Soybean the DNA. The SSR markers linked

Soybean is one of the most important grain legumes in the world in terms of production. It is also a promising source of vegetable oil, nutraceuticals and protein. Cultivation of soybean in India is recorded to be below average than the rest of the world. One of the major limitations for increasing the productivity of soybean is non-availability of resistant varieties for disease and pest. Soybean rust is a fungal disease caused by Phakopsora pachyrhizi, commonly known as Asian soybean rust. Under conducive condition it causes yield loss up to 80%.  Defoliation and early maturation are symptoms shown by infected plants. Application of fungicide is the only available treatment, but the fungus tends to develop resistance over it. Thus, there is need for developing soybean resistant varieties.


DNA molecular markers would prove to be powerful tool in developing soybean resistant varieties by selecting target traits for breeding. For this a mapping population would be develop by crossing a soybean rust resistance parent with a susceptible parent. The population will be artificially infected by rust inoculum and after few days’ disease score would be recorded on F2 population. Prior to infection, leaf samples will be collected to extract the DNA. The SSR markers linked to rust resistance would be identified by Bulked segregant analysis method. Over 500 primers will be screened on parents and F2 bulks. The identified putative markers would be screened on entire F2 mapping population for linkage analysis. Phenotypic and genotypic data would be corelated with the help of software based on recombinant frequency. Finally, the identified markers would be validated on rust resistance and susceptible soybean cultivars. Tightly linked molecular markers identified in this study would serve as an important tool for marker assisted selection of rust resistant soybean genotypes in soybean breeding programs.

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1.      Details of the Project:


A)    Introduction


               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.


b)     Hypothesis


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.


B)     Objectives


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.


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 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.





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