Supporting in situ conservation of the genetic diversity of crop wild relatives using genomic technologies.

The last decade has witnessed huge technological advances in genomics, particularly in DNA sequencing. Here, we review the actual and potential application of genomics in supporting in situ conservation of crop wild relatives (CWRs). In addition to helping in prioritization of protection of CWR taxa and in situ conservation sites, genome analysis is allowing the identification of novel alleles that need to be prioritized for conservation. Genomics is enabling the identification of potential sources of important adaptive traits that can guide the establishment or enrichment of in situ genetic reserves. Genomic tools also have the potential for developing a robust framework for monitoring and reporting genome-based indicators of genetic diversity changes associated with factors such as land use or climate change. These tools have been demonstrated to have an important role in managing the conservation of populations, supporting sustainable access and utilization of CWR diversity, enhancing accelerated domestication of new crops and forensic genomics thus preventing misappropriation of genetic resources. Despite this great potential, many policy makers and conservation managers have failed to recognize and appreciate the need to accelerate the application of genomics to support the conservation and management of biodiversity in CWRs to underpin global food security. Funding and inadequate genomic expertise among conservation practitioners also remain major hindrances to the widespread application of genomics in conservation.

Resolution of the Identity of 'Candidatus Liberibacter' Species From Huanglongbing-Affected Citrus in East Africa.

'Candidatus Liberibacter asiaticus', the bacterium associated with citrus Huanglongbing (HLB), was reported from Uganda and tentatively from Tanzania, posing a threat to citriculture in Africa. Two surveys of citrus expressing typical HLB symptoms were conducted in Uganda, Kenya, and Tanzania to verify reports of 'Ca. L. asiaticus' and to assess the overall threat of HLB to eastern and southern African citrus production. Samples were analyzed for the presence of 'Candidatus Liberibacter' species by real-time PCR and partial sequencing of three housekeeping genes, 16S rDNA, rplJ, and omp. 'Ca. L. africanus', the bacterium historically associated with HLB symptoms in Africa, was detected in several samples. However, samples positive in real-time PCR for 'Ca. L. asiaticus' were shown not to contain 'Ca. L. asiaticus' by sequencing. Sequences obtained from these samples were analogous to 'Ca. L. africanus subsp. clausenae', identified from an indigenous Rutaceae species in South Africa, and not to 'Ca. L. asiaticus'. Results indicate a nontarget amplification of the real-time assay and suggest that previous reports of 'Ca. L. asiaticus' from Uganda and Tanzania may be mis-identifications of 'Ca. L. africanus subsp. clausenae'. This subspecies was additionally detected in individual Diaphorina citri and Trioza erytreae specimens recovered from collection sites. This is the first report of 'Ca. L. africanus subsp. clausenae' infecting citrus and being associated with HLB symptoms in this host.

A large coronal loop in the Algol system.

The close binary Algol system contains a radio-bright KIV subgiant star in a very close (0.062 astronomical units) and rapid (2.86 day) orbit with a main sequence B8 star. Because the rotation periods of the two stars are tidally locked to the orbital period, the rapid rotation drives a magnetic dynamo. A large body of evidence points to the existence of an extended, complex coronal magnetosphere originating at the cooler K subgiant. The detailed morphology of the subgiant's corona and its possible interaction with its companion are unknown, though theory predicts that the coronal plasma should be confined in a magnetic loop structure, as seen on the Sun. Here we report multi-epoch radio imaging of the Algol system, in which we see a large, persistent coronal loop approximately one subgiant diameter in height, whose base is straddling the subgiant and whose apex is oriented towards the B8 star. This suggests that a persistent asymmetric magnetic field structure is aligned between the two stars. The loop is larger than anticipated theoretically, but the size may be the result of a magnetic interaction between the two stars.

Evaluation of the impact of fetal fibronectin test implementation on hospital admissions for preterm labour in Ontario: a multiple baseline time-series design.

