PCR and specifically random amplified polymorphic DNA (RAPD) analyses permit the analysis of hundreds of DNA samples per day after purified DNA is available. DNA preparation from the selected tissues, whether they are of animal, plant, or insect origins, is the timelimiting factor to fully exploit the potential of the technology. Numerous DNA isolation methods addressing this problem have been published, (l-s) but most of them, although simple, are designed either for animal or plant, or more often for specific species or tissues, and none has been reported for universal applications, including minute insects (<500 ~m). Here, we report a simple microextraction method for total DNA suitable for PCR-based DNA analyses in all species tested. The two key improvements are the use of a very high salt concentration in the extraction buffer that presumably "salts out" several PCR inhibitors, and the tissue disruption technique that renders the DNA extraction process simple, fast, and inexpensive. It provides highly repeatable results and uniform DNA yield. We use it routinely for extracting DNA from leaf tissues of Brassica plant species and from minute insects (smaller than 500 ~m) for both site-specific PCR amplification and RAPD analyses.The procedure is as follows: A 5-mmdiameter leaf disc for plant tissues, 10-20 mg of animal tissues, or a single intact insect (microhymenoptera; Trichogramma species) was immersed in 160 tzl of extraction buffer [200 mM Tris-HC1 (pH 8.0), 70 mM EDTA, 2 M NaC1, 20 mM sodium metabisulfite] in a microcentrifuge tube (Kontes). Tissue breakage was achieved by physical grinding using an electric motor-driven Kontes pestle until Technical no visible pieces of tissue remained. In the case of leaf tissues, the release of chlorophyll into the buffer was a good indication of effective breakage of the plant cell wall. Cells were lysed further by addition of 40 I~l of a 5% sarcosyl solution and were incubated at 60°C for 1 hr. The lysate was then centrifuged for 15 min at 16,000g to remove the cell debris. DNA was precipitated from the clear supernatant by the addition of 90 t~l of ]0 M ammonium acetate and 200 ~1 of isopropanol at room temperature for 15 min for plant and rat DNA but at -20°C for 2 hr for insect DNA because of the minute quantity of the latter. Total DNA was pelleted by centrifugation at 16,000g in the microcentrifuge for 15 min and then washed with 70% ethanol. The pellet was dried briefly and resuspended in 50 i~l of TE [10 mM Tris-HC1 (pH 8.0), 1 mM EDTA] with RNase added at 10 t~g/ ml. This protocol yields 500-750 ng of plant and rat DNA and -20 ng of insect DNA of sizes >23 kb, which is enough for at least 20 site-specific PCR or 50 RAPD reactions. Figure 1 shows the RAPD results with the DNA extracted from nine plant species, four different rat tissues, and four species of Trichogramma; not shown are similar results from the plant Brassica napus (canola) and two other species of microhymenoptera (Anaphes sordidatus and Anaphes sp. nov.). Both the quantity and quality o...

F2 segregation analyses of DNA restriction fragment length polymorphisms (RFLPs) detected between a cabbage line (No. 86-16-5) resistant to race 2 of Plasmodiophora brassicae (Woronin), the fungus responsible for clubroot disease, and a rapid cycling line (CrGC No. 85) was used to construct a detailed genetic map of Brassica oleracea. RFLP markers were random and seedling-specific cDNA clones. The 201 loci so far mapped in B. oleracea covered 1112 cM. They are assembled into nine major linkage groups and four small linkage groups. Twelve loci were found unlinked to any other markers. Twenty-one loci were detected with the 18 seedling-specific cDNAs. Two dominant QTLs for resistance to race 2 of the clubroot disease causal agent were also identified. Leaf morphology and biennial flowering appeared to segregate as single Mendelian traits, but only leaf morphology could be linked to other markers. This RFLP study in B. oleracea is providing additional information on genome organization and complements current RFLP mapping effort in B. napus.Key words: genetic mapping, Brassica oleracea, Plasmodiophora brassicae, breeding, clubroot resistance, DNA markers, RFLP.

F2 segregation analyses of DNA restriction fragment length polymorphisms (RFLPs) detected between two cultivars of canola ('Westar' × Topas') was used to construct a detailed genetic map of Brassica napus. RFLP markers were from a seedling-specific cDNA library. They were either randomly selected or previously characterized as seedling-specific cDNA clones. The 120 loci so far mapped in B. napus covered 1413 recombination units. They are assembled into 19 linkage groups. Seventeen loci were found unlinked to any other markers. Few polymorphisms were detected with the seedling-specific cDNAs and only seven loci could be mapped in this cross. Duplication of RFLP loci was extensive and reflects the amphidiploid nature of this species. However, several rearrangements of the linear order of duplicated loci could be seen. This RFLP study in B. napus provides important information on genome organization of functional DNA sequences and complements our current RFLP mapping effort in Brassica oleracea. The genetic markers of this map are currently being used in several breeding applications, such as tagging important agronomic traits and fingerprinting breeding lines and cultivars of canola, a major oilseed crop.Key words: genetic mapping, Brassica napus, breeding, restriction fragment length polymorphisms.

We have undertaken the construction of a Brassica napus genetic map with isozyme (4%), RFLP (26.5%) and RAPD (68%) markers on a 152 lines of a doubled-haploid population. The map covers 1765 cM and comprises 254 markers including three PCR-specific markers and a morphological marker. They are assembled into 19 linkage groups, covering approximatively 71% of the rapeseed genome. Thirty five percent of the studied markers did not segregate according to the expected Mendelian ratio and tended to cluster in eight specific linkage groups. In this paper, the structure of the genetic map is described and the existence of non-Mendelian segregations in linkage analysis as well as the origins of the observed distortions, are discussed. The mapped RFLP loci corresponded to the cDNAs already used to construct B. napus maps. The first results of intraspecific comparative mapping are presented.

Bulked segregant analysis was employed to identify random amplified polymorphic DNA (RAPD) markers linked to the restorer gene (Rfo) used in theOgura radish cytoplasmic male sterility of rapeseed. A total of 138 arbitrary 10-mer oligonucleotide primers were screened on the DNA of three pairs of bulks, each bulk corresponding to homozygous restored and male sterile plants of three segregating populations. Six primers produced repeatable polymorphisms between paired bulks. DNA from individual plants of each bulk was then used as a template for amplification with these six primers. DNA polymorphisms generated by four of these primers were found to be completely linked to the restorer gene with the polymorphic DNA fragments being associated either with the fertility restorer allele or with the sterility maintainer allele. Pairwise cross-hybridization demonstrated that the four polymorphic DNA fragments did not share any homology. Southern hybridization of labelled RAPD fragments on digested genomic DNA from the same three pairs of bulks revealed fragments specific to either the male sterile bulks or to the restored bulks and a few fragments common to all bulks, indicating that the amplified sequences are low copy. The four RAPD fragments that were completely linked to the restorer locus have been cloned and sequenced to develop sequence characterized amplified regions (SCARs). This will facilitate the construction of restorer lines used in breeding programs and is the first step towards map-based cloning of the fertility restorer allele.