Set Of Clones Research Articles

Sequencing and comparative genomics analysis in Senecio scandens Buch.-Ham. Ex D. Don, based on full-length cDNA library

Senecio scandens Buch.-Ham. ex D. Don, an important antibacterial source of Chinese traditional medicine, has a widespread distribution in a few ecological habitats of China. We generated a full-length complementary DNA (cDNA) library from a sample of elite individuals with superior antibacterial properties, with satisfactory parameters such as library storage (4.30 × 106 CFU), efficiency of titre (1.30 × 106 CFU/mL), transformation efficiency (96.35%), full-length ratio (64.00%) and redundancy ratio (3.28%). The BLASTN search revealed the facile formation of counterparts between the experimental sample and Arabidopsis thaliana in view of high-homology cDNA sequence (90.79%) with e-values <1e – 50. Sequence similarities to known proteins indicate that the entire sequences of the full-length cDNA clones consist of the major of functional genes identified by a large set of microarray data from the present experimental material. For other Compositae species, a large set of full-length cDNA clones reported in the present article will serve as a useful resource to facilitate further research on the transferability of expressed sequence tag-derived simple sequence repeats (EST-SSR) development, comparative genomics and novel transcript profiles.

Mobile cloud computing based privacy protection in location‐based information survey applications

AbstractNowadays, location‐based service (LBS) has become pervasive. Given its high utility value, LBS, however, presents serious privacy concerns for cautious users. In this paper, we investigate privacy preserving for location‐based information survey application, which calculates the geographic distribution of user's information. The design objective is twofold: (i) calculate an information distribution for a pool of mobile users and (ii) protect the location and value privacy of individual user, in the presence of malicious servers and possible corrupted users. Our proposed solution leverages a mobile cloud computing paradigm, in which each mobile device is replicated with a system‐level clone in cloud. The computing of distribution function is distributed among the set of cloud clones via a P2P protocol. We further enhance our basic scheme with the multiple aggregation mechanism, aiming to protect the correctness of the aggregate result from the active attacker. Compared to the approaches based on centralized server or aggregate proxy, our proposed scheme and its enhanced version are advantageous in avoiding single point of failure/attack, load balancing, and overhead reduction. Simulation results verify these advantages and the protection to the correctness of aggregate result and suggest that our proposed scheme is suitable for large‐scale applications. Copyright © 2014 John Wiley &amp; Sons, Ltd.

Each of the eight simian hemorrhagic fever virus minor structural proteins is functionally important

The simian hemorrhagic fever virus (SHFV) genome differs from those of other members of the family Arterivirus in encoding two adjacent sets of four minor structural protein open reading frames (ORFs). A stable, full-length, infectious SHFV-LVR cDNA clone was constructed. Virus produced from this clone had replication characteristics similar to those of the parental virus. A subgenomic mRNA was identified for the SHFV ORF previously identified as 2b. As an initial means of analyzing the functional relevance of each of the SHFV minor structural proteins, a set of mutant infectious clones was generated, each with the start codon of one minor structural protein ORF mutated. Different phenotypes were observed for each ortholog of the pairs of minor glycoproteins and all of the eight minor structural proteins were required for the production of infectious extracellular virus indicating that the duplicated sets of SHFV minor structural proteins are not functionally redundant.

A repetitive DNA element regulates expression of the Helicobacter pylori sialic acid binding adhesin by a rheostat-like mechanism.

