Accelerating wheat breeding for end-use quality with multi-trait genomic predictions incorporating near infrared and nuclear magnetic resonance-derived phenotypes.

KEY MESSAGE: Using NIR and NMR predictions of quality traits overcomes a major barrier for the application of genomic selection to accelerate improvement in grain end-use quality traits of wheat. Grain end-use quality traits are among the most important in wheat breeding. These traits are difficult to breed for, as their assays require flour quantities only obtainable late in the breeding cycle, and are expensive. These traits are therefore an ideal target for genomic selection. However, large reference populations are required for accurate genomic predictions, which are challenging to assemble for these traits for the same reasons they are challenging to breed for. Here, we use predictions of end-use quality derived from near infrared (NIR) or nuclear magnetic resonance (NMR), that require very small amounts of flour, as well as end-use quality measured by industry standard assay in a subset of accessions, in a multi-trait approach for genomic prediction. The NIR and NMR predictions were derived for 19 end-use quality traits in 398 accessions, and were then assayed in 2420 diverse wheat accessions. The accessions were grown out in multiple locations and multiple years, and were genotyped for 51208 SNP. Incorporating NIR and NMR phenotypes in the multi-trait approach increased the accuracy of genomic prediction for most quality traits. The accuracy ranged from 0 to 0.47 before the addition of the NIR/NMR data, while after these data were added, it ranged from 0 to 0.69. Genomic predictions were reasonably robust across locations and years for most traits. Using NIR and NMR predictions of quality traits overcomes a major barrier for the application of genomic selection for grain end-use quality traits in wheat breeding.

Physicochemical and functional characteristics of lentil starch.

The physicochemical properties of lentil starch were measured and linked up with its functional properties and compared with those of corn and potato starches. The amylose content of lentil starch was the highest among these starches. The crystallinity and gelatinization enthalpy of lentil starch were the lowest among these starches. The high amylose: amylopectin ratio in lentil starch resulted into low crystallinity and gelatinization enthalpy. Gelatinization and pasting temperatures of lentil starch were in between those of corn and potato starches. Lentil starch gels showed the highest storage modulus, gel strength and pasting viscosity than corn and potato starch gels. Peleg's model was able to predict the stress relaxation data of these starches well (R(2)>0.98). The elastic modulus of lentil starch gel was less frequency dependent and higher in magnitude at high temperature (60 °C) than at lower temperature (10 °C). Lentil starch is suitable where higher gel strengthened pasting viscosity are desired.

Interfacial and emulsifying properties of lentil protein isolate.

The dynamic interfacial tension (DIFT) at oil-water interface, diffusion coefficients, surface hydrophobicity, zeta potential and emulsifying properties, including emulsion activity index (EAI), emulsion stability index (ESI) and droplet size of lentil protein isolate (LPI), were measured at different pH and LPI concentration, in order to elucidate its emulsifying behaviour. Sodium caseinate (NaCas), whey protein isolate (WPI), bovine serum albumin (BSA) and lysozyme (Lys) were used as benchmark proteins and their emulsifying property was compared with that of LPI. The speed of diffusion-controlled migration of these proteins to the oil/water interface, was in the following order: NaCas>LPI>WPI>BSA>Lys, while their surface hydrophobicity was in the following order: BSA>LPI>NaCas>WPI>Lys. The EAI of emulsions stabilised by the above proteins ranged from 90.3 to 123.3 m(2)/g and it was 93.3 ± 0.2 m(2)/g in LPI-stabilised emulsion. However, the stability of LPI-stabilised emulsions was slightly lower compared to that of WPI and NaCas-stabilised emulsions at the same protein concentration at pH 7.0. The ESI of LPI emulsions improved substantially with decrease in droplet size when protein concentration was increased (20-30 mg/ml). Reduction of disulphide bonds enhanced both the EAI and ESI compared to untreated samples. Heat treatment of LPI dispersions resulted in poor emulsion stability due to molecular aggregation. The stability of LPI-stabilised emulsions was found to decrease in the presence of NaCl. This study showed that LPI can be as effective emulsifiers of oil-in-water emulsions as are WPI and NaCas at ≥20 mg/ml concentrations both at low and neutral pH. The emulsifying property of LPI can be improved by reducing the intra and inter-disulphide bond by using appropriate reducing agents.

Chromosomal loci associated with endosperm hardness in a malting barley cross.

