Scientia Horticulturae Parallel Consideration of Ssrs And

Scientia Horticulturae 198 (2016) 462–472 Contents lists available at ScienceDirect Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti Parallel consideration of SSRs and differentially expressed genes under abiotic stress for targeted development of functional markers in almond and related Prunus species a,1 a,1 a,1 a,1 Arghavan Alisoltani , Shekoufeh Ebrahimi , Sahar Azarian , Mahsa Hematyar , a,b,∗ c d e Behrouz Shiran , Hassan Jahanbazi , Hossein Fallahi , Sadegh Mousavi-Fard , a Fariba Rafiei a Department of Plant Breeding and Biotechnology, Faculty of Agriculture, Shahrekord University, Shahrekord, P.O. Box 115, Iran b Institute of Biotechnology, Shahrekord University, Shahrekord, P.O. Box 115, Iran c Agriculture and Natural Resources Research Center of Chaharmahal and Bakhtiari Province, Iran d Department of Biology, School of Sciences, Razi University, Bagh-e-Abrisham Kermanshah, Iran e Department of Horticultural Science, Faculty of Agriculture, Lorestan University, Khorramabad, P.O. Box 465, Iran a r t i c l e i n f o a b s t r a c t Article history: RNA-Seq approach is widely used to study plant transcriptome responses to different environmental Received 14 June 2015 stresses. RNA-Seq datasets have also become valuable resources to develop SSR markers and other types Received in revised form 8 October 2015 of markers in plant species. However, there are challenges such as the validation of SSR polymorphisms, Accepted 9 October 2015 and translation of these information into a functional approach for plant breeding programs. In our recent Available online 24 November 2015 work, the first de novo transcriptome assembly of almond have been reported in response to freezing stress, and thousands of differential expression (DE) genes have been identified. Here, for the first time, Keywords: we have suggested a parallel consideration of genes with DE under frost stress and SSR markers to find Almond Calmodulin functional markers in almond (Prunus dulcis Mill.) and other related Prunus species. The term “RNA-Seq SSR” was used in the current study, replacing the previous term “EST-SSR” (expressed sequence tagged), Differential expression Frost stress for the distinction between traditional EST sequencing and the new RNA-Seq methods. Eleven RNA-Seq Genetic diversity SSR markers were identified as polymorphic markers. Some of SSR loci were found on genes which are RNA-Seq SSR responsive in cold and other abiotic stresses, including calmodulin, trihelix transcription factor GT-1-like and delta-(8)-fatty-acid desaturase. Furthermore, these markers revealed high polymorphism in popu- lation of Prunus arabica, Prunus scoparia and Prunus haussknechtii. Our overall results suggest potential application of DE genes carrying SSR sequences as functional markers. The developed workflow and the new findings presented here are likely to open new opportunity for future genetic diversity, association studies and breeding projects of almond and other plants grown under environmental stresses. This workflow can also be applied to targeted validation and development of SNP and/or indel markers. © 2015 Elsevier B.V. All rights reserved. 1. Introduction (Prunus armeniaca), cherry (Prunus avium) and almond (Prunus dulcis Mill.). Spring frost is regarded as one of the main stress The genus Prunus belongs to Rosaceae family and contains factors, leading to significant decrease in crop productivity of several economically important species such as peach (Prunus per- almond and other fruit trees in Prunus genus (Mousavi et al., sica), Plum (Prunus cerasus), Chinese plum (Prunus mume), apricot 2014a; Salazar-Gutiérrez et al., 2014). Introducing new cultivars resistant to abiotic stresses are important to increase food production. Acquired plant tolerance to abiotic stresses can be achieved both by genetic engineering strategies and by conventional plant Abbreviations: DE, differential expression; EST, expressed sequence tag; MAS, breeding combined with the use of molecular markers in marker- marker assisted selection; RNA-Seq, RNA sequencing; SSR, simple sequence repeat. assisted selection (MAS) (Hajmansoor et al., 2013; Mousavi et al., ∗ Corresponding author at: Department of Plant Breeding and Biotechnology, 2014a; Roychoudhury et al., 2011). Among molecular markers, sim- Faculty of Agriculture, Shahrekord University, Shahrekord, P.O. Box 115, Iran. ple sequence repeats (SSRs) or microsatellites are the marker of Fax: +98 38 32324428. choice because they are co-dominant, more polymorphic and sta- E-mail addresses: [email protected], [email protected] (B. Shiran). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.scienta.2015.10.020 0304-4238/© 2015 Elsevier B.V. All rights reserved. A. Alisoltani et al. / Scientia Horticulturae 198 (2016) 462–472 463 ble. They are also much easier to assay compared with other types overcome these obstacles, Iiorizzo and colleagues offered compu- of molecular markers (Hajmansoor et al., 2013; Shiran et al., 2007). tational identifications of polymorphic SSR markers prior to actual SRRs are tandem repeats of 1–6 nucleotide motifs in nucleic laboratory verifications (Iorizzo et al., 2011). Zalapa et al. (2012) acid sequences, which are located on non-coding as well as cod- also suggested ways to reduce the cost for validating and screening ing regions of the plant genomes. EST-SSR is a type of SSR markers a growing number of SSRs. developed from expressed sequence tags (EST) libraries (Zalapa In addition to aforementioned solutions, we suggest that the et al., 2012). The polymorphism derived from EST-SSR may affect purposeful selection of SSR markers has the potential to yield many protein structure and function, which make them important for benefits beyond the random validation of SSRs. Since SSR sequences association, phylogenetic and evolutionary studies, quantitative on coding regions may alter protein structure and function leading trait loci (QTL) analysis, MAS as well as many other applications. SSR to phenotypic variations, it can help to identify candidate func- and EST-SSR markers have been applied in almond for generating tional genes and to increase the efficiency of MAS. To the best of linkage maps (Joobeur et al., 2000), phylogenetic analysis (Xu et al., our knowledge, there is no study on the parallel consideration of DE 2004), QTL mapping (Sánchez-Pérez et al., 2007), assessing the genes under stress and SSRs. Polymorphic SSRs on stress responsive genetic diversity (Rahemi et al., 2012; Shiran et al., 2007; Szikriszt genes can be important, and may be further translated to functional et al., 2011) and gene flow (Delplancke et al., 2012). EST-SSR mark- markers in breeding programs. Recently, we have conducted the ers have been also important to estimate the cross transferability holistic overview of gene expression in almond under frost stress, rate among Prunus species (Wang et al., 2012; Zhang et al., 2014). and a huge number of DE genes (Mousavi et al., 2014). However, development of SSR markers in almond and many In the current study, we have presented a pipeline for detec- other related species has been limited due to the lack of avail- tion and comparison of RNA-Seq SSRs under frost stress aimed able genomic information and EST libraries (Mousavi et al., 2014a; to develop informative markers. In addition to developing huge Zhang et al., 2014). Preparing and sequencing of the traditional EST number of SSR markers in almond, we proved the potential use libraries is a difficult, time-consuming and costly process. Whereas, of DE genes harboring SSR loci in analysis of genetic diversity in next generation sequencing (NGS) technology allows efficient iden- almond and related Prunus species. It is found that some of these tification of a large number of sequences at a fraction of the cost polymorphic markers are related to cold and other abiotic stress and efforts offered by traditional approaches. High-throughput responses such as calmodulin and trihelix transcription factor GT-1. RNA sequencing (RNA-Seq) is one of the NGS techniques, which These findings suggest the potential use of these markers in genetic is rapidly emerging as a major quantitative transcriptome profil- diversity, association studies and MAS breeding. ing approach (Wang et al., 2009). Large numbers of RNA-Seq data have been produced in Prunus species (approximately 100 datasets) 2. Material and methods as well as other genus in Rosaceae family (about 300 datasets) as recorded in NCBI-SRA (sequence read archive) database. Because 2.1. Data collection and SSR analysis of the high value of transferability (Wang et al., 2012; Zhang et al., 2014), RNA-Seq SSR markers can be used to explore the genetic The workflow of this study is presented in Fig. 1. RNA-Seq diversity, comparative genomics, evolution and functional studies data were obtained from our previous study on almond (P. dul- across almond and other Rosaceae species. cis Mill.) under frost stress (Mousavi et al., 2014a). Briefly, the Recently, the holistic overview of almond transcriptome has quality and trimming of datasets were conducted using FastQC been performed under frost stress condition, and more than 40,000 (Andrews, 2012) and FastX-toolkit (Gordon, 2011), respectively. contigs have been de novo assembled (Mousavi et al., 2014a). Frost Four paired-end sequencing libraries of almond were assembled injury is regarded as one the major limiting factor in the production using Trinity v1.3 (Grabherr et al., 2011), including ovary and anther of almond (Khanizadehi et al., 1989; Kodad et al., 2010; Rodrigo, tissues under normal and frost stress conditions. Annotation of de 2000). Almond is an early fruit tree, and it is usually exposed to −5 novo transcripts was performed using BLASTX (with E-value <10 ). late-spring frost, which could result in reduction or even abolishing Transcript quantification for RNA-Seq reads and DE analysis were the yield (Kodad et al., 2010; Samani et al., 2005). Frost stress could performed with RSEM (Li and Dewey, 2011) and EBSeq (Leng et al., damage trees from the early blooming stage to anthesis (Imani et al., 2013), respectively.

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