A molecular cytogenetic study of intergenomic recombination and introgression of chromosomal segments in lilies (Lilium)

Lilies (Lilium L.) are one of the most important ornamental bulbous crops for cut flower industry grown extensively in The Netherlands for last few decades. The genus Lilium consists of seven different sections with about 80 species. The species within genus Lilium (2n = 2x = 24) comprise a range of desirable and complementary characters. Besides being an important horticultural crop, lily (Lilium) also serves as an interesting model plant for molecular cytogenetic research and introgression breeding for several reasons like, i). Lily is a model crop for interspecific hybridization and it includes plants of different taxonomic species each of which possess valuable horticultural traits that need to be combined in the new cultivars. ii) Through careful selection n and 2n gametes can be obtained in interspecific hybrids. iii) The genomes of different species are so well differentiated genetically that the parental chromosomes can be clearly identified in the F1 hybrids as well as in the progenies through DNA in situ hybridization techniques. iv) The chromosomes are large enough and the number and position of homoeologous recombination sites can be clearly detected. v) The large and easily identified chromosomes in different lily species could be a potential source for the cytological mapping of the Lilium genomes. Taking advantage of these favourable attributes of lily, a molecular cytogenetic investigation was conducted to evaluate the amount of recombination and introgression of characters between Longiflorum - Asiatic (LA) and Oriental - Asiatic (OA) hybrids through the use of n and 2n gametes. For this purpose different F1 Longiflorum × Asiatic (LA) and Oriental × Asiatic (OA) hybrids were backcrossed with different Asiatic cultivars. Ovule and embryo rescue techniques were employed to get backcross (BC) progenies. Most of the F1 LA appeared to be sterile but some hybrids were able to produce only 2n gametes in considerable frequencies. However, in rare occasions it was also found that normal meiosis took place in few of the LA hybrids which resulted into the formation of normal n gametes. Ploidy level and intergenomic recombination was studied in LA interspecific hybrids in order to assess the possibility of functional n gametes and their potential use in introgression at diploid level in lily. A total of 104 BC1 LA interspecific lily hybrids were obtained, 27 diploids (2n = 2x = 24), 73 triploids (2n = 2x = 36) and 4 aneuploids (2x – 1, 2x + 2 or 2x + 3). Similarly, triploid BC1 (LAA) plants were backcrossed to diploid Asiatic parents. As a result 14 diploid BC2 progenies were produced. The intergenomic recombination and amount of introgression of respective genome (L and A) was assessed in these diploid genotypes through GISH (Genomic in situ Hybridization). Extensive intergenomic recombination was found among the chromosomes in LA hybrids. A large of amount of L- genome was transmitted from F1 LA hybrids to their subsequent BC1 progenies. However, very few segments of L- genome were introgressed from the BC1 diploid and triploid (LAA) plants to the BC2 progenies (Chapter 2). GISH identifies a considerable amount of recombination events amongst different interspecific lily hybrids (LA and OA) obtained from functional 2n gametes. Based on recombination sites on different chromosomes identified by GISH, cytological maps of three genomes of Lilium were constructed. For this purpose, BC progenies of two diploid interspecific hybrids of lily, viz., Longiflorum × Asiatic (LA) and Oriental × Asiatic (OA) were used. The BC progenies of LA hybrids consisted of both triploid (2n = 3x = 36) and diploid (2n = 2x = 24) with some aneuploid genotypes and those of OA hybrids mostly consisted of triploid (2n = 3x = 36) and some aneuploid genotypes. In LA hybrids 248 recombination sites were cytologically localized on 12 different chromosomes of each genomes (i.e., L and A). Similarly, 116 recombinant sites were marked on 12 chromosomes each from the BC progenies of OA hybrids (O and A genomes). The distances of the recombination sites from the centromeres are measured (in micrometres). Based on these recombination sites four cytological maps were constructed. Since an Asiatic parent was involved in both hybrids, viz., LA and OA, two maps were constructed for A genome which were indicated as Asiatic (L) and Asiatic (O) and one each for Longiflorum (A) and Oriental (A) genomes (Chapter 3). With a view to generate genetic variation via homoeologous recombination in BC progenies of LA and OA hybrids the most logical approach was the use of 2n gametes. 