Importance of the amino terminus in maintenance of oligomeric structure of sheep liver cytosolic serine hydroxymethyltransferase

The Role Of The Amino And Carboxyl-Terminal Regions Of Cytosolic Serine Hydroxymethyltransferase (SHMT) In Subunit Assembly And Catalysis Was Studied Using Sis Amino-Terminal (Lacking The First 6, 14, 30, 49, 58, And 75 Residues) And Two Carboxyl-Terminal (Lacking The Last 49 And 185 Residues) Deletion Mutants. These Mutants Were Constructed From A Full Length Cdna Clone Using Restriction Enzyme/PCR-Based Methods And Overexpressed In Escherichia Coli. The Overexpressed Proteins, Des-(A1-K6) SHMT And Des-(A1-W14)-SHMT Were Present In The Soluble Fraction And They Were Purified To Homogeneity. The Deletion Clones, For Des-(A1-V30)-SHMT And Des-(A1-L49)-SHMT Were Expressed At Very Low Levels, Whereas Des-(A1-R58)-SHMT, Des-/A1-G75)-SHMT, Des-(Q435-F483)-SHMT And Des-(L299-F483)-SHMT Mutant Proteins Were Not Soluble And Formed Inclusion Bodies. Des-(A1-K6)-SHMT And Des-(A1-W14)-SHMT Catalyzed Both The Tetrahydrofolate-Dependent And Tetrahydrofolate-Independent Reactions, Generating Characteristic Spectral Intermediates With Glycine And Tetrahydrofolate. The Two Mutants Had Similar Kinetic Parameters To That Of The Recombinant SHMT (Rshmt). However, At 55 Degrees C, The Des-(A1-W14)-SHMT Lost Almost All The Activity Within 5 Min, While At The Same Temperature Rshmt And Des-(A1-K6)-SHMT Retained 85% And 70% Activity, Respectively. Thermal Denaturation Studies Showed That Des-(A1-W14)-SHMT Had A Lower Apparent Melting Temperature (52 Degrees C) Compared To Rshmt (56 Degrees C) And Des-(A1-K6)-SHMT (55 Degrees C), Suggesting That N-Terminal Deletion Had Resulted In A Decrease In The Thermal Stability Of The Enzyme. Further Urea Induced Inactivation Of The Enzymes Revealed That 50% Inactivation Occurred At A Lower Urea Concentration (1.2+/-0.1 M) In The Case Of Des-(A1-W14)-SHMT Compared To Rshmt (1.8+/-0.1 M) And Des-(A1 -K6)-SHMT (1.7+/-0.1 M). The Apoenzyme Of Des-/A1-K6)-SHMT Was Present Predominantly In The Dimer Form, Whereas The Apoenzymes Of Rshmt And Des-(A1-K6)-SHMT Were A Mixture Of Tetramers (Approximate To 75% And Approximate To 65%, Respectively) And Dimers. While, Rshmt And Des-(A1-K6)-SHMT Apoenzymes Could Be Reconstituted Upon The Addition Of Pyridoxal-5'-Phosphate To 96% And 94% Enzyme Activity, Respectively Des-(A1-W14)-SHMT Apoenzyme Could Be Reconstituted Only Upto 22%. The Percentage Activity Refined Correlated With The Appearance Of Visible CD At 425 Nm And With The Amount Of Enzyme Present In The Tetrameric Form Upon Reconstitution As Monitored By Gel Filtration. These Results Demonstrate That, In Addition To The Cofactor, The N-Terminal Arm Plays An Important Role In Stabilizing The Tetrameric Structure Of SHMT.

