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EST-SSR Analysis and Cross-species Transferability Study in Lavandula
熏衣草属的EST-SSR 分析和跨物种转移的研究   

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Abstract

The genus Lavandula comprises of several economically important lavender species that are mainly cultivated worldwide for essential oil production. Identification of lavender species and their cultivars has been a huge bottleneck in lavender industries due to lack of appropriate identification mechanisms. Recent advances in modern technologies would help to address these identification issues through development of potential molecular markers, including simple sequence repeats (SSRs). SSRs can be developed from specific species, and can be potentially used for related species, which lack the source sequences to develop species-specific SSRs. Here, we describe the guidelines and steps of identifying and analyzing SSRs from expressed sequence tag (EST) sequences of lavender species. We also detail the validation procedures of selected EST-SSRs in distinguishing source (donor) species as well as related species.

Keywords: EST-SSR(EST-SSR), Lavandula(薰衣草), SSR(SSR), Lavender(薰衣草), Essential Oil(精油)

Materials and Reagents

  1. 1.5 ml centrifuge tube
  2. 1-200 µl volume pipette tips
  3. Leaf tissue (Okanagan Lavender and Herb Farm and The Greenery Garden Center, Kelowna, BC, Canada)
  4. DNA sequences (expressed sequence tag) (EST)
  5. Liquid N2 (Praxair)
  6. Genomic DNA Mini kit (Plant) GP100 (Geneaid Biotech Ltd., catalog number: GP100)
  7. 10x PCR buffer (NEB, catalog number: M0320S )
  8. Taq polymerase (NEB, catalog number: M0320S)
  9. dNTPs (Omega Bio-Tek, catalog number: TQAC135 )
  10. MgCl2 solution (NEB, catalog number: M0320S)
  11. Bovine serum albumin (BSA) (NEB, catalog number: B9000S )
  12. Custom-synthesized primers (Thermo Fisher Scientific, InvitrogenTM)
  13. SYBR® Safe DNA gel staining (Thermo Fisher Scientific, InvitrogenTM, catalog number: S33102 )
  14. 10x DNA loading dye (Ward’s Science, catalog number: 389115 )
  15. Acrylamide 40% solution (Acrylamide: Bis-Acrylamide 19:1) (Thermo Fisher Scientific, catalog number: BP1406-1 )
  16. 50 bp DNA ladder (NEB, catalog number: N3236S )
  17. 1 kb DNA ladder (FroggaBio, catalog number: DM010-R500 )
  18. Nuclease-free water (Thermo Fisher Scientific, catalog number: BP5611 )
  19. Ammonium persulfate (APS) (Thermo Fisher Scientific, catalog number: BP179-100 )
  20. NNN'N'-tetramethyl ethylenediamine (TEMED) (Sigma-Aldrich, catalog number: T9281 )
  21. Tris Base (Thermo Fisher Scientific, catalog number: BP152-1 )
  22. Glacial acetic acid (Thermo Fisher Scientific, catalog number: 351272-212 )
  23. Na2EDTA (VWR International, catalog number: CA71007-124 )
  24. Boric acid (Thermo Fisher Scientific, catalog number: B168-1 )
  25. PCR reaction mix (see Recipes)
  26. 0.5 M EDTA (see Recipes)
  27. 50x TAE buffer (see Recipes)
  28. 1x TAE buffer (see Recipes)
  29. 5x TBE buffer (see Recipes)
  30. 0.5x TBE working solution (see Recipes)
  31. 10% Ammonium per sulfate (APS) (v/w) (see Recipes)
  32. 1 M Tris (see Recipes)
  33. 10 mM Tris buffer, pH 8.0 (see Recipes)
  34. 1% agarose gel (Thermo Fisher Scientific, catalog number: BP160-500 ) (see Recipes)
  35. 6% polyacrylamide gel (see Recipes)

Equipment

  1. Desktop computer (Dell, model: precision T3610 Tower Workstation )
  2. Mortar and pastel (VWR International, Porcelain)
  3. Balance (Shimadzu Corporation, model: ELB300 )
  4. Freezer (-20 °C) (Frigidaire, model: FFFH20F2QW )
  5. Thermocycler (Thermo Fisher Scientific, ABI Applied BiosystemTM, model: Veriti® 96-Well Thermal Cycler )
  6. Horizontal gel electrophoresis (Thermo Fisher Scientific, OwlTM EasyCastTM, model: B2 Mini )
  7. Vertical gel electrophoresis (Bio-Rad Laboratories, model: Mini-protein Tetra system )
  8. Microcentrifuge (Eppendorf, model: 5417C )
  9. Microwave oven (Danby Designer)
  10. Water bath (Thermo Fisher Scientific, IsotempTM Digital-Control, model: 210 )
  11. Gel Imager (Mandel Scientific, model: Kodak Gel Logic 440 )
  12. Sterilmatic Autoclave (Thomas Scientific, model: STME-L )
  13. Spectrophotometer (Thermo Scientific, NanoDropTM, model: 2000 )

