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Development of SCAR and CAPS Markers Linked to the Beta Gene in Tomato

USDA-ARS, Vegetable Laboratory, Building 010-A, BARC-West, 10300 Baltimore Avenue, Beltsville, MD 20705

View larger version (63K): [in a new window]   Fig. 1. Cloning of OPAR181100 and UBC792830 RAPD markers linked to the B locus by the T/A cloning technique. OPAR18 and UBC792 amplified polymorphic fragments were excised from agarose gels and cloned into pGEM-T Easy vector. The same-sized fragments (indicated by arrows) were recovered from EcoRI digested plasmid DNA. Lanes m and M are 1-kbp and 100-bp DNA ladders, respectively. Lanes 1 to 4 and 6 to 9 are individuals of EcoRI digested cloned plasmid DNA with OPAR181100 and UBC792830 inserts, respectively. Lanes 5 and 10 are RAPD fragments amplified with primers OPAR18 and UBC792, respectively. Lane 11 is a pGEM-T Easy vector.

View larger version (90K): [in a new window]   Fig. 2. OPAR18 and UBC792 amplified RAPD products, SCAR18f/r and SCBC792f/r amplified SCAR products, and the amplified SCAR products subsequently digested with restriction enzymes. M is a 100-bp DNA ladder. Lanes 1 to 2 (OPAR18) and 7 to 8 (UBC792) are the RAPD products amplified from LA317 (BB) (lanes 1 and 7) and Floradade (bb) (lanes 2 and 8) parents. Lanes 3 to 4 and 9 to 10 are the SCAR18f/r and SCBC792f/r products amplified from LA317 (BB) (lanes 3 and 9) and Floradade (bb) (lanes 4 and 10). Lanes 5 and 6 are the SCAR18f/r SCAR products that were amplified and subsequently digested with RsaI for LA317 (BB) and Floradade (bb), respectively. Lanes 11 and 12 are the SCBC792f/r SCAR products that were amplified and subsequently digested with HinfI for LA317 (BB) and Floradade (bb) parents, respectively. Sizes of restriction enzyme digested fragments (Lanes 5–6 and 11–12) are indicated in Fig. 3 and 5.

View larger version (11K): [in a new window]   Fig. 3. Schematics of the 1067- and 779-bp fragments amplified by SCAR18f/r (a) and SCBC792f/r (b) primer pairs, respectively. BB and bb represent alleles from LA317 and Floradade, respectively. Short arrows at the ends represent the original 10-mer RAPD primers. Longer arrows indicate the locations of 22- or 24-mer SCAR primers. f and r represent forward and reverse SCAR primers, respectively. f1 is the forward SCAR primer used for the dominant SCBC682 marker. Solid triangles indicate the positions of RsaI or HinfI restriction sites.

View larger version (51K): [in a new window]   Fig. 4. Alignment of SCBC792f/r amplified nucleotide sequences from Floradade (bb) and LA317 (BB) parents. The original RAPD primer sequences are indicated in bold type and the SCBC792f, f1 and r primers are indicated by arrows. The HinfI restriction sites are indicated in the boxes. The nucleotide differences between bb and BB are indicated in bold italic type. A period denotes the absence of a nucleotide.

View larger version (90K): [in a new window]   Fig. 5. Two codominant CAPS markers, SCAR181067 (a) and SCBC792779 (b), linked to the B locus. Monomorphic SCAR18f/r and SCBC792f/r amplified products were digested with RsaI and HinfI, respectively. M is a 100-bp DNA ladder. P1, P2, and F1 are the LA317 (BB), Floradade (bb) parents, and F1 hybrid (Bb), respectively. BB, Bb, and bb are representative F2 individuals of high beta-carotene homozygotes, high beta-carotene heterozygotes, and low beta-carotene homozygotes, respectively.

View larger version (28K): [in a new window]   Fig. 6. A dominant SCAR marker, SCBC792682, linked to the B locus. M is a 100-bp DNA ladder. P1, P2, and F1 are the LA317 (BB) and Floradade (bb) parents, and the F1 hybrid (Bb), respectively. B_ and bb are representative high and low beta-carotene F2 individuals, respectively.