Cross-species amplification and phylogenetic reconstruction using fragaria microsatellite primers

Twenty-eight microsatellite primer pairs developed from Fragaria vesca ‘Rügen’ were applied to sixteen accessions representing eight diploid Fragaria species. The number of alleles generated, the power of discrimination and the percentage of accessions where no PCR product could be amplified were calculated for each locus for the thirteen non-F. vesca accessions. A phylogeny was then generated for the species accessions sampled, using the presence or absence of alleles at the polymorphic loci as character states. Despite the problems inherent in phylogeny reconstruction from microsatellite data, the phylogeny showed some congruence with a previously published phylogeny of Fragaria, based on nucleotide sequence data. However, relationships inferred from microsatellite allele data were relatively unresolved and poorly supported. The genetic basis of allelic polymorphisms at specific loci was investigated through direct sequencing of the PCR products amplified by three primer pairs. The potential utility of sequence data generated from microsatellite loci in evolutionary studies of closely related species groups is briefly explored.

Aspects of isolation underpinning mitotic behaviour in lupin protoplasts

This study reports on the influence of critical isolation factors on the subsequent culture of protoplasts of Lupinus albus L. Protoplasts were isolated from in vitro seedling cotyledons of five early maturing accessions in which protoplast yields and division frequencies appeared to be correlated as a high protoplast yield corresponded with a high division frequency. The overall difference among the accessions for mitosis was non- significant, although the highest yield and division frequency were observed in accession LA132, with Alban giving a significantly lower level. Accession Lucrop produced the lowest number of protoplasts, all of which collapsed during culture. Of the enzyme types used for tissue maceration, Pectolyase Y23, was significantly inferior to Macerase in terms of giving way to mitosis. The extent of division in Macerase- isolated protoplast population was 266% higher than that in the Pectolyase Y23- isolated one. The physiological maturity level of the explant, expressed in terms of developmental age, was optimal when 14 - 18- day- old seedling cotyledons were used for protoplast production and culture, rather than more mature ones, despite higher protoplast yields in the latter. On K8p medium, the protoplast division frequency was 129% greater when 18- day- old seedling cotyledons were used, than that with any other treatment. This work on protoplast culture of the potentially important lupin species, which is a pulse rich in dietary protein, oil and fibre, allows a further understanding of the biology, with an aim to advance lupin biotechnology.

In situ analysis and structural elucidation of sainfoin (Onobrychis viciifolia) tannins for high-throughput germplasm screening

A rapid thiolytic degradation and cleanup procedure was developed for analyzing tannins directly in chlorophyll-containing sainfoin (Onobrychis viciifolia) plants. The technique proved suitable for complex tannin mixtures containing catechin, epicatechin, gallocatechin, and epigallocatechin flavan-3-ol units. The reaction time was standardized at 60 min to minimize the loss of structural information as a result of epimerization and degradation of terminal flavan-3-ol units. The results were evaluated by separate analysis of extractable and unextractable tannins, which accounted for 63.6−113.7% of the in situ plant tannins. It is of note that 70% aqueous acetone extracted tannins with a lower mean degree of polymerization (mDP) than was found for tannins analyzed in situ. Extractable tannins had between 4 and 29 lower mDP values. The method was validated by comparing results from individual and mixed sample sets. The tannin composition of different sainfoin accessions covered a range of mDP values from 16 to 83, procyanidin/prodelphinidin (PC/PD) ratios from 19.2/80.8 to 45.6/54.4, and cis/trans ratios from 74.1/25.9 to 88.0/12.0. This is the first high-throughput screening method that is suitable for analyzing condensed tannin contents and structural composition directly in green plant tissue.

