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Plant Physiology Reports
Formerly known as 'Indian Journal of
Plant Physiology'
ISSN 2662-253X
Volume 24
Number 1
Plant Physiol. Rep. (2019) 24:104-111
DOI 10.1007/s40502-019-0434-8
Functional annotation of differentially
expressed genes under salt stress in
Dichanthium annulatum
Anita Mann, Naresh Kumar, Charu
Lata, Ashwani Kumar, Arvind Kumar &
B.L.Meena
1 23
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ORIGINAL ARTICLE
Functional annotation of differentially expressed genes under salt
stress in Dichanthium annulatum
Anita Mann
1
•Naresh Kumar
1
•Charu Lata
1
•Ashwani Kumar
1
•
Arvind Kumar
1
•B. L. Meena
1
Received: 31 July 2018 / Accepted: 8 January 2019 / Published online: 19 January 2019
ÓIndian Society for Plant Physiology 2019
Abstract Soil salinity is one of the important abiotic
stresses affecting plant growth and development. Halo-
phytes can be one of the options to explore the salt toler-
ance potential and to identify the potential gene(s) which
can be used in crop improvement programs. In view of this,
the present experiment was conducted on grass halophyte,
Dichanthium annulatum, which can tolerate soil salinity up
to EC 30 dS/m (*300 mM NaCl) to identify the
gene(s) for salt tolerance. The de novo assembly generated
267,196 transcripts and these assembled transcripts were
further clustered into 188,353 transcripts. An average of
64.47% of the transcripts was functionally annotated
against the viridiplantae databases since no genomic ref-
erence is available for Dichanthium. Gene ontology and
pathways analysis using KAAS database identified that
48.13% transcripts were involved in molecular function,
37.21% in cellular component and 14.66% in biological
processes. The annotation of these genes provides a path-
way analysis for their putative functions under salt stress
conditions.
Keywords Salt stress Halophytes Dichanthium Gene
Salt tolerance
Introduction
Soil salinity is one of the major problems worldwide,
especially in countries where irrigation is an essential input
for agriculture. Due to non-availability of good quality
water, farmers are forced to use saline water for irrigation
(Barrett-Lennard and Setter 2010). Since soil salinity
reduces the soil fertility and hence crop yield, there is an
urgent requirement for other alternates. One of such
approaches can be increasing crop production dramatically
by improving plant productivity under stress conditions.
But the genetic diversity for stress tolerance within tradi-
tional crops is too narrow to achieve this goal immediately
(Colmer et al. 2006). Therefore, the most feasible approach
is identification of stress tolerance genes in extremophiles
and then introducing them into traditional crops. Halo-
phytes, the plants growing under extreme saline conditions,
could be the best choice to identify salt tolerance genes.
Halophytes grow in a wide variety of saline habitats, from
coastal regions, salt marshes and mudflats, to inland
deserts, salt flats and steppes. Halophytes have evolved a
range of adaptations to tolerate seawater and survive under
higher concentrations of salts up to EC 50 dS/m
(*500 mM NaCl) (Kumar et al. 2018). These include
adjustment of internal water relations through ion com-
partmentation in cell vacuoles, accumulation of compatible
organic solutes, succulence, and salt-secreting glands and
bladders etc. (Flowers and Colmer 2008; Shabala 2013;
Agarwal et al. 2013). Optimal halophyte growth is
achieved at a concentration of around 50 mM NaCl for
monocots and between 100 and 200 mM for dicots (Glenn
et al. 1999; Flowers et al. 2014). Few reports are available
using comparative proteomics and other molecular tech-
nologies with the goal of identifying new salt-responsive
genes or proteins in halophytes (Pang et al. 2010; Wang
&Anita Mann
anitadgr13@gmail.com
1
ICAR-Central Soil Salinity Research Institute, Karnal,
Haryana 132001, India
123
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https://doi.org/10.1007/s40502-019-0434-8
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et al. 2013). Although much useful information related to
salt tolerance has been accumulated from the analysis of
salt-stressed glycophytes such as Arabidopsis thaliana, the
specific regulatory mechanisms that enable halophytes to
survive in extremely saline habits have yet to be com-
pletely elucidated. D. annulatum is a species of grass
commonly used as forage for livestock and also known as
marvel grass or Hindi grass. It is native to tropical Asia, the
Middle East and parts of Africa. In general, it is a perennial
grass often with stolons. The root system goes no deeper
than one meter. Plants can be diploid, tetraploid or hex-
aploid. It is tolerant of varied soil conditions, including
soils high in clay and sand, poorly drained soils, and soils
that are somewhat alkaline and saline. It forms a turf that
can stand up to grazing pressure. Hence, in the present
study, we have taken the grass halophyte, Dichanthium
annulatum, which can tolerate soil salinity up to EC 30 dS/
m(*300 mM NaCl) to exploit the de-novo RNA-seq
technology for functional annotation and gene ontology
under salinity stress. To our knowledge, this is the first
study conducted to understand the molecular basis of
salinity tolerance of grass halophytes, D. annulatum, using
the RNA-Seq approach.