Localisation of Plasmodium falciparum uroporphyrinogen III decarboxylase of the heme-biosynthetic pathway in the apicoplast and characterisation of its catalytic properties

Uroporphyrinogen decarboxylase (UROD) is a key enzyme in the heme-biosynthetic pathway and in Plasmodium falciparum it occupies a strategic position in the proposed hybrid pathway for heme biosynthesis involving shuttling of intermediates between different subcellular compartments in the parasite. In the present study, we demonstrate that an N-terminally truncated recombinant P. falciparum UROD (r(Δ)PfUROD) over-expressed and purified from Escherichia coli cells, as well as the native enzyme from the parasite were catalytically less efficient compared with the host enzyme, although they were similar in other enzyme parameters. Molecular modeling of PfUROD based on the known crystal structure of the human enzyme indicated that the protein manifests a distorted triose phosphate isomerase (TIM) barrel fold which is conserved in all the known structures of UROD. The parasite enzyme shares all the conserved or invariant amino acid residues at the active and substrate binding sites, but is rich in lysine residues compared with the host enzyme. Mutation of specific lysine residues corresponding to residues at the dimer interface in human UROD enhanced the catalytic efficiency of the enzyme and dimer stability indicating that the lysine rich nature and weak dimer interface of the wild-type PfUROD could be responsible for its low catalytic efficiency. PfUROD was localised to the apicoplast, indicating the requirement of additional mechanisms for transport of the product coproporphyrinogen to other subcellular sites for its further conversion and ultimate heme formation.

Nature and extent of genetic diversity of dengue viruses determined by 454 pyrosequencing

Dengue virus (DENV) populations are characteristically highly diverse. Regular lineage extinction and replacement is an important dynamic DENV feature, and most DENV lineage turnover events are associated with increased incidence of disease. The role of genetic diversity in DENV lineage extinctions is not understood. We investigated the nature and extent of genetic diversity in the envelope (E) gene of DENV serotype 1 representing different lineages histories. A region of the DENV genome spanning the E gene was amplified and sequenced by Roche/454 pyrosequencing. The pyrosequencing results identified distinct sub-populations (haplotypes) for each DENV-1 E gene. A phylogenetic tree was constructed with the consensus DENV-1 E gene nucleotide sequences, and the sequences of each constructed haplotype showed that the haplotypes segregated with the Sanger consensus sequence of the population from which they were drawn. Haplotypes determined through pyrosequencing identified a recombinant DENV genome that could not be identified through Sanger sequencing. Nucleotide level sequence diversities of DENV-1 populations determined from SNP analysis were very low, estimated from 0.009-0.01. There were also no stop codon, frameshift or non-frameshift mutations observed in the E genes of any lineage. No significant correlations between the accumulation of deleterious mutations or increasing genetic diversity and lineage extinction were observed (p>0.5). Although our hypothesis that accumulation of deleterious mutations over time led to the extinction and replacement of DENV lineages was ultimately not supported by the data, our data does highlight the significant technical issues that must be resolved in the way in which population diversity is measured for DENV and other viruses. The results provide an insight into the within-population genetic structure and diversity of DENV-1 populations.

Paracrine action of sFLT-1 secreted by stably-transfected Ehrlich ascites tumor cells and therapy using sFLT-1 inhibits ascites tumor growth in vivo

Background Vascular endothelial growth factor (VEGF) is known to play a major role in angiogenesis. A soluble form of Flt-1, a VEGF receptor, is potentially useful as an antagonist of VEGF, and accumulating evidence suggests the applicability of sFlt-1 in tumor suppression. In the present study, we have developed and tested strategies targeted specifically to VEGF for the treatment of ascites formation.Methods As an initial strategy, we produced recombinant sFLT-1 in the baculovirus expression system and used it as a trap to sequester VEGF in the murine ascites carcinoma model. The effect of the treatment on the weight of the animal, cell number, ascites volume and proliferating endothelial cells was studied. The second strategy involved, producing Ehrlich ascites tumor (EAT) cells stably transfected with vectors carrying cDNA encoding truncated form of Flt-1 and using these cells to inhibit ascites tumors in a nude mouse model. Results The sFLT-1 produced by the baculovirus system showed potent antiangiogenic activity as assessed by rat cornea and tube formation assay. sFLT-1 treatment resulted in reduced peritoneal angiogenesis with a concomitant decrease in tumor cell number, volume of ascites, amount of free VEGF and the number of invasive tumor cells as assayed by CD31 staining. EAT cells stably transfected with truncated form of Flt-1 also effectively reduced the tumor burden in nude mice transplanted with these cells, and demonstrated a reduction in ascites formation and peritoneal angiogenesis. Conclusions The inhibition of peritoneal angiogenesis and tumor growth by sequestering VEGF with either sFlt-1 gene expression by recombinant EAT cells or by direct sFLT-1 protein therapy is shown to comprise a potential therapy. Copyright (C) 2009 John Wiley & Sons, Ltd.

