conference schedule and program with abstracts - Horticulture ...

P-58
Characterization of grape cultivars through Expressed Sequence Tag Polymorphism
(ESTP)
M.F. Moura* 1 , H.E. Sawazaki 1 , A.R. Verdi 2 , C.L. Messias 3
1 Instituto Agronômico - IAC. Av. Barão de Itapura, Campinas, São Paulo, Brazil; 2 Instituto de
Economia Agrícola - IEA, São Paulo, São Paulo, Brazil; 3 College of Agriculture Engineering,
Feagri, UNICAMP, Campinas, São Paulo, Brazil
*Corresponding author: mouram@iac.sp.gov.br
The Brazilian cultivars ‘Maximo IAC 138’ (a hybrid of ‘Shiraz’ and ‘Seibel’ developed by Dr.
Santos Neto in the Instituto Agronômico de Campinas/IAC, SP, in 1946), ‘Rainha’ and
‘Madalena’ were characterized through Expressed Sequence Tag Polymorphism (ESTP) and
restriction sites. The foliar DNA was extracted using CTAB. The PCR was done using first with
eleven and then thirteen primers to find EST-SNPs (single nucleotide polymorphism) in the
grapevine genome. The amplicon sequencing was done after EXO-SAP purification, using the
Big Dye v. 3.0/ ABI PRISM 377 from Applied Biosystems, with three replicates. The sequences
analyzed with Sequencher 3.1 (Gene Codes Corporation) were compared using the Bioedit
v.7.05 and analyzed in silico for specific restriction sites (http://www.restrictionmapper.org/). It
was observed that one primer named 28 differentiated ‘Rainha’ from ‘Máximo’ in three regions,
while another primer, 126, differentiated ‘Madalena’ from ‘Rainha’ and ‘Maximo’ in two
regions and ‘Maximo’ from ‘Madalena’ in a different site. The primer 136 differentiated
‘Madalena’ from cultivars ‘Maximo’ and ‘Rainha’. The other primers seem not to have amplified
sequences with specific restriction site differences among cultivars. Through analysis “in silico”
we observed that with primer 136, the SduI enzyme differentiated ‘Rainha’ and ‘Maximo’ from
‘Madalena’ whereas with primer 28, the IMx enzyme differentiated ‘Maximo’ from ‘Rainha’.
The analyses carried out using the studied primers allowed the detection of sequences with
specific restriction sites and consequently, the differentiation of’ Maximo’, ‘Rainha’ and
‘Madalena’ cultivars.
137