Materials and methods
Plant material
Dichanthium annulatum plants were raised through root
cuttings in pots filled with sandy and moist soil in a screen
house at Division of Crop Improvement, ICAR—Central
Soil Salinity Research Institute, Karnal (29°430N, 76°580E,
and 245 m above the mean sea level), Haryana, India in 4
replications. The soil salinity in pots was created by ini-
tially using natural soil of EC 15.92 dS/m (150 mM NaCl)
brought from the highly saline experimental farm of CSSRI
at Nain, Panipat, Haryana. Further salinity level of EC 30
dSm
-1
(*300 mM NaCl) was maintained by irrigation
with saline water. The saline treatment was maintained in
three pots with one set of control plants irrigated with
normal tap water.
RNA library preparation
RNA was isolated from leaves using standard Trizol pro-
tocol. RNA sequencing libraries were prepared with Illu-
mina-compatible NEBNext
Ò
Ultra
TM
Directional RNA
Library Prep Kit (New England BioLabs, MA, USA). Total
RNA at final concentration of 100 ng to 1 lg was taken for
mRNA isolation, fragmentation and priming. Fragmented
and primed mRNA was further subjected to first strand
synthesis in the presence of Actinomycin D (Gibco, life
technologies, CA, USA) followed by second strand syn-
thesis. The double-stranded cDNA was purified using
HighPrep magnetic beads (Magbio Genomics Inc, USA).
Purified cDNA was end-repaired, adenylated and ligated to
Illumina multiplex barcode adapters as per NEBNext
Ò
Ultra
TM
Directional RNA Library Prep Kit protocol.
Adapter-ligated cDNA was purified using HighPrep
beads and was subjected to 15 cycles of Indexing-PCR
(37 °C for 15 min followed by denaturation at 98 °C for
30 s, cycling (98 °C for 10 s, 65 °C for 75 s) and 65 °C for
5 min) to enrich the adapter-ligated fragments. The final
PCR product (sequencing library) was purified with
HighPrep beads, followed by library quality control check.
Illumina-compatible sequencing library was quantified by
Qubit fluorometer (Thermo Fisher Scientific, MA, USA)
and its fragment size distribution was analysed on Agilent
2200 Tapestation.
De novo transcriptome sequencing
Sequencing for 150 bp length paired-end reads was per-
formed in an Illumina HiSeq sequencer (Genotypic Tech-
nologies, Bangalore) to produce on an average of 45.01
million raw sequencing reads. The reads were processed
for quality assessment and low-quality filtering before the
assembly. The raw data generated was checked for the
quality using FastQC.
Processed reads were assembled using graph-based
approach by Trinity program. Trinity combines the over-
lapping reads of a given length and quality into longer
contig sequences without gaps. The characteristic proper-
ties, including N50 length, average length, maximum
length, and minimum length of the assembled contigs were
calculated. The clustering of assembled transcripts based
on sequence similarity was performed using CD-HIT-EST
with 95% similarity between the sequences, which reduces
the redundancy without exclusion of sequence diversity
that is used for further transcript annotation and differential
expression analysis.
Functional annotation and gene ontology
Clustered transcripts were annotated using homology
approach to assign functional annotation using BLAST tool
against ‘‘viridiplantae’’ data from the UniProt database.
Transcripts were assigned with a homolog protein from
other organisms at E-value less than e-5 and minimum
similarity greater than 30%. Pathway analysis was done by
using KAAS Server. Oryza sativa japonica,Zea mays,
Musa acuminata and Dendrobium officinale were consid-
ered as reference organisms for pathway identification
having more than 60% similarity with the experimental
halophytic plant D. annulatum.
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Results and discussion
De novo sequence assembly of transcriptome
About 41.41 million reads from Dichanthium were gener-
ated and used for the downstream analysis in the present
experiment. Among all raw reads, on an average of 96.65%
of high-quality data (having a Phred-like quality score [
q30) was retained for every sample. The high-quality
RNA-seq reads were de novo assembled into transcripts
using Trinity, as the reference genomic sequence is not
available for Dichanthium. In absence of reference gen-
ome, the transcriptome coverage efficiency has been
assessed by relating the unique genes with the closest
available transcriptome in de novo sequencing (De
´lano-
Frier et al. 2011; Parchman et al. 2010). The Trinity
assembly of high quality reads resulted in 267,196 tran-
scripts and further clustering resulted in 188,353 transcripts
with an average length of 864 bp and N50 of 1100 bp.
Since the shorter sequences may lack a characterized pro-
tein domain or may be too short to show sequence matches,
resulting in false negative results, the contigs which were
less than 300 bp in length were excluded. Compared with
the other EST sequencing technologies, the RNA-Seq
provides assembled and annotated high quality reads and is
being used in a number of plants such as Spartina (Ferreira
de Carvalho et al. 2013; Gedye et al. 2010), Cynodon (Hu
et al. 2015), Sorghum (Dugas et al. 2011) and some others.
Various scientific approaches are being used to study the
expression pattern of genes under stress environment. To
reveal a variety of molecular responses against abiotic
stress in non-model plants on a transcriptomic scale, NGS
is regarded as the best method even when complete gen-
ome sequences are absent.
GO annotation
Since the fully-sequenced reference genome of D. annu-
latum is not available, hence the clustered high-quality
reads were blasted against the viridiplantae database. Of
the 188,353 clustered transcripts, 64.47%transcripts
(121,446) were annotated and remaining 35.52% (66,907)
transcripts could not be annotated due to lack of informa-
tion for Dichanthium in the NCBI database. The percentage
of annotated transcripts obtained in present study is within
the range of annotation percentage reported in other species
e.g. 64% in Artemia (De Vos et al. 2019), 61% in sugar
beet (Lv et al. 2018), 82% in Amaranthus (De
´lano-Frier
et al. 2011), 68% in Spartina (Ferreira de Carvalho et al.
2013) and Cicer (Garg et al. 2011). Similarly, 35% of
unique transcripts obtained in D. annulatum are in accor-
dance with the other reports as 8% unique transcripts in
Maize (Vega-Arreguı
´n et al. 2009), 7% in Ginseng and
Amaranthus (De
´lano-Frier et al. 2011; Sun et al. 2010),
13% in Spartina pectinata (Gedye et al. 2010) and 35% in
moth bean (Tiwari et al. 2018). The top BLAST hits for D.
annulatum showed 49.25% of sequence similarity to Z.
mays followed by Dichanthelium oligosanthes (14.36%),
O. sativa subsp. Japonica (12.77%), S. bicolor (6.46%),H.
vulgare subsp. Vulgare (3.05%), Setaria italic (2.41%),
Aegilops tauschii (1.80%), Saccharum hybrid cultivar
R570 (1.13%) and Arundo donax (1.11%) as presented in
Fig. 1.
The similarity distribution of Dichanthium sequences
w.r.t. reference showed that 55.07% of transcripts had a
similarity of more than 80%. Based on sequence similarity,
annotated transcripts were categorized into three ontologies
i.e. biological processes (BP), cellular component (CC) and
molecular function (MF). Among these ontologies, BP (1945
terms) was most abundant in terms of different categories
identified followed by MF (1486 terms) and CC (522 terms).
In the biological processes, genes involved in transcription
(3309), transcription regulation (3138), metabolic process
(1948), carbohydrate metabolic process (1490), translation
(1163) signal transduction (1118), defense response (726),
response to oxidative stress (448), response to salt stress
(190) and sodium ion transport (53) were highly represented.