Thiolytic screening method for exploring condensed tannin variation in a unique sainfoin germplasm bank

This EU funded 'HealthyHay'project stablished a sainfoin (Onobrychis vicifolia) germplasm bank at NIAB, Cambridge, with 306 accessions from around the world. A screening method was developed to characterise tannins by thiolytic degradation [1] directly in green plants for the first time. the method was validated by separate analysis of unextractable, extractable and purified tannins using thiolysis, HPLC-GPC and MALDI-TOF MS. Most tannins (58 to 73% of the total) could be recovered after Toyopearl HW50 fractionation with water, aqueous methanol and acetone. the greatest losses during purification occurred amongst larger molecular weight tannins with mean degree of polymerisation (mDP) > 18. The composition of water-,aqueous methanol- and acetone-soluble tannins differed considerably in their mDP and trans/cis ratios, but not in their prodelphinidin/orocyanidin (PD/PC) ratios.

Phylogenetic systematics of Sternbergia (Amaryllidaceae) based on plastid and ITS sequence data

The phylogenetics of Sternbergia (Amaryllidaceae) were studied using DNA sequences of the plastid ndhF and matK genes and nuclear internal transcribed spacer (ITS) ribosomal region for 38, 37 and 32 ingroup and outgroup accessions, respectively. All members of Sternbergia were represented by at least one accession, except S. minoica and S. schubertii, with additional taxa from Narcissus and Pancratium serving as principal outgroups. Sternbergia was resolved and supported as sister to Narcissus and composed of two primary subclades: S. colchiciflora sister to S. vernalis, S. candida and S. clusiana, with this clade in turn sister to S. lutea and its allies in both Bayesian and bootstrap analyses. A clear relationship between the two vernal flowering members of the genus was recovered, supporting the hypothesis of a single origin of vernal flowering in Sternbergia. However, in the S. lutea complex, the DNA markers examined did not offer sufficient resolving power to separate taxa, providing some support for the idea that S. sicula and S. greuteriana are conspecific with S. lutea

Genetic diversity of red-fleshed apples (Malus)

Anthocyanins are flavonoid pigments imparting red, blue, or purple pigmentation to fruits, flowers and foliage. These compounds are powerful antioxidants in vitro, and are widely believed to contribute to human health. The fruit of the domestic apple (Malus x domestica) is a popular and important source of nutrients, and is considered one of the top ‘functional foods’—those foods that have inherent health-promoting benefits beyond basic nutritional value. The pigmentation of typical red apple fruits results from accumulation of anthocyanin in the skin. However, numerous genotypes of Malus are known that synthesize anthocyanin in additional fruit tissues including the core and cortex (flesh). Red-fleshed apple genotypes are an attractive starting point for development of novel varieties for consumption and nutraceutical use through traditional breeding and biotechnology. However, cultivar development is limited by lack of characterization of the diversity of genetic backgrounds showing this trait. We identified and cataloged red-fleshed apple genotypes from four Malus diversity collections representing over 3,000 accessions including domestic cultivars, wild species, and named hybrids. We found a striking range of flesh color intensity and pattern among accessions, including those carrying the MYB10 R 6 allele conferring ectopic expression of a key transcriptional regulator of anthocyanin biosynthesis. Although MYB10 R 6 was strongly associated with red-fleshed fruit among genotypes, this allele was neither sufficient nor required for this trait in all genotypes. Nearly all red-fleshed accessions tested could be traced back to ‘Niedzwetzkyana’, a presumed natural form of M. sieversii native to central Asia.

Proanthocyanidin diversity in the EU 'HealthyHay' sainfoin (onobrychis viciifolia) germplasm collection

This study investigated 37 diverse sainfoin (Onobrychis viciifolia Scop.) accessions from the EU ‘HealthyHay’ germplasm collection for proanthocyanidin (PA) content and composition. Accessions displayed a wide range of differences: PA contents varied from 0.57 to 2.80 g/100 g sainfoin; the mean degree of polymerisation from 12 to 84; the proportion of prodelphinidin tannins from 53% to 95%, and the proportion of trans-flavanol units from 12% to 34%. A positive correlation was found between PA contents (thiolytic versus acid–butanol degradation; P < 0.001; R2 = 0.49). A negative correlation existed between PA content (thiolysis) and mDP (P < 0.05; R2 = −0.30), which suggested that accessions with high PA contents had smaller PA polymers. Cluster analysis revealed that European accessions clustered into two main groups: Western Europe and Eastern Europe/Asia. In addition, accessions from USA, Canada and Armenia tended to cluster together. Overall, there was broad agreement between tannin clusters and clusters that were based on morphological and agronomic characteristics.

