Genetic loci linking improved heat tolerance in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) to lower leaf and spike temperatures under controlled conditions

Heat stress adversely affects wheat production in many regions of the world and is particularly detrimental during reproductive development. The objective of this study was to identify novel quantitative trait loci (QTL) associated with improved heat tolerance in wheat (Triticum aestivum L.) and to confirm previous QTL results. To accomplish this, a recombinant inbred line (RIL) population was subjected to a three-day 38°C daytime heat stress treatment during early grain-filling. At maturity, a heat susceptibility index (HSI) was calculated from the reduction of three main spike yield components; kernel number, total kernel weight, and single kernel weight. The HSI, as well as temperature depression (TD) of the main spike and main flag leaf during heat stress were used as phenotypic measures of heat tolerance. QTL analysis identified 14 QTL for HSI, with individual QTL explaining from 4.5 to 19.3% of the phenotypic variance. Seven of these QTL co-localized for both TD and HSI. At all seven loci, the allele for a cooler flag leaf or spike temperature (up to 0.81°C) was associated with greater heat tolerance, indicated by a lower HSI. In a comparison to previous QTL results in a RIL population utilizing the same source of heat tolerance, seven genome regions for heat tolerance were consistently detected across populations. The genetic effect of combining three of these QTL, located on chromosomes 1B, 5A, and 6D, demonstrate the potential benefit of selecting for multiple heat tolerance alleles simultaneously. The genome regions identified in this study serve as potential target regions for fine-mapping and development of molecular markers for more rapid development of heat tolerant germplasm.     

Insecticidal activity of Arum maculatum tuber lectin and its binding to the glycosylated insect gut receptors

Mannose binding approximately 50 kDa homotetrameric lectin, purified from edible Arum maculatum tuber, was analyzed through SDS-PAGE and studied for its agglutination property using rabbit erythrocytes. Cross reactivity of the purified lectin was verified through western blot using Colocasia esculantum(Family, Araceae) tuber lectin antibody. The insecticidal activity of Arum maculatum tuber lectin (ATL) was tested against two economically important sucking pests, Lipaphis erysimi and Aphis craccivora, in an artificial diet. The LC(50) values for L. erysimi and A. craccivora were determined to be 21 microg/mL and 16 microg/mL, respectively. Addition of alpha-d-mannose in ATL-supplemented diet reduced the aphid mortality. Two major receptor proteins of ATL (approximately 40 kDa and approximately 35 kDa) were detected from the brush border membrane vesicle (BBMV) protein of L. erysimi and A. craccivora guts, respectively, using ligand-binding assay. Alpha-d-Mannose was found to be a deterrent to such binding of ATL to the BBMV receptors.     

Nodule-specific expression of Rhizobium meliloti symbiotic promoters P1 and P2 in chickpea-Rhizobium sp. symbiosis

Developmentally specific expression of Rhizobium spp. genes involved in symbiotic N₂ fixation is known to operate through cascade regulation of various nif and fix operons. Fusion constructs of lacZ under symbiotic promoters P1 (for nifHDK operon) and P2 (for fixABCX operon) of Rhizobium meliloti were mobilized into Rhizobium spp. (Cicer) strains Rcd301 and RCR13. The assays for -galactosidase activity to monitor the expression of lacZ under these promoters was performed in host backgrounds of Escherichia coli, R. meliloti, and Rhizobium spp. (Cicer). The enzyme assays indicated significant levels of expression from P1 and P2 promoters in chickpea rhizobia, specifically in symbiotic cells from nodules. However, as in R. meliloti, these promoters did not induce strong expression in free-living cells of Rhizobium spp. (Cicer). This indicates functional homology of R. meliloti promoters in rhizobium spp. (Cicer). Functional cross-reactivity of trans regulatory factors like NtrA, NtrC, and NifA between these rhizobia seems evident from the nodule-specific expression of P1 and P2 cis elements.     