A breeding objective for the malting barley industry is to produce lines with softer, plumper grain containing moderate protein content (9-12%) as they are more likely to imbibe water readily and contain more starch per grain, which in turn produces higher levels of malt extract. In a malting barley mapping population, 'Arapiles' × 'Franklin', the most significant and robust quantitative trait locus (QTL) for endosperm hardness was observed on the short arm of chromosome 1H, across three environments over two growing seasons. This accounted for 22.6% (Horsham 2000), 26.8% (Esperance 2001), and 12.0% (Tarranyurk 2001) of the genetic variance and significantly increased endosperm hardness by 2.06-3.03 SKCS hardness units. Interestingly, Arapiles and Franklin do not vary in Ha locus alleles. Therefore, this region, near the centromere on chromosome 1H, may be of great importance when aiming to manipulate endosperm hardness and malting quality. Interestingly, this region, close to the centromere on chromosome 1H, in our study, aligns with the region of the genome that includes the HvCslF9 and the HvGlb1 genes. Potentially, one or both of these genes could be considered to be candidate genes that influence endosperm hardness in the barley grain. Additional QTLs for endosperm hardness were detected on chromosomes 2H, 3H, 6H and 7H, confirming that the hardness trait in barley is complex and multigenic, similar to many malting quality traits of interest.

HIV expression is induced in dying cells.

Using HeLa cells stably transfected with an HIV-LTR-CAT construct, we demonstrated a peak in CAT induction that occurs in viable (but not necessarily cell-division-competent) cells 24 h following exposure to some cell-killing agents. gamma rays were the only cell-killing agent which did not induce HIV transcription; this can be attributed to the fact that gamma-ray-induced apoptotic death requires functional p53, which is not present in HeLa cells. For all other agents, HIV-LTR induction was dose-dependent and correlated with the amount of cell killing that occurred in the culture. Doses which caused over 99% cell killing induced HIV-LTR transcription maximally, demonstrating that cells that will go on to die by 14 days are the cells expressing HIV-LTR-CAT.

Regulation of thymus PCNA expression is altered in radiation-sensitive wasted mice.

Mice bearing the autosomal recessive mutation 'wasted' (wst/wst) express a disease syndrome characterized by neurologic dysfunction, immunodeficiency, and increased sensitivity to the killing effects of ionizing radiation relative to normal littermates (wst/-) and to parental control mice (BCF1, BALB/c, and C57BL/6). Many of these abnormalities, evident as early as 21 days of age, have been localized to thymic tissues and T-lymphocyte populations. Comparison of two-dimensional gel electrophoresis patterns of proteins from wst/wst and control mouse thymus revealed that an acidic protein with a molecular mass of approximately 30 kDa was consistently expressed at lower levels in wasted mice than in controls. Microsequencing of this protein revealed a sequence of 19 N-terminal amino acids identical to the sequence of murine proliferating cell nuclear antigen (PCNA). Northern blot analyses of PCNA expression in thymus and spleen demonstrated lower accumulation of PCNA-specific transcripts in wasted mice compared with that in controls. Because PCNA expression is associated with cell cycle progression, the percentages of thymic and splenic cells in each stage of the cell cycle were examined; there were no differences in the cell stage distribution of lymphocytes freshly isolated from wasted mice compared with littermate or parental controls. After activation with concanavalin A, however, splenocytes from wst/wst mice showed a lower percentage of cells in S phase compared with that in controls. Southern blots with PCNA probes showed that the PCNA loci from the wasted mice and their normal littermates have the same restriction maps. While differences in polymerase chain reaction (PCR) priming were obtained, these could be attributed to strain-specific differences in mouse PCNA pseudogenes. These results suggest the presence of an alteration in the pathway leading to PCNA expression in radiation-sensitive tissues of wasted mice.

The effects of 5-fluorouracil and doxorubicin on expression of human immunodeficiency virus type 1 long terminal repeat.

Previous work by many groups has documented induction of the human immunodeficiency virus (HIV) long terminal repeat (LTR) following exposure of cells to ultraviolet light and other DNA damaging agents. Our experiments set out to determine the relative activation or repression of the HIV-LTR in response to two classes of chemotherapeutic agents: Doxorubicin is a DNA damage-inducing agent, and 5-fluorouracil has an antimetabolic mode of action. Using HeLa cells stably transfected with a construct in which HIV-LTR drives expression of the chloramphenicol acetyl transferase reporter gene, we demonstrated an up to ten-fold induction following doxorubicin treatment at 24 h post-treatment. This induction was repressed by treatment with salicylic acid, suggesting a role for prostaglandin/cyclo-oxygenase pathways and/or NF-kappa B in the inductive response. Induction by 5-fluorouracil, in contrast, was more modest (two-fold at most) though it was consistently elevated over controls.

Physician factors affecting patient willingness to comply with exercise recommendations.

