UNIVERSITY OF AGRICULTURAL SCIENCES AND VETERINARY MEDICINE CLUJ-NAPOCA PHD SCHOOL OF ENGINEERING IN AGRICULTURAL SCIENCES FACULTY OF HORTICULTURE

Transcription

1 UNIVERSITY OF AGRICULTURAL SCIENCES AND VETERINARY MEDICINE CLUJ-NAPOCA PHD SCHOOL OF ENGINEERING IN AGRICULTURAL SCIENCES FACULTY OF HORTICULTURE Ing. Drd. Katalin SZABO Intra- and inter population variability study of the endemic subspecies of Astragalus excapus ssp. transsilvanicus by correlating molecular techniques with morphometric characters in order to develop conservation strategies [ ] SCIENTIFIC ADVISOR Prof. Ph.D. Doru PAMFIL Cluj Napoca

2 TABLE OF CONTENTS INTRODUCTION... 2 CHAPTER I. THE GENERAL FRAMEWORK OF THE RESEARCH ASPECTS OF ASTRAGALUS EXSCAPUS L THE IMPORTANCE OF EXAMINED SUBSPECIES BIODIVERSITY AND THE CONSERVATION OF BIOLOGICAL DIVERSITY THE ASSESSMENT OF GENETIC VARIABILITY BY SRAP TECHNIQUE OBIECTIVES OF THE RESEARCH... 7 CHAPTER II. THE METHODOLOGY OF EXPERIMENTATION AND THE BIOLOGICAL MATERIAL ANALISED BIOLOGICAL MATERIAL THE MORPHOMETRICAL CHARACTERS STUDIED HIGHLIGHTING THE GENETIC VARIABILITY BY SRAP TECHNIQUE... 9 CHAPTER III. RESULTS AND DISCUSSION RESULTS REGARDING THE INTRA- AND INTER POPULATION VARIABILITY ESTABLISHED UNDER MORPHOLOGICAL CHARACTERS RESULTS OF THE ANALYSIS OF MOLECULAR VARIANCE RESULTS OF MANTEL TEST CHAPTER VI. GENERAL CONCLUSIONS AND RECOMMENDATIONS SELECTIVE BIBLIOGRAPHY

3 INTRODUCTION Biodiversity conservation is a subject of great interest worldwide. It concerns not only biologists around the world, but the majority of scientists, who are intrigued by the intensive exploitation of all natural resources and the prospect of their rapid exhaustion. The development of strategies for biodiversity conservation has therefore become a national priority in many countries in order to maintain and protect endangered species. Conservation can be performed in situ, in the form of protected areas at the place of origin, or ex situ, in botanical gardens, research stations, specialized institutions or gene banks. Romania has the largest bio-geographical regions of Europe (Continental, Alpine, Pannonian, Pontic, and Steppe) with a great diversity of species, from the Carpathians to the Danube Delta. The relatively balanced distribution of landforms - 28% mountains, 42% hills and 30% plains - represents a unique feature in Europe and a rarity worldwide. Due to this fact, the Romanian flora includes 3,795 species and subspecies of vascular plants, of which 250 species are classified in various categories of vulnerability. Astragalus exscapus is a perennial plant of the Leguminosae family which is characterized by a specific form of rarity, namely the existence of small populations in distinctive habitats (Rabinowitz, 1981). The species belongs to the Red List of species threatened with extinction in The Carpathians, alongside other similar species like A. dasyanthus, A. frigidus, A. penduliflorus, A.pseudopurpureus, and A. Roemenie. Astragalus excapus L. ssp. transsilvanicus (Schur) Nyár. is a regional endemism mentioned in Annex 4b. GEO 57/2007 as a species of national interest requiring strict protection. The latest study conducted on bio-geographical steppe and forest steppe species in the Transylvanian Basin revealed that so far, about 80% of the populations of 2

