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A)   
Work Plan

 

i)                   
Plant materials and Inoculation

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A resistant plant and a susceptible plant
will be crossed to obtain F1 progenies and F2 population.
Then F1 seeds will be planted and also polymorphic microsatellite
locus analysis will be conducted between parents, in order to confirm that
plant arising from this cross will be hybrid.  
For preparing inoculums, the urediniospores will be obtained from the plants and it will be subjected to heat shock
treatment to break its dormancy. Its concentration will be determined with the
help of Neubauer chamber. Consequently, dilutions will be prepared with water as a solvent and will be sprayed on F2
population. No disease control will be applied.

 

ii)                
Evaluation
of rust resistance (Phenotypic analysis)

 

After few days the plants will be
assessed for resistance and classified according to their symptoms for typical
SR reaction. (Red-brown lesion will
denote resistance, while susceptibility will be indicated by TAN lesions). The
data obtained will be subjected to chi-square
test, at 5% level of significance to check the
segregation hypothesis.

 

 

iii)              
DNA
extraction and Bulk preparation.

Freeze dried leaf samples will be grounded in liquid nitrogen and DNA
will be extracted as per the protocol mentioned in DNeasy ® Plant
kit by Quiagen. Its concentration will be determined with the help of Nano Drop
2000/2000c. Accordingly, DNA dilutions will
be prepared to a final concentration of 10ng/ul with autoclaved Milli-Q water as diluent.

 

The bulked
segregant analysis method will be carried out to identify
SSR markers linked to rust resistance. 
For this purpose, DNA from10 highly resistant plants and 10 highly
susceptible plantswill be
pooled in equal proportions to form two bulks which contrasting traits.

 

iv)              
Identification
of molecular markers using Bulked segregant analysis

 

Approximately
500 SSR markers will be screened on contrasting parents and bulks with
contrasting traits, which will evenly cover the entire 20 chromosomes of
soybean. Amplification reaction will be carried out in Mastercycler PCR system
which will have a final volume of 20 ?L, containing 4.0?L DNA at 10 ng/?Lgenomic DNA of
soybean, 2.0 ?L of 10X PCR buffer, 0.4 ?L MgCl2 at
25 mM ?L-1, 2 ?L dNTPs at 2.0 mM ?L-1, 2
?L primer at 10 ?g/ul (Foward and Reverse mixture) and 0.2 ?LTaq (5 U ?L-1).
The program to be used for DNA amplification consists
of an initial denaturation at 94ºC for 5min followed by 40 denaturation cycles
at 95 ºC for 30 sec. Annealing temperature would be within the range of 54ºC –
60 ºC, with respect to the primers used. Extension would be carried out at 72°C
for 30 secand finally polymerization at 72°C for 7 min.

 

PCR products
will be resolved on 2% agarose gel. Gel would be stained with Ethidium bromide
and visualized on gel documentation system.

 

v)                 
Screening and
Analysis

 

The primers
which show polymorphism between parents and bulks would be used to screen the
entire mapping population and genotypic data will be recorded. Correlation
between Phenotypic and genotypic data of the mapping population will be
analyzed using mapping software and based on recombinant frequency distance
between the resistance gene and linked markers will be identified.

 

The tightly
linked molecular markers identified in the study would be screened onrust resistance
and susceptible soybean cultivars to see the effectiveness of the marker in
identifying rust resistance gene in different genotypes.

 

 

Timeline

 

Year
Plan

Achievable
targets

1st
Year

·        
Development of F2 mapping population
·        
Screening of  F2 population
for rust resistance.
·        
Collection of  F2
population’s leaf samples and extract DNA

2nd
Year

·        
Screening F3 population for phenotypic analysis.
·        
Screening parents and F2 population’s susceptible and
resistant bulks with SSR markers.

3rd
Year

·        
Screening entire F2  population with identified putative SSR markers
·        
Linkage analyis

4th
Year

·        
Validation of markers on different soybean cultivars

 

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