Study and validation of candidate red clover (Trifolium pratense L.) genes for leghaemoglobin, a key plant protein involved in the fixation of atmospheric nitrogen
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| Year of publication | 2018 |
| Type | Conference abstract |
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| Description | Symbiotic nitrogen fixation is a mutualistic relationship occurring between bacteria and plants, where bacteria provides nitrogen converted into organic compounds and plants in exchange supplies the bacteria with carbon and energy source. The most important and best researched symbiotic relationship occurs between rhizobacteria and plants of the family Fabaceae. Our study is focused to finding associations between the level of nitrogen fixation, for there are differences in efficiency in nitrogen fixation between genera and species, but also varies within species, and candidate genes for leghaemoglobins. Computational analysis based on Trifolium pratense sequence included in silico identification of 11 candidate Trifolium pratense genes for leghaemoglobins used for primer design. A large population of red clover genotypes was determined for nitrogen fixation ability by acetylene reduction assay for nitrogenase activity. Totally forty-eight plants (varieties Start, Global, Columbia and Vltavín) efficient and inefficient in nitrogen fixation were used for DNA isolation and comparative analyses using The Roche NimbleGen SeqCap EZ enrichment system to enrich targeted regions (100 kb in total) in the candidate genes. Enriched regions were sequenced on Illumina MiSeq System with the coverage 2000x. Moreover, leaf, root and nodule material obtained from contrast phenotypes was used for RNA isolation and relative quantification of candidate leghaemoglobin genes expression using real-time PCR. The results of this study include identification of candidate leghaemoglobin genes, their validation, expressional and computational analysis. |
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