Summary R/qtl is an extensible, interactive environment for mapping quantitative trait loci (QTLs) in experimental populations derived from inbred lines. It is implemented as an add-on package for the freely-available statistical software, R, and includes functions for estimating genetic maps, identifying genotyping errors, and performing single-QTL and two-dimensional, two-QTL genome scans by multiple methods, with the possible inclusion of covariates. Availability The package is freely available at http://www.biostat.jhsph.edu/~kbroman/qtl.

R/qtl: QTL mapping in experimental crosses

Recognizing the enormous potential of DNA markers in plant breeding, many agricultural research centers and plant breeding institutes have adopted the capacity for marker development and marker-assisted selection (MAS). However, due to rapid developments in marker technology, statistical methodology for identifying quantitative trait loci (QTLs) and the jargon used by molecular biologists, the utility of DNA markers in plant breeding may not be clearly understood by non-molecular biologists. This review provides an introduction to DNA markers and the concept of polymorphism, linkage analysis and map construction, the principles of QTL analysis and how markers may be applied in breeding programs using MAS. This review has been specifically written for readers who have only a basic knowledge of molecular biology and/or plant genetics. Its format is therefore ideal for conventional plant breeders, physiologists, pathologists, other plant scientists and students.

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An introduction to markers, quantitative trait loci (QTL) mapping and marker-assisted selection for crop improvement: The basic concepts

Importing and simulating data.- Data checking.- Single-QTL analysis.- Non-normal phenotypes.- Experimental design and power.- Working with covariates.- Two-dimensional, two-QTL scans.- Fit and exploration of multiple-QTL models.- Case study I.- Case study II.

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A Guide to QTL Mapping with R/qtl

Diversity Arrays Technology (DArT) can detect and type DNA variation at several hundred genomic loci in parallel without relying on sequence information. Here we show that it can be effectively applied to genetic mapping and diversity analyses of barley, a species with a 5,000-Mbp genome. We tested several complexity reduction methods and selected two that generated the most polymorphic genomic representations. Arrays containing individual fragments from these representations generated DArT fingerprints with a genotype call rate of 98.0% and a scoring reproducibility of at least 99.8%. The fingerprints grouped barley lines according to known genetic relationships. To validate the Mendelian behavior of DArT markers, we constructed a genetic map for a cross between cultivars Steptoe and Morex. Nearly all polymorphic array features could be incorporated into one of seven linkage groups (98.8%). The resulting map comprised ≈385 unique DArT markers and spanned 1,137 centimorgans. A comparison with the restriction fragment length polymorphism-based framework map indicated that the quality of the DArT map was equivalent, if not superior, to that of the framework map. These results highlight the potential of DArT as a generic technique for genome profiling in the context of molecular breeding and genomics.

http://www.pnas.org/content/101/26/9915.full.pdf

Diversity Arrays Technology (DArT) for whole-genome profiling of barley

Although the impact of tumor immunology on the clinical management of most cancers is still negligible, there is increasing evidence that anticancer immune responses may contribute to the control of cancer after conventional chemotherapy. Thus, radiotherapy and some chemotherapeutic agents, in particular anthracyclines, can induce specific immune responses that result either in immunogenic cancer cell death or in immunostimulatory side effects. This anticancer immune response then helps to eliminate residual cancer cells (those that fail to be killed by chemotherapy) or maintains micrometastases in a stage of dormancy. Based on these premises, in this Review we address the question, How may it be possible to ameliorate conventional therapies by stimulating the anticancer immune response? Moreover, we discuss the rationale of clinical trials to evaluate and eventually increase the contribution of antitumor immune responses to the therapeutic management of neoplasia.

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The anticancer immune response: indispensable for therapeutic success?

Map Manager QTX (QTX) is software for analysis of genetic mapping experiments in experimental plants and animals. It includes functions for mapping both Mendelian and quantitative trait loci. QTX is an enhanced version of Map Manager QT, rewritten with the aid of cross-platform libraries (XVT, Boulder Software Foundry, Inc.), which allow it to be compiled for multiple computer platforms. It currently is distributed for Microsoft Windows and Mac OS and is available at http://mapmgr.roswellpark.org/mmQTX.html.

Map Manager QTX, cross-platform software for genetic mapping.

The therapeutic efficacy of a total of 42 single-agent or combination protocols involving doxorubicin (Adriamycin, ADM) and tumor necrosis factor α (TNFα) were evaluated in the syngeneic murine lymphoma model, C57BL/6-EL4. Combination treatments were the most effective and the therapeutic effects were schedule-dependent; e.g. it was generally advantageous for ADM to precede TNFα administration. Two protocols selected for further study were 4 mg/kg ADM i.v. on days 1 and 8 plus TNFα, i.v., at either 16 000 U (7 μg)/injection, on days 1 and 8 or 4000 U (1.7 μg)/injection, on days 11–15. Survival of mice bearing one of four EL4 sublines having different in vitro drug sensitivities was assessed. These sublines were E10 (ADM-sensitive/TNFα-resistant), E16 (sensitive/sensitive), ER2 (ADM-resistant/TNFα-sensitive) and ER13 (resistant/resistant). Between 80% and 100% long-term survivors (i.e. tumor free on day 60) were obtained with the two treatments in mice bearing ADM-sensitive sublines, even though one of these sublines, E10, was resistant to TNFα in vitro. Induction of long-term survival appeared, therefore, to correlate with in vitro defined sensitivity/resistance to ADM, but not to TNFα. Treatment-induced modulations of tumoricidal immune effector functions were also examined. Taken together, the results indicated that induction of long-term survival involved complex interactions of: (1) ADM-induced tumor modifications, including, but not limited to, tumor debulking, (2) combination-treatment-induced modifications of splenic cytolytic T cell and macrophage activities, and (3) the restoration of thymus cellularity. Finally, when long-term survivors resulting from treatment of E10- or E16-bearing mice were implanted with ER2 on day 120, the majority survived, indicating that long-term immune memory, capable of recognizing drug resistant variants, had been established.

Doxorubicin plus tumor necrosis factor α combination treatments in EL4- lymphoma-bearing C57BL/6 mice

As reported previously, cyclophosphamide plus tumor necrosis factor-α treatment of C57BL/6 mice bearing advanced EL4 lymphoma induced approx. 60% long-term (i.e., >60 days) survivors. These mice developed protective immunity, as evidenced by 1) rejection (100% survival) of EL4 tumor re-implanted on day 60 (day 0 = initial tumor implantation); and 2) development of significant levels of specific EL4 tumor cell killing activity by both splenocytes and thymocytes. Using this model, age-related changes in functionally and phenotypically definable thymocyte subsets were assessed. In thymocytes from 90 to 308 day survivors, specific immune memory was long term; both CD4+ and CD8+ cells were required for the ex vivo stimulation of lytic activity, but the specific anti-EL4 cytotoxic effector was CD4−CD8+. On day 520, the surviving mice were randomized into 2 groups. One group received a second re-challenge with EL4 tumor cells and all survived. The other group was sacrificed on day 520. Their thymocytes, exposed to X-irradiated EL4, developed anti-EL4 lytic activity and, in comparison with thymocytes of young and age-matched control mice, were markedly enriched in CD4−CD8+CD44+ cells. On day 625, thymocytes from the survivors of the day 520 re-challenge were evaluated and were found to have developed specific anti-EL4 lytic activity. Phenotypically, they had returned toward the pattern seen in age-matched control mice although CD4−CD8+CD44+ cells remained increased. These mice were ≥2 years old, the median life span of C57BL/6 mice. Thus, mice cured of tumor by an immuno-modulating regimen rejected re-implanted primary tumor and maintained specific thymic anti-tumor immune memory for life. Int. J. Cancer 76:579–586, 1998.© 1998 Wiley-Liss, Inc.

Thymic anti-tumor effectors in mice cured of lymphoma by cyclophosphamide plus TNF-α therapy: Phenotypic and functional characterization up to 20 months after initial tumor inoculation

To investigate cytokine regulation in cells of freshly excised lymphoid tissues, rigorous quantitative reverse transcription/polymerase chain reaction (QRT-PCR) assays were developed to measure attomole (10−18 mol) amounts of the mRNA for seven cytokines: interleukin-1α (IL-1α), IL-1β, tumor necrosis factor α (TNFα), interferon γ (IFNγ), transforming growth factor β (TGFβ), IL-2 and IL-6 RNA was purified from single-cell suspensions of immune tissues (spleen, thymus and resident peritoneal cells) Data are presented demonstrating the utility of these assays for quantifying basal levels of all seven cytokine mRNAs in the freshly isolated splenocytes and thymocytes Studies to establish the usefulness of these assays for measuring changes in the levels of cytokine mRNA focused on IL-1α, IL-1β, TNFα and IL-2 in splenocytes, thymocytes and resident peritoneal cells Using the QRT-PCR assays developed, levels of cytokine mRNA could be quantified in RNA samples obtained both from freshly isolated cells and from cells following short-term (≤26 h) culture These measurements established basal in vivo levels of specific cytokine mRNAs and demonstrated specific modulation of their levels by cell culture and by the inclusion of immune stimulants (bacterial lipopolysaccharide or the plant lectin concanavalin A) in the culture These data provide new information on both basal and stimulated cytokine levels that is required for valid interpretations of the roles of cytokine expression in immune regulation

Measurement of low-abundance cytokine mRNA in cells of murine lymphoid organs: a new quantitative reverse transcription/polymerase chain reaction method.

Jane M. Meer

Papers

Map Manager QTX, cross-platform software for genetic mapping.

Doxorubicin plus tumor necrosis factor α combination treatments in EL4- lymphoma-bearing C57BL/6 mice

Thymic anti-tumor effectors in mice cured of lymphoma by cyclophosphamide plus TNF-α therapy: Phenotypic and functional characterization up to 20 months after initial tumor inoculation

Measurement of low-abundance cytokine mRNA in cells of murine lymphoid organs: a new quantitative reverse transcription/polymerase chain reaction method.