Dhia Bouktila

Full-genome identification and characterization of NBS-encoding disease resistance genes in wheat.

AbstractHost resistance is the most economical, effective and ecologically sustainable method of controlling diseases in crop plants. nIn bread wheat, despite the high number of resistance loci that have been cataloged to date, only few have been cloned, underlying the need for genomics-guided investigations capable of providing a prompt and acute knowledge on the identity of effective resistance genes that can be used in breeding programs. Proteins with a nucleotide-binding site (NBS) encoded by the major plant disease resistance (R) genes play an important role in the responses of plants to various pathogens. In this study, a comprehensive analysis of NBS-encoding genes within the whole wheat genome was performed, and the genome scale characterization of this gene family was established. From the recently published wheat genome sequence, we used a data mining and automatic prediction pipeline to identify 580 complete ORF candidate NBS-encoding genes and 1,099 partial-ORF ones. Among complete gene models, 464 were longer than 200 aa, among them 436 had less than 70xa0% of sequence identity to each other. This gene models set was deeply characterized. (1) First, we have analyzed domain architecture and identified, in addition to typical domain combinations, the presence of particular domains like signal peptides, zinc fingers, kinases, heavy-metal-associated and WRKY DNA-binding domains. (2) Functional and expression annotation via homology searches in protein and transcript databases, based on sufficient criteria, enabled identifying similar proteins for 60xa0% of the studied gene models and expression evidence for 13xa0% of them. (3) Shared orthologous groups were defined using NBS-domain proteins of rice and Brachypodium distachyon. (4) Finally, alignment of the 436 NBS-containing gene models to the full set of scaffolds from the IWGSC’s wheat chromosome survey sequence enabled high-stringence anchoring to chromosome arms. The distribution of the R genes was found balanced on the three wheat sub-genomes. In contrast, at chromosome scale, 50xa0% of members of this gene family were localized on 6 of the 21 wheat chromosomes and ~22xa0% of them were localized on homeologous group 7. The results of this study provide a detailed analysis of the largest family of plant disease resistance genes in allohexaploid wheat. Some structural traits reported had not been previously identified and the genome-derived data were confronted with those stored in databases outlining the functional specialization of members of this family. The large reservoir of NBS-type genes presented and discussed will, firstly, form an important framework for marker-assisted improvement of resistance in wheat, and, secondly, open up new perspectives for a better understanding of the evolution dynamics of this gene family in grass species and in polyploid systems.

Genetic variability of green citrus aphid populations from Tunisia, assessed by RAPD markers and mitochondrial DNA sequences

The green citrus aphid Aphis spiraecola (Patch) is one of the major pests of several plant species including economically important crops such as citrus. In this study, we used random amplified polymorphic DNA (RAPD) markers and mitochondrial cytochrome oxidase subunitu2003I sequences to assess the level and distribution of genetic diversity of A.u2003spiraecola populations reared from Rutaceae and Rosaceae in different regions in Tunisia. RAPD analysis conducted on 141 individuals with 5 primers revealed only 50 polymorphic RAPD markers, indicating a low genetic diversity that might result from the lack of sexual phase for this species in Tunisia. Analysis of molecular variance (amova) showed that the genetic structure was not associated with geographic location or year of collection (Pu2003=u20030.70 and 0.34, respectively); however, the host‐plant had a significant effect on the partitioning of the total genetic diversity (Pu2003<u20030.01). Multidimensional scaling analysis indicated that the distribution of genetic variability was significantly influenced by the host‐plant with no evidence of spatial differentiation. Based on 20 barcode sequences of the mitochondrial cytochrome‐c oxidase subunitu2003I (COI) gene, we revealed the occurrence of two haplotypes in association with the host‐plant. Results reported here suggest the occurrence of a limited gene flow between A.u2003spiraecola populations from Rosaceae and Rutaceae and, therefore, a possible host‐race status that could be considered in the development of an integrated controlling strategy.