Software

  1. BatchPrimer 3 (http://probes.pw.usda.gov/cgi-bin/batchprimer3/batchprimer3.cgi) (You et al., 2008)
  2. SSR mining server (https://www.rosaceae.org/bio/content?title=&url=/cgi-bin/gdr/gdr_ssr) (Jung et al., 2008)
  3. Perl script MIcroSAtelitte (MISA) identification tool (http://pgrc.ipk-gatersleben.de/misa/)
    Note: optional software, but not used in our study
  4. Blast2go online platform (https://www.blast2go.com/) (Conesa et al., 2005)
  5. OligoAnalyzer 3.1 (https://www.idtdna.com/calc/analyzer)

Procedure

  1. SSR identification from lavender EST libraries
    1. Expressed Sequence Tag (EST) databases containing approximately 23,000 sequences are developed for L. angustifolia and L. x intermedia plants (Lane et al., 2010; Demissie et al., 2012). The main steps of EST library development include total RNA isolation, mRNA purification and cDNA library construction, and partial sequencing of random cDNA clones at the 5' ends by Sanger sequencing.
    2. EST libraries are sorted manually based on the sources of two species, L. angustifolia and L. x intermedia, and are used for simple sequence repeats (SSRs) identification using web-based SSR analysis tools (Jung et al., 2008; You et al., 2008) (Figure 1).
    3. Sorted ESTs in FASTA format are further broken down manually into multiple files with maximum input sequences of 500 ESTs in BatchPrimer3 software (You et al., 2008). For each of input files, SSR screening parameters are set to pick up primer pairs and SSR motifs with minimum lengths of 12 bp for di-, tri- and tetra-nucleotides, 15 bp for penta-nucleotides as well as 18 bp for hexa-nucleotides. Then, additional parameter settings (minimum, optimum and maximum) are carried out for primer length (18, 21 and 25 bp), product size (120, 200 and 300 bp), GC content (40, 50 and 60%), Tm (50, 55 and 60 °C). Finally, screening of SSRs along with primer sets from input sequences are run, and the outputs are displayed as HTML format and saved as tab-delimited text or excel files for further analysis of SSR repeats.
    4. Sorted EST files are directly imported into the Genome Database for Rosaceae (GDR) SSR mining server (Jung et al., 2008), and the minimum SSR motifs screening parameters are set to 15 bp for mono- and penta-nucleotides, 12 bp for di-, tri- and tetra-nucleotides, as well as 18 bp for hexa-nucleotides. Other primer generating parameters are kept as default parameters of the software. The outputs are then generated as tab-delimited text file format, which are easily converted into excel files for further SSR repeats analysis. [Optional: automated SSR identification and potential primer designing can be made using Perl script MIcroSAtelitte (MISA) identification tool.]
    5. SSRs with potential primer flanking regions are further filtered out based on primers designed during SSR motif identification using the aforementioned tools.
    6. All identified SSRs with primer flanked regions are again characterized, targeting the length of repeats as longer category with SSR length of ≥ 18 bp for tri-nucleotides and ≥ 20 bp for di-, tetra-, penta and hexa-nucleotides, or with lower than those demarcations as shorter category.
    7. To predict the putative functional roles of the repeats under longer category, ESTs/unigenes containing SSRs with primer flanking regions are annotated against public databases using Blast2GO online platform (Conesa et al., 2005). In brief, ESTs containing repeats ≥ 18 bp are filtered and uploaded into Blast2GO, followed by blasting against the public database. Then, mapping of ESTs with Blast hit are continued prior to the final putative functional annotation. Lastly, based on the detected annotations, characterization of gene onthology (GO) annotation and putative functions of SSR containing ESTs are made.
    8. Depending on the annotated functions or random representation of identified repeats, unigenes/ESTs are selected and used to refine manually the designed primer pairs following accordingly the minimum, optimum and maximum parameters: primer length (18, 21 and 25 bp), product size (120, 200 and 300 bp), GC content (40, 50 and 60%), Tm (50, 55 and 60 °C) and others using OligoAnalyzer 3.1 online software. (Optional: primer designing can be done by any primer designing tools.)


      Figure 1. Overview of SSRs analysis from EST libraries of lavender species and validation of identified SSR on source as well as related Lavandula species. Major steps of SSR analysis from ESTs and subsequent validation include: (1) EST library development; (2) SSR motif identification using web-based mining tools; (3) SSR characterization, primer design and custom-synthesis of primers; (4) genomic DNA isolation; (5) PCR amplification of expected SSR motif-containing fragments; and (6) resolution of DNA fragments on polyacrylamide gel for scoring of amplicons as presence (1) or absence (0).

