Collecting wild Miscanthus germplasm in Asia for crop improvement and conservation in Europe whilst adhering to the guidelines of the United Nations' Convention on Biological Diversity.

BACKGROUND AND AIMS: Germplasm with diverse, agronomically relevant traits forms the foundation of a successful plant breeding programme. Since 1993, the United Nations has been advocating the implementation of the Convention on Biological Diversity (CBD) and the subsequent 2002 Bonn Guidelines as international best practice on germplasm collection and use. In 2006, a European team made an expedition to Asia to collect wild germplasm of Miscanthus, a C4 perennial rhizomatous grass, for breeding an environmentally adaptable, resilient and high-yielding bioenergy crop. We outline general aspects of germplasm collection, conservation, breeding and biomass production evaluation while following the CBD's guidelines, respecting biodiversity and conservation needs, and the ethical use of genetic resources. METHODS: Effective protocols, quarantine, methods for collecting seed and rhizomes, and a genebank for conservation were established. Versatile informatics and database architecture were used to assist in selection, flowering synchronization, crossing, evaluation, phenotyping and data integration. Approaches were formulated to comply with the CBD guidelines. KEY RESULTS: A total of 303 accessions of M. sinensis, M. sacchariflorus and M. floridulus were collected from 158 geographically and environmentally diverse locations. These species were shown to accumulate different amounts of aerial biomass due to combinations of stem count, height and thickness. Progeny from one interspecies cross accumulated more biomass in early trials and has shown double the yield performance in years 3-4 compared with the existing commercial cultivar M. × giganteus. An example of an F1 hybrid has already demonstrated the long-term potential of exploiting this collection for a breeding programme. CONCLUSIONS: By conforming to the CBD principles, the authors' international collaboration provides a practical example of implementing the CBD. The collection widened the genetic diversity of Miscanthus available to allow for breeding of novel hybrids that exhibit more diverse traits to increase yield and resilience for growth on marginal land and in climate-challenged environments.

Inside the 8p23.1 duplication syndrome; eight microduplications of likely or uncertain clinical significance.

The 8p23.1 duplication syndrome (8p23.1 DS) is a recurrent genomic condition with an estimated prevalence of 1 in 58,000. The core 3.68 Mb duplication contains 32 genes of which five are currently candidates for the phenotypic features. Here we describe four patients and five families with eight microduplications of 8p23.1 ranging from 187 to 1082 kb in size and one atypical duplication of 4 Mb. These indicate that a minimal region of overlap (MRO) in medial 8p23.1 can give rise to features of 8p23.1 DS including developmental delay, dysmorphism, macrocephaly and otitis media, but not congenital heart disease (CHD). This MRO spans 776 kb (chr8:10,167,881-10,943,836 hg19) and contains SOX7 and seven of the other 32 core 8p23.1 DS genes. In centromeric 8p23.1, microduplications including GATA4 can give rise to non-syndromic CHD but the clinical significance of two smaller centromeric microduplications without GATA4 was uncertain due to severe neurological profiles not usually found in 8p23.1 DS. The clinical significance of three further 8p23.1 microduplications was uncertain due to additional genetic factors without which the probands might not have come to medical attention. Variable expressivity was indicated by the almost entirely unaffected parents in all five families and the mildly affected sibling in one. Intronic interruptions of six genes by microduplication breakpoint intervals had no apparent additional clinical consequences. Our results suggest that 8p23.1 DS is an oligogenetic condition largely caused by the duplication and interactions of the SOX7 and GATA4 transcription factors.

Unbalanced jumping translocation involving 3q in myeloproliferative disease.

An 87-year-old woman was diagnosed with unclassified myeloproliferative disease having an acquired jumping translocation with the long arm of chromosome 3 translocating to the short arm telomeric region of chromosome 8 (major clone) and the long arm telomeric region of chromosome 10 (minor clone). Each abnormal clone was also associated with an extra copy of chromosome 8. Although there was no evidence of transformation to an acute leukemia, the patient deteriorated until her demise 7 months after disease presentation. There have been fewer than 70 cases of acquired jumping translocations reported in the literature. To our knowledge, this is the first acquired jumping translocation case to be reported in a patient with myeloproliferative disease.

