SALINITY STRESS
Genetic Variation for Salinity Tolerance in Pakistani Rice(Oryza sativa L.) GermplasmA. Sakina1, I. Ahmed1,2, A. Shahzad1,2, M. Iqbal1,2,3 & M. Asif3
1 Department of Plant Genomics and Biotechnology, PARC Institute of Advanced Studies in Agriculture, National Agricultural Research Centre,
Islamabad, Pakistan
2 National Institute for Genomics and Advanced Biotechnology, National Agricultural Research Centre, Islamabad, Pakistan
3 Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta Canada
Keywords
germination stage; hydroponic; rice
germplasm; salinity tolerance; seedling stage;
SSR marker
Correspondence
M. Iqbal
Department of Plant Genomics and
Biotechnology, PARC Institute of Advanced
Studies in Agriculture, National Agricultural
Research Centre, Park Road, Islamabad-
45500, Pakistan
Tel.: 092-346-8980868
Fax: 092-51-9255034
Email: [email protected]
First two authors contributed
equally to this work.
Accepted December 15, 2014
doi:10.1111/jac.12117
Abstract
Soil salinity is one of the major production constraints. Development and plant-
ing of salt-tolerant varieties can reduce yield losses due to salinity. We screened
185 rice genotypes at germination stage in petri dishes under control, 50, 100 and
150 mM salt stress, and at seedling stage in Yoshida’s hydroponic nutrient solu-
tion under control, 50 and 100 mM salt stress. At germination stage, 15 genotypes
including Nona Bokra, Sonahri Kangni, 7421, 7423 and 7467, whereas at seedling
stage, 28 genotypes including Nona Bokra, Jajai-77, KSK-133, KSK-282, Fakhr-
e-Malakand, Pakhal, IR-6, Khushboo-95, Shahkar and Shua-92 were found salt
tolerant. Basmati-370, Mushkan, Homo-46 and accessions 7436, 7437 and 7720
were sensitive to salinity at both germination and seedling stage. We further
screened a subset of 33 salt-tolerant and salt-sensitive genotypes with SSR mark-
ers. Four SSR markers (RM19, RM171, RM172 and RM189) showed significant
association with two or more of the studied traits under 50, 100 and 150 mM salt
stress. These markers may be further tested for their potential in marker-assisted
selection. The salt-tolerant genotypes identified in this study may prove useful in
the development of salt-tolerant rice varieties in adapted genetic background.
Introduction
Salt stress is one of the major abiotic stresses that severely
affect crop production throughout the world. More than
800 million hectares of the cultivable land in world is salt-
affected (FAO 2014). Plant growth is reduced under salt
stress primarily due to water deficiency and osmotic pres-
sure (Hu and Schmidhalter 2005). High salt concentration
in soil makes it difficult for plant roots to absorb water
(Munns and Tester 2008). Soil and water management
practices can be used for reduction of soil salinity, but these
are often associated with very high costs. Therefore, sus-
tainable crop production on saline soils requires cost-effec-
tive alternative approaches such as breeding of salt-tolerant
crop varieties. Crop adaptation to salinity is a great chal-
lenge for plant breeders and geneticists to meet the food
demands of ever increasing human population (Salam
et al. 2011). The presence of large genetic variability in a
crop species is a prerequisite to begin an effective breeding
programme.
Rice (Oryza sativa L.) exhibits sensitivity to salinity, and
its response to salinity varies with growth stage. Generally,
rice shows tolerance to salt stress during germination,
becomes sensitive during early seedling stage, gains toler-
ance during vegetative growth, becomes sensitive again
during reproductive and pollination stage, and exhibits an
increasing tolerance until maturity (IRRI 1967). Screening
of crops for salinity tolerance has been carried out for a
long time and different methodologies have been used for
this purpose (Khan et al. 1997, Zeng et al. 2002, Ali et al.
2007, Salam et al. 2011, Shahzad et al. 2012). Screening
under controlled conditions has given better results because
of reduced environmental effects. Germplasm screening at
seedling stage is simple than at vegetative or reproductive
stages (Gregorio et al. 1997). In addition, early growth
stages have shown better prediction of plant’s response to
© 2015 Blackwell Verlag GmbH 1
J Agro Crop Sci (2015) ISSN 0931-2250
salinity (Wang et al. 2011). Although all plant growth
stages are sensitive to salinity, seedling stage is considered
as foretelling of plant’s growth response to salinity (IRRI
1967).
Hydroponic evaluation is free of soil-related difficulties.
This method can reliably assess the response of genotypes
to salt stress and, therefore, identify salt-tolerant genotypes
(Bhowmik et al. 2009). Evaluation of plant response to salt
stress in different crop species in hydroponics culture has
been well documented (Xie et al. 2000, Zeng et al. 2002,
Ali et al. 2004, Akram et al. 2010, Kanawapee et al. 2011,
Mansuri et al. 2012, Shahzad et al. 2012). Zeng et al.
(2002) evaluated 12 rice genotypes for salt tolerance in
hydroponics and found genotypic differences for seedling
growth and other yield contributing parameters. Djana-
guiraman et al. (2003) evaluated four rice genotypes at ger-
mination and seedling stage and observed the differential
response of genotypes to salinity. They found a decreasing
trend in vigour index, shoot and root lengths and germina-
tion percentage with increase in salt concentration. Mah-
mood et al. (2000) tested 110 rice genotypes for salinity
tolerance and found a reduction in tillering and fresh
shoot/root biomass with increase in salinity. Their results
showed that 34 genotypes were highly sensitive, 33 were
moderately tolerant, and 38 were tolerant to salinity even at
100 mM salt level. Furthermore, they found that the bio-
mass of tolerant genotypes was significantly higher than the
sensitive genotypes and recommended using this trait for
identification of tolerant rice genotypes under salt stress.
Simple sequence repeat (SSR) markers have been exten-
sively used for the assessment of genetic diversity because
of high efficiency, ease of use, high reproducibility, codom-
inant behaviour and high polymorphism. SSRs can detect
high level of allelic diversity (Meti et al. 2013). Rahman
et al. (2010) screened 28 rice varieties with 7 SSR markers
that amplified a total of 82 alleles. The polymorphism
information content (PIC) value of SSRs in their study ran-
ged from 0.76 for marker RM153 to 0.91 for marker
RM335 with an average PIC of 0.86.