Development of Empirical Formulae to Determine Emission Rate from Various Opencast Coal Mining Operations

All major mining activities, particularly opencast coal mining,directly or indirectly contribute to the problem of air pollution.Therefore, air quality assessment and prediction arerequired to prevent and minimize the air quality deteriorationdue to various opencast coal mining operations. Determination ofemission rates for these activities is the first and foremostconcern. In view of the above, this study was undertaken to determinate emission rates and to develop empericalformulae to calculate emission rates of various opencast coalmining activities.To achieve the objectives, seven coal mining sites were selectedto generate site-specific emission data by considering miningpractices, method of working, geographical location,accessibility and above all resourceavailability. The study covers various mining activities andlocations including drilling, overburden loading and unloading,coal loading and unloading, the coal handling plant, the exposedoverburden dump, the stock yard, the workshop, the exposed pit surface roadsand haul roads.Based on the study, a set of twelve emperical formulae have beendeveloped for calculation of suspended particulate matter (SPM)emission rates from various opencast coal mining activities. Theemission of gaseous pollutants (sulphur dioxide and nitrogenoxide) has been found negligible for various mining activities.Therefore, observation for gaseous pollutants has been consideredfor overall mine activities and empirical formulae have subsequently beendeveloped.The developed empirical formulae were calculatedby a field study at another coal mine. The measured andcalculated values of emission rate were compared for eachactivity. Average correlation between the measured andcalculated values for different activities was estimated to be 85.6–99.9%, which indicates fairly good accuracy.Validation of the study was also carried out by means of thefugitive dust model (FDM) using the calculated emissionrate data from the empirical formulae for each mining activity of amine, meteorological data and other details as input.The average accuracy between measured and predictedvalues of concentration of SPM at certain receptor locations wasfound to be 79%. A user-friendly emission software called`EmissCalc' was developed to calculate emission rate, whichcan be used as input for different air quality models.     

Identification of quantitative trait loci (QTLs) associated with maintenance of wheat (Triticum aestivum Desf.) quality characteristics under heat stress conditions

The goal of this study was to identify quantitative trait loci (QTLs) associated with the maintenance of wheat grain quality following post-anthesis heat stress in a recombinant inbred line (RIL) population. The response to heat stress was measured using the sodium dodecyl sulfate sedimentation test (SDSS), a significant predictor of bread baking quality. SDSS scores were used to identify QTLs associated with grain quality and QTLs associated with quality stability were identified based on percent change in SDSS score between the heat stress and control treatments. Four QTLs were identified, located one each on linkage groups 1B, 1D, 4A, and 7A. The 1B, 1D, and 4A QTLs were associated with grain quality; the QTL on linkage group 7A was associated with quality stability. To confirm the detected QTLs, eighty advanced lines grown at three Texas nurseries were tested for relationships between allelic polymorphism at QTL linked markers and quality traits. Quality trait stability in the advanced lines was estimated using the coefficient of variability (CV%) of quality traits between nurseries. The analysis supported the relationship of the predicted QTLs on linkage groups 1B, 1D, and 4A with quality traits. The confirmed QTLs may be used in marker assisted selection (MAS) to develop wheat lines possessing superior quality traits. In addition, identification of genetic regions associated with this trait will aid the identification of the underlying genes.     

Prevalence,molecular fingerprinting and drug resistance profile of enterovirulent <Emphasis Type="Italic">Escherichia coli</Emphasis> isolates from free-ranging yaks of Tawang district,Arunachal Pradesh,India

Of 273 samples (rectal swab) collected from free-ranging yaks of Tawang district, Arunachal Pradesh, 42 Shiga toxin-producing Escherichia coli (STEC), six enteropathogenic E. coli (EPEC) and 27 enterotoxigenic E. coli (ETEC) strains were isolated. All the STEC and EPEC strains were further investigated for respective stx variants (for STEC only) and additional putative virulence factors. The 27 ETEC strains were also screened for characteristic enterotoxin gene(s) and colonization factors. Occurrence of ETEC was significantly (p < 0.05) higher in the diarrheic yaks and yaks of less than 1 year of age. Majority of enterovirulent E. coli isolates were resistant to amikacin, azithromycin, chloramphenicol, colistin, doxycycline, furazolidone, nalidixic acid, nitrofurantoin, streptomycin and tetracycline. Dendrogram, constructed with molecular fingerprinting profiles obtained from RAPD (Randomly Amplified Polymorphic DNA) and ERIC (Enterobacterial Repetitive Intergenic Consensus) PCR, placed the isolates in different clusters irrespective of their serotypes, virulence gene and drug resistance pattern. Collectively, the study indicates that yaks, being a potential reservoir of multidrug resistant STEC and EPEC, may represent significant risk to public health in this region. Higher recovery of ETEC isolates from yaks with diarrhea points out that ETEC may be a major determinant for repeated occurrence of diarrhea in yaks.     

