Mary Woodhead

How much effort is required to isolate nuclear microsatellites from plants

The attributes of codominance, reproducibility and high resolution have all contributed towards the current popularity of nuclear microsatellites as genetic markers in molecular ecological studies. One of their major drawbacks, however, is the development phase required to obtain working primers for a given study species. To facilitate project planning, we have reviewed the literature to quantify the workload involved in isolating nuclear microsatellites from plants. We highlight the attrition of loci at each stage in the process, and the average effort required to obtain 10 working microsatellite primer pairs.

Comparative analysis of population genetic structure in Athyrium distentifolium (Pteridophyta) using AFLPs and SSRs from anonymous and transcribed gene regions

To examine the performance and information content of different marker systems, comparative assessment of population genetic diversity was undertaken in nine populations of Athyrium distentifolium using nine genomic and 10 expressed sequence tag (EST) microsatellite (SSR) loci, and 265 amplified fragment length polymorphism (AFLP) loci from two primer combinations. In range‐wide comparisons (European vs. North American populations), the EST‐SSR loci showed more reliable amplification and produced more easily scorable bands than genomic simple sequence repeats (SSRs). Genomic SSRs showed significantly higher levels of allelic diversity than EST‐SSRs, but there was a significant correlation in the rank order of population diversities revealed by both marker types. When AFLPs, genomic SSRs, and EST‐SSRs are considered, comparisons of different population diversity metrics/markers revealed a mixture of significant and nonsignificant rank–order correlations. However, no hard incongruence was detected (in no pairwise comparison of populations did different marker systems or metrics detect opposingly significant different amounts of variation). Comparable population pairwise estimates of FST were obtained for all marker types, but whilst absolute values for genomic and EST‐SSRs were very similar (FST = 0.355 and 0.342, respectively), differentiation was consistently higher for AFLPs in pairwise and global comparisons (global AFLP FST = 0.496). The two AFLP primer combinations outperformed 18 SSR loci in assignment tests and discriminatory power in phenetic cluster analyses. The results from marker comparisons on A. distentifolium are discussed in the context of the few other studies on natural plant populations comparing microsatellite and AFLP variability.

Environmental and seasonal influences on red raspberry anthocyanin antioxidant contents and identification of quantitative traits loci (QTL)

Consumption of raspberries promotes human health through intake of pharmaceutically active antioxidants, including cyanidin and pelargonidin anthocyanins; products of flavonoid metabolism and also pigments conferring colour to fruit. Raspberry anthocyanin contents could be enhanced for nutritional health and quality benefits utilising DNA polymorphisms in modern marker assisted breeding. The objective was to elucidate factors determining anthocyanin production in these fruits. HPLC quantified eight anthocyanin cyanidin and pelargonidin glycosides: -3-sophoroside, -3-glucoside, -3-rutinoside and -3-glucosylrutinoside across two seasons and two environments in progeny from a cross between two Rubus subspecies, Rubus idaeus (cv. Glen Moy)xRubus strigosus (cv. Latham). Significant seasonal variation was detected across pigments less for different growing environments within seasons. Eight antioxidants mapped to the same chromosome region on linkage group (LG) 1, across both years and from fruits grown in field and under protected cultivation. Seven antioxidants also mapped to a region on LG 4 across years and for both growing sites. A chalcone synthase (PKS 1) gene sequence mapped to LG 7 but did not underlie the anthocyanin quantitative traits loci (QTL) identified. Other candidate genes including basic-helix-loop-helix (bHLH), NAM/CUC2-like protein and bZIP transcription factor underlying the mapped anthocyanins were identified.

Mapping QTLs for developmental traits in raspberry from bud break to ripe fruit

Protected cropping systems have been adopted by the UK industry to improve fruit quality and extend the current season. Further manipulation of season, alongside consideration of climate change scenarios, requires an understanding of the processes controlling fruit ripening. Ripening stages were scored from May to July across different years and environments from a raspberry mapping population. Here the interest was in identifying QTLs for the overall ripening process as well as for the time to reach each stage, and principal coordinate analysis was used to summarise the ripening process. Linear interpolation was also used to estimate the time (in days) taken for each plot to reach each of the stages assessed. QTLs were identified across four chromosomes for ripening and the time to reach each stage. A MADS-box gene, Gene H and several raspberry ESTs were associated with the QTLs and markers associated with plant height have also been identified, paving the way for marker assisted selection in Rubus idaeus.

