Genetic diversity and its effect on fitness in an endangered plant species, Dracocephalum austriacum L.

Abstract

The aim of this study was to estimate genetic diversity and assess its importance for plant fitness in a species belonging to the most endangered species in Europe, Dracocephalum austriacum L., and to select the most valuable populations for conservation of genetic diversity within the species in the studied regions. We analyzed allozyme variation of 12 populations in three distinct regions (Czech Karst, Moravia and Slovak Karst) in Central Europe. The results showed high genetic diversity within populations (80.14%) and relatively low differentiation among populations within regions (9.42%) and between regions (10.45%). Seed production was significantly higher in larger, genetically more diverse and less inbred populations. The results suggest that genetic diversity has important effect on seed production in this species and thus can be expected to have strong direct consequences for plant fitness and vitality of the whole populations. They also show large variation in genetic diversity between populations and indicate which populations should get a priority in attempts to conserve all the genetic diversity within the region.

Appendix: The gel staining procedures

The staining procedures followed Vallejos (1983) to visualize ADH, 6-PGDH, PGM, EST, IDH and Wendel and Weeden (1989) for SOD and SKDH with the following modifications: ADH (20 ml ethanol), 6-PGDH (0.1 M Tris–HCl pH 8.4, 30 mg 6-phosphogluconic acid), PGM (24 mg MgCl₂), EST (Na-phosphate buffer pH 6.45, 30 mg β-naphthylphosphate, 30 mg α-naphtylacetate), IDH (50 ml 0.1 M Tris–HCl pH 8.0, 50 mg isocitric acid, 80 mg MgCl₂), SKDH (30 ml 0.1 M Tris–HCl pH 8.4, 30 mg shikimic acid, 5 mg NADP, 6 mg MTT). Enzyme systems AAT and LAP were stained using the following methods. Two staining solutions were prepared for AAT: A (260 mg aspartic acid and 45 mg α-ketoglutaric acid dissolved in warm 20 ml 0.1 M Tris–HCl pH 8.4) and B (20 ml 0.1 M Tris–HCl pH 8.4, 50 mg Fast Blue BB Salt, 50 mg Fast Violet B). The solution A was prepared at least 15 min before the application. The gel was rinsed in water and then in buffer Tris–HCl pH 7. Solutions A and B were mixed and poured on the gel. The gel was incubated in the dark at 32°C until bands appeared. Then it was rinsed and fixed (1:1:3:5, glycerine, acetic acid, H₂O, methanol). The gel stained for LAP was rinsed in buffer 0.2 M Tris-maleate pH 6 and incubated 10 min with 45 mg l-leucyl-β-naphthylamide-HCl (in 50% acetone) and 60 mg MgCl₂ (both dissolved in 30 ml buffer). Afterwards solution of 25 mg Fast Black K Salt in 30 ml buffer was added and gel was incubated in the dark at 32°C until bands appeared. For SOD ingredients, 50 ml of 0.05 M Tris–HCl (pH 8.2), 5 mg of EDTA, 5 mg of NBT and 2 mg of riboflavin were combined and poured over the gel. This was incubated for 20 min in the dark at 32°C then removed and illuminated under a lamp until bands appeared on the blue background. A standard staining solution for ME was prepared by dissolving 150 mg malic acid in 25 ml 0.05 M Tris–HCl (pH 8.0) and adjusted to pH 7.5 with 1N NaOH; to this was added a solution of 10 mg of MTT, 5 mg of NADP and 2 mg of PMS in 25 ml of 0.05 M Tris–HCl (pH 8.0), and the resulting staining solution was poured over the gel. Afterwards, all gels were thoroughly rinsed in distilled water, dried between two cellophane sheets and stored.