This study was carried out to assess genetic variation in a
diverse collection of rice germplasm for salinity tolerance at
germination and seedling growth stages and to identify new
sources of salt tolerance in Pakistani rice germplasm for
improving salt tolerance in future rice varieties.
Materials and Methods
Plant material
The experimental material consisted of 185 rice genotypes
including commercial varieties, advance lines and landraces
from Pakistan, eight genotypes from India and 1 each from
Nepal, Japan and Philippines. Seeds of these genotypes
were acquired from the Genebank of Plant Genetic
Resources Institute, National Agricultural Research Centre,
Islamabad, Pakistan, and other rice research institutes of
Pakistan. Among the studied genotypes, Nona Bokra and
Kharai Ganja (Khan et al. 1987) and IR-6 from Pakistan
were salt-tolerant (Gurmani et al. 2006), whereas Basmati-
370 (Yadav et al. 2008) from Pakistan, Nipponbare (Jiang
et al. 2013) from Japan and Azucena (Awala et al. 2010)
from Philippines were previously reported as salt sensitive.
Due to large number of genotypes, screening at germina-
tion stage was carried out in three sets of 33, 76 and 76
genotypes, whereas screening at vegetative stage was carried
out in three sets of 70, 55 and 60. Some of the genotypes
did not germinate in three sets at germination stage testing
and two sets at vegetative stage (complete data given in
Tables S2, S3 and S5).
Screening of rice genotypes for salinity tolerance at
germination stage
A random sample of 10 seeds per genotype per replicate
were grown in Petri dish lined with two filter papers soaked
in either 10 ml distilled water (control) or 10 ml solution
of 50, 100 and 150 mM NaCl (salt treatments) and incu-
bated on a laboratory bench at 25 � 2 °C temperature and
10-h light period. The petri dishes were arranged in a com-
pletely randomized design (CRD) with three replications.
Germination of seeds was recorded on daily basis. The
number of seeds germinated was divided by the days from
first germination to calculate germination rate index (GRI)
using the following formula given by Maguire (1962):
GRI ¼ Xi
Yiþ Xii
Yiiþ . . .
Xn
Yn;
where
X = number of seeds germinated for the day.
Y = number of days from the first seed germinated.
i, ii,. . .n = No. of days.
Fresh weight of 7-day-old plumules and radicles was
recorded. Plumules and radicles were oven-dried at 70 °Cuntil constant weight to determine their dry weights.
Screening of rice genotypes for salinity tolerance at
vegetative growth stage
Screening at vegetative stage was carried out in a glass
house. Two-week-old pre-germinated uniform seedlings of
the 185 rice genotypes were transplanted in foam-plugged
holes (one plant per hole) in Styrofoam sheets floating over
200 l of Yoshida’s nutrient solution (Yoshida et al. 1976)
contained in a polyethylene lined iron tubs (100 9 100 9
30 cm). The salt treatments (0, 50 and 100 mM) were
applied in incremental manner (25 mM per day) as soon as
© 2015 Blackwell Verlag GmbH2
Sakina et al.
one new leaf emerged. The pH of hydroponic culture was
maintained at 5.0 (�0.5) and adjusted every day with 1 N
NaOH or 1 N HCl. The experiment was laid out in a com-
pletely randomized factorial design with three replications.
After 30 days of growth, plants were harvested, air-dried
and separated into shoots and roots for estimating their
biomass.
On the basis of relative performance during vegetative
growth stage at 50 and 100 mM salt stress, 13 tolerant, 11
moderately tolerant, six moderately sensitive and three sen-
sitive genotypes were selected of 185 genotypes. These 33
selected rice genotypes were tested under higher salinity
stress of 150 mM. Screening was performed using methods
described previously.
Molecular analysis using SSR markers
Genomic DNA of the 33 rice genotypes was isolated from
leaf tissues and was quantified following protocols
described in Shahzad et al. (2012). A total of 35 SSR primer
pairs were used to investigate genetic diversity and to find
association of these markers with salt tolerance in the
selected 33 genotypes. These markers were previously used
to study genetic variation for salt tolerance and the Saltol
QTL in rice (Lisa et al. 2004, Mohammadi-Nejad et al.
2008). Polymerase chain reaction (PCR) was performed
using protocol described by Shahzad et al. (2012). PCR-
amplified products were separated on 3 % agarose gels
stained with ethidium bromide and then visualized using
gel documentation system. Marker alleles were scored
either 1 for the presence or 0 for the absence. The amplifi-
cation products were used for pairwise comparison of
genotypes to measure the genetic similarity by Dice coeffi-
cients. Dice coefficients were computed using NTSYS-PC ver-
sion 2.1 software (Rohlf 2000). Similarity coefficients were
used to construct dendrogram using SAHN clustering
based on unweighted pair-group method with an arithme-
tic average (UPGMA) to conclude genetic relationships
between the genotypes.
Statistical analysis
Data for germination and vegetative growth stages were
analysed using two-way analysis of variance in GLM proce-
dure of MINITAB 13 (State College, PA, USA). Data of both
experiments were expressed as salt tolerance indices (STI)
which were measured by the following formula given by
Zeng et al. (2002):
STI ¼ Observations under Salinity
Means of the Controls� 100:
Salt tolerance indices values of all genotypes were calcu-
lated for all traits and treatments. All genotypes were cate-
gorized into four groups based on the average STI values
for all traits and treatments. As the range of average STI
values differed for the three set of studies, the range of each
set was divided into four groups and genotypes were
assigned into salt-tolerant, moderately salt-tolerant, mod-
erately salt-sensitive and sensitive groups based on their
average % STI values. Ward’s minimal variance cluster
analysis was used to make cluster group rankings on the
means of the STI to group the genotypes according to their
response under salt stress (Zeng et al. 2002).
