Implementation of two high through-put techniques in a novel application: detecting point mutations in large EMS mutated plant populations

Background  

The establishment of mutant populations together with the strategies for targeted mutation detection has been applied successfully to a large number of organisms including many species in the plant kingdom. Considerable efforts have been invested into research on tomato as a model for berry-fruit plants. With the progress of the tomato sequencing project, reverse genetics becomes an obvious and achievable goal.     

Utilization of nitrogen and energy by Holstein steers fed formaldehyde- and formic acid-treated alfalfa or orchardgrass silage at two intakes

Our objective was to measure the utilization of energy (E) and N by Holstein steers when fed alfalfa and orchardgrass silages offered at 65 and 90 g DM/kg live weight (LW).75 daily. Twelve steers adapted to the Beltsville respiration chambers were assigned to three Latin squares with 42-d periods. Steers in one square had permanent catheters in the portal and two mesenteric veins. Energy and N balance were measured during a 7-d collection of feces and urine that included a 3-d measurement of respiratory exchange. Energy and N variables were not different between catheterized and uncatheterized steers. Apparent digestibilities of DM, OM, CP, neutral detergent solubles and permanganate lignin were higher (P less than .01) and digestibilities of NDF and ADF, hemicellulose and cellulose were lower (P less than .01) for steers consuming alfalfa compared to orchardgrass silage. When fed alfalfa, steers' daily gross energy intake, DE, urine energy, ME, heat production and tissue energy retention were greater (P less than .01) and fecal energy losses were less (P less than .01) than when they were fed orchardgrass. Partial efficiency of ME use for tissue energy (TE) was greater (P less than .01) for steers when fed alfalfa (46.1%) than when fed orchardgrass (35.6%). Apparent ME (kcal/LW.75) required for maintenance of TE was similar for steers when fed alfalfa (133.9) and orchardgrass (131.2) silages. Nitrogen retention (g/d) was 48% greater (P less than .01) for steers when fed alfalfa (30.6) than when fed orchardgrass (20.7). This study demonstrates that steers used ME from alfalfa more efficiently for TE deposition than ME from orchardgrass.     

Early Pregnancy Diagnosis by Serum Progesterone and Ultrasound in Sheep Carrying Somatic Cell Nuclear Transfer-Derived Pregnancies

Early pregnancy diagnosis and monitoring play an important role following embryo transfer in sheep. The aims of the current study were to investigate (i) the pattern of serum progesterone profiles in sheep carrying somatic cell nuclear transfer (SCNT)‐derived (clone) pregnancies, and (ii) the frequency of pregnancy loss during development following SCNT embryo transfer. Sheep SCNT embryos were made using standard nuclear transfer techniques. Day 7 embryos were surgically transferred to oestrus‐synchronized recipients (n = 27). As a control, normal fertile ewes (n = 12) were bred by natural breeding. Serum was collected from all the ewes on the day of estrus (day 0 sample), 7 days post‐estrus (day 7 sample) and 19 days post‐estrus (day 19 sample) and every 10 days thereafter until lambing or pregnancy loss occurred. Serum progesterone (P4) was assessed using enzyme immunoassay. Pregnancy was confirmed by ultrasound scanning on day 35 of pregnancy followed by subsequent scanning every 10 days. In control ewes, pregnancy rate on day 35 was 83.3% (10/12), whereas in the ewes that received SCNT embryos, it was 22.2% (6/27; p < 0.05). The day 45 pregnancy rate in the control ewes was 83.3%, whereas in the SCNT embryo recipients it was 11.0% (p < 0.05). Hormone analysis revealed that SCNT embryo recipients exhibited a significantly lower P4 profiles at different time points in pregnancy compared to controls (p < 0.05). This study highlights the use of serum progesterone in combination with ultrasound for the investigation of embryo loss and crucial times during development of normal and SCNT embryos in sheep. Further, the serum P4 levels directly reflect the degree of placental development in these two groups.     