The salt stress-responsive transcripts observed under bio-
logical processes include dehydration responsive ele-
ment binding protein (DREB; DN58174_c6_g4_i3), Myb
family transcription factor EFM (DN72104_c1_g7_i1),
auxin response factor (DN26188_c0_g3_i1), AP2-EREBP-
type transcription factor (DN56877_c4_g3_i1), chaperonin-
like RbcX protein (DN60403_c1_g1_i4), glutathione per-
oxidase (DN64844_c1_g7_i1), L-ascorbate peroxidase
(DN61491_c0_g6_i3), peroxidase (DN64999_c3_g1_i2),
catalase (DN68555_c1_g1_i4), putative methionine sul-
foxide reductase (DN62432_c2_g3_i3) etc. It is already
established that salt stress leads to the overproduction of
reactive oxygen species (ROS) which are toxic to plant
growth and metabolism. To scavenge the ROS system, plan ts
adopt various mechanism such as activation of antioxidative
enzymes i.e. catalase, peroxidase, ascorbate peroxidase
etc.(Acosta-Motos et al. 2017; Das and Roychoudhury 2014;
Yoshida et al. 2014). In the present study, different tran-
scripts that belong to various antioxidative enzymes were
found to be expressed which may detoxify the ROS
components.
Within the molecular function ontology, ATP binding,
DNA binding, ADP binding, metal ion binding, protein
kinase activity, zinc ion binding, solute/proton antiporter
activity, calcium ion binding etc. were highly represented.
Some transcripts that are involved in the molecular func-
tion ontology are LRR receptor-like serine/threonine-pro-
tein kinase (DN47453_c0_g1_i1), asparagine synthetase
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(DN61786_c1_g1_i2), salt overly sensitive 1 (SOS1,
DN44879_c0_g1_i1), SOS2 (DN68935_c1_g2_i4) sodium/
hydrogen exchanger (DN68364_c0_g12_i7), cation/H
?
antiporter (DN61714_c4_g5_i1), K
?
efflux antiporter
6-like isoform X2 (DN72421_c2_g4_i1), plasma mem-
brane Na
?
/H
?
transporter (DN73047_c3_g5_i3), calcium-
binding EF-hand family protein (DN68613_c2_g4_i13),
calcineurin B-like7 (DN65265_c0_g3_i1), calmodulin-like
protein 1 (DN69677_c0_g1_i2). It has been reported in
earlier studies that these above-mentioned transcripts are
involved in salt tolerance mechanism (de Barajas-Lopez
et al. 2018; Himabindu et al. 2016). de Barajas-Lopez et al.
(2018) reported that protein kinases are involved in the salt
stress and showed that SOS2 (Salt Overly Sensitive 2),
SnRK3s (a.k.a. calcineurin B-like protein-interacting pro-
tein kinases, CIPKs) are Ser/Thr protein kinase acting in
the SOS pathway, providing salt tolerance. Likewise, many
reports in the literature showed that sodium/hydrogen
exchanger, cation/H
?
antiporter and K
?
efflux antiporter
6-like isoform X2 are involved in ion homeostasis (Pala-
valasa et al. 2017).
Cellular component ontology mainly represents genes
involved in integral component of the membrane (24,131),
nucleus (7293), cytoplasm (2735), plasma membrane
(2098), chloroplast (1441) and intracellular (1382). Like-
wise, Hu et al. (2015) reported the RNA-seq for gene
identification in bermudagrass (Cynodon dactylon) under
salt stress and found the 40,483 unigenes which they cat-
egorized into similarly three ontologies with 47 functional
groups. At the cellular level, 22.9% of transcripts represent
the integral component of the membrane which involves
the membrane transporters such as Potassium transporter
(DN64455_c0_g1_i4), Zinc transporter (DN62616_c2_g1_
i2), bidirectional sugar transporter SWEET (DN66098_
c1_g5_i21), transmembrane amino acid transporter family
protein (DN69618_c4_g5_i2), ABC transporter (DN56431_
c0_g2_i2), V-type proton ATPase subunit C (DN712
21_c2_g5_i1). In cytosol of plant cell, a high ratio of K
?
/
Na
?
is an important factor for maintaining ion homeostasis
under salt stress. It is reported that in high soil salinity, Na
?
may inhibit the potassium transporters by competing with
the K
?
for influx into plant roots (Assaha et al. 2017).
