Genetic diversity of red clover cultivars (Trifolium pratense L.) based on protein polimorphism

Abstract

Red clover is the second most important perennial forage legume. Based on morphological characters alone, it is difficult to distinguish accessories of red clover from each other because they have overlapping variations in terms of the major delimiting morphological and biological characters. The aim of this study was to analyze the genetic relationships of 32 red clover cultivars collected from European gene banks, as well as created in Serbia, based on seed storage proteins and isozymes. From 8 analyzed enzymic systems, there were no differences in zymograms for six enzymes only two enzymes were polymorphic. Two different allelic variants were found for enzyme shikimate dehydrogenase and three for phosphohexose isomeraze. The seed proteins in the area of higher molecular high weight, 55- 95 kDa, were identified as polimorphic. All the cultivars were placed into three clusters on the basis of Ward's distance range. The cluster pattern showed correlation between genetic diversity and geographic origin. Genetic distance between cultivars based on protein fingerprint could be used as a tool to control and protect intellectual property right over plant breeding material.

Crvena detelina je druga po značaju višegodišnja krmna leguminoza. U radu je proučavan proteinski polimorfizam sorti crvene deteline iz Srbije u poređenju sa sortama iz zapadne i severne Evrope koje se koriste kao standardni u procesu registracije i zaštite sorti poljoprivrednog bilja prema protokolu UPOV organizacije. Metodom horizontalne elektroforeze, na 11% skrobnom gelu, analizirani su enzimski sistemi: kisela fosfataza (ACP), šikimik dehidrogenaza (ShDH), malik enzim (ME), beta glukozidaza (ßGLU), izocitrat dehidrogenaza (IDH), malat dehidrogenaza (MDH), fosfoglukozo izomeraza (PGM), fosfoheksozo izomeraza (PHI). Polimorfizam proteina semena analiziran je 12% SDS PAGE elektroforezom iz homogenizovanog uzorka semena. Enzim PHI je imao tri, ShDH dve alelne varijante, dok su ostali enzimi bili monomorfni. Proteini semena su polimorfni, naročito proteini velikih molekulskih masa u opsegu 50-80 kDa. Multivarijacionom analizom (Ward's distance range) izdvojena su tri klastera, medu kojima se posebno izdvaja podgrupa koja obuhvata NS sorte crvene deteline, što ukazuje na srodnost materijala koji je korišćen u stvaranju ovih sorti. U odnosu na nivo ploidnosti takođe se izdvaja zaseban podklaster u kojem dominiraju tetraploidne forme crvene deteline sa severa Evrope. Polimorfizam proteina semena sorti crvene deteline bi mogao da se koristi kao dopunski pokazatelj u procesu njihove registracije i zaštite.