OBJECTIVE: To determine the impact of a health system-wide fetal fibronectin (fFN) testing programme on the rates of hospital admission for preterm labour (PTL). DESIGN: Multiple baseline time-series design. SETTING: Canadian province of Ontario. POPULATION: A retrospective population-based cohort of antepartum and delivered obstetrical admissions in all Ontario hospitals between 1 April 2002 and 31 March 2010. METHODS: International Classification of Diseases codes in a health system-wide hospital administrative database were used to identify the study population and define the outcome measure. An aggregate time series of monthly rates of hospital admissions for PTL was analysed using segmented regression models after aligning the fFN test implementation date for each institution. MAIN OUTCOME MEASURE: Rate of obstetrical hospital admission for PTL. RESULTS: Estimated rates of hospital admission for PTL following fFN implementation were lower than predicted had pre-implementation trends prevailed. The reduction in the rate was modest, but statistically significant, when estimated at 12 months following fFN implementation (-0.96 hospital admissions for PTL per 100 preterm births; 95% confidence interval [CI], -1.02 to -0.90, P = 0.04). The statistically significant reduction was sustained at 24 and 36 months following implementation. CONCLUSIONS: Using a robust quasi-experimental study design to overcome confounding as a result of underlying secular trends or concurrent interventions, we found evidence of a small but statistically significant reduction in the health system-level rate of hospital admissions for PTL following implementation of fFN testing in a large Canadian province.

Genetics and Genomics of African Rice (Oryza glaberrima Steud) Domestication.

African rice (Oryza glaberrima Steud) is one of the two independently domesticated rice species, the other one being Asian rice (Oryza sativa L.). Despite major progress being made in understanding the evolutionary and domestication history of African rice, key outstanding issues remain controversial. There appears to be an underlying difficulty in identifying the domestication centre and number of times the crop has been domesticated. Advances in genomics have provided unprecedented opportunities for understanding the genetic architecture of domestication related traits. For most of the domestication traits, the underlying genes and mutations have been identified. Comparative analysis of domestication genes between Asian and African rice has revealed that the two species went through an independent but convergent evolution process. The genetic and developmental basis of some of the domestic traits are conserved not only between Asian and African rice but also with other domesticated crop species. Analysis of genome data and its interpretation is emerging as a major challenge facing studies of domestication in African rice as key studies continue giving contradictory findings and conclusions. Insights obtained on the domestication of this species are vital for guiding crop improvement efforts.

Development and validation of diagnostic SNP markers for quality control genotyping in a collection of four rice (Oryza) species.

Morphological identification of closely related rice species, particularly those in the Oryza AA genome group, presents major challenges and often results in cases of misidentification. Recent work by this group identified diagnostic single nucleotide polymorphic (SNP) markers specific for several rice species and subspecies based on DArTseq next-generation sequencing technology ("DArTseq"). These SNPs can be used for quality control (QC) analysis in rice breeding and germplasm maintenance programs. Here, we present the DArTseq-based diagnostic SNPs converted into Kompetitive allele-specific PCR (KASPar or KASP) assays and validation data for a subset of them; these can be used for low-cost routine genotyping quality control (QC) analysis. Of the 224 species/subspecies' diagnostic SNPs tested, 158 of them produced working KASP assays, a conversion success rate of 70%. Two validation experiments were run with 87 of the 158 SNP markers to ensure that the assays amplified, were polymorphic, and distinguished the five species/subspecies tested. Based on these validation test results, we recommend a panel of 36 SNP markers that clearly delineate O. barthii, O. glaberrima, O. longistaminata, O. sativa spp. indica and japonica. The KASP assays provide a flexible, rapid turnaround and cost-effective tool to facilitate germplasm curation and management of these four Oryza AA genome species across multiple genebanks.

Epstein-barr virus-negative human malignant T-cell lines.

Two lymphoblastoid lines, CCRF-CEM and HSB-2, with properties of malignant cells, derived from children with leukemia secondary to lymphosarcoma, have T-cell properties and lack Epstein-Barr virus antigens.