Most abundant 10 terms from each functional ontology
of biological functions, molecular functions and cellular
components are represented as doughnut chart in Fig. 2and
some important genes/proteins involved mainly in salt
stress tolerance mechanisms are summarized in Table 1.
KEGG pathway analysis
The pathway analysis is an important part for functional
study of genes. Recently, a number of studies have been
reported on pathway analysis in salt stress (Naika et al.
2013; Zhang et al. 2018). To identify the active biological
pathways in Dichanthium, 27,431 unique transcripts were
annotated against the KAAS server. From unique pathways
identified, protein processing in the endoplasmic reticulum
(1274 transcripts) was the most abundant pathway and
anthocyanin biosynthesis (2 transcripts) was the least in
terms of the number of homologous transcripts. Along with
these pathways, we identified some other important path-
ways i.e. starch and sucrose metabolism (964 transcripts),
RNA transport (947 transcripts), MAPK signalling path-
way (763 transcripts), glutathione metabolism (697
Fig. 1 Species distribution of the top BLAST hits for the D. annulatum
Plant Physiol. Rep. (January–March 2019) 24(1):104–111 107
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transcripts) which may have a major role in salt stress
tolerance.
In plants, Endoplasmic reticulum (ER) is highly vul-
nerable to stress conditions where assembling and folding
of proteins take place. During salt stress, misfolded or
unfolded proteins can accumulate which may activate
various signalling pathways (Deng et al. 2013). Liu et al.
(2016) reported about 220 up-regulated transcripts between
seed-type and fibre-type Cannabis sativa in salt stress and
identified the DEGs that were involved in endoplasmic
reticulum protein processing pathway. The top 10 unique
pathways identified for all transcripts in Dichanthium are
presented in Fig. 3. Most of the expressed transcripts are
involved in glycolysis, purine metabolism, RNA transport,
endocytosis, starch and sucrose metabolism, plant hormone
signal transduction etc. Comparative proteomics of Thel-
lungiella leaves identified 209 salt-responsive proteins
(Wang et al. 2013). Functional classification of these pro-
teins into 16 categories indicated that the majority are
involved in carbohydrate metabolism, followed by those
involved in energy production and conversion, and then
those involved in the transport of inorganic ions. Pathway
analysis revealed that most of the proteins are involved in
starch and sucrose metabolism, carbon fixation, photosyn-
thesis, and glycolysis. Of these processes, the most affected
were starch and sucrose metabolism, which might be piv-
otal for salt tolerance. In our studies also, one of the
responsive pathways to salt tolerance includes starch and
sucrose metabolism including glycolysis. The complete
mechanism of these transcripts can be revealed by studying
the differential gene expression and validation by Real-
time PCR to design a schematic flowchart for salt tolerance
mechanism in grass halophyte, D. annulatum.
It has been observed that glycophytes and halophytes
might possess the same set of genes, but exhibit differential
expression and in most of the instances, the difference in
post-translational regulation between halophytes and gly-
cophytes distinguishes salt-sensitive or salt-tolerant phe-
notypes. Salinity tolerance involves complex responses at
the molecular, cellular, metabolic and physiological levels.
At the molecular level, genes encoding ion transporters,
transcription factors, protein kinases, and osmolytes are
able to confer salinity tolerance (Kasuga et al. 1999; Tuteja
2007). Pathways such as plant hormone signalling path-
way, SOS (salt overly sensitive) pathway, calcium-signal-
ing pathway, MAPK (mitogen-activated protein kinase)
signal transduction and transporters and proline metabo-
lism also have key roles in the salinity stress tolerance
(Shinde et al. 2018; Yao et al. 2018). In our studies also,
the functional annotation of Dichanthium sequences iden-
tified the similar transcripts involved in salinity tolerance.