  2. Validating and assessing cross-species transferability of SSRs
    1. Selected primer sets from aforementioned procedures are custom-synthesized and resuspended in 10 mM Tris (pH 8.0) buffer and stored at -20 °C until use.
    2. Fresh young leaves (~100 mg) per sample of both source and related species (Figures 1 and 2) are ground with liquid N2 to a fine powder with mortar and pestle, and transferred into 1.5 ml centrifuge tube containing 450 µl cell lysis buffer (GP1 or GPX1) and RNase A (5 µl) from the plant genomic DNA extraction kit. Note that GPX1 is used for samples of mature leaf tissues (Figure 2B), mostly rich in phenolic compounds. The samples are then incubated at 60 °C in water bath for 10-15 min, mixing by inverting every 5 min. The remaining procedures are done according to the kit's instructional manual. Finally, each of the samples is eluted with a total of 40 µl elution buffer with two round elution (25 µl and 15 µl, respectively) after 3-5 min incubations at room temperature. Eluted genomic DNA (gDNA) is quantified using NanoDrop 2000 spectrophotometer (1 µl elution buffer for blank and 1 µl gDNA for each sample). To check the gDNA integrity, 10 µl of the gDNA (100-300 ng) mixed with 1 µl of 10x loading dye and autoclaved water is loaded into each well of an agarose gel (1%) and run on horizontal gel electrophoresis with 1x TAE buffer at 80 V for 30-45 min (Figure 3). DNA image is then visualized and taken by the Gel Imager (Figure 3). Finally, a working solution is made for each sample into 50-70 ng/µl and stored at -20 °C until use.


      Figure 2. L. x intermedia cv Grosso plants for genomic DNA extraction. A. Young plants. B. Mature plant.


      Figure 3. Agarose gel (1%) resolution of genomic DNA samples (200 ng/well) isolated from young leaf tissues of L. x intermedia (lanes 1-5) and L. angustifolia (lanes 6-12) plants. M, 1kb DNA ladder (300 ng).

    3. PCR amplification for each primer set is performed by touch-down PCR using a two-stage amplification program (Figure 1). For the first stage, the Thermocycler is set to 15 min denaturation at 95 °C followed by 11 cycles of 30 sec denaturation at 95 °C, 30 sec annealing at 64-54 or 62-52 °C (depending on the primer type) with dropping of 1 °C in every cycle for annealing and 2 min at 72 °C for extension. For the second stage, the Thermocycler is programmed to perform 24 cycles at 95 °C for 30 sec, 54 °C or 52 °C for 30 sec and 72 °C for 2 min, and completed by a final extension at 72 °C for 10 min.
    4. The PCR amplified products are separated on a 6% polyacrylamide gel (Figure 4). Samples that are well mixed with loading dye (9 µl PCR products and 1 µl 10x loading dye) are loaded into gel wells using standard 1-200 µl volume pipette tips. One well is left empty to load a DNA ladder mix containing 0.35 µl of 50 bp DNA ladder (1 µg/ µl), 1 µl of 10x loading dye and 8.65 µl of autoclaved water. Once all samples and DNA ladder are carefully loaded, the gel is run in vertical gel electrophoresis using 0.5x TBE buffer at 160 V for approximately 45 min.
    5. The gel is then carefully transferred into a plastic container with 50 ml 0.5x TBE buffer supplemented with 1 µl SYBR safe and rocked for 30 min. After a quick rinse of the stained gel with fresh 0.5x TBE buffer, a gel image is taken using the Gel Imager (Figure 4).
    6. SSR primers from either L. angustifolia or L. x intermedia ESTs are validated for their polymorphism within the donor species (same as source species) as well as cross-species transferability to other related species. (Optional: A single amplicon of every targeted repeat can further be gel purified and sequenced to verify the authenticity of the designed primer sets that amplify the desire fragments.)
    7. Because of the complex polyploidy nature of Lavandula genome, we assess the polymorphism and cross-species amplification based on presence or absence of amplified SSR fragments. If the target fragments with expected sizes are detected, the data is recorded as “presence” (1). However, if the target fragments do not meet the expected size or there is no amplification, it is recorded as “absence” (0) for that particular fragment. As a demonstration, strong LAF15 amplicons across the given samples are assessed (Figure 4). Five fragments (~145, 155, 185, 200, 250 bp) from the two species are detected and scored. The scoring is made for each fragment across 15 samples as "1" for detected fragments and "0" for no or faint amplifications. For "~145 bp" and "~155 bp", the patterns of the scoring from sample-1 to sample-15 are "100100001110100" and "111111111111111" respectively. The remaining three fragments (~185, 200, 250 bp) are scored as "1000000000100100", "000100001100000" and "101000000000000", respectively. These scored data can be managed in excel sheet in line with the formats of the subsequent analysis software. A similar scoring pattern is also used for samples of related species. (Optional: Fluorescent dye-labeled primers can be custom-synthesized and used for automated capillary electrophoresis detection with the help of fluorescent dye tagged to the fragments during amplification with fluorescent dye-labeled primers. This option is also useful to detect the exact numbers and sizes of amplicons per a primer set.)


      Figure 4. Polyacrylamide gel (6%) resolution of EST-SSR (LAF15) amplicons from (A) samples of L. angustifolia (lanes 1-9) and L. x intermedia (lanes 10-15) plants, and from (B) samples of related species, including L. angustifolia (lane 1) and L. x intermedia (lane 3). M, 50 bp DNA ladder; lane 2, L. latifolia; lane 4, L. buchii; lane 5. L. dentata; lane 6. L. lusitanica; lane 7, L. x ginginsii; lane 8, L. stoechas.

Notes

Mature leaf tissue may contain large amounts of phenolic compounds that can co-purify with genomic DNA, and interfere with the subsequent PCR amplification reactions. Thus, extra caution must be taken on selection of tissue for DNA extraction. Young and fresh leaf tissues (Figure 2A) often give better DNA than mature and dry tissues.