Mutation in TERMINAL FLOWER1 reverses the photoperiodic requirement for flowering in the wild strawberry, Fragaria vesca

Photoperiodic flowering has been extensively studied in the annual short-day and long-day plants rice and Arabidopsis while less is known about the control of flowering in perennials. In the perennial wild strawberry, Fragaria vesca L. (Rosaceae), short-day and perpetual flowering long-day accessions occur. Genetic analyses showed that differences in their flowering responses are caused by a single gene, the SEASONAL FLOWERING LOCUS which may encode the F. vesca homolog of TERMINAL FLOWER1 (FvTFL1). We show through high-resolution mapping and transgenic approaches that FvTFL1 is the basis of this change in flowering behavior and demonstrate that FvTFL1 acts as a photoperiodically regulated repressor. In short-day F. vesca, long photoperiods activate FvTFL1 mRNA expression and short days suppress it, promoting flower induction. These seasonal cycles in FvTFL1 mRNA level confer seasonal cycling of vegetative and reproductive development. Mutations in FvTFL1 prevent LD suppression of flowering, and the early flowering that then occurs under LD is dependent on the F. vesca homolog of FLOWERING LOCUS T. This photoperiodic response mechanism differs from those described in model annual plants. We suggest that this mechanism controls flowering within the perennial growth cycle in F. vesca, and demonstrate that a change in a single gene reverses the photoperiodic requirements for flowering.

The effectiveness of botanic garden collections in supporting plant conservation: a European case study

Europe has the greatest concentration of botanic gardens in the world, they cultivate extensive collections of plants that include samples of European threatened plant species. This study looks at the effectiveness of these collections in supporting species conservation. A three part study is presented: (1) the results of a survey and assessment of threatened plants in botanic gardens, as defined by the Bern Convention; (2) case studies illustrating current issues in the ex situ management of European threatened plant species; and (3) presentation of policy recommendations on further improving botanic garden contributions to European plant conservation. The survey indicated that of 119 European botanic gardens in 29 European countries, 105 are cultivating 308 of the 573 threatened plant species listed by the Bern Convention. The survey identified 25 botanic gardens in 14 countries undertaking 51 conservation projects focused on 27 Bern listed species. In particular this survey has established that the majority of taxa are held in a small number of collections, dominated by non-wild origin accessions, and are not adequately documented. The majority of specimens in botanic gardens are cultivated out of the range country and not contributing to a specific conservation project. We review the genetic representation and documentation of origin in collections. Existing plant collections contain representatives of populations, now lost in the wild and maintain samples of at least nine European plant taxa identified as 'Extinct in the Wild'. However, inadequate standards of record keeping has compromised the conservation value of many collections. We highlight the dangers of hybridisation and disease in ex situ collections. The results suggest that botanic garden collections are skewed towards horticulturally robust and ornamental species and do not fully reflect priorities as defined by the Bern Convention. Recognising the limitations of traditional botanic garden collections we propose that botanic gardens more effectively utilise their two core competencies, namely scientific horticulture and public display and interpretation. The unique horticultural skills resident in European botanic gardens could be more effectively utilised through the application of horticulture to the management of wild populations.