4 A. exscapus ssp. transsilvanicus. disappeared. Currently, only 24 remaining populations have been identified in the Transylvanian Plain. In our research we aimed to investigate eight populations of A. exscapus ssp. transsilvanicus, to assess the degree of intra- and inter-population polymorphism. The study included both analysis of the morphometric characters and investigation of molecular markers technique. The overall objective of this thesis wanted to fit into the strategies of conservation of an endemic subspecies, unstudied to date at botanical and molecular level and which, in the absence of scientific papers or similar programs, can be a source of new information and a possible study model for other species. The materialization of this thesis was possible due to doctoral supervisor Prof. Ph.D. Doru PAMFIL, Head of Department of Biotechnology in the Faculty of Horticulture and Rector of the University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca. In this way I express my gratitude for the guidance of highly academic demeanor and for the moral and financial support that I received throughout doctoral internship. The analyzes were conducted in research laboratories of Biotechnologies platform of Life Sciences Institute of Veterinary Medicine Cluj-Napoca, where I met a competitive group of researchers whom I thank for honoring me with confidence and understanding. The research was cofinanced by the European Social Fund through the Sectoral Operational Program Human Resources Development Invest in people! With the contract code: POSDRU/159/1.5/S/ At the ending of a significat part of my professional development I would like to dedicate my regards to those whome contributed in some way to the finalisation of my thesys. 3

5 1. CHAPTER I. THE GENERAL FRAMEWORK OF THE RESEARCH 1.1. ASPECTS OF ASTRAGALUS EXSCAPUS L. Astragalus exscapus is part of the legume family, which is one of the largest families in the Magnoliopsida class with over 18,000 species classified in 650 genres (Polhill and Raven, 1981). Legumes are an important group of plants because they are used as crop or forage, but can also be used as green manure. The family is divided into three subfamilies: Papilionoideae, Caesalpinioideae and Mimosoideae. The Papilionoideae subfamily represents two thirds of all genera and species of the family, and contains the most important crop plants such as soybean (Glycine max), peas (Pisum sativum), chickpeas (Cicer arietinum), beans (Phaseolus vulgaris), lentil (Lens culinaris) and peanuts (Arachis hypogaea) and also Astragalus exscapus. (A) (B) Astragalus exscapus ssp. transsilvanicus (A) the aerial part (B) root system Source: original photo Perennial, hemicryptophyte with a height varying between 10 and 30 cm, blooming in May and June, A. exscapus is a species of plant which is considered rare, and is unique to the grassland area from oak to sessile forest level (Ciocârlan, 1988). 4

6 The subspecies Astragalus excapus L. ssp. transsilvanicus (Schur) Nyár. is a regional endemism mentioned in Annex 4b. GEO 57/2007 regarding the protected natural areas, the conservation of natural habitats, the wild flora and fauna, approved with amendments by Law no. 49/2011, as species of national interest, requiring strict protection THE IMPORTANCE OF EXAMINED SUBSPECIES Ecologically speaking, the Astragalus excapus L. is a very important link for the evolution of the endangered butterfly species Plebejus sephirus Kovacs (Szabo), being the main source of food for its larva and aft (Vojnits and Acs, 1995). At the same time, it is part of the Red List of endangered plant species in The Carpathians (Witkowski, 2003) and last, but not least, the swivel root system of this plant prevents soil erosion. In order to maintain biodiversity, there is more and more emphasis put on conservation of wild flora related with species of plant of economic interest (Rao and Hodgkin, 2002); in this context, the Astragalus exscapus shows interest for some breeding programs, constituting an important source of germplasm. The root system of the studied plant is very similar to that of Astragalus membranaceus, which is an herb with high content of various secondary metabolites. Thus, there is a possibility that A. exscapus. ssp. transsilvanicus presents healing abilities similar to this, the researches in this direction are underway BIODIVERSITY AND THE CONSERVATION OF BIOLOGICAL DIVERSITY The biodiversity from our days is the result of a long evolution of natural evolutionary processes, and it is increasingly exposed and threatened by human influence. Protecting biodiversity is in our own interest but especially of the future generations, because biological resources are vital for the survival of future civilizations. The Romanian flora includes 3795 species and subspecies of vascular plants due to relatively balanced distribution of landforms - mountains 28%, hills 42%, plateaus and 5

7 plains 30%. Of these, 250 species are classified into different IUCN categories of vulnerability. Figure no. 2 Bio-geographical regions in Europe sourse foto: The In situ and ex situ conservation are two major strategies used in the conservation of genetic resources of plants. Between these two strategies there is a fundamental difference: the ex situ conservation involves sampling, transfer and storage of the population of a certain species away from their original location, respectively in gene banks, botanical gardens, warehouses and / or laboratory, while in situ conservation (natural habitat) means setting interest varieties at origin, management and monitoring of areas in which these species belong (Negri and Veteläinen, 2009). 6

