CN109868330B - Lentil EST-SSR marker developed based on RNA-Seq and application - Google Patents
Lentil EST-SSR marker developed based on RNA-Seq and application Download PDFInfo
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Abstract
The invention discloses a lentil EST-SSR marker developed based on RNA-Seq and application thereof, wherein the invention develops 125 lentil EST-SSR markers in a large scale by a transcriptome sequencing assembly technology, provides forward and reverse primer sequences corresponding to the markers, provides a screening process of the markers and explains the application of the EST-SSR markers in the aspect of lentil germplasm resource genetic diversity analysis. The EST-SSR marker developed by the invention has high success rate and rich polymorphism, can amplify various types of strips in 94 portions of lentil germplasm DNA, shows good universality, has rich polymorphism of EST-SSR primers and greatly helps molecular assisted breeding of lentils.
Description
Technical Field
The invention relates to an EST-SSR marker of lentils developed based on RNA-Seq and application thereof, relating to the technical field of molecular markers.
Background
Lentils are annual self-pollinating, diurnal herbs and an important bean for cold seasons, diploid (2n ═ 2x ═ 14) plants with a genome size of about 4G. Lentils are the sixth world's edible bean. Lentils are important sources of protein, carbohydrates, vitamins and trace elements in the human diet, and are also high-value feeds for livestock. Lentils are beneficial to crop rotation due to their nitrogen fixation capacity. However, the single yield of lentils is still low due to the fact that they are often planted in poor soil and are affected by drought, high temperature stress and various diseases such as wilt, anthracnose, rust, neck rot, root rot and frost rot. According to the FAO data of 2016, the world average yield per unit area is only 1,152 kg/ha. Therefore, it is very urgent to improve the yield of lentils by using a suitable breeding strategy.
Some quality character genes are successfully introduced into the main stream variety of the lentils by the traditional breeding method, but the traditional method has poor effect and wastes time and labor when the quantity character genes are introduced, and the interaction of the environment or the genotype environment is generally great. The molecular marker has wide application in germplasm resource diversity research and molecular assisted breeding. Among them, Simple Sequence Repeat (SSR) is widely present in genome of eukaryote, and has a large number and high repeatability, and is one of the most commonly used molecular markers. EST-SSR is an SSR mined from expressed sequence tag libraries (ESTs) or transcriptome sequencing data, which is relatively easy to develop compared to genomic SSR.
At present, EST-SSR has been widely used in genetic studies of barley, cherry and sugarcane, but its application in lentils is less studied.
Disclosure of Invention
The invention overcomes the defects of the prior art and provides a lentil EST-SSR marker developed based on RNA-Seq and application thereof. The invention provides a large-scale development of 125 lentil EST-SSR markers by a transcriptome sequencing and assembling technology, and explains the application of the EST-SSR markers in the aspect of lentil germplasm resource genetic diversity analysis.
A group of lentil EST-SSR markers comprise forward primers and reverse primers corresponding to the following 125 sites:
note: and representing that the predicted value is not equal to the actual value, and listing the actual value.
Further, the lentil EST-SSR marker is applied to lentil germplasm genetic diversity analysis.
Further, the primer of the lentil EST-SSR marker is obtained by the following method:
1) selecting 6 parts of lentil materials with different sources, extracting RNA to establish a cDNA library, and sequencing on an Illumina platform to obtain original reads;
2) obtaining clean reads through data processing of the original reads obtained in the step 1), and assembling and splicing the clean reads to form Transcript and unigene;
3) designing an SSR primer pair by utilizing unigene obtained in the step 2), selecting 4 parts of hyacinth bean materials with different origins from 88 parts of hyacinth bean materials, extracting DNA as a PCR amplification template, carrying out PCR amplification, and screening an EST-SSR primer according to the amplification condition;
4) extracting DNA of 6 parts of the lentil material in the step 1) and 88 parts of the lentil material (94 parts in total) in the step 3) as a DNA template for PCR amplification, adding a fluorescent label to the EST-SSR primer screened in the step 3) to serve as a primer for PCR amplification, performing PCR amplification, and performing fluorescence-labeled capillary electrophoresis by using an amplification product to verify the EST-SSR primer screened in the step 3).
Further, the 6 parts of the lentil material in the step 1) is the whole plant of the lentil in the seedling stage.
Further, the data processing in step 2) includes the following steps: reads containing adapters, reads containing ploy-N, and low quality reads are deleted.
Further, the reaction system for PCR amplification in step 3): the total volume was 10. mu.l, and 2.0. mu.L (10 ng. mu.L) of DNA was contained-1) 1.0. mu.L (2.0 pmol. mu.L) of the primer set-1)、2×Premix TaqTM5.0 μ L and ddH2O 2.0μL。
Further, the reaction procedure of the PCR amplification in step 3): 5min at 94 ℃; 30s at 94 ℃, 45s at 52 ℃, 1min at 72 ℃ and 35 cycles; preserving at 72 deg.C for 10min and 4 deg.C.
Further, the reaction system for PCR amplification in step 4): a total volume of 15. mu.l containing 0.5. mu.L (10 ng. mu.L) of DNA-1) 1.5. mu.L (10 pmol. mu.L) of the primer set-1) 1.5. mu.L of 10xBuffer, 0.2. mu.L of Taq enzyme (2.5U), 1.5. mu.L of dNTP (2.5 mmol. multidot.L)-1)、0.75μL Mg2+And 10. mu.L of ddH2O。
Further, the reaction procedure of PCR amplification described in step 4): 5min at 94 ℃; 30 cycles of 94 ℃ for 30s, different annealing temperatures for 30s, and 72 ℃ for 30 s; 5min at 72 deg.C, 40min at 60 deg.C, and 4 deg.C.
Has the advantages that:
(1) the EST-SSR marker developed by the invention is large in RNA-Seq data volume and reliable. The invention assembles 217,836 trans-cript and 161,095 unigene according to the acquired lentil RNA-Seq data, and respectively totals 2.571 and 2.406 hundred million nucleotides.
(2) The EST-SSR marker developed by the invention has high success rate and abundant polymorphism. 480 primer screening is carried out on EST-SSR primers. The results showed that a total of 276 pairs of primers were satisfactory. And carrying out SSR fluorescence labeling capillary electrophoresis verification on 276 pairs of primers by utilizing 94 lentil germplasm pairs. The results showed that 125 primers were polymorphic and 43 primers were singlet, accounting for 26.04% and 8.96% of the 480 primers, respectively.
