CN115011720B - SNP molecular marker combination for constructing rubber tree variety DNA fingerprint, application and method - Google Patents
SNP molecular marker combination for constructing rubber tree variety DNA fingerprint, application and method Download PDFInfo
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Abstract
The invention discloses SNP molecular marker combination for constructing a rubber tree variety DNA fingerprint, application and a method thereof. The 50 SNP molecular markers screened by the invention have high specificity, sensitivity and resolution, are not influenced by environmental conditions, have accurate detection results and good repeatability and stability, and can comprehensively reveal the real level utilization of genetic diversity of the rubber tree; the DNA fingerprint library is established by utilizing the SNP marker combination, and the cluster analysis and variety identification can be carried out on the rubber tree varieties, so that the detection result is more accurate and efficient, theoretical basis is provided for the protection and utilization of rubber tree resources, 66 varieties of rubber trees are effectively distinguished for the first time, and the distinguishing rate of the invention on bred varieties can reach 100%. The single nucleotide marker loci of the invention have the advantages of simplicity, rapidness and low cost, and can be applied in large scale in production.
Description
Technical Field
The invention belongs to the technical field of molecular biology of rubber tree varieties, and particularly relates to SNP molecular marker combination for constructing a DNA fingerprint of a rubber tree variety, application and a method thereof.
Background
The Brazilian Amazon river basin of the original rubber tree is an important economic crop in tropical areas, and the produced natural rubber is an important strategic resource in China, and has irreplaceable functions in the fields of military, aerospace, ocean engineering, rail traffic and the like. Since the introduction of rubber tree in 1904 to China, the cultivated species of rubber tree, wild germplasm and homoplasmic materials saved in China so far total 6165 parts of germplasm materials of 5 species and 1 variety. For a long time, identification of rubber tree variety resources is mainly based on morphological character identification, and the classical and conventional identification method generally can complete the identification process after the rubber tree flowers. However, the rubber tree belongs to a perennial cross-pollinated tall tree, and can bloom and fruit only 5-6 years after field planting, and has the adverse factors of long sexual generation period, high heterozygosity of varieties and the like, so that the identification period is long in the character identification process, and the agronomic characters are easily influenced by environmental conditions and cultivation management measures. In addition, the traditional identification method is very difficult for identifying varieties with small character difference and relatively close relationship, and brings a plurality of inconveniences for identifying germplasm resources and varieties of rubber trees. Thus, there is a need for accurate molecular marker methods to identify rubber tree germplasm resources and varieties.
Molecular marker analysis methods are based on DNA polymorphisms such as RFLP, RAPD, AFLP, ISSR, SSR and SNP markers, which all produce DNA fingerprints with high individual specificity. Early RFLP, RAPD, AFLP molecular marker technology has been applied to DNA fingerprint database construction of a plurality of species, but the problems of poor stability, low polymorphism, difficult data integration and the like of the marker technologies in use often cannot be widely applied. Therefore, the international new variety protection consortium suggested SSR and SNP as the preferred DNA fingerprinting techniques. Because SNP markers have the advantages of complete genome coverage, high throughput, site specificity, co-dominant inheritance, low false detection rate, rapid reduction of development and detection cost, easy data integration and the like, the SNP markers become the main marker type for future genotype identification.
With the development of the second generation sequencing technology, the sequencing flux shows an exponential growth trend, and the sequencing cost is drastically reduced, so that powerful technical support is provided for detecting SNP markers at the whole genome level. How to formulate SNP locus screening principle, locus number, locus combination, judgment threshold and the like according to different identification targets becomes the key of screening SNP to construct fingerprint. The invention is based on SNP obtained by rubber tree resequencing, and the standardized flow of constructing the fingerprint by SNP is obtained through control of SNP sequencing quality, SNP locus quality control, judgment threshold setting of SNP screening and the like, so as to establish a molecular identity card of the rubber tree. At present, a complete fingerprint library of the rubber tree varieties is not established so as to identify the varieties.
Disclosure of Invention
The invention aims at overcoming the defects of the prior art and providing SNP molecular marker combination, application and method for constructing a rubber tree DNA fingerprint. Solves the problems of poor repeatability and low polymorphism of the prior art that co-dominant DNA markers such as RAPD, ISSR and the like are used; the invention also aims to provide the application of the polymorphic primer of the SNP molecular marker of the rubber tree nuclear genome in the identification of rubber tree varieties, the analysis of genetic structure and genetic diversity of resources, thereby providing a basis for the identification of rubber tree germplasm resources and cultivars from molecular level.
The first aspect of the invention provides a SNP molecular marker combination for constructing a rubber tree DNA fingerprint, the SNP molecular marker combination comprises lg01_54721919, and/or lg01_96036745, and/or lg02_ 96036745, and/or lg03_ 96036745, and/or lg04_ 96036745 and/or lg04_ 96036745, and/or lg05_ 96036745, and/or lg06_ 96036745, and/or lg07_ 96036745, and/or lg08_ 96036745 and/or lg04_ 96036745 and/or lg05_ 96036745 and/or lg06_ 96036745 and/or lg07_ 96036745, and/or lg08_ 96036745;
in the above table, "Sequence" is the nucleotide Sequence of the SNP molecular marker; the "[ ]" is marked as the position of SNP molecular marker locus, wherein the base is polymorphism of the locus.
It will be appreciated that in practical applications, a person skilled in the art may choose a suitable SNP molecular marker as appropriate for the application. For example, if the rubber tree to be tested is actually known to be sea reclamation 1 or RRIM623, one of lg01_96036745, lg03_104230492, lg04_16174898, lg04_33465133, lg04_71146652, lg04_96013598, lg07_57540924, lg07_72003724, lg09_50708986, lg09_64555870, lg11_54415, lg11_34802586, lg14_33209388, lg18_10766 or the like (refer to table 5 below) may be selected as the SNP molecular marker for identification, or two or more of them may be selected for multiplex identification. For example, it is actually known that the rubber tree to be tested is a sea reclamation 1, an RRIM623 or a Dafeng 95, but only one kind of SNP molecular marker may be used, such as Lm03_ 104230492, lm04_ 33465133, lg11_34802586, lg15_32001166 (refer to Table 5 below), or two or more kinds of SNP molecular markers may be used. When the number of the rubber trees to be tested is one of four or more, the person skilled in the art can select an appropriate SNP molecular marker for identification by referring to Table 5 below. When the rubber tree to be tested is completely unknown, the person skilled in the art can also identify the SNP molecular markers as batches.
Preferably, the method comprises the steps of, the SNP molecular marker combination comprises lg01_54721919, lg01_5237, lg02_5237, lg03_5237, lg04_5237, lg03_5237 LG 04-96036745, LG 05-96036745, LG 06-96036745, LG 07-96036745, LG 08-96036745, and LG 04-96036745, LG 05-96036745, lg05-96036745, lg06-96036745 LG 07_5237, LG 08_5237.
Preferably, the SNP molecular marker combination at least comprises lg03_104230492, lg02_8025843, lg01_54721919, lg15_32001166, lg07_72003724, lg05_88010730 and lg02_214754.
Preferably, the method comprises the steps of, the SNP molecular marker combination comprises lg01_54721919, lg01_5237, lg02_5237, lg03_5237, lg04_5237 LG 04-96036745, LG 05-96036745, LG 06-96036745, LG 07-96036745, LG LG 04-96036745, lg05-96036745 lg06_5237, lg07_5237.
In a second aspect of the present invention, there is provided a primer set for amplifying the SNP molecular marker combination according to the first aspect of the invention.
Preferably, the primer pairs for each SNP molecular marker are as shown in Table 3 below.
In a third aspect, the present invention provides a kit or gene chip comprising the primer set according to the second aspect of the present invention.
The fourth aspect of the invention provides an application of the SNP molecular marker combination of the first aspect of the invention, or the primer set of the second aspect of the invention, or the kit or the gene chip of the third aspect of the invention in genetic structure of a rubber tree variety population, and/or genetic diversity analysis of rubber tree resources, and/or identification of rubber tree varieties, and/or construction of a rubber tree DNA fingerprint.
The fifth aspect of the invention provides a construction method of a rubber tree variety DNA fingerprint library, which comprises the following steps: extracting total DNA of each rubber tree variety; amplifying by using the primer group of the SNP molecular marker in the second aspect of the invention; carrying out SNP locus polymorphism detection by utilizing the SNP molecular marker combination according to the first aspect of the invention, and carrying out genotyping on each rubber tree variety according to the detection result of the SNP locus, thereby constructing a rubber tree variety DNA fingerprint library; wherein the rubber tree variety is the rubber tree variety described in Table 2 below.
Preferably, SNP locus polymorphism detection is performed by using a SNaPshot method, and rubber tree variety identification, population genetic structure and genetic diversity analysis are performed.
Preferably, the SNP molecular marker combination comprises at least' lg03_104230492, lg02_8025843, lg01_54721919, lg15_32001166, lg07_72003724, lg05_88010730 and lg02_214754.
Preferably, the method comprises the steps of, the SNP molecular marker combination comprises lg01_54721919, lg01_5237, lg02_5237, lg03_5237, lg04_5237 LG 04-96036745, LG 05-96036745, LG 06-96036745, LG 07-96036745, LG LG 04-96036745, lg05-96036745 lg06_5237, lg07_5237.
In a sixth aspect, the present invention provides a rubber tree variety DNA fingerprint library constructed by the construction method according to the fifth aspect of the present invention.
The seventh aspect of the invention provides a method for identifying a rubber tree variety, which is characterized in that the primer group of the second aspect of the invention is adopted to carry out PCR amplification on total DNA of a sample to be detected, then SNP locus polymorphism detection is carried out, the genotyping of SNP molecular markers is determined according to the detection result of the SNP locus, and then the genotyping is compared with the genotyping in the rubber tree variety DNA fingerprint library of the sixth aspect of the invention.
Preferably, SNP site polymorphism detection is performed using the SNaPshot method.
The invention has the beneficial effects that:
the 50 SNP molecular markers screened by the invention have high specificity, sensitivity and resolution, are not influenced by environmental conditions, have accurate detection results and good repeatability and stability, and can comprehensively reveal the real level utilization of genetic diversity of the rubber tree; the DNA fingerprint library is established by utilizing the SNP marker combination, and the cluster analysis and variety identification can be carried out on the rubber tree varieties, so that the detection result is more accurate and efficient, theoretical basis is provided for the protection and utilization of rubber tree resources, 66 varieties of rubber trees are effectively distinguished for the first time, and the distinguishing rate of the invention on bred varieties can reach 100%. The single nucleotide marker loci of the invention have the advantages of simplicity, rapidness and low cost, and can be applied in large scale in production.
Drawings
FIG. 1 is a graph showing an example of the typing detection result of a part of the sample amplification products in example 2 of the present invention;
FIG. 2 is a UPGAM cluster analysis chart of the SNP-based rubber tree variety of example 2 of the present invention
FIG. 3 shows the identification rate of the core SNP to the variety of rubber tree in example 2 of the present invention.
Detailed Description
The invention will be further described with reference to specific embodiments in order to provide a better understanding of the invention. The specific techniques or conditions are not identified in the examples and are performed according to techniques or conditions described in the literature in this field or according to the product specifications. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1: screening and determination of rubber tree SNP loci
Genome re-sequencing was performed using 9 rubber tree varieties PR107, GT1, IAN873, RRIM600, 93-114, cloud-ground 77-4, hot-ground 7-33-97, hot-ground 8-79, hot-ground 628 as samples. Specific procedures are referred to "Du, H., yang, J., chen, B.et al target sequencing reveals genetic diversity, population structure, core-SNP markers, and fri shape-associated loci in pepper varieties.BMC Plant Biol 19,578 (2019)".
Based on the rubber tree genome re-sequencing data, the SNP sites were mined by comparison with the rubber tree reference genome (https:// www.ncbi.nlm.nih.gov/genome/: (a) a minimum allele frequency MAF >0.4; (b) a deletion rate <0.2; (c) a heterozygosity of <0.2; (d) upstream and downstream 200bp disorder variation; (e) two allele sites; (f) the chromosome distribution is clear; (g) evenly distributed across the genome.