Recipes

  1. PCR reaction mix, 25 µl
    0.3 µM forward primer
    0.3 µM reverse primer
    70 ng genomic DNA template
    2.5 µl 10x PCR buffer
    1.5 mM MgCl2
    0.5 µg BSA
    250 µM dNTPs
    1.25 U Taq polymerase
    Nuclease-free water
  2. 0.5 M EDTA (pH 8.0)
    Add 93.05 g of Na2EDTA in 400 ml of RO water
    Stir thoroughly and adjust pH 8.0 with pellet NaOH (the solution becomes transparent when the pH reaches to 8.0)
    Bring volume to 0.5 L with RO water
    Autoclave and store at RT
  3. 50x TAE buffer, 0.5 L
    121 g Tris base (MW = 121.1 g/mol)
    28.5 ml glacial acetic acid
    50 ml 0.5 M EDTA (pH 8.0)
    1. Dissolve 121 g Tris in 300 ml sterile reverse osmosis (RO) water with gently stirring
    2. Add EDTA and glacial acetic acid
    3. Bring the final volume to 0.5 L with sterile RO water
    4. Labeled properly and autoclave at 121 °C for 30 min
    5. Store at RT
  4. 1x TAE buffer, 4 L
    1. Measure 80 ml 50x TAE
    2. Bring the final volume to 4 L with RO water
    3. Store at RT
  5. 5x TBE buffer, 1 L, pH 8.3
    54 g Tris base (MW = 121.1 g/mol)
    27.5 g boric acid (MW = 61.83 g/mol)
    20 ml of 0.5 M EDTA (pH 8.0)
    1. Dissolve Tris base in 600 ml RO water with gentle stirring
    2. Add boric acid
    3. Add EDTA solution
    4. Adjust pH to 8.3 and bring up the final volume to 1 L
    5. Autoclave at 121 °C for 30 min and store at RT
  6. 0.5x TBE working solution, 2 L
    1. Measure 200 ml 5x TBE
    2. Bring the final volume to 2 L with RO water
    3. Store at RT
  7. 10% Ammonium per sulfate (APS) (v/w)
    Dissolve 1 g APS (MW = 228.2 g/mol) in 10 ml sterile RO water
    Store in 4 °C (prepared fresh after 30 days)
  8. 1 M Tris (pH 8.0), 0.5 L
    1. Dissolve 60.55 g Tris base in 400 ml
    2. Add 20 ml concentrated HCl
    3. Bring the final volume to 0.5 L with RO water
    Autoclave at 121 °C for 30 min and store at RT
  9. 10 mM Tris (pH 8.0) buffer, 100 ml
    Take 2 ml 1 M Tris (pH 8.0)
    Bring the final volume to 100 ml with sterilized RO water
    Store at 4 °C
  10. 1% agarose gel
    Add 0.5 g of Agarose into 250 ml Erlenmeyer flask containing 50 ml 1x TAE buffer
    Boil in microwave with frequent monitoring until it gets clear
    Cool down to ~50 °C
    Add 1 µl SYBR safe and mix gently
    Pour into the gel cast to solidify
  11. 6% polyacrylamid gel, 1.5 mm gel (10 ml)
    Add sequentially:
    1. 5.73 RO water
    2. 1.67 ml Acrylamide 40% solution [Acrylamide and Bis-Acrylamide (19:1)]
    3. 2.4 ml 5x TBE buffer
    4. 200 µl 10% APS
    5. 10 µl TEMED
    Mix thoroughly and pour into gel cast using pasture pipette (Avoid bubbles!)
    Wait till completely polymerized

Acknowledgments

This protocol was adapted from the previously published study, Adal et al. (2015). This work was supported through grants and/or in-kind contributions to SSM by UBC, Genome British Columbia, Natural Sciences and Engineering Research Council of Canada, Agriculture and Agri-Food Canada and the BC Ministry of Agriculture (through programs delivered by the Investment Agriculture Foundation of BC).

References

  1. Adal, A. M., Demissie, Z. A. and Mahmoud, S. S. (2015). Identification, validation and cross-species transferability of novel Lavandula EST-SSRs. Planta 241(4): 987-1004.
  2. Conesa, A., Gotz, S., Garcia-Gomez, J. M., Terol, J., Talon, M. and Robles, M. (2005). Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21(18): 3674-3676.
  3. Demissie, Z. A., Cella, M. A., Sarker, L. S., Thompson, T. J., Rheault, M. R. and Mahmoud, S. S. (2012). Cloning, functional characterization and genomic organization of 1,8-cineole synthases from Lavandula. Plant Mol Biol 79(4-5): 393-411.
  4. Jung, S., Staton, M., Lee, T., Blenda, A., Svancara, R., Abbott, A. and Main, D. (2008). GDR (Genome Database for Rosaceae): integrated web-database for Rosaceae genomics and genetics data. Nucleic Acids Res 36(Database issue): D1034-1040.
  5. Lane, A., Boecklemann, A., Woronuk, G. N., Sarker, L. and Mahmoud, S. S. (2010). A genomics resource for investigating regulation of essential oil production in Lavandula angustifolia. Planta 231(4): 835-845.
  6. You, F. M., Huo, N., Gu, Y. Q., Luo, M. C., Ma, Y., Hane, D., Lazo, G. R., Dvorak, J. and Anderson, O. D. (2008). BatchPrimer3: a high throughput web application for PCR and sequencing primer design. BMC Bioinformatics 9: 253.