For statistical analysis of SSR data, all scorable bands
were considered as single locus/allele. The loci were scored
as present (1) or absent (0). Polymorphism information
content (PIC) was calculated using the formula (Anderson
et al. 1993):
PIC ¼ 1�X
X2i
where
Xi = frequency of the ith allele of a particular locus
One-way analysis of variance was performed to test the
association of SSR data with STIs of different traits at 50
and 100 mM stress.
Results
Screening of genotypes at germination stage
In the first set of experiments, two-way analysis of variance
revealed significant (P ≤ 0.01) effects of genotype, treat-
ment and their interaction for fresh/dry radicle and plu-
mule weights (Table S1). At 50 mM NaCl stress, the STI of
fresh plumule weight showed a wide range of variation
between 113 % for Shadab and 27 % for IR-6 (Table S2).
For fresh radicle weight, the STI ranged between 99 % for
accession 7421 and 14 % for Palman Sufaid (Table S2).
Some genotypes showed better radicle and plumule growth
than other genotypes (Table S2) which may be due to their
ability to tolerate salt stress. The STI of dry plumule weight
at 50 mM salt stress was maximum (69 %) for Nona Bokra
and minimum (13 %) for Shaheen Basmati, whereas STI of
dry radicle weight was also maximum (81 %) for Nona Bo-
kra but minimum (11 %) for accession 7443 (Table S2).
At 100 mM NaCl stress, the minimum decrease in fresh
plumule weight (STI = 72 %) due to salt stress was
observed for the accession 7423, whereas the maximum
reduction (STI = 3.4 %) was found for accession 7461
(Table S2). Similarly, fresh radicle weight of accession 7467
was affected the least (STI = 90 %), whereas that of acces-
sion 7461 was affected the most (STI = 2.3 %) under salt
stress (Table S2). Accession 7467 showed maximum toler-
ance to reduction in dry plumule weight at 100 mM salt
stress (STI = 58 %), whereas accession 7461 exhibited the
© 2015 Blackwell Verlag GmbH 3
Genetic Variation for Salinity Tolerance in Rice
minimum tolerance for the same trait (STI = 2.2 %). Dry
radicle weights of the rice genotypes were affected more by
100 mM salt stress, with Swat-1 and 7423 showing the min-
imum decrease in this trait (STI = 25 %), whereas acces-
sion 7436 with maximum decrease in dry radicle weight
(STI = 7 %) (Table S2).
At 150 mM salt stress, STI of fresh plumule weight ran-
ged between 49 % for accession 7421 and 2 % for acces-
sions 7426, 7443, 7461 and Entry-144, whereas it ranged
from 22 % for accession 7421 and 1 % for accessions 7443,
DR-82 and Entry-126 for fresh radicle weight (Table S2).
STI for dry plumule weight varied from 51 % for accession
7423 and 2 % for KSK-282, Entry-144 and accession 7443,
whereas it ranged between 23 % for accession 7421 and
0.23 % for KSK-282 for dry radicle weight (Table S2). The
plumule and radicle fresh and dry weights decreased signif-
icantly with increasing salinity. However, the reduction in
STIs of the studied traits varied among genotypes. For
instance, Palman Sufaid showed the least reduction in STI
for the studied traits at 100 and 150 mM salt stress
although its STI values were relatively low as compared to
the tolerant genotypes (Table S2). Likewise, accession 7423
tolerated higher salt stress by showing <50 % reduction in
STI for fresh and dry shoot weights (Table S2).
In the second set of screening of genotypes at germina-
tion stage, the effect of genotype, treatment and their inter-
action was not significant (P ≥ 0.05) for fresh radicle/
plumule weights, but it was highly significant (P ≤ 0.01)
for dry radicle/plumule weights (Table S1). At 50 mM salt
stress, the STI of fresh plumule weight ranged between
118 % for Kangni-27 and 18 % for NPT-89. The STI for
fresh radicle weight ranged between 116 % for Pakhal and
16 % for NPT-89 (Table S2). The STI for dry plumule
weight ranged between 108 % for Purple Marker and 2 %
for Sarshar, whereas for dry radicle weight, it was maxi-
mum (107 %) for Lateefy and minimum (2 %) for
PK-386, NPT-89 and Sarshar (Table S2).
At 100 mM NaCl stress, STI for fresh plumule weight
ranged between 118 % for accession 7728 and 9 % for
NPT-89, whereas STI for fresh radicle weight ranged
between 120 % for Sugdasi Sadagulab and 6 % for NPT-89
(Table S2). STI for dry plumule weight was highest
(104 %) for accession 7726 and lowest (1 %) for Jhona
349. The STI for dry radicle weight ranged between 98 %
for IR-8 and 0.2 % for Basmati-C622 (Table S2).
At 150 mM NaCl stress, STI ranged between 109 % for
accession 7726 and 4 % for Mushkan for fresh plumule
weight, whereas STI for fresh radicle weight ranged
between 112 % for accession 7719 and 0.7 % for Sugdasi
Ratria (Table S2). For dry plumule weight, the highest STI
(102 %) was observed for accession 7703, whereas the low-
est (0.7 %) for Basmati-C622. For dry radicle weight, the
highest STI (91 %) was found for Mahlar-346 and the
lowest (0.2 %) for Sugdasi Ratria, Lateefy, Jajai-77 and
NPT-89 (Table S2).
In the third set of screening of genotypes at germination
stage, effect of genotype, treatment and their interaction
was highly significant for radicle and plumule fresh and dry
weights (Table S1). At 50 mM salt stress, STI for fresh plu-
mule weight was maximum (112 %) for accession 7428
and minimum (11 %) for accession 7437, whereas STI for
fresh radicle weight ranged between 108 % for accession
7428 and 9 % for accessions 7437 and 7777 (Table S2). STI
for dry plumule weight ranged between 107 % for acces-
sions 7428, 7447 and 7452 and 6.5 % for accession 7699,
whereas it ranged between 120 % for accession 7447 and
5 % for accession 7699 for dry radicle weight (Table S2).