8 1.4. THE ASSESSMENT OF GENETIC VARIABILITY BY SRAP TECHNIQUE The analysis of genetic diversity and relationships between or among populations, species or individuals is an important issue for many disciplines of biology fields. An important application of molecular markers is found in the field of genetics conservation to determine the variability of analyzed taxa, which is an important prerequisite for the development of conservation strategies. In order to assess the intra- and inter- population polymorphism, of A. exscapus ssp. transsilvanicus, from the wide variety of molecular techniques, we opted for the SRAP method (Sequence Related Amplified Polymorphism), forasmuch many recent studies reflect the sustainability of this technique and it has tehnical and economical advantages also comparing to other molecular markers (ex. RAPD, AFLP sau SSR) OBIECTIVES OF THE RESEARCH The overall objective of this thesis is to provide new data on genetic variability of the endemic subspecies Astragalus excapus L. ssp. transsilvanicus (Schur) Nyár., in order to develop a conservation strategy. To achieve the main scope of the study, during research were defined the following specific objectives: (i) identification of Astragalus exscapus ssp. transsilvanicus populations in the field; (ii) description of morphological characters of individuals from different populations, and the characterization of eco-geographical areas; (iii) sampling biological material for the molecular analysis and the isolation of DNA in order to achieve PCR amplification; (iv) analysis of intra- and inter-population variability using diversity indices by SRAP technique; (v) obtaining dendrograms based on the calculated genetic distances;(vi) correlating geographical and genetic distances by Mantel test; (vii) to develop the conservation strategies depending on the obtained results. 7

9 2. CHAPTER II. THE METHODOLOGY OF EXPERIMENTATION AND THE BIOLOGICAL MATERIAL ANALISED 2.1. BIOLOGICAL MATERIAL The biological material was represented by individuals of Astragalus exscapus L. subsp. transsilvanicus (Schur) Nyár. The specimens studied were collected from the following locations: Cluj county (Cojocna, Viişoara, Valea Florilor, Gădălin), Mureș County (Milăşel, Săbed, Sărmăşel Station) and Silivașu de Câmpie localised in Bistrița Năsăud county. The individuals of a population were sampled randomly, being chosen at a distance greater than 30 m of each other, so that the area occupied by this population to be well represented and characterized in terms of biodiversity. The number of representatives in a population varied between individuals / population analyzed, according to population size. A total of 126 plants were analyzed as it is shown in Table 1, with an average of individual / population. Table 1 Population symbol and localization of the studied Astragalus exscapus ssp. Transsilvanicus populations Simbol of Place Number of Longitude E Latitude N population analyzed individuals B Cojocna - Dl.Beleni (CJ) ' 52.38" 46 45' 57.65" G Gǎdǎlin (CJ) ' 50.40" 46 51' 28.38" M Milǎșel (MS) " " S Sǎbed (MS) " " SG Sǎrmǎșel Garǎ (MS) ' 45.04" 46 47' 03.11" SI Silivașu de câmpie (BN) ' 04.08" 46 46' 41.58" V Viișoara (CJ) " " VF Valea Florilor (CJ) ' 48.75" 46 40' 25.19" 8

10 2.2. THE MORPHOMETRICAL CHARACTERS STUDIED The morphometrical characters studied were represented by: plant height (mm), number of leaves / plant, number of flowers in the inflorescence and the inflorescence diameter (mm). The data were collected in May and June 2014 during the flowering period of the subspecies. The measurements were made with a ruler to determine the height of the plants, and with an electronic caliper to determine the diameter of the analyzed inflorescence. To test the equality of the environments between the populations, One-Way- ANOVA test (Fisher test) has been used which has been conducted with SPSS program. The test controls if there are significant differences between the characters averages on the observed populations HIGHLIGHTING THE GENETIC VARIABILITY BY SRAP TECHNIQUE After the morphometrical traits were measured, from each inventoried plant, 5 g of young leaves were sampled for DNA extraction, required for molecular analyzes. Within our experiences, the DNA isolation has been executed after the protocol of Lodhi et al. (1994) which has been improved by Rodica Pop et al. (2003). Because of the fact that there is no protocol established yet to Astragalus exscapus ssp. transsilvanicus DNA extraction, a preliminary testing was necessary. Following the tests and the DNA amount resulted after the test, we had concluded that the method of grinding plant material using the TissueLyser is better for the isolation of the DNA from samples of Astragalus exscapus ssp. Transsilvanicus than the classical grinding method in liquid nitrogen. The TissueLyser method has been generalized for the extraction protocol for all 126 samples. For the quantification of the extracted DNA, we used the ND 1000 (Nano Drop) spectrophotometer. In order to assess the intra- and inter-population polymorphism, in our research we used the SRAP technique (Sequence Related Amplified Polymorphism), developed by Li and Quiros in This technique is much simpler by comparison with other molecular 9

