Phylogenetic study of crambe species with issr markers

Abstract

Molecular genetic study with ISSR markers was carried out for six Crambe species. The phylogenetic tree showing similarity for Crambe and Brassica napus genotypes was constructed. The efficiency of ISSR markers application for interspecies plant biodiversity study was pointed out.

Introduction

The Crambe°L. genus belongs to Brassicaceae°family and consists of about 44 species [1]. These species are annual, biennial or perennial and have diverse application: as vegetable or forage plants, as oilseed, as the source of biofuels (seeds have up to 60% of erucic acid), in food industry for making pastry, in paint and varnish industry, in chemical industry and for arsenic elimination from soil and heavy metals (Cd, Pb, Cr) from water [1-5]. At the same time many of them are threatened species listed in the Red Data Book of Ukraine (vulnerable), European and EU 27 Red List(C. aspera), IUCN Red List (C. koktebelica, C. tataria, C. aspera, C. steveniana, C. тагійта) and in The World Red List (C. koktebelica) [6-7].

Genetic diversity analysis of plants is very important for biodiversity protection and preservation as well as breeding new varieties. The study of genetic structure of plant populations can provide information about the level of plant gene pool polymorphism to establish genetic similarity of both species and a particular plant within a population.

Various molecular genetic markers can be used for the purpose [8]. However, polylocus and polyallelic ISSR markers (based on Inter-Simple Sequence Repeats) are of great value to study population genetic diversity and phylogenetic relationships establishment [9].

The objective of the work was to develop molecular genetic system based on ISSR markers to estimate interspecies genetic similarity of crambe.

Materials and methods

The subject of the work was a set of 6 Crambe species: C. aspera, C. cordifolia, C. koktebelica, C. marilm, C. steveniana and C. tataria. Fresh apical leaves from plants grown in vitro (on hormone- free solid MS medium [10] at 24°С with a 16-h photoperiod and recurrent transplantation on the fresh medium every 30 days) were used for DNA extraction. In vitro culture was initialized from seeds obtained from M.M.°Gryshko National Botanical Garden, National Academy of Sciences of Ukraine. Total plant DNA was isolated with the modified CTAB method [11] 0.5 g of fresh tissue. Polymerase chain reaction (PCR) of 20 pl included 0.25 pM of forward and reverse primers (Metabion, Germany), 1^х Reaction Buffer B (Solis BioDyne, Estonia), 2 mM MgCl2, 0.2 pM of each deoxyribonucleotide-3- phosphate (Thermo Fisher Scientific, USA), 1 unit of FIREPol® DNA Polymerase (Solis BioDyne), and 30 ng of purified total plant DNA. Primer sequences for ISSR loci UBC827, UBC864, UBC890 [12], and A17898, B17899, IS-05, HB-10 [13] were utilized in the study.

Table 1 - Polymorphism of ISSR markers system used in the study

PCR products were separated by electrophoresis in 1.5% agarose gels in lithium borate buffer, 0.5 pg/ml ethidium bromide [14].Gels were visualized in UV-light and documented with a photosystem

Canon EOS 600D. Phylogenetic relationships was calculated by the unweighed pair-group method using arithmetic averages cluster analysis (UPGMA) with the software DARwin 6.0.010 [15].

Results and discussion

Seven preselected ISSR markers were used in the present study to identify six Crambe and one Brassica napus genotypes. Every marker system showed high level of polymorphism for every crambe sample (table 1). Some agarose gels are depicted on the figure 1.

According to the dendrogram the genotypes are distributed into two main clusters. The first cluster group includes four genotypes of Crambe species (C. maritima, C. orientalis, C. cordifolia and C. aspera) together with a Brassica napus genotype, which is located in the separate subcluster group. The second one contains two genotypes of C. tataria and C. steveniana.

In the research [16] some Turkish plant samples of Crambe species were analyzed. The high level of intraspecies and interspecies polymorphisms was pointed out in the work, though only C. maritima, C. orientalis and C. tataria genomes were studied there. It entirely correlates with our results.

Conclusion

The high efficiency of ISSR analyzes was defined for the study of Crambe species genetic diversity and their phylogenetic relationship establishment. Polylocus and polyallelic ISSR markers can provide with extensive characteristic of each plant sample. The selected system of markers will assist to carry out the complex research of a large number of crambe samples.

References

9

1. Branca F., Cartea E. Oilseeds. Brassica // Wild Crop Relatives: Genomic and Breeding Resources. - 2011. V. 10. - P 17- 36.
2. Red data book of Ukraine. Vegetable kingdom / ed. by. Didukh Ya. P. - K.: Globalconsulting, 2009. - p. 358.
3. Прахова Т.Я. Новая нетрадиционная масляничная культура - Крамбе абиссинская // Вестник Алтайского государственного аграрного университета. - 2013. - № 8 (106). - С. 8-10.
4. Artus N.N. Arsenic and cadmium phytoextraction potential of Crambe compared with indian mustard // J Plant Nutr. - 2006. - V. 29. - P. 667-679.
5. GonQalves A.C., Rubio F., Meneghel1 A.P., Coelho G.F., Dragunski D.C., Strey L. The use of Crambe abyssinica seeds as adsorbent in the removal of metals from waters // Rev. bras. eng. agric. ambient. - 2013. - V.17 (3). - P. 306-311.
6. The IUCN Red List of Threatened species. - http://www.iucnredlist.org
7. Bilz M., Kell S.P., Maxted N., Lansdown R.V. European Red List of Vascular Plants. Luxembourg: Publications Office of the European Union. 2011, 142 p.
8. Schulman A.H. Molecular markers to assess genetic diversity // Euphytica. - 2007. - V. 158. - P. 313-321.
9. Weicong Qi, Feng Lin, Yuhe Liu, Bangquan Huang, Jihua Cheng, Wei Zhang, Han Zhao High-throughput development of simple sequence repeat markers for genetic diversity research in Crambe abyssinica // BMC Plant Biology. - 2016. - 16:139.
10. Murashige T. and Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant., vol. 15, p. 473-497.
11. Stewart C.N., Via L.E. A rapid CTAB DNA isolation technique useful for RAPD fingerprinting and other PCR applications // BioTechniques. - 1993. - V. 14 (5). - Р. 748-749.
12. Werner E.T., Soares T.C.B., Gontijo A.B.P.L., Souza Neto J.D., Amaral J.A.T. Genetic stability of micropropagated plants of Crambe abyssinica Hochst using ISSR markers // Genetics and Molecular Research. - 2015. - V. 14(4). - P. 16450-16460.
13. Rasha M. A. Khalil, Soliman KH. A., Nahed A. K. F. R., Ibrahim S.A. Genetic polymorphism of some medicinal plants belonging to Brassicaceae using molecular markers // Egypt. J. Genet. Cytol. - 2010. - V. 39. - P. 41-55.
14. Singhal H., Ren Y.R., Kern S.E. Improved DNA electrophoresis in conditions favoring polyborates and Lewis acid complexation // PLoS One. - 2010. - 5, №6. -P. 1-6.
15. Perrier X., Flori A., Bonnot F. Data analysis methods. In: Hamon, P., Seguin, M., Perrier, X., Glaszmann, J.C.Ed., Genetic diversity of cultivated tropical plants. // Enfield, Science Publishers. Montpellier. - 2003. - P. 43 -76.
16. Tankahya-Hacxoglu B. Molecular diversity of the wild Crambe (Brassicaceae) taxa in Turkey detected by inter-simple sequence repeats (ISSRs) // Industrial Crops and Products. - 2016. - V. 80. - P. 214-219.