GnRH-1 mRNA, LH surges, steroid hormones, egg production, and intersequence pause days alter in birds exposed to longer wavelength of light in the later stages of production in Gallus gallus domesticus

The objective of this was to establish the effects of red spectrum of light (650 nm, treated n = 12) and normal spectrum of light (450 nm control = 12) on GnRH-I mRNA expression, amplitude and frequency of luteinizing hormone (LH), and egg production from 72-82 weeks of age in white leghorn hens. Birds exposed to red spectrum of wavelength significantly improved (P < 0.01) steroid hormone, and egg production improved over old laying 72 to 82 weeks. Weekly interval profiles followed the same pattern. At 77th weeks of age blood, samples from both the groups were collected at every 3 h for 36 h to study the pulsatile secretion of LH surges. Plasma LH concentration was higher (P < 0.01) in treated birds with more number of frequencies and amplitude LH surges in plasma of treated birds. LH frequencies were more pronounced and advanced during 36 h of sampling at 3 h interval in treated birds. Weekly interval of plasma LH, E2β, and P(4) concentrations increased (P < 0.01) in treated birds from 72 to 82 weeks of age. GnRH-I mRNA concentration was significantly (P < 0.01) higher in birds exposed to red spectrum of light compared to controls. It is hypothesized that exposure of birds to red spectrum of light-enhanced (P < 0.01) GnRH-I mRNA with more number of yellow yolky follicles was found in birds exposed to red spectrum of light during 77 days (72-82 weeks of age) of experimental period. It is concluded that higher levels of GnRH-I mRNA, LH, E2β, and P(4) concentration with lower incidence of pause days enabled the birds to lay more eggs even later in the productive period by modulating the wavelengths of light under normal husbandry conditions.     

Comparison of three enzyme-linked immunosorbant assays with the indirect immunofluorescent antibody test for the diagnosis of canine infection with Ehrlichia canis

The aim of this study was to compare three different enzyme-linked immunosorbant assays (recombinant major antigenic protein 2 (rMAP2)-ELISA, the Immunocomb (Biogal, Israel) and the Snap 3Dx assay (IDEXX Laboratories Inc., USA)) with the indirect immunofluorescent antibody test in detecting anti-Ehrlichia canis immunoglobulin-G (IgG) antibodies. Samples tested were collected from dogs suspected to be naturally infected with E. canis and from experimentally infected dogs.When qualitative results (positive/negative) were compared, there was an overall agreement of 81% (54/67) between the indirect immunofluorescence antibody (IFA) test and the rMAP2-ELISA. An overall agreement of 94% (63/67) was found between the IFA test and the Immunocomb, and an overall agreement of 91% (61/67) was found between the IFA test and the Snap 3Dx assay. In 50 of 67 (74.6%) samples tested, complete agreement in the qualitative results was found in all four tests. Sixteen of 17 samples with disagreement in the qualitative results were found to have IFA titers of 1:320 or less. The sensitivities and specificities of the tests were found to be 0.71 and 0.85 for the rMAP2-ELISA, 0.86 and 0.98 for the Immunocomb, and 0.71 and 1.00 for the Snap 3Dx assay.The tests performed in this study were found to be highly specific in detecting E. canis antibodies. Their sensitivity was found to be low with sera having IFA titers of < or =1:320, while high with sera having titers greater than 1:320. Repeating the serological tests 1-2 weeks after the first antibody assay may overcome the sensitivity problem with titers of < or =1:320.     

