Effect of storage regime on the stability of DNA used as a calibration standard for real-time polymerase chain reaction.

This article looks at storage factors influencing the stability of potential DNA calibration standards for use in quantitative polymerase chain reaction (PCR). Target sequences from the bacteria Campylobacter jejuni were cloned into a plasmid vector. Samples of these potential calibration standards were stored at +4, -20, and -80 degrees C as aqueous and lyophilized samples and were prepared as both single-use aliquots and multiple-use preparations. Results showed that the samples stored as single-use aqueous solutions at +4 degrees C and lyophilized samples stored at +4 and -20 degrees C were the most stable. Samples stored as frozen aqueous solutions at -20 degrees C were the least stable.

Deletion mapping of genetic regions associated with apomixis in Hieracium.

Although apomixis has been quoted as a technology with the potential to deliver benefits similar in scale to those achieved with the Green Revolution, very little is currently known of the genetic mechanisms that control this trait in plants. To address this issue, we developed Hieracium, a genus of daisies native to Eurasia and North America, as a genetic model to study apomixis. In a molecular mapping study, we defined the number of genetic loci involved in apomixis, and we explored dominance and linkage relationships between these loci. To avoid difficulties often encountered with inheritance studies of apomicts, we based our mapping effort on the use of deletion mutagenesis, coupled with amplified fragment length polymorphism (AFLP) as a genomic fingerprinting tool. The results indicate that apomixis in Hieracium caespitosum is controlled at two principal loci, one of which regulates events associated with the avoidance of meiosis (apomeiosis) and the other, an unlinked locus that controls events associated with the avoidance of fertilization (parthenogenesis). AFLP bands identified as central to both loci were isolated, sequenced, and used to develop sequence-characterized amplified region (SCAR) markers. The validity of the AFLP markers was verified by using a segregating population generated by hybridization. The validity of the SCAR markers was verified by their pattern of presence/absence in specific mutants. The mutants, markers, and genetic data derived from this work are now being used to isolate genes controlling apomixis in this system.

Bimodal patterns of floral gene expression over the two seasons that kiwifruit flowers develop.

Polymerase chain reaction fragments with homology to the Arabidopsis floral meristem identity genes LEAFY and APETALA1 have been isolated from kiwifruit (Actinidia deliciosa [A. Chev.] C. F. Liang and A. R. Ferguson) and have been named ALF and AAP1, respectively. Northern hybridisation analyses have shown that ALF and AAP1 have bimodal patterns of annual expression in developing first-order axillary buds and their subsequent shoots. This pattern of expression is consistent with the 2-year cycle of axillary bud, flower and fruit development observed in kiwifruit. The first period of expression was early in first-order bud development (late spring of the first growing season), when second-order meristems are initiated, and the second, approximately 10 months later, when those meristems differentiate flowers (late spring of the second growing season). In situ hybridisation analyses on axillary buds collected during late spring of the first growing season have shown ALF expression throughout the developing first-order buds and AAP1 expression was localised in developing second-order axillary meristems. During the spring of the second growing season, transcript accumulation for both ALF and AAP1 is localised in differentiating flowers. Our results show that important developmental events are occurring very early in kiwifruit first-order axillary bud development (spring of the first growing season) and it is likely that this includes floral commitment (evocation).