Theor Appl Genet (1984) 187-192 9 Springer-Verlag 1984 Changes in DNA composition in the evolution of Vicia species S. N. Raina and R. K. J. Narayan Department of Agricultural Botany, University College of Wales, Penglais, Aberystwyth, Dyfed SY23 3DD, Wales Received January 8, 1984; Accepted January 18, 1984 Communicated by D. yon Wettstein Summary. The composition of nuclear DNA in 3 Vicia species are compared. The species V. eriocarpa, V. jo- hannis and V. melanops are from three separate subge- neric sections of Vicia and show a fourfold variation in their amounts Of nuclear DNA. DNA melting exper- iments, buoyant density gradient analysis and Cot re- association experiments show that the quantitiative change in nuclear DNA between the three species is achieved by changes in the amounts of both repetitive and nonrepetitive DNA sequences. It is suggested that while the increase in the repetitive fraction is achieved by the proliferation of repetitive base sequences the in- crease in the nonrepetitive fraction is due to the steady accretion of highly diverged base sequences resulting from mutations, deletions, insertions and base sequence rearrangements among families of repetitive sequences. Key words: Vicia - nuclear DNA - Evolution - Base se- quence Introduction The genus Vicia (family Leguminosae) has about 120 species distributed in the temperate zones of both hemispheres. Ninetyfive percent of species within this genus are diploids with basic chromosome numbers 2 n-- 10, 12 or 14. Speciation and evolution within this genus has involved large changes in chromosome size and in nuclear DNA amounts (Martin and Shanks 1966; Raina and Rees 1983). The DNA variation is five fold among diploid species which suggests extensive amplifi- cation or deletion of base sequences during evolution. Despite this large scale variation in the amount of nu- clear DNA the distribution of DNA within the chromo- some complements of several diploid species have re- mained remarkably similar (Raina and Rees 1983). This would suggest constraints upon changes in DNA amounts. Similar constraints upon DNA distribution in chromosome complements during evolution are also re- ported in Lathyrus, (Narayan 1982; Narayan and Dur- rant 1983) Lolium and Festuca (Seal and Rees 1982). In the following investigation the molecular composition of the nuclear DNA of 3 Vicia species are compared. The results tell us about the changes in the composition of nuclear DNA during the evolution of Vicia species. Material and methods The three species V. eriocarpa (2 n = 14), V. johannis (2 n= 14) and V. melanops (2 n-- 10) used in this investigation come from three separate subgeneric sections, Cracca, Faba, and Vicia re- spectively. The difference in the amounts of nuclear DNA be- tween V. eriocarpa (4.5 pg) and V. melanops (20.04 pg) is four- fold. V.johannis (14.14 pg) has an intermediate DNA value. DNA extraction and purification Surface sterilised seeds of Vicia species were grown in the dark under axenic conditions for three to four weeks. Young shoots from seedlings were collected and ground to a fine powder un- der liquid nitrogen using mortar and pestle. The powder was suspended in an extraction buffer which contained 0.1 M NaC1, 10 mM EDTA, 50 mM tris pH 8.5, 1 M Na CIO4, 0.5% diethyl pyrocarbonate and 2% Sarkosyl. After lysis the solution was shaken with redistilled phenol and then with chloroform - octonol (24: 1). The aqueous extract which contained DNA was separated each time by centrifugation and this step was continued until no protein precipitate was visible in the in- terphase. The DNA extract was layered under two volumes of 98% ethyl alcohol. The DNA precipitate was spooled out, washed in 70% ethanol, air dried and then dissolved in 50 mM tris, 1 mM EDTA buffer (pH 8.5). The DNA was further purified by digesting with RNAse (75 ~tg per ml at 37 ~ for 1 h) and with pronase (100 Ixg/ml at 37 ~ for 3 h). To remove contaminating polysaccharides the