Effect of concentration and addition method of glycerol on the quality of cryopreserved mithun (Bos frontalis) spermatozoa

The effect of concentration and addition method of glycerol on the quality of cryopreserved mithun (Bos frontalis) spermatozoa was investigated. Semen samples were collected from five healthy mithun bulls through rectal massage method and cryopreserved in liquid nitrogen. The samples were diluted in Tris–egg yolk–glycerol extender, equilibrated for 4 h at 4 °C and loaded into 0.50‐ml straws. The straws were then frozen in liquid nitrogen vapour for 10 min and finally plunged into liquid nitrogen for storage. The required amount of glycerol was added into the diluted samples either in a single dose (3%, 4%, 5%, 6% or 7%; added at 37 °C immediately before equilibration) or in split doses (5%, 6% or 7%; the total amount was divided into four equal parts, and a part was added at 37 °C immediately before equilibration, and the remaining parts were added subsequently at 1, 2 and 3 h of equilibration at 4 °C). In the single‐dose addition method, following freeze‐thawing, greater (p < 0.05) motility (%) and proportion of live spermatozoa with intact acrosome (LSIA, %) in 5% glycerol (40.6 ± 1.7 and 43.4 ± 1.8 respectively) and lesser (p < 0.05) total morphological abnormalities (%) in 5% (14.1 ± 0.8) and 6% (13.7 ± 1.0) glycerol were observed compared to the other glycerol concentrations. In the split‐dose addition method, following freeze‐thawing, greater (p < 0.05) motility (%) and LSIA proportion (%) were found in 5% (50.2 ± 1.9 and 53.3 ± 1.8 respectively) compared to 6% or 7% glycerol, but the total morphological abnormalities were not different among the glycerol concentrations. In addition, in all the glycerol concentrations, better (p < 0.05) post‐freeze‐thaw motility and LSIA proportions were observed when glycerol was added in split doses compared to a single dose. In conclusion, Tris–egg yolk extender with 5% glycerol added in split doses was found most suitable for cryopreserving mithun sperm.     

Application of unmanned aerial systems for high throughput phenotyping of large wheat breeding nurseries

Background

Low cost unmanned aerial systems (UAS) have great potential for rapid proximal measurements of plants in agriculture. In the context of plant breeding and genetics, current approaches for phenotyping a large number of breeding lines under field conditions require substantial investments in time, cost, and labor. For field-based high-throughput phenotyping (HTP), UAS platforms can provide high-resolution measurements for small plot research, while enabling the rapid assessment of tens-of-thousands of field plots. The objective of this study was to complete a baseline assessment of the utility of UAS in assessment field trials as commonly implemented in wheat breeding programs. We developed a semi-automated image-processing pipeline to extract plot level data from UAS imagery. The image dataset was processed using a photogrammetric pipeline based on image orientation and radiometric calibration to produce orthomosaic images. We also examined the relationships between vegetation indices (VIs) extracted from high spatial resolution multispectral imagery collected with two different UAS systems (eBee Ag carrying MultiSpec 4C camera, and IRIS+ quadcopter carrying modified NIR Canon S100) and ground truth spectral data from hand-held spectroradiometer.

Results

We found good correlation between the VIs obtained from UAS platforms and ground-truth measurements and observed high broad-sense heritability for VIs. We determined radiometric calibration methods developed for satellite imagery significantly improved the precision of VIs from the UAS. We observed VIs extracted from calibrated images of Canon S100 had a significantly higher correlation to the spectroradiometer (r = 0.76) than VIs from the MultiSpec 4C camera (r = 0.64). Their correlation to spectroradiometer readings was as high as or higher than repeated measurements with the spectroradiometer per se.

Conclusion

The approaches described here for UAS imaging and extraction of proximal sensing data enable collection of HTP measurements on the scale and with the precision needed for powerful selection tools in plant breeding. Low-cost UAS platforms have great potential for use as a selection tool in plant breeding programs. In the scope of tools development, the pipeline developed in this study can be effectively employed for other UAS and also other crops planted in breeding nurseries.