Cloning Of Rice Dna And Identification Of Transfer-Rna Gene Clones

THE rapid development of recombinant DNA technology has brought forth a revolution in biology'>", it aids us to have a closer look at the 'way genes are organized, eS11 ecially in the complex eucaryotic genornes'<", Although many animal and yeast genes have been studied in detail using recombinant DNA technology, plant genes have seldom been targets for such studie., Germination is an ideal process to study gene expression .because it effects a . shift in the metabolic status of seeds from a state of 'dormancy to an active one. AJ;l understanding of gene organization and regulation darin.g germination can be accomplblted by molecular cloning of DNA from seeds lik.e rice. To study the status of histone, rRNA tRNA and other genes in the rice genome, a general method was developed to clone eucarvotic DNA in a' plasmid vector pBR 322. This essentially ~ involves the following steps. The rice embryo and plasmid pBR 322 DNAs were cut witll restriction endonuclease Bam Hi to generate stick.Y ends, The plasmid DNA was puosphatased, the DNA~ ware a~·tnealed and joined 'by T4 phage DNA ligase. The recombinant DNA molecules thus produced were transjerred into E. coli and colonies containing them Were selected by their sensitivity to tetracycline and resistance to ampicillin, Two clones were identified . 2S haVing tRNA genes by hybridization of the DNA in the clones \vitl1 32P-la.belled rice tRNAs.

Heparin versus 0.9% sodium chloride intermittent flushing for the prevention of occlusion in long term central venous catheters in infants and children

Background Guidelines and clinical practice for the prevention of complications associated with central venous catheters (CVC) around the world vary greatly. Most institutions recommend the use of heparin to prevent occlusion, however there is debate regarding the need for heparin and evidence to suggest 0.9% sodium chloride (normal saline) may be as effective. The use of heparin is not without risk, may be unnecessary and is also associated with increased cost. Objectives To assess the clinical effects (benefits and harms) of intermittent flushing of heparin versus normal saline to prevent occlusion in long term central venous catheters in infants and children. Search Methods The Cochrane Vascular Trials Search Co-ordinator searched the Specialised Register (last searched April 2015) and the Cochrane Register of Studies (Issue 3, 2015). We also searched the reference lists of retrieved trials. Selection criteria Randomised controlled trials that compared the efficacy of normal saline with heparin to prevent occlusion of long term CVCs in infants and children aged up to 18 years of age were included. We excluded temporary CVCs and peripherally inserted central catheters (PICC). Data Collection and Analysis Two review authors independently assessed trial inclusion criteria, trial quality and extracted data. Rate ratios were calculated for two outcome measures - occlusion of the CVC and central line-associated blood stream infection. Other outcome measures included duration of catheter placement, inability to withdraw blood from the catheter, use of urokinase or recombinant tissue plasminogen, incidence of removal or re-insertion of the catheter, or both, and other CVC-related complications such as dislocation of CVCs, other CVC site infections and thrombosis. Main Results Three trials with a total of 245 participants were included in this review. The three trials directly compared the use of normal saline and heparin, however, between studies, all used different protocols for the standard and experimental arms with different concentrations of heparin and different frequency of flushes reported. In addition, not all studies reported on all outcomes. The quality of the evidence ranged from low to very low because there was no blinding, heterogeneity and inconsistency between studies was high and the confidence intervals were wide. CVC occlusion was assessed in all three trials (243 participants). We were able to pool the results of two trials for the outcomes of CVC occlusion and CVC-associated blood stream infection. The estimated rate ratio for CVC occlusion per 1000 catheter days between the normal saline and heparin group was 0.75 (95% CI 0.10 to 5.51, two studies, 229 participants, very low quality evidence). The estimated rate ratio for CVC-associated blood stream infection was 1.48 (95% CI 0.24 to 9.37, two studies, 231 participants; low quality evidence). The duration of catheter placement was reported to be similar between the two study arms, in one study (203 participants). Authors' Conclusions The review found that there was not enough evidence to determine the effects of intermittent flushing of heparin versus normal saline to prevent occlusion in long term central venous catheters in infants and children. Ultimately, if this evidence were available, the development of evidenced-based clinical practice guidelines and consistency of practice would be facilitated.

