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Molecular Plant Breeding 2012, Vol.3, No.10, 103
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1-5, Pelita I-1 and PSBRc50, which were very
important for tracking
SalTol
with molecular markers.
The genotypes Kalimekri, Cheriviruppu, IR886-30-3-
1-4-2, Bhirpala, CR1015, Kajalsail, IR71907-3R-2-1-2,
IR71897-3R-1-1-2, IR55182-3B-14-3-2-3, IR58430-
6B-14-1-2, IR63295-AC211-3, IR64197-3B-8-2,
IR74099-3R-2-2, IR71991-3R-2-1, IR65192-4B-14-1,
IR60483-2B-17-2-1-2, IR72048-B-R-11-1-3-1-2B-2,
IR58443-6B-10-3 and IR71991-3R-2-6-1 were identified
as potential new sources of salinity tolerance other
than
SalTol
. These new findings can redirect breeding
strategies for salinity-tolerant rice to develop a new
generation of salinity-tolerant varieties.
3 Materials and Methods
3.1 Phenotyping of diverse rice genotypes
The usefulness of the identified molecular markers
linked to the
SalTol
QTL on chromosome 1 can be
confirmed when applied on a wider scale to 115
diverse rice genotypes (Table 1). These genotypes
were composed of 67 IRRI-developed elite lines for
salt tolerance, 18 IRRI-released modern varieties and
12 BRRI (Bangladesh Rice Research Institute)-released
modern varieties, along with 3 traditional Bangladeshi
varieties cultivated in coastal regions, 9 varieties of
Indian origin, 2 varieties from both Vietnam and
Indonesia, one variety from Pakistan and one from the
Philippines. We used 7 Pokkali accessions: Pokkali-1
(Acc. # IRGC8948), Pokkali-2 (Acc. # IRGC15238),
Pokkali-3 (Acc. # IRGC15388), Pokkali-4 (Acc. #
IRGC15602), Pokkali-5 (Acc. # IRGC15661), Pokkali-6
(Acc. # IRGC108921) and Pokkali-7 (Acc. #
IRGC15661) to identify the contributor of
SalTol
in
FL478 with other genotypes. All seeds were collected
from the IRRI Plant Breeding, Genetics, and
Biotechnology Division.
We screened 115 genetically diverse rice genotypes
under controlled environmental conditions in the
phytotron at IRRI with IR29 and FL478 as susceptible
and tolerant checks, respectively. Twenty pregerminated
seeds per genotype were transplanted in 100-hole
seedling floats. Seedling stage tolerance screening was
set up (Gregorio et al., 1997) using nutrient solution
described by Yoshida et al (1976). A salinity of EC 12
dSm
-1
was applied 4 days after transplanting. The
screening was conducted in the IRRI phytotron with
temperature maintained at 29
℃
/21
℃
day/night,
relative humidity of 70% during the day and natural
daylight. A modified standard evaluation score (Table
4) was used in rating the symptoms of salt damage
(Gregorio, 1997). Ratings of salt injury symptoms
were recorded 3 weeks after salinization when the
susceptible check IR29 was dead.
3.2 Molecular marker analysis
Previous reports (Islam et al., 2005; Islam et al.,
2011a) found that four SSR markers and one EST
marker were linked to the
SalTol
QTL on the
chromosome 1 segment. Niones (2004) also reported
one more SSR and EST marker linked to this QTL.
So, a total of five SSR markers and two EST
markers were used in this study.
Genomic DNA of the 115 genotypes was extracted
using the CTAB method described by Zheng et al
(1995). PCR was performed following the protocol
described by Temnykh et al (2000) using a PTC-100
dyad thermocycler machine (M J Research) with
384-well plates. Amplification products were resolved
by 8% polyacrylamide gel electrophoresis. Gels were
run for 2~3.5 hours at 100 volts and stained in
ethidium bromide and visualized under UV light.
The molecular weight for each SSR marker allele was
measured by using Alfa Imager software version 5.5.
Polymorphic information content (PIC) values of the
SSR markers were calculated according to the formula
of Anderson et al (1993). SSR marker alleles were
analyzed using the program Power Marker version
3.25 (Liu and Muse, 2005). Haplotype analysis was
conducted according to Bai et al (2003) and Liu and
Anderson (2003) using the best three SSR markers,
i.e., RM8094, RM3412 and RM493, to compare with
FL478 as a reference.
Acknowledgments
We thank Poverty Elimination through Rice Research
Assistance (PETRRA), the Generation Challenge Program
(GCP) and Challenge Program for Water and Food (CPWF)
for providing funds for doing this research. We also thank