1. i. Origin of Proposal
Human communities are greatly impacted by plant pathogens that cause serious epidemics in crop plants 1. One such example is an obligate fungus Phakopsorapachyrhizi Syd. & P. Syd which incites Soybean Rust (SBR) 2. SBR has been one of the most destructive diseases affecting the most important economic crop, soybean(Glycine max (L) Merr.). Soybean is a promising source of vegetable oil, nutraceuticals and protein.10 to 80% of yield losses are reported worldwide under the conducive environmental conditions for the development of disease 3. Defoliation and early maturation are observed in infected plants which ultimately leads to a reduction of weight and quality of grains. To combat this, resistant varieties of soybean should be planted but due to their limited availability, application of fungicides the only option left out for farmers. Nonetheless, fungicide treatments are high-priced and cause contamination of the environment. Over a period of time pathogens also tend to develop tolerance to certain fungicide, endangering the cultivation of soybean. Thus, the strategy adopted to sustain economically and environmentally is to search for resistant varieties. 4 For this purpose phenotypic screening of segregating population can be carried out but it is time-consuming and laborious. Hence, resistance genotypes in segregating population must be identified in the early breeding stages for successful breeding and cultivation of disease resistant varieties. This can be done with DNA marker-assisted selection. Microsatellites and SNPs (Single Nucleotide Polymorphism) are the most commonly used DNA markers for mapping the genomic regions in soybean. 5 Microsatellite markers (SSR) have a higher advantage over SNPs due to their high polymorphism, reproducibility, co-dominance and distributed across the genome. 6
ii. a) Rationale of the Study supported by cited literature
Most of the soybean growing countries have reported the presence of SBR. Amongst which Japan is the first country to report in 1903 7. During mid-century rust was confined to East Asia and Australia. But the disease started spreading to different countries since it disperses in the form of urediniospores through wind 8. In India, it was first accounted in 19709, in Puerto Rico in 1956 10, and in Hawai in 1994 11. By the beginning of the twentieth century, it was detected in Brazil, Paraguay, and Argentina 12 13. And by 2004, pathogen showed up in South America 14 followed by North America in 2005 15.
Some P. pachyrhizi populations have exhibited tolerance to fungicides16. Therefore the development of high yielding cultivars resistant to rust pathogen will be a sustainable approach to control SBR. Worldwide researchers have screened for resistance or tolerance to SBR 17 and identified seven different loci carrying dominant alleles: Rpp1 18, Rpp2 19, Rpp3 20, Rpp4 21, Rpp5 22, Rpp6 23 and Rpp1-b 24. However, these genes are not effective for all population of P. pachyrhizi25. Some researchers have identified recessive genes in association with SBR resistance 26.
Molecular markers are an essential tool to monitor the transfer alleles of interest for the development of resistant varieties 27. SSR’s have been used for mapping specific genes in soybean to determine the traits, QTLs, resistance to diseases and pest etc. 28. However, some P. pachyrhizi population have evolved the ability to prevail over single-gene resistance 29 30. Therefore, there is a need for identification of novel genes linked to SBR resistance for the development of resistance cultivar.
SBR is controlled by multiple genes.
c) Key Questions
· Establishing of mapping population.
· Assessing mapping population for rust.
· Identifying Molecular markers.
· Assessing mapping population with putativemolecular markers.
· Generating linkage map.
iii. Current status of research and development on the subject
Rpp1 and Rpp2 were incorporated in breeding programs from the time period 2000-2010. But within two years Brazil reported that the variety lost its effectiveness 31.Later on, a study conducted in the U.S. indicated that heterogenous fungal population and presence of one or more isolates gave the ability to overcome the resistance. This study also found that Rpp1 gene and Rpp6 gene, present in PI 200492 and PI 567102B respectively showed high levels of resistance. On comparison to susceptible (control), Rpp2, Rpp3, Rpp4 and Rpp5 varieties gave rise to incomplete resistance and moderate levels of rust development 32. According to one Study, type of lesions was no correlation to severity to rust33.PI 506764 (Hyuuga), contain resistance genes Rpp3 and Rpp5, but on classification, it showed same results as PI 200492 (Rpp1) and PI 462312 (Rpp3) lead to the observation that moderate resistance conferred by some genes is unable to discriminate between race-specific and race-nonspecific resistance 34.
