Report of the Tomato Genetics Cooperative Number 58

RESEARCH REPORTS TGC REPORT 58, 2008
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Primers, PTG190F1 and PTG190R1, for a CAPS marker were designed from the RFLP marker,
TG190 (36.3 cM for the potato-TXB 1992 map).
Forward primer, PTG190F1: 5‘-GCAGTACACTTCTCCTTATCATGTG-3‘
Reverse primer, PTG190R1: 5‘- AGTTTCAGTAGTTGTTCCAAATTCC-3‘
Primers, P7-43DF1 and P7-43DR1, for a co-dominant SCAR marker were designed from marker
cTOF-21-J12 (SGN-U321614, mRNA, BT014299, 43 cM), which matched exons in a sequence of
Vitis vinifera, AM427259.
Forward primer, P7-43DF1: 5‘- GGTAAAGAGATGCGATGATTATGTGGAG -3‘
Forward primer, P7-43DF3: 5‘- CACGGGATATGTTRTTGATAAGCATGT-3‘
Reverse primer, P7-43DR1: 5‘- GTCTTTACCACAGGAACTTTATCACC -3‘.
PCR protocol: DNA was extracted from fresh leaves of plants with MasterPure TM Plant Leaf
DNA Purification Kit (EPICENTRE ® Biotechnologies, Madison WI), and DNA adjusted to
approximately 15 ng/µl. PCR was carried out in 25-µl reactions containing 2.5 μl 2.5 mM dNTPs, 5 μl
10X buffer, 2.5 µl 25 mM MgCl2, 0.1 μl Taq polymerase (Promega Corp., Madison WI), 2.5 µl each
forward and reverse primer at 10 μM, 2.5 μl of 15 ng/μl DNA extract and H2O. PCR cycler
parameters were as follows: denaturation at 94ºC for 3 min, then 35 cycles at 94ºC for 30 sec,
annealing at 53ºC for 1 min, and extension at 72ºC for 1 min, followed by 72ºC for 10 min, then the
reaction was held at 4ºC. PCR reactions were performed in the MJ DNA Engine PT200
Thermocycler (MJ Research Inc., Waltham MA). Amplified fragments were separated by
electrophoresis through 1.5% or 2.0% agarose in 0.5X TBE buffer, then stained with ethidium
bromide, and visualized with UV light. For sequencing, ssDNA was digested in the PCR reactions
with shrimp alkaline phosphatase (Promega Corp.) and exonuclease I (EPICENTRE ®
Biotechnologies), and the PCR fragments were directly sequenced with Big Dye Sequencing Kit
and analyzed by the Biotechnology Center, University of Wisconsin-Madison.
The restriction enzyme digestion for the P7-43F1/R1 CAPS marker was a 20 μl reaction mixture
containing 13 μl water, 3 μl buffer D, 0.25 μl BSA, 1 μl NsiI (Promega Corp.), and 8 μl PCR reaction
mixture. The digestion for the PTG190F1/R1 CAPS marker was similar, using buffer B and AluI
(Promega Corp.). The reaction mixture was placed in a 37ºC water bath overnight. Analysis of
digestion was completed by electrophoresis through 1.5% agarose in 0.5X TBE buffer, then stained
with ethidium bromide, and visualized with UV light.
Germplasm: The cultivars M82-1-8 (H. Czosnek, Hebrew University of Jerusalem) and Purple
Russian (a heritage tomato, Seed Savers Exchange, Decorah IA) were the susceptible genotype (i-
3/i-3). L40, an F2 plant from Llanero F1 (resistant to begomoviruses, GenTropic Seeds, i-3/i-3) and
GMh6330 (i-3/i-3), a begomovirus-resistant inbred line from San Carlos University, were also used as
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