The Journal of Horticultural Science & Biotechnology
Vol. 87 No: 1
Data mining of ESTs to develop dbEST-SSRs for use in a polymorphism study of cauliflower (Brassica oleracea var. botrytis)
E. VAIDYA, R. KAUR and S.V. BHARDWAJ
Expressed sequence tags (ESTs) provide a valuable resource for developing simple sequence repeat (SSR; microsatellite) markers.
In this study, 3,364 EST sequences belonging to four varieties of Brassica oleracea (botrytis cauliflower), capitata (cabbage), italica (broccoli), and gemmifera (brussels sprout) and to Arabidopsis thaliana, derived from the NCBI website (http://www.ncbi.nlm.nih.gov/nucest), were screened for SSR motifs using two search programmes, MISA (http://pgrc.ipk-gatersleben.de/misa/misa.html) and SSRIT (http://www.gramene.org/db/markers/ssrtool). A total of 76 ESTs were found to contain tri-nucleotide or tetra-nucleotide repeat motifs, with AAC and ACAT being in greatest abundance.
Using PRIMER3 software (www.frodo.wi/mit.edu/primer3), 62 primer pairs were designed for further use as potential genic markers.
Of this set, 16 primer pairs were custom synthesised and used in polymorphism studies on cauliflower.
Annotation analysis using BLASTX revealed that a putative function could be assigned to 15 of the 16 EST-SSRs selected for primer design.
Thirteen of these 16 markers gave scorable bands, 11 of which revealed moderate-to-high polymorphism information content (PIC) values across 20 genotypes of cauliflower.
Eighteen genomic SSR primers were also used in this study, out of which 16 primer pairs amplified cauliflower DNA, eight primers being polymorphic.
Cluster analysis based on dendrograms formulated using the UPGMA algorithm revealed the genetic distances between genotypes, which so far have been based only on morphological traits.
The new set of EST–SSR markers described in this study should be useful for further studies on genetic diversity and genome analysis in this major crop species.
ISHS members & other users
(PDF 84707 bytes)
Go back to previous page