Characterization and selection of SSR markers for assessing the genetic diversity of proso millet (Panicum miliaceum L.) accessions

Background. Proso millet (Panicum miliaceum L.) is a nutrient-rich whole-grain crop with a short growing season, making it suitable for cultivation across diverse agro-ecological zones. Studying the genetic diversity of millet in germplasm collections is essential for genetic resources conservation and breeding practice. Due to their high polymorphism, SSR markers are widely used for the analysis of plant genetic material.

Materials and methods. Seven SSR markers were selected on the basis of published data, and their ability to detect genetic variability was assessed in 13 millet accessions from the VIR collection. The accessions originating from different agro-ecological zones were studied with PCR, followed by agarose gel electrophoresis. Genomic DNA was extracted from etiolated seedlings using a modified CTAB method. Genetic variability parameters were calculated: number of alleles (Na), effective number of alleles (Ne), expected heterozygosity (He), and polymorphic information content (PIC). Nei’s coefficient was used to construct a genetic distance matrix. Principal coordinate analysis (PCoA) and a phylogenetic tree (UPGMA) were made using DARwin 6.0.21.

Results. Out of the 7 selected SSR markers, four (GB-PMM-073, GB-PMM-085, GB-PMM-121, and GB-PMM-126) revealed polymorphism in the analyzed accessions, while the remaining three (SSR 28, SSR 109, and SSR 332) were monomorphic. A total of 19 allelic variants were detected. The average number of alleles per SSR-locus was 2.7, and the mean PIC value was 0.471. Two markers (GB-PMM-121, and GB-PMM-073) demonstrated a high level of informativeness (PIC > 0.6). The PCR products obtained from the 13 millet accessions were sequenced, making it possible to identify additional allelic variants in the sequence structure.

Conclusion. Preliminary results suggest that the identified polymorphic markers can be recommended for assessing and genotyping millet germplasm collections, and for characterization of breeding material.

1. Abdi S., Dwivedi A., Shashi, Kumar S., Bhat V. Development of EST-SSR markers in Cenchrus ciliaris and their applicability in studying the genetic diversity and cross-species transferability. Journal of Genetics. 2019;98(4):101. DOI: 10.1007/s12041-019-1142-x

2. Agarwal M., Shrivastava N., Padh H. Advances in molecular marker techniques and their replications in plant sciences. Plant Cell Reports. 2008;27(4):617-631. DOI: 10.1007/s00299-008-0507-z

3. Arkhestova D.Kh., Yakhutlova A.A., Khaudov A.D., Sokurova L.Kh., Kulemina T.V. Effectiveness of ISSR markers for detecting genomic variability in Panicum miliaceum L. accessions. Proceedings on Applied Botany, Genetics and Breeding. 2024;185(1):161-171. [in Russian]. DOI: 10.30901/2227-8834-2024-1-161-171

4. Balli D., Bellumori M., Masoni A., Moretta M., Palchetti E., Bertaccini B. et al. Proso millet (Panicum miliaceum L.) as alternative source of starch and phenolic compounds: A study on twenty-five worldwide accessions. Molecules. 2023;28(17):6339. DOI: 10.3390/molecules28176339

5. Bhat S., Nandini C.A.V., Srinathareddy S., Jayarame G., Prabhakar K. Proso millet (Panicum miliaceum L.) – a climate resilient crop for food and nutritional security: A review. Environment Conservation Journal. 2019;20(3):113-124. DOI: 10.36953/ECJ.2019.20315

6. Cho Y.I., Chung J.W., Lee G.A., Ma K.H., Dixit A., Gwag J.G. et al. Development and characterization of twenty-five new polymorphic microsatellite markers in proso millet (Panicum miliaceum L.). Genes and Genomics. 2010;32(3):267-273. DOI: 10.1007/s13258-010-0007-8

7. Cuevas H.E., Prom L.K. Evaluation of genetic diversity, agronomic traits, and anthracnose resistance in the NPGS Sudan Sorghum Core collection. BMC Genomics. 2020;21(1):88. DOI: 10.1186/s12864-020-6489-0

8. Elshire R.J., Glaubitz J.C., Sun Q., Poland J.A., Kawamoto K., Buckler E.S. et al. A robust, simple genotyping-by-sequencing (GBS) approach for high diversity species. PLoS One. 2011;6(5):e19379. DOI: 10.1371/journal.pone.0019379