At 100 mM NaCl stress, STI ranged between 107 % for
accession 7429 and 15 % for accession 7437 for fresh plu-
mule weight, whereas it ranged between 91 % for Entry-25
and 10 % for accession 7437 for fresh radicle weight
(Table S2). The STI for dry plumule weight was maximum
(95 %) for accession 7695 and minimum (6 %) for acces-
sion 7456, whereas the STI for dry radicle weight was high-
est (77 %) for accession 7705 but lowest (0.41 %) for
accession 7456 (Table S2). At 150 mM stress level, Entry-
26 showed maximum tolerance to reduction in fresh
plumule weight (STI = 61 %); accession 7429 showed
minimum tolerance (STI = 18 %) for the same trait,
whereas Entry-26 tolerated reduction in fresh radicle
weight the most (STI = 57 %) and accession 7458 the least
(STI = 6 %) (Table S2). The STI for dry plumule weight
was the highest (49 %) for accession 7705 but lowest
(0.6 %) for accession 7699. Dry radicle weight of accession
7705 was decreased by more than half (STI = 44 %) under
150 mM salt stress, whereas those of accessions 7456 and
7699 were only 0.4 % of that of control (Table S2). Based
on the per cent means of STIs for different growth param-
eters such as fresh/dry plumule and radicle weights, the 98
genotypes were grouped into four categories; 15 genotypes
were placed into tolerant group, 16 genotypes into moder-
ately tolerant, 27 genotypes into moderately sensitive and
39 genotypes into sensitive genotypes (Table 1). Of the
185 genotypes, 87 genotypes (11 in first set, 27 in second
set and 49 in third set) did not germinate at the three salt
stress levels and were, therefore, not grouped into any cat-
egory. The non-germination of these genotypes under salt
stress may be due to their higher salt sensitivity at germi-
nation stage.
Germination rate index varied among genotypes and
with salt treatments (Table S3). GRI of all genotypes
decreased with an increase in NaCl concentration, and the
maximum reduction in GRI was observed for the 150 mM
NaCl treatment (Table S3). GRI was maximum for acces-
sion 7451 (19.7), M3 and Shadab (18.9), accession 7423
(16.5) and DR-92 (13.5) under 0, 50, 100 and 150 mM salt
© 2015 Blackwell Verlag GmbH4
Sakina et al.
stress, respectively, whereas it was minimum for accession
7446 (0.23), accession 7437 (0.36), Sonahri Sugdasi (0.11)
and accession 7459 (0.06) under 0, 50, 100 and 150 mM salt
stress, respectively (Table S3).
Screening of rice genotypes for salinity tolerance at
vegetative growth stage
In the first set of experiment, two-way analysis of variance
indicated that effect of genotype, treatment and their inter-
action was highly significant (P ≤ 0.01) (Table S4). Seed-
lings grown in salinized conditions showed significant
decrease in the growth of shoots and roots of plants. The
STI for shoot length at 50 mM NaCl ranged between 101 %
for Sugdasi Ratria and 23 % for Basmati-198, whereas STI
for root length ranged between 113 % for Pakhal and 38 %
for Basmati-198 (Table S5). The STI for shoot dry weight
ranged between 86 % for IR-6 and 3 % for Basmati-370,
whereas that for root dry weight ranged between 29 % for
IR-6 and 0.9 % for Jhona-349 and Basmati-370 (Table S5).
The STI for shoot length at 100 mM NaCl ranged
between 83 % for Nona Bokra and 19 % for DR-83,
whereas STI for root length ranged between 127 % for
Nona Bokra and 35 % for Nipponbare (Table S5). At
100 mM NaCl, STI for shoot dry weight ranged between
48 % for Sugdasi Bengalo and 1.4 % for DR-83, whereas
STI for root dry weight ranged between 11.4 % for Sugdasi
Sadagulab and 0.6 % for Sonahri Kangni and Kashmir Bas-
mati (Table S5).
Effects of genotype, treatment and their interaction were
highly significant (P ≤ 0.01) for shoot/root length and
shoot/root dry weight for the second set of screening at
vegetative growth stage (Table S4). The STI for shoot
length at 50 mM NaCl ranged between 101 % for Entry-72
and 51 % for accession 7441. For root length, STI ranged
between 102 % for Entry-167 and 42 % for accession 7439
(Table S5). The STI at 50 mM NaCl ranged between 115 %
for accession 7452 and 24 % for accession 7441 for shoot
dry weight, whereas it ranged between 117 % for accession
7461 and 28 % for accessions 7439 and 7441 for root dry
weight (Table S5).
The STI for shoot length at 100 mM NaCl ranged
between 84 % for accession 7442 and 47 % for accession
7427, whereas for root length, the STI ranged between
116 % for accession 7463 and 55 % for accession 7430
(Table S5). At 100 mM NaCl stress, STI for shoot dry
weight ranged between 104 % for accession 7442 and
9.5 % for Entry-26, whereas for root dry weight, it ranged
between 115 % for accession 7442 and 9.3 % for Entry-26
(Table S5).
