Articles
TOWARDS THE IDENTIFICATION OF CANDIDATE GENES INVOLVED IN THE SIT TOMATO (LYCOPERSICON ESCULENTUM MILL.) MUTANT BY USING CDNA-AFLP ANALYSIS IN ISOGENIC LINES
Article number
618_9
Pages
95 – 103
Language
English
Abstract
Amplified fragment length polymorphism (AFLP) analysis, a PCR based technique, is capable of detecting more than 50 independent loci in a single PCR reaction.
The objective of the present study was to assess the polymorphic AFLP fragments between two isogenic lines (IL) of tomato (Lycopersicon esculentum Mill.), obtained from a backcross of an ABA-deficient mutant in a genetic background cv. Money Maker, both at genomic (gDNA) and cDNA level.
Eighty primer pairs were tested on cDNA detecting a total of ~ 8.300 AFLP fragments in the IL, with an average of 100 fragments obtained per primer pair, 164 fragments (1.7% of the total observed) were polymorphic, between the genotypes tested.
Thirty primer pairs tested on genomic DNA resulted in ~ 3.200 AFLP fragments (an average of 103 fragments per primer pair), only 9 fragments (0.28%) were polymorphic between the wild type and the isogenic ABA-deficient mutant sitiens. The AFLP primer pairs that show differential profiles between the ILs were used to isolate transcript-derived fragments (TDFs) in order to characterize cDNA clones potentially involved in the ABA pathway.
Generation of ESTs as differentially expressed sequences, between IL, is proposed as a way of developing ESTs around specific trait for an integrated approach to isolate genes of interest.
Starting from the 164 transcript-derived fragments (TDFs) detected, 23 fragments were isolated and sequenced.
The obtained sequences, compared against public databases, showed similarity with several EST tomato sequences of unknown functions.
The objective of the present study was to assess the polymorphic AFLP fragments between two isogenic lines (IL) of tomato (Lycopersicon esculentum Mill.), obtained from a backcross of an ABA-deficient mutant in a genetic background cv. Money Maker, both at genomic (gDNA) and cDNA level.
Eighty primer pairs were tested on cDNA detecting a total of ~ 8.300 AFLP fragments in the IL, with an average of 100 fragments obtained per primer pair, 164 fragments (1.7% of the total observed) were polymorphic, between the genotypes tested.
Thirty primer pairs tested on genomic DNA resulted in ~ 3.200 AFLP fragments (an average of 103 fragments per primer pair), only 9 fragments (0.28%) were polymorphic between the wild type and the isogenic ABA-deficient mutant sitiens. The AFLP primer pairs that show differential profiles between the ILs were used to isolate transcript-derived fragments (TDFs) in order to characterize cDNA clones potentially involved in the ABA pathway.
Generation of ESTs as differentially expressed sequences, between IL, is proposed as a way of developing ESTs around specific trait for an integrated approach to isolate genes of interest.
Starting from the 164 transcript-derived fragments (TDFs) detected, 23 fragments were isolated and sequenced.
The obtained sequences, compared against public databases, showed similarity with several EST tomato sequences of unknown functions.
Authors
A. Sciancalopore, G. Martelli, I. Greco, F. Cellini, F. Sunseri, M. Gallitelli
Keywords
Lycopersicon esculentum Mill., TDFs, transcript-derived fragments, ABA-deficient mutant, sitiens, GenEST
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