RESEARCH PROJECT UNDER PARB CGS SYSTEM
PROJECT ID NO. 18
Molecular mapping of quantitative trait loci
determining salinity tolerance at maturity stage in
indica rice
1. PROJECT TITLE
2. PARB THEME UNDER
WHICH THIS PROJECT
FALLS
Theme 1: Enhancing productivity on sustainable basis
of major farming system
3. PARB SUB-THEME UNDER
WHICH THIS PROJECT
FALLS
Sub-theme 1.1: Rice-wheat system
4. PARB PROJECT GROUP
FOR WHICH THIS
PROJECT MATCH
Project group 1.1.1: Improve salt tolerance of rice and
wheat
5. OBJECTIVE OF THE
PROJECT
(Mission statement)
Identification of microsatellite loci associated with
agronomic and physiological traits at maturity stage
under saline conditions in indica rice (Oryza sativa L.)
6. ORGANIZATION SUBMITTING THE PROJECT
a. Name of Host Organization:
University of the Punjab, Lahore
b. Host Institute/Division//Department: School of Biological Sciences
c. Administrative Contacts
i. Head of the Host Organization (VC/DG/DIRECTOR/etc.)
Name:
Title:
Telephone:
Email:
Prof. Dr. Mujahid Kamran
Vice Chancellor
042-9231098-9
vc@pu.edu.pk,
ii. Head of The Host Institution (Director/Chairman/Division Head etc.)
Name:
Title:
Telephone
Email:
Prof. Dr. Muhammad Akhtar
Director General
042-9230960
ma3@soton.ac.uk, sosb@cyber.net.pk
Signature with date and seal
2
7. I. COLLABORATING ORGANIZATION-1 (International)
a. Name of Organization:
Graduate School of Agricultural Sciences, Kobe University, Kobe, Japan
b. Institute/Division/Section/Department
Laboratory of Plant Breeding, Faculty of Agriculture
c. Administrative Contacts
i. Head of The Organization (VC/DG/DIRECTOR/etc.)
Name:
Dr. Chiharu Nakamura
Title:
Professor/ Dean
Telephone : +81-78-803-5928
Email:
nakamura@kobe-u.ac.jp
ii. Head of The Institution (Director/Chairman/Division Head etc.)
Name:
Dr Takashige Ishii
Title:
Associate Professor/ Head Lab. of Plant Breeding
Telephone:
+81-78-803-5825
Email:
tishii@kobe-u.ac.jp
Signature with date and seal
II. COLLABORATING ORGANIZATION-2 (Local)
a. Name of Organization:
Ayub Agriculture Research Institute, Faisalabad
b. Institute/Division/Section/Department
Soil Fertility Research Institute, Lahore
c. Administrative Contacts
i. Head of The Organization (VC/DG/DIRECTOR/etc.)
Name:
Dr. Muhammad Rashid
Title:
Director General
Telephone : 041-2651371, 2654359
Email:
dgaari@yahoo.com
ii. Head of The Institution (Director/Chairman/Division Head etc.)
Name:
Dr. Shahid Mehmood
Title:
Director
Telephone:
042-5222638
Email:
directorsfri@yahoo.com
Signature with date and seal
3
III. COLLABORATING ORGANIZATION-3 (Local)
a. Name of Organization:
Ayub Agriculture Research Institute, Faisalabad
b. Institute/Division/Section/Department
Soil Salinity Research Institute, Pindi Bhattian
c. Administrative Contacts
i. Head of The Organization (VC/DG/DIRECTOR/etc.)
Name:
Dr. Muhammad Rashid
Title:
Director General
Telephone : 041-2651371, 2654359
Email:
dgaari@yahoo.com
ii. Head of The Institution (Director/Chairman/Division Head etc.)
Name:
Dr. Shahzada Munawar Mehdi
Title:
Director
Telephone:
0547-531376
Email:
mehdi853@hotmail.com
Signature with date and seal
8. PROJECT MANAGER
Name:
Dr. Muhammad Arshad Javed
Title:
Assistant Professor
Organization/Institute: University of the Punjab/ School of Biological Sciences
Qualification and Relevant Experience (Attach CV):
PhD (16 years relevant experience), CV Attached
Telephone:
042-92311
Mobile:
0332-4122387
Fax:
042-9230980
E mail:
mirpur87@hotmail.com
Signature with date and seal
4
9. COLABBORATING SCIENTIST(S)
1) Name of the Team Leader-1:
Dr Takashige Ishii
Title:
Associate Professor
Qualification and Relevant Experience: Ph.D (CV Attached)
Telephone:
+81-78-8035825
Mobile:
+81-80-31054509
Fax:
+81-78-8035825
E mail:
tishii@kobe-u.ac.jp
Signature with date and seal
2) Name of the Team Leader-2:
Dr. Shahid. Mehmood
Director
Ph. D (CV Attached)
Title:
Qualification and Relevant Experience:
Telephone:
042-5222638
Mobile:
0321-4429919
Fax:
042-5222674
E mail:
directorsfri@yahoo.com
Signature with date and seal
3) Name of the Team Leader-3:
Mr. Ghulam Shabbir
Title:
Assistant Research Officer
Qualification and Relevant Experience: M. Sc (HONS) Agri. (CV Attached)
Telephone:
0547-531376
Mobile:
0321-4429919
Fax:
0547-531376
E mail:
NA
Signature with date and seal
5
10. APPROVED BY
Name:
Dr. Mubarik Ali
Designation:
Chief Executive
Approval Date of PARB Board of
Governors:
Signature with date and seal
14.4.2009
11. PROJECT DURATION
12. DATE OF COMMENCEMENT
13. TOTAL PROJECT COST (million rupees)
54 months
June 1, 2009
23.148
14. LOCATION OF THE PROJECT:
School of Biological Sciences, University of the Punjab, Lahore
15. BACKGROUND INFORMATION
i. Problem to be addressed
Soil/water salinity is one of the limiting factors in improving the rice productivity
world wide. Salinity tolerance in rice is a quantitative trait and it is influenced
strongly by environment. Any change in environment, such as temperature, humidity
or light, can dramatically change the transpiration rate and consequently the ion
uptake (Yeo et al., 1990; Flowers and Yeo, 1997; Zeng et al., 2001). Such changes
may alter the salt tolerance among the genotypes and precise selection for salt
tolerance is not possible. Moreover, response of rice plant to salinity varies with
developmental stage. Seedling stage and maturity stage are reported to be sensitive.