Association of candidate genes with flowering time and water-soluble carbohydrate content in Lolium perenne (L.).

We describe a candidate gene approach for associating SNPs with variation in flowering time and water-soluble carbohydrate (WSC) content and other quality traits in the temperate forage grass species Lolium perenne. Three analysis methods were used, which took the significant population structure into account. First, a linear mixed model was used enabling a structured association analysis to be incorporated with the nine populations identified in the structure analysis as random variables. Second, a within-population analysis of variance was performed. Third, a tree-scanning method was used, in which haplotype trees were associated with phenotypes on the basis of inferred haplotypes. Analysis of variance within populations identified several associations between WSC, nitrogen (N), and dry matter digestibility with allelic variants within an alkaline invertase candidate gene LpcAI. These associations were only detected in material harvested in one of the two years. By contrast, consistent associations between the L. perenne homolog (LpHD1) of the rice photoperiod control gene HD1 and flowering time were identified. One SNP, in the immediate upstream region of the LpHD1 coding sequence (C-4443-A), was significant in the linear mixed model. Within-population analysis of variance and tree-scanning analysis confirmed and extended this result to the 2118 polymorphisms in some of the populations. The merits of the tree-scanning method are compared to the single SNP analysis. The potential usefulness of the 4443 SNP in marker-assisted selection is currently being evaluated in test crosses of genotypes from this work with turf-grass varieties.

Coordinated expression of functionally diverse fructosyltransferase genes is associated with fructan accumulation in response to low temperature in perennial ryegrass.

* Fructan is the major nonstructural carbohydrate reserve in temperate grasses. To understand regulatory mechanisms in fructan synthesis and adaptation to cold environments, the isolation, functional characterization and genetic mapping of fructosyltransferase (FT) genes in perennial ryegrass (Lolium perenne) are described. * Six cDNAs (prft1-prft6) encoding FTs were isolated from cold-treated ryegrass plants, and three were positioned on a perennial ryegrass linkage map. Recombinant proteins were produced in Pichia pastoris and enzymatic activity was characterized. Changes in carbohydrate levels and mRNA levels of FT genes during cold treatment were also analysed. * One gene encodes sucrose-sucrose 1-fructosyltransferase (1-SST), and two gene encode fructan-fructan 6G-fructosyltransferase (6G-FFT). Protein sequences for the other genes (prfts 1, 2 and 6) were similar to sucrose-fructan 6-fructosyltransferase (6-SFT). The 1-SST and prft1 genes were colocalized with an invertase gene on the ryegrass linkage map. The mRNA levels of prft1 and prft2 increased gradually during cold treatment, while those of the 1-SST and 6G-FFT genes first increased, but then decreased before increasing again during a longer period of cold treatment. * Thus at least two different patterns of gene expression have developed during the evolution of functionally diverse FT genes, which are associated in a coordinated way with fructan synthesis in a cold environment.

Transformation of cytogenetically normal chronic myelomonocytic leukaemia to an acute myeloid leukaemia and the emergence of a novel +13, +15 double trisomy resulting in an adverse outcome.

A 58-year-old man was admitted with symptoms of lethargy and easy bruising for four months duration. Peripheral blood (PB) analysis revealed a white blood cell count (WBC) of 15.9 x 10(9)/l with monocytes 5.4 x 10(9)/l. Bone marrow (BM) was hypercellular with 15% blasts, monocytosis and trilineage dysplasia. Conventional cytogenetic analysis (G-banding) detected an apparently normal male karyotype (46,XY). A diagnosis of chronic myelomonocytic leukaemia (CMML) was made. After 3 years, PB analysis revealed a WBC count of 22 x 10(9)/l and a predominance of blasts. BM aspirate analysis also revealed 89% myeloid blasts and G-banding detected the emergence of an abnormal clone harbouring an extra copy of chromosomes 13 and 15. A diagnosis of disease transformation to acute myeloid leukaemia (AML) was made. Post chemotherapy BM aspirate was very hypocellular and the abnormal +13, +15 clone was still present suggesting primary refractory disease. A second course of chemotherapy was only administered for 24 hours due to complications. The abnormal +13, +15 clone was still present and it was decided that no further treatment apart from palliative care could be offered. The patient died 11 weeks later, five months after AML transformation. This is the first description of a cytogenetically normal CMML patient transforming to AML with the emergence of a unique +13, +15 double trisomy resulting in an adverse outcome.