9. FAO. Millet in Russia (Proso Rossii). Moscow: FAO; 2024. [in Russian] (ФAO. Просо России. Москва: ФАО; 2024). DOI: 10.4060/cd0716ru

10. Filyushin M.A., Shchennikova A.V., Kochieva E.Z. Antocyanidin-3-O-glucosyltransferase genes in pepper (Capsicum spp.) and their role in anthocyanine biosynthesis. Russian Journal of Genetics. 2023;59(5):517-529. [in Russian]. DOI: 10.31857/S0016675823050041

11. Flajsman M., Stajner N., Kocjan Acko D. Genetic diversity and agronomic performance of Slovenian landraces of proso millet (Panicum miliaceum L.). Turkish Journal of Botany. 2019;43(2):185-195. DOI: 10.3906/bot-1807-83

12. Gami P.B., Solanki S.D., Prajapati C.D., Patel M.S. Study of genetic architecture for yield related traits in pearl millet [Pennisetum glaucum (L.) R. Br.]. Pharma Innovation. 2021;10(12):128-133. DOI: 10.22271/tpi.2021.v10.i12b.9351

13. Hunt H.V, Vander Linden M., Liu X., Motuzaite-Matuzevicuite G., Colledge S., Jones M.K. Millets across Eurasia: chronology and context of early records of the genera Panicum and Setaria from archaeological sites in the Old World. Vegetation History and Archaeobotany. 2008;17 Suppl 1:5-18. DOI: 10.1007/s00334-008-0187-1

14. Kalia R.K., Rai M.K., Kalia S., Singh R., Dhawan A.K. Microsatellite markers: An overview of the recent progress in plants. Euphytica. 2011;177(3):309-334. DOI: 10.1007/s10681-010-0286-9

15. Kulemina T.V., Khoreva V.I., Shelenga T.V., Kurtseva A.F., Sidorenko V.S. The grain quality of milletlike cultures in conditions of the south Nonblack Earth Zone of Russia. Аgrarian Russia. 2010;(1):19-23. [in Russian]. DOI: 10.30906/1999-5636-2010-1-19-23

16. Liu S., Feuerstein U., Luesink W., Schulze S., Asp T., Studer B. et al. DArT, SNP, and SSR analyses of genetic diversity in Lolium perenne L. using bulk sampling. BMC Genetics. 2018;19(1):10. DOI: 10.1186/s12863-017-0589-0

17. Lu H., Zhang J., Liu K.B., Wu N., Li Y., Zhou K. et al. Earliest domestication of common millet (Panicum miliaceum) in East Asia extended to 10,000 years ago. Proceedings of the National Academy of Sciences of the United States of America. 2009;106(18):7367-7372. DOI: 10.1073/pnas.0900158106

18. Mohammadi S.A., Prasanna B.M. Analysis of genetic diversity in crop plants – Salient statistical tools and considerations. Crop Science. 2003;43(4):1235-1248. DOI: 10.2135/cropsci2003.1235

19. Nei M. Analysis of gene diversity in subdivided populations. Proceedings of the National Academy of Sciences of the United States of America. 1973;70(12):3321-3323. DOI: 10.1073/pnas.70.12.3321

20. Souza C.P.F., Ferreira C., de Souza E.H., Neto A.R.S., Marconcini J.M., da Silva Ledo C.A. et al. Genetic diversity and ISSR marker association with the quality of pineapple fiber for use in industry. Industrial Crops and Products. 2017;104:263-268. DOI: 10.1016/j.indcrop.2017.04.059

21. Suvi W.T., Shimelis H.A., Laing M.D., Mathew I., Shayanowako A.I.T. Assessment of the genetic diversity and population structure of rice genotypes using SSR markers. Acta Agriculturae Scandinavica, Section B – Soil and Plant Science. 2019;70(1):76-86. DOI: 10.1080/09064710.2019.1670859

22. Temnykh S., Park W.D., Ayres N., Cartinhour S., Hauck N., Lipovich L. et al. Mapping and genome organization of microsatellite sequences in rice (Oryza sativa L.). Theoretical and Applied Genetics. 2000;100(5):697-712. DOI: 10.1007/s001220051342

23. Zargar M., Dyussibayeva E., Orazov A., Zeinullina A., Zhirnova I., Yessenbekova G. et al. Microsatellite-based genetic diversity analysis and population structure of proso millet (Panicum miliaceum L.) in Kazakhstan. Agronomy. 2023;13(10):2514. DOI: 10.3390/agronomy13102514