Strain-specific markers of rhizobia from whole-genome sequencing data

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Abstract

Abstract. Field trials of rhizobial inoculants require simple and reliable methods for identifying the strains used to determine which strain has formed a nitrogen-fixing nodule. This task arises when testing the competitiveness of inoculant strains against local rhizobia strains, to track the fate of inoculant strains over long periods after the introduction of strains, and finally, such methods may be in demand when protecting the rights of strain owners and developers. The essence of the proposed identification method is to search for strain-specific DNA regions that are absent in other genomes of the same species and to construct a primer system for multiplex PCR, allowing simple, reliable and rapid identification of the strain. The advantages of this approach over other identification methods are, firstly, high reproducibility, and secondly, that the method is based on the detection of structural variants, the contribution of which to the evolution of rhizobia genomes is very high, while most genomic fingerprinting methods (AFLP, RAPD, REP, ERIC, etc.) are based on the detection of nucleotide polymorphisms in short fragments of the genome, but miss many events associated with genomic rearrangements and horizontal gene transfer. The use of the proposed method can also serve to monitor the evolutionary dynamics of rhizobial inoculant strains, especially in unique fragments of the genome, which is very important for R. leguminosarum, where the proportion of unique sequences is much higher than in other rhizobia.

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About the authors

E. E. Andronov

All-Russian Research Institute of Agricultural Microbiology

Author for correspondence.
Email: eeandr@gmail.com
Russian Federation, St. Petersburg, 196608