Abacá mosaic virus: a distinct strain of Sugarcane mosaic virus

Abacá mosaic virus (AbaMV) is related to members of the sugarcane mosaic virus subgroup of the genus Potyvirus. The ~2 kb 3′ terminal region of the viral genome was sequenced and, in all areas analysed, found to be most similar to Sugarcane mosaic virus (SCMV) and distinct from Johnsongrass mosaic virus (JGMV), Maize dwarf mosaic virus (MDMV) and Sorghum mosaic virus (SrMV). Cladograms of the 3′ terminal region of the NIb protein, the coat protein core and the 3′ untranslated region showed that AbaMV clustered with SCMV, which was a distinct clade and separate from JGMV, MDMV and SrMV. The N-terminal region of the AbaMV coat protein had a unique amino acid repeat motif different from those previously published for other strains of SCMV. The first experimental transmission of AbaMV from abacá (Musa textilis) to banana (Musa sp.), using the aphid vectors Rhopalosiphum maidis and Aphis gossypii, is reported. Polyclonal antisera for the detection of AbaMV in western blot assays and ELISA were prepared from recombinant coat protein expressed in E. coli. A reverse transcriptase PCR diagnostic assay, with microtitre plate colourimetric detection, was developed to discriminate between AbaMV and Banana bract mosaic virus, another Musa-infecting potyvirus. Sequence data, host reactions and serological relationships indicate that AbaMV should be considered a distinct strain of SCMV, and the strain designation SCMV-Ab is suggested.

Comparative immunogenicity of M. hyopneumoniae NrdF encoded in different expression systems delivered orally via attenuated S. typhimurium aroA in mice.

The Mycoplasma hyopneumoniae ribonucleotide reductase R2 subunit (NrdF) gene fragment was cloned into eukaryotic and prokaryotic expression vectors and its immunogenicity evaluated in mice immunized orally with attenuated Salmonella typhimurium aroA CS332 harboring either of the recombinant expression plasmids. We found that NrdF is highly conserved among M. hyopneumoniae strains. The immunogenicity of NrdF was examined by analyzing antibody responses in sera and lung washes, and the cell-mediated immune (CMI) response was assessed by determining the INF-[gamma] level produced by splenocytes upon in vitro stimulation with NrdF antigen. S. typhimurium expressing NrdF encoded by the prokaryotic expression plasmid (pTrcNrdF) failed to elicit an NrdF-specific serum or secretory antibody response, and IFN-[gamma] was not produced. Similarly, S. typhimurium carrying the eukaryotic recombinant plasmid encoding NrdF (pcNrdF) did not induce a serum or secretory antibody response, but did elicit significant NrdF-specific IFN-[gamma] production, indicating induction of a CMI response. However, analysis of immune responses against the live vector S. typhimurium aroA CS332 showed a serum IgG response but no mucosal IgA response in spite of its efficient invasiveness in vitro. In the present study we show that the DNA vaccine encoding the M. hyopneumoniae antigen delivered orally via a live attenuated S. typhimurium aroA can induce a cell-mediated immune response. We also indicate that different live bacterial vaccine carriers may have an influence on the type of the immune response induced.