11 marker methods (eg. AFLP), but the results between them are comparable; it has a higher repeatability than the RAPD technique; it uses primers of nucleotides and it is less expensive than the SSR markers. Following the preliminary testing of primers eight "Forward" primers and eight "Reverse" primers presented in table 2 - we have selected 14 combinations of SRAP primers that generated clearly visible DNA fragments and which presented high grades of polymorphism. Table. 2 Nucleotidic sequences of the tested SRAP primers Nucleotidic sequences of the tested FORWARD primers Nucleotidic sequences of the tested REVERSE primers Me1 F: TGA GTC CAA ACC GGA TA Em1 R: GAC TGC GTA CGA ATT AAT Me2 F: TGA GTC CAA ACC GGA GC Em2 R: GAC TGC GTA CGA ATT TGC Me3 F: TGA GTC CAA ACC GGA AT Em3 R: GAC TGC GTA CGA ATT GAC Me4 F: TGA GTC CAA ACC GGA CC Em4 R: GAC TGC GTA CGA ATT TGA Me5 F: TGA GTC CAA ACC GGA AG Em5 R: GAC TGC GTA CGA ATT AAC Me6 F: TGA GTC CAA ACC GGA CA Em6 R: GAC TGC GTA CGA ATT GCA Me7 F: TGA GTC CAA ACC GGA CG Em7 R: GAC TGC GTA CGA ATT CAA Me8 F: TGA GTC CAA ACC GGA CT Em8 R: GAC TGC GTA CGA ATT CAC The amplification of the samples has been performed after the protocol developed by Talebi et al (2012) which we adapted by reducing the reaction volume of 25 μl to 15μl, whilst maintaining the molar concentrations that have been specified in the protocol. The amplification program has been a specific one for SRAP technique and it is shown in the table below (Table 3). In order to separate and identify DNA fragments, we used the standard method of agarose gel electrophoresis. The procedure for the preparation of the gels has been as follows: 4 g agarose (Promega) were placed into 200 ml of 1X TAE buffer and then melted in a microwave oven until they resulted in a clear and transparent solution. The 10

12 solution was cooled to C and has been poured into a frame (Plexiglas plate) in which has been set in advance a comb ( with 40 teeth) which is necessary to form the wells after solidification of the gel. Table 3 PCR amplification program for SRAP tehnique Number of Temperature PCR stage Timing Number of cycle repetition C⁰ min Initial denaturation Denaturation Primers annealing 1 75 Extension Denaturation Primers annealing 1 72 Extension Final extension 10 For DNA migration, we use a 200V charger that ensured a steady stream at its terminals. For migration, the power supply has been programmed to a value of 115 V to 5 V/cm gel length, thus the migration time has been fixed at 2 hours. After the electrophoretic migration of the samples, the gel has been stained by immersion into a EtBr (ethidium bromide) solution (0.5 mg/ ml in 150 ml of 1X TAE) for 20 to 30 minutes on a mechanical shaker. The viewing of the amplification product has been performed under UV light, the images of gels were captured using BIOSPECTRUM AC Imaging System, manufactured by UVP, and subsequently analyzed with the TL120 (Total Lab) software. The presence of the band with the same size has been noted with 1, and its absence from the analyzed individuals, has been noted with 0, thus we obtained a binary matrix representing the basis for calculating the genetic distances between analyzed individuals. The software that has been used to calculate the genetic distances is Free Tree (Pavlicek et al., 1999) program which uses the Nei Li / Dice coefficient (Nei et al., 1979). 11