(3) The EST-SSR developed by the invention has stronger universality. The invention collects 94 lentils germplasm with different sources for EST-SSR marker detection analysis, wherein the 94 germplasm is derived from 4 large geographical areas: 82 parts of china from east asia (of which 10 parts are from xinjiang, 9 parts from inner mongolia, 7 parts from ningxia, 10 parts from the green sea, 11 parts from shanxi, 8 parts from shanxi, 9 parts from gansu, 9 parts from north of lake, 9 parts from Yunnan), 1 part from japan from east asia; 6 from west asia, (3 from turkish, 3 from syria); 4 parts from north america, (3 parts from canada and 1 part from the united states); 1 part of Morocco from North Africa. 125 pairs of EST-SSR primers can amplify various types of strips in 94 portions of lentil germplasm DNA, and show good universality.
(4) The EST-SSR primer has abundant polymorphism and is very helpful for the assisted breeding of lentil molecules.
Drawings
FIG. 1 shows the result of capillary electrophoresis of EST-SSR fluorescent primer P233 pair for part of test materials.
FIG. 2 is a graph of three-dimensional PCA analysis of 94 lentils based on 125 EST-SSR markers.
FIG. 3 is a UPGMA dendrogram based on EST-SSR markers.
Detailed Description
Materials and methods
1. Plant material
6 parts of lentils (Lens culiaris Medikus) were selected as test material, A0000008 from Shanxi, A0000094 from Qinghai, A0000170 from Shaanxi, A0000370 from Morocco, A0000669 from Canada, A0000677 from Japan, respectively. Three repeated samplings per material are made and labeled A0000008-1, A0000008-2, A0000008-3, A0000094-1, A0000094-2, A0000094-3, A0000170-1, A0000170-2, A0000170-3, A0000370-1, A0000370-2, A0000370-3, A0000669-1, A0000669-2, A0000669-3, A0000677-1, A0000677-2, and A0000677-3, while RNA-Seq is performed using Illumina HiSeq.
RNA-Seq 6 lentil germplasm and an additional 88 lentil germplasm were performed for simultaneous EST-SSR detection analysis. An additional 88 germplasm originated from 3 large geographical regions: 79 parts from china in east asia (of which 10 parts are from xinjiang, 9 parts from inner mongolia, 7 parts from ningxia, 9 parts from Qinghai, 10 parts from Shanxi, 7 parts from Shanxi, 9 parts from Gansu, 9 parts from Hubei, 9 parts from Yunnan); 6 from west asia, (3 from turkish, 3 from syria); 3 parts from north america, (2 parts from canada and 1 part from the united states).
The experimental sample is from the center of germplasm resources of crops in Shandong province, and from Jinan, China. The details are given in table 1.
Table 1.94 lentil germplasm origins.
2. Test method
(1) Lepidium melegueta Illumina sequencing and transcriptome Assembly
RNA of 6 whole plant (including roots, stems and leaves) materials of lentils at the seedling stage (4 weeks after sowing) is respectively extracted by a Trizol extraction method (Tiangen, Beijing, China), and 1.5 mu gRNA of each sample is taken as an RNA sample preparation material.
The RNA samples obtained above were subjected to NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA) to prepare a sequencing Library, and an index code was added to the attribute sequence of each sample. Briefly, mRNA was purified from total RNA using oligo-dT linked magnetic beads. Disruption with divalent cations was performed under high temperature conditions in NEBNext first strand synthesis reaction buffer (5X), first strand cDNA was synthesized using random hexamer primers and M-MuLV Reverse Transcriptase (RNase H-), followed by synthesis of second strand cDNA using DNA polymerase I and RNase H, conversion of the remaining overhanging ends to blunt ends by exonuclease/polymerase activity, adenylation of the 3' ends of the DNA fragments, and ligation of NEBNext adaptors with hairpin loop structures to prepare for hybridization. The library fragments were purified using the AMPure XP system (Beckman Coulter, Beverly, USA) to select cDNA fragments of 150-200 bp in length, co-treated with 3. mu.l of USER enzyme (NEB, USA) with adaptor-ligated cDNA fragments of appropriate size selection for 15min at 37 ℃ and thereafter inactivated for 5min at 95 ℃. Then, PCR was performed using Phusion High-Fidelity DNA polymerase, a universal PCR Primer and index (X) Primer, using the above-mentioned treated DNA fragment as a template. Finally, the PCR products were purified in AMPure XP system and library quality was assessed on Agilent Bioanalyzer 2100 system. Subsequently, clustering of index-coded samples was performed on the cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia). After cluster generation, the prepared library was sequenced on the Illumina Hiseq platform and double-ended reads (i.e., raw reads) were generated. The raw data is stored in order read archive (SRA) and is labeled as SUB5351043 in NCBI.
The raw data (raw reads) in FASTQ format obtained above is first processed by an internal Perl script. In this step, clean data is obtained by deleting reads containing adapter, reads containing ploy-N (unknown base proportion > 10%), and low quality reads (reads whose base number Q < ═ 20 accounts for 50% or more of the entire read) from the original data. Q20, Q30, GC-content and sequence repeat levels were also calculated for clean data. All downstream analyses are based on high quality cleaning data.
Transcriptome assembly was performed with min _ kmer _ cov set to 2with Trinity, and all other parameters set to default values, based on left. Data de-redundancy can be by Corset (https:// code. google. com/p/Corset-project /). Corset can perform hierarchical clustering on the transcript by using the comparison of the reads number and the expression pattern of the transcript to form clusters, reserve the longest transcript in each cluster, and perform subsequent analysis such as annotation according to the retention, namely unigene.
To obtain comprehensive gene function information, unigene function was annotated based on the following seven databases: NCBI non-redundant protein sequences (Nr, using diamond v0.8.22with e-value ═ 1e-5)；NCBI nucleotide sequences(Nt，using NCBI blast 2.2.28+with e-value＝1e-5)；Protein family(Pfam，using HMMER 3.0package and hmmscan with e-value＝0.01)；euKaryotic Ortholog Groups(KOG，using diamond v0.8.22with e-value＝1e-3)；SwissProt(using diamond v0.8.22with e-value＝1e-5)；Kyoto Encyclopedia of Genes and Genomes(KO，using KEGG Automatic Annotation Server with e-value＝1e-10)；Gene Ontology(GO，using Blast2GO v2.5 and self-writing script with e-value＝1e-6)。
(2) DNA extraction of lentil
The extraction of genomic DNA was carried out on fresh leaves of seedlings (4 weeks after sowing) of 94 parts of material (including 6 parts of RNA-Seq) using a novel plant genomic DNA extraction kit (DP320-03) of Tiangen Biochemical technology (Beijing) Inc., and laboratory treatment was carried out strictly according to the manufacturer's instructions. DNA quality and concentration were determined by NanoDrop One and the DNA was diluted to 25 ng/. mu.L and stored at-20 ℃ for use in PCR.