63 BC1 LA (LA × AA or AA × LA) and 53 OA (AA × OA) progeny plants were obtained after unilateral sexual polyploidization. 16 genotypes from F2 LA populations were obtained after bilateral sexual polyploidization through sib-mating of F1 LA hybrids. GISH was employed for the identification of the parental genomes, mode of origin of these progenies and measurement of the introgression in different interspecific lily hybrids. Most of the BC1 progeny plants (LA and OA) had originated through 2n gametes by First Division Restitution (FDR) mechanism. However, there were 12 genotypes in LA hybrids and four genotypes in OA hybrids that originated through 2n gametes formation as the result of Indeterminate Meiotic Restitution (IMR). A higher amount of recombination was found in LA hybrids as compared to OA hybrids. Intergenomic recombination was also determined in the sib-mated F2 LA population. In this case both parents had contributed gametes with the somatic number of chromosomes (i.e., 2n-2n) thus confirming the event of bilateral sexual polyploidization in interspecific LA hybrids. Based on these results, the relevance of interspecific lily hybrids obtained from uni- and bilateral sexual polyploidization leading to allotriploid and allotetraploid formation in interspecific lily hybrids is discussed in the context of introgression and mapping (Chapter 4). Molecular markers are an important tool for the construction of genetic linkage maps, as the first step in the genetic dissection of the required traits leading to crop improvement followed by the marker assisted breeding in different plants. Lilium has one of the largest genome in plant kingdom and genetic mapping in lilies is constrained by its large genome. DArT (Diversity Array Technology), a molecular marker technique can detect and type DNA variation at several hundred genomic loci in parallel without relying on genome sequence information. The DArT technique was developed for Longiflorum × Asiatic (LA) lily hybrids to enable an efficient and effective genetic mapping with the production of a large numbers of markers in microarrays-based assay. The restriction enzyme PstI + TaqI combination generated the largest frequency of polymorphic genomic representations for a genotyping array. Genomic representations from 88 F1 LA plants were used to assemble a DArT genotyping microarray. A total of 687 DArT markers were developed and 382 polymorphic markers were mapped on 14 main linkage groups which is two more then the haploid chromosome number (i.e. n = 12). The resulting linkage map with 382 DArT markers spanned 1329 cM (3.5 cM/marker on average). The results highlighted the potential of DArT as a genetic technique for genome profiling in the context of molecular breeding and genomics, especially in crops with large genome sizes where other techniques proved to be less useful (Chapter 5). The results of the present investigation are of practical implication. These results show the advantages of the n gametes and their subsequent progenies which opened a new approach of lily breeding ‘the analytic breeding’ in the allopolyploids. It also shows the possibility of using certain triploid hybrids for further breeding. A comparison has been made between different types of interspecific crosses, the amount of intergenomic recombination and introgressions of chromosomal segments obtained after unilateral sexual polyploidization. Furthermore, bilateral sexually polyploidization via sib-mated F1 hybrids producing 2n gametes has been proven. The use of allotetraploids obtained from bilateral sexual polyploidization could be a novel approach in the breeding of LA-hybrids. These allotetraploid with recombinant chromosomal segment may be a potential source to generate genetic variation in subsequent progenies. The molecular cytogenetic GISH and FISH techniques proved to be a powerful tool useful for the construction of cytogenetic maps in interspecific crosses in crops with large genomes sizes like lily. These techniques are also used for the identification and integration of genetic maps with chromosome maps. FISH also helps to monitor the introgressed chromosome segment or marker of interest in the subsequent progenies. Application of the DArT technique proved to be an effective method to construct genetic linkage maps especially crops (like Lilium) with large genome sizes where other techniques might be less useful. <br/