From the genome re-sequencing data of the 9 representative varieties, 50 SNP markers with high quality, single copy and high polymorphism are finally screened out and used for identifying rubber tree varieties from different sources, analyzing population genetic structures and analyzing genetic diversity of rubber tree resources. The sequence information of the 50 SNP molecular markers is shown in Table 1.
Table 1:
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in the above table, "Sequence" is the nucleotide Sequence in which the SNP molecular marker is located; the "[ ]" is marked as the position of SNP molecular marker locus, wherein the base is polymorphism of the locus.
Example 2: sequencing verification and application of rubber tree SNP marker
1 test material
The material to be tested is derived from a national rubber tree germplasm resource garden of the national institute of Tropical agricultural sciences rubber, 66 rubber tree varieties (table 2) are selected, 3 leaves of different plants are respectively collected from each variety, and the leaves are quickly frozen in a refrigerator at the temperature of minus 80 ℃ through liquid nitrogen for preservation, so that the DNA of the leaves of the rubber tree is extracted.
TABLE 2 information on 66 rubber tree varieties
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2 extraction and detection of total DNA of rubber tree
And extracting total DNA of leaves of each rubber tree variety by referring to a CTAB method, and detecting the total DNA quality of each variety by agarose gel electrophoresis and a spectrophotometer to ensure that the extracted total DNA of each rubber tree variety has good integrity, and the ratio of A260/A280 is between 1.6 and 2.0.
3 SNP primer design and screening
According to the sequence information of the 50 SNP molecular marker loci screened, a PCR reaction primer is designed by using primer design software PrimerPlax 2, 167 pairs of SNP primers are designed, and 50 pairs of SNP amplification primers with better specificity and higher polymorphism are obtained after screening, and the specific details are shown in Table 3. And single base extension primers were designed using Primer design software Primer3plus, see in particular Table 4.
TABLE 3 amplification primers
TABLE 4 extension primers
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In the table, in the extension primer, the capital is a specific sequence, and binds to the SNP region; the lowercase is a lengthened sequence, so that a plurality of types in one reaction can be accurately interpreted; the direction represents the sequencing direction.
4 typing experiment
4.1 PCR amplification reaction
The genomic DNA of 66 rubber tree varieties obtained by the extraction was used as a template, and the primers of Table 3 were used to amplify nucleotide fragments containing SNP sites: PCR master mix was placed in a 1.5mLEP tube and centrifuged at low speed with shaking. 8 pipettes were selected, 9. Mu.l of PCR master mix was added to each well of 384 well plates, and finally 1. Mu.l of template DNA (20 ng/uL) was added and mixed well, carefully covered with 384 Kong Fengban membrane, and each well was pressed firmly to prevent evaporation during the PCR procedure. Centrifuge at 1000rpm for 1min.
The PCR amplification system is as follows: 2X Taq PCR Master Mix 5.0.0. Mu.l, 1.0. Mu.l of genomic DNA (. About.20 ng), 1.0. Mu.l of a mixture of upstream and downstream primers (concentration: 10pmol/uL, ratio of upstream and downstream primers: 1:1), and dd H2O 3.0. Mu.l, and a total volume of the system of 10. Mu.L.
The PCR amplification procedure was: pre-denaturation at 94℃for 5min; denaturation at 94℃for 20s, annealing at 60℃for 30s, elongation at 72℃for 30s for 35 cycles; final extension at 72℃for 3min;16 ℃ for 1min; preserving at 4 ℃.
4.2 alkaline phosphatase treatment of the product
After the end of the PCR reaction, the PCR product was treated with SAP (shrimp alkaline phosphatase ) to remove free dNTPs in the system. An alkaline phosphatase treatment reaction solution was prepared in a new 1.5mLEP tube, and the SAP Mix reaction components were as follows: ddH 2 0.75. Mu.l, SAP (1U/uL) 0.5. Mu.l, exoI (5U/uL) 0.15. Mu.l, 10*SAP buffer 0.6. Mu.l, PCR product 4.0. Mu.l. The SAP mix reaction components were then added to 384 well PCR plates, after pipetting was completed, 384 Kong Fengban membranes were carefully covered and each well was pressed firmly against evaporation during the PCR procedure. Finally, after centrifugation, 384-well reaction plates were placed on a PCR instrument and the SAP reaction procedure was started. The SAP reaction procedure was as follows: 40min at 37 ℃; 15min at 85 ℃; preserving at 4 ℃.
4.3 Single base extension reaction
After the alkaline phosphatase treatment is finished, single-base extension reaction is carried out, and the reaction system is as follows: SNaPshot Mix 0.5. Mu.l, pooled PCR Products 3.0.0. Mu.l, pooled Primers (Table 4) 1.0. Mu.l, ddH 2 O0.5 μl. The reaction system is added into a 384-hole PCR reaction plate, after pipetting is completed, 384 Kong Fengban films are carefully covered, each hole is firmly pressed, evaporation and other phenomena are prevented during the PCR process, and the extension reaction process is carried out after centrifugation. The extension reaction procedure was: pre-denaturation at 95 ℃ for 30s; denaturation at 95℃for 5s, annealing at 52℃for 5s, extension at 60℃for 5s for 35 cycles; final extension at 72℃for 3min;16℃for 1min.
4.4 machine for feeding
2uL of SNaPshot reaction product was added to 8. Mu.L of deionized formamide containing 0.8% LIZ120, denatured at 95℃for 5 minutes, quenched at-20℃and sequenced on 3730 XL.
5 primer optimization adjustment
The raw data in the fsa format are exported from an ABI 3730xl instrument, classified and filed according to detection reaction, and respectively imported into GeneMarker analysis software for genotype data reading. And adjusting the proportion of the primers of each site according to the signal value and the peak-out condition of each site, so as to ensure that the signal value and the peak-out condition of each site are basically consistent.
6 quality control and analysis of results
Analyzing the formal experiment result, counting according to the conditions of the sample and the sites to obtain the parting result of each site of the sample, and deriving a PDF chart of each reaction result. An example of the result of typing detection of the amplification product of a part of the sample is shown in FIG. 1. And carrying out parameter analysis according to the result information to obtain detailed typing results, wherein genotypes of 50 SNP loci of 66 rubber tree varieties are shown in tables 5-9.
TABLE 5 genotype of 66 rubber tree varieties at 50 SNP loci (1)
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TABLE 6 genotype of 66 rubber tree varieties at 50 SNP loci (2)
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TABLE 7 genotype of 66 rubber tree varieties at 50 SNP loci (3)
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TABLE 8 genotype of 66 rubber tree varieties at 50 SNP loci (4)
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TABLE 9 genotype of 66 rubber tree varieties at 50 SNP loci (5)
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And constructing a phylogenetic tree by using a phasor R package according to the Nei genetic distance between samples by using a non-weighted group averaging method. The result of the UPGAM clustering analysis on 66 rubber tree varieties shows that some varieties with relatively close relativity are clustered (shown in figure 3), such as hot grinding 879, hot grinding 8-333 and hot grinding 87-4-26 of high-product lines are clustered; the cold-resistant strain is clustered by cloud-grinding 77-2, cloud-grinding 77-4 and GT1, which shows that 50 SNP markers can realize the differentiation of 66 rubber tree varieties.
The invention distinguishes 66 rubber tree varieties for the first time, and the distinguishing rate of the bred varieties can reach 100 percent. This shows that the detection of the single nucleotide marker loci of the invention has the advantages of simplicity, rapidness and low cost, and can realize large-scale application in production.
In order to further screen core SNP molecular marker loci, different numbers and different combinations of loci are selected from the 50 SNP molecular marker loci to distinguish 66 varieties, the distinguishing rate of the different numbers and different combinations of loci for 66 rubber tree varieties is counted, the number of loci is taken as the horizontal axis, and the maximum distinguishing rate of different locus combinations is taken as the vertical axis under the number of loci, and the diagram is shown in figure 3. As can be seen from FIG. 3, when the number of sites is 7, the discrimination rate for 66 varieties can reach 100%. The SNP molecular marker combinations achieving 100% discrimination are: lg03_104230492, lg02_8025843, lg01_54721919, lg15_32001166, lg07_72003724, lg05_88010730, and lg02_214754.
The above description of the specific embodiments of the present invention has been given by way of example only, and the present invention is not limited to the above described specific embodiments. Any equivalent modifications and substitutions for the present invention will occur to those skilled in the art, and are also within the scope of the present invention. Accordingly, equivalent changes and modifications are intended to be included within the scope of the present invention without departing from the spirit and scope thereof.
