A procedure for identifying reproducible RAPD markers from woody plant DNA is presented. The procedure relies on using a PCR buffer that contains 1% Triton-X-100 and 0.1 % gelatin [previously described for successful polymerase chain reaction (PCR) amplification of 16S/23S rRNA intergenic spacer regions from eubacteria], and amplification conditions of 50 cycles: 30 sec at 94C, 70 sec at 48C, and 120 sec at 72C. The combination of this buffer and these conditions amplified consistent fragments in higher amounts, as compared to other standard PCR buffers and conditions generally used for RAPD analysis. This procedure resulted in reliable RAPD patterns for all organisms tested. Chemical name used: α-[4-(1,1,3,3,-tetramethylbutyl)phenyl]-cohydroxypoly(oxy-l,2-ethanediyl) (Triton-X-l00).
Amnon Levi, Lisa J. Rowland, and John S. Hartung
K.S. Lewers, J.L. Maas, S.C. Hokanson, C. Gouin, and J.S. Hartung
Bacterial angular leafspot disease (Xanthomonas fragariae Kennedy and King) of strawberry (Fragaria species and F. ×ananassa Duch. cultivars) has become increasingly important to strawberry fruit and plant production. Strawberry cultivars and species vary in susceptibility to infection. However, little is known regarding epidemiology of the disease and resistance to infection. Two octoploid genotypes, a native F. virginiana (US 4808, tested as SG-89) and a F. virginiana (SG 26) × F. ×ananassa (`Earliglow') hybrid (US 4809, tested as 80-4-38), previously were found to be highly resistant to two differentially pathogenic strains of X. fragariae representing two of four genotypic strain groups. Our objective was to determine the number of genes involved with resistance for these two strawberry genotypes, whether strawberry resistance is conferred by dominant or recessive alleles, and whether or not the heritability is high enough for breeders to reliably make selections of resistant individuals in breeding populations. About 120 F1 seedlings from crosses of susceptible `Sweet Charlie' with each of the two resistant genotypes were clonally propagated and challenged with each of four X. fragariae strains. These strains were selected to represent four genotypes of X. fragariae defined by repetitive element based PCR: ATCC 33239, Xf-3, Xf-6, and Xf-1425. Plants were quantitatively rated on a scale of 0 (resistant) to 5 (susceptible) in replicated evaluations. High estimates for broad sense heritability support the conversion of the quantitative disease scores to qualitative scores and the classification of genotypes as resistant or susceptible. The qualitative ratings were used to estimate the number of genes involved with resistance. Some segregation ratios fit a 7S:1R ratio, and others fit a 15S:1R ratio, indicating that three or four unlinked loci could explain the inheritance of resistance in these populations. The high estimates for broad sense heritability show that resistant progeny can be selected with confidence, though large populations will be needed to identify enough resistant progeny from which to select for other important traits.
J.L. Maas, C. Gouin-Behe, J.S Hartung, and S.C. Hokanson
Bacterial angular leafspot disease (BALD) of strawberry, caused by Xanthomonas fragariae Kennedy & King, has dramatically affected commercial fruit and plant production throughout the world. Leaf lesions may kill leaves, while lesions on sepals make fruit unmarketable. The bacterium can kill stolon-tip plantlets that are being rooted for transplanting. Since plants become systemically infected, there is no adequate chemical control for BALD under conditions that favor development and spread of the disease. Strawberry is the only host and no cultivars or advanced selections have proven resistant to this disease. We screened 23 Fragaria ×ananassa, 13 F. chiloensis, 56 F. virginiana, and 2 F. vesca genotypes for resistance to two pathogenic isolates of X. fragariae (ATCC-33239, the original strain from Minnesota and Xf-3 from North Carolina). Leaves were inoculated by forcing bacterial suspensions into leaves under pressure with a syringe barrel and plunger. Plants were incubated in a moisture chamber for 3 days, followed by 1 week under mist and then placed on a greenhouse bench. Experiments were done twice for obviously susceptible reactions and three and four times for questionable and resistant reactions, respectively. Only two genotypes were found to show a resistant reaction: 80-4-38 (`Earliglow' (F. virginiana clone SG-26 from Georgia) and F. virginiana clone SG-89 (=Luby MS 7-7 from Minnesota). Each of these genotypes exhibited typical hypersensitive responses by walling-off inoculation areas. All other genotypes exhibited typical BALD symptoms 5 weeks after inoculation with both isolates.