SIMPLE: Stable Increased-throughput Multi-hop Protocol for Link Efficiency in Wireless Body Area Networks

In this work, we propose a reliable, power efficient and high throughput routing protocol for Wireless Body Area Networks (WBANs). We use multi-hop topology to achieve minimum energy consumption and longer network lifetime. We propose a cost function to select parent node or forwarder. Proposed cost function selects a parent node which has high residual energy and minimum distance to sink. Residual energy parameter balances the energy consumption among the sensor nodes while distance parameter ensures successful packet delivery to sink. Simulation results show that our proposed protocol maximize the network stability period and nodes stay alive for longer period. Longer stability period contributes high packet delivery to sink which is major interest for continuous patient monitoring.Comment: IEEE 8th International Conference on Broadband and Wireless Computing, Communication and Applications (BWCCA'13), Compiegne, Franc

M-GEAR: Gateway-Based Energy-Aware Multi-Hop Routing Protocol for WSNs

In this research work, we advise gateway based energy-efficient routing protocol (M-GEAR) for Wireless Sensor Networks (WSNs). We divide the sensor nodes into four logical regions on the basis of their location in the sensing field. We install Base Station (BS) out of the sensing area and a gateway node at the centre of the sensing area. If the distance of a sensor node from BS or gateway is less than predefined distance threshold, the node uses direct communication. We divide the rest of nodes into two equal regions whose distance is beyond the threshold distance. We select cluster heads (CHs)in each region which are independent of the other region. These CHs are selected on the basis of a probability. We compare performance of our protocol with LEACH (Low Energy Adaptive Clustering Hierarchy). Performance analysis and compared statistic results show that our proposed protocol perform well in terms of energy consumption and network lifetime.Comment: IEEE 8th International Conference on Broadband and Wireless Computing, Communication and Applications (BWCCA'13), Compiegne, Franc

Management of intercondylar femur fracture with distal femur locking compression plate: outcome analysis of 72 cases

Background: Supra-condylar and inter-condylar fractures of the distal femur account for 7% of all femoral fractures and have always been difficult to treat and regaining full knee function is often difficult. The purpose of this study is to evaluate the functional outcome, fracture healing, complications of distal femoral intercondylar fractures managed by locking compression plate.Methods: Total 72 patients of intercondylar femur fracture were operated by ORIF with distal femur-locking compression plate via the standard swashbuckler approach. The functional outcomes were analyzed using modified hospital for special surgery scoring system.Results: Muller type C2 fracture was the most common fracture type with 50 out of 72 patients. The average range of motion achieved was about 99.03°±24.73° (Closed fractures =105.83°±19.41°and open fractures = 89.50°±28.36°). There was also a significant difference in the duration of operative time, 84.28±18.32 minutes for closed fractures and 98.46±22.47 minutes for open fractures. The average duration for radiological union was 14.52±2.21 weeks for closed and 17.20±2.44 weeks for open fractures. The average knee score was 80.13±13.38 using modified Hospital for Special Surgery score.Conclusions: Closed fractures have a higher range of motion and a better knee score compared to open fractures, supporting the fact that soft tissue compromise also affects range of motion and post-op rehabilitation of the limb. The outcome seems to correlate with the nature of injury i.e. high vs low velocity, type of fracture, anatomic reduction, associated injuries, time elapsed since injury to fixation and the stability of fixation

Relevance of unilateral and bilateral sexual polyploidization in relation to intergenomic recombination and introgression in Lilium species hybrids