These annotations provide a valuable resource for dynamic
transcriptome changes in salt-tolerant research in halo-
phytes. Our results provide the genome sequence infor-
mation for the exploration of salt tolerance mechanisms in
this species and improve our knowledge of halophyte
response to salt stress. Currently, all overexpression studies
to improve salt tolerance have been based on the
Fig. 2 Frequency of top 10 abundant GO terms under biological process, molecular function and cellular component categories in D. annulatum
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Table 1 Transcripts involved in salt stress tolerance in D. annulatum
S. no. Transcript ID Protein name Pathways/functions
Biological functions
1. DN58174_c6_g4_i3 Dehydration responsive element binding protein 1A Transcription
2. DN72104_c1_g7_i1 Myb family transcription factor EFM Transcription
3. DN26188_c0_g3_i1 Auxin response factor Transcription regulation
4. DN56877_c4_g3_i1 AP2-EREBP-type transcription factor Transcription regulation
5. DN60403_c1_g1_i4 Chaperonin-like RbcX protein Response to salt stress
6. DN64844_c1_g7_i1 Glutathione peroxidase Response to oxidative stress
7. DN61491_c0_g6_i3 L-ascorbate peroxidase Response to oxidative stress
8. DN64999_c3_g1_i2 Peroxidase Response to oxidative stress
9. DN68555_c1_g1_i4 Catalase Response to oxidative stress
10. DN62432_c2_g3_i3 Putative methionine sulfoxide reductase Response to oxidative stress
11. DN62473_c2_g2_i3 ABA responsive element binding factor 1 Response to salt stress
12. DN58069_c0_g1_i2 Delta-1-pyrroline-5-carboxylate synthase Response to salt stress
13. DN65906_c5_g1_i4 Pyrroline-5-carboxylate reductase Response to salt stress
14. DN69314_c2_g5_i2 Heat shock 70 kDa protein Response to salt stress
15. DN60567_c2_g1_i3 Type IV inositol polyphosphate 5-phosphatase 11 Response to salt stress
Molecular functions
16. DN47453_c0_g1_i1 LRR receptor-like serine/threonine-protein kinase ATP binding
17. DN61786_c1_g1_i2 Asparagine synthetase ATP binding
18. DN44879_c0_g1_i1 SOS1 Solute: proton antiporter activity
19. DN68935_c1_g2_i4 SOS2 Protein kinase activity
20. DN68364_c0_g12_i7 Sodium/hydrogen exchanger Solute: proton antiporter activity
21. DN61714_c4_g5_i1 Cation/H
?
antiporter Solute: proton antiporter activity
22. DN72421_c2_g4_i1 K
?
efflux antiporter 6-like isoform X2 Solute: proton antiporter activity
23. DN73047_c3_g5_i3 Plasma membrane Na
?
/H
?
transporter Solute: proton antiporter activity
24. DN68613_c2_g4_i13 Calcium-binding EF-hand family protein Calcium ion binding
25. DN65265_c0_g3_i1 Calcineurin B-like7 Calcium ion binding
26. DN69677_c0_g1_i2 Calmodulin-like protein 1 Calcium ion binding
27. DN46215_c1_g2_i2 Zinc finger family protein Metal ion binding
28. DN61869_c0_g2_i4 C2C2-GATA transcription factor (GATA transcription factor 22) Zinc ion binding
29. DN53319_c0_g3_i2 Calcium-dependent protein kinase SK5 Protein kinase activity
30. DN46770_c0_g1_i1 Putative wall-associated receptor protein kinase family protein Protein Kinase Activity
Cellular components
31. DN64455_c0_g1_i4 Potassium transporter Integral component of membrane
32. DN62616_c2_g1_i2 Zinc transporter Integral component of membrane
33. DN66098_c1_g5_i21 Bidirectional sugar transporter SWEET Integral component of membrane
34. DN69618_c4_g5_i2 Transmembrane amino acid transporter family protein Integral component of membrane
35. DN56431_c0_g2_i2 ABC transporter Integral component of membrane
36. DN71221_c2_g5_i1 V-type proton ATPase subunit C Integral component of membrane
37. DN59535_c1_g2_i2 WAT1-related protein Plasma membrane component
38. DN58499_c4_g1_i1 Putative wall-associated kinase Plasma membrane component
39. DN63257_c2_g3_i4 Reticulon-like protein endoplasmic reticulum membrane
40. DN66070_c0_g1_i1 Leucine aminopeptidase 2 chloroplastic Cyoplasm
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assumption of a glycophytic pathway for salt tolerance and
since Dichanthium is still unexplored, the present work
using genes from halophytes will certainly be helpful in
future crop modelling.
Acknowledgements The authors are highly thankful to the Director,
ICAR-CSSRI, Karnal for providing necessary facilities to carry out
the research work. The first author also sincerely acknowledges the
ICAR-National Agricultural Science Fund (NASF), New Delhi for
funding this work.
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