Large-scale analysis of NBS domain-encoding resistance gene analogs in Triticeae

Proteins containing nucleotide binding sites (NBS) encoded by plant resistance genes play an important role in the response of plants to a wide array of pathogens. In this paper, an in silico search was conducted in order to identify and characterize members of NBS-encoding gene family in the tribe of Triticeae. A final dataset of 199 sequences was obtained by four search methods. Motif analysis confirmed the general structural organization of the NBS domain in cereals, characterized by the presence of the six commonly conserved motifs: P-loop, RNBS-A, Kinase-2, Kinase-3a, RNBS-C and GLPL. We revealed the existence of 11 distinct distribution patterns of these motifs along the NBS domain. Four additional conserved motifs were shown to be significantly present in all 199 sequences. Phylogenetic analyses, based on genetic distance and parsimony, revealed a significant overlap between Triticeae sequences and Coiled coil-Nucleotide binding site-Leucine rich repeat (CNL)-type functional genes from monocotyledons. Furthermore, several Triticeae sequences belonged to clades containing functional homologs from non Triticeae species, which has allowed for these sequences to be functionally assigned. The findings reported, in this study, will provide a strong groundwork for the isolation of candidate R-genes in Triticeae crops and the understanding of their evolution.

Genetic Variation and Invasion Pattern of the Arabian Rhinoceros Beetle, Oryctes agamemnon arabicus (Burmeister) (Coleoptera: Scarabaeidae), in Tunisia, Deduced from Mitochondrial DNA Sequences

The Arabian rhinoceros beetle, Oryctes agamemnon arabicus (Burmeister, 1847), is an invasive species, introduced to Tunisia from the United Arab Emirates and causing serious damage to date palm trees in southern Tunisia. Considering the economic importance of this pest, it is necessary to understand the genetic diversity within and among its populations and investigate its dispersal mode. In the present study, the mitochondrial gene encoding the cytochrome oxidase subunit I (COI) was used to analyse the extent of genetic variation between samples of O. agamemnon arabicus, collected from geographically distant locations in Tunisia. A 505 bp DNA fragment, resulting from PCR amplification, was sequenced. Representative sequences were aligned, revealing nine polymorphic sites that identified eight haplotypes. Haplotype diversity (Hd) and Tajimas D neutral test were 0.707 and -0.043, respectively, indicating a low level of mtDNA variability. The calculated gene flow value was Nm = 0.47, indicating a high amount of gene flow occurring between populations from Tozeur and those from Kebili, although both departments are separated by the Chott-El-Jerid salt lake. The presence of common haplotypes between Tozeur and Kebili as well as the clustering pattern obtained by the NJ method confirmed this result. Based on these facts and considering the limited flight of O. agamemnon arabicus and its slow reproduction, it is likely that its expansion in Tunisia was facilitated mainly by human transportation of infected offshoots.

Genetic Variability of the Tomato Leaf Miner (Tuta absoluta Meirick; Lepidoptera: Gelechiidae), in Tunisia, Inferred from RAPD-PCR

El minador de hojas de tomate Tuta absoluta Meyrick ha invadido el cultivo del tomate (Solanum lycopersicum L.) en Tunez desde 2008 y actualmente representa una importante amenaza para su produccion. En este estudio usamos la tecnologia de ADN polimorfico amplificado al azar-reaccion de cadena polimerasa (RAPD-PCR) para evaluar la variabilidad genetica dentro y entre siete poblaciones de T. absoluta, colectadas desde tomate en diferentes regiones de Tunez. Usando cinco primers RAPD-PCR y 108 individuos, se registraron 140 fragmentos polimorficos. Se generaron 335 fenotipos RAPD diferentes, entre los cuales 71 fueron redundantes y 264 unicos para una poblacion especifica. La estructura genetica de T. absoluta se investigo usando analisis de varianza molecular (AMOVA), distancias geneticas (Fst) y escalamiento multidimensional (MDS). Detectamos una alta diversidad genetica dentro y entre poblaciones en conjunto con una diferenciacion significativa entre poblaciones, sugiriendo que los genotipos fundadores podrian haber sido responsables de la introduccion de T. absoluta en Tunez. La presencia de fenotipos superpuestos probablemente indica eventos de migracion entre poblaciones, principalmente a traves de material vegetal infestado transportado por humanos.