Seasonal and dietary effects on Vitamin D deficiencies detected in wild boar from mid‐western Spain

Vitamin D (VitD) is involved in important mammalian physiological mechanisms, such as Ca–P metabolism, bone development and immunological response. VitD deficiencies are frequently detected in domestic animals and related to various health problems (e.g., rickets, bone deformation). However, knowledge about the status of VitD in wildlife species, such as the wild boar, is scarce. The aims of this work were to explore VitD status in wild boar populations from mid‐western Spain and to elucidate the influence of daylight exposure and food supplementation in levels of VitD. Serum concentration of VitD (measured as 25‐hydroxivitaminD) was assessed in 276 wild boar from 27 game estates located in mid‐western Spain using a commercial ELISA kit. In 19 out of 27 estates, the staff supplied a specific VitD‐enriched food (2,000 UI/Kg) ad libitum throughout the year, while in the remaining estates (8), no food was supplied. Blood samples were extracted from hunted animals (198) between October and February of hunting seasons 2016/2017 and 2017/2018, and from live wild boar (78) that were captured, sampled and released (March–September of 2017). The percentage of animals with VitD deficiency (<20 ng/ml), VitD insufficiency (20–30 ng/ml) and VitD sufficiency (>30 ng/ml) was estimated, and the relationship of these levels to factors like sex, age and season was assessed using chi‐square tests. Furthermore, associations between daylight exposure and supplemental food with VitD levels were explored using linear models. Of the studied wild boar population, 82.2% showed a VitD deficiency or insufficiency. VitD deficiencies were more frequent in animals sampled in winter and spring. Furthermore, levels of VitD positively correlated with daylight exposure and supplemental food intake. Ad libitum supplementation with VitD‐enriched food was insufficient to prevent VitD deficiencies in wild boar from November to April, probably because food consumption is lower during this period.     

Modelling soil-water-plant relationships in the Cerrado soils of Brazil: The case of maize (Zea mays L.)

A soil-water balance simulation model developed for the Cerrado soils of central Brazil is presented. The model calculates daily soil water evaporation, plant transpiration and soil-water balance for fourteen soil layers of 15 mm each. The model includes a subroutine to calculate capillary water movement. Computer simulations of daily soil water levels at five soil depths (15, 30, 45, 60 and 90 cm) for a field of maize are compared with actual field measurements over an 80-day period. Results indicated that the developed model can, in general, estimate the soil-water balance of the various depths within ± 10% of actual measurements.     

High throughput phenotyping for aphid resistance in large plant collections

ABSTRACT: BACKGROUND: Phloem-feeding insects are among the most devastating pests worldwide. They not only cause damage by feeding from the phloem, thereby depleting the plant from photo-assimilates, but also by vectoring viruses. Until now, the main way to prevent such problems is the frequent use of insecticides. Applying resistant varieties would be a more environmental friendly and sustainable solution. For this, resistant sources need to be identified first. Up to now there were no methods suitable for high throughput phenotyping of plant germplasm to identify sources of resistance towards phloem-feeding insects. RESULTS: In this paper we present a high throughput screening system to identify plants with an increased resistance against aphids. Its versatility is demonstrated using an Arabidopsis thaliana activation tag mutant line collection. This system consists of the green peach aphid Myzus persicae (Sulzer) and the circulative virus Turnip yellows virus (TuYV). In an initial screening, with one plant representing one mutant line, 13 virus-free mutant lines were identified by ELISA. Using seeds produced from these lines, the putative candidates were re-evaluated and characterized, resulting in nine lines with increased resistance towards the aphid. CONCLUSIONS: This M. persicae-TuYV screening system is an efficient, reliable and quick procedure to identify among thousands of mutated lines those resistant to aphids. In our study, nine mutant lines with increased resistance against the aphid were selected among 5160 mutant lines in just 5 months by one person. The system can be extended to other phloem-feeding insects and circulative viruses to identify insect resistant sources from several collections, including for example genebanks and artificially prepared mutant collections.