RT-PCR analysis and stress response capacity of transgenic gshI-poplar clones (Populus x canescens) in response to paraquat exposure.

Stress response capacity (Fv/Fm at 690 nm and F690/F735 at Fmax) of untransformed hybrid poplar, Populus x canescens (P tremula x P alba), and two transgenic lines overexpressing gamma-ECS (gamma-glutamylcysteine synthetase) either in the cytosol (cyt-ECS) or in the chloroplast (chl-ECS) was studied in response to the herbicide paraquat (4.0 x 10(-9) to 4.0 x 10(-6) M) for 21 days. Significant differences at sublethal (4.0 x 10(-7) M) and bleaching (4.0 x 10(-6) M) concentrations of paraquat were observed with about a two-fold and eight-fold decrease in the photosynthetic activity (Fv/Fm at 690 nm and F690/F735 at Fmax), respectively. None of the gshI transgenic lines (cyt-ECS, chl-ECS) with elevated GSH content exhibited significant tolerance to paraquat. Semiquantitative RT-PCR of the cyt-ECS clone was used for gene expression analysis of the nuclear encoded rbcS gene and the stress responsive gst gene. Expression of the constitutively expressed 26SrRNA ribosomal gene was probed as a control for all RT-PCR reactions. The relative intensities of gene expressions normalized to the level of 26SrRNA intensity showed a 50% decrease in the nuclear encoded rbcS expression and a 120% increase in the stress responsive gst gene expression of the paraquat treated (4.0 x 10(-7) M) samples of the transgenic poplar line (cyt-ECS).

3'CBFbeta deletion associated with inv(16) in acute myeloid leukemia.

Recent reports have shown that concomitant submicroscopic deletions can occur in association with chromosomal translocations/inversions in several leukemia subtypes. Detectable by fluorescence in situ hybridization (FISH), these losses of sequence include deletion of the 5' region of the ABL gene and the 3' region of BCR in chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL), as well as the 5' region of ETO in acute myeloid leukemia (AML) French-American-British type M2 associated with t(8;21), 3'MLL in AML and ALL, and 3' core-binding factor beta (CBFbeta) in AML associated with inv(16). While it has been widely reported that submicroscopic deletions of the derivative 9 in CML have an adverse prognostic impact, the clinical significance, if any, of deletions associated with t(8;21), inv(16)/t(16;16), or MLL rearrangement is yet to be determined. We analyzed a series of 39 patients diagnosed with AML who had cytogenetically detectable inv(16)/t(16;16) by using a FISH probe for the CBFbeta region to determine the incidence of the 3'CBFbeta deletion. Deletions were detected in three patients (8%), all associated with inv(16), bringing the number of cases reported so far to seven. The prognostic significance of this finding remains unclear.

AFLP analysis and improved phytoextraction capacity of transgenic gshI-poplar clones (Populus x canescens L.) for copper in vitro.

Clone stability and in vitro phytoextraction capacity of vegetative clones of P. x canescens (2n = 4x = 38) including two transgenic clones (ggs11 and lgl6) were studied as in vitro leaf disc cultures. Presence of the gshI-transgene in the transformed clones was detected in PCR reactions using gshI-specific primers. Clone stability was determined by fAFLP (fluorescent amplified DNA fragment length polymorphism) analysis. In total, 682 AFLP fragments were identified generated by twelve selective primer pairs after EcoRI-MseI digestion. Four fragments generated by EcoAGT-MseCCC were different (99.4% genetic similarity) which proves an unexpectedly low bud mutation frequency in P. x canescens. For the study of phytoextraction capacity leaf discs (8 mm) were exposed to a concentration series of ZnSO4 (10(-1) to 10(-5) M) incubated for 21 days on aseptic tissue culture media WPM containing 1 microM Cu. Zn2+ caused phytotoxicity only at high concentrations (10(-1) to 10(-2) M). The transgenic poplar cyt-ECS (ggs11) clone, as stimulated by the presence of Zn, showed elevated heavy metal (Cu) uptake as compared to the non-transformed clone. These results suggest that gshI-transgenic poplars may be suitable for phytoremediation of soils contaminated with zinc and copper.