OBJECTIVE: To evaluate how physician factors such as weight, exercise habits, and humanistic traits could influence patient willingness to comply with exercise recommendations. DESIGN: Survey questionnaire. SETTING: University-based Family Medicine Clinic. PATIENTS: 411 consecutive established patients of the Family Medicine Clinic. MAIN OUTCOME MEASURES: Selected Physician characteristics that patients believed would increase their willingness to comply with exercise recommendations. Results were compared with patient demographics to determine possible effects of physician characteristics on patients acceptance of exercise recommendations. RESULTS: Patients with higher education levels could be positively influenced by a physician being of appropriate weight, a regular exerciser, and a nonsmoker, and enlisting use of other experts, negotiating an exercise program, providing exercise counseling, and being their primary provider. Patients with higher income levels could be positively related to a physician's being of appropriate weight, and a nonsmoker, negotiating an exercise program, and enlisting use of other experts. Female patients could be positively influenced by physicians being well groomed, well dressed, accessible, and good listeners. Patients who regularly exercise could be positively influenced by a physician's appropriate weight and exercise regimen. CONCLUSIONS: Physicians may have a positive impact on patient willingness to comply by prescribing exercise and providing education and detailed guidance for all candidates. The study also showed that physicians' negotiating exercise programs and being good "exercise" role models is very important.

The effects of cisplatin and methotrexate on the expression of human immunodeficiency virus type 1 long terminal repeat.

Previous work by many groups has documented the induction of HIV-LTR (human immunodeficiency virus-long terminal repeat) following exposure of cells or whole animals to ultraviolet (UV) light and other DNA damaging agents. In these experiments we set out to determine whether exposure to the cancer chemotherapeutic agents methotrexate and cisplatin had any effect on the expression of the HIV-LTR. Using HeLa cells stably transfected with a construct in which HIV-LTR drives the expression of the reporter gene chloramphenicol acetyl transferase (CAT), we demonstrated induction of HIV-LTR 24-48 h following exposure to 50 microM cisplatin. When UV exposure (10 Jm-2) was coupled with cisplatin (50 microM) treatment (which also causes DNA damage), HIV-LTR induction was additive relative to either treatment alone. Methotrexate, which depletes the medium of tetrahydrofolate and does not induce DNA damage, induced HIV-LTR at later (6-7 days) time points than cisplatin or UV treatments. When methotrexate (128 microM) and UV (10 Jm-2) treatments were combined, the agents were synergistic with regard to HIV induction. For both drugs, though, induction was not due to generalized transcriptional activation since both cisplatin and methotrexate induced a repression of total transcription as measured in nuclear run-on assays.

The effects of multiple UV exposures on HIV-LTR expression.

Previous studies have shown that cellular stress agents such as UV radiation induce transcription from the long terminal repeat (LTR) of the human immunodeficiency virus (HIV). Using HeLa cells stably transfected with the HIV-LTR sequence, which transcriptionally drives the chloramphenicol acetyl transferase (CAT) reporter gene, we examined the effects of multiple exposures to UVC (254 nm) on HIV-LTR-CAT expression. Low doses (< or = 5 J m-2) had no effect on CAT expression, but up to 29-fold induction was observed with 10 J m-2 when cells were harvested 48 h after completion of the exposure. Little difference was noted in induction levels when cells were exposed to one 25 J m-2 dose, viable cells were harvested at 24 h, 48 h or 72 h, and cell lysates were assayed for CAT expression. Two sequential 12.5 J m-2 exposures, given 24 h apart, resulted in an additive effect on CAT expression; these two exposures produced CAT activity equivalent to that induced following a single 25 J m-2 dose. This additive effect was not evident at the lower doses (< or = 5 J m-2) or at the higher doses. Maximal induction was observed using doses from 25 to 37.5 J m-2. Multiple exposures with either the low (< or = 5 J m-2) or high doses (> 25 J m-2) did not result in an additive effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Salicylic acid inhibits ultraviolet- and cis-platinum-induced human immunodeficiency virus expression.

Previous studies have shown that exposure of HeLa cells stably transfected with an HIV-long terminal repeat-chloramphenicol acetyltransferase (HIV-LTR-CAT) construct to many DNA-damaging agents (such as UV light) induces expression from the HIV LTR. By culturing the cells with salicylic acid we demonstrated dose-dependent repression of this UV-or cis-platinum (cis-Pt)-induced HIV expression. While salicylic acid treatment, indomethacin treatment, UV exposure, or cis-Pt treatment alone decreased viability by up to 50%, equal numbers of viable cells were used for the CAT assays. Repression was evident if salicylic acid was administered 2 h before, at the same time as, or up to 6 h after exposure to the DNA-damaging agent. The kinetics were similar for UV- and for cis-Pt-induced HIV expression, and induction was dependent on the UV dose or cis-Pt concentration added to the culture. pH changes of the media alone in the absence of salicylic acid did not affect HIV expression. Indomethacin (100 microM) did not affect UV- or cis-Pt-induced HIV expression. These results suggest a role for the prostaglandins or the cyclo-oxygenase pathway or both in HIV induction mediated by DNA-damaging agents.