Characterization of novel wheat NBS domain-containing sequences and their utilization, in silico, for genome-scale R-gene mining

AbstractnIn crop improvement, the isolation, cloning and transfer of disease resistance genes (R-genes) is an ultimate goal usually starting from tentative R-gene analogs (RGAs) that are identified on the basis of their structure. For bread wheat, recent advances in genome sequencing are supporting the efforts of wheat geneticists worldwide. Among wheat R-genes, nucleotide-binding site (NBS)-encoding ones represent a major class. In this study, we have used a polymerase chain reaction-based approach to amplify and clone NBS-type RGAs from a bread wheat cultivar, ‘Salambo 80.’ Four novel complete ORF sequences showing similarities to previously reported R-genes/RGAs were used for in silico analyses. In a first step, where analyses were focused on the NBS domain, these sequences were phylogenetically assigned to two distinct groups: a first group close to leaf rust Lr21 resistance proteins; and a second one similar to cyst nematode resistance proteins. In a second step, sequences were used as initial seeds to walk up and downstream the NBS domain. This procedure enabled identifying 8 loci ranging in size between 2,115 and 7,653xa0bp. Ab initio gene prediction identified 8 gene models, among which two had complete ORFs. While GenBank survey confirmed the belonging of sequences to two groups, subsequent characterization using IWGSC genomic and proteomic data showed that the 8 gene models, reported in this study, were unique and their loci matched scaffolds on chromosome arms 1AS, 1BS, 4BS and 1DS. The gene model located on 1DS is a pseudo-Lr21 that was shown to have an NBS-LRR domain structure, while the potential association of the RGAs, here reported, is discussed. This study has produced novel R-gene-like loci and models in the wheat genome and provides the first steps toward further elucidation of their role in wheat disease resistance.

A rapid diagnostic technique of Bactrocera cucurbitae and Bactrocera zonata (Diptera: Tephritidae) for quarantine application

Backround n nThe melon fruit fly Bactrocera cucurbitae and the peach fruit fly Bactrocera zonata are serious pests, native to Asia, that have recently invaded Egypt. In Tunisia, no report of these species has yet been made, but pest risk data suggest that both Bactrocera species are likely to establish in other countries of the Mediterranean region. n n n nResults n nA quick method, based on a PCR-RFLP of the mitochondrial COI gene, has been developed to enhance species identification for quarantine purposes. The restriction enzymes DdeI and XmnI were used to digest COI PCR products, enabling a rapid diagnosis of B. cucurbitae and B. zonata and their distinction from Tunisias most devastating fruit pest, Ceratitis capitata. n n n nConclusion n nThe simplicity and relatively low cost of this molecular approach will replace the need to rear immature stages through to adults for identification and will facilitate rapid quarantine decisions providing greater plant protection.

Tracking MicroRNAs with a Potential for Virulence Regulation in the Pea Aphid, Acyrthosiphon pisum Harris (Hemiptera: Aphidae), and the Asian Citrus Psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae)