The effect of genotype, treatment and their interaction
was highly significant (P ≤ 0.01) for root/shoot length in
hydroponics culture at vegetative growth stage in the third
set of screening (Table S4). The effect of genotype, treat-
ment and their interaction was also significant on shoot
dry weight; however, effects of genotype, treatment and
their interaction were not significant (P ≥ 0.05) for root
dry weight (Table S4). The STI for shoot length at 50 mM
NaCl ranged between 114 % for accession 7511 and 34 %
for accession 7697, whereas for root length, it ranged
between 116 % for accessions 7465, 7510, 7513, 7727 and
7763 and 49 % for accession-7697 (Table S5). The STI for
Table 1 Categorization of rice genotypes on the basis of salt tolerance indices under salt stress at germination stage
Category Range of STI
# of
genotypes Genotypes
Set 1
Tolerant 40–60 % 4 7467, 7423, 7421, Nona Bokra
Moderately tolerant 30–39 % 6 Fakhre Malakand, Swat-1, 7451, KSK-282, Khushboo-95, Shadab
Moderately sensitive 20–29 % 5 Palman Sufaid, Shaheen Basmati, DR-82, 7426, Entry-126
Sensitive <19 % 7 Entry-144, IR-6, 7422, 7425, 7436, 7443, 7461
Set 2
Tolerant 40–55 % 6 Sonahri Kangni, 7438, 7703, 7704, 7726, 7728
Moderately tolerant 30–39 % 6 Kangni-27, M3, Purple Marker, 7440, 7702, 7719
Moderately sensitive 16–29 % 12 Dilrosh-97, IR-8, Kangni 9 Torh, Mahlar-346, Pakhal, PK 177, Sugdasi Ratria,
Sugdasi Sadagulab, 7720, 7721, 7724, 7727
Sensitive <15 % 24 Basmati-370, Basmati-C 622, Basmati 2000, Dokri Basmati, DR-92, DR-83,
PK-386, Homo-34, Homo 46, Jajai-77, Jhona 349, Kanwal-95, Kasalath,
KSK-133, Lateefy, Mushkan, NIAB-IR 9, NPT-89, Rachna Basmati, Sarshar,
Shahkar, Shua 92, Sugdasi Bengalo, Swat-2
Set 3
Tolerant ≥70 % 5 7449, 7695, 7705, Entry-25, 7428
Moderately tolerant 50–69 % 4 Entry-26, 7424, 7447, 7429
Moderately sensitive 30–49 % 10 7707, 7778, 7433, 7776, 7517, 7465, 7432, 7452, 7693, 7457
Sensitive <29 % 8 7437, 7777, 7699, 7458, 7456, 7720, 7459, 7779
© 2015 Blackwell Verlag GmbH 5
Genetic Variation for Salinity Tolerance in Rice
shoot dry weight at 50 mM NaCl stress ranged between
125 % for accession 7511 and 2.7 % for accession 7224,
whereas for root dry weights, it ranged between 112 % for
accession 7511 and 4.6 % for accession 7447 (Table S5).
The STI for shoot length at 100 mM NaCl ranged
between 99.9 % for accession 7511 and 19 % for DR-83,
whereas STI for root length ranged between 116 % for
accession 7425 and 26 % for accession 7704 (Table S5). At
100 mM NaCl stress, STI for shoot dry weight ranged
between 115 % for accessions 7511 and 7512, and 0.9 %
for accessions 7702 and 7704, whereas STI for root dry
weight ranged between 117 % for accession 7426 and
2.3 % for accessions 7699 and 7703 (Table S5). Salt stress
(50 mM) at vegetative stage resulted in a decrease in shoot
length in all genotypes except Sugdasi Ratria, 7429, Entry-
72, 7225, 7464, 7465, 7467, 7510, 7511, 7512 and 7513.
Shoot lengths of accessions 7511 and 7464 were not
affected even by 100 mM salt stress. In contrast to shoot
length, root lengths of most of the genotypes remained the
same under salt stress. However, root lengths increased at
higher salt stress (100 mM) in some of the genotypes such
as KSK-282, Nona Bokra, 7463, 7425, 7447, 7448, 7514,
7516 and 7694.
Based on means of STI for root/shoot lengths and root/
shoot dry weights, the 174 rice genotypes were grouped
into four categories. Among all the genotypes tested in
three sets of experiments at vegetative growth stage, 32
genotypes were placed into tolerant group, 52 genotypes
into moderately tolerant, 70 genotypes into moderately
sensitive and 20 into sensitive group (Table 2), whereas the
remaining genotypes did not germinate.
Root length was positively correlated with shoot and
root dry weights in all the three sets of experiments (Table
S6). Shoot length was also positively correlated with root
length, shoot and root dry weights in all experiments
except with root length in third set of vegetative growth
stage screening at 50 and 100 mM NaCl stress. Correlation
coefficients were highest between shoot dry weight and root
dry weight in all the experiments (Table S6).
On the basis of overall agronomic performance, 33
genotypes were selected for further molecular analysis. GRI
and STI of 33 salt-tolerant and salt-sensitive genotypes are
presented in Table 3. Among 35 SSR markers, 20 markers
did not show polymorphism. The remaining 15 SSR mark-
ers amplified 37 alleles (Table 4). The number of alleles
ranged from one to four alleles per locus with an average
Table 2 Classification of rice genotypes on the basis of salt tolerance indices (STI) under salt stress at vegetative growth stage
Category Range of STI
# of
genotypes Genotype
Set 1
Tolerant ≥50 % 16 Fakhre Malakand, IR-6, Jajai-77, Kharai Ganga, Khushboo-95, Kasalath,
KSK-133, KSK-282, Nona Bokra, Pakhal, Sarshar, Shahkar, Shua-92, Sugdasi
Bengalo, Sugdasi Ratria, Sugdasi Sadagulab
Moderately tolerant 40–49 % 25 Basmati-385, Basmati-C622, Dehradun Basmati, Dhera-Dun Basmati,
Dilrosh-97, DR-82, IR-8, JP-5, Kangni-27, Kangni 9 Torh, Ludan, Malhar-346,
NIAB-IR 9, Purple Marker, Pusa Basmati-1, Ranbir Basmati, Sada-Hayat, Sathra,
Shadab, Shaheen Basmati, Shandar, Sonahri Kangni, Sonahri Sugdasi, Super Basmati,
Swat-2,
Moderately sensitive 30–39 % 22 Azucena, Basmati-217, Basmati-2000, Dokri Basmati, DR-83, DR-92, Entry-126,
Entry-167, IR-36, Jhona-349, Kanwal-95, Kashmir Basmati, Lateefy, Mehak, NPT-89,
NPT-146, NPT-156, Palman Sufaid, PK-177, PK-386, Punjab Basmati-1, Rachna Basmati,
Sensitive <29 % 7 Basmati-198, Basmati-370, Basmati Pak, Homo-46, Mushkan, Nipponbare, Swat-1
Set 2
Tolerant ≥80 % 7 Entry-10, 7428, 7429, 7442, 7451, 7452, 7461
Moderately tolerant 66–79 % 13 Entry-8, Entry-72, Entry-94, Entry-136, 7427, 7431, 7433, 7435, 7443, 7450, 7453,
7454, 7463
Moderately sensitive 51–65 % 22 Entry-25, Entry-144, Entry-167, NPT 89, NPT 160, NPT 174, L3, M3, M5, 7430, 7432, 7434,
7436, 7437, 7438, 7439, 7456, 7457, 7459, 7458, 7460, 7462
Sensitive <50 % 3 Entry-26, 7441, 7440
Set 3
Tolerant ≥90 % 9 7425, 7426, 7464, 7465, 7467, 7510, 7511, 7512, 7513
Moderately tolerant 70–89 % 14 7225, 7254, 7421, 7422, 7423, 7424, 7445, 7448, 7466, 7514, 7516, 7719, 7727, 7781
Moderately sensitive 50–69 % 26 7444, 7447, 7517, 7518, 7520, 7677, 7678, 7693, 7694, 7697, 7698, 7699, 7700,
7701, 7705, 7706, 7708, 7709, 7710, 7722, 7762, 7763, 7773, 7778, 7779, 7782
Sensitive <50 % 10 7224, 7515, 7696, 7702, 7703, 7704, 7720, 7725, 7774, 7780
T, tolerant; MT, moderately tolerant; MS, moderately sensitive; S, sensitive.
