaaca gcctgcatat tttgatttag 60

ccatgaattt aaaagacaag tttgatctct tgagtactct cagaaatcat ggaattactc 120

<210> 47

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 47

gcctgcatat tttgatttag ccatgaattt aaaagacaag tttgatctct tgagtactct 60

cagaaatcat ggaattactc ctggttcaac ttatcagctt gatgatatcg aaaaggccat 120

<210> 48

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 48

tttgatttag ccatgaattt aaaagacaag tttgatctct tgagtactct cagaaatcat 60

ggaattactc ctggttcaac ttatcagctt gatgatatcg aaaaggccat aaagacagtt 120

<210> 49

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 49

catgaattta aaagacaagt ttgatctctt gagtactctc agaaatcatg gaattactcc 60

tggttcaact tatcagcttg atgatatcga aaaggccata aagacagttt ctataaaggt 120

<210> 50

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 50

ttgatctctt gagtactctc agaaatcatg gaattactcc tggttcaact tatcagcttg 60

atgatatcga aaaggccata aagacagttt ctataaaggt tccaagcctc aagtgcgtcg 120

<210> 51

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 51

tgagctggac aaacgctggc ctcaactgaa atatgagaac aactttggta gaaataaaca 60

atatctctgg aaaaatgaat tcctaaaaca tggaagctgt agtataaagc ggtaccaaca 120

<210> 52

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 52

aacaagaaca cactactgaa taactgcgcc cctggtgcaa catatcataa gatagacgat 60

ccaggtatgt tcaaacagat ggacgatcgg tggacagaac taacctcaga tgtaaaagat 120

<210> 53

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 53

agtaaaaaat atcaacgatt ctgggaacat gaattcttaa agcatggaac gtgttgtgag 60

ggtcatgata ctgaagaagc atattttaaa ttagccatgc gcttaaaaga cagatttgat 120

<210> 54

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 54

agcatggaac gtgttgtgag ggtcatgata ctgaagaagc atattttaaa ttagccatgc 60

gcttaaaaga cagatttgat cttttgacaa ttctcagagc tagtggaatt attcctggaa 120

<210> 55

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 55

atattttaaa ttagccatgc gcttaaaaga cagatttgat cttttgacaa ttctcagagc 60

tagtggaatt attcctggaa attattattc cattgacagc attcagaaag ccatcgaggg 120

<210> 56

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 56

cttttgacaa ttctcagagc tagtggaatt attcctggaa attattattc cattgacagc 60

attcagaaag ccatcgaggg agttactcga gcggttccaa atctatattg taatcctgat 120

<210> 57

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 57

gctagtggaa ttattcctgg aaattattat tccattgaca gcattcagaa agccatcgag 60

ggagttactc gagcggttcc aaatctatat tgtaatcctg atccaaataa cccaagaatg 120

<210> 58

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 58

catatcataa gatagacgat ccaggtatgt tcaaacagat ggacgatcgg tggacagaac 60

taacctcaga tgtaaaagat agtaaaaaat atcaacgatt ctgggaacat gaattcttaa 120

<210> 59

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 59

ggacgatcgg tggacagaac taacctcaga tgtaaaagat agtaaaaaat atcaacgatt 60

ctgggaacat gaattcttaa agcatggaac gtgttgtgag ggtcatgata ctgaagaagc 120

<210> 60

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 60

cgcagtaaga tttataataa attcacggat gacaaagaga agagcgatct gtacgaacgc 60

tggcctgacc tgaccatcac tgaatttgat tgtttagaca agcaagcttt ctggagccgt 120

<210> 61

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 61

gaatacataa agcatggcac gtgttgttca gacaagtttg atcgtgtgca atattttact 60

ttagccatgg ccttgaaaga caggttcgat cttttgaaat ctctgagaaa tcatggaatt 120

<210> 62

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 62

cagacaagtt tgatcgtgtg caatatttta ctttagccat ggccttgaaa gacaggttcg 60

atcttttgaa atctctgaga aatcatggaa ttattcgtgg atattcttat accgtccaaa 120

<210> 63

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 63

gacaagtttg atcgtgtgca atattttact ttagccatgg ccttgaaaga caggttcgat 60

cttttgaaat ctctgagaaa tcatggaatt attcgtggat attcttatac cgtccaaaag 120

<210> 64

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 64

agagcgatct gtacgaacgc tggcctgacc tgaccatcac tgaatttgat tgtttagaca 60

agcaagcttt ctggagccgt gaatacataa agcatggcac gtgttgttca gacaagtttg 120

<210> 65

<211> 120

<212> DNA

<213> Artificial sequence (unknown)

<400> 65

tgaatttgat tgtttagaca agcaagcttt ctggagccgt gaatacataa agcatggcac 60

gtgttgttca gacaagtttg atcgtgtgca atattttact ttagccatgg ccttgaaaga 120