(3) SSR detection and primer design
The transcriptome SSR detection was performed using MISA (MIcrosAtellite identification tool, http:// pgrc. ipk-gatersleen. de/MISA. html). The parameters are set as follows: the SSR repeat unit lengths are minimum mono-10, di-6, tri-5, tetra-5, penta-5, and hexa-5. In a composite SSR, the maximum disruption size allowed between two different SSRs is 100 bp. The microsatellite loci are screened out by utilizing Primer 3.0 software (https:/Source Memi. net/Projects/Primer 3/) and designing SSR Primer pairs according to flanking conserved sequences.
(4) EST-SSR primer screening
EST-SSR primer screening adopts 4 lentil germplasms (A0000036, A0000158, A0000667 and A0000376) with large source difference, 3 individuals are selected from each germplasm, and DNA is extracted from 12 individuals in total. The primers were synthesized by Beijing catalpi-technology Ltd. Premix Taq for PCR amplification and electrophoresisTM(TaKaRa TaqTMVersion 2.0) and DL500 DNAMarker were both purchased from TaKaRa Biotechnology (great company) co., Ltd. EST-SSR optimized reaction system is 10 mu L, including 2.0 mu L (10 ng. mu.L) of DNA-1) 1.0. mu.L (2.0 pmol. mu.L) of the primer set-1)、2×Premix TaqTM5.0 μ L and ddH2O2.0. mu.L. The PCR amplification reaction was performed on a TAdcvanced 96SG gradient Gene Amplifier (Biometra, Germany). The PCR reaction program is 94 ℃ for 5 min; 30s at 94 ℃, 45s at 52 ℃, 1min at 72 ℃ and 35 cycles; preserving at 72 deg.C for 10min and 4 deg.C. Amplification products were detected on 8% native polyacrylamide gel (acrylamide Acr: N '-N' methylenebisacrylamide Bis: 29: 1,1 XTBE buffer). Each sample was spotted with 1.5. mu.L of 50bp DNAladder as a molecular weight standard, electrophoresed at 260V for 1h30 min at 0.1% AgNO3Silver staining is carried out in the solution, and color development is carried out in NaOH solution.
(5) SSR fluorescence labeling capillary electrophoresis
Adding fluorescence labeling FAM (6-methoxy-fluorescein) or HEX (5-hexachloro-fluorore) to the 5' end of each pair of primers of the screened EST-SSRscein). The fluorescent primers used in the test were synthesized by Beijing catalpi Biotechnology Ltd. Sterilized ddH for primers2O is dissolved and diluted to 10. mu. mol. mu.L-1And (5) standby. The DNA used in the experiment was extracted from 94 portions of lentil material. Other reagents were purchased from TaKaRa Biotechnology (Dalian) co., Ltd. The PCR amplification by capillary electrophoresis adopts a reaction system of 15 mu L, which comprises: 0.5. mu.L of DNA (10 ng. mu.L)-1)、2μmol·L-11.5. mu.L of the primer set (10 pmol. mu.L)-1) 1.5. mu.L of 10xBuffer, 0.2. mu.L of Taq enzyme (2.5U), 1.5. mu.L of dNTP (2.5 mmol. multidot.L)-1)、0.75μL Mg2+And 10. mu.L of ddH2And O. The PCR reaction system is 94 ℃ for 5 min; 30 cycles of 94 ℃ for 30s, different annealing temperatures for 30s, and 72 ℃ for 30 s; 5min at 72 deg.C, 40min at 60 deg.C, and 4 deg.C. The PCR product was diluted 50-fold, 9.5. mu.L of diluted internal standard (GS500LIZ: HIDI ═ 1:120) and 0.5. mu.L of the diluted PCR product were added to each well of a 96-well plate, denatured at 95 ℃ for 5min, cooled on ice for 2min, and then subjected to automated fluorescence detection on an ABI 3730XL DNA analyzer (Applied Biosystems, Foster City, USA). The capillary electrophoresis procedure is pre-electrophoresis at 15kV for 2 min; 2kV voltage sample introduction for 10 s; electrophoresis at 15kV for 20 min. Finally, molecular internal standards (Size Standard) and Parameters (Parameters) were set, and Data Collection and image analysis were performed using Data Collection and Genemapper V4.0 software.
Second, data statistics and analysis
According to the Genemapper V4.0 analysis, the values of each allele of the respective fluorescent markers were read, and those below 500 were regarded as invalid peaks and discarded. And recording the size of each allele, storing the size into an Excel table, and performing corresponding conversion according to format requirements of different software. PCA analysis was performed using NTSYS-pc V2.10e. The genetic diversity parameters of each EST-SSR marker were calculated using PowerMarker V3.25, including: major allele frequency, allele factor, gene diversity, heterozygosity, Polymorphic Information Content (PIC), and the like. And (4) carrying out UPGMA method-based phylogenetic tree construction on the reference materials by combining MEGAV6.0 analysis software. The group Structure analysis of the reference material is carried out by using Structure V2.3.4, and the parameters are set as follows: length of burn Period 10000, Number of MCMC rep after Burnin 10000, Number of subgroups k (Number of events) 1-20, and Number of cycles (Number of events) 20. The optimal population structure and number of subpopulations were determined from the Delta K (Δ K) values (web address for online analysis http:// taylor0.biology. ucla. edu/struct _ harvest /).
Three, result in
(1) Lepidium melegueta Illumina sequencing and transcriptome assembly
18 small hyacinth bean samples A0000008-1, A0000008-2, A0000008-3, A0000094-1, A0000094-2, A0000094-3, A0000170-1, A0000170-2, A0000170-3, A0000370-1, A0000370-2, A0000370-3, A0000669-1, A0000669-2, A0000669-3, A0000677-1, A0000677-2 and A0000677-3 produced 0.677, 0.713, 0.746, 0.684, 0.816, 0.888, 0.890, 0.74755 9, 0.810, 0.756, 0.690, 0.704, 0.701, 0.737, 0.884, 0.991 and 0.961 billion rews by Illumina HiSeq system sequencing, respectively. The filtered sequencing data are 0.657, 0.684, 0.731, 0.657, 0.802, 0.742, 0.873, 0.876, 0.738, 0.798, 0.744, 0.681, 0.675, 0.673, 0.710, 0.852, 0.954 and 0.927 million clean reads, respectively. These clean reads were spliced into 217,836 Transcripts and 161,095 unigene, totaling 2.571 and 2.406 million nucleotides, respectively. The average length of Transcript is 1,180bp, and the average lengths of N50 and N90 are 2,075bp and 479bp respectively; the average length of the Unigene was 1,494bp, and N50 and N90 were 2,203bp and 714bp, respectively (Table 2).
Table 2 lentil transcriptome sequencing assembly results.
