Fine mapping and DNA fiber FISH analysis locates the tobamovirus resistance gene L3 of Capsicum chinense in a 400-kb region of R-like genes cluster embedded in highly repetitive sequences.

The tobamovirus resistance gene L(3) of Capsicum chinense was mapped using an intra-specific F2 population (2,016 individuals) of Capsicum annuum cultivars, into one of which had been introduced the C. chinense L(3) gene, and an inter-specific F2 population (3,391 individuals) between C. chinense and Capsicum frutescence. Analysis of a BAC library with an AFLP marker closely linked to L(3)-resistance revealed the presence of homologs of the tomato disease resistance gene I2. Partial or full-length coding sequences were cloned by degenerate PCR from 35 different pepper I2 homologs and 17 genetic markers were generated in the inter-specific combination. The L(3) gene was mapped between I2 homolog marker IH1-04 and BAC-end marker 189D23M, and located within a region encompassing two different BAC contigs consisting of four and one clones, respectively. DNA fiber FISH analysis revealed that these two contigs are separated from each other by about 30 kb. DNA fiber FISH results and Southern blotting of the BAC clones suggested that the L(3) locus-containing region is rich in highly repetitive sequences. Southern blot analysis indicated that the two BAC contigs contain more than ten copies of the I2 homologs. In contrast to the inter-specific F2 population, no recombinant progeny were identified to have a crossover point within two BAC contigs consisting of seven and two clones in the intra-specific F2 population. Moreover, distribution of the crossover points differed between the two populations, suggesting linkage disequilibrium in the region containing the L locus.

Direct connection of supercritical fluid extraction and supercritical fluid chromatography as a rapid quantitative method for capsaicinoids in placentas of Capsicum.

The fruits of Capsicum annuum L. are used worldwide as chili peppers and in folk medicines. The pungent components of C. annuum, which are irritants, are called capsaicinoids (CAPS), and the most abundant components are capsaicin, dihydrocapsaicin, and nordihydrocapsaicin. To analyze CAPS in the placentas of Capsicum fruits rapidly and safely, we used a directly connected system of supercritical fluid extraction and supercritical fluid chromatography (SFE/SFC). As a column for SFE/SFC, only a silica-type column was found to be suitable. The CAPS contents in placentas of C. annuum cv. Jalapeno (hot type) and C. annuum cv. Shishitoh (less-hot type) determined by the SFE/SFC method agreed well with those in the range of 0-13.81 mg g(-1) fr. wt determined by the usual extraction-HPLC method. The SFE/SFC method has the advantages of no need for pretreatment and no (or minimal) need for organic solvents. We conclude that this method is useful as a rapid (20 min) and safe screening test for the pungency of various Capsicum fruits.

Genetic analysis of stunted growth by nuclear-cytoplasmic interaction in interspecific hybrids of Capsicum by using RAPD markers.

When eight cultivars of Capsicum annuum were used as female parents in interspecific crosses with two accessions of C. chinense, dwarfism occurred in hybrids originating from 10 out of 16 combinations, while hybrids of the remaining 6 combinations grew normally. In contrast, when C. chinense was used as female parent, all of the hybrids showed severely stunted growth as if affected by a virus. These results suggested that the stunted growth expressed in the cross of C. chinense x C. annuum is caused by an interaction between nuclear gene(s) from C. annuum and the cytoplasm of C. chinense. To examine the number of nuclear gene(s) which cause(s) the stunted growth, we backcrossed F1 hybrids of C. annuum x C. chinense to C. chinense. About one-quarter of the progeny in the backcrossed hybrids of C. chinense x (C. annuum x C. chinense) showed the same stunted growth shown by the f1 hybrids of C. chinense x C. annuum, suggesting that two complementary genes of C. annuum cause the stunted growth. However, the higher abortion rates of ovules and lower germination percentage of seeds in C. chinense x C. annuum than in the selfed C. chinense implied that the genetic ratio of the stunted type would have been higher than that observed in the C. chinense x (C. annuum x C. chinense) progeny. We then attempted a linkage analysis between the stunted growth and randomly amplified polymorphic DNA (RAPD) of C. chinense x (C. annuum x C. chinense) progeny. A RAPD marker that associated with 94% of the stunted plants but not with 94% of the normal one was identified. This confirmed that a single nuclear gene of C. annuum which is linked to the RAPD marker with a recombination value of 6% causes the stunted growth in an interaction with the cytoplasm of C. chinense.