Sequence listing
<110> rubber institute of Tropical agricultural academy of sciences in China
<120> SNP molecular marker combination for constructing rubber tree variety DNA fingerprint, application and method
<160> 150
<170> SIPOSequenceListing 1.0
<210> 1
<211> 601
<212> DNA
<213> Artificial
<400> 1
aatccaaaaa aattggacca ttgaatttga accaaatcga acccgatcta ggacctgaac 60
tagattgaaa ccttggaggg ttcatggttt tgattctagt tcacccttta gatgaacaaa 120
aactggtggt tctgggccgc ttaaaactag aaccggattg gtggctgggc tttgggctag 180
gcatatgtgg tttgcaaaca aactaatggt aaaaaaataa aaataaaaaa aaaaaaaaag 240
atactaaaag agtgaaaagt agggaagggg ggaatcttgg gaattgctct aaatatagac 300
cgctaataat tgtttggtcc attatgtttc tttccatctc ttatgctttt tcgtacaagt 360
aatattgtgg atcaacaaat ttgtgattca tttggataaa tttcacccat ggtacctaaa 420
cttagggcat tataagtagt tctttggaga agttgttatc atataactga aacaccttca 480
aattattcaa tttctaattc cctcaagtag tttcccttat atttgattca aagatatatc 540
aacttccagt aagaaagtga ggtttgcaag ctctggtagg atctccctag tcaaattgtt 600
g 601
<210> 2
<211> 601
<212> DNA
<213> Artificial
<400> 2
aaatggtaat tgggattgcc ctaacagaag aaaagctcaa agctttaaaa gaaatagaca 60
aagagcagca acaagaagaa ggagaagggg aggcagagga agaacaagtg gttgagaaaa 120
gtggaggaga gagagaggga tttatagaga gagaagagga gggtaccatg atggcggtta 180
gtacaaatat agtagggggc gagggatgag ctgggtttgc tgatagatta atagttgtgt 240
aagaaaagga gaaagggagg gaattgaaat tggattaaag gaatcttaat tcctgaatgg 300
cacaatgaca aaggatattc ttcccaaata agcatccaga gaaacacccg agtatatgct 360
tctaaccaag cttgcagact ctgatcgctt ttactggttt tttgaaaata aaaaaataaa 420
taaatatatc gattgtgaac ctgtcactaa ttacagttga ctttttttta ttattgtata 480
tgagctaaaa ccattgataa gttgagtcaa ttttcttgtg ttctggagcg cctttttttt 540
ttttttataa ttagtgatgt tggtgaagcc tcaaatgact ttcatgtaga caggatgatt 600
a 601
<210> 3
<211> 601
<212> DNA
<213> Artificial
<400> 3
aaaaaaccta ctatattaaa aattcttttg aaagtaagca aaattttttg tctaatattg 60
tttggctagg gaatttttgg agtttatcaa ggtgcaagta accatgaaga attgcacaac 120
ttgatgaagg caacagttat gattcgcaga cttaaaaagg atgttctttc tgaacttcct 180
gtaaagcgtc gacagcaagt aaatactcta tttggttcta atatttgctg gttggatttc 240
agcaggtgtt tgtgctttgc gcatgctact agcgaacata atatatatat gctgtgaaat 300
agctgaatag tatagccatt aattatcaga gacatcaaaa taattttcaa atgctagaca 360
ttatttttta agcccccttc gggatgtgtt ttgcagtcca gctccttgag atctatgatg 420
gactggcttt gtacagttcc tttggcatta aatagctatc tactggaaaa attctcatgg 480
ggactggcaa aaattttcct aagtagtatt tttggatttt gtcatactgt agcatgcagt 540
caactgcatg aatgtttgct atcatcttta acttaacact gaaaaatgct tgtcttgtat 600
g 601
<210> 4
<211> 601
<212> DNA
<213> Artificial
<400> 4
aaaaaaaacc taacgatata taatataact ttatttagac tatataattt attataaaaa 60
ttaaaataaa aaagtttaaa agagggattt agtttaagag attaatcaaa taaaataatt 120
atatatttag gggacggcat gcctgcctac gtaatcttat aatgaaaaga aaaatgtgga 180
gaaagaccct tgaagaacga acaattgctc atatgaggtc cacgtgcatt acatattaaa 240
tatagattaa aattttaacc ttccacttct ttacatatyt gccctctttc ttgacagcta 300
gcttgtaatc ttycgctgta ctctgaccac tggtctccga tcgccatgtt gatggtgtat 360
aaactacaaa gtgcaaacca ttaatatatc tttcttttct tcacgtaaac cctaaatctc 420
ccctcttctc ttcacctgat ccgtctccaa agaagcagac tagtttactc aaaggtgcga 480
tctttttttt tttttaattt ttccccactc ttcttttgct taatgagaaa atggaagaaa 540
tgaatctaaa tcgaaatctt catcttctca actgtgtttt ttttttcgat tcgtttaact 600
c 601
<210> 5
<211> 601
<212> DNA
<213> Artificial
<400> 5
tcaatactat ggagatatag atcagatccc tcacatgtct tattggtgga gagcattgat 60
atacaatctg atttgacata tgaggaggaa ccagtaaaga tcttagcacg agaaatcaag 120
gaattaagga ataaaagaat tccattggtg aaagtacttt ggagaaacca caaagcgaag 180
gaagcgacgt gggagtgtga agagattagg agacaataat attcacagtt atttgaacta 240
ggtaaatttc gaggatgaaa tttatattaa gagggaaaga attgtaatgc cctcacttta 300
ggcagttctg tacattctac tactccgatg actaatgtct gttcagacgg ctgaaatgtt 360
tggaaccact cttaaattaa agtaaggagt cataatttaa tctaataaag atcaagtaag 420
gttgaaggaa aataaaagaa atagattata aattagttaa atgtgcataa gtcccagcga 480
tgagtaactc ccctaggaag ttggccacta agagttagag gactaaatta aattaatcaa 540
taagttaagg ggcaaataaa agattggagg actaccttga atagatgaaa aagttttggg 600
g 601
<210> 6
<211> 601
<212> DNA
<213> Artificial
<400> 6
aaaaaaaaga aattatagat tatatatata cacacacatt acttgcccag caaacgtcaa 60
aacgcatgtt ttggcaccgt ttttgtcacg aactaaaatc ctagcaccaa gaagtaaaac 120
cctttctaca aattccgcag aagcaaaaat cggaggatca aaattgccca tcaagaacga 180
agaattggcc tcagcaatgg ctgattcatc agtgaaatct gataagaaga agaagaagaa 240
gaggaagcta accagccccc aagataaacc aacgcctcca aagaagaagc acagaggtga 300
tgaaccagag aaagagagag aagtcgctgt aggtggaagc tcaaatcaga aagaatctta 360
ttctatgatt gaagcagagc ttccatggag gaatcttcaa cttattctat cacttcagag 420
caaagatatt gaccttcaaa agttagtatc tttcccatct cttcttcaca caaattgaat 480
ttttgagatt atttatttga ttttgaatgg aatgtaaata aattctgtac ataaagcaaa 540
tgctaatttg acaggaaggt ggaactagcc ttcagttatg tgaatttgag ggaaaaagga 600
g 601
<210> 7
<211> 601
<212> DNA
<213> Artificial
<400> 7
ttgaagttga aattgagaga ctaaaaaacg cttttcaata aaatacaatt ttagatcaag 60
ttaaattctt gtaataccca ataaacgttt ttttcaacaa tatcgacaac aataccaatc 120
caagtgtaat acttgaaacc aagtcattaa aactacatta gaaatatggt acaccttatt 180
aagagctgaa aagaccagaa aatgaagaaa ataaggatca aaataaattc aattgtacac 240
catccagcca cttaagtgta ggcagaaccc taaccagaaa taaggtaaaa ttttcatcag 300
caatggcatt ccccaccaca tccaattgtg tcaagttcaa gcctttcaac acaaaaaagg 360
cttcccaata atttgtcatg ctgaccccat caaccaaaat tctatcttgc tcccattcat 420
ttccaatcga attagacaaa ggagatgaac acagatatct ggttgtgtca atagagcgtg 480
cacctatctc attgtatgaa atatccagcg atttcaatga cttcagctgt ctcaatggct 540
ccaatgcagt aaaactagag agtttattct cactcaaact caaatgaatc agtagctgca 600
g 601
<210> 8
<211> 601
<212> DNA
<213> Artificial
<400> 8
tatattggct tcaacagatt gtggtgtttg tctctgtaat taatactaca ttaataaaat 60
agagaatttt ctctttttct ttgggagaca tgaattcata tttataatag cttagagtgc 120
ttgcagaagt tacagctttc tcaacaattt ctggcacatg atcgtacctt gaaaattcag 180
gacagtatgt tctgaattct cttttctatc aaactcttaa tcatgtgtct accgtactag 240
gagcagtggg taaaacgaac tcatccattc ccaagttcat taactgcttt ccaagctcat 300
ccctcaatct cctgctcatt ycaagtcttc tgcactctag ccttttgcgc aacaaatagc 360
acctcagcta ccttatctcc aaaaacgctc actttagcaa cagttcagct cagatcctct 420
gctaaatatc acttcaacaa catctcttct ggtgtagact tcttcaataa caagtgcatg 480
atagcacaaa cacagtacaa aaaaaaaaaa aaatacaaga taataaattt cataacatta 540
taaaaaaaat ttattaattt tgtaaaattt cattaataaa aaaaattaca gtcgctcaaa 600
a 601
<210> 9
<211> 601
<212> DNA
<213> Artificial
<400> 9
atttggcaat tggttgggaa gaaaaaaaat ttattgcatc acgccgacga tgagtgcatt 60
ggaaggcgga atccacgctc cttacgtgtc cttccatgtc tcacagctct gaatggaaaa 120
ctcaccgttc aaagtctgac ggctgaattg ttggacaggc taatcatgct gacttggagt 180
cctgcaattg gtactccaaa agtttattat ccagctaagt tatatataaa aaaatgcaac 240
ttaatttaat aattctcgat cgaatcacgg ccgtcatttc agagaaagga catgacgaga 300
aggaaagtga atcagagccg tcgaaataaa ataatgataa acgggattag gttaaaatgg 360
gaaaagaaac gaggtgtgtc tgccactggc aacgtctctg cacgctgctt aaaggccgaa 420
tcaatggtca gatcaaatca caggtaaatt tttgactttc aggaagaaaa taaaattaaa 480
tttaggaata aaattaaatt ttttaaataa taatcaaatt aaatttaggg ataaaataaa 540
aatttcaatc atttgatatg tgagatatac acagataagg cacatagtat atggatcagc 600
c 601
<210> 10
<211> 601
<212> DNA
<213> Artificial
<400> 10
aatcataacc ttaaaaaaag aaagtaactt cttgtatttc agttccatga tcataacact 60
tgctcgtcac acttttatgc ttcaatgaga aatctcaatg tcccaatcac tatttccaga 120
gggaattgtt attgtcacaa gattgtcctc ttctctgaat gtaaatccca cttctttgga 180
gtttaacaag caagattttg ggtttacact ggagtatgca ccaaacgtac cttcccctct 240
ccccttgatg cctattctac caccagactg ctcaattgac tctacagctc caccagaatt 300
gtacatgttt attaatccaa tagctgcgaa ctcaatattc tgattgtaaa cctgtgcata 360
tcaaacaaca ggggagaatt caaatacttg ttaatatgtg agctaaaccc caaagaactg 420
gccattccca acctaccttg ataggtgcta tggtaaagac atcacattgt agagttttta 480
acgaaatatc aaaagcttct tccttctgca atcgacatat agatcctgca aagttttaat 540
aacaaaaaaa gtataaaaat gtaatgtatc acatcaaagc aaatacattg gtcttgattt 600
t 601
<210> 11
<211> 601
<212> DNA
<213> Artificial
<400> 11
gtaaattcat ttttaaaatt tatgaattaa tttaattaat ttataagtta aaacaaacag 60
cctcttatga taaattatga caattaaaaa aaaactattt aattgtttta aagtttttat 120
aattaaaaca tgtcaaattt aagtttaaac tataaactat ttgttgatac cttttaatta 180
acatatttaa gaatacattt ttctcaacag aaatgttaaa taggccttgc aaattgataa 240
tgcctaacac tcgctctaac aactcgtgaa tatgcacctt cgtgaatatg caccttcaat 300
gagttcagat atttgattta ttcatgcacg taaatgtaat caaatgtatc ctttccaaat 360
ccaaatttca gaaatttctt aaatgtaaat atacagtttt catatatcgt tgaattgaac 420
ctcaatagat tttgagggaa agctacactt acttgcggga agaatgtgtc caactactgg 480
gaaatacgct ttcgaggctg gtaatcccac gtgctccaac cagcaagaac ttctaattcc 540
agcagcaacc cttcaatctt tgcaactaag ctctgatgcc actgctgtgc cattgtaaat 600
t 601
<210> 12
<211> 601
<212> DNA
<213> Artificial
<400> 12
atttcttttt agccaattcg catgtttaga atatatatat atatatatat atatatatat 60
atatatatat atatatatat atatatatat atatatatta aaagaaatag aaatcaagca 120
tgatagtatg agaattcaat taaattcttt tcttacaaat gagttattca aaaggaaatc 180
aataaggaaa tacgtatgta ttgttgcaaa tatacaagtg tagaattcct acatcggaaa 240
aatataggat gactgagaga catataaata ggaaagatcc aaaaactcat tgccttaagg 300
ttttgggtta aatgtgatgt caagctcatg agtgtgtcct tcacaagacc tatcaagaag 360
gattctccac aggcgctttg ggctccttag atttctaaca agaatatgca atcctaaaaa 420
atgcttgttt ccacttacac taaccaatca gaaaatttta tgcccaacag accataaatc 480
taaacaataa tgttctatga tatagtagta ctgttatcat tccatgcatt ggagcacact 540
aacctggtat aaatttattt aatgtagttc aaatcccaaa aaggggtata tataagttca 600
a 601
<210> 13
<211> 601
<212> DNA
<213> Artificial
<400> 13
gtgttcttct tgaaaagaga ggaaaaggtt cttaaaaatg ctagtgctaa tacaggcaga 60
gtaataaatc agcatataaa atattctcaa acaatctata tagtacgaaa acatttctgc 120
atgtccctgt gacagaagca gtaacaatgg gaaagttcag ggactttaaa catgttagtc 180
tggttttggt ataagattct cacaaaggcc ttgtccatca ttgagtataa tgacgcatca 240
tccaacttcc acaaaacagg aagttattat tcaaggcagc caaattttgg atgcagctca 300
caacaaatga agatgttgat gatgctaaga ttttggtgaa tgcaatatga gcacaaatta 360
tgttttacca acattaagcg taagggaact gcaagaatat tggaggatca aacagcaaga 420
gctttattcc tgtatgttat gttcttgtag tagtacgaat atctttcttt gtgcaaatta 480
cacaggattt aggcttcatt caggttactt ctccaaaggg cttgttaaag gtatatatct 540
cttgatgttc agcaagactg tagatcttta aatttttttc tttttcctat ttagaggaag 600
a 601
<210> 14
<211> 601
<212> DNA
<213> Artificial
<400> 14
tgcttctttt ttatttttct ttccctattt tattctttgt caaatagttt atttttgaaa 60
ttttataaat tttgctacac attattcaat ttgttgtaag tactgtccat ttgtgctgat 120
tcatgccaaa accttgcagg ctcttctttc aggatgctct gccggaggat tggcaactct 180
tattcattgt gataactttc gagatcttat gccaaaggat gctactgtaa aatgccttgc 240
tgatgcagga tttttccttg atgagtatgt ttgatatgaa tactgatact ttccacctca 300
gtatcttaag ttagcatgtc atatgagttg ggtgataccc atgtcatagt atttttgtac 360
catgtgctct taatcatctt ctaattatag aatttttatg tggatttaaa gatccccatt 420
ccccaaagtt tactttttta caccttggaa ttccaactcc cctttgcact atcctccttt 480
cttttatcat agtagaatta actacagtac gttagcaact taattcttct gcagagattt 540
tgttttccta gttttttcat caaaagttag atgtgtccgc tgaactgaat atatatatat 600
t 601
<210> 15
<211> 601
<212> DNA
<213> Artificial
<400> 15
tggaagagat cagtgaggaa gattttaata agcaggaggt taaggttgta aaagaatcga 60
aacctataga tggtaggttt tggacgatga gagatctgta caagtatcat cagatgggag 120
gaagatacgt ctccggcttg tataatcttg cttgggcaca ggcagtacag aataagcctc 180
taaatgagct atttgttgag gttgaacccg atgagaattc caagcgatcg tcgccgtcgt 240
cttctgtggc gtctgttaat agcaatagta acagcagcaa agaagacgag aagaagaaag 300
ttgataaagt tgtgattgat gatagcggcg atgagatgga tgataatatt gtggatgttg 360
acaaggagga aggtgaattg gaggagggtg agattgattt ggattttgac ccaggtgaga 420
aagctgctgg ggagggtaaa gaagggctgt ccaatagtga tgaaatgaat gttgatggct 480
tacagattga gtccaaggag aagaatttgg agaaggaaat caactccatt cgtgaggctc 540
tggagagtgt aacagtgatt gaagcacaca agtaagcaat ttgttgctgt tgtttcaata 600
a 601
<210> 16
<211> 601
<212> DNA
<213> Artificial
<400> 16
ggccaaatct cacagaccac ttccaagtcc caacaagaag gaaggccaaa agacgagctc 60
gctaaaacag ctggttatgt cggcgtagaa ccaagcaccc cattagtcct ctgggttgta 120
ctcgagggga gagagagatg ccaacccact tggtgggtgt tcttagggga gtgatagatt 180
cttaaacaag taaatcaaat agagtagatt gataaaatat ttaaataatt aattatatat 240
ataacaaaac aaagaatatg gtatacaagt attatattac catataaact aagtagaccc 300
ataatagtct gttgaatatt ttacgcttgg ggtaactggt agactaacca cgttcctaaa 360
acaggatata aaggtagttg aacaaaaagg catttttttt taattagaga gtgagaaaga 420
gggataaagg caagaaaaac acattgaaac gcaagcatct tgctccaaaa gaagtgaatc 480
tcatatttct ctggtgcttt attgactgca gataaaacac aagaaagccg ggaatcaaat 540
gattccatct caagagaaga aatctcaact cgacgccatt gctggtgaaa agagtcgatt 600
g 601
<210> 17
<211> 601
<212> DNA
<213> Artificial
<400> 17
ttatatattc ttttggattt tggaagtcaa gattcaactt ttgatttcat ttcctgctta 60
tattctggga tttgttttcc atagtttggt tgattttagt aattgaatat gggttcttga 120
gttgaaagtt attgagatct gagaattggg aaagctcagt gatttggata ggtgtcaatt 180
gccagtgtga aacttaagtt tgcttgctgt agctattcat gttcttggtg ctttgcttaa 240
ttagagtaca gcatgtattg agtgaagctc ttatttggct aagtgctcct ttgtcgttgg 300
atttcttgga tgtattacat tcttagttat gcttttattc actattcttt tctgtctttt 360
tcttggaatt cttggaatgc agcaagcaga tatttaaata tggtttaaaa cgactgattc 420
taaattctgt ttggtctcct aggcttttca ttttagcaat gaaactcttg ctttgaattg 480
attatgatgc tgcacctctt ctttgtcctt gaaattttga actgttttta atggtttttt 540
tttttttttt ttttgtttta tacatgcaat gaaaatttaa tttttttttt ttctgccata 600
t 601
<210> 18
<211> 601
<212> DNA
<213> Artificial
<400> 18
attattggtt tgaaatttgt ttctcggctg ttttttatct ctctatacgc cgtggtcttt 60
tgcatttata gttgagcttt ggataatttc ttatgtatct ctatctgtag gtctctgcat 120
atgaatagga tttatttttc cagttcaatt tctttattct gctgacattg cttttagttc 180
tcaaaatcaa cctttgagca catgtgaata tattttgcct aaaacttcac cattctgcat 240
gtattcaccc ctacacttga tttcctggtt catatatttg acatgattat aatatcggta 300
tttttggctc ccatgttgac atattgttgc aaactatagg ttgtattctt gttcatgctg 360
ggttgtgctt ctcttcatta ttaaagtctg tcttttctgt tttgcgtgat gaactctaat 420
tcatttttcc atttaaattt ctttgacttc attatggata attaacccag tttgatttga 480
atgctggtga gagttaagga aaactgaaaa ttactcttat gctgttgtga ttttcagaaa 540
atatcaactc aggccaggct aaatgatctt tttgggtaga atatatcccc tagaatgata 600
t 601
<210> 19
<211> 601
<212> DNA
<213> Artificial
<400> 19
ataaaaataa ttttcggttg gtttttattg aaaaactaaa attgaactaa attatcaaaa 60
aaatttggtt cagtatggtt cttcagttcg gttcaggatc cctaagaact gtgcacagct 120
ctgcttatac tatgtcaata ataatttaat tttaaagttc aattttgtag ataatgaccc 180
aaaaatagat tttcatttct ctaacaatcc ttattcgcat tattcttcag aaatatgaaa 240
tatgtggagc ataaaaaaga taacatttca ttaagataat caattgtaaa taaggtgaat 300
agtgagaaca tttactcaag aattagtttg gttgaacata tactcaagaa ttagtttggt 360
tgaagtaaga ggcttgcctg gcgttgatga aatccattat gcttgatcca tcaccgacct 420
tcaccactct tccattttcc atataaaaag ctccatcagc atactcgagt tcttccaatc 480
gatgggtcac ccataaagct gtaatttcat cagatgcaac caaggagttc ttgactgctt 540
taatcacccc aatctggcaa tcaatagcca acatgagtaa ataaatatta tcaccacatg 600
a 601
<210> 20
<211> 601
<212> DNA
<213> Artificial
<400> 20
gatatggaac caccaatcct gcataaactt gattctaact acaactaaga cgatgaacga 60
caatgacaat aattctgatg gcagcaaagg aaattttctt tgatattaga ttctgctttg 120
ttttcacatc tttttctgat tacacaggct tgaacgtctc aatcctggat gttcattaat 180
gtacttacct cattcaagac cgaaagacaa ctgcttttca agaatcaaga aaattcaaga 240
ttcacttatg aagaaacaga aatgaagaaa caacaaacat taaatccata cataaaggac 300
cagccaacat tgcataccta aggttggttg gttatattgt tgtatccgca ccatgaaatc 360
tttaatatca aaatggtttg aagccctcta atctatttcc agctatttct ttgctgcctt 420
ctctctctct ctctctctct ctctctatat atatataatt gaatatataa ttgaatcatg 480
atttggacaa accaccaaac ttcacttatg aagaagcaga aattattgag caccacatta 540
gttccactac tttgctgata ttcaaccggc ttttgtggct tttgtgatca aaaagttgct 600
g 601
<210> 21
<211> 601
<212> DNA
<213> Artificial
<400> 21
ttcctcccat atcaaatttc actattccct tgggatccaa atcagttgct gtcaggtcct 60
acttcttaga aagaatttgg tgacaaagtg acgactagtg tgtctgatca agtgatggga 120
gggtttaatt agatcttccc ttcaacacaa taagagggct ttacttcatt agttttattt 180
tttttagtta acttttccaa gttcttttgt tatttctctt atttcttcat taataaaata 240
tttatcttgt ttgagaacaa ggcttgaatg ttaaggattg agaacttcat gttgagaagt 300
taatgttctg caggacattg atgagtttaa gagaatgaag caaaggagaa aaaaaaatgc 360
tttttgaggc tcagattgat aaagcaatcc caagggatca gatgagatgg taattgtatt 420
gtagggaagt ggggtagagc ctagaggacc tggatcacct ataccagaag gcaccagaag 480
gatctgcagc agtatgagaa ttcaaatggt tctctggaag ttatccagct acgagctgat 540
aaggtggaga aatttctgaa aggccatgtg atcacggcaa ctgaatcttc tataaaaagg 600
g 601
<210> 22
<211> 601
<212> DNA
<213> Artificial
<400> 22
tagacttttg cccaataatg cttttgcgtt tggttttatg gatcccttct tgcttcatta 60
cactggtaaa aatgaacagt ttactgagac cttatataat attggtatca gtattatttc 120
cagtgttgag ggcaaatctt tgtcctttta gtgattttat ttgtttatac tgtggcaaat 180
tatgctcaag caatgatgtc atgtaactta atcaatgtca ttgcctgtgt catagtggtc 240
atatgttatt gcatattatg ttgtattcat ttacgccaag caactgtcat cagtagttct 300
catctatcat tagctcatgc ccatgctttg cttttgtatc ctacagaaat tttatgctct 360
tcaaaattag aacttgatca caagtttttg atcaataagt tagttgtcaa gatatgatac 420
tattttaatg tttgttttag ccagtgatag tataatgtgc ttttgaatca tctatactaa 480
tgtcattatt ccaggttggt ggtaatttaa ctgtggatgg tattgactat tatttatctg 540
aaacttttgg agaagggctg tacaagtcat gcaaagatgt taagtttggg acaatgaata 600
c 601
<210> 23
<211> 601
<212> DNA
<213> Artificial
<400> 23
ttaccaacta aatataacaa taatgatatt ataatcaaaa tgcttctgtt aaactagagc 60
ctcaatctct aaaaagttta ccctccctct catgccctct aaacgctaaa cattataccg 120
ttttagcgta ttgtttcttg tctctcttct ctatgtcctt tctgtcgata ttttcttagc 180
tagacttgca gggaaggaaa agtagatgtg cagtgcataa accctaatct ttctcggcag 240