© 2015 Blackwell Verlag GmbH6
Sakina et al.
of 2.5 alleles. The level of polymorphism among the 33
genotypes was detected by calculating polymorphism
information content (PIC) values for each of the 15 SSR
markers loci. The PIC value varied significantly for all SSR
loci and ranged from 0.14 (RM103) to 0.69 (RM143) with
an average PIC value of 0.51. Cluster analysis grouped the
33 genotypes into four clusters (Fig. 1). Cluster A com-
prised of 10 salt-tolerant/moderately tolerant and 3 salt-
sensitive/moderately sensitive genotypes, whereas cluster B
consisted of seven salt-tolerant/moderately tolerant geno-
types. Cluster C consisted of five salt-tolerant/moderately
tolerant and five salt-sensitive/moderately sensitive geno-
types, whereas cluster D consisted of one salt-tolerant/
moderately tolerant and two salt-sensitive/moderately sen-
sitive genotypes. Analysis of variance showed significant
association of some SSR marker alleles with various traits
at vegetative growth stage (Table 4). Alleles of marker
RM19 showed significant association with shoot length
and root and shoot dry weights at 50, 100 and 150 mM salt
stress (Table 4). Similarly, markers RM26, RM29, RM31,
RM72, RM103, RM124, RM125, RM150, RM171, RM172,
RM185, RM189 and RM208 showed significant association
with one or more of the studied traits at 50, 100 and
150 mM salt stress.
Discussion
Salt affects growth of crop plants by limiting the absorption
of water through roots. Salt stress has an immediate effect
on cell growth and enlargement, and high concentration of
salts can be extremely toxic (Munns and Tester 2008). Rice
is very sensitive to salinity at different growth stages,
Table 3 Average salt tolerance indices of different traits and germination rate indices (GRI) for 33 rice genotypes at vegetative growth stage
Genotype Group
Salt tolerance indices
GRIShoot length Root length Shoot dry weight Root dry weight
Fakhre Malakand T 58.6 97 41.2 10.4 14.2
IR-6 T 50 90.1 46.4 15.7 8.8
Khushboo-95 T 56.1 105.3 48.2 14.4 10.7
KSK-282 T 64.1 108.8 26.1 9 14
Nona Bokra T 89.2 114.8 37.8 18.5 4.5
7425 T 62.5 110 97.2 107 4.4
7426 T 53.5 108 108.8 110.6 14.8
7442 T 90.5 71.3 97 105.8 1.4
7451 T 85.5 84.3 83.5 72 13.1
7461 T 82.8 95.2 88.5 96.4 8.3
7467 T 93.3 107.1 67.4 99.1 13.7
7510 T 93.4 109.3 66.7 93.5 9.7
7512 T 100.1 84.8 117.7 110.3 1.3
Basmati-385 MT 57.6 106 14.3 8 9.1
DR-82 MT 58.2 109 22.2 7.1 13.9
JP-5 MT 48.2 100.8 9.7 3 0
Shadab MT 51.3 100.4 34.7 10.2 15.2
Shaheen Basmati MT 59.9 99.8 13 3.7 8.3
Super Basmati MT 55.1 105.2 23.8 5.7 2.8
7421 MT 51.4 105.5 45.7 107.4 15.5
7422 MT 44 104.6 47.9 104.4 6.8
7423 MT 51.2 103.6 56.8 79.2 15.4
7443 MT 85 75.5 31.5 83 6.5
7445 MT 55.8 99.8 77.5 76.2 0.8
Azucena MS 47.5 89.9 6.1 2 13.3
Entry-126 MS 42.9 74.5 3.5 2.1 11.6
Entry-144 MS 63 63.9 57 52.7 9.9
Palman Sufaid MS 54 72.1 11.2 1.5 13.4
7436 MS 63.9 71.3 46.5 42.9 6.8
7677 MS 51.2 106.4 38.8 31.2 3.8
Basmati-198 S 27.2 61.9 12.7 4.4 8.5
Swat-1 S 36.2 66.2 4.1 1.4 13.4
7696 S 54.9 98.2 19.6 18.5 14.7
STI values are average of 50, 100 and 150 mM salt treatments, whereas GRI values are average of 0, 50 and 100 mM treatments.
T, tolerant; MT, moderately tolerant; MS, moderately sensitive; S, sensitive; STI, salt tolerance indices.
© 2015 Blackwell Verlag GmbH 7
Genetic Variation for Salinity Tolerance in Rice
especially seedling stage. During germination, rice is more
tolerant than other growth stages (Khan et al. 1997).
Genetic variation for salinity tolerance has been reported in
rice.
