Family-based study designs are again becoming popular as new next-generation sequencing technologies make whole-exome and whole-genome sequencing projects economically and temporally feasible. Here we evaluate the statistical properties of linkage analyses and family-based tests of association for the Genetic Analysis Workshop 17 mini-exome sequence data. Based on our results, the linkage methods using relative pairs or nuclear families had low power, with the best results coming from variance components linkage analysis in nuclear families and Elston-Stewart model-based linkage analysis in extended pedigrees. For family-based tests of association, both ASSOC and ROMP performed well for genes with large effects, but ROMP had the advantage of not requiring parental genotypes in the analysis. For the linkage analyses we conclude that genome-wide significance levels appear to control type I error well but that “suggestive” significance levels do not. Methods that make use of the extended pedigrees are well powered to detect major loci segregating in the families even when there is substantial genetic heterogeneity and the trait is mainly polygenic. However, large numbers of such pedigrees will be necessary to detect all major loci. The family-based tests of association found the same major loci as the linkage analyses and detected low-frequency loci with moderate effect sizes, but control of type I error was not as stringent.

Regions containing origins of replication (oris) have been localized in the chloroplast genome by electron microscopic studies (1). Precise determination of DNA sequences that are required for a replication event would involve the use of in vitro and in vivo replication systems. Studies on plant organelle DNA replication have been greatly hampered by the lack of any in vivo DNA replication system and have heavily depended on less reliable in vitro replication systems. Furthermore, the presence of RNA editing system, existence of numerous transcription initiation sites, lack of selectable markers or reporter genes and poorly defined promoters have made accomplishment of plant mitochondrial transformation a formidable challenge. In order to circumvent the aforementioned problems which interfere with the expression of foreign genes, we Figure 1), cells were spun down in a microfuge and the supernatant was discarded. After washing once with Buffer A, cells were resuspended in buffer B (Sucrose 0.33 M, Tris-HCI 50 mM, pH 8.0, EDTA 0.2 M). The cell suspension was mixed with 0.5 mm glass beads in a 2 ml Bead-Beater tube and disrupted for 5 min in a Mini-Bead Beater (Bispec Products). The resultant homogenate was transferred to a fresh Eppendorf tube and the beads were rinsed with 0.5 ml of Buffer B to collect all of the homogenate. The pooled homogenate was spun at 78 xg for 1 min to pellet nuclei and cell debris; the resultant supernatant was centrifuged at 1500Xg for 5 min to pellet chloroplasts and the remaining supematant was spun at 13000 xg for 10 min to collect mitochondria. Pellets were resuspended in 250 1l of buffer B containing in addition 2 mg/ml of proteinase K. After incubating at room temperature for 10 min 100 ul of 3.5 M NaCl was added, and the final volume was adjusted to 0.5 ml. After adding 50 ,ul of 10% CTAB (hexadecyl trimethylammonium bromide), the mixture was incubated at 60°C for 1 hr. At this point, the mixture was extracted once with an equal volume of chloroform-isoamyl alcohol (24: 1). DNA in the upper aqueous phase was precipitated with 2/3rd volume of isopropanol at -20°C overnight. The pelleted DNA was resuspended in 400 td TE (Tris 10 mM, EDTA 1 mM, pH 8.0), extracted with phenol/chloroform and reprecipitated with 2 volumes of ethanol. In vivo replication products thus obtained were digested with appropriate restriction enzymes and separated on agarose gels. Gels were dried and exposed to X-ray film for autoradiography.In this study, in vivo DNA replication has been followed in exponentially growing cells, after permeabilizing with LPC. Sugarbeet cells treated with LPC are not morphologically different from untreated cells (as viewed under oil immersion in Zeiss Axioplan, magnification X 1400) except that nuclei always appear disorganized in LPC-treated cells. There is a significant difference in [32P]-dTTP incorporation among the three genomes between untreated and LPC-treated cells. The mitochondrial genome consistently shows maximal incorporation of total cellular DNA synthesis (...