(2) Polymorphism analysis of EST-SSR markers
Based on the RNA-Seq results, 26,449 pairs of EST-SSR primers were designed in total, and 480 pairs of primers were randomly selected for validation. And 4 parts of materials with larger source difference are utilized to carry out EST-SSR primer screening. The results showed that 276 out of 480 primers amplified clear bands, and the remaining primers amplified no or complex products. The primers were then verified by EST-SSR fluorescence-labeled capillary electrophoresis using a total of 94 lentil material pairs 276. The results showed that 125 pairs of primers were polymorphic (table 3), 43 pairs of primers were monomorphic, accounting for 26.04% and 8.96% of the 480 pairs of primers, respectively, and the remaining primers had poor capillary electrophoresis effects and were classified as primers with no amplification product or complex amplification product, and 125 labeled forward and reverse primers were obtained. FIG. 1 shows the result of capillary electrophoresis of a part of test materials by the fluorescent primer P233, and the reading of the allele is clear, accurate and reliable. The results in table 3 show that the major allele frequencies ranged from 0.1882 to 0.9946, with an average of 0.7143; the allelic factors range from 2 to 17, with an average value of 5.1520; gene diversity ranged from 0.0107 to 0.8836 with an average of 0.3800; heterozygosity ranging from 0 to 1, with an average value of 0.2541; PIC ranged from 0.0106 to 0.8727 with an average value of 0.3407. The results show that the 125 EST-SSR primers have abundant polymorphism and greatly help the lentil molecular assisted breeding.
TABLE 3.125 pairs of EST-SSR primers with polymorphisms
(3) Genetic diversity analysis of EST-SSR markers
PCA analysis
PCA analysis based on EST-SSR marker was performed on 94 tested lentils germplasm using NTSYS-pc V2.10e to obtain a three-dimensional space clustering map (FIG. 2). The front three-dimensional contribution rate is 98.45%. The results show that the spatial distribution of Chinese resources (black ellipses) and foreign resources (gray ellipses) is clearly separated, indicating that the relationship between the two is far and is significantly related to the geographical origin. Chinese resources are obviously gathered together and are mainly distributed on the right side of the cluster map, except 2 resources, which shows that the genetic basis of the Chinese resources is totally consistent and narrower, and is obviously different from foreign resources; and foreign resources present wide and uniformly distributed spatial characteristics, which shows that the genetic background of the foreign resources is wide and the diversity distribution of the foreign resources is more uniform than that of Chinese resources. Only 1 part of Japanese resources in foreign resources are distributed in Chinese resources, which shows that the genetic relationship between the germplasm and the Chinese resources is relatively close.
UPGMA clustering analysis
Calculating by using PowerMarker V3.25 to obtain genetic distance (Nei's, 1972), and drawing an EST-SSR mark-based UPGMA phylogenetic tree diagram (figure 3) for 94 lentil germplasm (comprising 6 RNA-Seq germplasm and 88 verification germplasm) by combining MEGAV6.0 analysis software, wherein open circles represent 6 lentil resources of RNA-Seq; filled circles represent the remaining 88 lentil resources; the circle without square box represents the Chinese lentil resource; the circles outlined by the boxes represent foreign lentil resources. The results showed that 94 lentil germplasm was divided into 2 large groups. The first group was divided into 2 subgroups, the first subgroup was only 3, all from the syria and turkey in west asia. The second subgroup is almost all chinese resources, only 1 japanese resource. The Chinese resources have two characteristics, namely, the genetic relationship among the resources of each province is very close, and the resources in each province are gathered together, such as Xinjiang, Shanxi, Yunnan, Hubei and the like, but the resources of each province are also crossed and permeated. This indicates that the genetic basis of Chinese resources is narrow overall, but the genetic relationship of resources between provinces is different. The second group was divided into 2 subgroups, mostly foreign resources. The first subgroup 3 from the united states and canada in north america, 1 from morocco in north africa, the second subgroup originating more in origin, 3 from syrian and turkey in west asia, 2 from singkiang and shanxi in china, and 1 from canada in north america. The genetic relationship between foreign resources is far, which indicates that the genetic basis is wider.
SEQUENCE LISTING
<110> lentil EST-SSR marker developed based on RNA-Seq and application
<120> germplasm resource center of agricultural institute of Shandong province
<130> 2019
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<170> PatentIn version 3.3
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<213> Artificial sequence
<400> 35
