Zbornik - Društvo genetičara Srbije

82 ZBORNIK ABSTRAKATA III KONGRESA GENETIÈARA SRBIJE III-Pos-11
Subotica, 30. novembar - 4. decembar 2004.
UTVRÐIVANJE POLIMORFIZMA MIKROSATELITSKIH
PRAJMERA KOD HEKSAPLOIDNE PŠENICE
Vladislava Galoviæ 1 , S. Denèiæ 1 i D. Jelovac 2
1
Nauèni institut za ratarstvo i povrtarstvo, Novi Sad
2
Institut za kukuruz «Zemun polje», Beograd-Zemun
Ukupna genomska DNK izolovana je iz sorti/linija po modifikovanoj metodi PLASCHKE
et al. (1995). Kao predstavnici Gatersleben wheat microsatellites (GWM) korišæena su dva
seta mikrosatelitskih prajmera, GWM165 i GWM539, koji su opisani po RODER et al.
(1998a, 1998b). Kineska sorta «Chinese Spring» je izabrana kao standard za analizu
parametara kao što su: GWM designacija, velièina fragmenata po"CS" standardu, rang
velièine alela i lokacija na hromozomu. PCR umnoavanje eljenih fragmenata je izvedeno
u zapremini od 30ul u Eppendorf PCR mašini po metodi Roder et al. (1998b).
Procedura provere PCR produkata na agaroznom gelu, priprema PCR produkata za
nanošenje na PAA (polyacrylamide gel) gel, uslovi elektroforeze kao i bojenje PAA gela
sprovedena je po GALOVIÆ et al. (2004). Statistièka obrada podataka je raðena u
programu NTSYSpc, modulom SIMQUAL za izraèunavanje genetièke distance.
Izraèunata je genetièka distanca po proceduri DICE (NEI and LI, 1979; DICE, 1945).
Prikaz UPGMA dendrograma uraðen je modulom SAHN (Sequential agglomerative
hierarical nested cluster analysis) po metodi SNEATH and SOKAL (1973). Oba, za
pšenicu specifièna prajmer seta umnoila su oèekivane fragmente (prema podacima o
DNK sekvenci). Polimorfnost oba primenjena prajmer seta potvrdila je ocenjena alelna
varijabilnost èime je detektovano ukupno 10 razlièitih alela za oba gwm lokusa. Na
osnovu dobijenih rezultata moe se zakljuèiti da je prajmer set gwm539 sa 7 razlièitih
alela pokazao veæu polimorfnost od prajmer seta gwm165 sa 3 razlièita alela kod
posmatranih genotipova.
DETERMINATION OF POLYMORPHISM OF
MICROSATELLITE PRIMERS IN HEXAPLOID WHEAT
Total genomic DNA was isolated from the cultivars and lines using a modification of the
method by PLASCHKE et al. (1995). The Gatersleben wheat microsatellites (GWM)
were represented by two sets of microsatellite primers, GWM165 and GWM539, which
were described according to RODER et al. (1998a, 1998b). The cultivar Chinese Spring
was used as the standard to analyze parameters such as GWM designation, fragment size
according to the CS standard, allele size rank and location on the chromosome. PCR multiplication
of desired fragments was performed at 30ul volume on an Eppendorf PCR
machine according to the method by Roder et al. (1998b).
The checking of the PCR products on agarose gel, their preparation for transfer onto
PAA (polyacrylamide gel) gel, electrophoresis conditions, and staining of the PAA gel
were all carried out according to GALOVIÆ et al. (2004). Statistical data processing was
done using the NTSYSpc program and the SIMQUAL module for genetic distance calculation.
Genetic distance was calculated according to DICE (NEI and LI, 1979; DICE,
1945). The UPGMA dendrogram was presented using the SAHN module (Sequential
Agglomerative Hierarchical Nested Cluster Analysis) according to SNEATH and
SOKAL (1973). Both of the wheat-specific sets of primers multiplied the expected fragments
(according to data on DNA sequence). The polymorphism of both primer sets was
confirmed by the estimated allelic variability and a total of 10 different alleles were detected
for the two gwm loci. The results showed that the gwm539 primer set with seven
different alleles exhibited a higher level of polymorphism than the gwm165 one with
three different alleles.