The aims and objectives of the proposed research are as follows:
· Development of mapping population.
· Screening of mapping population for rust at rust hotspot or by artificial inoculation.
· Identification of Molecular markers for rust using Bulked segregant analysis (BSA).
· Screening of mapping population with putative molecular markers.
· Linkage Analysis and Validation.
To carry out the stated objectives, soybean F2 or BC1 would be developed and established as a mapping population. For this, the plants could be cultivated in a hotspot or it can be artificially inoculated with Phakopsorapachyrhizi. Then the phenotype and molecular marker genotype will be analyzed. Further the number of recombinant individuals would be counted, and the genetic distance between the molecular marker and the target gene would be calculated in cM units to generate a genetic linkage map.
B) Work Plan
i) Plant materials and Inoculation
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 uredospores 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 SBR 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 NanoDrop 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 SBR resistance. For this purpose, DNA from10 highly resistant plants and 10 highly susceptible plants will be pooled in equal proportions to form two bulks which contrasting traits.
iv) Development of SSR markers and linkage analysis
Approximately 500 SSR markers will be screened 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 94 ºC/5min followed by 40 denaturation cycles at 95 ºC for 30 sec. Annealing temperature according to the marker. The final step will consist of polymerization at 72 °C for 7 min.
2% agarose gel stained with Ethidium bromide will be used for the genotypic analyses of PCR product. Then the results of phenotypic and genotypic analyses will be utilized to calculate the genetic distance between the marker and resistance gene using GeneMapper.
· Cultivate soybean.
· Screen F2 or BC1 for rust resistance.
· Collect F2 leaf samples and extract DNA
· Screen F3 for phenotypic and genotypic analysis.
· Screen Parents and F2 susceptible and resistant bulks with putative markers.
· Screen entire F2 with putative markers
· Construct Genetic Map
· Validate markers on different cultivars