T. S. Aksenova

All-Russian Research Institute of Agricultural Microbiology

Email: eeandr@gmail.com
Russian Federation, St. Petersburg, 196608

O. P. Onishchuk

All-Russian Research Institute of Agricultural Microbiology

Email: eeandr@gmail.com
Russian Federation, St. Petersburg, 196608

O. N. Kurchak

All-Russian Research Institute of Agricultural Microbiology

Email: eeandr@gmail.com
Russian Federation, St. Petersburg, 196608

V. I. Safronova

All-Russian Research Institute of Agricultural Microbiology

Email: eeandr@gmail.com
Russian Federation, St. Petersburg, 196608

A. G. Pinaev

All-Russian Research Institute of Agricultural Microbiology

Email: eeandr@gmail.com
Russian Federation, St. Petersburg, 196608

I. V. Evsyukov

All-Russian Research Institute of Agricultural Microbiology

Email: eeandr@gmail.com
Russian Federation, St. Petersburg, 196608

N. A. Provorov

All-Russian Research Institute of Agricultural Microbiology

Email: eeandr@gmail.com
Russian Federation, St. Petersburg, 196608

References

  1. Андронов Е. Е., Петрова С. Н., Чижевская Е. П., Коростик Е. В., Ахтемова Г. А., Пинаев А. Г. Влияние внесения генетически модифицированного штамма Sinorhizobium meliloti Ach-5 на структуру почвенного сообщества микроорганизмов // Микробиология. 2009. Т. 78. С. 525‒534.
  2. Andronov E. E., Chizhevskaya E. P., Korostik E. V., Akhtemova G. A., Pinaev A. G., Petrova S. N. Influence of introducing the genetically modified strain Sinorhizobium meliloti ACH-5 on the structure of the soil microbial community // Microbiology (Moscow). 2009. V. 78. P. 474‒482.
  3. Онищук О. П., Воробьев Н. И., Проворов Н. А. Нодуляционная конкурентоспособность клубеньковых бактерий: генетический контроль и адаптивное значение // Прикл. Биохимия и микробиология. 2017. Т. 53. С. 127‒135.
  4. Onishchuk O. P., Vorobyov N. I., Provorov N. A. Nodulation competitiveness of nodule bacteria: Genetic control and adaptive significance // Appl. Biochem. Microbiol. 2017. V. 53. P. 131‒139.
  5. Сафронова В. И., Чижевская Е. П., Андронов Е. Е. Разработка методики молекулярно-генетической паспортизации штаммов сельскохозяйственных микроорганизмов с помощью AFLP-фингерпринтинга // Сельскохозяйственная биология. 2012. Т. 47. № 6. С. 116‒121.
  6. Darling A. C., Mau B., Blattner F. R., Perna N. T. Mauve: multiple alignment of conserved genomic sequence with rearrangements // Genome Res. 2004. V. 14. P. 1394‒1403.
  7. De Bruijn F. J. Use of repetitive (repetitive extragenic palindromic and enterobacterial repetitive intergeneric consensus) sequences and polymerase chain reaction to fingerprint the genomes of Rhizobium meliloti isolates and other soil bacteria // Appl. Environ. Microb. 1992. V. 58. P. 2180‒2187.
  8. Epstein B., Tiffin P. Comparative genomics reveals high rates of horizontal transfer and strong purifying selection on rhizobial symbiosis genes // Proc. Biol. Sci. 2021. V. 288. Art. 20201804.
  9. Freiberg C., Felllay R., Bairoch A., Broughton W. J., Rosenthal A., Perret X. Molecular basis of symbiosis between Rhizobium and legumes // Nature. 1997. V. 387. P. 394‒401.
  10. Guo X., Flores M., Mavingui P., Fuentes S. I., Hernandez G., Davila G., Palacios R. Natural genomic design in Sinorhizobium meliloti: novel genomic architecture // Genome Res. 2003. V. 13. P. 1810‒1817.
  11. Harrison S. P., Mytton L. R., Scøt L., Dye M., Cresswell A. Characterization of Rhizobium isolates by amplification of DNA polymorphisms using random primers // Can. J. Microbiol. 1992. V. 38. P. 1009‒1015.
  12. Jorrin B., Palacios J. M., Peix Á., Imperial J. Rhizobium ruizarguesonis sp. nov., isolated from nodules of Pisum sativum L. // Syst. Appl. Microbiol. 2020. V. 43. Art.126090.
  13. Laguerre G., Geniaux E., Mazurier S. I., Rodríguez Casartelli R., Amarger N. Conformity and diversity among field isolates of Rhizobium leguminosarum bv. viciae, bv. trifolii, and bv. phaseoli revealed by DNA hybridization using chromosome and plasmid probes // Can. J. Microbiol. 1992. V. 39. P. 412‒419.
  14. Laguerre G., Allard M. R., Revoy F., Amarger N. Rapid identification of Rhizobia by restriction fragment length polymorphism analysis of PCR-amplified 16S rRNA genes // Appl. Environ. Microb. 1994. V. 60. P. 56‒63.
  15. Provorov N. A., Andronov E. E., Kimeklis A. K., Onishchuk O. P., Igolkina A. A., Karasev E. S. Microevolution, speciation and macroevolution in rhizobia: genomic mechanisms and selective patterns // Front. Plant. Sci. 2022. V. 13. Art. 1026943.
  16. Rahimah A. R., Cheah S. C., Rajinder S. Freeze drying of oil palm (Elaeis guineensis) leaf and its effect on the quality of extractable DNA // J. Oil Palm Res. 2006. V. 18. P. 296‒304.
  17. Rosselli R., La Porta N., Muresu R., Stevanato P., Concheri G., Squartini A. Pangenomics of the symbiotic Rhizobiales. Core and accessory functions across a group endowed with high levels of genomic plasticity // Microorganisms. 2021. V. 9. Art. 407.
  18. Safronova V., Sazanova A., Belimov A., Guro P., Kuznetsova I., Karlov D., Chirak E., Yuzikhin O., Verkhozina A., Afonin A., Tikhonovich I. Synergy between rhizobial co-microsymbionts leads to an increase in the efficiency of plant–microbe interactions // Microorganisms. 2023. V. 11. Art. 1206.
  19. Vos P., Hogers R., Bleeker M., Rejans M., Van de Lee T., Hornes M., Frijters A., Pot J., Peleman J., Kupier M., Zabeau M. AFLP: a new technique for DNA fingerprinting // Nucleic Acids Res. 1995. V. 23. P. 4407‒4414.
  20. Young J. P., Crossman L. C., Johnston A. W., Thomson N. R., Ghazoui Z. F., Hull K. H., Wexler M., Curson A. R., Todd J. D., Poole P. S., Mauchline T. H., East A. K., Quail M. A., Churcher C., Arrowsmith C., Cherevach I., Chillingworth T., Clarke K., Cronin A., Davis P., Fraser A., Hance Z., Hauser H., Jagels K., Moule S., Mungall K., Norbertczak H., Rabbinowitsch E., Sanders M., Simmonds M., Whitehead S., Parkhill J. The genome of Rhizobium leguminosarum has recognizable core and accessory components // Genome Biol. 2006. V. 7. R34. https://doi.org/10.1186/gb-2006-7-4-r34
  21. Young J. P.W., Moeskjær S., Afonin A., Rahi P., Maluk M., James E. K., Cavassim M. I.A., Rashid M. H., Aserse A. A., Perry B. J., Wang E. T., Velázquez E., Andronov E. E., Tampakaki A., Flores Félix J. D., Rivas González R., Youseif S. H., Lepetit M., Boivin S., Jorrin B., Kenicer G. J., Peix Á., Hynes M. F., Ramírez-Bahena M.H., Gulati A., Tian C. F. Defining the Rhizobium leguminosarum species complex // Genes. 2021. V. 12. Art. 111. https://doi.org/10.3390/genes12010111

Supplementary files

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3. Fig. 1. Alignment of chromosomes of the genomes of strains RCAM1365 and RCAM0626. The three largest unique fragments for each chromosome are marked with an asterisk.

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4. Fig. 2. Amplification of marker fragments for strain RCAM0626 (a) and RCAM1365 (b). The numbers correspond to the primers in Tables 2 and 3.

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5. Fig. 3. Multiplex PCR with 10 pairs of primers for marking strains: a ‒ strain RCAM1365 (fragments 1, 2, 3, 5, 6, 7 according to Table 2 are marked with red dots); b ‒ strain RCAM0626 (fragments 1, 2, 4, 5, 6, 8 according to Table 3 are marked with red dots). Strains RCAM0626, RCAM1365 and 11 strains of R. leguminosarum of different origin were used as controls.

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