tggcccgatc aactcatttt ctctctcaat tcaaggtcaa ttttactctc gttttcattt 300
tattatcaat tttgttatca ttgataatcg caatctttaa gttgtttttg cttcagtaac 360
ctacaatatg actgaagacg tcgagatgaa ggaacttcct gctccctcca actccgccac 420
ctcctcttcc ccttccacac tcaaccgtaa gcctatttct gggttttctt caattttttg 480
atttcttgct tgatttctga tttgggtatg tgagtttttc tgttttgtag atttgaagga 540
gatcgcttca ctgattgaga ctggtgccta tgcccgggag gtgcggcgga tagtgcgggc 600
t 601
<210> 24
<211> 601
<212> DNA
<213> Artificial
<400> 24
tattgcagaa gactgagaaa tcaatgaaag atcactcctt ccaaatccca taagacctga 60
agcccctcca aagagacctc tattattcct gccacatcca aatatgaaat tgtccacagg 120
ggttgttcct aaattgagat gctccgtccc tagctcaccc ctggtatagg aaccatcacc 180
atagttaaca acatagttac aggttggtgg gttgctccca cagactcctg aattcccagt 240
tgcaaattgg agggattggc aaattgatga attacataaa actgtttcgt acgaagggga 300
tactgaaggg ttgaaaagag ggtcttgttg attataacat agtctgcaag gttgacattg 360
aacccatgtt aaatcacttc ctgtgtctac tatcactgtc atgtttcgac cacctaattc 420
tactgtaaca atgtagttca atgtctgaag tcttacacca gaaactaatg ggatttgact 480
atgtacagaa tcttcacttt tgccatgaac actctttttt attcgagatt gaagtgaccg 540
aacttgaaaa ttgtcaagaa tcaaactttc ttgtagcttc ttgttccagt cagtaatctg 600
g 601
<210> 25
<211> 601
<212> DNA
<213> Artificial
<400> 25
ggccaacata ttaggtactt aaaaaaaaaa aaaaaatcac cttgtcataa ccccgcccca 60
ccccccaacc cccatccccc acccacgcca ccccacatgc cccccaccca taggcttgga 120
cgcttattgc cctatatcct caggcatatc ttaagaagac cttagcaata aaagcgtcct 180
atatatgagt tcccctccat ttcaggccct tctatctatc catttatggt gccataatcc 240
acaagcttct caggcaatta gaggaaatta acgtgaaatg acaacttgca aaaatgccga 300
aaatgaattt caccaataac aattattagt ttggcaagta aattagtgcc tcctgcagtt 360
agatgcaagc gactacagaa gattcaagat cgatgtcaaa ttgccagatc tggcttcaca 420
tattcaaaca ttgaagtaag aaggttcatt tattagattt tctagatgat gatgatgatt 480
ggatggatca taatgacatt gaattgtatg atcataatca tggctgctgt ctggttcgga 540
gttgaaagct gaccgatcta actcagaatc caaaccggta aactttgatt gaccaaaacc 600
a 601
<210> 26
<211> 601
<212> DNA
<213> Artificial
<400> 26
gtgcatctat tatggtcatc atgttggaaa aacatagaat gatcaacttc actcctagac 60
attccaaatt gttagactac attactgaat cgactaaatc atgctctcgg aattacttta 120
aaccatacag tgattgtcat agatgacata ataagccaga cttctccctg aacaacaaac 180
cctggtggtt gctttttata tatttccttg gctagttcac catgtaaaaa ggtattttta 240
atatccaatt gatagagtgt tcagtgatta attgtagcta atgaaatggg tagtcgaacc 300
aaggcaatct tagccactgg agagaaagta tcactatagt caagcccaaa gatctacgta 360
tatcctttgg caacaagatg agctttaagc ttatcaattt tttcgttagg acccacctta 420
actgtaaaaa tccatcgaca tccaacaata gatttatcta gtggtaaaag taccaagtac 480
caagtctcat ttgtatggag catagtcatc tcctcaatca ttgcatctcg ccaccctgga 540
tggtccaatg cttctctaac cttctttggt attgttggaa tctcaatccc tacattaaag 600
g 601
<210> 27
<211> 601
<212> DNA
<213> Artificial
<400> 27
taaaaatgga aaaccatcta ttctttttaa ttgaaattgt ttttgacaag cagggagagt 60
gcaatatgga aggcaacatt caaaagatta gatttatcta tgatattagc ctatgtttta 120
tataatcaat gatgtaatat aatataatta taatttttat tttaatattt attttatttg 180
tgatggtaat tataacaaag gagttataat tgcaatgata gtgtaatctc ctcaacaaac 240
cattgccatc ggcatccaaa attagagcag actagccaat tttgataaat aattctaagt 300
tggtttctat caaagttgat tcatatcgac tttaatttca aattagactg atctgattct 360
gggctaaaac caaattgtga tcatgattac cataggtatt accatgattc aagttagacc 420
taaaattgaa tcaaaaccaa attgatcaag ctgaagatca aaattagtca atattttatg 480
agctcaaaca ttgtgggagg gtcggtctct ctccccttag ccgaattgcg ttgcaaccaa 540
tggtttagac cgatgatata attaagttaa aaccaccttt ttaaaaaaaa aattatttta 600
a 601
<210> 28
<211> 601
<212> DNA
<213> Artificial
<400> 28
gagaaaatag tgatgtatct ttaagtgtct ctcatatgac ttcaaataag cagttggtcg 60
acatgtttac tctaagtagc aaattattcc atactagagt ttgcaagtta ggcatgtgta 120
atatacatta accaacttga aggggagtat tagagtccct aacttgtagg atttaattgt 180
ttattctttc ctatttgtat ttattcttct cgattgtatt tattagtttc ctattgtttt 240
aatttcccta aaatacttgt gtataagtag gatccatgga aaagtttgaa ttacagagaa 300
atacataaaa tttcctccaa attagtggtg tccacaactc atttaacgag ttggcaagtc 360
aagatgcaca taaaacaacc attgaagatg gataacataa ttatttattt ggttaatatt 420
ttttatcatt ttatcttcaa aatcacaaat ttaaatctga cattttttaa aaaattactc 480
ttaagtagaa taattaaata tttatatggg gttaagttag agtttagtgg tgaacctgta 540
acacaatata atatccatgt tccaacataa taaccatgtg aaaacacaaa agagagggcc 600
a 601
<210> 29
<211> 601
<212> DNA
<213> Artificial
<400> 29
cgcaggttgg taatccaagc aaagcaaatg gcaggaaatc caaacctcat ctctctgaag 60
tttagtacca ttgaagcttg tgcctatatt ctagttacta tgttctcata ctgttaccta 120
catagggaaa aatgccaaat tatgatcaat cctgacagct tcctagaagc acgcaccatc 180
ctgtaacctg cgcatgctcc aacatgccaa attcaatcaa catcatcacc agaaaagggc 240
aatggtaatg cacgtggtag aggctggagc tccacaaaca tcacataatt caacagaatg 300
tcaagaatct catccactta tatcaacatt agattaatca tatcattgcc attaattatt 360
atgcttaatg aacataaatt gaaatatcca agcaccagga aagatccata gttgtatcca 420
agcaccgttt attataaagc agaaagttgc tgtacatggc acatgaaaaa tacctatagc 480
caactgaaat caagtgagac gttccttgtt tcccaaatcc cttttaaatg tgctctacat 540
ggcaaaaagg ctaatttcac gcataaaatc aaaatatgat agagctttga gaagagttat 600
t 601
<210> 30
<211> 601
<212> DNA
<213> Artificial
<400> 30
caaggggctt gcaaaaagaa taagtcttaa taacatccca aaattgagct tcaagttcag 60
tatagccttc agggcccatc tcctatgcca cacacacaca cacacacaca cacacagaga 120
aatagagaga tactgctatt gaccgctcca ccgcctgaat gaaatcaatt ttttccacta 180
ataatttgtt ttcccttcca gccaacaaag cagcttcatt tactagattt gcaagatcag 240
ccctgcaaaa tataaaatgg aaaaagtcac ttccgataca ttagctaaaa gtaggacatt 300
gaagtctctt aatcaggagg aaatttaccc agtaaaacca gtggtcatag atgcaatgtc 360
actaaggtca acatcctcgc caagaggaag ctctttcttg gaagcatgca cttttaaaat 420
ggcttctctt ccattcctat caggcgtttc cacctataga gtatagacca aatgagataa 480
atgtctgaga caactattta gtggaactag cttgcctgtc aatcttgacc atccaaaatg 540
tgaagaagct gaaatacaaa ccataaccac acgatcaaat cttcctggtc ggcgaagtgc 600
a 601
<210> 31
<211> 601
<212> DNA
<213> Artificial
<400> 31
attgtaatta aactttgaga ttataagtaa acttgtaatt tattatttat gaatttctat 60
tatgaatgca atagatgata ccttttggag atcgcataaa taattgtgaa tgatgtgata 120
cagtagaata tgatatggaa aagtgtgaaa tgaatataaa cataaaaaca ggtaactagt 180
ggaactcgct agatgctaac aaaacagagg aggctctgtc tgggtttcta taaaaaaaaa 240
aaattctaca tatagatttt ctgttttaaa tgatattaaa atttacaatg gacatgataa 300
gacaagatag ggtgctccag caccgaatgt ggcacttctt gctcgactat acagtagaca 360
agtaaggggc gtcacacatt atgagttgcg tgagtaatgc tctttgtgag cttcaccttg 420
tgtcaagtag tattagagta ggaaaatcac cccaactgag atgacttacc ttgatggagg 480
tggcaactaa tagtggttct tctgatggtg atcgggaagc taaaaccgtg atcattagaa 540
caaaatgttg cgattaagga agtgacattt ggagcaacat atggatctca aattcatgca 600
t 601
<210> 32
<211> 601
<212> DNA
<213> Artificial
<400> 32
aatttagtaa gcagaattaa acaatggaaa ttcaatatat atttatttat atgtaaataa 60
aaaaataact atagacgaac ttacgagtat gtcagagaca attctgggct gctttagagg 120
cttgagaata aaacgaacta ctcgagaaat taaaacaatc aatataatct caaacaagac 180
gaggctgaag gagtaatcca aagggttttc tccataaaaa acacctaatg gatggggagc 240
atggattttt tgacatatta gtctaactgg agaaatgact tgctctacat gcaaagaggc 300
tcgaggctta gcttccatcg tattgtgcca ttcatctctg atttgagaat gataaactgg 360
gaaactccta accatgacaa gtacctgtaa tttcgaaacc atagaaaaaa aaaatgaaga 420
agaaaagaat agaaccacag atagaggcga gagtcattgc tgttaataat ggacaagcgt 480
atttttgggg gtgcttagca gtcttcaacg tttaccattt tccccctatt tttagtctat 540
catccacgtc aaggttgttg ggtttctcaa ctcagatgca cgcagcaaaa gtacaattta 600
a 601
<210> 33
<211> 601
<212> DNA
<213> Artificial
<400> 33
agtattctct gactgtgtct ctactttttt tttatttttg ttttaaaact aatccacgta 60
ttattatgtg gttgggttgc cataccaact atgattctta attggtttag cagcttcact 120
ataaaatttg tcattatata ataaggaaaa agatgtaatt ataataaatt aacataaaat 180
ttttgcaata aggatcatat atttataatt ttaaatttga agtttcttct aaaaataaat 240
atactcaaga attttaagaa agaaacaagt aatcttattt ctggtggcaa ataatatcat 300
ttaccggttg gctaataata taaaaacaaa taccgcaatt gatatataac cgaatggtat 360
atggatgaag ctataaaatt cgtagggttg caaacagcaa tgttgcttaa gattagtagt 420
ataagaaatg gttaattaag agaatacatt aattagaatt aaaacaacaa agcagtgtag 480
aataattggt tccaagtatc aggaagccca ggaaggcacc aatggctaca gtctgctgat 540
cggtaagggt tagccctctg ctcagggctc aagtcaccac tatatatgga tggatggcaa 600
t 601
<210> 34
<211> 601
<212> DNA
<213> Artificial
<400> 34
aatatagatt acaaaataat gtggataata ttccaagcta tagacaaagt ctcctatcat 60
taaattcaag gaaattttag aatttttcta ctaaacttat ttttataaag taaatattat 120
tttatcaata attattcttt tacaattgta aaaaatttaa cctaatagtg aaatagataa 180
aattatagtt tttaaatttg gttcaatatg agagaatcaa tgtgatgcat ctggttgatg 240
agccatgtac aaaggttcgc tgatgttctt ggagagaggt tggggtgaac gagttatgac 300
atttgttgct tcacgaggac atttagtcta aacgggtcgg aaatgatgca tcacacattg 360
gttatgcaag gagattttgg gtgttatata taggtttgta aacctcttgt gagctagctt 420
ttgaggtgaa tttaagtaga tgtatatcat ttggtatcag agccccactc tgtatgggtt 480