Sexual polyploids were induced in diploid (2n = 2x = 24) interspecific F1 hybrids of Longiflorum × Asiatic (LA) and Oriental × Asiatic (OA) Lilium hybrids by backcrossing to Asiatic (AA) parents as well as by sib-mating of the F1 LA hybrids. A majority of the BC1 progenies were triploid and the progenies from sib-mating were tetraploid or near tetraploids. Genomic in situ hybridization (GISH) technique was applied to assess the intergenomic recombination in the BC1 populations of LA and OA hybrids obtained after unilateral sexual polyploidization. A total of 63 LA (LA × AA and AA × LA) and 53 OA hybrids were analysed. LA hybrids were originated through the functioning of 2n gametes either as 2n eggs or 2n pollen while those of OA hybrids originated through functional 2n pollen of diploid OA genotype. In both type of crosses, a majority of the progenies had originated through First Division Restitution (FDR) mechanism of functional 2n gamete either with or without a cross over. However, there were nine LA- and four OA-genotypes where Indeterminate Meiotic Restitution (IMR) was the mechanism of 2n gamete formation. Based on GISH, total amount of introgression of Longiflorum and Oriental genome into Asiatic genome was determined. Most of the BC progenies exhibited recombination and the amount of recombination was higher in LA hybrids as compared to OA hybrids. Intergenomic recombination was also determined cytologically in the 16 plants of sib-mated LA hybrids where both parents had contributed 2n gametes. Based on these results the nature of interspecific lily hybrids obtained from uni- and bilateral sexual polyploidization leading to allotriploid and allotetraploid formation is discussed in the context of introgression and intergenomic recombinatio

99mTc-Nannocolloid Localization of Lymphorenal Fistula Causing Chyluria

Chyluria is an abnormal condition in which chyle appears in the urine because of a fistulous communication between the lymphatics and the urinary tract. It is not life-threatening and spontaneous regression is reported in 50% of cases. Lymphangiography has been the main imaging modality for localization of the site of fistula, but it is invasive and requires expertise. Lymphoscintigraphy using Tc-99m labelled colloid is a safe, non-invasive, reproducible technique, which bears less radiation exposure. A 67-year-old male presented with 7-month history of chyluria following a spinal surgery. Bilateral lower limb lymphoscintigram revealed sluggish lymph flow in the left lower limb and visualization of tracer in the left kidney consistent with lymphorenal fistula. Subsequent cystography revealed appearance of chylous urine from left ureter. Patient refused surgery

Antigen presenting cells (APCs) from thermally injured and/or septic rats modulate CD4+ T cell responses of naive rat

AbstractRegulation of immune response is marked by complex interactions among the cells that recognize and present antigens. Antigen presenting cells (APCs), the antigen presenting cell component of the innate immune response plays an important role in effector CD4+ T cell response. Thermal injury and/or superimposed sepsis in rats' leads to suppressed CD4+ T cell functions. We investigated modulations of CD4+ T cell function by APCs (purified non-T cells) from thermally injured and/or septic rats. Rats were subjected to 30% total body surface area scald burn or exposed to 37 °C water (Sham burn) and sepsis was induced by cecal-ligation and puncture (CLP) method. At day 3 post-injury animals were sacrificed and CD4+ T cells and APCs from mesenteric lymph nodes (MLN) were obtained using magnetic microbead isolation procedure. APCs from injured rats were co-cultured with sham rat MLN CD4+ T cells and proliferative responses (thymidine incorporation), phenotypic changes (Flow cytometry), IL-2 production (ELISA) and CTLA-4 mRNA (RT-PCR) were determined in naive rat CD4+ T cells. The data indicate that APCs from thermally injured and/or septic rats when co-cultured with CD4+ T cells suppressed CD4+ T cell effector functions. This lack of CD4+ T cell activation was accompanied with altered co-stimulatory molecules, i.e., CD28 and/or CTLA-4 (CD152). In conclusion, our studies indicated that defective APCs from thermally injured and/or septic rats modulate CD4+ T cell functions via changes in co-stimulatory molecules expressed on naive CD4+ T cells. This altered APC: CD4+ T cell interaction leads to suppressed CD4+ T cell activation of healthy animals