Heterologous signals allow efficient targeting of a nuclear-encoded fusion protein to plastids and endoplasmic reticulum in diverse plant species

Approximately 30% of plant nuclear genes appear to encode proteins targeted to the plastids or endoplasmic reticulum (ER). The signals that direct proteins into these compartments are diverse in sequence, but, on the basis of a limited number of tests in heterologous systems, they appear to be functionally conserved across species. To further test the generality of this conclusion, we tested the ability of two plastid transit peptides and an ER signal peptide to target green fluorescent protein (GFP) in 12 crops, including three monocots (barley, sugarcane, wheat) and nine dicots (Arabidopsis, broccoli, cabbage, carrot, cauliflower, lettuce, radish, tobacco, turnip). In all species, transient assays following microprojectile bombardment or vacuum infiltration using Agrobacterium showed that the plastid transit peptides from tomato DCL (defective chloroplast and leaves) and tobacco RbcS [ribulose bisphosphate carboxylase (Rubisco) small subunit] genes were effective in targeting GFP to the leaf plastids. GFP engineered as a fusion to the N-terminal ER signal peptide from Arabidopsis basic chitinase and a C-terminal HDEL signal for protein retention in the ER was accumulated in the ER of all species. The results in tobacco were confirmed in stably transformed cells. These signal sequences should be useful to direct proteins to the plastid stroma or ER lumen in diverse plant species of biotechnological interest for the accumulation of particular recombinant proteins or for the modification of particular metabolic streams.

Diversity in the sunflower: Puccinia helianthi pathosystem in Australia

Sunflower rust caused by Puccinia helianthi is the most important disease of sunflower in Australia with the potential to cause significant yield losses in susceptible hybrids. Rapid and frequent virulence changes in the rust fungus population limit the effective lifespan of commercial cultivars and impose constant pressure on breeding programs to identify and deploy new sources of resistance. This paper contains a synopsis of virulence data accumulated over 25 years, and more recent studies of genotypic diversity and sexual recombination. We have used this synopsis, generated from both published and unpublished data, to propose the origin, evolution and distribution of new pathotypes of P. helianthi. Virulence surveys revealed that diverse pathotypes of P. helianthi evolve in wild sunflower populations, most likely because sexual recombination and subsequent selection of recombinant pathotypes occurs there. Wild sunflower populations provide a continuum of genetically heterogeneous hosts on which P. helianthi can potentially complete its sexual cycle under suitable environmental conditions. Population genetics analysis of a worldwide collection of P. helianthi indicated that Australian isolates of the pathogen are more diverse than non-Australian isolates. Additionally, the presence of the same pathotype in different genotypic backgrounds supported evidence from virulence data that sexual recombination has occurred in the Australian population of P. helianthi at some time. A primary aim of the work described was to apply our knowledge of pathotype evolution to improve resistance in sunflower to sunflower rust. Molecular markers were identified for a number of previously uncharacterised sunflower rust R-genes. These markers have been used to detect resistance genes in breeding lines and wild sunflower germplasm. A number of virulence loci that do not recombine were identified in P. helianthi. The resistance gene combinations corresponding to these virulence loci are currently being introgressed with breeding lines to generate hybrids with durable resistance to sunflower rust.