gcagccgcaa ctaaaaccaa 20
<210> 36
<211> 20
<212> DNA
<213> Artificial sequence
<400> 36
tggccgaatc tgcacaatct 20
<210> 37
<211> 22
<212> DNA
<213> Artificial sequence
<400> 37
tccaattcat gcagtggagt ga 22
<210> 38
<211> 20
<212> DNA
<213> Artificial sequence
<400> 38
aaatgcttgc taggggacgg 20
<210> 39
<211> 20
<212> DNA
<213> Artificial sequence
<400> 39
tggggacaac aacgagactt 20
<210> 40
<211> 20
<212> DNA
<213> Artificial sequence
<400> 40
cccttcctgc aaaaacaccg 20
<210> 41
<211> 21
<212> DNA
<213> Artificial sequence
<400> 41
acttgtttcg gtccaacgac t 21
<210> 42
<211> 21
<212> DNA
<213> Artificial sequence
<400> 42
ccatggttaa agctgccatg g 21
<210> 43
<211> 20
<212> DNA
<213> Artificial sequence
<400> 43
cccaatcacc aggggtaaca 20
<210> 44
<211> 25
<212> DNA
<213> Artificial sequence
<400> 44
tgttggttgt tgataagttg tgtgt 25
<210> 45
<211> 23
<212> DNA
<213> Artificial sequence
<400> 45
agtgttggtg tacgaatttg tgt 23
<210> 46
<211> 22
<212> DNA
<213> Artificial sequence
<400> 46
tcggttgaca ctagagtaga ga 22
<210> 47
<211> 22
<212> DNA
<213> Artificial sequence
<400> 47
agatttgtac catccctcac ca 22
<210> 48
<211> 20
<212> DNA
<213> Artificial sequence
<400> 48
tcaggctgtg aacaagctcc 20
<210> 49
<211> 25
<212> DNA
<213> Artificial sequence
<400> 49
tctctcttca aggaacaatg aacct 25
<210> 50
<211> 21
<212> DNA
<213> Artificial sequence
<400> 50
tggttggtta agcaagtgac g 21
<210> 51
<211> 20
<212> DNA
<213> Artificial sequence
<400> 51
cctctctcca agactcccgt 20
<210> 52
<211> 20
<212> DNA
<213> Artificial sequence
<400> 52
caacgacatc tggcaaagca 20
<210> 53
<211> 27
<212> DNA
<213> Artificial sequence
<400> 53
acacaattac aaagagctat aaactct 27
<210> 54
<211> 21
<212> DNA
<213> Artificial sequence
<400> 54
ggtggtgagc ttattggtgg a 21
<210> 55
<211> 20
<212> DNA
<213> Artificial sequence
<400> 55
aagaaggaaa gaggaggcgc 20
<210> 56
<211> 20
<212> DNA
<213> Artificial sequence
<400> 56
gcgagagaga ggcacagttt 20
<210> 57
<211> 24
<212> DNA
<213> Artificial sequence
<400> 57
gcataattcc aaaacaaagg ctgg 24
<210> 58
<211> 22
<212> DNA
<213> Artificial sequence
<400> 58
tcccgtagtt ttcttgttcc tt 22
<210> 59
<211> 20
<212> DNA
<213> Artificial sequence
<400> 59
catccactct ctctcgcgac 20
<210> 60
<211> 20
<212> DNA
<213> Artificial sequence
<400> 60
gagagtgaca cgcgagagag 20
<210> 61
<211> 20
<212> DNA
<213> Artificial sequence
<400> 61
tggaagaagc atgccaaggg 20
<210> 62
<211> 26
<212> DNA
<213> Artificial sequence
<400> 62
atttctcatt acacgttaca gtacat 26
<210> 63
<211> 20
<212> DNA
<213> Artificial sequence
<400> 63
acctcagcga ccgcatttaa 20
<210> 64
<211> 22
<212> DNA
<213> Artificial sequence
<400> 64
tgttttgaaa cctggatggg ga 22
<210> 65
<211> 20
<212> DNA
<213> Artificial sequence
<400> 65
agttttaccg tggccctgtg 20
<210> 66
<211> 20
<212> DNA
<213> Artificial sequence
<400> 66
cctctcacct cgcatgaaca 20
<210> 67
<211> 23
<212> DNA
<213> Artificial sequence
<400> 67
tgccaaagaa aacaagaaga gca 23
<210> 68
<211> 20
<212> DNA
<213> Artificial sequence
<400> 68
ttttcggcta gctccacgtt 20
<210> 69
<211> 20
<212> DNA
<213> Artificial sequence
<400> 69
gcaaacttgt tggctgccat 20
<210> 70
<211> 26
<212> DNA
<213> Artificial sequence
<400> 70
actcaagaac attacttttt ccctgt 26
<210> 71
<211> 20
<212> DNA
<213> Artificial sequence
<400> 71
cccaacccgg ttcagaaaag 20
<210> 72
<211> 20
<212> DNA
<213> Artificial sequence
<400> 72
gagcaacaca caacaccgac 20
<210> 73
<211> 20
<212> DNA
<213> Artificial sequence
<400> 73
cttcctcctc ctccattcgc 20
<210> 74
<211> 20
<212> DNA
<213> Artificial sequence
<400> 74
gtcggttctt ccacctctcc 20
<210> 75
<211> 20
<212> DNA
<213> Artificial sequence
<400> 75
gtcggtggag ggagaaaagg 20
<210> 76
<211> 20
<212> DNA
<213> Artificial sequence
<400> 76
gcgacgcaaa cagctaacaa 20
<210> 77
<211> 20
<212> DNA
<213> Artificial sequence
<400> 77
gcccaagtta gaagagggct 20
<210> 78
<211> 20
<212> DNA
<213> Artificial sequence
<400> 78
cccctcttct ttccctctcc 20
<210> 79
<211> 20
<212> DNA
<213> Artificial sequence
<400> 79
ttgatcggcg aacttcacca 20
<210> 80
<211> 21
<212> DNA
<213> Artificial sequence
<400> 80
acattctact tttgcaacgg c 21
<210> 81
<211> 20
<212> DNA
<213> Artificial sequence
<400> 81
gcggcacatc acttggacta 20
<210> 82
<211> 22
<212> DNA
<213> Artificial sequence
<400> 82
acctaataat ctccgaggga cg 22
<210> 83
<211> 20
<212> DNA
<213> Artificial sequence
<400> 83
acccaccatt ttgacgaggt 20
<210> 84
<211> 20
<212> DNA
<213> Artificial sequence
<400> 84
cgaatgaatc cgctgcacag 20
<210> 85
<211> 20
<212> DNA
<213> Artificial sequence
<400> 85
cgtacaccac tgacacgact 20
<210> 86
<211> 20
<212> DNA
<213> Artificial sequence
<400> 86
agaacttggg ccaattgggt 20
<210> 87
<211> 20
<212> DNA
<213> Artificial sequence
<400> 87
tgaggagcgt caatgttgct 20
<210> 88
<211> 20
<212> DNA
<213> Artificial sequence
<400> 88
ccgggcttca tgcgaatcta 20
<210> 89
<211> 20
<212> DNA
<213> Artificial sequence
<400> 89
agcccttttg ccttggtcta 20
<210> 90
<211> 22
<212> DNA
<213> Artificial sequence
<400> 90
cagattccaa cagtgctcaa gc 22
<210> 91
<211> 20
<212> DNA
<213> Artificial sequence
<400> 91
agctttgaca ctgctcccaa 20
<210> 92
<211> 21
<212> DNA
<213> Artificial sequence
<400> 92
actgccagta agttgacacc a 21
<210> 93