13 For the processing and the graphical presentation of the dendrograms, we have used TreeView program (Page, 1996). The analysis of molecular variance intra- and inter population (AMOVA) has been carried through the GenAlEx 6501 (Peakall and Smousse, ). Also, the same analysis program has been used for the Mantel test, respectively for the principal coordinate analysis (PCoA). 3. CHAPTER III. RESULTS AND DISCUSSION 3.1. RESULTS REGARDING THE INTRA- AND INTER POPULATION VARIABILITY ESTABLISHED UNDER MORPHOLOGICAL CHARACTERS By testing the equality of the averages between the observed characters, it has been verified whether there are significant differences among the analyzed populations of Astragalus exscapus ssp. transsilvanicus, by applying the Fisher test. On the basis of obtained results, the null hypothesis has been rejected because the calculated F value has been greater than the tabular one. Thus, it can be argued that the plant height, number of leaves per plant, number of flowers per plant and flourish diameter varies significantly from one population to another, with a probability of 99%. Studied character Plant height Number of leaves / plant Analysis of Variance Sum of squares Degrees of freedom Mean square s 2 =SP/GL F Table no.14 Sig. Between population , , Among populations , ,721 Total , Between population 6180, ,

14 Studied character Number of leaves / plant Number of flowers / plant Inflorescence diameter Analysis of Variance Sum of squares Degrees of freedom Mean square s 2 =SP/GL F Table no.14 Among populations 31921, ,523 Total 38102, Between population 48864, , Among populations , ,272 Total , Between population 50274, , Among populations , ,857 Total , Sig. The average values of the four morphological characters measured in the eight populations of Astragalus exscapus from Transylvanian Plain, have allowed the preparation of some graphics; based on these characters, it was possible to make assessments on the growth vigor of the plant (adding the number of leaves per plant and plant height), and their generative capacity (referring to the total number of flowers in inflorescence and to the diameter of inflorescence). The highest average value in terms of plant height has been recorded in individuals originating from the Valea Florilor population (298.6 mm), compared with the lowest average (158.8 mm) measured in individuals in the population of Beleni as represented in figure no

15 The average values of leaves per plant Average plant heighti (mm) Ing. Katalin SZABO Beleni Gadalin Milasel Sabed Sarmasel Gara Silivasu de Campie Valea Florilor Viisoara Figure no. 3 Graphical representation of average values on plant height for the studied populations Experimental data on the average number of leaves per plant (Figure no. 4) highlights the greatest average value (39.5) in population of the Valea Florilor and the population of Silivaşu de Câmpie shows the lowest values (17.8). The average of the analyzed individuals from Beleni population (19.4) has been close to that of the population of Silivaşu de Câmpie. The results of the Fisher test point out that among the 8 of the analyzed populations, there are significant differences in terms of plant height Beleni Gadalin Milasel Sabed Sarmasel Gara Silivasu de Campie Valea Florilor Viisoara Figure no. 4 Graphical representation of average values on number of leaves per plant for each studied population 14

16 The average value of inflorescence diameter (mm) The average values of flowers per plant Ing. Katalin SZABO Regarding the number of flowers in blossom (Figure no. 5) the highest average values have been recorded in individuals from Săbed resort (76.7), respectively in the population of Valea Florilor (73.8), while lower average values have been observed in the individuals of the population of Gădălin (22.2) Beleni Gadalin Milasel Sabed Sarmasel Gara Silivasu de Campie 73.8 Valea Florilor 41.4 Viisoara Figure. no. 5 Graphical representation of average values on number of flowers per plant for the studied populations Also, the largest average values of the inflorescence diameter (Figure 6) were measured in the same two populations as in the case of the average number of flowers per plant, these are: the resort of Săbed (102.3 mm) and the station Valea Florilor (100.3 mm) Beleni Gadalin Milasel Sabed Sarmasel Gara Silivasu de Campie Valea Florilor Viisoara Figure. no. 6 Graphical representation of average values on inflorescence diameter for each studied population 15