Seedling stage sensitivity can be avoided by transplanting the older seedlings (about
30 days old) but sensitivity at maturity stage can not be escaped. This results in low
rice productivity in saline areas.
Rice-wheat system (Kallar area in Punjab) is situated in world’s biggest
irrigation system which resulted in the development of salinity in soil and water. An
area of one million hectares under rice cultivation is reported to be salt affected
(Qureshi et al., 1991) and it accounts for 40 – 70 % reduction in rice yield (Aslam et
al., 1995). Development of salinity tolerant rice cultivar is the most feasible slow due
to complex genetic background.
ii. Relevance of the Project to the problem to be addressed
Salinity tolerance in rice is thoroughly investigated in Pakistan but much of the
work was conducted to determine the effects of salinity by screening the genotypes at
seedling stage (Javed et al., 2006) or maturity stage (Javed et al., 2003; Aslam et al.,
6
1995; Qureshi et al., 1991). Limited work was carried out to find the genetic control
in rice (Mahmood et al., 2004). Salinity tolerance is quantitative trait. Quantitative
trait loci (QTLs) analysis for salinity tolerance has been reported at seedling stage
(Lee et al., 2007; Javed MA 2006; Lin et al., 2004). However, the mapping of
quantitative trait loci determining the rice productivity has not been investigated yet.
Therefore, this research project is proposed to find the QTLs associated with plant and
yield traits in saline conditions. Instead of screening the rice genotypes on the basis of
morph-physiological parameters, DNA based screening could give the precise results
with less time, cost and labour. Thus, the development of salinity tolerant rice
cultivars could be accelerated.
iii. Literature review preferably for the last 5 years.
Several studies indicated that salinity tolerance is a complex character involving
interactions of a number of component traits which are physiological in nature and
may be inherited independently (Yeo and Flowers, 1999; Chaubey and Senadhira,
1994). The genetic studies of salt tolerance in rice (Moeljopawiro and Ikehashi, 1981;
Gregorio and Senadhira, 1993; Mahmood et al., 2004) revealed, both, additive and
dominant effects. Plant breeding, in its conventional form, is based on phenotypic
selection of superior genotypes within segregating progenies obtained from crosses.
Application of this methodology often encounters difficulties related to genotype and
environment interactions (Munns et al., 2006). In addition, several phenotypic
procedures are expensive, time consuming or sometime unreliable for particular traits.
Salinity tolerance shows strong influence of environment. Therefore, marker assisted
selection (MAS) is an approach that has been developed to avoid the problems
connected with conventional breeding by changing the selection criteria from
selection of phenotypes towards selection of genes, either directly or indirectly.
Molecular markers are not environmentally regulated and are unaffected by the
conditions in which the plants are grown. Moreover, these are detectable in all stages
of plant growth. With the availability of an array of molecular markers and genetic
maps, MAS has become possible both for the traits governed by major genes as well
as for quantitative trait loci (QTLs). The usefulness of a given molecular marker
depends on its capability in revealing polymorphism in the nucleotide sequence
allowing discrimination between molecular marker alleles. These polymorphisms are
revealed by molecular techniques such as restriction fragment length polymorphisms
(RFLP), amplified fragment length polymorphism (AFLP), microsatellite or single
sequence length polymorphism (SSR) etc. Several studies related to molecular
genetics of salt tolerance have been conducted in many crops including rice (Zhang et
al., 1995; Mano and Takeda, 1997; Lin et al., 1998; Foolad, 1999; Flowers et al.,
2000;; Koyama et al., 2001;; Yao et al., 2005). Microsatellite markers detect simple
sequence length polymorphism (SSLP) and are rapidly replacing restriction fragment
length polymorphism (RFLPs) for many kinds of genetic studies, largely because of
their technical simplicity, the small amount of starting DNA required, the relatively
low cost for the user, rapid turn-around time and high power of genetic resolution.
QTL analysis for salinity tolerance has been reported at seedling stage in indica and
japonica backgrounds (Lee et al., 2007; Javed et al., 2006; Lin et al., 2004; Parasad et
al., 2000) but the association of quantitative trait loci with rice productivity in saline
7
conditions has not been investigated yet. Therefore, identification of QTLs
determining the salinity tolerance in rice for plant and yield traits at maturity is
proposed.
iv. Achievements and related research in hand, if any
Pokkali is a traditional indica rice cultivar and possesses salinity tolerant (Javed
et al, 2006; Xie et al., 2000). Basmati rice is sensitive to salinity and performs poorly
in saline fields. KS 282 is a long grain non aromatic variety. KS 282 is reported to be
a high yielding variety in saline conditions (Aslam et al., 1995; Qureshi et al., 1991).
Rice plant mapping populations derived from crosses between salinity tolerant and
salinity sensitive cultivars are one of the basic prerequisites to identify the major/
minor QTLs associated with salinity tolerance. Therefore, Basmati rice varieties (Bas.
370, Bas 385, Bas 2000, Bas 198, and Pak Basmati) were crossed with salt tolerant
cultivars during Kharif 2008 and F1seeds of following crosses has been harvested:
Basmati 370 x KS 282
Basmati 385 x KS 282
Basmati 2000 x KS 282
Basmati 370 x Pokkali
Basmati 385 x Pokkali
F2 population, from parent lines showing maximum polymorphism, will be used
in proposed investigation to identify the quantitative trait loci associated with
agronomic traits under saline conditions.
Seedling stage is very sensitive to salinity. Quantitative loci analysis has been
carried out and a total of twenty four microsatellite loci have been identified by single
point analysis controlling salinity tolerance at this satge in indica rice (Javed 2006).
Mapped F2 population was derived from a cross between Shaheen Bas and Pokkali in
those studies. Thus, it will be an important rice breeding objective to incorporate
salinity tolerance at seedling and maturity stages in Basmati rice by pyramiding the
identified QTLs.
16. PROJECT PLAN
a.Scientific/technical methodology (give details):
Plant materials
Theoretically, F2 is the most informative type of population for gene mapping and
genetic analysis, and have been used directly for QTL analysis. However, it is usually
difficult to assess the reliability of the data due to the inability to carry out replicated field
tests with F2 populations for data collection. To partly resolve this problem, several
studies used F3 families in place of the F2 individuals frequently referred as F2:3
populations (Wu et al., 2009) in making field measurements of the quantitative traits.