<211> 22
<212> DNA
<213> Artificial sequence
<400> 93
gtgtcagttg agtcacattg ca 22
<210> 94
<211> 20
<212> DNA
<213> Artificial sequence
<400> 94
cccaggaagt acagacgcaa 20
<210> 95
<211> 22
<212> DNA
<213> Artificial sequence
<400> 95
tgattgtgga tcacgtctgt ca 22
<210> 96
<211> 20
<212> DNA
<213> Artificial sequence
<400> 96
tcgtgctctg atgccatctt 20
<210> 97
<211> 20
<212> DNA
<213> Artificial sequence
<400> 97
gggacatgga ttccgtggtt 20
<210> 98
<211> 20
<212> DNA
<213> Artificial sequence
<400> 98
gattcgccac cacaaaaccc 20
<210> 99
<211> 20
<212> DNA
<213> Artificial sequence
<400> 99
gggcgtgagt ggtgacttta 20
<210> 100
<211> 23
<212> DNA
<213> Artificial sequence
<400> 100
tggctgctca attttgtgtt agg 23
<210> 101
<211> 20
<212> DNA
<213> Artificial sequence
<400> 101
aacacgtgca actctttgga 20
<210> 102
<211> 21
<212> DNA
<213> Artificial sequence
<400> 102
cgacgtcgtt tcgttctttc g 21
<210> 103
<211> 20
<212> DNA
<213> Artificial sequence
<400> 103
tcctactgat ccaagggcca 20
<210> 104
<211> 20
<212> DNA
<213> Artificial sequence
<400> 104
gccttcttct ccggcagatt 20
<210> 105
<211> 20
<212> DNA
<213> Artificial sequence
<400> 105
cccaactcgc gatctctctc 20
<210> 106
<211> 20
<212> DNA
<213> Artificial sequence
<400> 106
gcttggagaa cgtagacgct 20
<210> 107
<211> 20
<212> DNA
<213> Artificial sequence
<400> 107
gtttaacggg taggtgggca 20
<210> 108
<211> 20
<212> DNA
<213> Artificial sequence
<400> 108
atggctcgga gatgcaaaca 20
<210> 109
<211> 20
<212> DNA
<213> Artificial sequence
<400> 109
tcgtgcaatt gccagtacaa 20
<210> 110
<211> 24
<212> DNA
<213> Artificial sequence
<400> 110
tggttaaagg gggagaaaat tagt 24
<210> 111
<211> 20
<212> DNA
<213> Artificial sequence
<400> 111
ggtgcacgtg aaatgtgact 20
<210> 112
<211> 21
<212> DNA
<213> Artificial sequence
<400> 112
tcccatactt gtcttccagc t 21
<210> 113
<211> 20
<212> DNA
<213> Artificial sequence
<400> 113
agcgacccca atcaaatcca 20
<210> 114
<211> 20
<212> DNA
<213> Artificial sequence
<400> 114
ggtcaggagt gggagctcta 20
<210> 115
<211> 25
<212> DNA
<213> Artificial sequence
<400> 115
ttctcagtag atattctgtg tctgt 25
<210> 116
<211> 20
<212> DNA
<213> Artificial sequence
<400> 116
tttgctggat atggtgccaa 20
<210> 117
<211> 20
<212> DNA
<213> Artificial sequence
<400> 117
agtggaacct gaagctacca 20
<210> 118
<211> 20
<212> DNA
<213> Artificial sequence
<400> 118
gcacgctgtt tcttgaccaa 20
<210> 119
<211> 20
<212> DNA
<213> Artificial sequence
<400> 119
actgtcctgg gtcagttcga 20
<210> 120
<211> 20
<212> DNA
<213> Artificial sequence
<400> 120
ttttcctggt ggatccgctg 20
<210> 121
<211> 20
<212> DNA
<213> Artificial sequence
<400> 121
cttcctccac aagccacact 20
<210> 122
<211> 21
<212> DNA
<213> Artificial sequence
<400> 122
tgaggttctg tttggagttg t 21
<210> 123
<211> 20
<212> DNA
<213> Artificial sequence
<400> 123
tcgaggagta attcggggga 20
<210> 124
<211> 21
<212> DNA
<213> Artificial sequence
<400> 124
ggcagaacca agtgaaatcc g 21
<210> 125
<211> 22
<212> DNA
<213> Artificial sequence
<400> 125
tcgaagaagc ctcacatatg aa 22
<210> 126
<211> 20
<212> DNA
<213> Artificial sequence
<400> 126
ttggcagtga aacgaaagcg 20
<210> 127
<211> 20
<212> DNA
<213> Artificial sequence
<400> 127
agagatggag gagacgcaga 20
<210> 128
<211> 21
<212> DNA
<213> Artificial sequence
<400> 128
ggggaaaaac aagcaacctg g 21
<210> 129
<211> 20
<212> DNA
<213> Artificial sequence
<400> 129
aaacgggtaa caagttgcgc 20
<210> 130
<211> 22
<212> DNA
<213> Artificial sequence
<400> 130
tgtgcatcta taactggaac ga 22
<210> 131
<211> 20
<212> DNA
<213> Artificial sequence
<400> 131
aacaaccacg atcccagcaa 20
<210> 132
<211> 20
<212> DNA
<213> Artificial sequence
<400> 132
aggttccgtt tttcccagct 20
<210> 133
<211> 22
<212> DNA
<213> Artificial sequence
<400> 133
gcaaatcacc aacaacaatg ca 22
<210> 134
<211> 20
<212> DNA
<213> Artificial sequence
<400> 134
catggtgtcg gagctagtcc 20
<210> 135
<211> 20
<212> DNA
<213> Artificial sequence
<400> 135
tgagttgatg atcgtcgccg 20
<210> 136
<211> 21
<212> DNA
<213> Artificial sequence
<400> 136
tggctataca atggcgagag g 21
<210> 137
<211> 20
<212> DNA
<213> Artificial sequence
<400> 137
agggaggtga tcggagtgaa 20
<210> 138
<211> 20
<212> DNA
<213> Artificial sequence
<400> 138
ggagctaaca ccacaccaca 20
<210> 139
<211> 20
<212> DNA
<213> Artificial sequence
<400> 139
gccaaatgcc cttctttccc 20
<210> 140
<211> 20
<212> DNA
<213> Artificial sequence
<400> 140
tggtgctttt gacgatgagt 20
<210> 141
<211> 21
<212> DNA
<213> Artificial sequence
<400> 141
acgggttcat ctgagaactc c 21
<210> 142
<211> 20
<212> DNA
<213> Artificial sequence
<400> 142
cattccccta aacccgaccc 20
<210> 143
<211> 22
<212> DNA
<213> Artificial sequence
<400> 143
tggaacaatg ctatgtgtgt ga 22
<210> 144
<211> 22
<212> DNA
<213> Artificial sequence
<400> 144
tggtgagaaa actgttagtg ga 22
<210> 145
<211> 20
<212> DNA
<213> Artificial sequence
<400> 145
acccatctct tcgagctcct 20
<210> 146
<211> 20
<212> DNA
<213> Artificial sequence
<400> 146
gcatttggag tggacggaga 20
<210> 147
<211> 20
<212> DNA
<213> Artificial sequence
<400> 147
ttccattttc tcccaccgga 20
<210> 148
<211> 25
<212> DNA
<213> Artificial sequence
<400> 148
tgcttttgga gattctattg gagga 25
<210> 149
<211> 20
<212> DNA
<213> Artificial sequence
<400> 149