In the present study, salt stress significantly reduced vari-
ous growth attributes of rice genotypes studied. However,
the extent of growth reduction under salt stress was depen-
dent on genotypes. Salt-sensitive genotypes showed more
reduction in their biomass as compared to tolerant geno-
types. Fresh and dry plumule and radicle weights of all
genotypes were decreased with increase in salt stress. How-
ever, salt-sensitive genotypes showed greater reduction in
fresh and dry plumule and radicle weights. Root length was
also decreased with an increase in salt stress. Djanaguir-
aman et al. (2003) reported significant decrease in root and
shoot lengths, and vigour index with increase in salt con-
centration. However, reduction of vigour index in their
study was minimum at all salt stress levels in tolerant geno-
types as compared to sensitive ones. Roots have direct con-
tact with soil for water and minerals uptake, so root
characters can effectively be used as selection criteria in
breeding for salinity tolerance. In present study, shoot
Table 4 Simple sequence repeat (SSR) markers, PIC values and association of marker alleles with different traits at 50, 100 and 150 mM salt stress
given to 33 rice genotypes at vegetative growth stage
Marker
PIC
Alleles
50 mM 100 mM 150 mM
Value SL RL SDW RDW SL RL SDW RDW SDW RDW
RM19 (12) 0.57 RM19a ns ns ns *** ns ns ** *** * ns
RM19b ns ns ns ns ns ns ns ns ns ns
RM19c * ns ns *** ** ns *** *** * *
RM26 (5) 0.59 RM26a ns ns * ns ns * * ns * *
RM26b ns ns * ns ns * * ns * *
RM26c ns ns ns *** ns ns *** *** * *
RM29 (2) 0.41 RM29a ns ns ns ns ns * ns ns ns ns
RM31 (5) 0.48 RM31a * ns ns ns * * ns ns ns ns
RM72 (8) 0.63 RM72a * ns ** * ns ns * ns * *
RM72b ns ns * ns ns ns ns ns ns ns
RM72c * ns ns ns ns ns ns ns ** *
RM103 (6) 0.14 RM103a ns ns ns ns * ** ns ns ns ns
RM103b ns ns ns ns * ** ns ns ns ns
RM124 (4) 0.63 RM124a ns ns ns ns ns ns ns ns ns ns
RM124b ns ns ns ns ns ns ns ns ns ns
RM124c ns ns ns ns ns ns ns ns ns ns
RM124d * ns ns ns ** ns * ns ns ns
RM125 (7) 0.68 RM125b ns ns ns ns ns ns ns * ns ns
RM125c ns ns ns ns ns ns ns ns ns ns
RM125d ns ns ns ns ns ns ns ns ns ns
RM143 (3) 0.69 RM143a ns ns * ns ns * ns ns ns ns
RM143b ns ns ns ns ns ns ns ns ** **
RM143c ns ns ns ns ns ns ns ns ** **
RM150 (6) 0.48 RM150b * ns ns ns ns ns ns ns ns ns
RM150c * ns ns ns ns ns ns ns ns ns
RM171 (10) 0.52 RM171b ** ns ns * * ns ** * ns ns
RM171c ns ns ns ns ns ns ns ns ns ns
RM171d * ns ns ns * ns ns ns ns ns
RM172 (7) 0.32 RM172a * ns ns * * ns * * ns ns
RM172b ns ns ns ns ns ns ns ns ns ns
RM185 (4) 0.47 RM185a ns ns ns ns ns ns ns ns ns ns
RM185b ns ns ns ns ns ns ns ns ns ns
RM189 (9) 0.64 RM189b ** ns ns ** *** ns ** ** ns ns
RM189c ns ns ns ns * * ns ns ns ns
RM189d ** ns ns *** ** ns *** *** ** *
RM208 (2) 0.44 RM208a ns ** * ns ns ** ns ns * *
RM208b ns * * ns ns ** ns ns ns ns
Numbers in parenthesis within the marker column indicates chromosomal location of the marker.
SL, shoot length; RL, root length; SDW, shoot dry weight; RDW, root dry weight; ns, not significant.
Significant at ***P ≤ 0.001; **P ≤ 0.01; *P ≤ 0.05.
© 2015 Blackwell Verlag GmbH8
Sakina et al.
length was highly affected by salinity than root length. This
might be due to the reason that plants, especially those of
drought or salt-tolerant species, tend to propagate their
roots deeper to absorb more water during osmotic stress.
These results are in agreement with the findings of Haq
et al. (2009) who reported differential response of rice
genotypes under salinity stress. They found maximum
reduction of shoot fresh weight under salinity in Azucena
and minimum reduction in shoot dry weight in Moro-
berekan. They reported maximum shoot fresh/dry weight
ratio in Moroberekan, whereas minimum in Nipponbare.
An increase in plant height during stress results in an
increase in plant’s biomass. Fresh and dry biomass, espe-
cially at seedling stage, has been found associated with salt
tolerance in crop plants and can, therefore, be used as an
indicator of salt tolerance or sensitivity. In present study,
total dry biomass showed a greater reduction in sensitive
genotypes than tolerant genotypes. The sensitive genotypes
exhibited various symptoms of salt injury such as yellowing
of leaf, reduction in root and shoot growth and ultimately
dying of seedlings at vegetative growth stage. Mansuri et al.
(2012) evaluated 15 rice genotypes for salt tolerance and
reported growth reduction, rolling and drying of leaves and
reduction in seedling height under saline conditions. They
also found reduced root/shoot dry weight under salinity
stress and reported higher biomass in tolerant genotypes
compared to sensitive ones. They concluded that biomass
was positively correlated with salt stress tolerance and,
therefore, can be used as selection criterion for salt toler-
ance (Mansuri et al. 2012). Our results also showed a
reduction in shoot and root growth of rice genotypes under
salinity stress that resulted in reduced shoot and root
lengths. Bhowmik et al. (2009) also reported significant
reduction in plant height and dry matter under salt stress
in 11 rice genotypes.
Dry matter production is a reliable indicator of perfor-
mance under salt stress as it is associated with higher grain
yield under saline conditions (Maas 1986). Various plant’s
traits such as shoot and root fresh and dry weights are asso-
ciated with salt tolerance at early growth stages and can be
used as selection criteria for salt tolerance (Ashraf et al.
1999). We found strong positive correlation between differ-
ent growth parameters such as shoot/root length and their
dry weights. Our results are in agreement with the findings
of Ashraf et al. (1999). They suggested root and shoot dry
weights as selection criteria in breeding for salt tolerance.