gaaaggttcc ctatgttagt gtatgctcta gagcatatca ttgtattgta tcttgttgta 540
gactcttatt ttatgatgag tatgtgcatt gaggccgtga gaaacccgat gatgaaaaat 600
t 601
<210> 35
<211> 601
<212> DNA
<213> Artificial
<400> 35
gctgcttttg tccgctgtca tgtgcgcatt ctcagccaaa caatgcccca actgtggcct 60
tacctcagtt ccctacccac tcagcacaca ccccacctgc ggagaccaat catacaagat 120
acggtgcaac gcaggtgtac tcattttcga cacgcttaac aattcctatc cgatattttc 180
catcaatccg tcgattcaac gattggtaat acaacctgca aatctgttat caaatacctg 240
catttcatct gattttatcc accaaggcat ccagttgagc tcttccctac cgttcaacat 300
taccggcgag aacactgtca tgctttttaa ctgctccgag tcgatacttg atcaaccgtt 360
gaattgttcg ccgagtagct tgtgccactt atacgttgag ggagaagagt actctcggtg 420
cagagattca tcgttgtgtt gtacattcaa agcgggcggg tcagcgtcgg cctataggat 480
acgcgtccta gaaaacgggt gtagggctta tagaagtttc attggtttgg attggggttt 540
gccagtggat aagtggccaa agccgggtgt ggagatccag tgggtgcttc ctctggaacc 600
a 601
<210> 36
<211> 601
<212> DNA
<213> Artificial
<400> 36
gcaaagctta gaaagcttag atttatcaca caataatttt ttggtgaaat tccttgcaca 60
ttggcaaatc tctttgagct gagttactta gacttgagta acaataaact agagggtcgc 120
ataccaagtg gtcctcagat ggatcggatg aacgacccaa attcttatgc caataacagt 180
ggattgtgtg gaatgcaaat taaagtatca tgtgagaagg tgccttctga accaaagctg 240
aaagaggaaa agaccaagaa aagcaatagt tgggagatat ggttttcatg ggaaatggca 300
atgattggat atccttctgg atttttgtca acagttttag ttatgtatgt cattggctac 360
tttaatatta caccacagcc aggtagaaga aaaagaagaa gaagccctgt taggcatggt 420
ctttttggtt tttgacgcat ttgatatatg ttcttagtgt ttgttgttat aaaaaaaggg 480
cttacaaggt cttcatgcta tgtccttaat tgttgcactg ctgcttccct atttcctttg 540
ttacctcaga agattgttgc ccttttgttt ttgtatgttg tgatgtatct cctaccaccc 600
c 601
<210> 37
<211> 601
<212> DNA
<213> Artificial
<400> 37
aaattttgaa ttgtagtagt tatgatgtaa tcctcttaat tgtaggaaga cttgttcttt 60
tgaaattttg taagcttgct ttaaaaaggt ggagcttatc attaataaaa taagcaaaat 120
ttctatacaa aagtgttctt atgagattgg aggattctct ctcaacgagc actcaattaa 180
ttcttcacaa taggtttctt taagaaagca ggaacgtgaa tcagggaaag tccaagtact 240
tgaggtagat ttctaggtca ttgtcctgtg atgagatcct attcaagtgc acataacttg 300
atatcagagt tggttgtcat gggtgaacct tagttaccta atatgctgta ttcctaagtc 360
atggtatggg tattgggtac gcggtatgca gtacaagaat taatgaaata taaggtactt 420
taggggtaga cttaattggt gcattaattt gcttcgatgt ttgtcaaatt atatttattc 480
agtatataaa ttatatttat atagaattat taatttttta attttattta atataatgtt 540
tataaaaaaa ttaaaaatta attagtatga taatttttta acataatata cacattattt 600
a 601
<210> 38
<211> 601
<212> DNA
<213> Artificial
<400> 38
cattcttgcc gaaacttaac aatacaacgg ttcttagtaa ctctagatgt gtaatgcaat 60
tggtaaggtt gtgttgctct ttcgtcaatg gtcatcggtt tagatcgtga gaataagggg 120
aaggccgcgt acccttgccc gtctttcaca aaagttactc tataaagtta aaattttgag 180
ttagattgct ctgcaaaatc ctccattagg ttagctggtg gttcagggta atttggagag 240
ggacgcatag agtccagttg tactgaagac aggtgaagca attgaatcca cttgctcttg 300
caattgagag attatgtcaa tctcttctac acctgtctac ccaatgtcaa acgtaagtaa 360
tcaaagacct agattttaac gtcccaatta taatttctaa tctatctcta tttctcagaa 420
ccaatggctc aaggaattca ccttatacaa aactaaagag ctgacccaca catacacttg 480
taaataaaat tctgcaatta caattttttc gctcgattca aatcacgaac acgcaaccca 540
tcgatctgtg atttttagca gaggctctgc gtttggtttt gctgcttatt gtagcacctg 600
g 601
<210> 39
<211> 601
<212> DNA
<213> Artificial
<400> 39
tagaaatatt ctttataatt gaacaatata ttcttattat atagtatagt tttatgttat 60
aaattatgaa cttgtagaca atcattatca ttgtgcatgt gttgaaactc acttaaattt 120
aatgaaaaaa ttaaataata caaaggggta gcgggttaaa ttaaaaggtt agaatatatt 180
aatataaaat gtcataatct gacaactata ataagaggag taaattaacc aaaaagaaaa 240
aaaagaaaaa agaaagaaaa ggatggagac aaagaatgac cggtggaaga aaaatagtta 300
tgcgaagtat taggtgcact tctcatcgta aaaataaaat aaatccttaa attttaaagc 360
tgtagggtct aacaagtaac aaccctttag aaaaagaatc gaccgaatcg gtcaatttgt 420
atcgattctc ctgattcact atcaaattgt aagattcgat cacgattcag gtacatttgg 480
gatttacccc atagacttga atcattgtga tagagtgaag aacagcaatt ataaacaatc 540
agaatagaaa tggaactgag ttagtattat gaaacataga atgctcaaac agcaaataca 600
a 601
<210> 40
<211> 601
<212> DNA
<213> Artificial
<400> 40
tctaggcaac acacagaaag ctaaagctag gccaagagaa gataacagaa acaagatggg 60
ttctctatcc ggtgaatcaa agtatcatgt agttctgttc cctttcatgt caaaaggcca 120
cactatccct attctccagc tagcccgtct ccttcttcgt cgccagatct ctgttaccat 180
ctttactact ccagccaatc gtcccttcat agctaaatat ctgtccaaca cagctgcctc 240
aatcattgag ctgtccttcc ctggaaacgt ccctgaaata ccttctggca ttgaaaacac 300
agataagctc ccttccattt ctctgtttcc ttattttgcc ttctccacca agctcatgca 360
acctgagttt gaacgtgcac ttgcgtatct tccatctgta aatttcatgg tttctgatgg 420
attcctgtgg tggactctgg agtctgcaat caaattcggt tttccaagat tggttttctt 480
tggcatgtcc aatcatgcta tgtgtatggg caaagctatt aatgagaaca ggcttttttt 540
tgggcctgag tcggatgaag agttaattac agtgattcca tttccatgga taaaggtcac 600
t 601
<210> 41
<211> 601
<212> DNA
<213> Artificial
<400> 41
tatgatgttt attattgttc acaaatccac aattttcctt cgcattacta attatgaatt 60
aagagtaatc aaatattaca aaggtcaatc caaaagcatt aatttgcatt aataaagaaa 120
actaatacaa aatttgatag agtaaggaac ttaagcgaat ggaaagaaaa taatctaaaa 180
ttgtattaag aacttgagat tacagtaatg gaagtacacg agcaattctc ttactctcct 240
ctctaattct caatgcaaat atagcaagaa aagggggaaa aaaccttcca aacactctag 300
ggagaatgct tggcccagca ataatgccaa aataaaagag ctaagccctc ccgccattaa 360
gaattttgcc cccaagttag tattatagtt ggaaaataca attactctct catgcccccc 420
attctacctt ttctctccca tatataattc tcgtagctgt tgcatcacat gcacccgaga 480
attgctccaa aggagttgag ctaagctggc caaccaactg caatccttcc agaactttct 540
tgcttcatct agaatacacc atatccctaa caatagtacc cgctcaaaat agccttgtcc 600
t 601
<210> 42
<211> 601
<212> DNA
<213> Artificial
<400> 42
tggcaaagtc ccaacttctt tgcatacttc caagcaaatg caagcaatgc tggctttctt 60
ggagagatgc tttgttcagg cctgaatatt gttggcttca actggatttc atctccagcc 120
gcaactgagc tagaatccct tgtcatagat tggatgggaa aactgttgaa gcttccgccc 180
tcatttctct tctctggtaa tggaggcggt gtcttgcatg gcagcacatg tgaggctgta 240
gtgtgcactt tggctgcagc aagagataag gccttgaaaa ggatgggatg ggataaaatt 300
acaaagttgg tggtttatgc ctctgatcaa actcatgcca ctctcttcaa gggtaccaaa 360
atagtaggta tcccatcctc taatatccgt tccctcccta cttcattttt atctgggttt 420
tccctgccac ctcaaacact tcaagaagca atcgaaaatg atataaaatc tgggttttat 480
ccattgtttc tttgtgccac tgtgggaaca actgcctgcg gagcagttga tcctatacga 540
gaactagggg aaattgctac gaaatacaac ttatggttcc acattgacgc agcttatgct 600
g 601
<210> 43
<211> 601
<212> DNA
<213> Artificial
<400> 43
atgcgatagc gtcgtcaatg tcgatgaggg accatcggca acagccctgc attacctgta 60
ccaccttcaa catcttagcc ccgatataca agcgcctcaa ccttgacaac aatcaaaatt 120
cccgtgaaag cgattgcaga gcctattggc ttgccagaaa caaccggatt ttggattcct 180
tattgcacga gagatcttcc atcatttgtc tccaggtttt tcttggcttt atcaattcaa 240
atcctttact atcgttcaat cattattatt tctttattgt tgtttattcc tttttggcgg 300
ggatgacaaa ggaattctgg ttggggaacg aagagctggt gaacatgtac gagaagagac 360
tgggtgacgc tggttatgtc aatttccagc ttgcgcgcac caacaaccgc ggtgatggta 420
ctcttctttt cgccatcttt ttttcctcct tttaattttt tttaatctct tcttgtgaca 480
tataagcgat cgaatttttt tgttttattt gcttttttgg atgttttgct ttcttttaaa 540
tctcattgcc taacggtaat tttagaaaaa gaaggtacag agggtttaaa tttaaataaa 600
g 601
<210> 44
<211> 601
<212> DNA
<213> Artificial
<400> 44
aggaagtctt caaagacaat atcgacatta ttgatcaaga ggtagaagca ataattctgg 60
aatttttgaa ctaatccaaa taaatgggtt gccttcgagg agagtccctt gaaacttttt 120
acactactgc ataaagaata ttattatggg tatgtgtcaa tgtcatatca tttaactttt 180
tcttctgcct ttgtgtccct ttctcttcta tttctccttc tctttgcttc ttattttttc 240
tcttttttgt aactttaatt tttgtcttgt gttcaggctt tgttagttat gacaagcgcc 300
cgatacaagg ctgtttggga ggtttatcac acaaatgggg atctggtaag ggtggtgagt 360
acaaaggaga ttgacatgaa agagagatat cttctgaccc ttattgaggc aaaaaggagg 420
actactgaac caatacgcta agcagatggc cttcaattat agatcaatgc attagaggag 480
atggctgaga ggcaccgaaa gaggattgta gaattggaaa tagaatgggg ggatatgatg 540
agtaaataag tgtggatgga caagaagata caaagaacac gaggtcttct caacgagagc 600
c 601
<210> 45
<211> 601
<212> DNA
<213> Artificial
<400> 45
tattgataga tgagaattgg gtggccaagg tttcagactt tggtttgtct agatgggtcc 60
caccagcgac tcccaaacgc acgttagcac tgtagttagg ggaagttttg ggtacgtgga 120
tccagagtac taccgtcggc aacacctcac ggaaaaatct gatgtgtact catttggagt 180
agttctgttt gagttgctct gtgctaggcc gccagtgatg ccaggactac caaaagagca 240
agtcaattta gctgattggg ctcggatttg ctgtagaaga ggagccattg atcagataat 300
ggacccctat ttgaggggta atattgaccc tgtctgtttg gagaaatttg gagagattgc 360
tgagagttgc ctgcgtgacc aaggaacaca aaggccaaca atgagcgatg tggtttgtgg 420
gcttgagttt gctttgcagc ttcaagagac tgctgagaac actagaaata gtattgatag 480
agtgagtgag gagcaggaaa gtccattgtt gttgcgtgga gaagctataa ctactgatga 540
tgatgacttg tttagcgtct caggtggaca caagcatgga tccaagagca cgattagtag 600
t 601
<210> 46
<211> 601
<212> DNA
<213> Artificial
<400> 46
aaagaacatt attacttcat ttttctgctt attggtatat ttgttatttg attcaaacta 60