Genetic variation at flowering time loci in wild and cultivated barley

The worldwide spread of barley cultivation required adaptation to agricultural environments far distant from those found in its centre of domestication. An important component of this adaptation is the timing of flowering, achieved predominantly in response to day length and temperature. Here, we use a collection of cultivars, landraces and wild barley accessions to investigate the origins and distribution of allelic diversity at four major flowering time loci, mutations at which have been under selection during the spread of barley cultivation into Europe. Our findings suggest that while mutant alleles at the PPD-H1 and PPD-H2 photoperiod loci occurred pre-domestication, the mutant vernalization non-responsive alleles utilized in landraces and cultivars at the VRN-H1 and VRN-H2 loci occurred post-domestication. The transition from wild to cultivated barley is associated with a doubling in the number of observed multi-locus flowering-time haplotypes, suggesting that the resulting phenotypic variation has aided adaptation to cultivation in the diverse ecogeographic locations encountered. Despite the importance of early-flowering alleles during the domestication of barley in Europe, we show that novel VRN alleles associated with early flowering in wild barley have been lost in domesticates, highlighting the potential of wild germplasm as a source of novel allelic variation for agronomic traits.

Exploring variation in proanthocyanidin composition and content of sainfoin (Onobrychis viciifolia)

To maximise the potential benefits to ruminants from sainfoin, plant breeding should focus on developing varieties with predictable condensed tannin (CT) profiles. Little is known about whether and to what extent accession and environment influence sainfoin CT structures. We sought to investigate the likely extent of accession and environment effects on CT characteristics of sainfoin. Four single-flowering (Communis) accessions and two multiple-flowering (Bifera) accessions, grown at three sites and collected at two harvest times were used. Sainfoin CTs were characterised by thiolytic degradation and by high-performance liquid chromatography-gel permeation chromatography (HPLC-GPC). Also, CT concentration measured earlier by the HCl-butanol method was compared with that from thiolysis

In vitro effects of extracts and purified tannins of sainfoin (Onobrychis viciifolia) against two cattle nematodes

Sainfoin (Onobrychis viciifolia) is a condensed tannin (CT)-containing legume and has anthelmintic potential against gastrointestinal nematodes of ruminants. This study investigated in vitro effects of acetone/water extracts and derived CT fractions from different types of sainfoin (i.e. accessions) against larvae of Cooperia oncophora and Ostertagia ostertagi by applying the larval feeding inhibition assay (LFIA). Seven sainfoin accessions were extracted and tested with L1 larvae at 10 and 40 μg extract/ml. In addition, CT in extracts from 4 accessions were fractionated according to polymer size and tested by LFIA at two concentrations (2 and 10 μg CT fraction/ml). All sainfoin extracts caused significant inhibition of L1-feeding of both C. oncophora and O. ostertagi with varying intensity compared to the control (phosphate buffered saline). For both nematode species the in vitro effect was positively correlated with CT content in the extracts, but not with any of the structural CT parameters. In contrast, the 16 CT fractions revealed significant correlations between in vitro effect and CT content, polymer size (mean degree of polymerisation, mDP) and monomeric composition (prodelphinidin percentage, % PD). These differences between crude extracts and purified fractions may stem from the fact that extracts contain complex CT mixtures, which may mask and thus suppress CT structural effects. This study provides the first indication that, apart from CT and % PD content, polymer size also contributes to anthelmintic activity of CTs. The results, therefore, suggest that the inter-accession variability in CT content and composition needs to be taken into account in future plant breeding programmes which seek to enhance the anthelmintic properties of sainfoin

Shoot calcium and magnesium concentrations differ between subtaxa, are highly heritable, and associate with potentially pleiotropic loci in Brassica oleracea