Similar results of significant correlation between plant bio-
mass and plant height have been previously reported
(Bhowmik et al. 2009, Mansuri et al. 2012). Increase in
plant height allows plants to produce more biomass (Peng
et al. 1999). Zhang et al. (2004) also found that increase in
plant height resulted in increased biomass production in a
double-haploid (DH) population.
Clustering based on SSR marker grouped the selected salt-
tolerant/moderately tolerant and salt-sensitive/moderately
sensitive genotypes into four clusters. However, 3 of the 4
clusters had both tolerant/moderately tolerant and sensitive/
moderately sensitive genotypes. Cluster B had all salt-toler-
ant/moderately tolerant genotypes, whereas cluster A had 10
tolerant/moderately tolerant and three sensitive/moderately
sensitive genotypes. Despite the inability of the 15 polymor-
phic SSR markers to group salt-tolerant and salt-sensitive
genotypes into different clusters, these markers were able to
group 17 of the 23 tolerant genotypes closely. Markers
RM19, RM26, RM29, RM31, RM72, RM103, RM124,
RM125, RM150, RM171, RM172, RM185, RM189 and
Fig. 1 Genetic similarity among 33 rice geno-
types based on Simple sequence repeat (SSR)
marker data.
© 2015 Blackwell Verlag GmbH 9
Genetic Variation for Salinity Tolerance in Rice
RM208 showed significant association with one or more of
the studied traits at 50 and 100 mM salt stress. Therefore,
these markers may be further tested either on segregating
populations or recombinant inbred populations derived
from tolerant and sensitive rice varieties for their potential
in marker-assisted selection for salt tolerance. Pervaiz et al.
(2009) assessed genetic variability in 35 Asian rice cultivars
using 32 SSR markers. They found considerable polymor-
phism between Basmati and coarse rice varieties as indicated
by the amplification of 144 alleles in 35 rice cultivars. Num-
ber of alleles in their study ranged from 2 (for markers
RM10, RM13, RM19) to 13 (for marker RM70) with an
average of 4.5 alleles per locus. In our study, PIC values
showed a significant positive linear correlation with the
number of alleles at SSR locus. Rahman et al. (2010)
screened 28 local rice varieties with 7 primer pairs and found
82 alleles. Marker RM335 produced the maximum number
of alleles (15) and had the highest PIC value (0.91). Kanawa-
pee et al. (2011) investigated genetic diversity in 30 rice
genotypes using RAPD and SSR markers and found higher
level of polymorphism in SSR than in RAPD markers.
We classified the studied rice genotypes as tolerant, mod-
erately tolerant, moderately sensitive and sensitive to salt
stress on the basis of relative shoot and root lengths, and
shoot and root fresh and dry biomass production. The
accessions, which acquired high STI values for the above-
mentioned parameters, were considered salt tolerant, while
those that had less STI values were considered salt sensitive.
However, response to salt stress of the studied genotypes
varied with growth stage. Some genotypes, such as Nona
Bokra, KSK-282, Kangni-27, Fakhre Malakand, Khushboo-
95, Shadab, Sonahri Kangni and Purple marker, and some
accessions were tolerant at both germination and vegetative
growth stage. However, a number of genotypes, including
Basmati-370, Basmati-2000, DR-82, DR-83, Homo-46,
Mushkan, Lateefy, Jhona-349, Rachna Basmati and Palman
Sufaid, and some accessions were sensitive to salt stress at
both germination and vegetative growth stage. Some rice
genotypes including IR-6, Jajai-77, KSK-133, Pakhal,
Sarshar, Shaheen Basmati, Shua-92 and Swat-2 were sensi-
tive to salt stress at germination stage but tolerant at vege-
tative stage. Similarly, some genotypes including Swat-1,
7438, 7440, 7447, 7702, 7703 and 7704 were tolerant at
germination stage but sensitive at vegetative growth stage.
So these genotypes behaved differently to salt stress at dif-
ferent growth stages. Variability in response to salt stress of
rice at different developmental stage has been reported pre-
viously (Akbar and Neue 1987, Lutts et al. 1995).
Conclusion
We found significant genetic variation for salt tolerance
at germination and vegetative growth stage in a large
collection of Pakistani rice germplasm including commer-
cial varieties, advanced lines and landraces. We were also
able to identify rice genotypes that were either tolerant to
salt stress at both germination and vegetative growth stage
or one of these stages. These genotypes offer a valuable
genetic resource for local as well as international rice breed-
ers for use in breeding for salt tolerance. These sources can
be exploited in a planned manner to widen the genetic base
of existing rice varieties against salt tolerance. The SSR
markers used in this study could not classify the selected
genotypes into different salt-tolerant categories. However,
we observed an association between a representative set of
the studied genotypes with SSR markers. These markers
may be useful in screening for salt tolerance in rice germ-
plasm. However, the association of these markers with salt-
tolerant genes/quantitative trait loci needs to be confirmed
in segregating populations or any other suitable mapping
populations so that the potential of these markers in mar-
ker-assisted selection schemes can be determined.
Acknowledgements
The financial support from Higher Education Commission
of Pakistan and Pakistan Agricultural Research Council is
gratefully acknowledged. The authors also acknowledge and
appreciate the support of Dr. M. Aashiq Rabbani and Direc-
tor, Gene Bank of Plant Genetic Resources Program, NARC,
Islamabad, for providing rice seeds used in this study.
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Table S1 Percent sums of squares for different growth
parameters of rice grown under salt stress at germination
stage.
Table S2 Salt tolerance indices of agronomic parameters
in rice under different salinity stress levels (mM) at germi-
nation stage.
Table S3 Germination rate indices (GRI) for rice geno-
types tested under 0, 50, 100 and 150 mM NaCl stress at
germination stage.
Table S4 Percent sums of squares for different growth
parameters of rice under salt stress at vegetative growth
stage.
Table S5 Salt tolerance indices of four traits in rice under
different salinity stress levels at vegetative growth stage.
Table S6 Correlation among four agronomic traits in
rice at vegetative growth stage under 0, 50 and 100 mM salt
stress.
© 2015 Blackwell Verlag GmbH12
Sakina et al.