简介

拉丁族 包括几种经济上重要的薰衣草品种,主要在全世界种植精油生产。 由于缺乏适当的鉴定机制,薰衣草物种及其栽培品种的鉴定一直是薰衣草工业的巨大瓶颈。 现代技术的最新进展将有助于通过开发潜在的分子标记物(包括简单序列重复(SSR))来解决这些鉴定问题。 SSR可以从特定物种开发,并且可以潜在地用于相关物种,其缺乏源序列以开发物种特异性SSR。 在这里,我们描述从薰衣草物种的表达序列标签(EST)序列鉴定和分析SSR的指南和步骤。 我们还详细选择EST-SSR在区分源(供体)物种以及相关物种的验证程序。

关键字:EST-SSR, 薰衣草, SSR, 薰衣草, 精油

材料和试剂

  1. 1.5ml离心管
  2. 1-200μl体积移液器吸头
  3. 叶组织(Okanagan薰衣草和草本农场和绿地花园中心,基隆拿,加拿大BC)
  4. DNA序列(表达序列标签)(EST)
  5. 液体N <2>(Praxair)
  6. 基因组DNA迷你试剂盒(Plant)GP100(Geneaid Biotech Ltd.,目录号:GP100)
  7. 10×PCR缓冲液(NEB,目录号:M0320S)
  8. Taq聚合酶(NEB,目录号:M0320S)
  9. dNTP(Omega Bio-Tek,目录号:TQAC135)
  10. MgCl 2溶液(NEB,目录号:M0320S)
  11. 牛血清白蛋白(BSA)(NEB,目录号:B9000S)
  12. 定制合成的引物(Thermo Fisher Scientific,Invitrogen TM
  13. SYBR 安全DNA凝胶染色(Thermo Fisher Scientific,Invitrogen TM ,目录号:S33102)
  14. 10×DNA加载染料(Ward's Science,目录号:389115)
  15. 丙烯酰胺40%溶液(丙烯酰胺:双丙烯酰胺19:1)(Thermo Fisher Scientific,目录号:BP1406-1)
  16. 50bp DNA梯(NEB,目录号:N3236S)
  17. 1kb DNA梯(FroggaBio,目录号:DM010-R500)
  18. 无核酸酶水(Thermo Fisher Scientific,目录号:BP5611)
  19. 过硫酸铵(APS)(Thermo Fisher Scientific,目录号:BP179-100)
  20. NNN'N'-四甲基乙二胺(TEMED)(Sigma-Aldrich,目录号:T9281)
  21. Tris Base(Thermo Fisher Scientific,目录号:BP152-1)
  22. 冰乙酸(Thermo Fisher Scientific,目录号:351272-212)
  23. Na 2 EDTA(VWR International,目录号:CA71007-124)
  24. 硼酸(Thermo Fisher Scientific,目录号:B168-1)
  25. PCR反应混合物(参见配方)
  26. 0.5 M EDTA(见配方)
  27. 50x TAE缓冲区(请参阅配方)
  28. 1x TAE缓冲区(请参阅配方)
  29. 5x TBE缓冲区(参见配方)
  30. 0.5x TBE工作解决方案(参见配方)
  31. 10%过硫酸铵(APS)(v/w)(参见配方)
  32. 1 M Tris(见配方)
  33. 10mM Tris缓冲液,pH8.0(见Recipes)
  34. 1%琼脂糖凝胶(Thermo Fisher Scientific,目录号:BP160-500)(参见Recipes)
  35. 6%聚丙烯酰胺凝胶(见配方)

设备

  1. 台式机(Dell,型号:Precision T3610塔式工作站)
  2. 砂浆和粉彩(VWR国际,瓷器)
  3. Balance(Shimadzu Corporation,型号:ELB300)
  4. 冷冻器(-20℃)(Frigidaire,型号:FFFH20F2QW)
  5. 热循环仪(Thermo Fisher Scientific,ABI Applied Biosystem公司,型号:Veriti 96孔热循环仪)
  6. 水平凝胶电泳(Thermo Fisher Scientific,Owl TM EasyCast TM ,型号:B2 Mini)
  7. 垂直凝胶电泳(Bio-Rad Laboratories,型号:Mini-protein Tetra系统)
  8. 微量离心机(Eppendorf,型号:5417C)
  9. 微波炉(Danby Designer)
  10. 水浴(Thermo Fisher Scientific,Isotemp TM数字控制,型号:210)
  11. Gel Imager(Mandel Scientific,型号:Kodak Gel Logic 440)
  12. Sterilmatic高压灭菌器(Thomas Scientific,型号:STME-L)
  13. 分光光度计(Thermo Scientific,NanoDrop ,型号:2000)