To grow mouse mammary epithelial cells in culture.

Normal mouse mammary epithelial cells from Balb/c mice were successfully cultivated on tissue culture plastic with lethally irradiated LA7 feeder cells. The feeder cells also promoted colony formation from single mouse mammary cells, and the fraction of cells that formed colonies was proportional to the density of feeder cells. The mouse mammary cells could be passaged at least 8-12 times as long as new feeder cells were added at each passage. The cells now in culture have doubled in number at least 30 times, but the in vitro lifespan is not yet known. The cultures of mouse cells maintained by this technique never became overgrown with fibroblasts and numerous domes formed in the cultures.

Model simulation of the cretaceous ocean circulation.

Three-dimensional numerical ocean circulation model experiments that were designed to evaluate the circulation characteristics for the mid-Cretaceous ( approximately 100 million years ago) show that the primary direction of flow through the Tethys Ocean was eastward; in contrast, a westward flowing circumglobal Tethys current has been a consistent feature of earlier reconstructions of Cretaceous ocean circulation. The model studies demonstrate that (i) ocean circulation is sufficiently sensitive to the role of continental positions, sea level, and climate to limit the application of modern analogs to past circulations, and (ii) reconstructions based on limited biogeographic data may not provide unique surface circulation patterns.

Evidence for competitive displacement of Ceratitis cosyra by the invasive fruit fly Bactrocera invadens (Diptera: Tephritidae) on mango and mechanisms contributing to the displacement.

Bactrocera invadens Drew, Tsuruta & White (Diptera: Tephritidae) invaded Kenya in 2003. Before the arrival of B. invadens, the indigenous fruit fly species Ceratitis cosyra (Walker) was the predominant fruit fly pest of mango (Mangifera indica L.). Within 4 yr of invasion, B. invadens has displaced C. cosyra and has become the predominant fruit fly pest of mango, constituting 98 and 88% of the total population in traps and mango fruit at Nguruman, respectively. We tested two possible mechanisms responsible for the displacement namely; resource competition by larvae within mango fruit and aggression between adult flies. Under interspecific competition, larval duration in B. invadens was significantly shorter (6.2 +/- 0.6-7.3 +/- 0.3 d) compared with C. cosyra (8.0 +/- 1.2-9.4 +/- 0.4 d). Pupal mass in C. cosyra was affected by competition and was significantly reduced (7.4 +/- 0.3-9.6 +/- 0.6 mg) under competitive interaction compared with the controls (12.1 +/- 1.5-12.8 +/- 1.1 mg). Interspecific competition also had a significant adverse effect on C. cosyra eclosion, with fewer adults emerging under co-infestation compared with the controls. Interference competition through aggressive behavior showed that fewer C. cosyra (3.1 +/- 0.8) landed on mango dome compared with the controls (14.2 +/- 1.5) when adults were mixed with B. invadens adults in Plexiglas cages. Similarly the number of times C. cosyra was observed ovipositing was significantly lower (0.2 +/- 0.2) under competitive interaction compared with the controls (6.1 +/- 1.8). Aggressive encounters in the form of lunging/ head-butting and chasing off other species from the mango dome was higher for B. invadens compared with C. cosyra. Our results suggest that exploitative competition through larval scrambling for resources and interference competition through aggressive behaviors of the invader are important mechanisms contributing to the displacement of C. cosyra by B. invadens in mango agroecosystems.

Advances in Molecular Genetics and Genomics of African Rice (Oryza glaberrima Steud).

