Genetic Relationships Among Medicago sativa L. Clones Commonly Grown in Central Anatolia

Amplified fragment length polymorphism (AFLP) markers were used to investigate genotypic variability among 34 alfalfa clones (M. sativa) using 15 primer combinations with restriction enzymes EcoRI, PstI and MseI. 34 unique AFLP fragments were observed. The 15 primer pairs produced a total of 1002 fragments of which 460 were polymorphic. The number of polymorphic fragments detected per primer combination ranged 7 to 67. Furthermore, 22 clone-specific markers were also detected in the 13 clones. Data analysis was performed with NTSYSpc version 2.1 software. Genetic distance values ranged 5.9374 to 1.1453. Fifteen clones which showed the highest genetic variation were selected for producing synthetic variety of Alfalfa.

Alfalfa is distributed worldwide and grows in highly contrasting environments.This extensive geographical adaptation promotes genetic variation and give breeders possibility of using highly diverse genotypes in breeding programs (Maureira et al. 2004).Synthetic variety breeding is most effective and intensive method to improve perennial forage crops like alfalfa through polycross.Classical breeding studies require long time to select individual clones for synthetic variety production (Moreno-Gonzales and Cubero 1994).
Knowledge about genetic variability in species is important for optimal use of genetic resources in plant breeding programs.The use of molecular markers especially AFLP (Amplified fragment length polymorphism) markers help to select genetic dissimilarity potential parents for production of synthetics (Kidwell et al. 1994a).Some studies in alfalfa have detected positive associations between DNA marker diversity and hybrid yield (Kidwell et al. 1994a, Kidwell et al. 1994b, Osborn et al. 1998, Segovia-Lerma et al. 2003).AFLP is frequently used for the identification of molecular markers because of certain advantages over other techniques, such as high level of identified polymorphism, high reproducibility, and relative technical simplicity (Vos et al. 1995).
The aim of the study was to genetically evaluate alfalfa plants, collected from different regions of Turkey

Material and Methods
Plant material: Thirty four individual plants were collected from different parts of Central Anatolia to breed a new synthetic alfalfa variety compatible with climatic conditions of Central Anatolia (Table 1).Initially, the plants were phenotypically evaluated in terms of important agronomic characteristics.Thereafter, they were multiplied using shoot tip cuttings using perlite and vermiculite (3:1) as rooting medium.
DNA extraction and AFLP analysis: DNA was isolated from leaf tissue of two weeks old seedling as described by Doyle and Doyle (1987).
AFLP analysis was done following Vos et al. (1995) with minor modifications.Thirty four alfalfa clones were analyzed with seventeen primer combinations.Two primer pairs (P55 + CGA/ M55+ Amplification of the generated fragments was performed in two consecutive amplification cycles with primers containing one, or three selective nucleotide extensions (Table 2).First, preamplification was performed using two primers P00/M00 or E00+C/ M00 with following PCR conditions: 60 s at 94 ºC; 30 s at 60 ºC; 60 s at 72 ºC; this was followed by 7 min at 72 ºC extension, for 26 cycles.Selective amplification was conducted using three PstI, five EcoRI and nine MseI primers.Each primer contained three selective nucleotide extensions at the 3′ end (Table 2).PstI/EcoRI primer was labeled by phosphorylating the 5" end with [ 33 P]ATP.
The selective amplification was performed for 36 cycles with the following cycle profile: a 30s DNA denaturation step at 94 ºC, a 30s annealing step at 65 ºC and a 1 min extension step at 72 ºC.The annealing temperature of 65 ºC in the first cycle was subsequently reduced in each cycle by 0.7 ºC for next 12 cycles, and was continued at 56 ºC for remaining 23 cycles.All amplification reactions were performed in a Biometra T-Gradient thermocycler.
Table 2. Sequences of adapters, preamplification and selective amplification primers employed.
Data analysis: All genotypes were scored for presence or absence of polymorphic AFLP fragments and the data were entered into a binary matrix as discrete variables ("1" for presence and "0" for absence of a homologous fragment).Only distinct, reproducible, well-resolved fragments were scored.Data were analyzed with NTSYSpc version 2.1 (Numerical Taxonomy and Multivariate Analysis System, Version 2.1) (Rohlf 2000).

Results and Discussion
A large range of variation was obtained using different AFLP primer combinations.Fifteen AFLP primer pairs revealed 460 polymorphic bands among 1002 scorable bands (45.90 % polymorphism).The minimum number of polymorphic bands were produced by E26+M62 and P57+M50 (7 bands), whereas the maximum number of polymorphic bands were produced by E17+M61 and E25+M53 (67 bands) (Table 3).
There was a marked difference between EcoRI/MseI primers and PstI/MseI primers in the number of the visible bands observed.EcoRI/MseI primers produced a slightly larger number of fragment compared to PstI/MseI primers.No significant differences were recorded in percentage of polymorphic bands produced by both of EcoRI/MseI (21.86-54.09%)and PstI/MseI primers ((16-73.62%)Each clone presented a unique AFLP pattern.There were clone-specific markers (present in one clone but absent in the others).Twenty two (22) clone specific bands for 13 clones were detected (Table 4).This revealed that for identification of a given clone, specific amplification profiles obtained with single primer/clone combination can be used.Distribution of the clonespecific markers according to primer combinations is shown in Table 4.
Genetic similarity and diversity analysis among thirty four alfalfa clones was performed using the data analysis software, NTSYSpc version 2.1 software (Numerical Taxonomy and Multivariate Analysis System) (Rohlf, 2000).The genetic distance matrix was obtained using "Nei72"algorithm (Table 5).A dendrogram was constructed using the unweighted pair group method average (UPGMA) clustering (Figure 1).According to the genetic distance matrix presented in Table 5,    The locations of the clone pairs showing high genetic distance indicate implications of different ecogeoprachic characteristics on development of plants.
A synthetic variety is developed by intercrossing a number of genotypes known for superior combining ability with high genetic distance.Therefore, synthetic variety is made up of genotypes previously tested for their ability to produce a superior progeny when crossed in all combinations in agreement with Ferreira et al. (1995) who emphasised that heterosis and the combining ability of parents depend directly on the genetic diversity between them and the chance of finding promising combinations is better when more divergent material is used.
The feasibility of using AFLP DNA markers in future marker-based assessments of genetic diversity in alfalfa was supported by the observation that hierarchical patterns of diversity among the germplasms were associated with their geographic, origins.Use of these primers for automated AFLP analysis could be used as a high-throughput system for accurately characterizing genetic diversity among large numbers of alfalfa populations for breeding purpose.This information should also prove useful in designing strategies to more efficiently manipulate heterosis in alfalfa.This approach could subsequently be refined to include individual genotype analysis for more detailed characterization of specific populations of interest.

_______________________________________________________ 1 The
Scientific and Technical Council of Turkey, Research Institute for Genetic Engineering and Biotechnology, P.O.Box 21, 41470 Gebze-Kocaeli indicates EcoRI adapter sequences; P indicates PstI adapter sequences ; M indicates MseRI adapter sequences.

Table 1 .
The locations of the clones used in AFLP analysis.

Table 3 .
AFLP primer combinations generating polymorphic products after Pst I/Mse I and EcoR I/Mse I enzyme digestion, and distribution of AFLP markers.

Table 4 .
Distribution of the clone-specific markers obtained from AFLP reactions according to primer combinations.

Table 5 .
Genetic distance matrix based on Nei 72.

Table 6 .
Selected clones for synthetic variety breeding Medicago sativa L. clones commonly grown in Central Anatolia"