A stay-green mutation of Lolium perenne affects NO3 - uptake and translocation of N during prolonged N starvation.

Apparent Km and Vmax for net NOS″ uptake and short-term translocation patterns of recently absorbed N were compared in a stay-green mutant and wild-type selection line of Lolium perenne L. by means of a series of depletion studies using 18 NO3 , performed over 12 d under conditions of progressively increasing N deprivation. In view of the greater retention of N in senescent leaves of the stay-green phenotype, it was predicted that NOS″ uptake would be up-regulated relative to the normal line, and that a proportionally higher fraction of recently absorbed N would be allocated to young leaves. It was shown that the stay-green trait had significant phenotypic consequences for plant N relations, with higher 'sink strength' of shoots for recently absorbed N, and higher Vmax for NO3 uptake compared with those of normal plants. The stay-green mutation had no effect on the Km of the nitrate uptake system. Although the N-use efficiency might he expected to be lower in stay-green than in normal plants, there were no differences in rates of dry matter production.

Tetrasomy 13 as the sole cytogenetic abnormality in acute myeloid leukemia M1 without maturation.

We report a case of acute myeloid leukemia (AML) M1 showing a 48,XY,+13,+13 karyotype. Treatment was according to the Medical Research Council AML14 trial protocol with two courses of DAT chemotherapy. Postchemotherapy bone marrow examination failed to show complete remission or cytogenetic normalization. Despite having resistant disease, the patient initially remained clinically well although requiring regular blood transfusions for anemia. However his leukocyte count gradually increased and he became symptomatic. He was treated subsequently with FLAG but died approximately 2 weeks later, 6 months after first presenting. Tetrasomy 13 as the sole cytogenetic abnormality has not been reported previously in M1 AML and has only been reported in three other AML cases, all with an immature phenotype and poor outcome.

Differences in growth and nitrogen productivity between a stay-green genotype and a wild-type of Lolium perenne under limiting relative addition rates of nitrate supply.

The stay-green mutation of the nuclear gene sid inhibits chlorophyll degradation during leaf senescence in grasses. Decreased productivity is expected under conditions of limited external N availability, due to the higher retention of N in senescent tissues. However, this has not been reported when plants are grown at limiting low external concentrations of N. In the present study a different approach was taken, based on the relative addition rate method for defining limiting N supply. Wild-type and stay-green genotypes of Lolium perenne L. were grown for 30 days in flowing solution culture and then supplied with NO3- on an hourly basis over 35 days at relative addition rates (RARs) of 0.03, 0.06, 0.09 and 0.12 day-1, ranging from severe N limitation to optimal supply. Plants were steady-state labelled with 15NO3- prior to RAR treatments, then switched to 14NO3- to allow measurement of the re-distribution of N absorbed prior to RAR control. Following acclimation, relative growth rates (RGRs) approached the corresponding RAR, but were significantly lower for stay-green than wild-type at RARs of 0.03 and 0.06 day-1. Tiller numbers were lower in stay-green plants after 35 days at all RARs except 0.12 day-1. Concentrations of total N in senescent laminae of stay-green plants exceeded those in wild-type plants by a similar margin (4.8-6.8 mg g-1 DW) irrespective of RAR. Maximum nitrogen productivity (Pn) was 3.9 g DW g-1 N day-1 (Nmin = 7.1 mg g-1 DW) in wild-type plants, and 5.1 g DW g-1 N day-1 (Nmin = 10.7 mg g-1 DW) in stay-green plants. The higher N productivity of stay-green plants indicated these plants used a smaller pool of metabolically available N more efficiently in biosynthesis compared with wild-type plants. The retention of N, absorbed prior to RAR treatments, in senescent laminae was significantly higher in stay-green plants at RAR of 0.03 day-1 after day 21 (i.e. 20% versus 15% of the total N recovered). However, in terms of the whole N economy of the plant the margin represented only 1.7% of the total N content on day 35.

Breeding for Bio-ethanol Production in Lolium perenne L.: Association of Allelic Variation with High Water-Soluble Carbohydrate Content.