However, Li et al. (2000) demonstrated to obtain replicated data from F2 mapping
populations.
Two F2 plant populations will be selected for molecular mapping in the proposed
studies. Selection criteria will be maximum polymorphism between the parent lines of
these populations.
8
Salinity Blocks
The phenotyping of segregating population misleads the plant breeder in natural field
conditions due to change in environment and uneven salinity distribution within the same
piece of land. To avoid the experimental error and have precise results, construction of
salinity block is proposed. Salinity block will be constructed along with a glass roof and
surrounding fence. Two salinity blocks will be constructed, each of 20 x 40 feet in
dimensions. Glass roof will not create any hindrance in light reception and dilution of salt
by rainfall could be avoided. A survey will be conducted in saline areas of Punjab to find
a piece of land with desired electrical conductivity (6-8 dS/m). Soil from the selected
fields will be brought to fill the salinity blocks.
Phenotyping the F2 populations for agronomic traits at maturity
Pre germinated seeds will be grown in seedling beds (seedling to seedling and row to
row distances 1 and 3 inches, respectively). Five hundreds seedlings of each population
will be grown and maintained for thirty days. At the time of transplantation, each seedling
will be separated into three plantlets by peeling off the tillers during the early tillering
stage. Two plantlets of similar genetic background will be, considered as two replications,
transplanted in salinity blocks. Where as third seedling will be transplanted in normal soil.
Plant to plant and row to row distance will be maintained at nine and twelve inches,
respectively. These populations will be grown till maturity and data for following
physiological and agronomic traits will be recorded;
I. Physiological traits
Dried plant material will be used to make solutions, as described by DionisioSese et al. (1998), for the measurement of following traits:
1) Sodium contents in flag and subtending leaves
2) Potassium contents in flag and subtending leaves
3) Sodium Potassium ratio in flag and subtending leaves
4) Sodium contents in root
5) Potassium contents in root
6) Sodium and potassium ratio in root
7) Calcium contents in flag leaf and roots
8) Calcium sodium ratio in flag leaf and roots
II. Agronomic traits
1) Plant height (cm)
2) Days to 50 % flowering
3) Number of tillers per plant
4) Panicle weight (g)
5) Panicle length (cm)
6) Number of spikelets per panicle
7) Panicle fertility (%)
8) Days to maturity
9) 1000 grain weight (g)
10) Yield per plant (g)
9
Polymorphism survey of parental cultivars
The DNA of parental lines/ cultivars will be extracted from the leaves of fifteen days
old seedlings as described by Ikeda et al. (2001). Polymorphism survey will be carried out
using microsatellite markers, randomly distributed through out the rice genome. F2
populations will be selected as mapping population based on maximum polymorphism
between the parent lines/ cultivars of those crosses. The polymorphic microsatellite
markers will be used further for the genotyping of F2 populations.
Phenotyping the F3 populations for agronomic traits at maturity
After data collection, the seeds from each plant of these two populations will be
preserved. Randomly five seedlings from each candidate F2 line will be selected and
grown in saline and normal conditions till maturity. Data for physiological and agronomic
traits, as of F2 populations, will be recorded.
Genotyping the F2 population
Plant DNA extraction
Several protocols for plant DNA extraction hve been reported (Dellaporta et al., 1983;
Lange et al., 1998), however, the DNA extraction will be carried out as described by Ikeda
et al. (2001) because of its simplicity and convenience. A few pieces of young rice
seedlings (5mm) will be taken in 1.5 ml microtube. After adding 200 µl of TE buffer (10
mM Tris-HCl, 1 mM EDTA, pH 8.0) in microtubes, the leaf pieces will be grinded
thoroughly. The microtubes will be placed in boiling water for 15 minutes. After while,
800 µl of TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0) will be added. The
microtube contents will be mixed well and centrifuged at 14000 rpm for three minutes.
The supernatant will be collected and used as template DNA for PCR.
PCR amplification
PCR will be performed in 50μl reaction containing 5 µl of extracted DNA, 0.2 μM of
each primer, 100 μM of each dNTP (dATP, dCTP, dGTP and dTTP), 10 mM Tris-Cl (pH
8.3), 50 mM KCl, 1.5 mM MgCl2, 0.1 % Tritionx-100, and 1 unit Taq DNA polymerase.
Amplifications will be carried out as follows:
94 ºC for 5 minutes followed by 35 cycles of 94 ºC for 1 minute, 55 ºC for 1 minute,
72 ºC for two minutes and ending with 5 minutes at 72 ºC for the final extension.
Microsatellite primers
The unique sequences flanking microsatellite repeats have already been reported by
Wu and Tanksley (1993), Panaud et al. (1996) and Temnykh et al. (2000, 2001). They
designed to produce well-matched primers, 17-22 nucleotides long, with a G C contents
around 50 % (melting temperature approximately 60 ˚C), a low frequency of primer
dimers and preferably G or C rich at 3′ end. Primers will be selected to produce a PCR
product in the range of 80-320 bps.
Non-radioactive detection of microsatellite alleles
The amplified products, containing microsatellite regions, will be electrophoreses in 4
% polyacrylamide denaturing gel with 0.5X TBE buffer. Banding patterns will be
visualized by non-radioactive silver staining method as prescribed by Panaud et al. (1996).
10
Mapping of microsatellite markers and further construction of molecular linkage
maps
Mapping of microsatellite markers will be carried out based on polymorphic survey of
parental lines/cultivars of respective F2 population. The molecular linkage map will be
constructed by multipoint analysis (Lander and Green, 1987), using the program
MapMaker v. 2.0 (Lander et al., 1987), based on the genotype data of each F2 population.
Map distances between the microsatellite loci will be presented in centiMorgan (cM),
using the Kosambi function (Kosambi, 1944). Marker order of microsatellites will be
followed after Temnykh et al. (2001), and framework will be constructed first. The
markers with no information will be then integrated into the framework map by using
“compare” command in MapMaker program and information of physical location
available on http://www.gramene.org
Data analysis and identification of QTLs
Statistical analyses will be performed using qGene (Nelson, 1997). QTL mapping will
be conducted on the basis of F2 data by regression of trait performance on marker
genotype using standard analysis of variance (ANOVA) procedures. QTL mapping ranges
from simplest method of single point analysis (Sax, 1923) to more sophisticated methods
such as interval mapping (Lader and Botstein, 1989). However Composite Interval
mapping (CIM) is the most advanced and precise method of QTL mapping (Zeng, 1994).