Effects of gamma rays, ultraviolet radiation, sunlight, microwaves and electromagnetic fields on gene expression mediated by human immunodeficiency virus promoter.

Previous work by our group and others has shown the modulation of human immunodeficiency virus (HIV) promoter or long terminal repeat (LTR) after exposure to neutrons and ultraviolet radiations. Using HeLa cells stably transfected with a construct containing the chloramphenicol acetyl transferase (CAT) gene, the transcription of which is mediated by the HIV-LTR, we designed experiments to examine the effects of exposure to different types of radiation (such as gamma rays, ultraviolet and sunlight irradiations, electromagnetic fields and microwaves) on HIV-LTR-driven expression of CAT. These results demonstrated ultraviolet-light-induced transcription from the HIV promoter, as has been shown by others. Exposure to other DNA-damaging agents such as gamma rays and sunlight (with limited exposures) had no significant effect on transcription mediated by HIV-LTR, suggesting that induction of HIV is not mediated by just any type of DNA damage but rather may require specific types of DNA damage. Microwaves did not cause cell killing when cells in culture were exposed in high volumes of medium, and the same cells showed no changes in expression. When microwave exposure was carried out in low volumes of medium (so that excessive heat was generated) induction of HIV-LTR transcription (as assayed by CAT activity) was evident. Electromagnetic field exposures had no effect on expression of HIV-LTR. These results demonstrate that not all types of radiation and not all DNA-damaging agents are capable of inducing HIV. We hypothesize that induction of HIV transcription may be mediated by several different signals after exposure to radiation.

Low doses of neutrons induce changes in gene expression.

Studies were designed to identify genes induced in fibroblasts after exposure to low-dose neutron radiation but not after gamma rays. Our past work had shown similar modulation of transcripts for alpha-tubulin, beta- and gamma-actins, ornithine decarboxylase and interleukin 1 after exposure to either neutrons or gamma rays. However, differences in the expression of beta-protein kinase C and c-fos genes were observed, with both being induced after exposure to gamma rays but not neutrons. Recently we have identified two genes that are induced after exposure to neutrons but not gamma rays: Rp-8 (a gene associated with apoptosis) and the long terminal repeat (LTR) of the human immunodeficiency virus (HIV). Induction of Rp-8 mRNA was demonstrated in Syrian hamster embryo (SHE) fibroblasts and was found to be induced in cells exposed to neutrons administered at low (0.005 Gy/min) and high dose rate (0.12 Gy/min). No induction of other genes associated with apoptosis such as Rp-2, bcl-2 and Tcl-30 was observed. The induction of transcription from the LTR of HIV was demonstrated in HeLa cells bearing a transfected construct of the chloramphenicol acetyl transferase (CAT) gene driven by the HIV-LTR promoter. Measurements of CAT activity and CAT transcripts after irradiation demonstrated an unresponsiveness to gamma rays over a broad range of doses (0.1-3 Gy). Twofold induction of the HIV-LTR was detected after exposure to neutrons (0.48 Gy) administered at low (0.05 Gy/min) but not high (0.12 Gy/min) dose rates. Ultraviolet-mediated HIV-LTR induction, however, was inhibited by exposure to low-dose-rate neutron irradiation. These results are interesting in light of reports that Rp-8 is induced during apoptosis and that HIV causes apoptosis.

Modulation of expression of virus-like elements following exposure of mice to high- and low-LET radiations.

Modulation of virus expression has been reported following exposure to a variety of cellular stresses, including UV radiation and heat-shock. The experiments reported here were designed to examine expression of endogenous VL30 (virus-like 30 S) elements following exposure of whole mice to ionizing radiations. Whole mice were exposed to doses of neutrons (50 cGy) or gamma-rays (300 cGy) shown to be equally efficient in cancer production in the whole animal, and tissues were harvested at 10 and 60 min following completion of the exposure. RNA extracted from these tissues and from tissues of untreated controls was examined for VL30 RNA accumulation by dilution dot blot and Northern blot analyses. These studies revealed that neutrons repressed VL30 RNA accumulation evident within 10 min following exposure in brain, gut, thymus and spleen but not in liver, in which VL30 RNA was unaffected by radiation exposure. During this same time interval, gamma-rays induced VL30 expression in gut and brain and to a lesser extent in liver. These experiments suggest the presence of a differential molecular response following whole-body exposure to high- versus low-LET radiations. In addition, this work demonstrates that ionizing radiations may affect expression of murine endogenous viral sequences.