During persistent infection, optimal expression of bacterial factors is required to match the ever-changing host environment. The gastric pathogen Helicobacter pylori has a large set of simple sequence repeats (SSR), which constitute contingency loci. Through a slipped strand mispairing mechanism, the SSRs generate heterogeneous populations that facilitate adaptation. Here, we present a model that explains, in molecular terms, how an intergenically located T-tract, via slipped strand mispairing, operates with a rheostat-like function, to fine-tune activity of the promoter that drives expression of the sialic acid binding adhesin, SabA. Using T-tract variants, in an isogenic strain background, we show that the length of the T-tract generates multiphasic output from the sabA promoter. Consequently, this alters the H. pylori binding to sialyl-Lewis x receptors on gastric mucosa. Fragment length analysis of post-infection isolated clones shows that the T-tract length is a highly variable feature in H. pylori. This mirrors the host-pathogen interplay, where the bacterium generates a set of clones from which the best-fit phenotypes are selected in the host. In silico and functional in vitro analyzes revealed that the length of the T-tract affects the local DNA structure and thereby binding of the RNA polymerase, through shifting of the axial alignment between the core promoter and UP-like elements. We identified additional genes in H. pylori, with T- or A-tracts positioned similar to that of sabA, and show that variations in the tract length likewise acted as rheostats to modulate cognate promoter output. Thus, we propose that this generally applicable mechanism, mediated by promoter-proximal SSRs, provides an alternative mechanism for transcriptional regulation in bacteria, such as H. pylori, which possesses a limited repertoire of classical trans-acting regulatory factors.

Clonal structure and dynamics of peripheral Populus tremula L. populations

Abstract: Native Populus tremula L. populations at the south-western limit of the species’ range are threatened by the anthropic reduction of their habitats and by climate change. In these areas, P. tremula forms small and multiclonal populations. Knowledge of the structures and dynamics of these populations is essential to design effective conservation programs. The clonal spatial and ramet age distributions of three natural populations in north-western Spain were studied. Trees with diameter > 3 cm at breast height were mapped and their age identified. Multilocus genotypes based on 11 isoenzymatic loci were obtained for a sample of 90-106 evenly distributed trees per population. Clonal assignment of the remaining trees in each stand was interpolated using geostatistical methods. All three stands were multiclonal, with higher clonal diversity than that reported in northern populations, and no genotype was detected in more than one stand. Most clones exhibited aggregated distributions, but some scattered clones were also found. The clonal structures of the stands are consistent with the hypothesis that recruitment is absent inside the stand. A combined analysis of ages and genotypes revealed cycles of regrowth in which each clone experienced varying degrees of success in generating new ramets. These degrees of success could be the consequence of varying abilities for vegetative propagation among genotypes. Geostatistical interpolation can be a useful tool for the elaboration of clonal maps, thus reducing the costs of sampling and genotyping, but further research is needed to define the optimum scale of sampling for accurate clonal assignment. For P. tremula conservation programs based on clonal material, small plantations with relatively few clones (approximately 10) provide an appropriate strategy if the metapopulation is maintained at a sufficient level of diversity. The set of clones used for plantations and ex-situ collections should consider both the most abundant and the least frequent clones to avoid a biased selection.

Spontaneous Reversion of the Angiogenic Phenotype to a Nonangiogenic and Dormant State in Human Tumors

The angiogenic switch, a rate-limiting step in tumor progression, has already occurred by the time most human tumors are detectable. However, despite significant study of the mechanisms controlling this switch, the kinetics and reversibility of the process have not been explored. The stability of the angiogenic phenotype was examined using an established human liposarcoma xenograft model. Nonangiogenic cells inoculated into immunocompromised mice formed microscopic tumors that remained dormant for approximately 125 days (vs. <40 days for angiogenic cells) whereupon the vast majority (>95%) initiated angiogenic growth with second-order kinetics. These original, clonally derived angiogenic tumor cells were passaged through four in vivo cycles. At each cycle, a new set of single-cell clones was established from the most angiogenic clone and characterized for in vivo for tumorigenic activity. A total of 132 single-cell clones were tested in the second, third, and fourth in vivo passage. Strikingly, at each passage, a portion of the single-cell clones formed microscopic, dormant tumors. Following dormancy, like the original cell line, these revertant tumors spontaneously switched to the angiogenic phenotype. Finally, revertant clones were transcriptionally profiled and their angiogenic output determined. Collectively, these data demonstrate that the angiogenic phenotype in tumors is malleable and can spontaneously revert to the nonangiogenic phenotype in a population of human tumor cells. Leveraging the rate of reversion to the nonangiogenic phenotype and tumor dormancy may be a novel anticancer strategy.

Developing a cell suspension system for Musa-AAA-EA cv. ‘Nakyetengu’: a critical step for genetic improvement of Matooke East African Highland bananas

Embryogenic cell suspensions of triploid East African Highland bananas (Musa AAA-EA) were initiated and generated using cooking cultivar ‘Nakyetengu’ belonging to the Nakabululu clone set. Immature male flowers produced embryogenic calli consisting of embryos and friable tissue after 4 mo culture on a modified MA1 callus induction medium. Friable calli were initiated and maintained in liquid MA2 medium. A cell growth rate of 1.5–2.0 sedimented cell volume (SCV) per month was observed. Embryo development was observed at 2.18 × 103 embryos per mL SCV. Germination of these embryos was observed at 2.8% and 6.2% for two cell suspension lines. Plant regeneration efficiency was 60–100%, all producing normal plants with a shoot and roots at weaning. In the field, somatic cell-derived plants were all normal morphology and comparable to control plants during vegetative and reproductive stages. This study is a breakthrough for recalcitrant East African Highland banana and offers a system that can provide essential raw materials for associated germplasm improvement through genetic engineering approaches.

Sugarcane Specific Drought Responsive Candidate Genes Belonging to ABA Dependent Pathway Identified from Basic Species Clones of Saccharum sp. and Erianthus sp.

Development of drought tolerant sugarcane cultivars has long been a major breeding objective in view of depleting water source and longer dry spells. Identification of specific candidate genes would be an efficient approach to the genetic improvement of sugarcane for drought tolerance. As the involvement of abscisic acid (ABA) in mediating drought stress has been well demonstrated, in the present study eleven putative candidate genes from orthologous species belonging to ABA dependent pathway viz ABF 2, ABF 3, ATHB-7, CIPK 14, CDPK 18, LEA 3, MYB 2, RD 28, RGS 1, SNRK 2.5 and TPS 2 were screened on a set of clones belonging to tolerant species viz. Saccharum spontaneum, S. barberi, S. sinense, S. robustum and Erianthus and the susceptible species of S. officinarum. Out of these, ten genes except ATHB-7 were amplified in Saccharum and Erianthus species. The genes ABF 2, CIPK 14, LEA 3, MYB 2, RD 28, RGS1 and SNRK 2.5 were absent in almost all the susceptible clones of S. officinarum and were present in the tolerant species clones implicating their role in imparting drought tolerance in sugarcane. Number of genes detected in S. officinarum ranged from 2 to 6 whereas drought tolerant species clones exhibited 4–10 genes. The clones SES 106 B and Iritty 2 (S. spontaneum), Pathri (S. barberi) and IK 76–99 (Erianthus) were found as the repositories of ten genes to be advantageously used in breeding for drought tolerance in sugarcane. The sequencing of the polymorphic bands and BLAST search would lead to the identification of new drought responsive genes in sugarcane for use as novel and functional candidate genes for improving drought tolerance in sugarcane through marker-assisted introgression breeding and genetic transformation approach.

Structured diversity using EST-PCR and EST-SSR markers in a set of wild blueberry clones and cultivars

Blueberries are perennial, vegetatively propagated out-crossing shrubs with great potential of health benefits. In tetraploid blueberry (Vaccinium spp.), genetic analyses have focused more on diversity, whereas genetic structure has been studied less extensively. This study investigated the genetic structure and diversity in 28 wild clones, six cultivars and two selections of lowbush, half-high and highbush blueberries. Summary statistics, structure estimation and clustering by neighbour-joining (NJ), principal coordinate analysis (PCoA) and by the analysis of molecular variance (AMOVA), using 10 expressed sequence tag-polymerase chain reaction (EST-PCR) and two EST-simple sequence repeat (SSR) primer pairs, were performed to characterize and discriminate the genotypes. A total of 213 markers were detected. Wide genetic diversity was evident from high values of expected heterozygosities, Shannon's index and polymorphism information content, and from AMOVA. Structure analysis subdivided the lowbush blueberries into three distinct groups leaving the half-high and highbush blueberries into one cluster which was in agreement with the NJ clustering and PCoA. Twelve EST-primer pairs efficiently differentiated wild and cultivated blueberries, making this technology valuable for genome mapping and for long-term conservation of blueberry resources for the benefit of germplasm conservation and breeding purposes.

Randomized, replicated, staggered clonal-row (R2SCR) seed orchard design

Spatial randomization of clones across a seed orchard’s grid is commonly applied to promote cross-fertilization and minimize selfing. The high selection differential attained from advanced-generation breeding programs sets high premier on the genetic gain and diversity delivery from seed orchards, thus clonal allocation is important and even more challenging when clones share common ancestry. Evidences of low selfing in many conifers’ seed orchards, as a result of their high genetic load, inbreeding depression, and polyembryony are abundant and call for orchards’ design re-evaluation, specifically when randomization is associated with added managerial burden. Clonal-rows represent a viable option for simplifying orchards management; however, they are often associated with elevated correlated matings between adjacent clones. Here, we propose a modified clonal-row design that replicates, staggers, and randomizes the rows, thus doubling the number of adjacent clones and providing different set of neighboring clones at each replication, thus allowing accommodating related parents more readily than any single-tree arrangement. We present a novel algorithm packaged in user-friendly software for executing various seed orchards’ designs. The developed program is interactive and suitable for any orchard size and configuration, accommodates any number of clones that are allocated to rows with variable length (ranging from a single tree to any even number) and pre-set separation zone between ramets of the same clone. The program offers three deployment modes (equal, linear, and custom) each with multiple layouts determined by the number of iterations requested. The resulting layouts are ranked based on four criteria including: (1) the number of empty positions, (2) deviation between expected and observed clone size, (3) minimum inbreeding, and (4) a neighborhood index that expresses the efficiency of clonal distribution.

An Investigation into the Characteristics of Merged Code Clones during Software Evolution

SUMMARY Although code clones (i.e. code fragments that have similar or identical code fragments in the source code) are regarded as a factor that increases the complexity of software maintenance, tools for supporting clone refactoring (i.e. merging a set of code clones into a single method or function) are not commonly used. To promote the development of refactoring tools that can be more widely utilized, we present an investigation of clone refactoring carried out in the development of open source software systems. In the investigation, we identified the most frequently used refactoring patterns and discovered how merged code clone token sequences and

Sugarcane-specific drought responsive candidate genes belonging to ABA-independent pathway identified from tolerant and susceptible clones ofSaccharumandErianthusspecies

Drought is a major limitation to sustainable sugarcane production worldwide and the study of candidate genes provide a productive approach for establishing the basic responses of plants to drought. In present study, 14 putative candidate genes belonging to abscisic acid (ABA) independent pathway were screened on a set of tolerant species clones belonging to Saccharum spontaneum, S. barberi, S. sinense, S. robustum and Erianthus and the susceptible species clones of S. officinarum. Out of these, 11 genes viz., DREB 1A, DRF 1, Hep2, HRD, NAC 2, NIT 1, PIN 3, SHN 1, S1Z 1, Snac 1 and Wrky 38 were present in Saccharum and Erianthus, highlighting the role of these genes in drought tolerance. The number of candidate genes detected in S. officinarum ranged from zero to three. Three typical clones viz., Awela 68, Laukona 15 and 57 NG 215 did not show the presence of any of the drought specific genes studied, showing their pure S. officinarum nature, while the rest are suspected to be introgressed forms evolved in their place of origin/diversity. All the drought tolerant species clones exhibited eight to eleven genes indicating the role of these genes in imparting drought tolerance in sugarcane. All genes were uniformly present in drought tolerant species clones viz., Iritty 2, S. spontaneum (Coimbatore) and SES 600 (S. spontaneum), NG 77–59 (S. robustum), Kewali and Khatuia (S. barberi), Ikhri (S. sinense) and IK 76–62 and IK 76–48 (Erianthus sp.). These clones could serve as potential donors in pre-breeding activities for genetic improvement of drought tolerance in sugarcane and the eleven sugarcane specific drought responsive genes can serve as novel and functional candidate gene markers in marker-assisted introgression breeding programmes.

Functional Early Hematopoietic Progenitor Cells Derived From Mouse Embryonic Stem Cells and Induced Pluripotent Stem Cells

We have previously reported a method to produce early hematopoietic progenitor cells from C57BL/6J-derived mouse embryonic stem cells (mESCs). After co-culture on OP9 stromal cells for one week, four different C57Bl/6 mESC lines consistently differentiated into hematopoietic progenitors, as determined by immunophenotyping; we detected cells that mark as KLS (Lin- Kit+ Sca+), CMPs, GMPs, and MEPs (but not SLAMs) from all four lines. In addition, functional progenitors for erythrocytes, monocytes, and mast cells (by morphology and immunophenotyping) were detected after another week of culture in methylcellulose with hematopoietic cytokines (SCF, IL-3, IL-6, and Epo). These findings were replicated using four different lots of fetal bovine serum, and with three different lots of OP9 cells from ATCC. We injected 1x106 “OP9-induced” progenitor cells retroorbitally into unconditioned NSG mice, and detected multilineage hematopoietic engraftment (myeloid compartments marked by CD34, CD11b, Kit, and Gr-1, lymphoid compartments marked by CD3 and B220, and erythroid compartments marked by Ter119) in the bone marrow and/or spleens of 10 out of 19 recipients at 3 months.Using the OP9 co-culture system, we have differentiated miPSC clones from three independent iPSC experiments, using an integrating polycistronic lentivirus expressing OCT4, SOX2, and KLF4 as the reprogramming vector. One set of miPSC clones was produced from mouse embryonic fibroblasts (MEFs) from pooled C57BL/6J embryos, and two sets were made from adult mouse fibroblasts derived from a single animal, producing 6, 12, and 12 independent iPS clones for analysis, respectively. All thirty clones had pluripotent features, as determined by alkaline phosphatase staining and immunophenotyping (SSEA1, Oct4, and Nanog). We have injected the OP9-induced progenitors derived from one miPSC clone into NSG mice; thus far, 2 out of 14 recipients have demonstrated engraftment in the peripheral blood. However, the efficiency of hematopoietic progenitor generation with OP9 induction (based on the immunophenotyping and progenitor assays noted above) was highly variable for miPSCs from all three experiments. Among all three sets of miPSC clones, 18/30 exhibited differentiation efficiencies comparable to wild-type B6 ESCs, 5/30 clones exhibited moderately reduced differentiation efficiencies, 5/30 clones exhibited markedly reduced differentiation efficiencies, and 2/30 clones (from two different iPSC experiments) did not produce any detectable hematopoietic progenitors with OP9 induction. These phenotypes were stable and highly reproducible. The 2 clones that did not yield any hematopoietic progenitors had robust pluripotency marks, and one that was injected into the hindflank of NSG mice produced cystic teratomas. We found that 2% DMSO pretreatment of mESCs for 24 hours prior to OP9 co-culture improved the differentiation efficiency of wild-type B6 ESCs by 50% (Chetty et al. Nature Methods 10(6):553-6, 2013), but it did not rescue the phenotype of miPSC clones that did not produce hematopoietic progenitors. We are currently performing exome sequencing on the 24 miPSC clones from the adult fibroblast reprogramming experiments to determine whether phenotypic heterogeneity is due to specific mutations in the iPSC clones (Young et al. Cell Stem Cell 10(5):570-82, 2012).In summary, we have developed a simple system to derive functional early hematopoietic progenitor cells from mouse embryonic stem cells and/or induced pluripotent stem cells. OP9-induced progenitor cells engraft into NSG mice without the need for forced expression of HoxB4 (Wang et al. Proc Natl Acad Sci USA 102(52):19081-6, 2005). We have detected functional heterogeneity in miPSC clones derived from the same parental cells, which could be due to genetic variation in the founding cell from which each clone was derived, to different integration sites of the OSK lentivirus in each clone, or to as yet undefined epigenetic mechanisms. Exome sequencing may help to resolve this issue. Regardless, this approach could be a valuable tool for studying the hematopoietic development of a variety of mESC lines and/or miPSC lines derived from genetically altered mice. Disclosures:No relevant conflicts of interest to declare.

High-Throughput Sequencing Of T Cell Receptors Detects Signatures Of T Cell Repertoire That Predict MRD Status In Patients With Mantle Cell Lymphoma Undergoing Immunotransplantation

Background A clinical trial is ongoing at Stanford for MCL patients in first remission that interdigitates an autologous CpG-stimulated tumor cell vaccine with autologous peripheral blood stem cell transplant (PSCT) (NCT00490529). In this trial, blood samples collected before and after vaccination and serially post-transplant are assayed for minimal residual disease (MRD) and for T cell repertoire using the LymphoSIGHT™ sequencing method (Faham et al., Blood 2012). We identified a set of T cell clones that appear to be responding to the vaccine, and therefore we investigated whether the number of these clonotypes was correlated with MRD status. Methods Using universal primer sets, we amplified rearranged IgH variable (V), diversity, and joining (J) gene segments from genomic DNA. Amplified products were sequenced to obtain &gt;1 million reads. The B cell tumor-specific sequence was identified for each patient based on its high frequency in the original tumor biopsy. The presence of the tumor cells was then monitored in serial blood samples with a sensitivity of 1 cell per million leukocytes. The same blood samples were used for amplification, sequencing and analysis of the entire TCRβ repertoire. To facilitate identification of tumor vaccine-induced TCRβ clonotypes, we sequenced the TCRβ repertoire immediately before and after the administration of both the priming vaccination and a booster vaccination. We developed a metric called the vaccine response score (V score). This metric is calculated for each clonotype and reflects the increase in frequency after the initial vaccination AND after the boost. The formula for calculating V score is: V = F1 x F2 x square root [1/ (|F1 – F2| + 1)], where F1 and F2 represent the fold-change of the priming and boost vaccinations, respectively. Clonotypes with a V score &gt;10 were deemed to be vaccination-induced by virtue of these frequency changes. Results In a series of 12 vaccinated patients, the number of clonotypes with V score ≥ 10 ranged between 0 and 262, with a median of 57. We utilized an antigen-specific analysis to validate that clones with high V scores (≥ 10) were in fact tumor-specific. For this analysis, we incubated peripheral blood mononuclear cells (PBMCs) with the tumor and then sequenced the TCRβ repertoire from cells obtained after culture. Clones that were enriched after culture compared to pre-stimulation PBMCs were deemed to be antigen-specific. These clones that are antigen-specific are highly likely to have a high V score compared to a random frequency-matched set of clones (P two tailed = 1.8 x 10-10), providing further evidence that clones with a high V score are tumor-specific. We then analyzed the relationship between V score and clinical outcome. Patients could be stratified into two groups with “high” (&gt; 25) or “low” (&lt;25) numbers of vaccine-responsive clonotypes. Patients in the high V score group, who had larger numbers of putative tumor-specific T cells, were more likely to have sustained molecular remission during the first-year post-transplant compared with patients in the low V score group (P = 0.018) (Figure 1). Conclusions T cell repertoire analysis identified clonotypes responding to the vaccination, and the presence of these vaccine-specific clonotypes correlates with MRD positivity at the important landmark of one year post-PSCT. Further analysis of additional patients enrolled on the MCL trial is ongoing. This data underscores the prognostic relevance of the sequencing-based V score metric and provides a novel approach for assessment of cancer immunotherapy responses. Disclosures: Faham: Sequenta: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