软件

  1. BatchPrimer 3( http://probes.pw .usda.gov/cgi-bin/batchprimer3/batchprimer3.cgi )(您等。,2008)
  2. SSR挖掘服务器( https ://www.rosaceae.org/bio/content?title =& url =/cgi-bin/gdr/gdr_ssr )(Jung等人,2008) >
  3. Perl脚本MIcroSAtelitte(MISA)识别工具( http://pgrc.ipk- gatersleben.de/misa/
    注意:可选软件,但我们的研究中未使用
  4. Blast2go在线平台( https://www.blast2go.com/)(Conesa et al 。,2005)
  5. OligoAnalyzer 3.1( https://www.idtdna.com/calc/analyzer

程序

  1. 薰衣草EST文库的SSR鉴定
    1. 为 L开发了包含大约23,000个序列的表达序列标签(EST)数据库。 angustifolia 和 L。 x intermedia 植物(Lane ,2010; Demissie等人,2012)。 EST文库开发的主要步骤包括总RNA分离,mRNA纯化和cDNA文库构建,以及通过Sanger测序在5'端随机cDNA克隆的部分测序。
    2. 基于两种物种的来源EST手工分选EST文库。 angustifolia 和 L。 x intermedia ,并且用于使用基于网络的SSR分析工具的简单序列重复(SSR)鉴定(Jung等人,2008; You 。,2008)(图1)。
    3. 在FASTA格式中排序的ESTs在BatchPrimer3软件中进一步手动分解成多个文件,最大输入序列为500个EST(You 等。,2008)。对于每个输入文件,设置SSR筛选参数以挑选具有对于二,三和四核苷酸为12bp,对于五核苷酸为15bp以及对于六核苷酸为18b的最小长度的引物对和SSR基序,核苷酸。然后,对引物长度(18,21和25bp),产物大小(120,200和300bp),GC含量(40,50和60%),Tm (50,55和60℃)。最后,运行SSR与来自输入序列的引物组的筛选,并且输出以HTML格式显示并保存为制表符分隔的文本或excel文件,用于进一步分析SSR重复。
    4. 将排序的EST文件直接导入蔷薇科(GDR)SSR挖掘服务器的基因组数据库(Jung等人,2008),并设置最小SSR基序筛选参数对于单核苷酸和五核苷酸为15bp,对于二,三和四核苷酸为12bp,以及对于六核苷酸为18bp。其他引物生成参数保留为软件的默认参数。然后将输出生成为制表符分隔的文本文件格式,这些文件格式很容易转换为excel文件以进行进一步的SSR重复分析。 [可选:可以使用Perl脚本MIcroSAtelitte(MISA)识别工具进行自动SSR识别和潜在引物设计。]
    5. 使用上述工具基于在SSR基序识别期间设计的引物进一步过滤出具有潜在引物侧翼区的SSR。
    6. 所有鉴定的具有引物侧翼区域的SSR再次表征,将重复序列的长度作为更长类型,SSR长度对于三核苷酸≥18bp,对于二,四,五和六核苷酸≥20bp,比那些划分为较短类别。
    7. 为了预测更长类别下的重复的推定的功能作用,使用Blast2GO在线平台(Conesa等人,2005)对包含具有引物侧翼区的SSR的EST/unigenes进行公开数据库注释。简而言之,包含≥18bp重复序列的ESTs被过滤并上传到Blast2GO中,然后针对公共数据库进行爆破。然后,在最后推定的功能注释之前继续具有Blast命中的EST的映射。最后,基于检测到的注释,基因神经(GO)注释的特征和SSR含有EST的推定功能。
    8. 根据所标注的功能或鉴定的重复的随机表示,选择unigenes/EST,并用于手动精制设计的引物对,随后相应地最小,最佳和最大参数:引物长度(18,21和25bp),产物大小120,200和300bp),GC含量(40,50和60%),Tm(50,55和60℃)和其它使用OligoAnalyzer 3.1在线软件。 (可选:底漆设计可以通过任何底漆设计工具完成。)


      图1.来自薰衣草物种的EST文库的SSR分析概述以及来源和相关的薰衣草属物种上鉴定的SSR的验证。来自ESTs和随后的SSR分析的主要步骤验证包括:(1)EST文库开发; (2)使用基于网络的挖掘工具的SSR基序识别; (3)SSR表征,引物设计和引物的定制合成; (4)基因组DNA分离; (5)预期的含有SSR基序的片段的PCR扩增;和(6)解析聚丙烯酰胺凝胶上的DNA片段,用于将扩增子打分为存在(1)或不存在(0)。
  2. 验证和评估SSRs的物种间转移性
    1. 从上述程序中选择的引物组是定制合成的并且重悬于10mM Tris(pH 8.0)缓冲液中并储存在-20℃直至使用。
    2. 将来源和相关物种(图1和2)的每个样品的新鲜嫩叶(?100mg)用液体N 2用研钵和杵研磨成细粉,并转移到1.5ml离心机管含有来自植物基因组DNA提取试剂盒的450μl细胞裂解缓冲液(GP1或GPX1)和RNA酶A(5μl)。注意GPX1用于成熟叶组织样品(图2B),主要富含酚类化合物。然后将样品在60℃水浴中孵育10-15分钟,每5分钟反转混合。其余程序根据试剂盒的说明手册进行。最后,在室温下3-5分钟温育后,用总共40μl洗脱缓冲液洗脱每个样品,两次圆形洗脱(分别为25μl和15μl)。使用NanoDrop 2000分光光度计(每个样品1μl洗脱缓冲液作空白和1μlgDNA)定量洗脱的基因组DNA(gDNA)。为了检查gDNA完整性,将10μl与1μl10x负载染料和高压灭菌水混合的gDNA(100-300ng)加载到琼脂糖凝胶(1%)的每个孔中,并用1x TAE的水平凝胶电泳缓冲液在80V下30-45分钟(图3)。 DNA图像然后可视化并由凝胶成像仪(图3)。最后,将每个样品的工作溶液制备成50-70ng /μl,并在-20℃下储存,直到使用