The isolation of RNA from raspberry (Rubus idaeus) fruit

Previous attempts to extract high-quality, total RNA from raspberry (Rubus idaeus) fruits using published protocols have proven to be unsuccessful. Even the use of protocols developed for the extraction of RNA from other fruit tissue has resulted in low yields (1) or the isolation of degraded RNA (2). Here, we report on the development of a quick and simple method of extracting total RNA from raspberry fruit. Using this method, high yields of good quality, undegraded RNA were obtained from fruit at all stages of ripening. The RNA is of sufficient quality for northern analysis and cDNA library construction.

Isolation of RNA from blackcurrant (Ribes nigrum L.) fruit

Extraction of high-quality RNA from blackcurrant fruit has hitherto proved difficult, probably owing to high levels of phenolic and polysaccharide components in the berries. The procedure described here is a modification of one described for grape berries, and yields RNA suitable for in vitro translations, RNA blot analysis, and cDNA library construction.

The development of a genetic linkage map of blackcurrant (Ribes nigrum L.) and the identification of regions associated with key fruit quality and agronomic traits

The first genetic linkage map of blackcurrant (Ribes nigrum L.) was constructed using AFLP, SSR (genomic and EST-derived) and SNP markers, in a mapping population derived from two diverse breeding clones of blackcurrant from the SCRI breeding programme. Cluster analysis of the population revealed that the individuals within the population formed two distinct sub-populations, with segregation ratios consistent with one sub-population having the two intended parents, and the other being selfed segregants. The latter sub-population improves the map by providing a more informative estimate of recombination frequency than the crossed sub-population for some marker configurations, and also revealed the presence of two unlinked loci affecting viability. Several important phenological, agronomic and fruit quality traits were evaluated in the mapping population, and QTLs affecting these are located on the linkage map. This provides a framework for the development of marker-assisted breeding strategies for blackcurrant, to improve breeding efficiency and time to cultivar.

Morphological, ecological and genetic evidence for distinguishing Anastrophyllum joergensenii Schiffn. and A. alpinum Steph. (Jungermanniopsida: Lophoziaceae)

Abstract The liverwort Anastrophyllum joergensenii Schiffn., reported from Norway, Scotland, Alaska and the Sino-Himalaya is shown to consist of two distinct species, A. alpinum Steph. (treated before as a synonym of A. joergensenii) in the Himalaya, western China, Alaska and Scotland, and A. joergensenii Schiffn. s. str. in Norway, Scotland and western China. The two species are distinguished on genetic characters, size, leaf and perianth characters, and appear to have different ecological preferences. Anastrophyllum alpinum, although the more widespread of the two in Scotland, is there known only as non-fertile plants, whereas in the Sino-Himalaya fertile populations and sporophytes are not infrequent; in contrast, the rarer A. joergensenii can produce perianths in Scotland, Norway and Yunnan but androecia and sporophytes are unknown. The differences between the two are detailed and the ecology and distribution outlined.

The isolation of genomic DNA from blackcurrant (ribes nigrum L.)

A method is described for isolating DNA of high molecular mass (Mr) from blackcurrant and other soft-fruit species. Following a hexacethylytimethyl ammonium bromide (CTAB)-based extraction procedure, samples are treated with a glycosidic hydrolase mixture and RNase, and then purified. The suitability of this DNA for Southern analysis and genomic-library construction is demonstrated.

Cloning and characterisation of the cDNA clones of five genes that are differentially expressed during ripening in the fruit of blackcurrant (Ribes nigrum L.)

Summary Measurements of respiration rate show that blackcurrant ( Ribes nigrum cv. Ben Alder) fruit do not exhibit a respiratory climacteric during ripening, and that ripe fruit produce only very low levels of ethylene. Differential screening of a cDNA library constructed from RNA extracted from blackcurrant fruit enabled the isolation of the cDNA clones of five genes that showed gready enhanced steady-state transcript levels in fully ripe fruit compared with green fruit. The expression patterns of the corresponding genes were also determined in other tissues of the blackcurrant plant. The sequences of the cDNA clones were compared with known sequences in databases. Four of the clones were identified tentatively on the basis of sequence similarity. pRIB 1 was similar to a protein found in the fruit of kiwifruit, pRIB3 was similar to other plant metaUothionein-like proteins, pRIB6 to members of the cysteine proteinase family and pRIB7 to a mitochondrial RNA splicing protein. It was not possible to identify pRIB5 on the basis of sequence similarity. Southern analysis indicated that all of these genes were present in the blackcurrant genome at low copy number. Sequences similar to RIB 3 and RIB 5 are not present in the genomes of red raspberry ( Rubus idaeus ) or Tayberry ( Rubus loganobaccus ). The nucleotide sequence data reported appears in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the following accession numbers: AJ007576 (pRIBl), AJ007577 (pRIB3), AJ007578 (pRIB5), AJ007579 (pRIB6) & AJ007580 (pRIB7).