Research article Expressed sequence tag based identification and expression analysis of some cold inducible elements in seabuckthorn (Hippophae rhamnoides L.) Rajesh Ghangal a , Saurabh Raghuvanshi b , Prakash C. Sharma a, * a University School of Biotechnology, Guru Gobind Singh Indraprastha University, Sector-16C, Dwarka, New Delhi 110075, India b Interdisciplinary Center for Plant Genomics, Department of Plant Molecular Biology, University of Delhi South Campus, Benito Juarez Marg, New Delhi 110021, India article info Article history: Received 21 September 2011 Accepted 14 October 2011 Available online 21 October 2011 Keywords: Seabuckthorn ESTs Gene expression Cold stress Microsatellites abstract A cDNA library was constructed from the mature leaves of seabuckthorn (Hippophae rhamnoides). Expressed Sequence Tags (ESTs) were generated by single pass sequencing of 4500 cDNA clones. We submitted 3412 ESTs to dbEST of NCBI. Clustering of these ESTs yielded 1665 unigenes comprising of 345 contigs and 1320 singletons. Out of 1665 unigenes, 1278 unigenes were annotated by similarity search while the remaining 387 unannotated unigenes were considered as organism specific. Gene Ontology (GO) analysis of the unigene dataset showed 691 unigenes related to biological processes, 727 to molecular functions and 588 to cellular component category. On the basis of similarity search and GO annotation, 43 unigenes were found responsive to biotic and abiotic stresses. To validate this observation, 13 genes that are known to be associated with cold stress tolerance from previous studies in Arabidopsis and 3 novel transcripts were examined by Real time RT-PCR to understand the change in expression pattern under cold/freeze stress. In silico study of occurrence of microsatellites in these ESTs revealed the presence of 62 Simple Sequence Repeats (SSRs), some of which are being explored to assess genetic diversity among seabuckthorn collections. This is the first report of generation of transcriptome data providing information about genes involved in managing plant abiotic stress in seabuckthorn, a plant known for its enormous medicinal and ecological value. Ó 2011 Elsevier Masson SAS. All rights reserved. 1. Introduction Seabuckthorn (Hippophae sp.) is a hardy, deciduous shrub belonging to family Elaeagnaceae. Hippophae rhamnoides is native to northwestern Europe through central Asia to the Altai moun- tains, western and northern China and in northern Himalayas in India [1]. In India, seabuckthorn is widely distributed in cold desert region covering approximately 11,500 ha in Leh district of Jammu and Kashmir state [2] with the highest Importance Value Index in Lahaul-Spiti valley of Himachal Pradesh [3]. Since ancient times, seabuckthorn has been used in traditional medicines to treat bowel irregularities, gastric ulcers, skin infection/wounds, influenza, cough, and cold as referred in classic Tibetan medicinal texts including “the RGyud Bzi” (The four books of Pharmacopoeia). Medicinal importance of seabuckthorn has been attributed to the presence of carotenoids, tocopherols, lipids, tannins, ascorbic acid, flavonoids, sterols, triterpenes, organic acids, phytosterols, poly- phenols, etc. in berries, seeds, leaves, roots, young branches and bark [4]. Total flavonoids of Hippophae (TFH) content is helpful in treating anemic-cardiopathy, angina pectoris, hyperlipidemia and prevents formation of atherosclerosis and thrombus [5]. Apart from medicinal uses, seabuckthorn is also considered to be an ecologi- cally important plant as it prevents soil erosion and conserves water and soil nutrients [6]. Moreover, seabuckthorn is known to harbor Frankia, a nitrogen fixing actinomycetes in its roots and therefore has been used in land reclamation as an initial colonizer in inundated lands [7]. Gulati et al. [8] reported the presence of phosphate-solubilizing fluorescent pseudomonas from the rhizo- sphere of seabuckthorn, which might help in the release of fixed and poorly soluble form of phosphorus. Seabuckthorn being desert inhabitable contributes to ecosystem welfare, promoting species richness as its fruits and leaves provide food to many bird and animal species [9]. Seabuckthorn is considered to be drought resistant with toler- ance to soil salinity and extreme temperatures ranging from 43  C to þ40  C [10e12]. Although seabuckthorn has been exploited for medicinal purposes at a large scale, very few studies have been conducted to elucidate the mechanisms or sources (i.e. genes/ proteins) helping the plant in combating various abiotic stresses [13e15]. Keeping this in mind, we generated EST data for this * Corresponding author. Tel.: þ91 1125302306; fax: þ91 1128035243. E-mail address: prof.pcsharma@gmail.com (P.C. Sharma). Contents lists available at SciVerse ScienceDirect Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy 0981-9428/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2011.10.005 Plant Physiology and Biochemistry 51 (2012) 123e128