1. Details of the Project:
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 Phakopsora pachyrhizi Syd. & P. Syd which causes soybean rust 2. Rust 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 lead to 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 rust. 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 America14 followed by North America in 200515.
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 rust. Worldwide researchers have screened for resistance or tolerance to rust 17 and identified seven different loci carrying dominant alleles: Rpp1 18, Rpp2 19, Rpp3 20, Rpp4 21, Rpp5 22, Rpp6 23and Rpp1-b 24. However, these genes are not effective for all population of P. pachyrhizi 25. Some researchers have identified recessive genes in association with rust 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 rust resistance for the development of resistant cultivars.
SSR markers linked to rust resistance gene would be identified.
c) Key Questions
· Establishing of mapping population.
· Assessing mapping population for rust.
· Identifying Molecular markers.
· Assessing mapping population with putative molecular 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 development32. According to one Study, type of lesions was no correlation to severity to rust 33. 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. Rust cause around 10 – 90% of yield loss. Initially, Rust 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. In another study, on screening, two lines NRC 80 and MAUS 417 were found to be moderately susceptible to natural epiphytotic conditions at Meghalaya 37. Recently 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. tomentella 38.
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 markersto 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 rust 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 population 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 P. pachyrhizi. 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.