tggaattggc tgctgcaaat tgatatccgt tagttctctg ttaagtgctt ttgttggact 120
gcgagaatca agatttcttg cttcttgcat cttcaagcta tagaatactg tttgaaatct 180
tcaccatgtg ttgcccctaa tcccattgaa ctcagcttcc tggaaaattg ttgttccata 240
ttctaatgaa ttcaaatcaa aatgttcgtg ttttcttgcc tcgccctttt tccttcaaat 300
gtcaacagga ctgtttttca ttcattttcc tcagataggg ttcacatact gttttctttt 360
ctactaataa tcaatctttt ctttttttgg ttaagagaat tttgtctggc aggctgtagc 420
ttgttactct cttggtacat ttatgtgttt tactgggatc atcctttaac ttctgaatta 480
tgttgctaat ccaaaatcat gacatgtaac tactgaagtt tctttatcct acttgattcc 540
attgaaatga aactatagtt agaatattgt caattacgtg ttttatacct catatattct 600
g 601
<210> 47
<211> 601
<212> DNA
<213> Artificial
<400> 47
atgcttagtc aacgtgggac tgtggcatca agtacattgt ttgttcctta tcttttgtta 60
tgtgctacgc ataagaaatt ggagatattc gttttgtttg aggaaaaatg ataagagtac 120
aattcattga gccaattata tttgtgcata ttggctgatc ttctattatt ttctgtgtca 180
tgttgaaatt attttttgta attacttgct gtttgggatg aaatatattt gtggcttcat 240
agtttttaag atgttgcaat attttatgta attactacaa ttttgcttta gagtttgaaa 300
agttgttatg ttatattcta ctttacagtc cactatgtgg aaataagacc ttgggtttgg 360
ctactaggta ctggtgtatc ttctctactc ctactcacaa ttcgcttact gaatttcaga 420
tggagtggat gcatgaagcg atcgtatgat aaaggtgtta cttggacaaa gagagaacaa 480
cttcctcctg gtatattagg accaataaag aacaaggtat aatttgaaaa tgaaagccat 540
cataatggat gtttatatca ggaaggatgc agctttattg ttactgatgg gatacccctg 600
a 601
<210> 48
<211> 601
<212> DNA
<213> Artificial
<400> 48
taaaaataaa aaaattaaat aattaatttt attaaattga agggactaat taatatttaa 60
aaaaaaaaag ggggacaaag cttaaaccag ctaaaatgtc atatcctgcg ccgaccttgt 120
actactgttt tgcgtaaaaa actattccgc gtgtcagata acataagccg tagtcttgtc 180
ggtttgtagt gtcttatcat agtcggtgtt tttacaggcg acacttgcaa gcgacccctc 240
acaccaaacg tgtccgtcaa ttcttctctt caagatactt ctctctcttt ggatacggtc 300
aaatttcggc ttctctcttt tcaacctttt ttttcaagta cttccataaa ctaacaatga 360
cttgagcata gacttttctt tcttggctac tactactgtt gaagcggaca ttgaggtgaa 420
agctagtgca cactgtgtga tattttcttc tttcctattt ttggtttttt atttttttta 480
gcttaaattt gatcttgaac tcttagcaga agagaggaat accatcgtcg tttggttagg 540
agctgggtgg ttaggtggtc ggtcctcttg ctttcaaaga gcacttcttt acaagttggc 600
t 601
<210> 49
<211> 601
<212> DNA
<213> Artificial
<400> 49
ggccaattct ctcagacact cttcttgcct atattttgca cttatcatca aagctgtgtt 60
accagtatct gtcctggaat ttatatcaca acctgaatct attagacatt taaggatagt 120
tggtaagcct aaacgggcag ccatgtgtat gggccggaac tcggtttttt gagttttgac 180
agggtactcc acattggcac cacagctcag tagcacattg acagctccag cattaccaca 240
gagaatggcg tggtggagga gagttcttcc atggtgagta gtgtttgggg aggtgtgttc 300
gagaagcatg cgcaatatag cacccgtagt ttcaaaatac tccactgcac accaggtaat 360
tgtgtagggc tcagctagtc ctgcgcccac tcgaaactct tctccagtag tagtgtccca 420
tgaccatgct cctagactca ctttgacatc tgtcctggca ccatcctgtt ttatcatttt 480
gataccaatc agaataacca ttagtaaagc aatattgaga gaaggatagg gattcctttt 540
ttcaaaaaaa aaaaaaagaa gaactaacat aataacaagg agagaagtac ggctctatta 600
t 601
<210> 50
<211> 601
<212> DNA
<213> Artificial
<400> 50
tttaatgctc taatgaacta attaccatgc tttcgctcct acaattgata tttagggtta 60
atattcattg cattttatta gtgttttgtc tcttttatgt ttattttatg tgtttttagt 120
aatattttag aaaatttctt attccaaccc aattttttga tttttatatt ttattatgtg 180
tttttatcta gttcttgaac taaggacaaa tttggaagag atctgtaagt caaaaagtga 240
agaacggaaa gccagaagaa ttacacaggt catgtaacct accctatgta acctcctgga 300
ccccgtgcaa ccttctagac aaaagaaaat taaaaaattc aagccataaa aaattatacg 360
aggtacccag tgtaacgtta caaggcctgt gtactgtccc cggaaaaaat tctaacttaa 420
tttctcttct ttctaactcg atccggatgg acttataaga tttttaggac tctgaaataa 480
ggatttttac actagatata aatataataa gtcaggattt aggggttagg aaccttatta 540
cattaaaaag gaccaattta cattcaagag tgtgcattaa cttcaagaaa ggcataggag 600
a 601
<210> 51
<211> 20
<212> DNA
<213> Artificial
<400> 51
gccgcttaaa actagaaccg 20
<210> 52
<211> 20
<212> DNA
<213> Artificial
<400> 52
aagtttaggt accatgggtg 20
<210> 53
<211> 20
<212> DNA
<213> Artificial
<400> 53
gtggttgaga aaagtggagg 20
<210> 54
<211> 20
<212> DNA
<213> Artificial
<400> 54
atcagagtct gcaagcttgg 20
<210> 55
<211> 20
<212> DNA
<213> Artificial
<400> 55
tgtaaagcgt cgacagcaag 20
<210> 56
<211> 20
<212> DNA
<213> Artificial
<400> 56
ggaactgtac aaagccagtc 20
<210> 57
<211> 20
<212> DNA
<213> Artificial
<400> 57
aagacccttg aagaacgaac 20
<210> 58
<211> 20
<212> DNA
<213> Artificial
<400> 58
agtctgcttc tttggagacg 20
<210> 59
<211> 20
<212> DNA
<213> Artificial
<400> 59
gacatatgag gaggaaccag 20
<210> 60
<211> 20
<212> DNA
<213> Artificial
<400> 60
gccgtctgaa cagacattag 20
<210> 61
<211> 20
<212> DNA
<213> Artificial
<400> 61
tgcccagcaa acgtcaaaac 20
<210> 62
<211> 20
<212> DNA
<213> Artificial
<400> 62
gcgacttctc tctctttctc 20
<210> 63
<211> 20
<212> DNA
<213> Artificial
<400> 63
attgtacacc atccagccac 20
<210> 64
<211> 20
<212> DNA
<213> Artificial
<400> 64
agccattgag acagctgaag 20
<210> 65
<211> 20
<212> DNA
<213> Artificial
<400> 65
gcttagagtg cttgcagaag 20
<210> 66
<211> 20
<212> DNA
<213> Artificial
<400> 66
tggagataag gtagctgagg 20
<210> 67
<211> 20
<212> DNA
<213> Artificial
<400> 67
tctgacggct gaattgttgg 20
<210> 68
<211> 20
<212> DNA
<213> Artificial
<400> 68
tttaagcagc gtgcagagac 20
<210> 69
<211> 20
<212> DNA
<213> Artificial
<400> 69
tctccccttg atgcctattc 20
<210> 70
<211> 20
<212> DNA
<213> Artificial
<400> 70
cgattgcaga aggaagaagc 20
<210> 71
<211> 20
<212> DNA
<213> Artificial
<400> 71
tgcctaacac tcgctctaac 20
<210> 72
<211> 20
<212> DNA
<213> Artificial
<400> 72
gaagttcttg ctggttggag 20
<210> 73
<211> 20
<212> DNA
<213> Artificial
<400> 73
gtgtagaatt cctacatcgg 20
<210> 74
<211> 20
<212> DNA
<213> Artificial
<400> 74
agatttatgg tctgttgggc 20
<210> 75
<211> 20
<212> DNA
<213> Artificial
<400> 75
caatgggaaa gttcagggac 20
<210> 76
<211> 20
<212> DNA
<213> Artificial
<400> 76
gctcttgctg tttgatcctc 20
<210> 77
<211> 20
<212> DNA
<213> Artificial
<400> 77
tcgagatctt atgccaaagg 20
<210> 78
<211> 20
<212> DNA
<213> Artificial
<400> 78
aaggaggata gtgcaaaggg 20
<210> 79
<211> 20
<212> DNA
<213> Artificial
<400> 79
taatcttgct tgggcacagg 20
<210> 80
<211> 20
<212> DNA
<213> Artificial
<400> 80
tctcacctgg gtcaaaatcc 20
<210> 81
<211> 20
<212> DNA
<213> Artificial
<400> 81
tggttatgtc ggcgtagaac 20
<210> 82
<211> 20
<212> DNA
<213> Artificial
<400> 82
ggttagtcta ccagttaccc 20
<210> 83
<211> 20
<212> DNA
<213> Artificial
<400> 83
aggtgtcaat tgccagtgtg 20
<210> 84
<211> 20
<212> DNA
<213> Artificial
<400> 84
aagcctagga gaccaaacag 20
<210> 85
<211> 20
<212> DNA
<213> Artificial
<400> 85
ctctatctgt aggtctctgc 20
<210> 86
<211> 20
<212> DNA
<213> Artificial
<400> 86
tgaagagaag cacaacccag 20
<210> 87
<211> 20
<212> DNA
<213> Artificial
<400> 87
gtgcacagct ctgcttatac 20
<210> 88
<211> 20
<212> DNA
<213> Artificial
<400> 88
tttcatcaac gccaggcaag 20
<210> 89
<211> 20
<212> DNA
<213> Artificial
<400> 89
tacacaggct tgaacgtctc 20
<210> 90
<211> 20
<212> DNA
<213> Artificial
<400> 90
ggcagcaaag aaatagctgg 20
<210> 91
<211> 20
<212> DNA
<213> Artificial
<400> 91
cttcaacaca ataagagggc 20
<210> 92
<211> 20
<212> DNA
<213> Artificial
<400> 92
ccatctcatc tgatcccttg 20
<210> 93
<211> 20
<212> DNA
<213> Artificial
<400> 93
ttgcctgtgt catagtggtc 20
<210> 94
<211> 20
<212> DNA
<213> Artificial
<400> 94
gtacagccct tctccaaaag 20
<210> 95
<211> 20
<212> DNA
<213> Artificial
<400> 95
tcttagctag acttgcaggg 20
<210> 96
<211> 20
<212> DNA
<213> Artificial
<400> 96
ttacggttga gtgtggaagg 20
<210> 97
<211> 20
<212> DNA
<213> Artificial
<400> 97
atcccataag acctgaagcc 20
<210> 98
<211> 20
<212> DNA
<213> Artificial
<400> 98
caagaccctc ttttcaaccc 20
<210> 99
<211> 20
<212> DNA
<213> Artificial
<400> 99
taggcttgga cgcttattgc 20
<210> 100
<211> 20
<212> DNA
<213> Artificial
<400> 100
cttctgtagt cgcttgcatc 20
<210> 101
<211> 20
<212> DNA
<213> Artificial
<400> 101
cgactaaatc atgctctcgg 20
<210> 102
<211> 20
<212> DNA
<213> Artificial
<400> 102
cgtagatctt tgggcttgac 20
<210> 103
<211> 20
<212> DNA
<213> Artificial
<400> 103
ctcctcaaca aaccattgcc 20
<210> 104
<211> 20
<212> DNA
<213> Artificial
<400> 104
cgaccctccc acaatgtttg 20
<210> 105
<211> 20
<212> DNA
<213> Artificial
<400> 105
gttggtcgac atgtttactc 20
<210> 106
<211> 20
<212> DNA
<213> Artificial
<400> 106
ggacaccact aatttggagg 20
<210> 107
<211> 20
<212> DNA
<213> Artificial
<400> 107
aagggcaatg gtaatgcacg 20
<210> 108
<211> 20
<212> DNA
<213> Artificial
<400> 108
ggaaacaagg aacgtctcac 20
<210> 109
<211> 20
<212> DNA
<213> Artificial
<400> 109
gatactgcta ttgaccgctc 20
<210> 110
<211> 20
<212> DNA
<213> Artificial
<400> 110
gcttccaaga aagagcttcc 20
<210> 111
<211> 20
<212> DNA
<213> Artificial
<400> 111
aaaacagagg aggctctgtc 20
<210> 112
<211> 20
<212> DNA
<213> Artificial
<400> 112
gatgccagga ctaccaaaag 20
<210> 113
<211> 20
<212> DNA
<213> Artificial