      图2. x intermedia cv Grosso植物进行基因组DNA提取。 A.幼苗。 B.成熟植物

      图3.从L的幼叶组织分离的基因组DNA样品(200ng /孔)的琼脂糖凝胶(1%)分离。 x intermedia (lane 1-5)和 L。 angustifolia (泳道6-12)植物。 M,1kb DNA梯(300ng)。

    3. 使用两阶段扩增程序通过接触式PCR(touch-down PCR)进行每个引物组的PCR扩增(图1)。对于第一阶段,将热循环仪设置为在95℃变性15分钟,随后是11个循环:在95℃变性30秒,在64-54或62-52℃退火30秒(取决于引物类型)在每个退火循环中降低1℃,在72℃延伸2分钟。对于第二阶段,将热循环仪编程为在95℃30秒,54℃或52℃30秒和72℃2分钟进行24个循环,并且通过在72℃最终延伸完成10分钟。
    4. PCR扩增产物在6%聚丙烯酰胺凝胶上分离(图4)。使用标准的1-200μl体积移液管吸头将与加载染料(9μlPCR产物和1μl10x加样染料)充分混合的样品加载到凝胶孔中。一个孔留空以装载含有0.35μl50bp DNA梯(1μg/μl),1μl10×装载染料和8.65μl高压灭菌水的DNA梯状混合物。一旦所有样品和DNA梯度被小心加载,凝胶在使用0.5x TBE缓冲液在160V下进行垂直凝胶电泳约45分钟。
    5. 然后将凝胶小心地转移到具有50ml 0.5×TBE缓冲液的塑料容器中,所述缓冲液补充有1μlSYBR safe并摇动30分钟。用新鲜的0.5×TBE缓冲液快速冲洗染色的凝胶后,使用凝胶成像仪拍摄凝胶图像(图4)。
    6. 来自L的SSR引物。 angustifolia 或 L。 x中间体EST是在供体物种(与源物种相同)以及跨物种可转移性与其他相关物种之间的多态性验证的。 (任选:每个靶向重复序列的单个扩增子可进一步被凝胶纯化和测序,以验证扩增期望片段的设计引物组的真实性。)
    7. 由于Lavandula基因组的复杂的多倍体性质,我们基于存在或不存在扩增的SSR片段来评估多态性和跨物种扩增。如果检测到具有期望大小的目标片段,则将数据记录为"存在"(1)。然而,如果靶片段不满足预期大小或没有扩增,则将其记录为该特定片段的"不存在"(0)。作为演示,评估跨越给定样品的强LAF15扩增子(图4)。检测来自两个物种的五个片段(?145,155,185,200,250bp)并记分。对15个样品中的每个片段进行评分,对于检测的片段为"1",对于无或微弱的扩增为"0"。对于"?145bp"和"?155bp",从样品-1到样品-15的记分的模式分别是"100100001110100"和"111111111111111"。剩余的三个片段(?185,200,250bp)分别记为"1000000000100100","000100001100000"和"101000000000000"。这些评分数据可以根据后续分析软件的格式在Excel表中管理。类似的评分模式也用于相关物种的样品。 (任选:荧光染料标记的引物可以定制合成并用于自动毛细管电泳检测,在荧光染料标记的引物扩增过程中用荧光染料标记片段,该选项也可用于检测每个引物组的扩增子的确切数目和大小。)


      图4.来自(A)L的样品的EST-SSR(LAF15)扩增子的聚丙烯酰胺凝胶(6%)分辨率。 angustifolia (1-9道)和。 (泳道10-15)植物和来自(B)相关物种的样品,包括L。 angustifolia (泳道1)和。 ×intermedia (泳道3)。 M,50bp DNA梯;泳道2,L。 latifolia ;泳道4,L。 buchii ;第5道。 dentata ;第6道。 lusitanica ;泳道7,L。 x ginginsii ;泳道8,L。 stoechas 。

笔记

成熟的叶组织可以含有大量的酚类化合物,其可以与基因组DNA共纯化,并干扰随后的PCR扩增反应。因此,必须特别注意选择用于DNA提取的组织。年轻和新鲜的叶组织(图2A)通常给出比成熟和干燥组织更好的DNA。

