熏衣草属的EST-SSR 分析和跨物种转移的研究

Ayelign M. Adal; Zerihun A. Demissie; Soheil S. Mahmoud

doi:10.21769/BioProtoc.1891

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Peer-reviewed

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

Ayelign M. Adal

ZD Zerihun A. Demissie

SM Soheil S. Mahmoud email

发布: 2016年08月05日第6卷第15期 DOI: 10.21769/BioProtoc.1891 浏览次数: 9696

评审: Samik BhattacharyaYi ZhangAnonymous reviewer(s)

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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)

Materials and Reagents

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

Equipment

Desktop computer (Dell, model: precision T3610 Tower Workstation )
Mortar and pastel (VWR International, Porcelain)
Balance (Shimadzu Corporation, model: ELB300 )
Freezer (-20 °C) (Frigidaire, model: FFFH20F2QW )
Thermocycler (Thermo Fisher Scientific, ABI Applied Biosystem^TM, model: Veriti^® 96-Well Thermal Cycler )
Horizontal gel electrophoresis (Thermo Fisher Scientific, Owl^TM EasyCast^TM, model: B2 Mini )
Vertical gel electrophoresis (Bio-Rad Laboratories, model: Mini-protein Tetra system )
Microcentrifuge (Eppendorf, model: 5417C )
Microwave oven (Danby Designer)
Water bath (Thermo Fisher Scientific, Isotemp^TM Digital-Control, model: 210 )
Gel Imager (Mandel Scientific, model: Kodak Gel Logic 440 )
Sterilmatic Autoclave (Thomas Scientific, model: STME-L )
Spectrophotometer (Thermo Scientific, NanoDrop^TM, model: 2000 )

Software

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

Procedure

English

中文翻译

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如何引用

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.