ggcaagaagt atggtgggct 20
<210> 150
<211> 20
<212> DNA
<213> Artificial sequence
<400> 150
aatgcccagt ctgctgagtc 20
<210> 151
<211> 20
<212> DNA
<213> Artificial sequence
<400> 151
gctaaggtcc cacacacaga 20
<210> 152
<211> 22
<212> DNA
<213> Artificial sequence
<400> 152
tgaaacactt ctcctaccac ca 22
<210> 153
<211> 20
<212> DNA
<213> Artificial sequence
<400> 153
ccgaacttct cccttcagca 20
<210> 154
<211> 20
<212> DNA
<213> Artificial sequence
<400> 154
tttgtgttcg cgtttccgtc 20
<210> 155
<211> 20
<212> DNA
<213> Artificial sequence
<400> 155
acacctgctg catttcaagg 20
<210> 156
<211> 20
<212> DNA
<213> Artificial sequence
<400> 156
accacaatcc tgtcttggca 20
<210> 157
<211> 20
<212> DNA
<213> Artificial sequence
<400> 157
aaacagagca accaccacca 20
<210> 158
<211> 20
<212> DNA
<213> Artificial sequence
<400> 158
tggtgcaaac attgtgagcg 20
<210> 159
<211> 20
<212> DNA
<213> Artificial sequence
<400> 159
aacctccact tcccctcact 20
<210> 160
<211> 20
<212> DNA
<213> Artificial sequence
<400> 160
agagttcctc cacctcctcc 20
<210> 161
<211> 20
<212> DNA
<213> Artificial sequence
<400> 161
ctaatgaggg aaggcagggg 20
<210> 162
<211> 20
<212> DNA
<213> Artificial sequence
<400> 162
acacgaacac tacagcaggg 20
<210> 163
<211> 20
<212> DNA
<213> Artificial sequence
<400> 163
cgttcctcag ctgccttctt 20
<210> 164
<211> 20
<212> DNA
<213> Artificial sequence
<400> 164
cagctgctga caccaaggta 20
<210> 165
<211> 20
<212> DNA
<213> Artificial sequence
<400> 165
ctcctccacc accggtttac 20
<210> 166
<211> 21
<212> DNA
<213> Artificial sequence
<400> 166
ccaggtgttg ggggagatat g 21
<210> 167
<211> 20
<212> DNA
<213> Artificial sequence
<400> 167
ttgaagggaa catcacgggg 20
<210> 168
<211> 20
<212> DNA
<213> Artificial sequence
<400> 168
acaaagaagg agcggcaaga 20
<210> 169
<211> 20
<212> DNA
<213> Artificial sequence
<400> 169
cctttacagc ctgccagtga 20
<210> 170
<211> 20
<212> DNA
<213> Artificial sequence
<400> 170
cacggcaacc acgtgaaaat 20
<210> 171
<211> 20
<212> DNA
<213> Artificial sequence
<400> 171
caaccctact tcccgcactt 20
<210> 172
<211> 21
<212> DNA
<213> Artificial sequence
<400> 172
ctcatgctcc gttgttgttc a 21
<210> 173
<211> 22
<212> DNA
<213> Artificial sequence
<400> 173
tggcactttt gttaagaggt gg 22
<210> 174
<211> 21
<212> DNA
<213> Artificial sequence
<400> 174
agccatacac tcagacacac a 21
<210> 175
<211> 22
<212> DNA
<213> Artificial sequence
<400> 175
tcacctttga gctcaatcac ca 22
<210> 176
<211> 20
<212> DNA
<213> Artificial sequence
<400> 176
acccacatgc ctctctctct 20
<210> 177
<211> 20
<212> DNA
<213> Artificial sequence
<400> 177
caccggattc cccaatcgaa 20
<210> 178
<211> 21
<212> DNA
<213> Artificial sequence
<400> 178
tgaggatcac caatggaagc a 21
<210> 179
<211> 20
<212> DNA
<213> Artificial sequence
<400> 179
agatgtgggg cgaatttgga 20
<210> 180
<211> 22
<212> DNA
<213> Artificial sequence
<400> 180
tctcctcttc aacatggttc gt 22
<210> 181
<211> 20
<212> DNA
<213> Artificial sequence
<400> 181
tcacttcatc gtgctgttgc 20
<210> 182
<211> 20
<212> DNA
<213> Artificial sequence
<400> 182
ggttgccaac taagcttccc 20
<210> 183
<211> 20
<212> DNA
<213> Artificial sequence
<400> 183
atgctctaca ccaactgcgg 20
<210> 184
<211> 20
<212> DNA
<213> Artificial sequence
<400> 184
gcggtggcac ttgagataca 20
<210> 185
<211> 21
<212> DNA
<213> Artificial sequence
<400> 185
tctgcttgga ttgccttacg t 21
<210> 186
<211> 22
<212> DNA
<213> Artificial sequence
<400> 186
tccatttgga cgataccatg ct 22
<210> 187
<211> 20
<212> DNA
<213> Artificial sequence
<400> 187
cacgcatcac cattgcgaaa 20
<210> 188
<211> 20
<212> DNA
<213> Artificial sequence
<400> 188
tagggacagg aaggatgggg 20
<210> 189
<211> 20
<212> DNA
<213> Artificial sequence
<400> 189
caacaggtgc caaaactccg 20
<210> 190
<211> 21
<212> DNA
<213> Artificial sequence
<400> 190
agcttcacaa acgaagaccc a 21
<210> 191
<211> 21
<212> DNA
<213> Artificial sequence
<400> 191
tggctgttac tccaagttgc a 21
<210> 192
<211> 21
<212> DNA
<213> Artificial sequence
<400> 192
agagtttaag cagctggagc a 21
<210> 193
<211> 21
<212> DNA
<213> Artificial sequence
<400> 193
ggtgtggaat tgggaattgc t 21
<210> 194
<211> 22
<212> DNA
<213> Artificial sequence
<400> 194
tccctttcct ccattttccc tc 22
<210> 195
<211> 20
<212> DNA
<213> Artificial sequence
<400> 195
tcggctaaga acttgctccc 20
<210> 196
<211> 20
<212> DNA
<213> Artificial sequence
<400> 196
ttcgtagtga ggagacccgt 20
<210> 197
<211> 20
<212> DNA
<213> Artificial sequence
<400> 197
taaaagcttt gccgcgtctg 20
<210> 198
<211> 20
<212> DNA
<213> Artificial sequence
<400> 198
caccatcttt tcccacccca 20
<210> 199
<211> 20
<212> DNA
<213> Artificial sequence
<400> 199
tccggcgtga aactctttga 20
<210> 200
<211> 20
<212> DNA
<213> Artificial sequence
<400> 200
acgtgatggc tagtcttcca 20
<210> 201
<211> 20
<212> DNA
<213> Artificial sequence
<400> 201
tgattgctgc tggacctgtt 20
<210> 202
<211> 20
<212> DNA
<213> Artificial sequence
<400> 202
ttgccgccac tacttccaat 20
<210> 203
<211> 20
<212> DNA
<213> Artificial sequence
<400> 203
gtcccctccg gtgagatttc 20
<210> 204
<211> 20
<212> DNA
<213> Artificial sequence
<400> 204
tccacatgcc acgtcactac 20
<210> 205
<211> 20
<212> DNA
<213> Artificial sequence
<400> 205
cttggattgt ttgcgtgcga 20
<210> 206
<211> 20
<212> DNA