Sentinel lymph node scintigraphy and radioguided dissection in breast carcinoma: an initial experience at Aga Khan University Hospital

Objective: To evaluate the efficacy of SLN biopsy using imaging and gamma probe in breast cancer and to establish this technique at Aga Khan University Hospital.Methods: Thirty two patients (mean age 33-76 yrs) with operable breast carcinoma (4 with post-neo adjuvant therapy) with clinically negative axilla were studied. In 28 patients simultaneous axillary dissection was performed. Sentinel lymph nodes (SLN) scintigraphy was performed a day before surgery by injecting Tc-99m labeled nannocolloid sub-dermally in the peri-areolar region in 24 and peri-tumoral in remaining 8. First lymph node (LN) to appear to on the scan was labeled as SLN and marked on the skin. Blue dye was also injected in all patients and blue and hot LN was explored in the axilla using gamma probe.Results: The sentinel LN was identified in 31 patients (96.9% success rate) while in one patient (3.1%) SLN was not visualized on the scan (negative study) and this was a post-neo-adjuvant therapy case. The blue dye successfully localized the sentinel LNs in all 32 cases The gamma probe guided localization was successful in all 31 while in one case with post-neo-adjuvant chemotherapy it failed. In 9 out of 32 (28.1%) cases SLN was positive for metastasis and in 6 out of these 9 (66.6%) the SLN was the only metastatic node. This includes the post-neo-adjuvant case as well. There was no case of skip metastasis, i.e. negative SNL and positive other axillary nodes. In remaining 23 (71.9%) cases SLN was negative for metastasis and in all, axilla was free of disease (NPV of 100%).CONCLUSION: Lymphoscintigraphy with gamma probe guided SNL biopsy is safe, simple and highly reliable technique. With blue dye technique it reduces the blindness of the procedure if performed independently but increases the cost. Finally, this can accurately stage the axilla, possibly allowing axillary dissection to be foregone in patients where the SLN is clear

Comparing the Reaction Rates of Plasmonic (Gold) and Non-Plasmonic (Palladium) Metal Particles in Photocatalytic Hydrogen Production

Both Pd and Au metal particles are used in photocatalytic hydrogen generation. Yet while both act as electron sink only gold is poised to respond to visible light due to its plasmonic response. In order to quantitatively gauge their relative contribution into the reaction, the photocatalytic H2 production, from Au/TiO2 and Pd/TiO2 catalysts was studied under UV and UV–Vis light. While under UV light excitation, a weak dependence on the work function of the metal is observed, under UV–Vis light, Au is found to be twice more active than Pd. Under identical UV–Vis light irradiation, the turn over frequency calculated from XPS at.% is found to be 2.8 and 1.8 s−1 for Au and Pd, respectively. The effect is far more pronounced when the rates are normalized to the number of particles of each metal. Both the semiconductor TiO2 (UV light) and the plasmonic metal (visible light) need to be excited for the enhancement to occur; visible light alone causes a negligible reaction rate. Photocurrent measurements further confirmed the difference in the photocatalytic activity under UV and UV–Vis light excitation. Moreover, because of the presence of Au particles responding to visible light the reaction rate is enhanced due to “light penetration depth” effect

Hybridizing Micro - B4C with Carbon nanotubes to Enhance the Mechanical Properties of Aluminium Matrix Composites

In present work, the effects of hybridizing micron sized B4C particles with multi walled carbon nanotubes on the microstructural and mechanical properties of Al - B4C composite were investigated. Microstructure reveals grain refinement ascribed to the presence of uniformly distributed micron sized B4C particles with multi walled carbon nanotubes. The Al - (B4C + MWCNT) hybrid composite indicates the alliance of mechanical properties such as hardness, tensile strength and ductility. The enhanced attribute of Al - (B4C + MWCNT) hybrid composite when compared to Al - B4C is the increased content and uniform distribution of MWCNTs. © Published under licence by IOP Publishing Ltd