Identification of differentially-expressed genes potentially implicated in drought response in pitaya (Hylocereus undatus) by suppression subtractive hybridization and cDNA microarray analysis

Drought is one of the most severe threats to the growth, development and yield of plant. In order to unravel the molecular basis underlying the high tolerance of pitaya (Hylocereus undatus) to drought stress, suppression subtractive hybridization (SSH) and cDNA microarray approaches were firstly combined to identify the potential important or novel genes involved in the plant responses to drought stress. The forward (drought over drought-free) and reverse (drought-free over drought) suppression subtractive cDNA libraries were constructed using in vitro shoots of cultivar ‘Zihonglong’ exposed to drought stress and drought-free (control). A total of 2112 clones, among which half were from either forward or reverse SSH library, were randomly picked up to construct a pitaya cDNA microarray. Microarray analysis was carried out to verify the expression fluctuations of this set of clones upon drought treatment compared with the controls. A total of 309 expressed sequence tags (ESTs), 153 from forward library and 156 from reverse library, were obtained, and 138 unique ESTs were identified after sequencing by clustering and blast analyses, which included genes that had been previously reported as responsive to water stress as well as some functionally unknown genes. Thirty six genes were mapped to 47 KEGG pathways, including carbohydrate metabolism, lipid metabolism, energy metabolism, nucleotide metabolism, and amino acid metabolism of pitaya. Expression analysis of the selected ESTs by reverse transcriptase polymerase chain reaction (RT-PCR) corroborated the results of differential screening. Moreover, time-course expression patterns of these selected ESTs further confirmed that they were closely responsive to drought treatment. Among the differentially expressed genes (DEGs), many are related to stress tolerances including drought tolerance. Thereby, the mechanism of drought tolerance of this pitaya genotype is a very complex physiological and biochemical process, in which multiple metabolism pathways and many genes were implicated. The data gained herein provide an insight into the mechanism underlying the drought stress tolerance of pitaya, as well as may facilitate the screening of candidate genes for drought tolerance.

Combining Next-Generation Sequencing and Immune Assays: A Novel Method for Identification of Antigen-Specific T Cells

In this study, we combined a novel sequencing method, which can identify individual clonotypes based on their unique T cell receptor (TCR) rearrangement, with existing immune assays to characterize antigen-specific T cell responses. We validated this approach using three types of assays routinely used to measure antigen-specific responses: pentamers which enable identification of T cells bearing specific TCRs, activation marker expression following antigen stimulation and antigen-induced proliferation to identify cytomegalovirus (CMV) specific clonotypes. In one individual, 8 clonotypes were identified using a pentamer reagent derived from the CMV pp65 protein. The same 8 clonotypes were also identified following sequencing of cells that upregulated an activation marker following incubation with an identical peptide derived from pp65. These 8 and an additional 8 clonotypes were identified using a more sensitive CFSE-based proliferation assay. We found clear sequence homology among some of the clonotypes identified, and the CDR3 region in one clonotype was identical to a previously published pp65-specific clonotype sequence. Many of these CMV-specific clonotypes were present at frequencies below 10−5 which are undetectable using standard flow-cytometric methods. These studies suggest that an immune response is comprised of a diverse set of clones, many of which are present at very low frequencies. Thus, the combination of immune assays and sequencing depicts the richness and diversity of an immune response at a level that is not possible using standard immune assays alone. The methods articulated in this work provide an enhanced understanding of T cell-mediated immune responses at the clonal level.

