PCR identification of rpgip1 transgene in Pisum sativum L. | PCR identifikacija transgena rpgip1 pri grahu (Pisum sativum L.)

English. Recent efforts to increase Ascochyta blight resistance of pea have focused on the introduction of foreign genes by genetic engineering. The rpgip1 gene from Rubus idaeus was introduced by Agrobacterium-mediated transformation into Pisum sativum, cv. Baroness with the aim to increase pea resistance to fungal diseases. Notwithstanding this success, practical applications have to be preceded by the development of analytical methods for screening. Singleplex and multiplex PCR assays were employed to test primer efficiency in identifying the rpgip1 transgene in 11 pea genotypes. Five from ten primer combinations were effective in identifying transgene or insert sequences. PCR amplification using five other primer pairs revealed unspecific amplicons. According to in silico analyses, they arose from retrotransposons and pea genes including homologues of rpgip1. Two sets of primers were prepared with the aim of simultaneous amplification of different rpgip1 fragments. Fingerprints were sums of bands observed from individual pairs so the utility of multiplex assays was demonstrated. An additional advantage of multiplex PCR was clear differentiation between the transgene and endogenous pgip genes present in the donor species, R. idaeus. Sequencing of two PCR products confirms that no substantial rearrangements at the rpgip1 transgene arose during development of transgenic plants. However, a deletion occurred at 59 bp in the PGIP+VST line and a substitution at 392 bp in the PGIP line. The frequency of point mutations was not high (1.1 x 10-3) and comparable with the frequency expected for host genes based on the neutral theory of molecular evolution.

Slovenian. Novejši dosežki pri povečanju odpornosti graha na Ascochyta so povezani z uvajanjem tujih genov s pomočjo genskega inženiringa. Gen rpgip1 iz malinjaka (Rubus idaeus) je vključen  v grah, cv. Baroness, s transformacijo z bakterijo Agrobacterium, da bi se povečalo odpornost graha na to glivično bolezen. Pred praktično uporabo te metode je potrebno razviti načine za spremljanje dedovanja tega transgena. Enojna in multipleks PCR sta bili uporabljeni za testiranje učinkovitosti začetnikov in za identificiranje transgena rpgip1 pri 11 genotipih graha. Pet od desetih začetnikov je bilo uporabnih za identifikacijo transgenov ali za vključevanje sekvenc. PCR namnoževanje z drugimi petimi začetniki je dalo nespecifične namnožke. Glede na in silico analize so ti nastali zaradi retrotranspozonov in grahovih genov, ki vključujejo homologe rpgip1. Dva seta začetnikov sta bila pripravljena za istočasno namnoževanje različnih odlomkov rpgip1. Elektroferogrami so bili vsote črt individualnih parov, tako je prikazana uporabnost  multipleksnega poskusa. Dodatna prednost multipleksnega PCR je razločna diferenciacija med transgenom in genom pgip prisotnim v donorski vrsti R. idaeus. Sekvenciranje dveh PCR produktov potrjuje, da ni pri  rpgip1 bistvenega prerazporejanja tekom razvoja transgenih rastlin. Toda pojavila se je delecija pri 59 bp v liniji PGIP+VST in substitucija pri 392 bp v liniji PGIP. Relativna pogostnost točkovnih mutacij ni bila visoka (1.1 x 10-3) in je bila primerljiva  z pogostnostjo pri gostiteljivih genih, glede na nevtralno teorijo molekulske evolucije.