Trapping to monitor tephritid movement: Results, best practice, and assessment of alternatives

Movement of tephritid flies underpins their survival, reproduction, and ability to establish in new areas and is thus of importance when designing effective management strategies. Much of the knowledge currently available on tephritid movement throughout landscapes comes from the use of direct or indirect methods that rely on the trapping of individuals. Here, we review published experimental designs and methods from mark-release-recapture (MRR) studies, as well as other methods, that have been used to estimate movement of the four major tephritid pest genera (Bactrocera, Ceratitis, Anastrepha, and Rhagoletis). In doing so, we aim to illustrate the theoretical and practical considerations needed to study tephritid movement. MRR studies make use of traps to directly estimate the distance that tephritid species can move within a generation and to evaluate the ecological and physiological factors that influence dispersal patterns. MRR studies, however, require careful planning to ensure that the results obtained are not biased by the methods employed, including marking methods, trap properties, trap spacing, and spatial extent of the trapping array. Despite these obstacles, MRR remains a powerful tool for determining tephritid movement, with data particularly required for understudied species that affect developing countries. To ensure that future MRR studies are successful, we suggest that site selection be carefully considered and sufficient resources be allocated to achieve optimal spacing and placement of traps in line with the stated aims of each study. An alternative to MRR is to make use of indirect methods for determining movement, or more correctly, gene flow, which have become widely available with the development of molecular tools. Key to these methods is the trapping and sequencing of a suitable number of individuals to represent the genetic diversity of the sampled population and investigate population structuring using nuclear genomic markers or non-recombinant mitochondrial DNA markers. Microsatellites are currently the preferred marker for detecting recent population displacement and provide genetic information that may be used in assignment tests for the direct determination of contemporary movement. Neither MRR nor molecular methods, however, are able to monitor fine-scale movements of individual flies. Recent developments in the miniaturization of electronics offer the tantalising possibility to track individual movements of insects using harmonic radar. Computer vision and radio frequency identification tags may also permit the tracking of fine-scale movements by tephritid flies by automated resampling, although these methods come with the same problems as traditional traps used in MRR studies. Although all methods described in this chapter have limitations, a better understanding of tephritid movement far outweighs the drawbacks of the individual methods because of the need for this information to manage tephritid populations.

Rapid isolation and detection of erythropoietin in blood plasma by magnetic core gold nanoparticles and portable Raman spectroscopy

Isolating, purifying, and identifying proteins in complex biological matrices is often difficult, time consuming, and unreliable. Herein we describe a rapid screening technique for proteins in biological matrices that combines selective protein isolation with direct surface enhanced Raman spectroscopy (SERS) detection. Magnetic core gold nanoparticles were synthesised, characterised, and subsequently functionalized with recombinant human erythropoietin (rHuEPO)-specific antibody. The functionalized nanoparticles were used to capture rHuEPO from horse blood plasma within 15 minutes. The selective binding between the protein and the functionalized nanoparticles was monitored by SERS. The purified protein was then released from the nanoparticles’ surface and directly spectroscopically identified on a commercial nanopillar SERS substrate. ELISA independently confirmed the SERS identification and quantified the released rHuEPO. Finally, the direct SERS detection of the extracted protein was successfully demonstrated for in-field screening by a handheld Raman spectrometer within 1 minute sample measurement time.

Identification and immunogenicity of an immunodominant mimotope of Avibacterium paragallinarum from a phage display peptide library

Avibacterium paragallinarum is the causative agent of infectious coryza. The protective antigens of this important pathogen have not yet been clearly identified. In this paper, we applied phage display technique to screen the immunodominant mimotopes of a serovar A strain of A. paragallinarum by using a random 12-peptide library, and evaluated the immunogenicity in chickens of the selected mimotope. Polyclonal antibody directed against A. paragallinarum strain 0083 (serovar A) was used as the target antibody and phage clones binding to this target were screened from the 12-mer random peptide library. More than 50% of the phage clones selected in the third round carried the consensus peptide motif sequence A-DP(M)L. The phage clones containing the peptide motif reacted with the target antibody and this interaction could be blocked, in a dose-dependent manner, by A. paragallinarum. One of the peptide sequences, YGLLAVDPLFKP, was selected and the corresponding oligonucleotide sequence was synthesized and then inserted into the expression vector pFliTrx. The recombinant plasmid was transferred into an expression host Escherichia coli GI826 by electroporation, resulting in a recombinant E. coli expressing the peptide on the bacterial surface. Intramuscular injection of the epitope-expressing recombinant bacteria into chickens induced a specific serological response to serovar A. A. paragallinarum. The chickens given the recombinant E. coli showed significant protection against challenge with A. paragallinarum 0083. These results indicated a potential for the use of the mimotope in the development of molecular vaccines for infectious coryza.