African rice (Oryza glaberrima) has a pool of genes for resistance to diverse biotic and abiotic stresses, making it an important genetic resource for rice improvement. African rice has potential for breeding for climate resilience and adapting rice cultivation to climate change. Over the last decade, there have been tremendous technological and analytical advances in genomics that have dramatically altered the landscape of rice research. Here we review the remarkable advances in knowledge that have been witnessed in the last few years in the area of genetics and genomics of African rice. Advances in cheap DNA sequencing technologies have fuelled development of numerous genomic and transcriptomic resources. Genomics has been pivotal in elucidating the genetic architecture of important traits thereby providing a basis for unlocking important trait variation. Whole genome re-sequencing studies have provided great insights on the domestication process, though key studies continue giving conflicting conclusions and theories. However, the genomic resources of African rice appear to be under-utilized as there seems to be little evidence that these vast resources are being productively exploited for example in practical rice improvement programmes. Challenges in deploying African rice genetic resources in rice improvement and the genomics efforts made in addressing them are highlighted.

Role of genomics in promoting the utilization of plant genetic resources in genebanks.

Global efforts have seen the world's plant genetic resources (PGRs) conserved in about 1625 germ plasm repositories. Utility of these resources is important in increasing the resilience and productivity of agricultural production systems. However, despite their importance, utility of these resources has been poor. This article reviews the real and potential application of the current advances in genomic technologies in improving the utilization of these resources. The actual and potential application of these genomic approaches in plant identification, phylogenetic analysis, analysing the genetic value of germ plasm, facilitating germ plasm selection in genebanks as well as instilling confidence in international germ plasm exchange system is discussed. We note that if genebanks are to benefit from this genomic revolution, there is need for fundamental changes in the way genebanks are managed, perceived, organized and funded. Increased collaboration between genebank managers and the user community is also recommended.

Jug r 4, a legumin group food allergen from walnut (Juglans regia Cv. Chandler).

Allergy to walnut is the most frequently reported tree nut allergy in the United States. Walnut 2S albumin, a vicilin-like protein, and a lipid transfer protein allergen have previously been described. Our objective was to clone and express a cDNA encoding a legumin group protein, assess IgE-binding with sera from walnut allergic patients, and investigate cross-reactivity with selected nuts. Primers were used to obtain the cDNA by 5' and 3' rapid amplification of cDNA ends from walnut mRNA. The cDNA was subcloned into the pMAL-c2X vector and the recombinant fusion protein, named rJug r 4, was expressed in Escherichia coli. The obtained cDNA encoded a precursor protein with a predicted molecular weight of 58.1 kD, which showed significant sequence homology to hazelnut and cashew legumin allergens. Serum IgE from 21 of 37 (57%) patients bound the rJug r 4 fusion protein. In vitro cross-reactivity was demonstrated with hazelnut, cashew, and peanut protein extracts.

Antigenic stability of pecan [Carya illinoinensis (Wangenh.) K. Koch] proteins: effects of thermal treatments and in vitro digestion.

Rabbit polyclonal antibody-based inhibition ELISA as well as immunoblotting analyses of proteins extracted from variously processed pecans (cv. Desirable) indicate that pecan proteins are antigenically stable. Pecan antigens were more sensitive to moist heat than dry heat processing treatments. SDS-PAGE and immunoblotting analysis of the native and heat-denatured proteins that were previously subjected to in vitro simulated gastric fluid digestions indicate that stable antigenic peptides were produced. Both enzyme-to-substrate ratio and digestion time were influential in determining the stability of pecan polypeptides. The stable antigenic polypeptides may serve as useful markers in developing assays suitable for the detection of trace amounts of pecans in foods.

Detection of T-cell lymphoma-associated antigens on cord blood lymphocytes and phytohemagglutinin-stimulated blasts.

Absorption studies demonstrate that T-cell lymphoma-associated antigens detected by rabbit antisera to human T-lymphoblast cell lines are present in suspensions of cord blood lymphocytes and phytohemagglutinin-stimulated adult blood lymphocytes in amounts similar to those found in T-cell lymphoma tumor cell suspensions. Smaller amounts of antigen activity are found in suspensions of tonsil cells, thymocytes, and unstimulated adult blood lymphocytes. Little or no antigen activity is found in suspensions of lymphoblasts from patients with other types of leukemia or from B-cell lines. T-cell depletion removes antigen activity from suspensions of normal lymphocytes. These findings suggest that T-cell lymphoma-associated antigens may be fetal antigens expressed by activated T-cells.