Increasing the extractable sugar yield from perennial crops is one strategy to generate renewable fuels such as bio-ethanol. Lolium perenne L. (perennial ryegrass) can contain significant (>30% dry matter) water-soluble sugars in the form of polymeric fructan which is readily extracted, broken down and fermented to bio-ethanol. A population of L. perenne generated from four parents which differed in water-soluble carbohydrate (WSC) content was subjected to multiple rounds of selection and recombination on the basis of early spring WSC content to produce a high WSC, and a low WSC population. A control population was generated by selecting the same number of plants at random. The alleles present at six candidate gene loci were analysed before and after selection and correlated to WSC content. Significant differences in the allele frequency of L. perenne soluble-acid invertase1:4 were observed between the three populations with one haplotype significantly associated with the high WSC C2S+ population (after three rounds of selection and two rounds of recombination). Moreover, WSC content was also associated with biomass accumulation. Thus, in addition to a 2.84-fold increase in WSC yield, the C2S+ population also had 1.48-fold more biomass per plant, resulting in 3.9-fold higher WSC yield per plant than the control population.

Jumping translocation in acute monocytic leukemia (M5b) with alternative breakpoint sites in the long arm of donor chromosome 3.

An 86-year-old man presented with acute hepatic failure, worsening thrombocytopenia, and anemia having been diagnosed and managed expectantly with cytogenetically normal RAEB-1. After 20 months a diagnosis of disease transformation to acute monocytic leukemia (M5b) was made. Conventional G-banded analysis of unstimulated bone marrow cultures demonstrated a jumping translocation (JT) involving proximal and distal breakpoints on donor chromosome 3 at bands 3q1?2 and 3q21, respectively. Recipient chromosomes included the long-arm telomeric regions of chromosomes 5, 10, 14, 16, and 19. A low-level trisomy 8 clone was also found in association with both proximal and distal JT clones. Conventional G-banded analysis of unstimulated peripheral blood cultures detected the proximal 3q1?2 JT clone involving recipient chromosome 10 several weeks after transformation to acute monocytic leukemia. Interestingly, JTs involving recipient chromosomes 5, 14, 16, and 19 were not detected in this peripheral blood sample. Palliative care was administered until his demise 2.2 months after disease transformation. There have been fewer than 70 cases of acquired JTs reported in the literature, including one myeloproliferative neoplasm and five acute myeloid leukemias involving a single breakpoint site on donor chromosome 3. Our case is unique as it is the first acquired case to demonstrate a JT involving alternative pericentromeric breakpoint sites on a single donor chromosome consisting of a proximal breakpoint at 3q1?2 and a more distal breakpoint at 3q21.

Outgrowth endothelial cells: characterization and their potential for reversing ischemic retinopathy.

PURPOSE: Endothelial progenitor cells (EPCs) have potential for promoting vascular repair and revascularization of ischemic retina. However, the highly heterogeneous nature of these cells causes confusion when assessing their biological functions. The purpose of this study was to provide a comprehensive comparison between the two main EPC subtypes, early EPCs (eEPCs) and outgrowth endothelial cells (OECs), and to establish the potential of OECs as a novel cell therapy for ischemic retinopathy. METHODS: Two types of human blood-derived EPCs were isolated and compared using immunophenotyping and multiple in vitro functional assays to assess interaction with retinal capillary endothelial cells and angiogenic activity. OECs were delivered intravitreally in a mouse model of ischemic retinopathy, and flat mounted retinas were examined using confocal microscopy. RESULTS: These data indicate that eEPCs are hematopoietic cells with minimal proliferative capacity that lack tube-forming capacity. By contrast, OECs are committed to an endothelial lineage and have significant proliferative and de novo tubulogenic potential. Furthermore, only OECs are able to closely interact with endothelial cells through adherens and tight junctions and to integrate into retinal vascular networks in vitro. The authors subsequently chose OECs to test a novel cell therapy approach for ischemic retinopathy. Using a murine model of retinal ischemia, they demonstrated that OECs directly incorporate into the resident vasculature, significantly decreasing avascular areas, concomitantly increasing normovascular areas, and preventing pathologic preretinal neovascularization. CONCLUSIONS: As a distinct EPC population, OECs have potential as therapeutic cells to vascularize the ischemic retina.