17 Regarding the morphological features analyzed, we can conclude that the most vigorous populations, with a high capacity for regeneration, are found in Săbed and Valea Florilor populations. Important to note is that the measurements have been conducted during a period of two weeks, in all eight populations, to minimize the influence of the time factor. However dissimilarities between populations may be due to different climatic conditions; therefore it has been necessary a more accurate analysis through modern molecular techniques, which offer more precise data about the analyzed individuals. Following the isolation of DNA from samples of Astragalus exscapus ssp. transsilvanicus by the method of grinding the plant material using the TissueLyser II (Retsch, Quiagen), DNA samples were obtained with corresponding quantity and quality. Each sample has been brought to the concentration of 50ng / ml DNA, by making aliquots according to the work methodology described by the authors of SRAP technique (Li and Quiros,2001). After the working stages have been completed and - based on analysis of electrophoretic profiles of individuals the specific molecular marking and the binary matrix have been obtained, we calculated the genetic distances on which we built the intra- and inter-population dendrograms. Although the greatest dissimilitude according to calculated genetic distances, is between the populations of Valea Florilor and Săbed, we can observe that in the dendrogram these two populations are not located at the extremities; moreover, according to dendrogram, individuals from population of Sărmăşel Gară can be considered a solitary group (Fig. no. 7) 16

18 Figure no. 7 Dendrogram of the studied populations Given the fact that the hierarchical grouping of the eight populations of the analyzed Astragalus exscapus ssp. transsilvanicus is not conclusive, the investigation has been continued in terms of genetic diversity indices (Table no. 5). The percentage of polymorphic bands / population ranged from 72.22% to 85.42% with an average of 77.69%. The mean genetic diversity (He) expressed by the diversity index Nei (Nei, 1973) has been 0,228. Shannon diversity index ranged between and values. The most valuable populations in terms of genetic variability have proved to be the Gădălin (PBP = 85.42%, Ao = 1.743, Ae = 1.403, I = 0.382, He = 0.247), and the Silivaşu de Câmpie populations (PBP = 85.42%, Ao = 1.764, Ae = 1.403, I = 0.382, He = 17

19 0.247) as opposed to those of Cojocna-Beleni hill resort (PBP = 72.22%, Ao = AE = 1.349, I = 0.324, He = 0.210) Tabel no.5 Parameters of genetic diversity of Astragalus exscapus ssp. transsilvanicus populations obtained by SRAP technique Name of population PBP N A o A e I He Cojocna - Dl.Beleni 72.22% (CJ) Gǎdǎlin (CJ) 85.42% Milǎșel (MS) 75.69% Sǎbed (MS) 72.92% Sǎrmǎșel Garǎ (MS) 78.47% Silivașu de câmpie 85.42% (BN) Valea Florilor (CJ) 72.92% Viișoara (CJ) 78.47% Average 77.69% Where PBP - the percentage of polymorphic bands, N - number of analyzed individuals, Ao - observed allele number / locus, Ae number of effective alleles, I - Shannon diversity index, He - expected heterozygosity or Nei diversity index (wiyh th presumption of Hardy -Weinberg balance). The number of alleles observed Ao fall between the values of (Cojocna population, Belen Hill) and 1764 (population of Silivaşu de Cămpie). The highest value of expected heterozygosis has been registered in Gădălin population (0.247) RESULTS OF THE ANALYSIS OF MOLECULAR VARIANCE The results obtained after the analysis of intra- and inter population molecular variance are represented in the table below (Table no. 6). The data indicates that 83% of the total variation is found within the populations, while 17 percent indicates variability between the populations; these results are in concordance with those obtained by Vicente et al. (2011) in the study conducted in Spain over five populations of Astragalus nitidiflorus, an endemic species threatened with extinction. 18

20 Tabel no.6 The analysis of molecular variance of Astragalus exscapus ssp. transsilvanicus Source of variation GL SP MS Estimated variation Total Variation(%) Between populations % Among populations % Total % Stat Value P(rand >= data) PhiPT Our results are also in compliance with the results obtained by Wall et al. (2014) in research on endangered species of Astragalus michauxii performed by microsatellite, where the percentage of intra-population variability has amounted to the value of 91.34% of total variation RESULTS OF MANTEL TEST The correlation of the genetic distances with the geographical distances has been performed with GenAlEx 6501 (Peakall and Smousse, ) software. The matrix of geographical distances (Table no.7) indicates that the lowest geographical distance has been recorded between Sărmăşel Gară and Silivaşu de Câmpie (5.52 km), and the largest geographical distance has been recorded between Gădălin and Săbed resorts (50.43 km). Tabel no.7 Geographical distance matrix of Astragalus excapus ssp. transsilvanicus populations Silivașu Beleni Gădălin Milășel Săbed Sărmășel Gară de Câmpie Valea Florilor Viișora Beleni Gădălin Milășel Săbed Sărmășel Gară Sil. de Campie Valea Florilor Viișora

