We report the first gene-based linkage map of Lupinus angustifolius (narrow-leafed lupin) and its comparison to the partially sequenced genome of Medicago truncatula. The map comprises 382 loci in 20 major linkage groups, two triplets, three pairs and 11 unlinked loci and is 1,846 cM in length. The map was generated from the segregation of 163 RFLP markers, 135 gene-based PCR markers, 75 AFLP and 4 AFLP-derived SCAR markers in a mapping population of 93 recombinant inbred lines, derived from a cross between domesticated and wild-type parents. This enabled the mapping of five major genes controlling key domestication traits in L. angustifolius. Using marker sequence data, the L. angustifolius genetic map was compared to the partially completed M. truncatula genome sequence. We found evidence of conserved synteny in some regions of the genome despite the wide evolutionary distance between these legume species. We also found new evidence of widespread duplication within the L. angustifolius genome.

Accepted ManuscriptThis is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. hours in soaking solutions containing 10-50 mM octopamine, 0.1-1.0 mg/mL FITC, and 0.5-6 mM spermidine did not affect vitality. Spermidine phosphate salt hexahydrate rather than spermidine or spermidine trihydrochloride increased uptake of FITC by nematodes, and this resulted in more effective gene silencing. Silencing pat-10 and unc-87 genes of P. thornei and P. zeae resulted in paralysis and uncoordinated movements in both species, although to a higher degree in P. thornei.There was also a greater reduction in transcript of both genes in P. thornei indicating that it may be more susceptible to RNAi. For P. thornei treated with dsRNA of pat-10 and unc-87 there was a significant reduction (77-81%) in nematode reproduction on carrot mini discs over a 5 week period. The results show that RLNs are clearly amenable to gene silencing, and that in planta delivery of dsRNA to target genes in root lesion nematodes should confer host resistance.Moreover, for the two genes, dsRNA derived from either nematode species silenced the corresponding gene in both species. This implies cross-species control of nematodes via RNAi is possible.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

No abstract

The root-knot nematode Meloidogyne javanica induces giant cells and feeds from them during its development and reproduction. To study the cellular processes underlying the formation of giant cells, laser microdissection was used to isolate the contents of early-stage giant cells 4 and 7 days post-infection (dpi) from tomato, and cDNA libraries from both stages were generated with 87 [250 expressed sequence tag (EST) clones] and 54 (309 EST clones) individual transcripts identified, respectively. These transcripts have roles in metabolism, stress response, protein synthesis, cell division and morphogenesis, transport, signal transduction, protein modification and fate, and regulation of cellular processes. The expression of 25 selected transcripts was studied further by real-time quantitative reverse transcriptase-polymerase chain reaction. Among them, 13 showed continuous up-regulation in giant cells from 4 to 7 dpi. The expression of two transcripts was higher than in controls at 4 dpi and remained at the same level at 7 dpi; a further five transcripts were highly expressed only at 7 dpi. The Phi-1 protein gene, a cell cycle-related homologue in tobacco, was expressed 8.5 times more strongly in giant cells than in control cells at 4 dpi, but was reduced to 6.7 times at 7 dpi. Using in situ hybridization, the expression of the Phi-1 gene was preferentially localized in the cytoplasm of giant cells at 4 dpi, together with a pectinesterase U1 precursor gene. The identification of highly expressed transcripts in developing giant cells adds to the knowledge of the plant genes responsive to nematode infection, and may provide candidate genes for nematode control strategies.

Control of plant-parasitic nematodes (PPNs) via host-induced gene silencing (HIGS) involves rational selection of genes and detailed assessment of effects of a possible knockdown on the nematode. Some genes by nature may be very important for the survival of the nematode that knockdown may be resisted. Possible silencing and effects of 20 such genes involved in the RNA interference (RNAi) pathways of Meloidogyne incognita were investigated in this study using long double-stranded RNAs (dsRNAs) as triggers. Two of the genes, ego-1 and mes-2, could not be knocked down. Expression of six genes (xpo-1, pash-1, xpo-2, rha-1, ekl-4, and csr-1) were significantly upregulated after RNAi treatment whereas for 12 of the genes, significant knockdown was achieved and with the exception of mes-2 and mes-6, RNAi was accompanied by defective phenotypes in treated nematodes including various degrees of paralysis and abnormal behaviors and movement such as curling, extreme wavy movements, and twitching. These abnormalities resulted in up to 75% reduction in infectivity of a tomato host, the most affected being the J2s previously treated with dsRNA of the gfl-1 gene. For 10 of the genes, effects of silencing in the J2s persisted as the adult females isolated from galls were under-developed, elongated, and transparent compared to the normal saccate, white adult females. Following RNAi of ego-1, smg-2, smg-6, and eri-1, reduced expression and/or the immediate visible effects on the J2s were not permanent as the nematodes infected and developed normally in tomato hosts. Equally intriguing was the results of RNAi of the mes-2 gene where the insignificant change in gene expression and behavior of treated J2s did not mean the nematodes were not affected as they were less effective in infecting host plants. Attempt to silence drsh-1, mut-7, drh-3, rha-1, pash-1, and vig-1 through HIGS led to reduction in nematode infestation by up to 89%. Our results show that genes may respond to RNAi knockdown differently so an exhaustive assessment of target genes as targets for nematode control via RNAi is imperative.

No abstract

(2015) De novo analysis of the transcriptome of Pratylenchus zeae to identify transcripts for proteins required for structural integrity, sensation, locomotion and parasitism. Molecular Plant Pathology, 17 (4). pp. 532-552. This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/mpp.12301 Accepted ArticleThis article is protected by copyright. All rights reserved.Accession numbers: BioProject ID PRJNA268047; SRR1657910 (Short Read Archive); SUB990140 (Transcriptome Shotgun Assembly temporary BioSample submission ID). SummaryThe root lesion nematode Pratylenchus zeae, a migratory endoparasite, is an economically important pest of major crop plants (e.g. cereals, sugarcane). It enters host roots, migrates through root tissues and feeds from cortical cells, and defends itself against biotic and abiotic stresses in the soil and in host tissues. We report de novo sequencing of the P. zeae transcriptome using 454 FLX, and identification of putative transcripts encoding proteins required for movement, response to stimuli, feeding and parasitism. Sequencing generated 347,443 good quality reads which were assembled into 10,163 contigs and 139,104 singletons: 65% of contigs and 28% of singletons matched sequences of free-living and parasitic nematodes. Three quarters of annotated transcripts were common to reference nematodes, mainly representing genes encoding proteins for structural integrity and fundamental biochemical processes. Over 15,000transcripts were similar to Caenorhabditis elegans genes encoding proteins with roles in mechanical and neural control of movement, responses to chemicals, mechanical and thermal stresses. Notably, 766 transcripts matched parasitism genes employed by both migratory and sedentary endoparasites in host interactions, of which three studied hybridised to the gland cell region, suggesting that they might be secreted. Conversely, transcripts for effectors reported to be involved in feeding site formation by sedentary endoparasites were conspicuously absent. Transcripts similar to those encoding some Accepted ArticleThis article is protected by copyright. All rights reserved.secretory-excretory products at the host interface of Brugia malayi, the secretome of Meloidogyne incognita and products of gland cells of Heterodera glycines were also identified. This P. zeae transcriptome provides new information for genome annotation and functional analysis of possible targets for control of pratylenchid nematodes.