This method is more precise and effective at mapping QTL compared to single point
analysis and interval mapping when linked QTLs are involved. Therefore, QTL
Cartographer (Basten et al., 1994, 2001) will be used to identify the QTL association with
salinity tolerance at maturity stage in indica rice. Mapping analyses will be carried out for
the replicated phenotyping data of each F2 population, separately. A separate analysis will
be conducted using the phenotyping data of F3 population as well. This will advance the
genetic background knowledge of salinity tolerance in rice at maturity stage.
Molecular screening of indigenous rice gene pool for salinity tolerance
The field performance of indigenous rice in saline conditions has been reported in
Pakistan (Aslam et al., 1995; Qureshi et al., 1991). However, the potential of indigenous
rice gene pool for salinity tolerance has not been exploited through molecular tools.
Plant materials will be obtained from Rice Research Institute, Kala Shah Kaku (seeds
of released rice cultivars only because it has been banned to supply the breeding materials
to other research organization by Director General (Research) AARI, Faisalabad). Plant
Genetic Resources Institute (NARC, Islamabad) is the only institute in Pakistan providing
local/ exotic plant germplasm to research organizations. There are about 4000 accessions
of rice at PGRI. Accessions of Basmati rice collected from different zones of Punjab
province will be obtained. Genetic purity of these accessions will be tested by
microsatellite markers. Pure accessions from PGRI and plant materials from RRI, Kala
Shah Kaku will be grown in salinity blocks at School of Biological Sciences, University
of The Punjab. DNA of these will be extracted from the leaves of 15 days old seedlings.
Data will be recorded for agronomic and physiological traits at maturity stage. Identified
QTLs, associated with these traits under saline conditions, will be used for molecular
screening of indigenous rice gene pool to validate the association of these QTLs with
microsatellite markers.
11
Production of salt tolerant rice cultivars is one of the breeding objectives of Economic
Botany Division, Soil Salinity Research Institute, Pindi Bhattian. Several kinds of rice
material (commercial cultivars/ advanced lines/ segregating populations) are screened in
saline conditions (salinity blocks and saline fields) to determine the potential of respective
material for salinity tolerance. Selected plant materials will be grown in salinity blocks
and saline fields (EC 6-8 dS/m). The DNA of plant materials will be extracted from leaves
of 15 days old seedlings. The data will be recorded for agronomic traits by Economic
Botany Division at SSRI. However, molecular screening and data for physiological traits
will be carried at School of Biological Sciences, University of The Punjab and Soil
Fertility Research Institute Lahore, respectively.
Final report submission
Final report will be submitted to PARB, the last activity in the proposed research
project.
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Yeo AR, ME Yeo, SA Flowers and TJ Flowers 1990. Screening of rice (Oryza sativa L.)
genotypes for physiological characters contributing to salinity resistance, and their
relationship to overall performance. Theo Appl Genet 79: 377-384.
Yeo AR and TJ Flowers 1993. Varietal differences in the toxicity of sodium ions in rice
leaves. Physiol Plant 59: 189–195.
Zeng L and MC Shannon 2000. Salinity effects on seedling growth and yield components
of rice. Crop Sci. 40: 996-1003.
Zeng L, MC Shannon and SM Lesch. 2001. Timing of salinity stress: affects on rice
growth and yield components. Agric water Manag 48: 191-206.
Zeng ZB 1994. Precision mapping of quantitative trait loci. Genetics 136: 1457–1468.
Zhang GY, G Yan, SL Chen and SY Chen. 1995. RFLP tagging of salt tolerance gene in
rice. Pl Sci 110: 227-234.
b. Milestones:
Item
Description
Achievement indicators
Risk involved
Completion
Scientists Involved
date
Polymorphism November 30, Dr. M. Arshad Javed*
2013
Dr. Takashige Ishii
level of
Dr. Shahid Mehmood
parental lines/
Mr. Ghulam. Shabbir
cultivars
Cost
(Rs.
million)
23.148
Objective Identification of microsatellite
loci associated with agronomic
and physiological traits at
maturity stage under saline
conditions in indica rice (Oryza
sativa L.)
Microsatellite loci
showing association with
salinity tolerance at
maturity stage in indica
rice
Output-1 Synthesis of F2 populations
Seeds of different F2
populations
Seedlings in seedling boxes
Nil
Nil
July 31, 2009
//
0.002
DNA in microtubes
Nil
July 31, 2009
//
0.010
Plants in the pots
Nil
Nov. 15, 2009
//
0.003
Images of microsatellite
banding pattern of parent
cultivars and F1 seedlings
Appropriate F2
populations for mapping
Images of banding
patterens of microsatellite
markers throughout the rice
genome
Tables and figures showing
the facts for the selection
of appropriate mapping
populations
Nil
March 31, 2010
//
0.035
Nil
June 30, 2010
Nil
May 30, 2010
//
0.0500
Nil
June 30, 2010
//
0.100
Activity-1 Growing F1 seeds along with
parent cultivars in seedling boxes
Activity-2 DNA extraction of parent cultivars
and F1 seedlings
Activity 3 Growing parent cultivars and F1
seedlings till maturity.
Activity 4 Confirmation of true hybrid
seedlings to get F2 true seeds
Output-2 Selection of mapping
populations
Activity-1 Screening for polymorphic
microsatellte markers throughout
the rice genome between the
parental cultivars
Activity-2 Polymorphism survey, analysis
and further selection of
appropriate mapping populations
March 31, 2010 Dr. M. Arshad Javed
Dr. M Arshad Javed
0.050
0.600
15
June 30, 2010
Two salinity blocks with
uniform medium to
Nil
screen the rice genotypes
for salinity tolerance at
an EC 6-8 dS/m
//
Salinity block construction
Two salinity blocks, each
Nil
of 20 x 40 feet in size
//
Survey and selection of soil having Enough soil of desired
Nil
an EC 6-8 dS/m and filling of
salinity level
salinity blocks with that soil
May 30, 2011
Phenotyping F2 genotypes
Data of agronomic and
Nil
physiological traits under
saline conditions
Oct. 31, 2010
Growing F2 populations in salinity Replicated F2 populations
Nil
blocks and normal field conditions in salinity blocks and
till maturity
normal field
Jan. 31, 2011
Recording the data for agronomic
Data of F2 populations
Nil
traits e.g. yield and yield
components
April 31, 2011
Analyses of plant samples for
Physiological data of F2
Nil
physiological parameters. (Plant
populations
samples will be 9000 and analyses
of each sample will be carried out
for 9 physiological traits)
May 30, 2011
Statistical analysis
Tables and figures e.g.