Morphological, biological, and biochemical characteristics of human bladder transitional cell carcinomas grown in tissue culture and in nude mice.

The morphological, biological, biochemical, and karyotypic characteristics of four human bladder transitional cell carcinoma lines, SW-780, SW-800, SW-1738, and SW-1710, were investigated. In tissue culture, each cell line presented a distinct phenotypic expression. All but line SW-1710 grew when transplanted in the nude mouse. Light and electron microscopic studies showed morphological characteristics similar to the tumors of origin, being independent of the passages in tissue culture medium, tumor cell extracts, and the plasma of nude mouse-grown tumors, showing isoenzyme quantitative distribution typical for each cell line. In addition, each cell line exhibited a unique genetically determined enzyme phenotypic profile which, along with the karyotypic analysis, makes their identification feasible. These characteristics make the described tumor lines a valuable tool in studying various aspects of the biology of human bladder transitional cell carcinoma.

Enhanced G2 chromatid radiosensitivity, an early stage in the neoplastic transformation of human epidermal keratinocytes in culture.

A deficiency in DNA repair, manifest as enhanced chromatid radiosensitivity during the G2 phase of the cell cycle, together with a proliferative stimulus such as that provided by active oncogenes may be necessary and sufficient for the malignant neoplastic transformation of human keratinocytes in culture. Normal epidermal keratinocytes established as continuous cell lines by transfection with pSV3-neo or infection with adeno 12-SV40 hybrid virus developed enhanced G2 chromatid radiosensitivity after 18 passages in culture. In contrast to cells from primary or secondary culture, these cells could be transformed to malignant neoplastic cells by infection with Kirsten murine sarcoma virus containing the Ki-ras oncogene or in one line by the chemical carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine; both of these agents produced a marked proliferative response. Cytological heterogeneity and karyotypic instability characterized the cells during their progression to neoplasia. These results are interpreted in terms of a mechanism for neoplastic transformation.

Establishment and characterization of four new human non-small cell lung cancer cell lines.

Four new human non-small cell lung cancer cell lines have been established in vitro. These cell lines have been characterized by (a) growth of a tumor in nude mice with histopathology similar to that of the primary, (b) isoenzyme patterns phenotypically human and distinct from each other, (c) distinguishing karyotypic findings, (d) growth rate determinations, and (e) presence of epidermal growth factor receptors. Each of the cell lines will form colonies when directly seeded into a flask without soft agar. The development and availability of the four cell lines may facilitate in vitro studies of the biology of this common cancer. Their clonogenic potential may be of value in the study of sensitivity to antineoplastic agents. Their low passage level may mean that their antigens still resemble those of the primary tumor.

Isolation and characterization of a spontaneously immortalized human breast epithelial cell line, MCF-10.

Two sublines of a breast epithelial cell culture, MCF-10, derived from human fibrocystic mammary tissue exhibit immortality after extended cultivation in low calcium concentrations (0.03-0.06 mM) and floating transfers in low calcium (MCF-10F), or by trypsin-Versene passages in the customary (normal) calcium levels, 1.05 mM (MCF-10A). Both sublines have been maintained as separate entities after 2.3 years (849 days) in vitro and at present have been in culture for longer than 4 years. MCF-10 has the characteristics of normal breast epithelium by the following criteria: (a) lack of tumorigenicity in nude mice; (b) three-dimensional growth in collagen; (c) growth in culture that is controlled by hormones and growth factors; (d) lack of anchorage-independent growth; and (e) dome formation in confluent cultures. Cytogenetic analysis prior to immortalization showed normal diploid cells; although later passages showed minimal rearrangement and near-diploidy, the immortal cells were not karyotypically normal. The emergence of an immortal culture in normal calcium media was not an inherent characteristic of the original tissue from which MCF-10 was derived since reactivated cryo-preserved cells from cultures grown for 0.3 and 1.2 years in low calcium were incapable of sustained growth in normal calcium.