食谱

  1. PCR反应混合物,25μl
    0.3μM正向引物
    0.3μM反向引物
    70 ng基因组DNA模板
    2.5μl10x PCR缓冲液
    1.5mM MgCl 2·h/v 0.5μgBSA
    250μMdNTPs
    1.25 U Taq聚合酶
    无核酸酶水溶液
  2. 0.5 M EDTA(pH 8.0)
    在400ml RO水中加入93.05g Na 2 EDTA 充分搅拌并用沉淀NaOH调节pH8.0(当pH达到8.0时溶液变得透明) 用RO水将体积调至0.5 L / 高压灭菌并在RT储存
  3. 50x TAE缓冲液,0.5μL
    121克Tris碱(MW = 121.1克/摩尔) 28.5ml冰醋酸
    50ml 0.5M EDTA(pH8.0)
    1. 轻轻搅拌下将121g Tris溶于300ml无菌反渗透(RO)水中
    2. 加入EDTA和冰醋酸
    3. 用无菌RO水将终体积调至0.5 L
    4. 正确标记并在121℃高压灭菌30分钟
    5. 在RT存储
  4. 1x TAE缓冲液,4μL
    1. 测量80 ml 50x TAE
    2. 用RO水
      使最终体积为4 L
    3. 在RT存储
  5. 5x TBE缓冲液,1L,pH 8.3 54克Tris碱(MW = 121.1克/摩尔) 27.5g硼酸(MW = 61.83g/mol) 20ml 0.5M EDTA(pH8.0)
    1. 在温和搅拌下将Tris碱溶解在600ml RO水中
    2. 加入硼酸
    3. 加入EDTA溶液
    4. 将pH调节至8.3,并将最终体积调至1 L
    5. 在121℃下高压灭菌30分钟并在室温下贮存
  6. 0.5x TBE工作溶液,2 L
    1. 测量200 ml 5x TBE
    2. 用RO水
      使最终体积为2 L
    3. 在RT存储
  7. 10%过硫酸铵(APS)(v/w)
    将1g APS(MW = 228.2g/mol)溶解在10ml无菌RO水中
    储存于4°C(30天后新鲜制备)
  8. 1M Tris(pH 8.0),0.5L
    1. 将60.55g Tris碱溶解在400ml
    2. 加入20ml浓HCl
    3. 用RO水使最终体积为0.5 L
    在121℃下高压灭菌30分钟并在室温下贮存
  9. 10mM Tris(pH8.0)缓冲液,100ml
    取2 ml 1M Tris(pH 8.0)
    用灭菌RO水将最终体积调至100 ml 存储在4°C
  10. 1%琼脂糖凝胶 将0.5g琼脂糖加入含有50ml 1x TAE缓冲液的250ml锥形瓶中 在微波炉煮沸,频繁监测,直到它清除
    冷却至?50°C
    加入1μlSYBR安全,轻轻混匀
    倒入凝胶中凝固
  11. 6%聚丙烯酰胺凝胶,1.5mm凝胶(10ml) 顺序添加:
    1. 5.73 RO水
    2. 1.67ml丙烯酰胺40%溶液[丙烯酰胺和双丙烯酰胺(19:1)]
    3. 2.4 ml 5x TBE缓冲液
    4. 200μl10%APS
    5. 10μlTEMED
    充分混合并用牧草吸管注入凝胶(避免气泡!)
    等待完全聚合

致谢

此协议改编自以前发表的研究,Adal等人。 (2015)。这项工作是通过UBC,不列颠哥伦比亚省基因组,加拿大自然科学和工程研究理事会,加拿大农业和农业食品部和农业部的补助和/或对SSM的实物捐助(通过投资提供的计划BC省农业基金会)。

参考文献

  1. Adal,AM,Demissie,ZA and Mahmoud,SS(2015)。  新颖的Lavandula EST-SSR的鉴定,验证和种间转移性。 Planta 241(4):987-1004。
  2. Conesa,A.,Gotz,S.,Garcia-Gomez,JM,Terol,J.,Talon,M.and Robles,M.(2005)。  Blast2GO:用于在功能基因组学研究中进行注释,可视化和分析的通用工具。 生物信息学 21(18):3674-3676。
  3. Demissie,ZA,Cella,MA,Sarker,LS,Thompson,TJ,Rheault,MR和Mahmoud,SS(2012)。  来自Lavandula的1,8-桉树脑合成酶的克隆,功能表征和基因组组织/em> 79(4-5):393-411。
  4. Jung,S.,Staton,M.,Lee,T.,Blenda,A.,Svancara,R.,Abbott,A.and Main,D.(2008)。  GDR(蔷薇科基因组数据库):蔷薇科基因组学和遗传学数据的综合网络数据库。 em> Nucleic Acids Res 36(数据库问题):D1034-1040
  5. Lane,A.,Boecklemann,A.,Woronuk,GN,Sarker,L.and Mahmoud,SS(2010)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm。 nih.gov/pubmed/20043174"target ="_ blank">用于调查Lavandula angustifolia中精油生产调节的基因组资源 Planta 231 ):835-845。
  6. 你,FM,Huo,N.,Gu,YQ,Luo,MC,Ma,Y.,Hane,D.,Lazo,GR,Dvorak,J.and Anderson,OD(2008)。< a class = ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/18510760"target ="_ blank"> BatchPrimer3:用于PCR和测序引物设计的高通量网络应用程序。 em> BMC Bioinformatics 9:253.
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Adal, A. M., Demissie, Z. A. and Mahmoud, S. S. (2016). EST-SSR Analysis and Cross-species Transferability Study in Lavandula. Bio-protocol 6(15): e1891. DOI: 10.21769/BioProtoc.1891.
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