Nil
correlation tables etc
Construction of molecular
Molecular linkage map
indica x indica March 31, 2012
linkage map
background,
may result in
low
polymorphism
July 31, 2010
DNA extraction of F2 populations Extracted DNA in
Nil
microtubes
Output-3 Construction of salinity blocks
Activity-1
Activity-2
Output-4
Activity-1
Activity-2
Activity 3
Activity 4
Output-5
Activity-1
Dr. M Arshad Javed
1.200
//
1.0
//
0.2
Dr. M Arshad Javed*
Dr. Shahid Mehmood
0.800
Dr. M Arshad Javed
0.050
//
0.120
Dr. Shahid Mehmood
0.600
Dr. M Arshad Javed
0.030
Dr. M Arshad Javed
2.500
//
0.100
16
Activity-2 Selection of microsaellite markers
for genotyping the F2 populations
throughout the rice genome
Activity-3 Genotyping of F2 populations
Nil
Sep. 30, 2010
//
0.400
Nil
Sep. 30, 2011
//
1.450
Activity-4
Nil
Dec. 31, 2011
//
0.200
Nil
Jan. 31, 2012
//
0.300
Nil
March 31,
2012
//
0.050
Nil
June 30, 2012
Dr. M Arshad Javed*
Dr. Shahid Mehmood
1.200
Nil
Nov. 30, 2011
//
0.150
Nil
March 31,
2012
//
0.300
Nil
March 31,
2012
Dr. Shahid Mehmood
0.750
Activity-5
Activity-6
Output-6
List and images of
polymorphic microsatellite
markers with fine bands
Images and scored data
showing homoheterozygousity at all the
mapped microsatellite loci
throughout the rice genome
Construction of Molecular
Images showing the
Linkage Maps for each
positions of microsatellite
populations
loci on different linkage
groups
Integrating the microstaellite
Scored data showing
markers in between the loci having homo-heterozygosity at all
a distance more than 20 cM (Centi the mapped microsatellite
Morgan) in both molecular linkage loci throughout the rice
groups
genome
Re-construction of Molecular
Positions of microsatellite
Linkage Maps using new scoring
loci on different linkage
by MapMaker
group
Phenotyping of F3 populations
Data of agronomic and
physiological traits
Activity-1 Growing 5 plants from each 200
F2 genotype in saline and normal
field till maturity
Activity-2 Recording the data for agronomic
traits e.g. yield and yield
components
Activity 3 Analyses of plant samples for
physiological parameters. (Plant
samples will be 12000 and analyses
of each sample will be carried out
for 9 physiological traits)
Rice populations in
salinity blocks and normal
field
Analysed data in the form
of tables/ figures.
//
17
Output-7 Quantitative trait loci analysis
for yield, yield components and
physiological traits determine
the rice productivity in saline
conditions using the phenotyping
data of F2 and F3 genotypes
Activity-1 Analyses of phenotypic data of F2
populations
Identified microsatellite
loci associated with
salinity tolerance in rice
(Oryza sativa L.)
Nil
May 31, 2012
Dr. Takashige Ishii*
Dr. M Arshad Javed
0.500
Statistical analyses (tables
and figs)
//
Dr. Takashige Ishii*
Dr. M Arshad Javed
0.100
Activity-2 Analyses of phenotypic data of F3
populations
Activity-3 Quantitative trait loci analysis
using F2 replicated data
Statistical analyses (tables
and figs)
Locations on different
chromosomes and
associations of
microsatellite loci with
traits which determine the
rice productivity in saline
conditions
Locations on different
chromosomes and
associations of
microsatellite loci with
traits which determine the
rice productivity in saline
conditions
Data of molecular
screening of fine/ coarse
rice cultivars/ advanced
lines with reference to
identified QTLs
Statistical
analyses
(tables and
figures)
Nil
//
//
0.125
Nil
//
//
0.150
Nil
//
//
0.125
Nil
Aug. 31, 2013
Dr. M Arshad Javed*
Dr. Shahid Mehmood
Mr. Ghulam Shabbir
1.600
Activity-4 Quantitative trait loci analysis
using F3 data
Output 8
Molecular screening of Basmati/
non Basmati rice cultivars/
advanced lines/ segregating
populations/ indigenous rice
germplasm for QTLs associated
with agronomic and physiological
traits in saline conditions
18
Activity-1 Collection of fine/coarse rice
germplasm from Rice Research
Institutes and Plant Genetic
Resources, NARC, Islamabad
Activity 2 Growing indigenous rice cultivars/
varieties/ advanced lines/
segregating populations till
maturity in salinity blocks at SBS
Activity 3 DNA extraction of plant materials
from experiment at School of
Biological Sciences and SSRI
Pindi Bhattian
Activity 4 Phenotyping of rice germplasm at
SBS
Activity 5 Following activities will be
carried out at Soil Salinity
Research Institute, Pindi Bhattian,
Hafizabad.
i. Growing indigenous rice
cultivars/ varieties/ advanced
lines/ segregating populations
(about 100 plants) in salinity
blocks/ saline fields (EC 6-8
dS/m)
ii. Recording data for agronomic
traits at maturity.
iii. Plant samples for physiological
analyses
Activity 6 Analysis of physiological traits at
maturity stage (plant samples from
SBS and SSRI) at Soil Fertility
Research Institute Lahore
Seeds of rice germplasm
Nil
April 30, 2012
//
0.050
Rice in the salinity blocks
Nil
Nov 30, 2012
//
0.050
DNA in microtubes
Nil
Aug. 30, 2012
//
0.100
Data of agronomic traits
Nil
April 30, 2013
//
0.150
Rice germplasm in the
salinity blocks
Nil
April 30, 2013
Mr. Ghulam. Shabbir
0.300
Data of physiological traits
Nil
May 31, 2013
Dr. Shahid Mehmood
0.500
19
Nil
August 30,
2013
Output-9 Final report
Images of microsatellite
bands showing identified
QTLs associated with
respective traits
Project completed
Nil
Activity 1 Submission of final report
Submission
Nil
November 30, Dr. M Arshad Javed
2013
November 30, Dr. M Arshad Javed
2013
Activity 7 Validation of identified QTLs
associated with salinity tolerance
at maturity stage
Salaries
Equipments
Management cost and incentives etc
Put * on the incharge of the activity. Put share of each scientist in ( ), if different than equal.