<400> 113
acgaggctga aggagtaatc 20
<210> 114
<211> 20
<212> DNA
<213> Artificial
<400> 114
atgactctcg cctctatctg 20
<210> 115
<211> 20
<212> DNA
<213> Artificial
<400> 115
gtaatcttat ttctggtggc 20
<210> 116
<211> 20
<212> DNA
<213> Artificial
<400> 116
taccgatcag cagactgtag 20
<210> 117
<211> 20
<212> DNA
<213> Artificial
<400> 117
gatgcatctg gttgatgagc 20
<210> 118
<211> 20
<212> DNA
<213> Artificial
<400> 118
gggaaccttt caacccatac 20
<210> 119
<211> 20
<212> DNA
<213> Artificial
<400> 119
tacaagatac ggtgcaacgc 20
<210> 120
<211> 20
<212> DNA
<213> Artificial
<400> 120
taagtggcac aagctactcg 20
<210> 121
<211> 20
<212> DNA
<213> Artificial
<400> 121
agatggatcg gatgaacgac 20
<210> 122
<211> 20
<212> DNA
<213> Artificial
<400> 122
cctaacaggg cttcttcttc 20
<210> 123
<211> 20
<212> DNA
<213> Artificial
<400> 123
gattctctct caacgagcac 20
<210> 124
<211> 20
<212> DNA
<213> Artificial
<400> 124
gcaccaatta agtctacccc 20
<210> 125
<211> 20
<212> DNA
<213> Artificial
<400> 125
ccagttgtac tgaagacagg 20
<210> 126
<211> 20
<212> DNA
<213> Artificial
<400> 126
ggttgcgtgt tcgtgatttg 20
<210> 127
<211> 20
<212> DNA
<213> Artificial
<400> 127
gagacaaaga atgaccggtg 20
<210> 128
<211> 20
<212> DNA
<213> Artificial
<400> 128
gctgttcttc actctatcac 20
<210> 129
<211> 20
<212> DNA
<213> Artificial
<400> 129
gtcaaaaggc cacactatcc 20
<210> 130
<211> 20
<212> DNA
<213> Artificial
<400> 130
acgcaagtgc acgttcaaac 20
<210> 131
<211> 20
<212> DNA
<213> Artificial
<400> 131
agtaatggaa gtacacgagc 20
<210> 132
<211> 20
<212> DNA
<213> Artificial
<400> 132
attctcgggt gcatgtgatg 20
<210> 133
<211> 20
<212> DNA
<213> Artificial
<400> 133
ctggctttct tggagagatg 20
<210> 134
<211> 20
<212> DNA
<213> Artificial
<400> 134
gatcagaggc ataaaccacc 20
<210> 135
<211> 20
<212> DNA
<213> Artificial
<400> 135
cttagccccg atatacaagc 20
<210> 136
<211> 20
<212> DNA
<213> Artificial
<400> 136
tcgtacatgt tcaccagctc 20
<210> 137
<211> 20
<212> DNA
<213> Artificial
<400> 137
ttcttctgcc tttgtgtccc 20
<210> 138
<211> 20
<212> DNA
<213> Artificial
<400> 138
gccatctgct tagcgtattg 20
<210> 139
<211> 20
<212> DNA
<213> Artificial
<400> 139
gatgccagga ctaccaaaag 20
<210> 140
<211> 20
<212> DNA
<213> Artificial
<400> 140
tgctcctcac tcactctatc 20
<210> 141
<211> 20
<212> DNA
<213> Artificial
<400> 141
tgttgcccct aatcccattg 20
<210> 142
<211> 20
<212> DNA
<213> Artificial
<400> 142
ggatgatccc agtaaaacac 20
<210> 143
<211> 20
<212> DNA
<213> Artificial
<400> 143
gtgcatattg gctgatcttc 20
<210> 144
<211> 20
<212> DNA
<213> Artificial
<400> 144
tcatgcatcc actccatctg 20
<210> 145
<211> 20
<212> DNA
<213> Artificial
<400> 145
cataagccgt agtcttgtcg 20
<210> 146
<211> 20
<212> DNA
<213> Artificial
<400> 146
atcacacagt gtgcactagc 20
<210> 147
<211> 20
<212> DNA
<213> Artificial
<400> 147
gagagttctt ccatggtgag 20
<210> 148
<211> 20
<212> DNA
<213> Artificial
<400> 148
ggaatcccta tccttctctc 20
<210> 149
<211> 20
<212> DNA
<213> Artificial
<400> 149
acaggtcatg taacctaccc 20
<210> 150
<211> 20
<212> DNA
<213> Artificial
<400> 150
ggttcctaac ccctaaatcc 20
Claims (4)
1. A SNP molecular marker combination for constructing a rubber tree variety DNA fingerprint, characterized in that the SNP molecular marker combination comprises lg03_104230492, lg02_8025843, lg01_54721919, lg15_32001166, lg07_72003724, lg05_88010730, and lg02_214754;
wherein, "Sequence" is the nucleotide Sequence of the SNP molecular marker; the "[ ]" is marked as the position of SNP molecular marker locus, wherein the base is polymorphism of the locus.
2. The use of the SNP molecular marker combination of claim 1 in the genetic structure of a population of rubber tree varieties, and/or genetic diversity analysis of rubber tree resources, and/or identification of rubber tree varieties, and/or construction of a DNA fingerprint of rubber tree varieties as set forth in the following table:
3. a construction method of a rubber tree variety DNA fingerprint library is characterized in that total DNA of each rubber tree variety is extracted; amplifying by using a primer pair; carrying out SNP locus polymorphism detection by using the SNP molecular marker combination of claim 1, and carrying out genotyping on each rubber tree variety according to the detection result of the SNP locus, thereby constructing a rubber tree variety DNA fingerprint library; the primer pair is as follows:
；
The rubber tree varieties are those described in the following table:
4. the identification method of the rubber tree variety is characterized in that a primer group is adopted to carry out PCR amplification on the total DNA of a sample to be detected, SNP locus polymorphism detection is carried out by utilizing the SNP molecular marker combination of claim 1, the genotyping of the SNP molecular markers is determined according to the detection result of each SNP locus in the combination, and then the genotyping is compared with the genotyping in the rubber tree variety DNA fingerprint library constructed by the construction method of claim 3; the primer group is as follows:
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311487416.6A CN117467794A (en) | 2022-05-31 | 2022-05-31 | SNP molecular marker for distinguishing rubber tree varieties and variety identification method |
| CN202210613434.3A CN115011720B (en) | 2022-05-31 | 2022-05-31 | SNP molecular marker combination for constructing rubber tree variety DNA fingerprint, application and method |
| CN202311482430.7A CN117487950A (en) | 2022-05-31 | 2022-05-31 | Construction method of rubber tree variety DNA fingerprint library |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210613434.3A CN115011720B (en) | 2022-05-31 | 2022-05-31 | SNP molecular marker combination for constructing rubber tree variety DNA fingerprint, application and method |
Related Child Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
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| JP2017184651A (en) * | 2016-04-04 | 2017-10-12 | 株式会社ブリヂストン | Variety discrimination methods of para rubber tree, and kits for variety discrimination of para rubber tree |
| CN110241252A (en) * | 2019-07-30 | 2019-09-17 | 中国农业科学院郑州果树研究所 | SNP marker for constructing peach DNA fingerprinting combines and application and method |
| CN111808983A (en) * | 2020-07-31 | 2020-10-23 | 中国热带农业科学院橡胶研究所 | Rubber tree variety standard DNA fingerprint spectrum library and construction method and special primer thereof |
| CN113846177A (en) * | 2021-07-30 | 2021-12-28 | 中国热带农业科学院橡胶研究所 | SNP molecular marker for rubber tree secondary emulsion tube array number and application thereof |
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| JP2009165385A (en) * | 2008-01-15 | 2009-07-30 | Bridgestone Corp | Method for identifying kind of hevea brasiliensis |
| WO2010034961A1 (en) * | 2008-09-23 | 2010-04-01 | Sumatra Biosciences Pte Ltd | Methods of obtaining hevea bras iliensis plants |
| CN101603090A (en) * | 2009-02-27 | 2009-12-16 | 中国热带农业科学院橡胶研究所 | A kind of method for identifying rubber tree varieties of quickly, efficiently and accurately |
| JP2013198447A (en) * | 2012-03-26 | 2013-10-03 | Bridgestone Corp | Method for detecting hevea brasiliensis white rot disease microbe, method for diagnosing hevea brasiliensis white root disease and primer for detecting hevea brasiliensis white root disease microbe |
| CN105838809B (en) * | 2016-05-19 | 2019-04-02 | 中国热带农业科学院橡胶研究所 | One kind SNP marker relevant to rubber tree latex dust quantity and its application |
| CN105861498B (en) * | 2016-05-19 | 2018-12-14 | 中国热带农业科学院橡胶研究所 | One kind SNP marker relevant to rubber tree dry incineration method and its application |
| CN105950729B (en) * | 2016-05-19 | 2019-04-16 | 中国热带农业科学院橡胶研究所 | One kind SNP marker relevant to rubber tree stem girth and its application |
| CN117467794A (en) * | 2022-05-31 | 2024-01-30 | 中国热带农业科学院橡胶研究所 | SNP molecular marker for distinguishing rubber tree varieties and variety identification method |
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| CN110241252A (en) * | 2019-07-30 | 2019-09-17 | 中国农业科学院郑州果树研究所 | SNP marker for constructing peach DNA fingerprinting combines and application and method |
| CN111808983A (en) * | 2020-07-31 | 2020-10-23 | 中国热带农业科学院橡胶研究所 | Rubber tree variety standard DNA fingerprint spectrum library and construction method and special primer thereof |
| CN113846177A (en) * | 2021-07-30 | 2021-12-28 | 中国热带农业科学院橡胶研究所 | SNP molecular marker for rubber tree secondary emulsion tube array number and application thereof |
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| CN117467794A (en) | 2024-01-30 |
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