Abstract

Background

   Leymus chinensis (Trin.) Tzvel. is a high saline-alkaline tolerant
   forage grass genus of the tribe Gramineae family, which also plays an
   important role in protection of natural environment. To date, little is
   known about the saline-alkaline tolerance of L. chinensis on the
   molecular level. To better understand the molecular mechanism of
   saline-alkaline tolerance in L. chinensis, 454 pyrosequencing was used
   for the transcriptome study.

Results

   We used Roche-454 massive parallel pyrosequencing technology to
   sequence two different cDNA libraries that were built from the two
   samples of control and under saline-alkaline treatment (optimal stress
   concentration-Hoagland solution with 100 mM NaCl and 200 mM NaHCO[3]).
   A total of 363,734 reads in control group and 526,267 reads in
   treatment group with an average length of 489 bp and 493 bp were
   obtained, respectively. The reads were assembled into 104,105 unigenes
   with MIRA sequence assemable software, among which, 73,665 unigenes
   were in control group, 88,016 unigenes in treatment group and 57,576
   unigenes in both groups. According to the comparative expression
   analysis between the two groups with the threshold of “log2 Ratio ≥1”,
   there were 36,497 up-regulated unegenes and 18,218 down-regulated
   unigenes predicted to be the differentially expressed genes. After gene
   annotation and pathway enrichment analysis, most of them were involved
   in stress and tolerant function, signal transduction, energy production
   and conversion, and inorganic ion transport. Furthermore, 16 of these
   differentially expressed genes were selected for real-time PCR
   validation, and they were successfully confirmed with the results of
   454 pyrosequencing.

Conclusions

   This work is the first time to study the transcriptome of L. chinensis
   under saline-alkaline treatment based on the 454-FLX massively parallel
   DNA sequencing platform. It also deepened studies on molecular
   mechanisms of saline-alkaline in L. chinensis, and constituted a
   database for future studies.

Introduction

   Leymus chinensis (Trin.) Tzvel., a perennial rhizome grass of the tribe
   Gramineae family with an allotetraploid species(2n = 4x = 28),
   naturally grew on alkaline-sodic soils in northern China [45][1],
   [46][2]. It's also an economically and ecologically important grass
   plant that contains many extremely valuable stress resistance genes
   [47][3]. Because of its high drought and saline–alkaline tolerance
   [48][4], [49][5], [50][6], L. chinensis plays an important role in the
   establishment of artificial grassland and in the protection of
   environment, which has received considerable attention in recent
   decades [51][7], [52][8]. Despite such advances, the genome of L.
   chinensis hasn't been published, little is known about its reference of
   genetic information on-line, and few studies have been reported on
   saline-alkaline of L. chinensis on molecular level. Therefore, the
   studies on molecular mechanisms of saline-alkaline in L. chinensis have
   far-reaching significance.

   These years, a lot of studies have been reported on abiotic stresses of
   plants. The saline-alkaline stress is one of the main abiotic stresses,
   which is more seriously harmful than any single salt and alkaline
   stress on plants. Maybe salinity and alkalinity have a cooperative
   effect when they simultaneously stress on plants, which also had been
   demonstrated in L. chinensis and other species [53][9], [54][10],
   [55][11]. The mechanisms of abiotic stresses on plants are complex and
   diverse, even involve multiple complex physiological and metabolic
   pathways, which mostly include synthesis of extrusion and
   compartmentalization of sodium ions, response to abiotic stress,
   pathogen defense and adjustment of ion homeostasis [56][12], [57][13].
   These mechanisms involve the expression of a cluster of genes and
   interaction among their gene products rather than individual genes, and
   the gene expression affected by many internal and external factors
   [58][14]. Therefore, the more comprehensive understanding of abiotic
   stress tolerance mechanisms need to be based on the gene expression
   level.

   Over the past decades, the significant progress has been made in
   genome-wide gene expression profiling (GEP) by the development and
   application of differential display [59][15], as well as the large
   scale analysis of differential gene expression technology, such as cDNA
   libraries cloning technology [60][16], [61][17], [62][18], SAGE
   [63][19], Microarray technology [64][20], [65][21], and others.
   However, each of the above techniques has its disadvantages, such as
   high false positive rates, low level expression abundance,
   time-consuming and intensive labor [66][22]. As the first
   next-generation technology to reach the market, the development of the
   454 Life Sciences (454; Branford, CT, USA; now Roche, Basel) sequencing
   platform (the 454 Sequencer) provides a compelling case study for the
   establishment of a new disruptive technology [67][23]. Moreover, 454
   the 454-FLX massively parallel DNA sequencing platform is an effective
   next generation sequencing technology to better understand the
   transcriptome of unknown genome plant [68][24]. Meanwhile, massively
   parallel DNA sequencing platforms have become available which reduce
   the cost of DNA sequencing by over two orders of magnitude, making
   global transcriptome analysis inexpensive, and widespread [69][25].
   Furthermore, a lot of studies on the comparative high throughput
   sequencing of plant transcriptome in many model and non model species,
   such us maize, grapevine, eucalyptus, olive genotype and cucumber
   flower have been reported [70][26], [71][27], [72][28], [73][29].

   To gain a global view of the molecular mechanisms of saline-alkaline in
   L. chinensis, a transcriptome study on the two samples of control and
   saline-alkaline treatment (Hoagland solution with 100 mM NaCl and 200
   mM NaHCO[3]) was performed to make a comparative gene expression
   analyses. Basing on barley, rice and wheat which closely relate to L.
   chinensis as references, we present a bioinformatic exploration,