21 Genetic distances Ing. Katalin SZABO From the Mantel test results we can infer the fact that there is no positive correlation between the two analyzed matrices; more accurate, the genetic distances are not correlated with the geographical distances. Similar results were obtained by Alexander et al. (2004) after studying eight populations of Astragalus oniciformis by ISSR technique. Also, Vicente et al., (2011) have conducted the Mantel test in research of about five populations of Astragalus nitidiflorus, and the results refuted a positive correlation between genetic and geographic distances (Fig. no. 8) Testul Mantel y = x R² = Y Linear (Y) Geographic distances (km) Figure no.8 Correlation of genetic distances with geographical distances Although there is no positive correlation between genetic and geographic distances, the principal coordinates analysis (Fig. no. 9) indicates two main groups of individuals: a group is comprising individuals from Gădălin, Cojocna - Beleni, Milăşel and Silivaşu de Câmpie populations, while the other group is composed of Săbed, Sărmăşel Gară, Valea Florilor and Viişoara populations. The high variability can be seen in individuals of the Gădălin population where individuals may be spreading more broadly, while at the population of Săbed population can be observed a more severe grouping. 20

22 Coord. 2 Ing. Katalin SZABO Principal Coordinates (PCoA) Coord. 1 Beleni Gadalin Milasel Sabed Sarmasel Gara Silivasu de Campie Valea Florilor Viisoara Figure no. 30 Principal coordinate analysis of eight populations of Astragalus exscapus ssp. transsilvanicus An explanation for the grouping of individuals in this manner may be the genetic migration of one population into another by biological vectors such as pollinators (eg. Bombus hortorum, B. pascuorum) or zoocoria, given the fact that Astragalus exscapus ssp. transsilvanicus multiplies exclusively through seeds (Becker et al., 2011). Another explanation could be the fact that before the fragmentation process of the habitats by human intervention, there existed a single homogeneous population of Astragalus exscapus ssp. transsilvanicus, which gradually, due to the isolation of the habitats, has suffered a process of adaptation to the new conditions of growth and development (personal communication). 21

23 4. CHAPTER VI. GENERAL CONCLUSIONS AND RECOMMENDATIONS The anthropogenic impact (the overgrazing and the fragmentation of the areas) has a negative effect over the habitats of this subspecies, because it determines changes in size of population. Consequently, in addition to in situ conservation, it is necessary the ex situ conservation of the subspecies, using modern methods such as the introduction of germplasm in gene banks. In the selection of populations for ex situ preservation, there are considered the population with highest genetic variability because this index is positively correlated with the survival potential of the species. In case of analyzed subspecies by molecular marking with SRAP primers, the largest genetic variability has been recorded in populations of Gădălin and Silivaşu de Câmpie. The genetic data from our research provides a more thorough basis for future investigations. By determining the number of migrants / generation there could be estimated the state of genetic balance between the populations of Astragalus exscapus ssp. transsilvanicus; in this regard it would be recommended the review of all existing populations with co-dominant markers. Regarding the methods of subspecies conservation in gene banks, a relevant study about seed germination and the behavior of subspecies under field conditions would be necessary. Also, the in vitro propagation of the subspecies and the Rhizobium bacteria investigations would be interesting subjects to study. As about the content of isoflavone glycosides in roots of Astragalus exscapus ssp. transsilvanicus identified in Astragalus membranaceus also the chromatogram obtained through HPLC technique confirms their existence in the analyzed extract. 22

