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Subcellular fractionation and mtDNA extraction

A combination of mtDNA purification protocols was adopted (Scotti et.al 2001 and Gianniny et.al 2004). Briefly, this method yields 5 µg of mtDNA/g of tomato fresh tissue (etiolated seedlings). Quality control is performed by PCR amplification of serial dilutions of the extracted mtDNA with primers designed on actin (nuclear-), rubisco rbcL (chloroplastic-) and coxII (mitochondrial-specific) genes. mtDNA with less than 10 % of nuclear and chloroplastic contaminations is fragmented by sonication/hydrodynamic and/or enzymatic shearing in a range of 1000-3000 pb and purified from an agarose gel

Library construction and standard nomenclature>

Purified mtDNA fragments are repaired and ligated into the pMOS-Blue (pMOSBlue Blunt Ended Cloning Kit, Amersham Bioscience) and pZERO-2 vectors (Invitrogen) following the manufacture instructions. Selected clones are classified and stored in freeze media in 96-well plates according the following nomenclature: SlmiNNZZZXY. This code accounts for:
Sl: Solanum lycopersicon LA1706 (seeds provided by the C.M. Rick Tomato Genetics Resource Center, UC Davis).
mi: mitochondrial library
NN: library #
ZZZ: plate #
XY: well place in the plate
Individual clones are free available (only shipping is charge) upon request.

In house pipeline for sequence analysis

Sequence reads are generated in an ABI 3101 instruments by a single reaction using the M13 forward and reverse primers. Output information is automatically deposited into the INTA01 server. The following pipeline has been designed for information processing:
Base calling by Phred software.
Sequence vector masking by comparison with pMOS-Blue vector sequence using Cross_match with default options.
sequence validation and trimming by SeqClean.
contigs (and singletons) are generated by CAP3 software, with minimum overlap of 40 nt and identity percentage of 90%.
contigs (and singletons) are blasted against the following DB: