GENETIC FINGERPRINT ESTABLISHED FOR THE SELECTED ALFALFA GENOTYPES USING MOLECULAR MARKERS

1. GENETIC FINGERPRINTESTABLISHED FOR THE SELECTED ALFALFA GENOTYPES USING MOLECULAR MARKERS

2. OBJECTIVES • Assessment intra- and interspecific variability at molecular level • The work methods used were: • Molecular techniques of genetic variability detection (extraction of genomic DNA, enzymatic amplification through the PCR reaction using 13RAPD, 3SSR and 5ISSR primers, analysis of the reaction products through agarose gel electrophoresis); • Statistical-mathematical methods of result processing and interpretation.

3. Assessment of interspecific variability using ISSR molecular markers Polymorphism rate for the alfalfa genotypes using ISSR primers

4. UPGMA clustering of alfalfa genotypes using the ISSR primers

5. We identified 22 alleles present in all the 30 genotypes studied. These alleles may be considered ISSR specific markers for alfalfa

6. Assessment of intergenotypical variability using RAPD molecular markers Polymorphism rate for the alfalfa genotypes using RAPD primers

7. UPGMA clustering of alfalfa genotypes using RAPD primers

8. Specific RAPD alleles identified in studied alfalfa genotypes The 20 alleles identified in all the 30 genotypes studied may be considered RAPD specific markers for alfalfa

9. The results of the analyses performed using two primer categories proved that we identified, in some genotypes from the collection studied, specific DNA fragments These bands may be used as DNA markers in the identification of alfalfa genotypes

10. In order to assess the intraspecific genetic diversity, we tested 16 ISSR primers and used only three of them: AFca 11, AFct 32 and AFct 45.

11. Estimated heterozygoty, effective number of alleles and diversity index in alfalfa genotypes studied using SSR primers

12. CONCLUSIONS • Genomic DNA amplification from 30 genotypes with 5 ISSR primers, generated 117 amplicons, of which 95 were polymorphic markers, the mean – 18.2; • 22ISSR markers, present in all the 30 genotypes studied, may be considered specific for alfalfa • Variance analysis for the alfalfa studied genotypes, from the viewpoint of the bands generated by the ISSR primers, recorded high and significant values in the cultivars: Cosmina, Sigma, Super and the: F 1615-04, F 1109-99, F 105-90 lines etc • The results obtained with the help of the five ISSR primers, prove the existence of a high genetic variability among the genotypes analyzed, which could be efficiently explored in alfalfa breeding programs. • -20fragments amplified in all the 30 genotypes studied may be considered specific RAPD markers for alfalfa;

13. The RAPD analysis with the help of the 13 primers shows the existence of a high genetic variability among the genotypes studied, which could be efficiently explored in alfalfa breeding programs; The results of the genetic diversity assessment at loci level showed the highest frequent heterozygosity in F 907-97 (52.70 %), Cosmina (51.30 %), F 1822-06 (51.30 %), Satelit (46.70 %) and Dorina (43.30 %) genotypes; The highest frequent allele homozygosity at loci level was observed in F 1615-04 (83.30 %), Sigma (82.00 %), Alina (75.30 %), Stolo 13 (74.70 %) and F 1206-00 (74.70 %) genotypes; Also, in the studied genotypes, we observed the existence of a direct correlation between the estimated heterozygosity and the effective number of alleles, so that the genotypes having several alleles/locus record a higher frequency of heterozygosity, too;

14. THANK YOU !