Dr. M Arshad Javed
0.450
0.130
0.130
4.263
3.567
6.743
16.
PROJECT STAFF DESCRIPTION:
Additional staff requirements and their proposed qualifications
Name of post
No. of posts with
justification
Research
One
associate
Justification:
Following duties will
(Only Host
performed by research
Institute)
associates;
1. Screening and
phenotyping of
segregating
populations in
saline conditions
2. Data collection for
plant and yield
related traits
3. Polymorphic
survey of
microsatellites in
parental lines
4. DNA extraction of
F2 population
5. PCR,
Electrophoresis,
non-radioactive
silver staining etc.
6. Scoring of bands
Daily wages
1. Growing rice
labour
nursery and
(Host institute:
transplantation
2-3 man days/
2. Help in lab work
day,
(e.g. washing,
counting of seeds
Collaborating
institutes:
etc.) and data
variable
collection etc.
numbers of
man days will
be required
depending on
rice growing
season and data
collection)
Proposed
qualifications
Master
degree in
Agriculture
sciences/
Plant
Sciences/
Molecular
Biology
Ability to
read and
write easy
English will
be preferred
Experience
Pay Package
Research
experience
in rice
salinity
tolerance
and
molecular
biology
will be
preferred
Rs.
40,000/month
+ 7 % annual
increment
and one
additional
salary for
each
completed
year on
completion of
the project
Familiar
with
growing
crop plants
Rs. 250/ day
+ 10 % bonus
of wages
21
17.
SUMMARY OF THE BUDGET (Details as Annexure)
Item of Expenditure
Research Phase
(Host and collaborating institutes)
(Million Rs.)
Demon. Phase
Year 1 Year 2 Year 3 Year 4
Year 5
Total
Rs million
Salaries
Resaerch Associate (1)
School of Biological Sciences (Host Institute)
0.48
0.530
0.581
0.631
0.555
2.777
0.20
0.05
0.000
0.728
0.297
0.099
0.000
0.926
0.297
0.099
0.000
0.977
0.198
0.074
0.074
0.977
0.099
0.000
0.000
0.654
1.089
0.322
0.074
4.263
0.690 0.710
0.420 0.000
0.230 0.000
1.340 0.710
6.290
2.055
0.230
8.575
Daily paid labour
School of Biological Sciences (Host Institute)
Soil Fertility Research Institute (Colleborator II)
Soil Salinbity Rsearch Institute (ColleboratorIII)
Sub Total (A)
Operating
2.270 1.540 1.080
0.250 0.625 0.760
0.000 0.000 0.000
Sub Total (B) 2.520 2.165 1.840
School of Biological Sciences (Host Institute)
Soil Fertility Research Institute (Colleborator II)
Soil Salinbity Rsearch Institute (ColleboratorIII)
Equipment
School of Biological Sciences (Host Institute)
Sub Total (C)
3.567
3.567
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
3.567
3.567
0.000
0.425
0.425
0.000
0.000
0.000
0.390
0.000
0.390
0.000
0.000
0.000
0.000
0.000
0.000
0.390
0.425
0.815
0.000
0.000
0.000
0.000
0.250
0.250
0.000
0.000
0.000
0.000
0.250
0.250
1.629
0.106
0.075
0.000
1.810
0.592
0.000
0.181
0.000
0.773
0.587
0.063
0.215
0.000
0.864
0.380
0.000
0.124
0.076
0.579
0.341
0.000
0.000
0.000
0.341
3.528
0.169
0.594
0.076
4.367
0.407
0.027
0.019
0.000
0.453
0.091
0.091
9.593
0.148
0.000
0.045
0.000
0.193
0.039
0.039
4.096
0.147
0.016
0.054
0.000
0.216
0.043
0.043
4.580
0.095
0.000
0.031
0.019
0.145
0.029
0.029
3.071
0.085
0.000
0.000
0.000
0.085
0.017
0.017
1.807
0.882
0.042
0.149
0.019
1.092
0.219
0.219
23.148
Overseas Travel
School of Biological Sciences (Host Institute)
Kobe University, Japan (Collaborator I)
Sub Total (D)
International colleboration
Kobe University, Japan (Collaborator I)
Sub Total (E)
Management Cost (25% of the project cost)
School of Biological Sciences (Host Institute)
Kobe University, Japan (Collaborator I)
Soil Fertility Research Institute (Colleborator II)
Soil Salinbity Rsearch Institute (ColleboratorIII)
Sub Total (F)
Incentive for Scientists (5% project cost)
School of Biological Sciences (Host Institute)
Kobe University, Japan (Collaborator I)
Soil Fertility Research Institute (Colleborator II)
Soil Salinbity Rsearch Institute (ColleboratorIII)
Sub Total (G)
Incentive for PM (1% of the project cost)
Sub Total (H)
G. Total (A+B+C+D+E+F+G)
22
18. BUDGET INSTALMENTS
Instalment
Host
Collaborating
(Half Yearly)
Institute
Institute I
st
5.657
0.000
1
Collaborating Collaborating
Institute II
Institute III
0.190
0.000
Total
(Rs. Million)
5.847
2nd
2.986
0.558
0.204
0.000
3.748
rd
1.825
0.000
0.440
0.000
2.265
4th
1.321
0.000
0.511
0.000
1.832
th
1.536
0.000
0.515
0.000
2.051
th
1.589
0.328
0.612
0.000
2.529
th
1.015
0.000
0.324
0.235
1.574
th
1.008
0.000
0.324
0.164
1.497
th
1.807
18.742
0.000
0.886
0.000
3.120
0.000
0.399
1.807
23.148
3
5
6
7
8
9
Total
19.INTERNATIONAL COLLABORATION
Analyses of quantitative traits are often complicated due to gene-environment
interactions. Multiple research approaches will be practised to identify the quantitative
trait loci (QTL) associated with salinity tolerance at maturity stage. Emphasis will be
given to map the stable and major QTLs. Multiple analyses of phenotypic, derived from
different populations, and genotyping data will be carried out in the Laboratory of Plant
Breeding, Faculty of Agriculture, Kobe University, Kobe, Japan
.