DArT markers: Diversity analyses and mapping in Sorghum bicolor

The sequential nature of gel-based marker systems entails low throughput and high costs per assay. Commonly used marker systems such as SSR and SNP are also dependent on sequence information. These limitations result in high cost per data point and significantly limit the capacity of breeding programs to obtain sufficient return on investment to justify the routine use of marker-assisted breeding for many traits and particularly quantitative traits. Diversity Arrays Technology (DArT™) is a cost effective hybridisation-based marker technology that offers a high multiplexing level while being independent of sequence information. This technology offers sorghum breeding programs an alternative approach to whole-genome profiling. We report on the development, application, mapping and utility of DArT™ markers for sorghum germplasm. Results: A genotyping array was developed representing approximately 12,000 genomic clones using PstI+BanII complexity with a subset of clones obtained through the suppression subtractive hybridisation (SSH) method. The genotyping array was used to analyse a diverse set of sorghum genotypes and screening a Recombinant Inbred Lines (RIL) mapping population. Over 500 markers detected variation among 90 accessions used in a diversity analysis. Cluster analysis discriminated well between all 90 genotypes. To confirm that the sorghum DArT markers behave in a Mendelian manner, we constructed a genetic linkage map for a cross between R931945-2-2 and IS 8525 integrating DArT and other marker types. In total, 596 markers could be placed on the integrated linkage map, which spanned 1431.6 cM. The genetic linkage map had an average marker density of 1/2.39 cM, with an average DArT marker density of 1/3.9 cM. Conclusion: We have successfully developed DArT markers for Sorghum bicolor and have demonstrated that DArT provides high quality markers that can be used for diversity analyses and to construct medium-density genetic linkage maps. The high number of DArT markers generated in a single assay not only provides a precise estimate of genetic relationships among genotypes, but also their even distribution over the genome offers real advantages for a range of molecular breeding and genomics applications.

Mapping quantitative trait loci for resistance to Pratylenchus thornei from synthetic hexaploid wheat in the International Triticeae Mapping Initiative (ITMI) population

Root-lesion nematode (Pratylenchus thornei) is a serious pathogen of wheat in many countries. The International Triticeae Mapping Initiative (ITMI) population of recombinant inbred lines (RILs) was assessed for resistance to P. thornei to determine the chromosome locations of the resistance genes. The ITMI population is derived from a cross between the resistant synthetic hexaploid wheat W-7984 and a susceptible bread wheat cultivar Opata 85. Two years of phenotypic data for resistance to P. thornei were obtained in replicated glasshouse trials. Quantitative trait locus (QTL) analysis was performed using available segregation and map data for 114 RILs. A QTL on chromosome 6DS showed consistent effects for reduced nematode numbers (partial resistance) across years and accounted for 11% and 23% of the phenotypic variation. A second QTL for P. thornei resistance on chromosome 2BS accounted for an additional 19% and 5%. Restriction fragment length polymorphism (RFLP) and simple sequence repeat (SSR) markers associated with the QTLs are physically located in regions rich in major genes at the distal ends of the short chromosome arms of 6D and 2B. SSR markers with potential for marker-assisted selection of P. thornei resistance effective in different genetic backgrounds have been identified.