The pea aphid, Acyrthosiphon pisum (Hemiptera: Aphidae), and the Asian citrus psyllid, Diaphorina citri (Hemiptera: Psyllidae), are two major pests that conjunct in their virulence pathways against hosts by settling a gene-for-gene interaction model. The most effective controlling method of these pests is the deployment of host resistance (R) genes, which strongly depends on both host and insect genotypes. MicroRNAs (miRs) are noncoding RNAs of ∼22 nucleotides inducing sequence-specific post-transcriptional gene silencing. The identification and analysis of miRs is nowadays a useful reverse genetics tool for studying insect virulence and host resistance pathways. in the present study, we utilized 15 virulence-regulatory miRs of the Hessian fly, Mayetiola destructor (Diptera: Cecidomyiidae), to search for homologies across 103 A. pisum mature miRs and 86 D. citri miR-like expressed sequence tags (ESTs), mined from several databases. Our results showed that reference miRs from M. destructor could be grouped with their most similar microRNA counterparts from A. pisum and D. citri within eight phylogenetic clades, and that seven of A. pisum miRs clustered with M. destructor ones at a similarity threshold of 90 %. Such structural similarities strongly suggest that different avirulence (Avr) genes from M. destructor, A. pisum and D. citri would be tightly regulated by a regulatory network including similar miRs. Our study offers a promising way to further explore the structural similarity between miRs toward their potential use in Integrated Pest Management (IPM).

Structure of the Black Bean Aphid Aphis fabae (Hemiptera: Aphididae) Complex, Inferred from DNA Barcoding

The black bean aphid, Aphis fabae Scopoli is a polyphagous aphid causing significant damage to several cultivated and uncultivated plants. In the present work, we have used COI barcoding to distinguish between three subspecies of A. fabae, frequently found in Tunisia, namely A. f. fabae, A. f. solanella and A. f. cirsiiacanthoidis. Results of our study revealed that COI barcoding was efficient to generate haplotypes discriminating A. f. fabae from the two remaining subspecies. Indeed, among seven haplotypes, identified and named H1–H7, five were found exclusively in A. f. fabae, one represented exclusively A. f. cirsiiacanthoidis, and one was shared by A. f. cirsiiacanthoidis and A. f. solanella. However, the differentiation of Aphis f. fabae from A. f. solanella and A. f. cirsiiacanthoidis was not further supported by a phylogenetic distinction, as it was revealed by neighbour-joining and maximum parsimony phylogenies. Such a coherent genetic structure of the A. fabae complex, not governed by the subspecies factor, as inferred from our present mitochondrial (mt)DNA data, is in agreement with previous analyses based on genomic DNA. Therefore, we hypothesize that the phenotypical and physiological processes having led to differentiation between subspecies would have arisen from weak selection pressures, thus resulting in little genetic change between subspecies. In combination with additional still-to-come data from additional markers, results of this study will be important to constitute a groundwork for pest integrated management.

Large-scale bioinformatic analysis of the regulation of the disease resistance NBS gene family by microRNAs in Poaceae.

In the present study, we have screened 71, 713, 525, 119 and 241xa0mature miRNA variants from Hordeum vulgare, Oryza sativa, Brachypodium distachyon, Triticum aestivum, and Sorghum bicolor, respectively, and classified them with respect to their conservation status and expression levels. These Poaceae non-redundant miRNA species (1,669) were distributed over a total of 625xa0MIR families, among which only 54 were conserved across two or more plant species, confirming the relatively recent evolutionary differentiation of miRNAs in grasses. On the other hand, we have used 257xa0H.xa0vulgare, 286T.xa0aestivum, 119xa0B.xa0distachyon, 269xa0O.xa0sativa, and 139xa0S.xa0bicolor NBS domains, which were either mined directly from the annotated proteomes, or predicted from whole genome sequence assemblies. The hybridization potential between miRNAs and their putative NBS genes targets was analyzed, revealing that at least 454xa0NBS genes from all five Poaceae were potentially regulated by 265xa0distinct miRNA species, most of them expressed in leaves and predominantly co-expressed in additional tissues. Based on gene ontology, we could assign these probable miRNA target genes to 16xa0functional groups, among which three conferring resistance to bacteria (Rpm1, Xa1 and Rps2), and 13xa0groups of resistance to fungi (Rpp8,13, Rp3, Tsn1, Lr10, Rps1-k-1, Pm3, Rpg5, and MLA1,6,10,12,13). The results of the present analysis provide a large-scale platform for a better understanding of biological control strategies of disease resistance genes in Poaceae, and will serve as an important starting point for enhancing crop disease resistance improvement by means of transgenic lines with artificial miRNAs.