Development of an Unbiased Antigen-Mining Approach To Identify Novel Vaccine Antigens and Diagnostic Reagents for Bovine Tuberculosis

Previous experiments for the identification of novel diagnostic or vaccine candidates for bovine tuberculosis have followed a targeted approach, wherein specific groups of proteins suspected to contain likely candidates are prioritized for immunological assessment (for example, with in silico approaches). However, a disadvantage of this approach is that the sets of proteins analyzed are restricted by the initial selection criteria. In this paper, we describe a series of experiments to evaluate a nonbiased approach to antigen mining by utilizing a Gateway clone set for Mycobacterium tuberculosis, which constitutes a library of clones expressing 3,294 M. tuberculosis proteins. Although whole-blood culture experiments using Mycobacterium bovis-infected animals and M. bovis BCG-vaccinated controls did not reveal proteins capable of differential diagnosis, several novel immunogenic proteins were identified and prioritized for efficacy studies in a murine vaccination/challenge model. These results demonstrate that Rv3329-immunized mice had lower bacterial cell counts in their spleens following challenge with M. bovis. In conclusion, we demonstrate that this nonbiased approach to antigen mining is a useful tool for identifying and prioritizing novel proteins for further assessment as vaccine antigens.

Stable MSAP Markers for the Distinction of Vitis vinifera cv Pinot Noir Clones

Grapevine is one of the most economically important fruit crops. Molecular markers have been used to study grapevine diversity. For instance, simple sequence repeats are a powerful tool for identification of grapevine cultivars, while amplified fragment length polymorphisms have shown their usefulness in intra-varietal diversity studies. Other techniques such as sequence-specific amplified polymorphism are based on the presence of mobile elements in the genome, but their detection lies upon their activity. Relevant attention has been drawn toward epigenetic sources of variation. In this study, a set of Vitis vinifera cv Pinot noir clones were analyzed using the methylation-sensitive amplified polymorphism technique with isoschizomers MspI and HpaII. Nine out of fourteen selective primer combinations were informative and generated two types of polymorphic fragments which were categorized as "stable" and "unstable." In total, 23 stable fragments were detected and they discriminated 92.5% of the studied clones. Detected stable polymorphisms were either common to several clones, restricted to a few clones or unique to a single clone. The identification of these stable epigenetic markers will be useful in clonal diversity studies. We highlight the relevance of stable epigenetic variation in V. vinifera clones and analyze at which level these markers could be applicable for the development of forthright techniques for clonal distinction.

IDENTIFIKASI PATOTIPE GLOBODERA ROSTOCHIENSIS MENGGUNAKAN KLON DIFERENSIAL

Identification of Globodera rostochiensis using differential clones. Potato cyst nematode, Globodera rostochiensis, is a relatively new pathogen in Indonesia that becomes a constraint to potato production. To manage the parasite effectively, it is very important to identify the pathotype of G. rostochiensis populations. Therefore, this research was carried out to identify G. rostochiensis pathotypes. Four G. rostochiensis samples, consisting of three samples from East Java and one sample from Central Java, were identified using a set of differential clones. The result showed that G. rostochiensis samples from East Java were new pathotype, whereas the sample from Central Java was Ro1 pathotype.

Starch Characteristics of Modern and Heirloom Potato Cultivars

In a number of ways, modern potato breeding efforts differ from those that created heirloom cultivars. As a result of the rapid expansion of the potato processing industry in the mid twentieth century, potato breeders shifted their focus from fresh market varieties to those intended for the fry and chip markets. Selection for higher dry matter content and lower reducing sugar levels in tubers was successful. This study sought to determine whether modern processing cultivars differ from heirloom cultivars for two major characteristics of tuber starch, amylose content and starch granule morphology. Since breeding efforts for processing potatoes have focused on the maintenance of tuber quality during storage, these parameters were measured in both fresh and stored tubers. Twenty cultivars selected to span the range of cultivar release dates in the U.S. were grown at Hancock, WI in 2009 and 2010 and evaluated for tuber amylose content and measurements of starch granule surface area, length, width, length to width ratio, and smoothness. These traits were found to be stable across years and during storage. Significant differences in the amylose content and starch granule morphology exist among cultivars, but there is no clear distinction between modern and heirloom cultivars. Genetic variation for amylose content and starch granule morphology was found within the set of clones sampled for this study. However, germplasm with higher amylose content will need to be identified and utilized in breeding if the nutritional benefits associated with high fiber and low glycemic index are to be realized.