24 SELECTIVE BIBLIOGRAPHY 1. Alexander, J. A., Aaron Liston, And Steve J. Popovich, 2004, Genetic diversity of the narrow endemic Astragalus Oniciformis (Fabaceae), American Journal of Botany 91(12): Bădărău Al. S., Dezsi St., Comes O., 2000, Cercetări biogeografice asupra speciilor stepice -silvostepice de Astragalus L. din depresiunea Transilvaniei, Studia universitatis Babes-Bolyai, Geographia, XLV, Bădărău Al. S., Dezsi St., Man T., 2001, Cercetări biogeografice asupra speciilor stepice -silvostepice de Astragalus L. din depresiunea Transilvaniei (II), Studia universitatis Babes-Bolyai, Geographia, XLVI, Becker, T., Voss, N., & Durka, W., 2011, Pollen limitation and inbreeding depression in an old rare bumblebee-pollinated grassland herb. Plant Biology (Stuttgart, Germany), 13(6), doi: /j x 5. Ciocârlan V., 1987, Flora ilustrată a României. Vol. 5, 383 pag. Editura. Academiei Române 6. Li G., Quiros C. F.,2001, Sequence-related amplified polymorphism (SRAP), A new marker system based on a simple PCR reaction: its application to mapping and gene tagging in Brassica, Theor. Appl. Genet., 103 p Lodhi M. A., Guang-Ning Z., F. N. F. Weeden, B.I. Reisch, 1994, A simple and efficient method for DNA extraction from grapevine cultivars, Vitis species and Ampelopsis, Plant Molecular Biology Reporter 12(1), pag Negri, V., Maxted, N. and Veteläinen, M., 2009, European landrace conservation: an introduction. In: Veteläinen, M., Negri, V. and Maxted, N. (eds.) European landraces: on-farm conservation, management and use. Pp Bioversity Technical Bulletin 15. Bioversity International, Rome 9. Nei M, Li WH., 1979, Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc Natl Acad Sci USA 76:

25 10. Ordonanţa de Urgenţă a Guvernului României Nr. 57 din 20 iunie 2007 privind regimul ariilor naturale protejate, conservarea habitatelor naturale, a florei şi faunei sălbatice 11. Page, R.D.M., 1996, TREEVIEW: An application to display phylogenetic trees on personal computers. Computer Applications in the Biosciences 12: Peakall, R., & Smouse, P. E., 2012, GenAlEx 6.5: genetic analysis in Excel. Population genetic software for teaching and research--an update. Bioinformatics (Oxford, England), 28(19), doi: /bioinformatics/bts Peakall,R. and Smouse,P.E., 2006, GenAlEx 6: genetic analysis in Excel. Population genetic software for teaching and research. Mol. Ecol. Notes, 6, Polhill, R.M. și Raven, P.H. (eds) Advances in Legume Systematics. Royal Botanic Gardens, Kew 15. Pop Rodica, M. Ardelean, D. Pamfil, Ioana Gaboreanu, 2003, The efficiency of different DNA isolation and purification protocols in ten cultivars of Vitis vinifera Buletinul USAMV-CN 59: Rabinowitz, D., 1981, Seven forms of rarity. pp in The Biological aspects of rare plant conservation. Ed. by H. Synge. Wiley 17. Rao, V., și Hodgkin, T., 2002, Genetic diversity and conservation and utilization of plant genetic resources. Plant Cell, Tissue and Organ Culture, Talebi, M., M. Kazemi and B.E. Sayed-Tabatabaei, 2012, Molecular diversity and phylogenetic relationships of Pistacia vera, Pistacia atlantica subsp.mutica and Pistacia khinjuk using SRAP markers. Biochemical Systematics and Ecology 44: Vicente MJ, Segura F and Aguado M., 2011,. Genetic diversity of Astragalus nitidiflorus, a critically endangered endemic of SE Spain, and implications for its conservation. Biochem. Syst. Ecol. 39: Vojnits A. M.,Eszter Acs, 1995, A population of the Hungarian Zephyr Blue, Plebejus sephirus kovacsi (Lepidoptera: lycaenidae), Holarctic Lepidoptera, Vol. 2 No. 12(1):

26 21. Vos P, Hogers R, Bleeker M, Reijans M, van de Lee T, Hornes M, Frijters A, Pot J, Peleman J, Kuiper M, Zabeau M, 1995, AFLP: a new technique for DNA fingerprinting, Nucleic Acids Res 23: Wall. Wade A., Norman A. Douglas, William A. Hoffmann, Thomas R. Wentworth, Janet B. Gray, Qiu-Yun Jenny Xiang, Brian K. Knaus, Matthew G. Hohmann, 2014, Evidence of population bottleneck in Astragalus michauxii (Fabaceae), a narrow endemic of the southeastern United States, Conserv Genet 15: Witkowski Z. J., Król W., Solarz W., 2003 Carpathian List Of Endangered Species, WWF and Institute of Nature Conservation, Polish Academy of Sciences, Vienna- Krako 25