<213> Artificial sequence
<400> 206
cagggttcac gagttaggca 20
<210> 207
<211> 20
<212> DNA
<213> Artificial sequence
<400> 207
atttcatcgc cgacaccgaa 20
<210> 208
<211> 20
<212> DNA
<213> Artificial sequence
<400> 208
tgtcggagga tctcgaacct 20
<210> 209
<211> 20
<212> DNA
<213> Artificial sequence
<400> 209
catcatcagc ggaaatcggc 20
<210> 210
<211> 20
<212> DNA
<213> Artificial sequence
<400> 210
tcagtgaggt tgcgggaaaa 20
<210> 211
<211> 19
<212> DNA
<213> Artificial sequence
<400> 211
<210> 212
<211> 20
<212> DNA
<213> Artificial sequence
<400> 212
gtggttgcaa ggaatgtggc 20
<210> 213
<211> 21
<212> DNA
<213> Artificial sequence
<400> 213
cctccggtca aagattcagg t 21
<210> 214
<211> 21
<212> DNA
<213> Artificial sequence
<400> 214
acactgctaa ctaccggatc a 21
<210> 215
<211> 22
<212> DNA
<213> Artificial sequence
<400> 215
acgtgttctt tatgtgtggt ca 22
<210> 216
<211> 20
<212> DNA
<213> Artificial sequence
<400> 216
tgctgagggt tctgggtact 20
<210> 217
<211> 20
<212> DNA
<213> Artificial sequence
<400> 217
cccagcacta aaacaagcgg 20
<210> 218
<211> 22
<212> DNA
<213> Artificial sequence
<400> 218
tgctacatgt gttttatggc ac 22
<210> 219
<211> 20
<212> DNA
<213> Artificial sequence
<400> 219
aatcgtcaac ggcggttagt 20
<210> 220
<211> 20
<212> DNA
<213> Artificial sequence
<400> 220
cccgcgcaca tgcattaaaa 20
<210> 221
<211> 20
<212> DNA
<213> Artificial sequence
<400> 221
ctccgacacg acgaggtatg 20
<210> 222
<211> 21
<212> DNA
<213> Artificial sequence
<400> 222
tccatcgctc aaactcacac a 21
<210> 223
<211> 20
<212> DNA
<213> Artificial sequence
<400> 223
atgtaggtcc tggcaaaggc 20
<210> 224
<211> 21
<212> DNA
<213> Artificial sequence
<400> 224
gcctctcatg aagctgatgg a 21
<210> 225
<211> 20
<212> DNA
<213> Artificial sequence
<400> 225
agggagaatg cgtttgaggg 20
<210> 226
<211> 20
<212> DNA
<213> Artificial sequence
<400> 226
acagcacgac caaccatgaa 20
<210> 227
<211> 20
<212> DNA
<213> Artificial sequence
<400> 227
ttgcaggttc cccatgtgaa 20
<210> 228
<211> 20
<212> DNA
<213> Artificial sequence
<400> 228
acataagcaa cacccttcga 20
<210> 229
<211> 23
<212> DNA
<213> Artificial sequence
<400> 229
tgcgtggttc atctacatat cca 23
<210> 230
<211> 20
<212> DNA
<213> Artificial sequence
<400> 230
taggaaccaa caaggctcgg 20
<210> 231
<211> 20
<212> DNA
<213> Artificial sequence
<400> 231
atgttcccca tgtgaaccgt 20
<210> 232
<211> 20
<212> DNA
<213> Artificial sequence
<400> 232
acataagcaa cacccttcga 20
<210> 233
<211> 20
<212> DNA
<213> Artificial sequence
<400> 233
caaagcgctt tgtgggagac 20
<210> 234
<211> 20
<212> DNA
<213> Artificial sequence
<400> 234
tgatcaggac gtgccatcag 20
<210> 235
<211> 20
<212> DNA
<213> Artificial sequence
<400> 235
ctgacagaaa acgcgctgag 20
<210> 236
<211> 20
<212> DNA
<213> Artificial sequence
<400> 236
ttcgtctacc cgatccggta 20
<210> 237
<211> 20
<212> DNA
<213> Artificial sequence
<400> 237
acgcacatac aagacccaca 20
<210> 238
<211> 20
<212> DNA
<213> Artificial sequence
<400> 238
agaccacgac ggaggaacta 20
<210> 239
<211> 20
<212> DNA
<213> Artificial sequence
<400> 239
tcgaacactc tgaaaggccc 20
<210> 240
<211> 20
<212> DNA
<213> Artificial sequence
<400> 240
ttgaggcacc tgactgcttt 20
<210> 241
<211> 20
<212> DNA
<213> Artificial sequence
<400> 241
gctgaagagg ctgcaattgc 20
<210> 242
<211> 20
<212> DNA
<213> Artificial sequence
<400> 242
acctagtgaa ccaccttgct 20
<210> 243
<211> 20
<212> DNA
<213> Artificial sequence
<400> 243
ttccaacgac cttccccatg 20
<210> 244
<211> 23
<212> DNA
<213> Artificial sequence
<400> 244
acatctccaa accaagcaaa aga 23
<210> 245
<211> 20
<212> DNA
<213> Artificial sequence
<400> 245
agggttctgc acttgttgtt 20
<210> 246
<211> 21
<212> DNA
<213> Artificial sequence
<400> 246
agccacaatc acaaatcagc a 21
<210> 247
<211> 20
<212> DNA
<213> Artificial sequence
<400> 247
tggtccactc ctccagagag 20
<210> 248
<211> 20
<212> DNA
<213> Artificial sequence
<400> 248
ttctgaccct caaaccggtg 20
<210> 249
<211> 20
<212> DNA
<213> Artificial sequence
<400> 249
gtctgcatga gtgtgggtga 20
<210> 250
<211> 20
<212> DNA
<213> Artificial sequence
<400> 250
attgttcgta cccccacacc 20
Claims (2)
1. A group of lentil EST-SSR markers are characterized by comprising forward primers and reverse primers corresponding to the following 125 sites:
2. use of a lentil EST-SSR marker as claimed in claim 1 in analysis of lentil germplasm genetic diversity.
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| CN201910323093.4A CN109868330B (en) | 2019-04-22 | 2019-04-22 | Lentil EST-SSR marker developed based on RNA-Seq and application |
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| WO2003085133A2 (en) * | 2002-04-08 | 2003-10-16 | Centre For Dna Fingerprinting And Diagnostics | Novel fissr-pcr primers and method of genotyping diverse genomes of plant and animal systems including rice varieties |
| CN102070704A (en) * | 2010-09-17 | 2011-05-25 | 中国疾病预防控制中心病毒病预防控制所 | Entire gene sequence of severe fever with thrombocytopenia syndrome virus (SFTSV) and application |
| CN104736721A (en) * | 2012-03-20 | 2015-06-24 | 陶氏益农公司 | Molecular markers for low palmitic acid content in sunflower (helianthus annus), and methods of using the same |
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