20.INTRNATIONAL TRAVELS
i. HOST INSTITUTE
a) Name of scientist(s) visiting institute and number of visits
Dr. Muhammad Arshad Javed, (PM) No. of Visit (1)
b) Name of institute(s) to be visited
Laboratory of Plant Breeding, Faculty of Agriculture, Kobe University,
Kobe, Japan
c) Purpose of each visit

To conduct multiple analyses to identify the QTLs associated with salinity
tolerance at maturity stage using the phenotyping data from replicated F2
populations as well as phenotyping data obtained in F3 population under
the supervision of Dr. Ishii.

Identification of major and minor QTLs and to discuss the further potential
utilization of these QTLs in rice breeding programs to incorporate salinity
tolerance in promising rice lines/varieties.
ii. COLLABORATING INSTITUTE
a) Name of scientist(s) visiting institute and number of visits
Dr Takashige Ishii
No. of visit
(1)
b) Name of institute(s) to be visited
School of Biological Sciences, University of the Punjab, Lahore
23
c) Purpose of each visit

To get guideline and benefits from his expertise in making precise
phynotyping under saline conditions and genotyping procedure to
minimize the experimental error

To provide an opportunity to Pakistani rice researchers to interact with one
of the pioneers of microsatellite development in Cornell University
21. IMPORT OF TECHNOLOGIES
(Not applicable)
22. COMMERCIALIZATION AND BENEFIT TO END USERS
i) Method of transferring results:
a) The facility of molecular screening will be extended for other research
institutes to screen their breeding material for salinity tolerance
b) Results of proposed studies will be presented in international and national
conferences
c) Seminar will be arranged for the plant researchers to disseminate the
knowledge related to molecular screening for salinity tolerance in rice.
d) The findings will be published in reputed scientific journals.
e) A final report will be submitted to PARB at the end of the project.
ii) Agency/company/consultants involved in adaptation and adoption
a) Rice research institutes /organizations in Punjab and Pakistan
b) NARC, Islamabad
c) Ayub Agricultural research Institute, Faisalabad
d) Private seed business and research organizations e.g. Guard rice, Emkey seeds
etc.
iii) Expected benefits to end users
a) Plant scientists will get an opportunity to know the genetic background of
salinity tolerance.
b) The quantitative trait loci (QTLs) associated with salinity tolerance at maturity
satge will enhance the breeder’s capability to identify the desirable genotypes
in segregation populations at DNA level, without environmental effects.
c) Molecular screening for salinity tolerance will save the capital, time and labour
resources in screening the segregating populations.
d) Pyramiding of major QTLs will accelerate the development of salt tolerant rice
cultivars.
e) Salinity tolerance will be incorporated in promising rice lines/cultivars through
marker assisted backcrossing. Ultimately, the marginal soils would be put
under rice cultivation. Fine mapping of these loci will lead to clone the genes
as well.
23. FINAL REPORT SUBMISSION (date)
Final report will be submitted on November 30, 2013
24
Annexure for Host Institution, School of Biological Sciences (SBS)
DETAILED ACTIVITY WISE COSTS (Attach basis of calculation as annexure)
(Million Rupees)
Budget
Research phase
Demonstration phase
Code Item of Expenditure
Year 1 Year 2 Year 3 Year 4 Year 5 Total
A. Salaries
One Research Associate @ Rs. 40000/PM with
Annual Incremnet of 7% of initial pay
0.480 0.530 0.581 0.631 0.341 2.563
One pay/completed year for Research associate
0.000 0.000 0.000 0.000 0.214 0.214
Daily wages labour (Rs 250/man day+10% bonus) 0.198 0.297 0.297 0.198 0.099 1.089
Sub Total (A) 0.678 0.827 0.878 0.829 0.653 3.866
B. Operational
Research Material & Supplies
Fertilizer
0.005 0.010 0.015 0.025 0.000 0.055
Irrigation
0.010 0.015 0.020 0.020 0.000 0.065
Selfing bags/tags/labels etc
0.015 0.025 0.025 0.025 0.010 0.100
Pesticides
0.005 0.010 0.015 0.025 0.000 0.055
Glass wares
0.250 0.125 0.100 0.075 0.000 0.550
Plastic wares
0.150 0.110 0.090 0.080 0.500 0.930
Chemicals
0.450 0.300 0.300 0.100 0.000 1.150
Microsatellite primers (Rice markers)
1.100 0.650 0.250 0.090 0.000 2.090
Travelling Allowance
0.100 0.050 0.050 0.050 0.050 0.300
POL
0.100 0.075 0.075 0.075 0.050 0.375
Stationery
0.020 0.030 0.030 0.040 0.040 0.160
Repair of equipments/machinery
0.000 0.075 0.050 0.025 0.000 0.150
Communication costs (postage/phone/fax/internet) 0.030 0.040 0.040 0.040 0.040 0.190
Advertisement costs
0.020 0.015 0.010 0.010 0.010 0.065
Printing costs
0.015 0.010 0.010 0.010 0.010 0.055
Sub Total (B) 2.270 1.540 1.080 0.690 0.710 6.290
C. Machinery and equipment
Salinity Blocks with glass roof+sorrounding fence 1.000 0.000 0.000 0.000 0.000 1.000
Cold cabinet (2)
0.100 0.000 0.000 0.000 0.000 0.100
EC meter (1)
0.085 0.000 0.000 0.000 0.000 0.085
pH meter (1)
0.060 0.000 0.000 0.000 0.000 0.060
Seed counter
0.350 0.000 0.000 0.000 0.000 0.350
0.045 0.000 0.000 0.000 0.000 0.045
Hot plate (1)
Genequencer + power supply unit + accessories
0.525 0.000 0.000 0.000 0.000 0.525
PCR machine multiplex (96 well)