Calcium (Ca) and magnesium (Mg) are the most abundant group II elements in both plants and animals. Genetic variation in shoot Ca and shoot Mg concentration (shoot Ca and Mg) in plants can be exploited to biofortify food crops and thereby increase dietary Ca and Mg intake for humans and livestock. We present a comprehensive analysis of within-species genetic variation for shoot Ca and Mg, demonstrating that shoot mineral concentration differs significantly between subtaxa (varietas). We established a structured diversity foundation set of 376 accessions to capture a high proportion of species-wide allelic diversity within domesticated Brassica oleracea, including representation of wild relatives (C genome, 1n = 9) from natural populations. These accessions and 74 modern F-1 hybrid cultivars were grown in glasshouse and field environments. Shoot Ca and Mg varied 2- and 2.3-fold, respectively, and was typically not inversely correlated with shoot biomass, within most subtaxa. The closely related capitata (cabbage) and sabauda (Savoy cabbage) subtaxa consistently had the highest mean shoot Ca and Mg. Shoot Ca and Mg in glasshouse-grown plants was highly correlated with data from the field. To understand and dissect the genetic basis of variation in shoot Ca and Mg, we studied homozygous lines from a segregating B. oleracea mapping population. Shoot Ca and Mg was highly heritable (up to 40). Quantitative trait loci (QTL) for shoot Ca and Mg were detected on chromosomes C2, C6, C7, C8, and, in particular, C9, where QTL accounted for 14 to 55 of the total genetic variance. The presence of QTL on C9 was substantiated by scoring recurrent backcross substitution lines, derived from the same parents. This also greatly increased the map resolution, with strong evidence that a 4-cM region on C9 influences shoot Ca. This region corresponds to a 0.41-Mb region on Arabidopsis (Arabidopsis thaliana) chromosome 5 that includes 106 genes. There is also evidence that pleiotropic loci on C8 and C9 affect shoot Ca and Mg. Map-based cloning of these loci will reveal how shoot-level phenotypes relate to Ca 21 and Mg 21 uptake and homeostasis at the molecular level.

Diversity in expression of phosphorus (P) responsive genes in Cucumis melo L.

Background: Phosphorus (P) is a major limiting nutrient for plant growth in many soils. Studies in model species have identified genes involved in plant adaptations to low soil P availability. However, little information is available on the genetic bases of these adaptations in vegetable crops. In this respect, sequence data for melon now makes it possible to identify melon orthologues of candidate P responsive genes, and the expression of these genes can be used to explain the diversity in the root system adaptation to low P availability, recently observed in this species. Methodology and Findings: Transcriptional responses to P starvation were studied in nine diverse melon accessions by comparing the expression of eight candidate genes (Cm-PAP10.1, Cm-PAP10.2, Cm-RNS1, Cm-PPCK1, Cm-transferase, Cm-SQD1, Cm-DGD1 and Cm-SPX2) under P replete and P starved conditions. Differences among melon accessions were observed in response to P starvation, including differences in plant morphology, P uptake, P use efficiency (PUE) and gene expression. All studied genes were up regulated under P starvation conditions. Differences in the expression of genes involved in P mobilization and remobilization (Cm-PAP10.1, Cm-PAP10.2 and Cm-RNS1) under P starvation conditions explained part of the differences in P uptake and PUE among melon accessions. The levels of expression of the other studied genes were diverse among melon accessions, but contributed less to the phenotypical response of the accessions. Conclusions: This is the first time that these genes have been described in the context of P starvation responses in melon. There exists significant diversity in gene expression levels and P use efficiency among melon accessions as well as significant correlations between gene expression levels and phenotypical measurements.

Shoot yield drives phosphorus use efficiency in Brassica oleracea and correlates with root architecture traits

The environmental and financial costs of using inorganic phosphate fertilizers to maintain crop yield and quality are high. Breeding crops that acquire and use phosphorus (P) more efficiently could reduce these costs. The variation in shoot P concentration (shoot-P) and various measures of P use efficiency (PUE) were quantified among 355 Brassica oleracea L. accessions, 74 current commercial cultivars, and 90 doubled haploid (DH) mapping lines from a reference genetic mapping population. Accessions were grown at two or more external P concentrations in glasshouse experiments; commercial and DH accessions were also grown in replicated field experiments. Within the substantial species-wide diversity observed for shoot-P and various measures of PUE in B. oleracea, current commercial cultivars have greater PUE than would be expected by chance. This may be a consequence of breeding for increased yield, which is a significant component of most measures of PUE, or early establishment. Root development and architecture correlate with PUE; in particular, lateral root number, length, and growth rate. Significant quantitative trait loci associated with shoot-P and PUE occur on chromosomes C3 and C7. These data provide information to initiate breeding programmes to improve PUE in B. oleracea.