India ranks fifth in the world with regards to the cultivation of Soybean. SBR cause around 10 – 90% of yield loss. Initially, SBR was found in Northeastern states, hills of U.P. and West Bengal 35. Now rust is known to occur in almost all states. To overcome this disease, researchers came up with a botanical fungicide, neem seed kernel extract along with hexaconazole to reduce the severity of disease 36. On screening, two lines NRC 80 and MAUS 417 were found to be moderately susceptible to natural epiphytotic conditions at Meghalaya 37. A species, Glycine tomentella was found to contain Rpp resistance genes same as Soybean. Researchers from Palampur, Himachal Pradesh had participated in this study conducted by University of Illinois and United Soybean Board project and demonstrated the capability to backcross resistance genes into Soybean from G. tomentella38.
iv. The relevance and expected outcome of the proposed study:
SSR markers are widely distributed across host genome. Moreover, it is highly reproducible, reliable, easy to analyze, and co-dominant hence they are ideal molecular markers to monitor the transfer alleles of interest for the development of resistant varieties 6
In the proposed proposal we aim to divulge the pattern of inheritance of soybean resistance to rust and screen molecular markers that are linked to resistance genes to improve breeding schemes for SBR resistance varieties.
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.
C) 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
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7. Duration of the Project: 4 years
8. Budget of the Project
1 Laboratory equipment:
Laminar Air Flow
Sigma – Aldrich
Total cost of equipments: Rs. 19,00,000
Preparation of Media, buffer, and gel for electrophoresis is needed for the project which explains the need for refrigerator, autoclave, microwave, magnetic stirrer, pH meter and weighing balance. Few sample of plants needs to maintain in the laboratory which justifies the necessity for growth chamber. Prepare media and PCR mixture, has to be done under sterile condition, to avoid contamination, which clarifies the need for Laminar Air flow hood. Minispin, Vortex, Nano drop, multi-block heater and Ice maker are needed for extraction and quantification of DNA. PCR system to amplify the DNA. For electrophoresis and visualization of gel, electrophoresis unit and gel doc are required. To calculate genetic distance, software like GeneMapper is needed, this justifies the need for a computer.
Number of personnel
Justification for Manpower and salaries /wages: –
Senior and Junior Scientists will be engaged in research work. Lab technician, required for
assistance in research work and to maintain the laboratory in good conditions. Helper would be required to assist in field work.
The concerned institute would cater the salaries of all experts.
DNeasy® Plant kit
Glass wares and Plastic wares
Microfuge tubes and tips
(Mortar and pestle, aluminum foil, cotton, Tissue roll, Gloves)
Total cost of consumables: Rs. 6,00,000
Justification for consumables: –
The budget includes reagents for media preparation, PCR mixture and electrophoresis. Majority of the chemicals will be acquired from Himedia, Sigma and SRL. The cost also includes DNA extraction reagents, glass wares and plastic wares which will be utilized throughout the project. Microfuge tubes and tips would be required for DNA extraction and PCR, which will be procured from Tarsons. Miscellaneous will be purchased in bulk quantity from the local retailer.
4 Other Requirements
The total cost of other requirements: Rs. 10,00,000
Finance required for the electricity bill, water bill, communication bill, insurance etc. are included in contingencies. An amount is also kept aside for maintaining and validating instruments. Field amount consists of an amount of manure, fertilizers, farming equipment etc.
* Field will be provided by the concerned institute.
Total budget of the project = Rs. 19,00,000 + 6,00,000 + 10,00,000
= Rs. 35,00,000