0.625 0.000 0.000 0.000 0.000 0.625
Silver staining shaker (1)
0.045 0.000 0.000 0.000 0.000 0.045
Silver staining trays (6)
0.042 0.000 0.000 0.000 0.000 0.042
Mini leaf crusher and grinder (1)
0.035 0.000 0.000 0.000 0.000 0.035
25
Manual pipettes (4)
Multichannel pipettes (2)
Multi0channel syringe and accessories (2)
Water boiling chamber (1)
Gel dryer (1)
Laboratory cabinets (2)
Digital camera (1)
Computer (1) + accessories
Sub Total (C)
(D) Overseas Travel
Dr M Arshad Javed (PM)
Sub Total (D)
Project cost E = (A+B+C+D)
Management Cost(MC) 25% of the project cost
TOTAL F = (E + MC)
Incentive for Scientists (5% of the project cost)
Incentive for PM (1% of the project cost)
Incentive for PM (1% of the Collab.1 Budget)
Incentive for PM (1% of the Collab.2 Budget)
Incentive for PM (1% of the Callab.3 Budget)
Sub Total (G)
TOTAL PROJECT COST (F+G)
0.075
0.090
0.180
0.050
0.075
0.065
0.020
0.100
3.567
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.075
0.090
0.180
0.050
0.075
0.065
0.020
0.100
3.567
0.000
0.000
6.515
1.629
8.144
0.407
0.081
0.005
0.004
0.000
0.498
8.641
0.000
0.000
2.367
0.592
2.959
0.148
0.030
0.000
0.009
0.000
0.187
3.146
0.390
0.390
2.348
0.587
2.935
0.147
0.029
0.003
0.011
0.000
0.190
3.125
0.000
0.000
1.519
0.380
1.899
0.095
0.019
0.000
0.006
0.004
0.124
2.023
0.000
0.000
1.363
0.341
1.704
0.085
0.017
0.000
0.000
0.000
0.102
1.807
0.390
0.390
14.113
3.528
17.641
0.882
0.176
0.008
0.030
0.004
1.101
18.742
26
Annexure for Collaborator I, Kobe University, Japan
DETAILED ACTIVITY WISE COSTS (Attach basis of calculation as annexure)
(Million Rupees)
Budget
Research phase
Demonstration phase
Code Item of Expenditure
Year 1 Year 2 Year 3 Year 4 Year 5
Total
(A) Overseas Travel
Dr Takashige Ishii (Japanese Colleborator)
International Colleboration with Kobe
University for the analyses of phenotypic and
genotypic data for the identification of QTLs
associated with salinity tolerance in indica rice
0.425 0.000 0.000 0.000 0.000
0.000
Sub Total (A) 0.425
Management Cost(MC) 25% of the project cost
0.106
TOTAL B = (A + MC) 0.531
0.027
Incentive for Scientists (5% of the project cost)
Sub Total (C) 0.027
0.000
0.000
0.000
0.000
0.000
0.000
0.250
0.250
0.063
0.313
0.016
0.016
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.425
0.250
0.675
0.169
0.844
0.042
0.042
27
Annexure for Collaborator II, Soil Fertility Research Institute (SFRI)
DETAILED ACTIVITY WISE COSTS (Attach basis of calculation as annexure)
(Million Rupees)
Budget
Research phase
Demonstration phase
Code Item of Expenditure
Year 1 Year 2 Year 3 Year 4 Year 5 Total
A. Salaries
Daily wages labour (Rs 250/man day+10% bonus) 0.050 0.099 0.099 0.074 0.000 0.322
Sub Total (A) 0.050 0.099 0.099 0.074 0.000 0.322
B. Operational
Research Material & Supplies
Plastic and paper bags/tags/labels etc
0.050 0.075 0.025 0.025 0.000 0.175
Glass wares
0.050 0.050 0.075 0.075 0.000 0.250
Plastic wares
0.050 0.050 0.075 0.070 0.000 0.245
Chemicals
0.025 0.300 0.435 0.100 0.000 0.860
Travelling Allowance
0.025 0.025 0.025 0.025 0.000 0.100
POL
0.025 0.075 0.050 0.035 0.000 0.185
Stationery
0.005 0.025 0.025 0.045 0.000 0.100
Repair of equipments/machinery
0.000 0.000 0.020 0.025 0.000 0.045
Communication costs (postage/phone/fax/internet) 0.020 0.020 0.020 0.020 0.000 0.080
Printing costs
0.000 0.005 0.010 0.000 0.000 0.015
Sub Total (B) 0.250 0.625 0.760 0.420 0.000 2.055
Project cost C = (A+B) 0.300 0.724 0.859 0.494 0.000 2.377
Management Cost(MC) 25% of the project cost
0.075 0.181 0.215 0.124 0.000 0.594
TOTAL F = (C + MC) 0.375 0.905 1.074 0.618 0.000 2.971
0.019 0.045 0.054 0.031 0.000 0.149
Incentive for Scientists (5% of the project cost)
Sub Total (D) 0.019 0.045 0.054 0.031 0.000 0.149
TOTAL PROJECT COST (C+D)
0.394 0.950 1.127 0.648 0.000 3.120
28
Annexure for Collaborator III, Soil Salinity Research Institute (SSRI)
DETAILED ACTIVITY WISE COSTS (Attach basis of calculation as annexure)
(Million Rupees)
Budget
Research phase
Demonstration phase
Code Item of Expenditure
Year 1 Year 2 Year 3 Year 4 Year 5 Total
A. Salaries
Daily wages labour (Rs 250/man day+10% bonus) 0.000 0.000 0.000 0.074 0.000 0.074
Sub Total (A) 0.000 0.000 0.000 0.074 0.000 0.074
B. Operational
Research Material & Supplies
Fertilizer
0.000 0.000 0.000 0.020 0.000 0.020
Irrigation
0.000 0.000 0.000 0.020 0.000 0.020
Selfing bags/tags/labels etc
0.000 0.000 0.000 0.030 0.000 0.030
Pesticides
0.000 0.000 0.000 0.020 0.000 0.020
Plastic wares
0.000 0.000 0.000 0.010 0.000 0.010
Travelling Allowance
0.000 0.000 0.000 0.050 0.000 0.050
POL
0.000 0.000 0.000 0.025 0.000 0.025
Stationery
0.000 0.000 0.000 0.030 0.000 0.030
Communication costs (postage/phone/fax/internet) 0.000 0.000 0.000 0.020 0.000 0.020
Printing costs
0.000 0.000 0.000 0.005 0.000 0.005
Sub Total (B) 0.000 0.000 0.000 0.230 0.000 0.230
Project cost C = (A+B) 0.000 0.000 0.000 0.304 0.000 0.304
Management Cost(MC) 25% of the project cost
0.000 0.000 0.000 0.076 0.000 0.076
TOTAL D = (C + MC) 0.000 0.000 0.000 0.380 0.000 0.380
Honoraria/Incentives for Scientists (5% of the project cost) 0.000 0.000 0.000 0.019 0.000 0.019
Sub Total (E) 0.000 0.000 0.000 0.019 0.000 0.019
TOTAL PROJECT COST (D+E)
0.000 0.000 0.000 0.399 0.000 0.399