Impact of defoliation frequency on regrowth and carbohydrate metabolism in contrasting varieties of Lolium perenne.

The aims of the study were to gain a better understanding of fructan metabolism regulation during regrowth of Lolium perenne, and to evaluate the role of fructans of remaining tissues as well as carbon assimilation of new leaf tissues in refoliation. Two varieties that contrast for carbohydrate metabolism, Aurora and Perma, were subject to severe and frequent or infrequent defoliations before regrowth. Aurora, which had a greater content of fructans in leaf sheaths than Perma before defoliation, produced more leaf biomass within the 4 days following the first cut. At the end of the regrowth period, Aurora produced more leaf biomass than Perma. Photosynthetic parameters, which were barely affected by defoliation frequency, could not explain these differences. Fructan synthesising activities [sucrose:sucrose 1-fructosyltransferase (1-SST) and fructan:fructan 6G-fructosyltransferase (6G-FFT)], declined after defoliation. In elongating leaf bases, corresponding transcript levels did not decline concomitantly, suggesting a post-transcriptional regulation of expression, while in leaf sheaths the gene expression pattern mostly followed the time-course of the enzyme activities. Regulation of Lp1-SST and Lp6G-FFT gene expression depends, therefore, on the sink-source status of the tissue after defoliation. During the phase of reserve accumulation, fructosyltransferase activities together with corresponding transcripts increased more in frequently defoliated plants than in infrequently defoliated plants.

An assessment of the ability of the stay-green phenotype in lolium species to provide an improved protein supply for ruminants.

The stay-green phenotype results from a naturally occurring mutation in which senescent leaves retain their chlorophyll and the associated apoprotein, LHCPII. Protection of this protein pool could deliver grass with enhanced protein content and could decrease the extent of protein degradation by plant proteases in the rumen. This would enhance the efficiency of protein utilization in livestock to the benefit of the environment. Field plots of stay-green and wild-type Lolium perenne were defoliated at intervals to simulate grazing. There were variations in foliar protein content and proteolysis throughout the year, but no significant differences between genotypes when material was analysed fresh or after it was cut and dried to simulate hay-making, which possibly induced senescence. In a subsequent experiment with stay-green and wild-type L temulentum, increased protein retention and decreased protein degradability were observed in stay-green leaves that were allowed to senescence naturally and extensively on the plant. That there is no difference between the two L. perenne genotypes suggests that as a field crop in grazed pastures the stay-green genotype would not confer a nutritional advantage in terms of protein degradability. It is possible that grazing promotes a high proportion of non-senescent to senescent leaf material within the sward and thus any advantage conferred by the stay-green phenotype would be effectively masked by an abundance of mature foliage. It is suggested that the stay-green trait would be of benefit in areas where agricultural practice permits extensive natural senescence to occur.

An enhanced molecular marker based genetic map of perennial ryegrass (Lolium perenne) reveals comparative relationships with other Poaceae genomes.

A molecular-marker linkage map has been constructed for perennial ryegrass (Lolium perenne L.) using a one-way pseudo-testcross population based on the mating of a multiple heterozygous individual with a doubled haploid genotype. RFLP, AFLP, isoenzyme, and EST data from four collaborating laboratories within the International Lolium Genome Initiative were combined to produce an integrated genetic map containing 240 loci covering 811 cM on seven linkage groups. The map contained 124 codominant markers, of which 109 were heterologous anchor RFLP probes from wheat, barley, oat, and rice, allowing comparative relationships between perennial ryegrass and other Poaceae species to be inferred. The genetic maps of perennial ryegrass and the Triticeae cereals are highly conserved in terms of synteny and colinearity. This observation was supported by the general agreement of the syntenic relationships between perennial ryegrass, oat, and rice and those between the Triticeae and these species. A lower level of synteny and colinearity was observed between perennial ryegrass and oat compared with the Triticeae, despite the closer taxonomic affinity between these species. It is proposed that the linkage groups of perennial ryegrass be numbered in accordance with these syntenic relationships, to correspond to the homoeologous groups of the Triticeae cereals.