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2. MATERIAL AND METHODS
An enriched microsatellite library was screened for two microsatellite repeat sequences (CT and
GT) and was constructed from the Brazilian variety IAC-UNA according to Billotte et al (1999) and Kijas et al
(1994). The characterization of the library has already been described (Benchimol et al. 2005, in submission).
Primer designing was performed using PrimerSelect (DNAStar, Inc) with the following conditions:
a) amplification DNA size from 150 pb to 350 pb; b) GC content between 40-60%; c) Temperature
annealing (Ta) between 45 and 60ºC; d) primer length between 18 and 22 pb; e) no hairpins or dimmers.
Twenty primers were designed for microsatellite loci and were tested for 14 genotypes, including
Andean (A) and Mesoamerican (M) gene pools. Total DNA was extracted from the following accessions:
‘Sanilac’ (M), ‘Baetão’ (M), ‘Red Kidney’ (A), ‘Jamapa’ (M), ‘Flor de Mayo’ (M), ‘Tu’ (M), ‘Carioca Comum’
(M), ‘Jabola’ [CB] (A), ‘Fradinho Cruzeiro’ (Vigna spp), ‘87-JP-12’ (A), ‘BAT-93’ (M), ‘Jalo EEP-558’
(A), ‘IAC-UNA’ (M), ‘CAL-143’ (A). PCR reactions were carried out in a total volume of 25 µl final
volume containing 50 ng of template DNA, 0.2µM of forward and reverse primer, 100 µM of each
dNTP, 2.0 mM MgCl 2 , 10 mM Tris-HCl, 50 mM KCl, and 0.5 U Taq DNA Polymerase (Invitrogen, SP.,
Brazil). Reactions were performed using the following conditions: 1 min. at 94 o C; then, 30 cycles of
[1 min. 94 o C, 1 min. at Ta, 1 min at 72 o C], followed by 5 min at 72 o C. Amplification products were
checked by electrophoresis on 3% agarose gels and then loaded on 6% w/v denatured polyacrylamide
gels using a 10 bp ladder as a size standard, and silver stained according to Creste et al (2001).
The polymorphism information content (PIC) value was calculated by the following formula:
PIC = 1-, where fi is the frequency of the i allele (marker) for the i th SSR locus (Lynch and Walsh
1998).
3. RESULTS AND DISCUSSION
Fifty microsatellite were polymorphic for the twenty analyzed loci (Table 1), four of them being
monomorphic and one without amplification. Thirteen primers were perfect dinucleotide motifs, one was
imperfect (FJ 20) and six presented compound motifs. The number of alleles ranged from 01 to 03, with
average 2.07 alleles per locus. PIC values ranged from 0.14 to 0.65. The higher PIC (0.65) was founded
in FJ 14, that present the biggest repetition motif, (GA) 10 , and the most elevated number of alleles (03).
The primer pairs were also used to amplify Vigna spp., accession “Fradinho Cruzeiro” using the same
conditions optimized for Phaseolus vulgaris acessions. These results showed that microsatellites well
succedded in amplifying microsatellite loci for Phaseolus. This new microsatellites can be used for genetic
and QTL mapping, marker-assisted selection and germplasm characterization in common bean, and
they consist in an important tool for genetic improvement in common bean breeding programs.
REFERENCES
Benchimol LL, Campos T, Carbonell SAM., Colombo CA, Gouvea LRL, Chioratto AF, Risterucci AM, Souza AP
(2005) Development of Simple Sequence Repeat (SSR) markers in common bean (Phaseolus vulgaris L.) from a
genomic enriched library. (Submitted to Plant Breeding)
Documentos, IAC, Campinas, 79, 2007
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